Biochimie 71 (1989) 125-135 © Soci6t6 de Chimie biologique/Elsevier, Paris

125

Study of a 8-hydroxy ketone-hemiketal equilibrium by 2D-NMR exchange spectroscopy for the antibiotic griso- rixin: involvement in cationic transport through membranes

Jean-Claude BELOEIL l, Nelly MORELLET 1, Corinne PASQUIER l, G6rard DAUPHIN 2. and Georges JEMINET 2

1Laboratoire de R~sonance Magndtique Nucldaire, Institut de Chimie des Substances Naturelles, F-91190 Gif-sur-Yvette; and 2Laboratoire de Chimie Organique Biologique, Unitd de Recherche Associde au CNRS 485, Universit(. Blaise Pascal, F63177 Aubidre Cedex, France

(Received 10-1-1988, accepted after revision 15-3-1988)

Summary m Study of a 8-hydroxyketone-hemiketal equilibrium in the polyether antibiotic grisorixin was performed with 2D-NMR spectroscopy.. The efficiency of 13C chemical exchange spectroscopy for the assignment of 1H and 13C resonances, in the 2 forms, was shown, making possible a conformational investigation of both forms.

This equilibrium was observed for grisorixin in solvents of varying polarity, such as CD2C12, C D C I 3, CDaCN , o r CD3OD, but not in C6Ol2 or C6D6 . Other related antibiotics with the same terminal hetero- cycle were described only in the closed hemiacetalic structure.

The low ionic fluxes measured in a bulk chloroformic membrane for grisorixin were explained by this equilibrium, which competed unfavorably with the cation capture process at the water-chloroform inter- face. This equilibrium would not be present in aphospholipidic bilayer membrane Containing the iono- phore, published experimental results are taken into account.

Th . . . . . . l ;nr t n , , ) n m o r i c ' Pquilihrium observed for grisorixin could be linked to the specific axial • g a y . , I , I ~ . ~ . ~ . ~ a a L ~ A . . . . . . . . . . . . . . . . . . ~ . ,

stereochemistry of the C7-C8 bond, which creates tension in the globular conformation.

2D-NMR exchange spectroscopy / grisorixin / hydroxyketone-hemiketal tautomerism

Introduction

Polyether antibiotics of the nigericin group are a large and growing class of naturally occurring acid ionophores, which have recently been review- ed [1]. Structurally, they consist of oxygenated heterocycles linked together to fo rm an open- chain multidentate system. Many of them have a 6-membered hemiketal ring in the terminal position. Accordingly, keto-hemiketal tautome- rism would be expected to occur at this site. Despite numerous 1H and 13C t f11j NMR studies in the polyether antibiotic field, under a variety of experimental conditions, to our knowledge such a tautomerism has never been explicitly reported.

The ionophore properties of grisorixin 1 [2] (Fig. 1) (which is closely related to nigericin 2 R - CH2OH), were recently studied by our group in a bulk chloroform membrane by the U-tube system [3]. We knew grisorixin to be an efficient K + carrier in biological membranes, comparable to nigericin [4], and this property was confirmed in planar bilayer lipid membranes [5]. Hence, the surprisingly low ionic fluxes measured for this compound with K + in chloroform prompted us to examine its structure in this organic phase.

We describe here a study of the behavior of the acid form of grisorixin in polar solvents by 1D and 2D high-field NMR techniques. The familiar 1H [6] and the less common 13C [7] exchange 2D spectroscopy were used.

*Author to whom correspondence shouM be addressed.

126 J.C. Beloeil et al.

M ,~ Me M e M e M e M e e ~,,F~ 1 7 . I m s l g t • z'l j p 1t

40 le II - - - ,3 , , - . , , , , . _ g'cCZ • '~ ' -= '~ ~ , , - ~ ,,zl ~ ! \ A, I e I

. , " . _ l Z ~ H n H . O H

~ f ; " It, , ~_ 1 R : M e H H H H

COOH 2 R= CH2OH 1

M,," Me

1 R_-Me O H

Fig. 1. Ketone-hemiketal exchange of grisorixin R = Me ! closed form (40%), 1' open form (60%), 2 nigericin (closed form only).

!a this paper, we emphasize that the use of chloroform as a model membrane for ion trans- port studies, though very useful in the ionopho- rous antibiotic field, should be systematically accompanied by an N M R of the carrier structure in the same phase and must be coupled with measurements in more relevant model membra- nes to avoid imprecise conclusions.

Materials and methods

P r o d u c t i o n o f gr isorix in acid 1

Grisorixin was from the stock sample of the Labora- toire de Chimie Organique Biologique, Aubi/~re, [2]. It was obtained from a culture of the strain Strepto- coccus griseus 2142 N6.

• V Z v J t l i ~ l J l ~ l . , i ~ l U

All the spectra were recorded on a Bruker WM 400 spectrometer. CDCI 3 was purified by filtration on K2CO 3. 0.5 cm 3 of a 200 mM solution of grisorixin acid was used for ~H and ~3C spectra.

- t H - IH chemical shift correlation (COSY) The applied pulse sequence was (lr/2)-(tO-(~r/4)- (FID, t2); the spectral width in F1 and F2 4000 Hz; the number of data points in t2 was 2048; and 512 increments were recorded. Before Fourier transfor- mation, the data were multiplied with an unshifted sine bell. The rr /2 pulse width was 9.5 p,s.

- 1H- 13C chemical shift correlation The applied pulse sequence was (~ /2 , 1H)-(h/2)-(7r, 13C)-(tl/2)-(D1)-(rr/2, IH, 7r/2, 13C)-D2)-(BB, SH; FID, t2) with D1 = 0.00357 s and D2 = 0.001785 s. The spectra width in F1 was 1650 Hz and in F2 11905 Hz; the number of data points in t2 was 4096, and 256 increments were recorded. Before Fourier transformation, the data were multiplied with an unshifted sine bell in F2 and Lorentz-Gauss in F1. The 7r/2 pulse width was 7.6/zs and the decoupler ~ ' /2 pulse width was 10/zs.

- t H - ; H chemical exchange correlation The applied pulse sequence was (Tr/2)-(tl)-(Tr/2) - (tm)-(-rr/2)-(FID, t2) with t m = 0.3 S (pseudo-random variation of 20%). The spectral width in F1 and F2 was 4000 Hz; the number of data points in tz was 2048, and 512 increments were recorded. Before Fourier transformation the data were multiplied with an unshifted sine bell.

- t3C- t3C chemical exchange correlation The applied pulse sequence was (~-/2, 13C)-(tl)- (7r/2, 13C)- tm)-(Tr, 13C)-(FID-t2) with tm -- 0.1 S. Broad band ~H decoupling was applied throughout. The spectral width in F1 and F2 was 11,905 Hz. The number of data points in t2 was 2048, and 512 incre- ments were recorded. Before Fourier transformation the data were multiplied with an unshifted sine bell.

Results

The IH and 13C 1D NMR spectra of the chloro- form solution of the acid form of grisorixin (Fig. 2a, b) are very complex. The number of signals in the ~3C spectrum points to the occurrence of 2 exchanging forms of the molecule in the slow exchange limit (Fig. 2b).

~H 2D exchange spectroscopy correlates signals through exchange of magnetization. There are 2 main possibilities: nuclear-Overhau- ser effect, and chemical exchange. In our case, we checked that in solvents there is no chemical exchange for grisorixin, with the same timing as the pulse sequence, cross-correlation peaks were not detectable. COSY-like peaks from 0 quan- tum coherences were strongly attenuated by pseudo-random variation of the mixing time, and superimposition of COSY and exchange 2D spectra eliminated any confusion. The use of the ~3C version of this experiment, already reported [7], has several advantages: for example, because of the low proportion of the ~3C isotope, there are no COSY-like peaks or cross-correla- tion peaks in the exchange spectrum.

2D-NMR exchange spectroscopy of ionophore grisorixin 127

, ' , " " I , , . " ' 1 " " " • " " ' " ' • I • " • " • = " ~' ' " ' I " . . . . . . . " I " ~' " " " "

4 .0 3 .0 2 .0 t .O PPH

a)

• • • | • • i • • • • • 71

200 .0 PPN

1 b,

100.0

Fig. 2. Grisorixin ~H and ~3C NMR spectra, a ~H specfrum of I + 1' in CDCI3. b ~3C spectrum of 1 + 1' in CDCI~.

We set out first to identify the exchanging species, then to obtain information on the conformation in solution of these molecules. A rough value of the mean lifetime was obtained through line-width measurements on the 1H spectrum, which showed that exchange was very slow compared with ~H and 13C experiments at 9.3 T. Therefore, COSY (Fig. 3a) and 1H-13C shift correlation experiments (Fig. 4) consisted of the superimposition of 2 spectra.

Two strategies were possible for the assign- ment of the ~3C and 1H NMR spectra by 2D spec- troscopy. The first consisted of a 8-8 ~3C correla- tion (INADEQUATE), leading to the complete assignment of ~3C NMR resonances, followed by IH-13C chemical-shift correlation for total assignment of the 1H NMR spectrum [23]. The second consisted of the parallel use of homo- and heteronuclear chemical-shift correlation ~H-~H

and IH-13C [8]. We chose the latter strategy to include exchange experiments because it required far less grisorixin; furthermore, the 8-8 ~3C correlation spectrum was very complex when the number of signals was considered.

For each form of the molecule 1 and 1' the COSY spectrum (Fig. 3a) shows 4 groups of correlated signals corresponding to coupled proton groups separated by quaternary carbons. Ascription of these groups to each form of the molecule was obtained from signal integration. The 2 forms are present in the ratio 60:40. Carbon assignments were deduced from lH-13C chemical shift correlations (Fig. 4). ~H and 13C exchange experiments (Fig. 3b, 5), allowed the first probable assignment to be confirmed. ~H and 13C exchange 2D spectra give the correlation between 2 signals A and B through chemical exchange: the closer the A and B sites are to the

128 J.C. Beloeil et al.

i .' 11 ,: ;, , ,,, ]- I | 1 ! I O__l I I I I I ! ,

4 , I I ~ , I I o I I o

I I o I I I o I

I I

I n

! o ! i

, ,, !

o I ! n ,p

o I

If,, I: !

1 ! ! i '-

I o ! !

i I I

! o I I

! !

' 121', I i

I ! ! ! !

I I

, I . I -f

PPN 1 . 7 ] | . S i . 5

i

!

ID

J

a

b

Fig. 3. a COSY (400 MHz, CDCI3) spectrum of grisorixin 1 + 1' (low-field region) presented as a contour plot (/t LH in 2 dimensions), b Corresponding exchange spectrum (', open form).

part of the molecule modified by the exchange reaction, the higher the value of VA-V B. From this and from comparison with grisorixin spectra in solvents where there is no exchange (C6D6, C6D12 ) (Table I) and with grisorixin salt spectra [8b], it is clear that only ring F is modified during the exchange reaction and one of the 2 exchang- ing forms is the classical hemiacetal form (Fig. 1).

The occurrence of a carbonyl carbon (13C = 213.9 ppm), without disappearance of H25, and chemical shift variation of Me30 (alH, 1.29 ppm-2.12 ppm), shows that ring F has open- ed into a ketonic form (Fig. 1). For example, where a prime denotes the ketone form, correla- tions between H28-Me31 , H28'-Me31' , H24_H25, Ha4'-H25' are clearly seen in the COSY spectrum (Fig. 3a) and corresponding exchange correla- tion between Meao-Me30, Me30-Me30', Meat Me31', HE4-H24', HEs-H25' (Fig. 3b) can be found in 1H exchange spectra. The 13C exchange spec- trum shows the marked variation of the chemical shift of the carbons of ring F, C28-C28' , C25-C25' (Fig. 5). It is noteworthy that because of the broadening of IH signals by multiple scalar cou- plings, certain correlations are not seen in the 1H exchange 2D spectrum, for example HEs-H28'. Conversely, in laC exchange spectra (Fig. 5) practically all exchanging pairs are visible, and so we can say that the ~3C version is markedly more informative then the ~H version, and is especially useful for the assignment of quater- nary carbons and singlet methyls, _Aeenrding tn the method described above, signals of the 88 XH and 80 carbons of the 2 forms were assigned (Table II).

Discussion

After exterJsive study of grisorixin acid 1 in CDCI3, its behavior in other solvents was studi- ed. In dipolar solvents such as CD3CN, slightly dipolar solvents such as CDzCI2 and protic solvents (CD3OD), grisorixin exhibits the 2 exchanging forms described above. These results show that unavoidable traces of acid in CDCI 3 do not p;ovide the sole explanation for the behavior of grisorixin in CDCI 3. Conversely, in nonpolar solvents, such as C6D 6 (and C6D12 ) (Table I), only the closed hemiacetal form of ring F is encountered. Thus as expected [9], it is the pola- rity of the solvent that favors this ring-chain tautomerism. Except for proximity effects due to ring F opening, IH and 13C chemical shifts (Tables I, II) and IH-IH scalar coupling cons-

8 ,=t

_

Z

~ .

0

,-I =.

>d

• --

+ o 8 °

I 1H

I

,

lsA

,se

a I

I I

. ...

...--

m

I I

I I

I I

I ii

j |

I='=

='11

I

I I

I "-

-"

I -

-

I

i I

~ I

a i

i I

I I

~ i

I I

i I

I I

' I

I I

i i

I I

I -

-

~ I

m~

| I

I I

I I

I I

I I

a I

I I

m

m

m

i ,,

m=

,.,

N

e. I

.a~

..

=,.-

.

m

m

lllil •

•

ma

~.

I,

•

D

u

I

m

! '

1.0

1 ~.

0

~'P

N

I 2.

0 I

1.0

I,j

n

~=

=-

--

-

N C

~

~ q

l' 1ira

~J

EI

N

--I --I'

,ira

-° t

N

0 o

~

÷ x

°~

L

E

,Z

N ~ E

°~

c~E

E~

~ L

~ 0

2D-NMR exchange spectroscopy of ionophore grisorixin 131

tants (Table Il l) of the 2 forms of grisorixin acid are very similar to the NMR parameters of griso- rixin potassium salt [8b]. Thus, the conforma- tions of the 2 forms of grisorixin are very close to the globular form of grisorixin potassium salt [8bl.

Table 1.1Ha and ~3Cb chemical shifts of grisorixin 1 in C6D6".

No. C 8 13C 8 IH

1 176.4 2 44.6 2.66 3 73.5 3.97 4 27.9 1.58

5A-5B 26.5 1.81-1.37 6A-6B 23.9 1.99-1.09

7 68.7 4.03 8A-8B 36.1 2.77-1.06 9 60.9 4.47

10A-10B 31.7 2.34-0.92 11 77.7 3.15 12 37.6 1.70 13 108.7 14 39.4 2.16 15A-15B 43.0 1.87-1.77 16 81.8 17 82.7 3.47 18A- 18B 25.8 2.07-1.63 19A-19B 30.7 2.46-1.37 20 83.6 21 86.0 4.27 22 35.3 2.10 23A-23B 32.5 2.52-1.36 24 77.8 4.37 25 74.4 4.39 26 33.2 1.35 27A-27B 37.7 1.83-1.41 28 40.5 1.70 29 97.4 3(1 27.3 1.85 31 17.2 1.28 32 17.4 0.78 33 15.7 0.86 34 20.6 "1.09 35 28.5 1.94 36 13.3 1.12 37 13.5 1.15 38 11.0 0.90 39 13.1 1.11 40 57.6 3.54

"8 ~H attribution by COSY spectrum for 1 in C6D~2 and S ECSY spectrum for I in C 6 D 6 •

hl3C attribution by J-modulated spin echo spectrum and 2D tH-13C chemical shift correlation. • Close values were obtained in C~D~2 and are not given.

Other related antibiotics with the same struc- ture on the terminal anomeric carbon, and a similar linked heterocycle sequence, such as mutalomycin [10], etheromycin [11], K41 [12], 6016 [13], emericid [14], carriomycin [15], A. 204A [16], septamycin [17], and A. 28695B [18], and even nigericin [19] which is very akin to gri- sorixin, do not show this equilibrium, judging from the published NMR spectra. The stereo- chemistry of the chain segments linked to the heterocycles is identical for all the above-mention- ed antibiotics, except for grisorixin and nigeri- cin, where substituents in position 3 and 7 of ring A are trans to each other. The axial position of bond 7-8 in grisorixin may destabilize the hemia- cetal form of ring F through an increase of tension in the globular form required to achieve head-to-tail chelation, which occurs in the crys- talline acid form [20] (Fig. 6). Nigericin has 2 hydroxyl groups and hence there are 2 opportu- nities for stabilization of the conformation by additional hydrogen bonds.

6r~ .O s\AZ f"" ' F . . . . . .

Fig. 6. Schematic representation of the crystalline acid form of grisorixin, with an enclosed water molecule [20].

Further, a general feature has recently emer- ged from X-ray studies of model hydroxylacto- nes and hemiketals showing ring-chain tautome- rism [21]; namely, the specific orientation of a lone pair of exocyclic oxygen almost exactly antiperiplanar to the endocyclic C-0 bond. This geometrical requirement can be met in grisorixin (Fig. 7), given the direction of the head-to-tail hydrogen bonding, providing an effect comple- mentary, to the conformational strain already mentioned.

132 J.C. Beloeil et al.

Table Ii. IH and ~3C chemical shifts of grisorixin I + 1' in CDCI3.

Closed form I Open form 1'

No. C 8 IH 813C 8 IH 813C

1 176.12 175.61 2 2.33 44.16 2.33 44.09 3 3.77 73.15 3.73 73.67 4 1.80 27.90 1.78 27.62

5A-5B 1.98-1.50 26.19 1.98-1.50 26.19 6A-6B 1.99-1.13 23.75 1.98-1.12 23.45

7 4.07 68.98 4.07 68.79 8A-8B 2.48- 0.96 35.56 2.45 - 0.96 35.79 9 4.15 . 60.42 4.10 60.42 10A- 10B 2.23-0.99 31.89 2.23-1.04 31.74 11 3.39 77.95 3.34 78.9(t 12 1.75 37.24 1.75 37.01 13 108.23 107.63 14 2.09 39.11 2.09 39. i9 15A-15B 1.74-1.56 42.21 1.74-1.58 42.13 16 81.81 89.'71 17 3.54 82.35 3.46 83.45 18A- 18B 1.82-1.68 25.71 1.82-1.62 27.57 19A- 19B 2.15-1.36 30.74 2.26-1.36 31.96 20 83.63 84.71. 21 4.05 85.97 3.94 85.76 22 2.24 35.12 2.24 35.56 23A-23B 2.28- i.37 32.67 2.28- i.36 3i .74 24 4.33 77.54 4.22 78.97 25 3.97 74.40 3.57 70.43 26 1.24 32.95 1.24 33.40 27A-27B ! .40- ! .31 37.20 2.18-1.03 38. i3 28 1.49 39.67 2.73 45.93 29 96.93 213.90 30 1.29 26.91 2.12 26.66 31 0.89 16.81 1.07 17.33 32 0.84 17.50 0.82 16.67 33 0.87 15.61 0.87 15.59 34 1.11 22.51 1.16 24.48 35 1.42 27.74 1.38 28.23 36 0.88 13.15 0.88 12.94 37 1.01 12.84 1.04 13.10 38 0.91 10.90 0.92 11.21 39 1.02 12.94 1.02 13.20 40 3.33 57.53 3.41 57.35

8 in ppm from internal TMS. Codings A and B refer to the proton at lowest and highest field side, respectively.

2D-NMR exchange spectroscopy of ionophore grisorixin 133

Table I l l . A p p a r e n t coupling constants J (Hz)* of grisorixin 1 in C6D 6 and grisorixin 1 (closed form) + 1 (open form) in CDCI3.

1H J J J

C6D6(1 ) CDCI 3 (1) CDCI 3 (1')

2, 3a 10.3 10.5 10.5 2, Me39 7.0 7.1 7.1

3a ,4e 2.8 2.5 2.7 4e, Me38 7.0 7.0 7.0 7e, 6Aa 5.8 - - 7e, 6Be 1.0 - - 7e, 8A 13.1 13.5 13.5 7e, 8B 3.7 - - t~A, 8B 13.2 13.5 13.5 8A, 9a 4.8 4.5 4.5 8B, 9a 11.0 11.0 - % , 10Ae 2.0 2.0 - 9a, 10Ba 11.0 11.0 -

10A, 10B 14.0 - - 10Ae, l l e 3.0 3.0 3.0 10Ba, l l e 3.0 3.0 3.0

l i e , 12a 3.0 3.0 3.0 12a, Me 37 7.2 7.0 7.0 14, ! 5 A 12.0 - - 14, 15B 8.9 12.0 12.0 14, Me36 7.0 6.5 6.5

15A, 15B 12.0 12.0 12.0 17, 18A 5.3 10.0 11.5 17, 18B 30.2 5.8 4.0

18A, 18B 11.0 - - 18A, 19A 11.4 - - 18A, 19B 1.0 - - 19A, 18B 3.2 - - 19A. i 9 ~ 11.4 - - 21,22 4.0 4.0 4.0 "~ 23A 7.0 - - 2 , , ~ _ _ .z., 23B 1.0 22, Me33 7.0 7.0 7.0

23A, 23B 12.3 12.0 12.0 23A, 24 9.0 9.0 9.5 23B, 24 7.0 6.5 6.5

24, 25a 3.0 2.5 2.1 25a, 26a 10.0 9.0 10.2

26, Me32 6.2 6.0 7.0 26, 27A 12.0 - -

28a, Me3! 6.7 - - - 9.0 28, 27A - 6.0 28, 27B

*Coupling constant determination by 1D, 2D spectra and selective irradiation, a, Axial; e, equatorial.

134 J.C. Beloeil et al.

C29 C28

H !

Me30 Fig. 7. Newman projection down the O-C,9 "anomeric'" bond.

Conclusion

2D spectroscopy is gradually becoming a ~'outine technique. In the study of a chemical exchange in the slow exchange limit, difficulty in interpre- tation is increased 2-fold (twice the number of ~H and ~3C assignments). We have shown that ~3C chemical exchange experiments can be of great help in addressing this problem by allowing the 2 exchanging forms to be identified in the ~3C spectrum and, through heteronuclear chemical shift correlation, in the ~H spectrum.

Considering the transport problem through a bulk chloroformic membrane, cation capture a n d ,-nmp!exation at the water-chloroform inter- face during the transport process ought to be quite different for the 2 forms, which very proba- bly explains the low ionic fluxes measured [3]. In this connection, we have shown in previous works that the opening of the terminal hemiace- talic cycle F by sodium borohydride reduction, giving a dihydro derivative, either for grisorixin [4] or for nigericin [22], orastically reduced the ionophoric properties of the natural metabolite. We explained that the terminal heterocycle F plays an important role in the transporting capa- bility of the skeleton [22]. However, in mito- chondrial membrane, for instance, there was no marked difference in K ÷ efflux induced by griso- rixin [4] or nigericin [22] under the same experi- mental conditions. Likewise, Amblard et al. [5] measured a high flux for K ÷ in bilayer lipid membranes (BLM) with grisorixin. This led us to conclude that in a more relevant phospho- lipidic membrane the hydroxyketone-hemiketal equilibrium observed in chloroform was not markedly present, with the bilayer membrane, for this purpose, being closer to a nonpolar aprotic solvent.

References

1 Westley J.W. (1982) In: Polyether Antibiotics: Naturally Occurring Acid lonophores (Westley J.W., ed.), Vol. 1 (Biology), Marcel Dekker, New York, pp. 1-465

2 Gachon P., Kergomard A., Staron T. & Este- ve C. (1975) J. Antibiotics 28,345-350

3 Bolte J., Caffarel-Mendes S., Demuynck C. & Jeminet G. (1983) Stud. Phys. Theor. Chem. 24. 247-253

4 Chapel M., Jeminet G., Gachon P., Debise R. & Durand R. (1979) J. Antibiotics 32,740-745

5 Amblard G., Sandeaux R., Sandeaux J. & Gavach C. (1985)J. Membr. Biol. 88, 15-23

6 (a) Jeener J., Meier B.H., Bachman P. & Ernst R.R. (1979)J. Chem. Phys. 71,4546-4549 (b) Huang Y., Macura S. & Ernst R.R (1981) J. Am. Chem. Soc. 103, 5327-5333

7 Benn R. (1982) Angew. Chem. Int Ed. 21, 626-627

8 ta) Beloeil J.C., Delsuc M.A., Lallemand J.Y., Dauphin G. & Jeminet G. (1984)J. Org. Chem. 4a, 1797-1800 (b) Cuer A., Dauphin G., Jeminet G., Be- loeil J.C. & Lallemand J.Y. (1985) Nouv. J. Chem. 9, 437-441

9 (a) Whiting J.E. & Edward J.T. (1971) Canad. J. Chem. 49, 3799-3806 (b) Hurd C.D. & Saudners W.H. (1952) J. Am. Chem. Soc. 74, 5324-5329

10 (a) Fehr T., King D.H. & Kuhn M. (1977) J. Antibiotics 3u, 9u3-go/ (b) Fehr T., Keller-Juslen C., Loosli H.R., Kuhn M. & Von Wartburg A. (1979) J. Antibiotics 32, 535-536

11 Anteunis M.J.O. & Rodios N.A. (1978) Bull. Soc. (?him. Belg. 87,753-764

12 (a) Shiro M., Nakai H., Nagashima K. & Tsu- ji N. (1978) J.C.S. Chem. Comm. 682-683 (b) Tsuji N., Nagashima K., Terni Y. & Tori K. (1979) J. Antibiotics 32, 169-172

13 Otake N., Ogita T., Nakayama H., Sato S. & Saito Y. (1978)J.C.S. Chem. Comm. 875-876

14 (a) Rodios N.A. & Anteunis M.J.O. (1978) Bull. Soc. Chem. Belg. 87,447-457 (b) Le Cocq C. & Lallemand J.Y. (1981) J.C.S. Chem. Comm. 150-152

15 Rodios N.A & Anteunis M.J.O. (1978) Bull. Soc. Chim. Belg. 87,437-446

16 Anteunis M.J.O. & Verhegge G. (1977) Bull. Soc. Chim. Belg. 86, 353-366

17 Rodios N.A. & Anteunis M.J.O. (1978) J. Anti- biotics 31,294- 301

18 Dorman D.E., Hamill L., Occolowitz J.L., Terui Y., Tori K. & Tsuyi N. (1980)J. Antibiotics 33,252-255

19 Rodios N.A. & Anteunis M.J.O. (1977) Bull. Soc. Chim. Belg. 86, 917-929

2D-NMR exchange spectroscopy of ionophore grisorixin 135

20 Alleaume M. (1974) Abstracts of Second Euro- pean Crystallographic Meeting, European Com- mittee of Crystallography, Keszthely, Hungary, 405-407

21 Chadwick D.J. & Whittleton S.N. (1984) J. Chem. Res. 398-399

22 David L., Chapel M., Gandreuil J., Jeminet G. & Durand R. (1979) Experientia 35, 1562-1563

23 Ajaz A.A., Robinson J.A. & Turner D.L. (1987) J. Chem. Soc. Perkin Trans. I, 27-36