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Biosynthetic Investigations on Pyridazomycin.

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the use of the stereochemically pure compounds anti-1 and anti2, a large number of stereoisomers of anti-5 are possible. These
isomers are only in part due to the exolendo attack that occurs
in the Diels-Alder reaction. In our case, the adducts are solely
bicyclic compounds. with possible formation of the isomers
shown in Scheme 2. Here the s y n and anti designations indicate
the relative positions of the oxygen and vinylene bridges.
- H
....I I I
- H
: ... .-..
1986, 51.4160-4175.
[3] S. Wegener, K . Mullen, Chcjm. Ber. 1991, i24. 2101 -2103; Mucroniulrculcs
1993. 26, 3037 3040; T. Horn, K. Miillen. ibid. 1993, 26, 3472-3475.
(41 H. Schultz. H. Lehmann. M. Rein. M. Hanack. Strucr. Bondrnp (Berlin) 1991,
74. 41: C. Feucht, T. Linssen, M. Hanack. Chetn. Eer. 1994. 127. 113-117.
[5] M. Hanack, K . Haberroth, M. Rack, CImn. Err. 1993. 126. 1201 -1204: A.
Ferencz. R. Ries. G . Wegner. Angew. Ckem. 1993, iO5. 1251-1253; Angcw.
Chon Int. Ed EngI. 1993, 32. 11 84- 1187.
[6] M. Rack. M . Hanack. unpublished: M. Rack. Dissertation, Universitit
Tubingen. 1994.
[7] R. Gabioud, P. Vogel, TEtrahedron 1980, 36. 149-154.
[8] The measured UVWIS spectra of oligomers 5-9 are equivalent to those of the
starting monomers 1 and 2. Aggregation of 5-9 WAS not observed.
[9] In all reactions only a single product was obtained. The decrease in the yield
can he attributed t o the formation of higher molecular weight oligomers, which
remain on the coluinn during chromatographic purification.
[lo] Formation of ring structures was not observed in any of the conversions. The
direct reaction of the .YW isomers of 1 and 2 (equimolar) at ambient pressure
also did not lead to the formation of ring structures.
[ l l ] Determination of the molar masses was conducted using an API Ill TAGA
6000E mass spectrometer from the company Sciex. Samples in MeOHiCH,CI,
( 1 : l ) were injected with an injection pump at a continuous flow rate of
5 pLmin-'.
1121 Molecular weights were determined by matrix-supported laser desorption mass
spectrometry using a VESTEC Laser-Tec focus (BenchTop 11) instrument.
Measurement conditions: positive ion mode, 26 K V acceleration current; matrix: sinapic acid and 2.5-dihydroxybenzoic acid.
[13] Assuming ideal stereochemistry in the Diels-Alder reaction for anti-5 (svnrwk-H). the molecule would have a length of approximately 6.3 nm.
1141 E. Agostinelli. D. Attanasio. I. Collamati, V. Fares. Inorg. Chem. 1984, 23.
1 162- 1 165
1151 X-ray structure data of anti-1 : Enraf-Nonius CAD-4 diffractometer. room
temperature. Cu,, radiation. j . = 1.5405 structure solution with SHELX 86;
triclinic, space group PT. u = 9.32411). h =12.381(2), i =12.720(2) A.
2 =7290(1), /J' =73.92(1), 7 = 85.05(1) , V=1348.66A3. Z = 2, pidlrd=
1.395 gcm-', 6002 reflections of which 4878 with I > . i p ( l ) were observed.
R = 0.055. R, = 0.056. Further details of the crystal structure investigation
may be obtained from the Fachinformationszentrum Karlsruhe. D-76344
Eggenstein-Leopoldshafen ( F R G ) . on quoting the depository number CSD58252
1161 Assignment of the two configurational isoiners of 2 WAS verified by comparing
the 'H NMR spectra of rmti-2 and the product of the reaction of anti-1 with 3.
1171 P R. Ashton. G. R . Brown, N. S. Isaacs, D. Giufrida, F. H. Kohnke. J. P.
Mathias. A . M . Z. Slawin. D. R. Smith. J. F. Stoddart. D. J. Williams, J Am.
Ci7en7 So<. 1992, 114. 6.730-6353.
[ l X ] A. Bax, M . F. Summers. J. A m . Cliem. Su(c 1986. 108, 2093-2094.
(%~tn.Tech. Luh. 1990, 38, 8- 13; W. Christophel, L. C. Miller, J. Org. Chem.
.... 1 1 I
o n t i / endo - H
o n t i / ex0
- H
Scheme 2 Possible stereoisomers formed in the Diels-Alder reaction of bicyclic
enophile 2 and dienophile I
In comparable cycloaddition reactions the primary products
obtained are solely the synlendo-H isomers." 'I In our case, only
a single isomer of anri-5 should therefore be obtained; however,
there are more peaks in the N M R spectra of anti-5 than would
be expected from a single isomer. Based on the signal doubling
in the I3C N M R spectrum of anti-5, we conclude that at least
two isomers are formed. Determination of the exact structure of
these isomers would require that they be separated, crystallized,
and subjected to X-ray structure analysis. For the exact assignment of the I3C N M R signals of anti-5, C/H-COSY and C/Hcorrelated long range coupled N M R spectra were obtained.['*]
We are presently working on aromatization procedures for
the oligomers described here.
E,xperimental Procedure
5: 150 mg (0.16 mmol) of 1 and 80 mg (0.07 mmol) of 2 were dissolved in 20 mL of
toluene and stirred 7 d under reflux. After the reaction mixture had cooled. the
toluene was removed under racuum. The green residue was taken up in a small
amount of CH,CI,. precipitated by addition of hexane, and centrifuged. This process was repeated twice in order to remove residual I and 2. Further purification was
accomplished by column chromatography on alumina (deactivated with 5 % H,O)
eluting uith CHCI,. After chromatographic purification the compound was a p i n
precipitated in CH,Cl,/hexane and dried under vacuum a t 75 C. Compound 5 &as
obtained as an olive-green powder (145 ing, 73OA).
6 : 80 mg (0.26 pmol) of 5 and 12 mg (77 pmol) of 3 were dissolved in 10 inL of
toluene and stirred 48 h under reflux. The workup proceeded analogously as in the
preparation of 5. Compound 6 was obtained as an olive-green powder (70 mg.
87 "1"
7 9: Reaction and workup were analogous to those described for 5 and 6, with
yields of 65. 81. and 36%, respectivelq.
4: Reaction procedure identical to that for 5, except that equimolar amounts of I
and 2 were used. Purification was possible by continuous extraction with CH,CI,.
after which the solvent was removed under vacuum.
Received: March 3. 1994 [Z 6725 IE]
German version: Angen.. Cheni. 1994, 106. 1712
[I] S. Kivelson, 0. L. Chapman. Phrs. Re!,. B . Cond<,n.\. Muttcr 1983, 2K. 7263;
J L. Bredas. R. H. Baughman. .I Chem. P l i ~ s .1985. 83, 1316 1322, A. K .
Bakhshi. J. Ladik. S j n t h . Mrr. 1989, 30. 115 121: P. Gomez-Romero. Y . 4 .
Lee, M. Kertezs, Inorg. Clicm. 1988, 27, 3672-3675: K. Mullen, PUFCAppl.
C / w n . 1993. 0.5 ( I ) , 89-96.
[2] J. K. Stille. K. Plummer. J Org. C%rm. 1961, 26, 4026-4029: M. Wagner. W.
Wohlfahrt, K. Miillen, Chirnru 1988, 42, 377-379; A. D. Schliiter. Nadir.
VCH 0-69451 Weinhrim, 1994
Biosynthetic Investigations on Pyridazomycin""
Heike Bockholt, John M. Beale, and Jiirgen Rohr*
Dedicated to Professor Heinz G. Floss
on the occasion of his 60th birthday
The antifungal antibiotic Pyridazomycin 1, produced by the
soil bacterium Streptomyces violaceoniger ssp. griseofuscus
(strain TU 2557), is the only natural product containing a pyridazine ring."] The biosyntheses of this unusual nitrogen heterocyclic compound is of interest because of the features of the
[*I Priv.-Doz. Dr. J. Rohr. Dr. H. Bockholt
Iiistitut fur Organische Chemie der UniversitPt
Tammannstrasse 2. D-37077 Gottingen ( F R G )
Te1et:ax- Int code + (551)39-9660
Prof. Dr. J. M. Beale
Institute for Medicinal and Natural Product Chemistry
College of Pharmacy, University of Texas.
Austin, TX. 78712 (USA)
[**I This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds
der Chemischen Industrie. and NATO.
057U-0833;Y4;1515-i648 S 1U.00f ,2510
Angew. Clicm Inr. Ed. Engl. 1994. 33, No. 18/16
N - N bond and the amino acid side chain linked to a quarternary positively charged nitrogen atom. Structurally related
compounds with partially o r fully saturated pyridazine rings
occur in biologically active cyclopeptides, for example the moiety 2 in luzopeptin A,['] and piperazic acid (3) in citropeptin,
himastatin, variapeptin, and others.[31An N-N bond also appears in the pyrimidine nucleotide biosynthesis inhibitor pyrazofurin (4)i4'as well as in several noncyclic azoxy compounds,
such as valanimycin (5).rs-y1 Because of the N+-bound side
I ornithine / proline
+ ornithine
:oxalate etc.:
glycine /urea etc
ornithine / proline
chain. I exhibits chemical lability at a pH >6.5, limiting chemical derivatization approaches to improve the biological activity. Thus, we expected to gain information from the biosynthetic
studies which allowed selective biological derivatizdtion approaches, such as precursor-directed biosynthesis."O1 Here we
present the results of our initial feeding experiments from which
emerges an interesting novel biosynthetic pathway of general
For the biogenetic origin of all carbon and heteroatoms in
pyridazomycin 1 from building blocks o f the primary
metabolism. four principal working hypotheses were considered. in which I and 11 each require two, and 111 and IV, three
building blocks, preferably amino acids (Scheme 1 ) . A seemingly insurmountable microbiological problem, namely the low
production of pyridazomycin by Streptonyes violriceoniger of
0.75 mg L - '. was solved by strain selection experiments, extensive studies of the fermentation process (using a 1 L-fermentor),
and, in particular, by avoiding an alkaline pH during the isolation procedure." 'I This finally enabled us to isolate pyridazomycin 1 in amounts of 20-30 m g L - ' .
The above-mentioned four hypotheses were examined by a
series of feeding experiments with stable, isotopically labeled
precursors in such a way that basic information was made available quickly (Table 1 ) . Feeding experiments with '3C-labeled
acetate (universal precursor of the intermediates o f the citric
acid cycle and putative direct precursor of the C , building block
in hypothesis 111) and [1,3-i3C,]glycerol (universal precursor of
the carbohydrate metabolism and of amino acids deriving from
pyruvate, such as alanine, valine, o r leucine) provided "scrambling". in other words unspecific, indirect enrichments of several carbon atoms of 1, in particular C - 4 , C-5', C-6', and C-7'.
Both precursors are definitely not immediately incorporated.
.4irg<m (~'l!?!!!.
In:. Ed. Engl. 1994. 33. N o . 15/16
Scheme 1. Hypothetical precursors I-IV ofpyridazomycin 1. The numbers refer to
the remaining carbon atoms of a precursor in 1.
The C, amino acid glutamic acid serves as a metabolic precursor
of the four amino acids ornithine, glutamine, proline, and
arginine, in other words it has also the characteristic of a general
The feeding experiment with [5-' 3C]glutamic acid showed a
weak, but significant 13Cenrichment in the C-5 position of the
pyridazomycin side chain. Since C-7' of the carboxamide side
chain was not enriched, and consecutive feeding experiments
Table 1. Incorporation experiments on pyridazomycin 1 with . S : r q ~ ~ o n ! ~ VwI O\ luceonrger [a]
Specific incorporation
Enrichmenl in positions
[1.2-' 'CJacetate [b]
[1 .3-13C,]glycerol[c]
[S-"C]glutamic acid[d]
[5-"C]ornithine [el
[5-'5N]ornithine [f]
[2-"C]gIycine [g]
[2-'JC.'sN]glycine [h]
c - 4 , c-5'. c-6'. c-7'
c - 4 , c-5'.c-h'. c-7'
C-3'. N-2'
[a] Additionally, feeding experiments with [3-"C]-/I-alanine, ~-[?-~'C]leucine.
[I13C]glycine, ~,r-[4-"C]aspartic acid. ["C]urea, [S-l'CH,]methionine. l.-[S"N]glutamine, [2-' 5N]glutamine, and [2.3-2H,]succin~cacid, respectively. did not
result in the enrichment of any of the atoms of 1: [b] 12.2 mmol (99% "C): [c]
2.7 mmol ( 5 0 % "C); [d] 3.4mmol (99% I T ) : [el 5.9 mmol (99% I T ) : [f]
3.0 mmol(99%, "N); due to poor yield, the only signal observable In the "N N M R
spectrum IS N-1'; [g] 6.7mmol (99% " C ) . [h] 3.3 mmol (99% "C. 99% "N):
'J(C,N) = 2.6 Hz.
VCH ~rlugsjiesell~chrrft
rnhH, D-694Sl Weinhein!, 1994
0570-0833'Y4; 1515-1649 $ 10.00 + 25;O
with differently I 'N-labeled glutamine samples gave no incorporations, hypotheses I (or IV) for glutainine as one of the
building blocks of the aromatic ring had to be rejected.
Feeding experiments with [5-13C]- and [5-15N]ornithine,
however, confirmed the apparent idea that this amino acid
serves directly as the immediate building block of the side chain.
The former showed a significantly better incorporation than
[5-i3C]glutamic acid. The "N N M R spectrum of 1 isolated
after the experiment with [5-' 'Nlornithine shows that the nitrogen atom N-1' of the pyridazine ring derives directly from the
5-NH2 group of this amino acid. These incorporation experiments along with several additional negative results (see Table 1.
footnote [a]) finally ruled out the hypotheses I - I I I , thus, the
[5C + 4C + 1 C] hypothesis (IV) was favored and examined with
regard to the C, pool. Within this phase of the studies a feeding
experiment with [2-'3C]glycine (also useful as source of the tetrahydrofolic acid C,-metabolism) gave a high specific incorporation (ca. 10%) into the expected position. namely C-3' of 1 .
Thus glycine is the second biosynthetic building block of pyridazomycin 1. An additional feeding experiment with [23C.' 'Nlglycine confirmed this conclusion and furthermore
proved that N-2' also derives from this amino acid (I5N NMR)
and that the C-N bond of glycine remains intact, proven by the
observed coupling ('JC,R= 2.6 Hz) in the I3C N M R spectrum
of 1. Since the feeding experiment with [l-'3CC]glycine did not
yield a "C-enriched sample of 1, the origin of C - 4 from the
carboxyl group of glycine was ruled out.
The results of the three feeding experiments with labeled
glycine also allow a conclusion about the remaining building
block of 1: The incorporation of glycine is performed under
decarboxylation, that is, under release of an electron pair. This
requires an electrophilic carbon, preferably a carbonyl group. of
the remaining building block to serve as an acceptor. Thus. we
assume that oxaloacetate 6 is the most likely candidate for the
remaining building block, because its functionalities permit
both necessary bond formations, an aldol condensation of its
carbonyl group with glycine (+ C-3'-C-4 bond of 1) and an
amide bond formation of its (activated) 7-carboxyl group with
the h-NH, of ornithine (+C-6'-N-l' bond of 1. Scheme 2).
Even though an incorporation experiment with D,L-[4-I3C]aspartate, which we expected to be transaminated into oxalo-
acetate 6, failed, we still favor the [5C + 4C + 1 C] hypothesis
with 6 as the most likely third building block of pyridazomycin.
The negative result may be caused by meinbrane permeability
problems or the inability of the organism to transaminate aspartate into oxaloacetate. Thus, the formation of 6 in Streptom-yces
violuceoniper may be performed only by the citric acid cycle,
whose metabolization into the uncertain carbon atoms of 1 is
indicated by the feeding experiments with [I ,2-' 3C,]acetate and
[1.3-r3C,]glycerol. Both compounds can serve as sources of the
citric acid cycle, the former directly and thus more immediate
than the latter, which has to be metabolized by glycolysis and
the pyruvate decarboxylase complex. The specific incorporation
rates"'] (Table 1 ) reflect these circumstances. Presently we
are working on a synthesis of 3C-labeled oxaloacetate parallel
to the establishment of a cell-free system to examine 6 directly
as a precursor. We are also looking for the stereochemical course
of the glycine incorporation into 1. Future aspects of our
biosynthesis studies on 1 will focus on the N - N bond formation
the order of the bond connection between the
given biosynthetic building blocks as well as on the enzymes
involved ("pyridazomycin synthase", see Scheme 2).
Esperimental Procedure
Cultivation of the microorganism: S t ~ q ~ r o r n ~ c i~iolucvunger
(TU 2557) was cultivated on slants (20% agar-agar, 10% malt extract,4% glucose.4% yeast extract.
pH 7.0. adjusled before outoclaving) at 28 'C until sporulation occurred. then stored
at 4 C (for max. foiir months). The preculture for the ferineiitations has inoculated
directly from the slants and shaken on a rotary shaker (type BS 4, B. Braun.
Melsungen, FRG) in 750 mL Erlenmeyer flasks fitted with three baffles. filled with
100 mL medium (1.5% soybran meal. 2.5% mannitol, pH 6.8 before autoclaving)
nd 250 rpm. 100 mL of the preculture was used to inoculate the
1 L fermentor (type ISF 100. Infors GmbH. Basel. Switzerland, filled with 1 L of the
same medium as used for the preculture plus additional 30 m m o l L - ' ornithine
hydrochloride. except the feeding experiments with labeled ornithine and glutamic
acid. respectively. 28 C. acration: 1.6 L m i n - ' . 700 rpm). harvcsting about 64 h
after inoculation.
Isolation of pyridazomycin I : The culture filtrate of the fermentations was exposed
to an acidic ion exchange resin (Dowex 50) and washed with about 1.8 L deinineraliLed watcr,elution with NH: HCOO- (pH 5;gradient 0.3 n(1.5 L ) . 0 . 5 ~ ( 0 . 7L),
and 0 . 7 ~(1 L). The fractions conraining I were evaporated in sacuo at 40 'C and
chromatographed on silica gel (column 60 x 3 cm, nBuOH:CH,COOH:H,O =
3 : 2 : 2 ) . followed b) chromatography on Sephadex G-10 (column i 0 0 x 2 . 5 cm,
M e O H : H 2 0 = 8:2. pH 4.0. adjusted with 0.1 M HCI) and lyophiliaation of the
pyridazomycin-containing fractions.
F-eeding experiments- The labeled compounds were dissolved in sterile Mater
(100 mL) and added in four equal portions every six hours or continuously beginning at 25 h (after inoculation) to the growing 1 L-culture (1 L-fermentor) of S.
viuluwotir,qer (Tii 2557)
Labeled compounds: All the 'H-. l 3 C - , and/or "N-labeled compounds. if not
rynthesized (see below). Mere obtained from the Cambridge Isotope Laboratories.
Cambridge. MA. USA. The variously labeled ornithine samples were synthesized
according to Gould ct i l l . [I41 from 2-chloroethanol. K'.'CN (99 'YO "C) and KC"N
(99% "N). respectively. and diethylacetamido malonate. The labeled glycerol was
synthesizcd from bromoacetic acid and K"CN (99% IzC) via cyanoacetic acid.
diethyl malonate. and diethyl 2-acetoxy malonate (oxidation with PbOAc,). The
[.l-"C]/~-alanine was synthesized by hydrogenation (12 h, 25 C. 90 mg Pt as catalyst. in ethanol (30 mL). acidified uithconcentrated HCI (0.2 mL))ofthe previously
mentioned labeled c)mmacetic acid (1.5 g) and purified by chromatography on
silica gel (column, 3Ox3cm. I-propanol:H,O = 4.1. yield: 0.3 g).
Received: February 26, 1994 [Z6715IE]
German version. Angeu.. Chrrn. 1994, 106. 1733
oxaloacetate (7)
? pyridazomycin synthase
Scheme 2. Proposed mechanism of the biosynthesis of pyridazomycin 1
[I] R . Grote. Y Chen. A . Zeeck. Z. Chen, H . Zdhner. P. Mischnick-Lubhecke. W.
A. Konig. J. Aiirihior. 1988. 4 / . 595-601.
[2] P. Hughes, .I.Cliirdq. J. Org Clwn. 1989.54. 3260-3264. and references therein.
[3] a) M. Nakayawa. Y. Haynkawa. K. Furihata, H. Seto. J Afrrihioi. 1990. 43.
477-484: b) K . Isshiki, T. Sawa. H. Naganawa. Y. Koizumi. N. Matsuda, M.
Hamada. T. Takeuchi. M. lijima. M . Osono, T Masuda. M . Ishizuka, ihrd.
1990. 43, 1195-1198; c ) J. E. Leet. D. R. Schroeder, B. S Krishnan, J A.
Matson, r h d . 1990,43.961-966:d ) 0. D. Hensens. R. P. Borris. L. R. Koupal,
C . G. Caldwell. S A. Currie. A. A. Haidri, C. F. Homnick. S S. Honeycutt, S.
M. Lindenmayer. C. D. Schwartz. B. A. Weissberger. H. B. Woodruff. D. I-.
(racemic mixture). Very recently, the kinetic resolution of the
two chiral isomers has been demonstrated.[”] Effort has also
been invested in the prediction of the electronic structure of the
moIecuIe.[’2s 131
Owing to the scarcity of the material-the total worldwide
production of pure C,, is probably in the 10-100 mg range
few computational predictions have ever been checked experimentally. In particular. very little information is currently available on solid-state and structural properties of C,, or
(&-containing compounds.[’41 In a scanning tunneling microscopy (STM) study of various higher fullerenes, Li et aI.[”]
have shown that pure C,, appears to form closely packed layers.
A crystal structure determination of either pure C,, or of any
C,,-containing compound has, however, not yet been reported.
In this paper we describe the preparation and structure determination of the first C,,-containing van der Waals compound.
C76(S8)6.This material is a further representative of the family
of fullerene-sulfur adducts, which have the general formula
CZn(S8)mr[’‘-19]and which are formed by a combination of
fullerene molecules with S, rings. Its structure is closely related
to that of the compound C70(S8)6.[1s1
Just as in the C,, compound, which in the absence of a well-ordered C,, single crystal
allowed the first molecular structure study of C,, in the solid
state, C,,(S,), permits the determination of C-C bond lengths
in an only weakly (van der Waals) matrix-perturbed fullerene
molecule by diffraction methods. The material also offers, for
the first time, the chance to perform macroscopic measurements
on a structurally well-defined C,,-containing crystalline solid.
C,, was isolated from fullerene-containing soot prepared by
the Kratschmer-Huffman method (electric arc evaporation of
graphite in an inert gas atmosphere).[’] Toluene soot extract was
Preparation and Structure of C76(S& :
separated by liquid chromatography on an activated
A First Step in the Crystallographic
alumina column with 5 YOand I 5 % toluene in hexane as eluent.
Investigation of Higher Fullerenes**
Amount and composition of the resulting C,,-enriched starting
material used for further purification by high-performance liqRudi H. Michel, Manfred M. Kappes, Peter Adelmann,
uid chromatography (HPLC) has already been described elseand Georg Roth”
where,[”] together with a more quantitative discussion of the
whole procedure. Briefly, HPLC isolation of C,, was carried
“Higher ful1erenes”-those molecular allotropes of carbon
out with a Buckyclutcher column.L211
Three consecutive purifiwith more than 70 atoms per molecule-have recently become
three-component eluent
of great interest to preparative chemists and theoreticians. They
( S O YO toluene, 40% hexane, and 10 YO dichloromethane.
are minor constituents of the “Kratschmer-Huffman soot”[”
6 mLmin- I ) in the first cycle and a different composition (28 Yo
which contains predominantly C,, and C,,. C,, is the smallest
55 YOhexane. and 17 % dichloromethane, 6 mLmin- I )
among the more abundant (C,,, C,,. C,,, C,J “higher fulleroptimized for C,,/C,, resolution in the second and third cycle.
enes” and small quantities of it have been separated chromatoThe resulting purity was better than 99% C,, .[221 Exposure to
graphically by a number of groups.[”‘] The topology and
light was minimized during the whole separation process. Expomolecular structure of C,, has been studied theoretically by
sure to air was minimized in the third separation stage by purgvarious methods.[’- l o ] A detailed prediction of C - C bond
ing with nitrogen. Third stage C,, solutions, once separated,
lengths in the C,, molecule based on a b initio SCF-HartreeFock calculations has been published by Colt and S c ~ s e r i a . ” ~ were stored at below 0 “C prior to eluent removal during which
heating above 40 ’C was avoided. The solid material was recrysThere are two possible isolated pentagon ring isomers of C,,;
tallized from CS,, stored under nitrogen. and keptltransferred
these have D,and Jahn-Teller distorted Tdsymmetry. The most
at low temperature prior to use.
stable isomer of C,, is predicted to be the D, form. Ettl et al.I51
Given the very small amount of material available--a few
confirmed this molecular symmetry by I3C NMR measurehundred
pg in the present case--the growth of C,,(S& single
ments on C 7 , in solution. As a chiral species, C,, exists as a
crystals of appreciable size and quality poses quite a challenge.
“right-handed” and a “left-handed” form. These are energetiThe following procedure finally proved to be successful :
cally degenerate and expected to occur in equal proportions
Crystals were grown by slow evaporation (under vacuum) of
a solution of C,, and sulfur (in excess) in CS, at room temper[*IDr. G. Koth. Dr. P. Adelmann
Kerriforscliuiigsl.entrum Karlsruhe
ature. A capillary (1 mm internal diameter, one end open) conI nst i t u t I-ur Nti kleare Festkorperphysi k
taining about I mm3 of the solution was dipped with the closed
Portfjch 3640, D-76021 Karlsruhe (FRG)
end into a “CS,-heat bath” and the total pressure adjusted such
Telefax: Int. code + (7247)82-4624
that the CS, bath was gently boiling. This stabilizes both the
R. H . Michel. Prof. M. M. Kappes
temperature and the CS, vapour pressure above the solution.
lnatitut fur Physikalische Chemie der Universirdt Karlsruhe ( F R G )
The outer container was slightly heated from below to introduce
[**I M. M. K. acknowledges support from the Bundesministerium fur Forschung
und Technologie under “Pilotprogramm Fullerene”.
a small temperature gradient. This helps to avoid unwanted
Zink. L. Zitano. J. M. Fieldhouse, T. Rollins, M . S. Springer. J. P. Springer, ibid.
1991.44.149 254.
[4] a) J, G . Buchanan. M. R. Hamblin. G. S. Sood. R. H . Wightman. J. C%m?.Sur.
Uicnr. C‘orninioi. 1980. 91 7-91X: b) C. S. J. Walpole. R. Wrigglesworth. Nut.
Prod Rqi. 1989. 6 , 311-346.
[S] a ) M . Ymia~o.T. Takeuchi. H. Umerawa. N . Sakata. H . Hayashi. M. Hori. J.
Anrrhior. 1986.39.1263-1269. and references therein; b) R. J. Parry. Y. Li. E-L.
1992, 114. 10062-10064.
Lii. J. .4rir. C/reni. SJC.
[6] Y. Takahashi. M. Nakayama, 1. Watanabe, T. Deushi. H. Ishiwata. M. Shiratsuchi. G. Otani. J. Anrihior. 1989. 42, 1541-1546. and references therein.
[7] a) T. Kameyarna. A. Takahashi, H. Matsumoto, S. Kurasawa. M. Hamada. Y
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[I31 We :“istime an N - N bond formation by an N-oxidation of ornithine. for
example. initiated through an ornithine-.Vs-hydroxylase[5 b].
[I41 J. Wityak. V. I . Palaniswamy, S. J. Gould. J. Luhdlcvl Conipd. 1985. 22. 1155
A n g c ~ Chcnr.
Inr. E d EngI. 1994. 33, No. lS:16
; VCH V~rluRsRPsrllsrhuJrrnbH. D-694S1 Weinhrrm. 1994
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investigation, biosynthetical, pyridazomycin
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