The biosynthesis of 3-(trans-2-nitrocyclopropyl)alanine, a constituent of the signal metabolite hormaomycin

Article abstract of DOI:10.1002/ejoc.200400493

Feeding experiments with Streptomyces griseoflavus using deuterium-labeled racemic 3,3-[D₂]- (6b), 4,4-[D₂]- (6c), 5,5-[D ₂]- (6d), and 6,6-[D₂]-lysine (6e), and 3-amino-5-(2-amino-1,1-dideuterioethyl)-4,5-dihydrofuran-2-one dihydrochloride (34·2HCl) were carried out in order to obtain detailed information about the hitherto unknown biosynthetic pathway from lysine to the unusual amino acid 3-(trans-2′-nitrocyclopropyl)alanine [(3-Ncp)Ala] (2), which is a building block of hormaomycin 1a. The corresponding lysine dihydrochlorides were prepared in 33, 24, 19, and 30% overall yield, respectively, along a new efficient general synthetic route applying an alkylation of the lithium enolate of O'Donnel's glycine equivalent 7 as a key step. In the attempted preparation of 5,5-[D₂]-4-hydroxylysine (29), the respective γ-lactone (34·2 HCl) was obtained in five steps with 10% overall yield. The distribution of isotope labels in hormaomycins 1b-d led to the formulation of a reasonable cyclization mechanism of 2-amino-4-hydroxy-6-(hydroxyimino)hexanoic acid, an ω-oxime analogue of 4-hydroxylysine. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005.

Full text of DOI:10.1002/ejoc.200400493

FULL PAPER
The Biosynthesis of 3-(trans-2-Nitrocyclopropyl)alanine, a Constituent of the
Signal Metabolite Hormaomycin
Melanie Brandl,[a] Sergei I. Kozhushkov,[a] Boris D. Zlatopolskiy,[a] Petra Alvermann,[a]
Bernadette Geers,[a] Axel Zeeck,*[a] and Armin de Meijere*[a]
Keywords: Natural products / Amino acids / Synthetic methods / Isotopic labeling / Biosynthesis
Feeding experiments with Streptomyces griseoflavus using
deuterium-labeled racemic 3,3-[D2]- (6b), 4,4-[D2]- (6c), 5,5-
[D2]- (6d), and 6,6-[D2]-lysine (6e), and 3-amino-5-(2-amino-
1,1-dideuterioethyl)-4,5-dihydrofuran-2-one dihydrochloride
(34·2HCl) were carried out in order to obtain detailed in-
an alkylation of the lithium enolate of OЈDonnel’s glycine
equivalent 7 as a key step. In the attempted preparation of
5,5-[D2]-4-hydroxylysine (29), the respective γ-lactone (34·2
HCl) was obtained in five steps with 10% overall yield. The
distribution of isotope labels in hormaomycins 1b−d led to
formation about the hitherto unknown biosynthetic pathway the formulation of a reasonable cyclization mechanism of 2-
from lysine to the unusual amino acid 3-(trans-2Ј-nitrocyclo-
propyl)alanine [(3-Ncp)Ala] (2), which is a building block of
hormaomycin 1a. The corresponding lysine dihydrochlorides
were prepared in 33, 24, 19, and 30% overall yield, respect-
ively, along a new efficient general synthetic route applying
amino-4-hydroxy-6-(hydroxyimino)hexanoic acid, an ω-ox-
ime analogue of 4-hydroxylysine.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
Introduction
Among the natural products containing a cyclopropyl
group, the peptide lactone hormaomycin 1a, isolated from
Streptomyces griseoflavus (strain W 384), which shows some
selective antibacterial activity and was identified as a signal
metabolite of streptomycetes,[1,2] is especially intriguing.
The molecule of 1a contains two moieties of the previously
unknown 3-(trans-2Ј-nitrocyclopropyl)alanine [(3-Ncp)Ala]
(2). The synthesis of the two diastereomeric building blocks
of 1[3] and an improved approach to their synthetic precur-
sors[4] have previously been reported. The amino acid 2,
along with hypoglycines A 3 and B 4,[5] as well as 3-(trans-
2Ј-aminocyclopropyl)alanine [(3-Acp)Ala] in belactosine A
5,[6] belongs to the group of very rare 4,5-methanoamino
acids, the biosynthesis of which has not yet attracted
much attention.[7]
Results and Discussion
First Feeding Experiments with Labeled Methionine and
Lysines
In the first feeding experiment with (S)-[13C-methyl]-
methionine performed to elucidate the biosynthesis of Hor- maomycin 1a, no 13C enrichment in any position of the
cyclopropyl rings of the isolated metabolite was observed.[8]
This excluded the first conceived possibility that the cyclo-
[a]
Institut für Organische und Biomolekulare Chemie der Georg-
propyl ring in (3-Ncp)Ala would be formed by cyclopro-
panation of a nitroethenyl derivative by (S)-adenosylme-
thionine, which in some cases acts as a methylene donor in
the biosynthesis of cyclopropane derivatives.[7] Based on a
August-Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
Fax: (internat.) ϩ49-(0)551-399475
E-mail: Armin.deMeijere@chemie.uni-goettingen.de
azeeck@gwdg.de
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DOI: 10.1002/ejoc.200400493
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A. Zeeck, A. de Meijere et al.
FULL PAPER
simple count of its C atoms, the proteinogenic diamino acid
lysine 6a might be a reasonable biosynthetic precursor to 3-
(nitrocyclopropyl)alanine (2), in which case the amino
group at C-6 would have to be converted into the nitro
group at C-2Ј in 2. Indeed, a successful feeding experi-
ment[8] with commercially available racemic [1-13C]-lysine
to give hormaomycin with a 13C label in both (3-Ncp)Ala
moieties rigorously proved this hypothesis. It is noteworthy
that the 13C enrichment at C-1, 10.4% in (3-Ncp)Ala I and
45.7% in the (3-Ncp)Ala II, was not the same. This experi-
ment, however, could not prove whether the nitro group of
(3-Ncp)Ala originates from the ω-amino group of lysine or
not. This information was obtained from a feeding experi-
ment with [6-15N]-lysine: in this case the isolated hormao-
mycin showed in its FAB and MALDI-TOF mass spectra
a significant increase in intensity of the [Mϩ ϩ2] peak rela-
tive to those of unlabeled hormaomycin 1a. The cleanly ob-
servable splitting of the respective C-2Ј signals in its 13C
NMR spectrum due to 15NϪ13C coupling (1J ϭ 14 Hz) also
corroborates that the 15N enrichment had taken place at
both nitro groups of 1a. Thus, the ω-amino group of lysine
is retained in an oxidized form.
In view of these results we decided to synthesize selec-
tively deuterium-labeled racemic lysines in order to obtain
more detailed information about the intermediates along
the biosynthetic pathway to the (3-Ncp)Ala residues in 1.
Feeding experiments with appropriately deuterium-labeled
racemic 3,3-[D2]- (6b), 4,4-[D2]- (6c), 5,5-[D2]- (6d), and 6,6-
[D2]-lysine (6e) were carried out, and the loss or the preser-
vation of deuterium in the different positions of the (3-
Ncp)Ala moieties in the isolated hormaomycins were used
to propose a cyclization mechanism, which is compatible
with all of the experimental facts.
method, see Exp. Sect.), respectively, by applying essentially
the same set of standard procedures.
By adding a solution of lithium aluminumdeuteride to
the cyano ester 8, selective reduction of the ester function
could be achieved. Two different methods, treatment with
either iodine in the presence of triphenylphosphane and
imidazole according to Corey et al.[15] or with the iodine-
bis(diphenylphosphanyl)ethane reagent,[16] could be em-
ployed to transform the hydroxy to an iodo substituent in
the presence of a tetrahydropyranyloxy group or convert
the latter directly into an iodo substituent. The alkylation
of lithiated acetonitrile with the iodide 13 under modified
conditions[17] gave the desired product 14c, along with
about 18% of the bis-alkylated nitrile 15, which could be
separated by chromatography from 14c only after deprotec-
tion. Because of this, the transformation of 14c into 10c
was done in two steps (Scheme 1). The overall yields of the
Preparation of Various Deuterium-Labeled Lysines
Although the deuterium-labeled racemic 3,3-[D2]-
(6b),[9,10] 4,4-[D2]- (6c),[10] 5,5-[D2]- (6d),[11a] and 6,6-[D2]-
lysine (6e)[10,11] had previously been prepared, more ef-
ficient and general approaches to all four compounds were
elaborated. Firstly, in all four cases the incorporation of
deuterium should be achieved by reduction of an ester func-
tionality with lithium aluminum deuteride, which is the le-
ast expensive source of deuterium. Secondly, one of the
most versatile methods to introduce an amino acid frag-
ment into a molecule, namely by nucleophilic substitution
of an appropriate leaving group such as a halide, with the
lithium enolate of 7 (OЈDonnel’s glycine equivalent[12]), ap-
peared to be most attractive. Thirdly, catalytic hydrogen-
ation of a cyano group ought to be applicable to install a
6-amino substituent in 6bϪd.
The appropriate starting materials, the deuterated 4-iodo-
butyronitriles 10bϪd were each synthesized in a few steps
from commercially available ethyl 3-cyanopropanoate 8,
THP-protected ethyl glycolate 11 (easily prepared from in-
expensive glycolic acid in two steps[13]) and THP-protected
ethyl 3-hydroxypropanoate 16 (obtained from freshly pre-
Scheme 1. Preparation of dideuterium-labeled 4-iodobutyronitriles
10bϪd: a) LiAlD4 (inverse addition of a solution in THF over 1 h,
Et2O, 20 °C, 2 h; b) Ph3P, imidazole, I2, Et2O/MeCN, 0 °C, 2 h; c)
LiCH2CN, THF, 0 °C, 40 min; d) PPTS (pyridinium p-toluenesul-
fonate), MeOH, 50 °C, 1.5 h; e) LiAlD4, Et2O, 34 °C, 2 h; f) NaCN,
DMSO, 40 °C, 3 h; g) I2, dppe [1,2-bis(diphenylphosphanyl)-
pared ethyl 3-hydroxypropanoate[14] using a common ethane], CH2Cl2, 0 Ǟ 20 °C, 2 h
124
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Biosynthesis of a Constituent of the Signal Metabolite Hormaomycin
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Scheme 2. Preparation of the dideuterium-labeled lysines 6bϪd: a)
7-Li, THF, Ϫ78Ǟ20 °C, 56 h; b) Et2O/1 n aqueous HCl, 20 °C,
1Ϫ3 h; c) Boc2O, MeOH, 20 °C, 12 h; d) Raney Ni, H2 (4 bar),
NH3/MeOH, 20 °C, 15 hϪ3 d; e) 1 n aqueous HCl, 20 °C, 2 d
Scheme 3. Preparation of 6,6-dideuteriolysine 6e: a) LiAlD4, Et2O,
34 °C, 2 h; b) Ph3P, imidazole, I2, Et2O/MeCN, 0 °C, 2 h; c)
KNBoc2, DMF, 20 °C, 4 h; d) I2, dppe, CH2Cl2, 0Ǟ20 °C, 2 h; e)
7-Li, THF, Ϫ78Ǟ20 °C, 56 h; f) 1 n aqueous HCl, Et2O, 20 °C, 2 d
prepared iodonitriles 10bϪd were 55, 33 and 31%, respec-
tively.
The 6-amino substituent en route to lysine had previously
The nucleophilic substitution on all the iodonitriles been introduced by nucleophilic substitution of iodide in
10bϪd with the lithium enolate of OЈDonnel’s glycine a protected 4-iodobutanol like 24 with potassium bis(tert-
equivalent 7 gave high yields (70, approximately 100 and butoxycarbonyl)amide.[22] This method turned out to be
94%) of the protected amino acids 19bϪd (Scheme 2). An rather efficient when applied to 24. In spite of the fact that
optimized sequence of all the substituent transformations the resulting 25 was partially deprotected upon transform-
was developed for the nonlabeled iodonitrile 10a, prepared ation to the iodide 26 with the I2·dppe reagent
in an analogous manner to compound 10b.
Surprisingly, deprotection of the nonlabeled 19a (X ϭ after further conversion via 28 was 30%.
(Scheme 3),[23] the overall yield of 6e as the dihydrochloride
Y ϭ Z ϭ H) by treatment with 1 n aqueous HCl solution
These new general synthetic approaches to isotopomers
followed by Pd/C-catalyzed hydrogenation under acidic 6bϪe appear to be more efficient than those previously
conditions yielded pipecolinic acid hydrochloride[18] as the reported,[9Ϫ11] in which the lysines 6b,[10] 6c,[10] and 6e[11b]
sole product, and no reaction was observed with the pro- were prepared over 7, 7, and 8 steps, respectively, in overall
tected nonlabeled 19a upon attempted hydrogenation under yields of 2.5, 2.3 and 17%, respectively.[11c]
the same conditions. Yet, when the diphenylmethyleneam-
The previously reported preparation of racemic nonlab-
ino group was first deprotected and reprotected with a tert- eled 4-hydroxylysine by photochlorination of lysine in hy-
butoxycarbonyl group,[19] the cyano group in the resulting drochloric acid solution[24] could not be reproduced.[25a]
nonlabeled amino acid derivative 20a (X ϭ Y ϭ Z ϭ H) Therefore, an alternative six-step preparation of 29 starting
was smoothly transformed into an aminomethyl moiety in from the known tert-butyl 2-(tert-butoxycarbonylamino)-4-
very high yield upon Raney Ni-catalyzed hydrogenation un- oxobutyrate (30)[26] was developed (Scheme 4).
der basic conditions (methanolic ammonia).[20] These con-
Because of its reversibility, the aldol reaction of the alde-
ditions were also used for the dideuterionitriles 19bϪd and, hyde 30 with lithiated trideuterioacetonitrile turned out to
after the usual one-pot deprotection, the dideuteriolysines at best furnish a mixture of 30 and 31 in 48 and 42% yield,
6bϪd were obtained as dihydrochlorides in 33, 24, and 19% respectively, even when the transformation was performed
overall yield, respectively.
in the presence of anhydrous cerium(iii) chloride.[27] How-
Because the very same sequence could not be used for the ever, as the starting material 30 can easily be recycled from
synthesis of 6,6-dideuteriolysine 6e, it was prepared along a the reaction mixture, the overall yield of 30 can be increased
similar route in six steps from the known THP-protected by several repetitions of the aldol reaction. After THP-pro-
methyl γ-hydroxybutyrate 22[21] (Scheme 3).
tection of the hydroxy group in 31 and Raney Ni catalyzed
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A. Zeeck, A. de Meijere et al.
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Further information was obtained from a feeding experi-
ment with 4,4-[D2]-lysine 6c. The ESI mass spectrum of the
isolated hormaomycin shows, besides the [M ϩ Naϩ] peak
of the unlabeled product, a significant increase in the [M ϩ
Naϩ ϩ2] peak, which corroborates the introduction of only
1
two deuterium labels. The H NMR spectrum of 1c clearly
shows a decrease in the intensity of the cleanly visible signal
for 1Ј-H of (3-Ncp)Ala I (δ ϭ 0.33 ppm), while the signal
of 1Ј-H of (3-Ncp)Ala II overlaps strongly with other sig-
nals. Also, the signals of C-1Ј of (3-Ncp)Ala I and (3-
Ncp)Ala II had lower intensities relative to the correspond-
ing signals in the 13C NMR spectrum of undeuterated 1a.
This experiment proved that during the biosynthesis of (3-
Ncp)Ala only one hydrogen from C-4 of lysine is abstrac-
ted.
In order to see whether C-5 of lysine is involved in the
formation of the cyclopropyl group of (3-Ncp)Ala, a feed-
ing experiment with 5,5-[D2]-lysine 6d was carried out. In
this case, the spectroscopic data indicates that a total of
four deuterium labels [two for each (3-Ncp)Ala moiety]
were retained in the isolated hormaomycin 1d to a signifi-
cant degree (about 30Ϫ35% according to the ESI mass
spectrum). In the 1H NMR spectrum of 1d, the signal inten-
sities for all four 3Ј-protons are significantly decreased, and
in the 13C NMR spectrum a significant decrease in signal
intensity for C-3Ј of both (3-Ncp)Ala residues is observed
as well as an upfield shift for the respective signals of C-1Ј
and C-2Ј. This experiment shows that none of the hydro-
gens at C-5 of lysine is lost during the biosynthesis of (3-
Ncp)Ala. In addition, the result strictly excludes the forma-
tion of a double bond between C-4/C-5 or C-5/C-6 at an
intermediate stage.
In contrast to the preceding feeding experiments, all of
the labels of 6,6-[D2]-lysine 6e had been lost in the hormao-
mycin 1 isolated from an analogous feeding experiment.
This means that during the biosynthesis of (3-Ncp)Ala 2
either both hydrogens from C-6 are lost, or an exhaustive
exchange of D with H takes place.
In order to test whether 4-hydroxylysine is a biosynthetic
intermediate en route from lysine 6a to (3-Ncp)Ala, a feed-
ing experiment with the racemic 5,5-[D2]-4-hydroxylysine
Scheme 4. Attempted preparation of racemic 5,5-dideuterio-4-hy-
droxylysine (29): a) LiCD2CN, CeCl3, THF, Ϫ78 °C, 20 min; b)
DHP (3,4-dihydro-2H-pyran), PPTS (pyridinium p-toluenesulfon-
ate), CH2Cl2, 20 °C, 24 h; c) Raney Ni, H2 (4 bar), NH3/MeOH,
20 °C, 3 h; d) Boc2O, MeOH, 20 °C, 9 h; e) 1 n aqueous HCl,
MeOH, 20 °C, 2 d
hydrogenation of the cyano group in 32 under basic con-
ditions, the second amino group was also protected with a
tert-butyloxycarbonyl (Boc) group to give the completely
protected hydroxydiamino acid 33. With this exhaustive
protection, the 4-hydroxylysine derivative 33 could more
easily be purified by chromatography, and, in addition, the
spontaneous cyclization to the γ-lactone by way of a free
hydroxy function under the basic conditions of hydrogen-
ation that was observed in a similar preparation,[25a] could
be prevented. However, upon complete deprotection of 33
under acidic conditions in a one-pot operation, the lactone
3-amino-5-(2-amino-1,1-dideuterioethyl)-4,5-dihydrofuran-
2-one dihydrochloride (34·2HCl) was obtained as the sole
product.[28]
Feeding Experiments with Deuterium-labeled Lysines
The first feeding experiments were carried out with the (29) or the synthetically more easily accessible lactone 34
3,3-[D2]-lysine 6b to rigorously exclude the possibility that was of prime interest.[29] The feeding of 34 indeed led to
the biosynthesis of (3-Ncp)Ala might proceed via an inter- deuterium-labeled hormaomycin 1d as proved by ESI mass
mediate with a double bond between C-3 and C-4, which and D NMR spectra of the isolated product. The fact that
could serve as a precursor to the nitrocyclopropyl ring in the lactone 34 was accepted as a substrate in the biosynthe-
close analogy to the postulated biosynthetic pathway to hy- sis of hormaomycin as well as the loss of one deuterium
poglycine A 3.[7] The loss of even one deuterium label would label from C-4 of 4,4-[D2]-lysine makes 4-hydroxylysine
confirm this hypothesis. However, the deuterium-labeled (and/or its lactone) a highly probable intermediate.
hormaomycin 1b which was obtained from this feeding
experiment, according to its ESI mass spectrum, had a mo-
lecular mass which was 4 units higher than that of 1a. The
Conclusion
1
2H-, 13C- and H NMR spectra confirmed that both deu-
terium labels had been introduced in both (3-Ncp)Ala moi-
According to the results of the feeding experiments de-
eties to the same extent. This proved that no hydrogen is scribed, one may conclude that (i) lysine is definitely the
lost from C-3 of lysine during the conversion into (3- precursor of both (3-Ncp)Ala moieties, (ii) both hydrogens
Ncp)Ala. Thus, the C-3 position is not involved in the con- at the C-3 and C-5 of lysine are retained all along the way
struction of the nitrocyclopropyl ring.
to the nitrocyclopropyl ring, (iii) complete loss or exchange
126
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Biosynthesis of a Constituent of the Signal Metabolite Hormaomycin
FULL PAPER
of both hydrogens at C-6 of lysine as well as loss of one
hydrogen at C-4 of lysine occurs, and (iv) the lactone 34 of
4-hydroxylysine (29) is an acceptable substrate, and thus 29
is the most probable intermediate in the biosynthesis of (3-
Ncp)Ala from lysine.
Hormaomycin 1a most probably is biosynthesized by a
non-ribosomal peptide synthetase (NRPS, hormaomycin
synthetase). The non-proteinogenic amino acids usually are
substrates of a multienzyme complex and must be com-
pletely biosynthesized before they are assembled to a pep-
tide. This had been proved in the case of 3-(2Ј-nitrocyclo-
propyl)alanine (2) by feeding a deuterium-labeled racemic
sample.[2b,30] Thus, there must be a continuous biosynthetic
route from lysine to (3-Ncp)Ala.
Taking into account also that (i) racemic 2-(3Ј-aminocy-
clopropyl)alanine is not a substrate for the hormaomycin
synthetase and does not affect the biosynthesis of hormao-
mycin 1a,[30] and (ii) 6-nitronorleucine [(S)-lysine with the
oxidized ω-amino group] is accepted by Streptomyces
griseoflavus strain W 384 as a substrate, but led to novel
hormaomycin analogues with intact nitronorleucine (conse-
quently nitronorleucine-containing intermediates have
never been observed in the culture broth at any time with-
out addition of this amino acid),[31] both precursors cannot
be an intermediate along the route. On the basis of all this,
a reasonable biosynthetic pathway for (3-Ncp)Ala that in-
cludes a cyclization mechanism can now be proposed
(Scheme 5).
In the first step, (S)-lysine (S)-6a must undergo hydroxyl-
ation at C-4 to give 4-hydroxylysine (2S)-29 which, in turn,
would probably be oxidized at its ω-amino group to give the
nitroso compound (2S)-35a,[32] or cyclized to the lactone 34
followed by oxidation to give the nitrosolactone 37a as an
intermediate. One conceivable route would be via the cat-
ionic intermediate (2S)-36 or the azabicyclobutonium rela-
tive (2S)-38 of the azahomoallyl cation (2S)-36 formed by
oxime tautomer (2S)-35b of (2S)-2-amino-4-hydroxy-6-ni-
trosohexanoic acid [(2S)-35a]. Homoallyl cations are cap-
able of cyclizing to cyclopropylmethyl cations;[33] however,
in the case of (2S)-36, such a cyclization would lead to a
thermodynamically highly unfavorable intermediate with a
positive charge on nitrogen. However, such a ring closure
might be facilitated by nucleophilic assistance through at-
tack of water on the azabicyclobutonium cation (2S)-38[34]
Scheme 5. Proposed biosynthetic pathway to 3-(2Ј-nitrocyclopropyl)-
alanine (2S,1ЈR,2ЈS)-2
followed by dehydration of the formed intermediate 39 to ment with 6,6-[D2]-lysine (6e). In both tentative pathways
give 3-(2Ј-nitrosocyclopropyl)alanine (41). the total exchange of deuterium of 6,6-[D2]-lysine 6e can
A more likely alternative pathway to 41 would be via the also be accounted for by equilibration between the tautom-
oxime anion (2S)-40 formed upon deprotonation of the ox- eric nitroso and oxime intermediates 35a/35b and 37a/
ime tautomer (2S)-37b of the nitrosolactone (2S)-37a 37b.[37] Epimerization of (S)-(3-Ncp)Ala to yield the (R)-(3-
which, by nucleophilic attack, would open the ring of the Ncp)Ala residue in 1a needs an external epimerase before
lactone moiety in 40 with simultaneous formation of the the assembly or more likely an internal epimerase as part
cyclopropyl ring (see ref.[35]) and subsequent reprotonation. of the hormaomycin synthetase.[2b]
Final oxidation of the nitroso group in 41 to the nitro group
As a result of these efforts a new biosynthetic pathway
would then afford (S)-(3-Ncp)Ala 2. The latter transform- has been established by which lysine is converted into the
ation could also occur as an NADP-dependent oxi- 3-(2Ј-nitrocyclopropyl)alanine (2). Whether this pathway
dation[36a] of the oxime (2S)-42, tautomeric to 41,[36b] to can be a model for a biomimetic synthesis of (3-Ncp)Ala,
afford the amino acid 2 in its aci-form (2S)-43. This would and the characterization of the enzyme for these steps,
explain the loss of the deuterium label in the feeding experi- should be attractive projects for the future.
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A. Zeeck, A. de Meijere et al.
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C-3Ј), 35.0 (Ϫ, C-2), 60.3 (Ϫ, OCH2CH3), 61.9 (Ϫ, C-3), 62.8 (Ϫ,
C-6Ј), 98.6 (ϩ, C-2Ј), 171.5 (Cquat, CϭO) ppm.
Experimental Section
General: 1H and 13C NMR spectra were recorded at 250/500/600
(1H) and 62.9/125.7/150.8 MHz [13C, additional DEPT (Distor-
tionless Enhancement by Polarization Transfer)] with a Bruker AM
250, a Varian VXR 500, and a Varian Inova 600 instrument,
respectively, in CDCl3 solution, CHCl3/CDCl3 as internal reference
(if not otherwise specified); δ in ppm, J in Hz. MS (EI): Finnigan
MAT 95 spectrometer (70 eV); DCI-MS: Finnigan MAT 95,
200 eV, reactant gas NH3. ESI-MS: Finnigan LCQ. IR: Bruker IFS
66 (FT-IR) spectrometer, measured as KBr pellets, oils between
KBr plates. M.p.: Büchi 510 capillary melting point apparatus, un-
corrected values. TLC: MachereyϪNagel precoated sheets,
0.25 mm Sil G/UV254. Column chromatography: Merck silica gel,
grade 60, 230Ϫ400 mesh. Starting materials: Anhydrous diethyl
ether and THF were obtained by distillation from sodium benzo-
phenone ketyl, MeOH from magnesium methoxide, DMF and
DMSO from CaH2, MeCN and C2H2 from P4O10. tert-Butyl N-
(diphenylmethylene)glycinate (7),[12a] ethyl (tetrahydropyran-2-
yloxy)acetate (11),[13] methyl 4-(tetrahydropyran-2-yloxy)butyrate
(22),[21] and tert-butyl 2-(tert-butoxycarbonylamino)-4-oxobutyrate
(30)[26] were prepared according to published procedures. All other
chemicals were used as available commercially (Merck, Acro˜s,
BASF, Bayer, Hoechst, Degussa AG, and Hüls AG). Organic ex-
tracts were dried with anhydrous MgSO4. Microbiology: Shaking
incubator: Braun BS4. Fermenter, Braun Biostat M. Media (per
liter of demineralized water): M2Ca: malt extract (10 g), glucose
(4 g), yeast extract (4 g), agar-agar (20 g), CaCO3 (0.5 g), pH 7.0;
M6: d-mannitol (20 g), soybean flour (20 g), meat extract (20 g),
NaCl (2 g), l-valine (0.3 g), ZnSO4·6 H2O (0.5 g), pH 7.3; M10:
d-mannitol (50 g), l-asparagine (3 g), K2HPO4 (1 g), NaCl (25 g),
MgSO4·7 H2O (50 mg), CaCl2·2 H2O (50 mg), CH3COONa
(420 mg), meso-inositol (100 mg), trace elements solution: CaCl2·2
H2O (8 g), MnCl2·2 H2O (5 g), ZnCl2 (50 mg), CuCl2·2 H2O
(50 mg), FeCl2·6 H2O (50 mg), vitamins solution: thiamine hydro-
chloride (1 g), calcium d-pantothenate (1.2 g), flavine mononucleo-
tide (1 g), nicotinic acid (2.3 g), pyridoxine hydrochloride (12 g), p-
aminobenzoic acid (200 mg), vitamin B12 (100 mg), folic acid
(10 mg), biotin (6 mg). For the dilution demineralized water was
used. After adjusting the pH with 0.5 m NaOH or 0.5 m HCl, all
media were sterilized at 121 °C for 30 min. After autoclaving, the
vitamins solution (1 mL/L) was added to M10 after sterile fil-
tration. All ingredients were purchased from Merck, Gibco, Difco,
Sigma, Riedel de Haen and Henselwerk GmbH. (RS)-[1-13C]-Ly-
sine (99%) and (RS)-[6-15N]-lysine (99%) were purchased from
CamproScientific.
Reduction of Esters 8, 11, 16 and 22 with LiAlD4. General Procedure
(GP) 1: GP1a: To a stirred solution of the corresponding ester
(50 mmol) in anhydrous diethyl ether (150 mL) was added LiAlD4
(25 mmol) as a solution in THF at ambient temperature over a
period of 1 h under Ar. After this, the reaction mixture was stirred
at the same temperature for an additional 2 h, cooled to 10 °C, the
reaction was quenched with saturated Na2SO4 solution, and the
organic solution was dried and concentrated under reduced press-
ure. The resulting alcohols were purified by column chromatogra-
phy or used without further purification. GP1b: To a vigorously
stirred suspension of LiAlD4 (25 mmol) in anhydrous diethyl ether
(200 mL) was added dropwise a solution of the corresponding ester
(47.5 mmol) in Et2O (50 mL) under Ar. The resulting mixture was
stirred under reflux for 2 h and then worked up according to GP1a.
4,4-Dideuterio-4-hydroxybutyronitrile (9b): Alcohol 9b[38] (1.87 g,
81%) was obtained from the cyano ester 8 (3.35 g, 26.3 mmol) and
LiAlD4 (14.8 mmol, 16.1 mL of a 0.92 n solution in THF) accord-
ing to GP1b, after column chromatography (30 g of silica gel, 3 ϫ
1
15 cm column, Et2O, Rf ϭ 0.34) as a colorless oil. H NMR: δ ϭ
1.67 (t, J ϭ 7.0 Hz, 2 H, 3-H), 2.32 (t, J ϭ 7.0 Hz, 2 H, 2-H), 3.56
(s, 1 H, OH) ppm. 13C NMR: δ ϭ 13.0 (Ϫ, C-2), 27.2 (Ϫ, C-3),
58.6 (quint, JCD ϭ 22.0 Hz, C-4), 119.5 (Cquat, CN) ppm.
1,1-Dideuterio-2-(tetrahydropyran-2-yloxy)ethanol (12): Alcohol
12[39] (28.71 g, 97%) was obtained from the ester 11 (37.64 g, 0.2
mol) and LiAlD4 (0.1 mol, 91.7 mL of a 1.09 n solution in THF)
according to GP1b as a viscous liquid which was used without
further purification. 1H NMR: δ ϭ 1.46Ϫ1.54 (m, 4 H, 4Ј-H, 5Ј-
H), 1.61Ϫ1.78 (m, 2 H, 3Ј-H), 3.25 (s, 1 H, OH), 3.41Ϫ3.50 (m, 1
H, 6Ј-H), 3.59 (d, J ϭ 11.3 Hz, 1 H, 2-H), 3.70 (d, J ϭ 11.3 Hz, 1
H, 2-H), 3.80Ϫ3.88 (m, 1 H, 6Ј-H), 4.51 (t, J ϭ 2.6 Hz, 1 H, 2Ј-H)
ppm. 13C NMR: δ ϭ 19.7 (Ϫ, C-4Ј), 25.0 (Ϫ, C-5Ј), 30.5 (Ϫ, C-
3Ј), 61.1 (quint, JCD ϭ 19.9 Hz, C-1), 62.9 (Ϫ, C-6Ј), 70.1 (Ϫ, C-
2), 99.8 (ϩ, C-2Ј) ppm.
1,1-Dideuterio-3-(tetrahydropyran-2-yloxy)propanol (17): Alcohol
17 (12.15 g, 99%) was obtained from the ester 16 (15.2 g,
75.1 mmol) and LiAlD4 (1.66 g, 39.5 mmol) according to GP1b as
1
a viscous liquid. H NMR: δ ϭ 1.42Ϫ1.52 (m, 4 H, 4Ј-H, 5Ј-H,),
1.59Ϫ1.70 (m, 4 H, 2-H, 3Ј-H), 3.36Ϫ3.47 (m, 3 H, 3-H, OH),
3.71Ϫ3.82 (m, 2 H, 6Ј-H), 4.40Ϫ4.51 (m, 1 H, 2Ј-H) ppm. 13C
NMR: δ ϭ 19.4 (Ϫ, C-4Ј), 25.0 (Ϫ, C-5Ј), 30.4 (Ϫ, C-3Ј), 31.7 (Ϫ,
C-2), 59.4 (quint, JCD ϭ 26.0 Hz, C-1), 62.3 (Ϫ, C-3), 65.2 (Ϫ, C-
6Ј), 98.9 (ϩ, C-2Ј) ppm. MS (CI): m/z (%) ϭ 180 (95) [M ϩ NH4ϩ],
163 (5) [M ϩ Hϩ].
Ethyl 3-(Tetrahydropyran-2-yloxy)propanoate (16): To a solution of
freshly prepared ethyl 3-hydroxypropanoate[14] (54.34 g, 0.46 mol)
in anhydrous CH2Cl2 (180 mL) was added 3,4-dihydro-2H-pyran
(DHP, 53.0 g, 0.63 mol) and then, under vigorous stirring and in-
tensive cooling, two drops of concentrated hydrochloric acid was
added. After the strongly exothermal reaction was completed, the
reaction mixture was stirred at ambient temperature for an ad-
ditional 1 h, then the reaction was quenched with NaHCO3 (15.0 g,
0.18 mol), and the mixture stirred for 30 min. The mixture was
filtered, dried, concentrated under reduced pressure and distilled in
vacuo to give 16 (73.2 g, 79%) as a colorless liquid, b.p. 94Ϫ96 °C
(0.6 mbar). 1H NMR: δ ϭ 1.20 (t, J ϭ 7.1 Hz, 3 H, CH3),
1.45Ϫ1.57 (m, 4 H, 4Ј-H, 5Ј-H), 1.59Ϫ1.76 (m, 2 H, 3Ј-H), 2.53 (t,
J ϭ 6.0 Hz, 2 H, 2-H), 3.41Ϫ3.57 (m, 1 H, 6Ј-H), 3.61Ϫ3.67 (m, 1
H, 6Ј-H), 3.75Ϫ3.82 (m, 1 H, 3-H), 3.84Ϫ3.98 (m, 1 H, 3-H), 4.11
1,1-Dideuterio-4-(tetrahydropyran-2-yloxy)butanol (23): Alcohol 23
(12.15 g, 99%) was obtained from methyl 4-(tetrahydropyran-2-
yloxy)butyrate (22) (10.1 g, 50.0 mmol) and LiAlD4 (2.10 g,
50.0 mmol) according to GP1b as a viscous liquid. IR: ν˜ ϭ 3429
cmϪ1, 2941, 2869, 1352, 1121, 1076, 1033. 1H NMR: δ ϭ 1.51Ϫ1.59
(m, 4 H, 2-H, 3-H), 1.62Ϫ1.81 (m, 6 H, 3Ј-H, 4Ј-H, 5Ј-H), 2.15 (br.
s, 1 H, OH), 3.37Ϫ3.53 (m, 2 H, 4-H), 3.74Ϫ3.89 (m, 2 H, 6Ј-H),
4.56 (t, J ϭ 3.3 Hz, 1 H, 2Ј-H) ppm. 13C NMR: δ ϭ 19.5 (Ϫ, C-
4Ј), 25.3 (Ϫ, C-5Ј), 26.5 (Ϫ, C-3), 29.8 (Ϫ, C-2), 30.6 (Ϫ, C-3Ј),
62.0 (quint, JCD ϭ 20.0 Hz, C-1), 62.3 (Ϫ, C-4), 67.5 (Ϫ, C-6Ј),
98.8 (ϩ, C-2Ј) ppm. MS (CI): m/z (%) ϭ 370 (3) [2M ϩ NH4ϩ],
194 (100) [M ϩ NH4ϩ], 177 (3) [M ϩ Hϩ]. For the NMR spectra
of the non-deuterated compound see ref.[40]
(q, J ϭ 7.1 Hz, 2 H, OCH2), 4.55Ϫ4.58 (m, 1 H, 2Ј-H) ppm. 13C Preparation of Iodides 10b؊d, 13, 18, 24 and 26. General Procedure
NMR: δ ϭ 14.1 (ϩ, CH3), 19.1 (Ϫ, C-4Ј), 25.3 (Ϫ, C-5Ј), 30.3 (Ϫ, (GP) 2: GP2a: Iodine (5.07 g, 20 mmol) was added in small por-
128
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 123Ϫ135

Biosynthesis of a Constituent of the Signal Metabolite Hormaomycin
FULL PAPER
tions over a period of 30 min to an efficiently cooled (0 °C) solution
of the respective alcohol (10.1 mmol), Ph3P (4.71 g, 18 mmol) and
imidazole (1.29 g, 18.9 mmol) in a mixture of anhydrous MeCN
(20 mL) and Et2O (30 mL) under Ar. Stirring was continued for
2 h at 0 °C, the reaction mixture was diluted with Et2O (150 mL),
washed with 20% Na2S2O3 aqueous solution (2 ϫ 100 mL) and
brine (100 mL), dried, and concentrated under reduced pressure.
The residue was thoroughly extracted with hexane (100 mL) by vig-
orous stirring for 1 h in the dark. The solution was filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography or used without further purification.
GP2b: To a solution of 1,2-bis(diphenylphosphanyl)ethane (dppe)
(12.0 mmol) in anhydrous CH2Cl2 (30 mL) was added a solution
of iodine (12.0 mmol) in CH2Cl2 (60 mL) at Ϫ10 °C over a period
of 30 min under Ar. After additional stirring for 30 min, a solution
of the corresponding THP-protected alcohol (10.0 mmol) in
CH2Cl2 (15 mL) was added dropwise at 0 °C, and the resulting
mixture was warmed to ambient temperature over a period of 2 h.
The reaction mixture was poured into hexane/Et2O (2:1) mixture
(250 mL), filtered through a pad of Celite (3 cm), and concentrated
under reduced pressure. The product was purified by column chro-
matography.
C-6), 98.5 (ϩ, C-2) ppm. For the NMR spectra of a non-deuterated
compound see ref.[43]
2-(3,3-Dideuterio-3-iodopropoxy)tetrahydropyran (18): Iodide 18
(20.38 g, 100%) was obtained from the alcohol 17 (12.15 g,
74.9 mmol), PPh3 (19.6 g, 74.7 mmol), imidazole (5.10 g,
74.9 mmol) and I2 (19.0 g, 74.9 mmol) according to GP2a as a
slightly yellow oil and used without further purification. 1H NMR:
δ ϭ 1.40Ϫ1.52 (m, 4 H, 5-H, 4-H), 1.60Ϫ1.78 (m, 2 H, 3-H), 1.98
(t, J ϭ 5.8 Hz, 2 H, 2Ј-H), 3.31Ϫ3.47 (m, 2 H, 6-H), 3.67Ϫ3.82
(m, 2 H, 1Ј-H), 4.51 (t, J ϭ 3.3 Hz, 1 H, 2-H) ppm. 13C NMR:
δ ϭ 3.11 (quint, JCD ϭ 24.9 Hz, C-3Ј), 19.2 (Ϫ, C-4), 25.2 (Ϫ, C-
5), 30.3 (Ϫ, C-3), 33.0 (Ϫ, C-2Ј), 61.9 (Ϫ, C-1Ј), 66.5 (Ϫ, C-6), 98.5
(ϩ, C-2) ppm. For the NMR spectra of a non-deuterated com-
pound see ref.[44]
2-(4,4-Dideuterio-4-iodobutoxy)tetrahydropyran (24): Iodide 24
(10.97 g, 77%) was obtained from the alcohol 23 (8.81 g,
50.0 mmol), PPh3 (13.1 g, 50.0 mmol), imidazole (3.40 g,
50.0 mmol) and I2 (12.7 g, 50.0 mmol) according to GP2a as a col-
orless oil after column chromatography [50 g of silica gel, 2 ϫ
1
10 cm column, hexane/Et2O, 2:1, Rf ϭ 0.35 (10:1)]. H NMR: δ ϭ
1.47Ϫ1.66 (m, 4 H, 4-H, 5-H), 1.68Ϫ1.82 (m, 4 H, 3-H, 2Ј-H), 1.91
(t, J ϭ 7.3 Hz, 2 H, 3Ј-H), 3.35Ϫ3.53 (m, 2 H, 1Ј-H), 3.70Ϫ3.88
(m, 2 H, 6-H), 4.56 (t, J ϭ 3.2 Hz, 1 H, 2-H) ppm. 13C NMR: δ ϭ
19.6 (Ϫ, C-4), 25.4 (Ϫ, C-5), 30.4 (Ϫ, C-2Ј), 30.5 (Ϫ, C-3Ј), 30.7
(Ϫ, C-3), 62.3 (Ϫ, C-1Ј), 66.2 (Ϫ, C-6), 98.8 (ϩ, C-2) ppm. The
intensity of the CD2 carbon signals was too low. For the NMR
4,4-Dideuterio-4-iodobutyronitrile (10b): Iodide 10b[41] (9.65 g, 68%)
was obtained from the alcohol 9b (6.24 g, 71.6 mmol), PPh3 (19.7 g,
75.1 mmol), imidazole (5.12 g, 75.2 mmol) and I2 (19.1 g,
75.3 mmol) according to GP2a as a slightly yellow oil after column
chromatography (150 g of silica gel, 6 ϫ 15 cm column, hexane/
Et2O, 1:1, Rf ϭ 0.35). IR: ν˜ ϭ 2942 cmϪ1, 2246, 1438, 1421, 1192,
spectra of the non-deuterated compound see ref.[45]
.
3,3-Dideuterio-4-(tetrahydropyran-2-yloxy)butyronitrile (14c): Un-
der Ar, a solution of acetonitrile (6.13 g, 7.8 mL, 149.3 mmol) in
anhydrous THF (100 mL) was added dropwise to a solution of
nBuLi (149.3 mmol, 60.7 mL of a 2.46 n solution in hexane) in
THF (60 mL) at Ϫ78 °C over a period of 1 h. After an additional
stirring for 1 h at the same temperature, the resulting fine suspen-
sion was cannulated to a stirred solution of the iodide 13 (33.53 g,
129.9 mmol) in anhydrous THF (100 mL) over a period of 20 min,
while maintaining the temperature of the reaction mixture at 0 °C.
The mixture was stirred at the same temperature for an additional
15 min, and the reaction was quenched with water (20 mL) and
partitioned between diethyl ether and brine (200 mL of each). The
inorganic phase was extracted with diethyl ether (3 ϫ 80 mL), the
combined organic layers were washed with brine (100 mL), dried
and concentrated under reduced pressure. The residue was purified
by column chromatography (250 g of silica gel, 7 ϫ 20 cm column,
hexane/Et2O, 1:1.5, Rf ϭ 0.40) to give a nonseparable mixture
(22.2 g) which consisted, according to 1H and 13C NMR spec-
troscopy, of 14c (estimated yield ca. 70%) and 3,3-dideuterio-2-[1,1-
dideuterio-2-(tetrahydropyran-2-yloxy)ethyl]-4-(tetrahydropyran-2-
1
1108, 1046, 1016, 993. H NMR: δ ϭ 2.04 (t, J ϭ 6.6 Hz, 2 H, 3-
H), 2.44 (t, J ϭ 6.6 Hz, 2 H, 2-H) ppm. 13C NMR: δ ϭ 3.1 (quint,
JCD ϭ 23.2 Hz, C-4), 17.9 (Ϫ, C-2), 28.1 (Ϫ, C-3), 118.0 (Cquat
,
ϩ
CN) ppm. MS (CI): m/z (%) ϭ 232 (100) [M ϩ NH4 ϩ NH3],
215 (63) [M ϩ NH4ϩ].
3,3-Dideuterio-4-iodobutyronitrile (10c): Iodide 10c (7.08 g, 75%)
was obtained from the alcohol 9c (4.18 g, 48.0 mmol), PPh3
(13.11 g, 50.0 mmol), imidazole (3.47 g, 51.0 mmol) and I2 (12.73 g,
50.2 mmol) according to GP2a as a slightly yellow oil after column
chromatography (120 g of silica gel, 4.5 ϫ 20 cm column, hexane/
1
Et2O, 1:1, Rf ϭ 0.35). H NMR: δ ϭ 2.51 (s, 2 H, 2-H), 3.27 (s, 2
H, 4-H) ppm. 13C NMR: δ ϭ 2.83 (Ϫ, C-4), 18.2 (Ϫ, C-2), 28.4
(quint, JCD ϭ 20.3 Hz, C-3), 118.3 (Cquat, CN) ppm. MS (CI):
ϩ
m/z (%) ϭ 232 (100) [M ϩ NH4 ϩ NH3], 215 (57) [M ϩ NH4ϩ].
2,2-Dideuterio-4-iodobutyronitrile (10d): Iodide 10d[42] (7.05 g, 50%)
was obtained from the protected alcohol 14d (12.15 g, 71.0 mmol),
dppe (23.9 g, 60.0 mmol) and I2 (15.0 g, 59.1 mmol) according to
GP2b after column chromatography (120 g of silica gel, 4 ϫ 15 cm
1
1
column, hexane/Et2O, 1:1, Rf ϭ 0.28) as a colorless oil. H NMR:
yloxy)butyronitrile (15) (yield approximately 18%). 14c and 15: H
δ ϭ 2.07 (t, J ϭ 6.8 Hz, 2 H, 3-H), 3.23 (t, J ϭ 6.8 Hz, 2 H, 4-H)
ppm. 13C NMR: δ ϭ 3.2 (Ϫ, C-4), 17.6 (quint, JCD ϭ 20.8 Hz, C-
2), 28.2 (Ϫ, C-3), 118.1 (Cquat, CN) ppm.
NMR: δ ϭ 1.49Ϫ1.59 (m, 4 H, tetrahydropyranyl-H), 1.61Ϫ1.77
(m, 2 H, tetrahydropyranyl-H), 3.41Ϫ3.51 (m, 2 H, CH2O),
3.74Ϫ3.89 (m, 2 H, CH2O), 4.53Ϫ4.58 (m, 1 H, CHO) (common
signals); 2.43 (s, 2 H, 2-H, 14c), 2.99 (s, 1 H, 2-H, 15). 14c: 13C
NMR: δ ϭ 13.9 (Ϫ, C-2), 19.3 (Ϫ, C-4Ј), 25.2 (Ϫ, C-5Ј), 25.4
(quint, JCD ϭ 14.8 Hz, C-3), 30.34 (Ϫ, C-3Ј), 62.2 (Ϫ, C-4), 64.6
(Ϫ, C-6Ј), 98.9 (ϩ, C-2Ј), 119.4 (Cquat, CN) ppm. For the properties
of the non-deuterated compound 14b see ref.[46]. 15 (a mixture of
two diastereomers): 13C NMR: δ ϭ 19.1 (Ϫ, 2 CH2), 25.1 (Ϫ, 2
CH2), 25.17 (ϩ, 2 CH), 30.30 (Ϫ, 2 CH2), 61.8, 62.3, 63.7, 63.8 (Ϫ,
CH2O), 98.3, 99.1 (ϩ, CHO), 121.6 (Cquat, 2 CN) ppm. The signals
of CD2 carbon could not be detected because of their low intensity.
2-(2,2-Dideuterio-2-iodoethoxy)tetrahydropyran (13): Iodide 13
(33.53 g, 67%) was obtained from the alcohol 12 (28.70 g,
193.7 mmol), PPh3 (50.80 g, 193.7 mmol), imidazole (13.21 g,
194 mmol) and I2 (49.16 g, 193.7 mmol) according to GP2a after
column chromatography (120 g of silica gel, 4 ϫ 15 cm column,
hexane/Et2O, 10:1, Rf ϭ 0.32) as a colorless oil. 1H NMR: δ ϭ
1.40Ϫ1.63 (m, 4 H, 5-H, 4-H), 1.64Ϫ1.84 (m, 2 H, 3-H), 3.43Ϫ3.52
(m, 1 H, 6-H), 3.66 (d, J ϭ 10.8 Hz, 1 H, 1Ј-H), 3.77Ϫ3.81 (m, 1
H, 6-H), 3.89 (d, J ϭ 10.8 Hz, 1 H, 1Ј-H), 4.63 (t, J ϭ 3.3 Hz, 1
H, 2-H) ppm. 13C NMR: δ ϭ 3.20 (quint, JCD ϭ 20.7 Hz, C-2Ј), 3,3-Dideuterio-4-hydroxybutyronitrile (9c): The mixture of 14c and
19.1 (Ϫ, C-4), 25.2 (Ϫ, C-5), 30.4 (Ϫ, C-3), 62.1 (Ϫ, C-1Ј), 67.9 (Ϫ, 15 (22.17 g) obtained as indicated above was stirred in MeOH
Eur. J. Org. Chem. 2005, 123Ϫ135
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
129

A. Zeeck, A. de Meijere et al.
(150 mL) with PPTS (2.0 g, 7.96 mmol) at 50 °C for 1.5 h. After ene)glycinate (7) (7.16 g 24.2 mmol) and nBuLi (24.2 mmol,
FULL PAPER
cooling, the mixture was concentrated under reduced pressure, the
residue was taken up with CH2Cl2 (5 mL) and separated by column
chromatography (200 g of silica gel, 4.5 ϫ 30 cm column, Et2O,
9.84 mL of a 2.46 n solution in hexane) according to GP3 as a
colorless oil after column chromatography (200 g of silica gel, 6 ϫ
20 cm column, hexane/Et2O, 1:1, Rf ϭ 0.33). H NMR: δ ϭ 1.45
1
Rf ϭ 0.33) to give 9c (7.58 g, 67% over two steps) as a colorless oil. [s, 9 H, C(CH3)3], 1.96Ϫ2.07 (m, 2 H, 3-H), 2.30 (s, 2 H, 5-H). 3.96
1H NMR: δ ϭ 2.39 (s, 2 H, 2-H), 3.23 (s, 1 H, OH), 3.60 (s, 2 H, (dd, J ϭ 5.0, 7.5 Hz, 1 H, 2-H), 7.15Ϫ7.19 (m, 2 H, Ph-H),
4-H) ppm. 13C NMR: δ ϭ 13.2 (Ϫ, C-2), 27.0 (quint, JCD
19.8 Hz, C-3), 59.5 (Ϫ, C-4), 119.6 (Cquat, CN) ppm.
ϭ
7.30Ϫ7.39 (m, 3 H, Ph-H), 7.45Ϫ7.47 (m, 3 H, Ph-H), 7.62Ϫ7.66
(m, 2 H, Ph-H) ppm. 13C NMR: δ ϭ 16.9 (Ϫ, C-5), 21.9 (quint,
JCD ϭ 30.3 Hz, C-4), 27.9 [ϩ, C(CH3)3], 32.1 (Ϫ, C-3), 64.7 (ϩ,
C-2), 81.2 [Cquat, C(CH3)3], 119.4 (Cquat, CN), 127.5, 127.9, 128.1,
2,2-Dideuterio-4-(tetrahydropyran-2-yloxy)butyronitrile (14d): To a
suspension of NaCN (5.60 g, 114 mmol) in anhydrous DMSO
(180 mL) was added the iodide 18 (20.4 g, 75.0 mmol) at ambient
temperature over a period of 20 min under Ar. After an additional
stirring at 40 °C for 3 h, the reaction mixture was poured into ice-
cold water (200 mL), and the resulting mixture extracted with Et2O
(3 ϫ 100 mL). The combined ethereal phases were washed with
H2O (3 ϫ 100 mL) and brine (100 mL), and dried and concentrated
under reduced pressure. Column chromatography of the residue
(100 g of silica gel, 4 ϫ 15 cm column, hexane/Et2O, 1:1, Rf ϭ 0.27)
128.6, 130.3 (ϩ, Ph-C), 136.2, 139.1 (Cquat, Ph-C), 170.49 (Cquat
,
CϭN), 170.52 (Cquat, CϭO) ppm. MS (CI): m/z (%) ϭ 729 (19)
[2M ϩ Hϩ], 365 (100) [M ϩ Hϩ].
tert-Butyl 5-Cyano-5,5-dideuterio-2-(diphenylmethylenamino)pen-
tanoate (19d): Compound 19d (12.26 g, 94%) was obtained from
the iodide 10d (7.05 g, 35.8 mmol), tert-butyl N-(diphenylmethyl-
ene)glycinate (7) (11.64 g, 39.4 mmol) and nBuLi (39.4 mmol,
24.0 mL of a 1.64 n solution in hexane) according to GP3 as a
colorless oil after column chromatography (120 g of silica gel, 4 ϫ
1
gave 14d (8.06 g, 63%) as a colorless oil. H NMR: δ ϭ 1.41Ϫ1.51
1
15 cm column, hexane/Et2O, 1:1, Rf ϭ 0.33). H NMR: δ ϭ 1.44
(m, 4 H, 5Ј-H, 4Ј-H), 1.59Ϫ1.73 (m, 2 H, 3Ј-H), 1.83 (t, J ϭ 6.0 Hz,
2 H, 3-H), 3.34Ϫ3.46 (m, 2 H, 6Ј-H), 3.69Ϫ3.79 (m, 2 H, 4-H),
4.49 (t, J ϭ 3.3 Hz, 1 H, 2Ј-H) ppm. 13C NMR: δ ϭ 13.5 (quint,
JCD ϭ 21.0 Hz, C-2), 19.1 (Ϫ, C-4Ј), 25.0 (Ϫ, C-5Ј), 25.3 (Ϫ, C-3),
30.2 (Ϫ, C-3Ј), 62.0 (Ϫ, C-4), 64.5 (Ϫ, C-6Ј), 98.6 (ϩ, C-2Ј), 119.3
(Cquat, CN) ppm. MS (CI): m/z (%) ϭ 189 (38) [M ϩ NH4ϩ], 172
(100) [M ϩ Hϩ]. For the properties of the non-deuterated com-
[s, 9 H, C(CH3)3], 1.65Ϫ1.69 (m, 2 H, 4-H), 1.96Ϫ2.08 (m, 2 H, 3-
H), 3.94Ϫ3.99 (m, 1 H, 2-H), 7.15Ϫ7.19 (m, 2 H, Ph-H), 7.28Ϫ7.38
(m, 3 H, Ph-H), 7.43Ϫ7.48 (m, 3 H, Ph-H), 7.63Ϫ7.66 (m, 2 H,
Ph-H) ppm. 13C NMR: δ ϭ 16.4 (quint, JCD ϭ 22.0 Hz, C-5), 21.7
(Ϫ, C-4), 27.8 [ϩ, C(CH3)3], 32.1 (Ϫ, C-3), 64.7 (ϩ, C-2), 81.0
[Cquat, C(CH3)3], 119.3 (Cquat, CN), 127.4, 127.8, 128.4, 128.5,
130.2 (ϩ, Ph-C), 136.1, 139.0 (Cquat, Ph-C), 170.0 (Cquat, CϭN),
170.4 (Cquat, CϭO) ppm. MS (CI): m/z (%) ϭ 729 (23) [2M ϩ Hϩ],
365 (100) [M ϩ Hϩ].
pound see ref.[46]
.
Coupling of Iodides 10b؊d and 26 with tert-Butyl N-(Diphenylmeth-
ylene)glycinate (7). General Procedure (GP) 3: To a stirred solution
of protected glycine 7 (40 mmol) in anhydrous THF (300 mL) was
added dropwise nBuLi (40 mmol as a solution in hexane) at Ϫ78
°C over a period of 1 h under Ar. After stirring for an additional
1 h at this temperature, a solution of iodide (40 mmol) in THF
(50 mL) was added to the suspension of the lithio compound at the
same temperature over 15 min. The mixture was stirred for 20 h at
Ϫ78 °C, then warmed to 20 °C over a period of 24 h and stirred
for 12 h at ambient temp. After this, the mixture was poured into
ice-cold water (100 mL), extracted with diethyl ether (3 ϫ 60 mL),
and the combined organic layers were washed with H2O (80 mL),
brine (80 mL), dried and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel deac-
tivated with triethylamine.
Change of a Protective Group in Imines 19b؊d. General Procedure
(GP) 4: To a solution of the respective imine (18.9 mmol) in Et2O
(200 mL) was added 1 n aqueous HCl solution (100 mL), and the
resulting mixture was vigorously stirred with TLC-monitoring. The
aqueous phase was washed with Et2O (2 ϫ 50 mL), brought to a
pH of about 10 with concentrated ammonia solution, and the sol-
vents were evaporated under reduced pressure. The residue was
taken up with EtOAc (200 mL), and the solution was washed with
aqueous 10% ammonia solution (2 ϫ 50 mL), brine (50 mL), and
dried and concentrated under reduced pressure. The crude amine
was dissolved in anhydrous MeOH (150 mL), and to this solution
was added in one portion di-tert-butyl pyrocarbonate (Boc2O)
(28.0 mmol). The resulting solution was stirred at ambient tem-
perature for 12 h and concentrated under reduced pressure. The
residue was taken up with diethyl ether (180 mL), washed with H2O
(3 ϫ 50 mL), dried and concentrated under reduced pressure. The
product was purified by column chromatography.
tert-Butyl 5-Cyano-3,3-dideuterio-2-(diphenylmethylenimino)penta-
noate (19b): Compound 19b (12.43 g, 70%) was obtained from the
iodide 10b (9.62 g, 48.8 mmol), tert-butyl N-(diphenylmethyl-
ene)glycinate (7) (14.4 g, 48.8 mmol) and nBuLi (49.0 mmol,
29.7 mL of a 1.65 n solution in hexane) according to GP3 as a
colorless oil after column chromatography (200 g of silica gel, 6 ϫ
15 cm column, hexane/Et2O, 1:1, Rf ϭ 0.37). 1H NMR: δ ϭ 1.45
[s, 9 H, C(CH3)3], 1.64Ϫ1.68 (t, J ϭ 7.1 Hz, 2 H, 4-H), 2.30 (t, J ϭ
7.1 Hz, 2 H, 5-H), 3.95 (s, 1 H, 2-H), 7.15Ϫ7.19 (m, 2 H, Ph-H),
7.29Ϫ7.38 (m, 3 H, Ph-H), 7.44Ϫ7.46 (m, 3 H, Ph-H), 7.63Ϫ7.67
(m, 2 H, Ph-H) ppm. 13C NMR: δ ϭ 14.1 (Ϫ, C-5), 22.0 (Ϫ, C-4),
28.0 [ϩ, C(CH3)3], 31.6 (quint, JCD ϭ 22.0 Hz, C-3), 64.8 (ϩ, C-
2), 81.3 [Cquat, C(CH3)3], 119.5 (Cquat, CN), 127.7, 128.1, 128.6,
128.7, 130.4 (ϩ, Ph-C), 136.4, 139.3 (Cquat, Ph-C), 170.6 (Cquat, Cϭ
N), 170.7 (Cquat, CϭO) ppm. MS (CI): m/z (%) ϭ 729 (23) [2M ϩ
Hϩ], 365 (100) [M ϩ Hϩ].
tert-Butyl 2-(tert-Butoxycarbonylamino)-5-cyano-3,3-dideuteriopen-
tanoate (20b): Compound 20b (13.27 g, 94%) was obtained from
19b (17.1 g, 46.9 mmol) according to GP4 after chromatographic
purification (200 g of silica gel, 6 ϫ 20 cm column, hexane/Et2O,
2:1, Rf ϭ 0.31) as a colorless solid, m.p. 72Ϫ74 °C (hexane). 1H
NMR: δ ϭ 1.42, 1.45 [s, 9 H, C(CH3)3], 1.71 (t, J ϭ 6.9 Hz, 2 H,
4-H), 2.38 (t, J ϭ 6.9 Hz, 2 H, 5-H), 4.16 (d, J ϭ 8.3 Hz, 1 H, 2-
H), 5.11 (d, J ϭ 8.3 Hz, 1 H, NH) ppm. 13C NMR: δ ϭ 16.7 (Ϫ,
C-5), 21.2 (Ϫ, C-4), 27.9, 28.2 [ϩ, C(CH3)3], 31.3 (quint, JCD
ϭ
21.1 Hz, C-3), 52.8 (ϩ, C-2), 79.8, 82.4 [Cquat, C(CH3)3], 119.1
(Cquat, CN), 155.3 (Cquat, NCϭO), 171.1 (Cquat, CϭO) ppm. MS
(CI): m/z (%) ϭ 618 (20) [2M ϩ NH4ϩ], 318 (100) [M ϩ NH4ϩ],
301 (5) [M ϩ Hϩ]. C15H24D2N2O4 (300.4): calcd. C 59.98, H(D)
9.39, N 9.32; found C 60.29, H(D) 9.19, N 9.15.
tert-Butyl 5-Cyano-4,4-dideuterio-2-(diphenylmethylenamino)pen-
tanoate (19c): Compound 19c (8.83 g, 100%) was obtained from the tert-Butyl 2-(tert-Butoxycarbonylamino)-5-cyano-4,4-dideuteriopen-
iodide 10c (4.93 g, 25.0 mmol), tert-butyl N-(diphenylmethyl- tanoate (20c): Compound 20c (5.68 g, 78%) was obtained from 19c
130
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2005, 123Ϫ135

Biosynthesis of a Constituent of the Signal Metabolite Hormaomycin
FULL PAPER
(8.83 g, 24.2 mmol) according to GP4 after chromatographic purifi-
1.42 [s, 9 H, C(CH3)3], 1.44Ϫ1.61 (m, 1 H, 3-H), 1.62Ϫ1.80 (m, 1
H, 3-H), 2.63 (s, 2 H, 6-H), 4.19Ϫ4.23 (m, 1 H, 2-H), 5.18 (br. s, 1
cation (200 g of silica gel, 6 ϫ 20 cm column, hexane/Et2O, 2:1,
Rf ϭ 0.27) as a colorless solid, m.p. 77Ϫ79 °C (hexane). 1H NMR: H, NH), 5.22 (br. s, 2 H, NH2) ppm. 13C NMR: δ ϭ 22.2 (Ϫ, C-
δ ϭ 1.41, 1.45 [s, 9 H, C(CH3)3], 1.65 (dd, J ϭ 7.2, 13.8 Hz, 1 H,
3-H), 1.91 (dd, J ϭ 5.2, 13.8 Hz, 1 H, 3-H), 2.37 (s, 2 H, 5-H) 4.15
4), 27.9, 28.2 [ϩ, C(CH3)3], 30.3 (Ϫ, C-3), 33.0 (quint, JCD
ϭ
22.0 Hz, C-5), 41.7 (Ϫ, C-6), 53.8 (ϩ, C-2), 79.4, 81.6 [Cquat
,
(ddd, J ϭ 5.0, 5.2, 7.2 Hz, 1 H, 2-H), 5.11 (d, J ϭ 5.0 Hz, 1 H, NH) C(CH3)3], 155.3 (Cquat, NCϭO), 171.9 (Cquat, CϭO) ppm.
ppm. 13C NMR: δ ϭ 16.5 (Ϫ, C-5), 21.8 (quint, JCD ϭ 22.0 Hz, C-
Deprotection of Protected Amino Acids 21b؊d. General Procedure
(GP) 6: The protected amino acids 21bϪd were taken up with 1 n
aqueous HCl solution (250 mL), and the mixture vigorously stirred
at ambient temperature for two days. After evaporation of the sol-
vent, the residue was dried in a desiccator over P2O5 under reduced
pressure to give amino acid dihydrochlorides in pure form.
4), 27.9, 28.2 [ϩ, C(CH3)3], 31.8 (Ϫ, C-3), 52.9 (ϩ, C-2), 79.8, 82.4
[Cquat, C(CH3)3], 119.1 (Cquat, CN), 155.3 (Cquat, NCϭO), 171.1
(Cquat, CϭO) ppm. C15H24D2N2O4 (300.4): calcd. C 59.98, H(D)
9.39, N 9.32; found C 60.10, H(D) 8.97 N 9.16.
tert-Butyl 2-(tert-Butoxycarbonylamino)-5-cyano-5,5-dideuteriopen-
tanoate (20d): Compound 20d (3.94 g, 69%) was obtained from 19d
(6.90 g, 18.9 mmol) according to GP4 after chromatographic puri-
fication (100 g of silica gel, 4.5 ϫ 15 cm column, hexane/Et2O, 2:1,
Rf ϭ 0.27) as a colorless solid, m.p. 70Ϫ72 °C. IR (KBr): ν˜ ϭ
3273 cmϪ1, 2978, 1735, 1703, 1397, 1366, 1155. 1H NMR: δ ϭ 1.39,
1.42 [s, 9 H, C(CH3)3], 1.60Ϫ1.78 (m, 2 H, 4-H), 1.81Ϫ1.98 (m, 2
H, 3-H), 4.08Ϫ4.21 (m, 1 H, 2-H), 5.15 (br. s, 1 H, NH) ppm. 13C
NMR: δ ϭ 16.4 (quint, JCD ϭ 22.5 Hz, C-5), 21.4 (Ϫ, C-4), 27.9,
rac-2,6-Diamino-3,3-dideuteriohexanoic Acid (rac-3,3-Dideuteriolys-
ine) Dihydrochloride (6b·2 HCl): Compound 6b·2 HCl (2.04 g, 91%)
was obtained from 21b (3.08 g, 10.1 mmol) according to GP6 as a
colorless powder, m.p. 168Ϫ170 °C. 1H NMR (D2O): δ ϭ
1.28Ϫ1.35 (t, J ϭ 7.6 Hz, 2 H, 4-H), 1.55 (tt, J ϭ 7.6, 7.6 Hz, 2 H,
5-H), 2.84 (t, J ϭ 7.6 Hz, 2 H, 6-H), 3.91 (br. s, 1 H, 2-H) ppm.
13C NMR (D2O): δ ϭ 23.7 (Ϫ, C-4), 28.7 (Ϫ, C-5), 31.0 (quint,
JCD ϭ 21.10 Hz, C-3), 41.5 (Ϫ, C-6), 55.0 (ϩ, C-2), 174.5 (Cquat
,
28.2 [ϩ, C(CH3)3], 32.0 (Ϫ, C-3), 52.9 (ϩ, C-2), 79.8, 82.3 [Cquat
,
,
CϭO) ppm. MS (CI): m/z (%) ϭ 166 (35) [M Ϫ 2HCl ϩ NH4ϩ],
149 (100) [Mϩ Ϫ 2HCl ϩ H].
C(CH3)3], 119.1 (Cquat, CN), 155.3 (Cquat, NCϭO), 171.1 (Cquat
CϭO) ppm. MS (CI): m/z (%) ϭ 618 (25) [2M ϩ NH4ϩ], 318 (100)
[M ϩ NH4ϩ]. C15H24D2N2O4 (300.4): calcd. C 59.98, H(D) 9.39,
N 9.32; found C 60.21, H(D) 9.35, N 9.26.
rac-2,6-Diamino-4,4-dideuteriohexanoic Acid (rac-4,4-Dideuteriolys-
ine) Dihydrochloride (6c·2 HCl): Compound 6c·2 HCl (3.69 g, 96%)
was obtained from 21c (5.29 g, 17.4 mmol) according to GP6 as a
Raney Ni-Catalyzed Hydrogenation of Nitriles 20b؊d and 32. Gen-
eral Procedure (GP) 5: To a solution of the respective nitrile
(10 mmol) in MeOH (150 mL) was added a 3.5 n ammonia solu-
tion in MeOH (50 mL) and freshly prepared Raney Ni (3.0 g), and
the resulting mixture was hydrogenated at ambient temperature in
a Parr apparatus under a pressure of 4 bar of hydrogen with TLC-
monitoring for the indicated time. The mixture was then filtered
through a pad of Celite (3 cm) and concentrated under reduced
pressure. The residue was taken up with EtOAc (150 mL), and the
solution was washed with aqueous 10% ammonia solution (2 ϫ
50 mL), dried and concentrated under reduced pressure again.
1
colorless powder, m.p. 178Ϫ180 °C. H NMR (D2O): δ ϭ 1.50 (t,
J ϭ 7.4 Hz, 2 H, 5-H), 1.72 (dd, J ϭ 6.1, 16.4 Hz, 1 H, 3-H), 1.82
(dd, J ϭ 6.1, 16.4 Hz, 1 H, 3-H), 2.80 (t, J ϭ 7.4 Hz, 2 H, 6-H),
3.88 (dd, J ϭ 6.1, 6.1 Hz, 1 H, 2-H) ppm. 13C NMR (D2O): δ ϭ
23.1 (quint, JCD ϭ 20.5 Hz, C-4), 28.5 (Ϫ, C-5), 31.5 (Ϫ, C-3), 41.2
(Ϫ, C-6), 55.0 (ϩ, C-2), 174.4 (Cquat, CϭO) ppm.
rac-2,6-Diamino-5,5-dideuteriohexanoic Acid (rac-5,5-Dideuteriolys-
ine) Dihydrochloride (6d·2 HCl): Compound 6d·2 HCl (1.98 g, 94%)
was obtained from 21d (2.90 g, 9.53 mmol) according to GP6 as a
colorless powder, m.p. 173Ϫ175 °C. 1H NMR (D2O): δ ϭ
1.32Ϫ1.35 (m, 2 H, 4-H), 1.79Ϫ1.81 (m, 2 H, 3-H), 2.83 (s, 2 H,
6-H), 3.90 (dd, J ϭ 5.6, 5.6 Hz, 1 H, 2-H) ppm. 13C NMR (D2O):
δ ϭ 26.7 (Ϫ, C-4), 31.0 (quint, JCD ϭ 20.9 Hz, C-5), 34.7 (Ϫ, C-
3), 44.5 (Ϫ, C-6), 58.8 (ϩ, C-2), 177.5 (Cquat, CϭO) ppm. MS (CI):
m/z (%) ϭ 149 (100) [Mϩ Ϫ 2HCl ϩ H], 147 (90) [Mϩ Ϫ 2HCl Ϫ
H], 130 (80) [Mϩ Ϫ 2HCl Ϫ H2O].
tert-Butyl 6-Amino-2-(tert-butoxycarbonylamino)-3,3-dideuteriohex-
anoate (21b): Compound 21b (3.08 g, 100%) was obtained from 20b
(3.05 g, 10.2 mmol) according to GP5 (reaction time 3 d) as a
slightly yellow oil. 1H NMR: δ ϭ 1.27Ϫ1.40 (m, 4 H, 4-H, 5-H),
1.41, 1.43 [s, 9 H, C(CH3)3], 2.65 (t, J ϭ 6.6 Hz, 2 H, 6-H),
4.10Ϫ4.15 (m, 1 H, 2-H), 5.03 (br. s, 1 H, NH), 5.25 (br. s, 2 H,
NH2) ppm. 13C NMR: δ ϭ 22.5 (Ϫ, C-4), 28.2, 28.6 [ϩ, C(CH3)3],
30.2 (quint, JCD ϭ 22.0 Hz, C-3), 33.6 (Ϫ, C-5), 41.7 (Ϫ, C-6), 54.0
(ϩ, C-2), 79.7, 81.9 [Cquat, C(CH3)3], 155.6 (Cquat, NCϭO), 172.3
(Cquat, CϭO) ppm.
N,N-Bis(tert-butoxycarbonyl)-1,1-dideuterio-4-(tetrahydropyran-2-
yloxy)butylamine (25): To a suspension of potassium bis(tert-bu-
toxycarbonyl)amide (4.72 g, 18.5 mmol) in anhydrous DMF
(40 mL) was added iodide 24 (5.15 g, 18.0 mmol) in one portion
under Ar. The resulting mixture was stirred at ambient temperature
for 4 h, poured into a mixture of ice-cold water (150 mL) and di-
ethyl ether (50 mL). The aqueous layer was extracted with Et2O
(50 mL), and the combined organic phases were washed with H2O
(2 ϫ 50 mL) and brine (50 mL), and dried and concentrated under
reduced pressure. The product was purified by column chromatog-
raphy (170 g of silica gel, 4.5 ϫ 20 cm column, hexane/Et2O, 2:1,
Rf ϭ 0.44) to give 25 (6.49 g, 96%) as a colorless oil. IR: ν˜ ϭ 2941
tert-Butyl 6-Amino-2-(tert-butoxycarbonylamino)-4,4-dideuteriohex-
anoate (21c): Compound 21c (5.29 g, 98%) was obtained from 20c
(5.33 g, 17.73 mmol) according to GP5 (reaction time 2 d) as a
1
slightly yellow oil. H NMR: δ ϭ 1.14 (t, J ϭ 6.8 Hz, 2 H, 5-H),
1.33, 1.35 [s, 9 H, C(CH3)3], 1.48 (dd, J ϭ 7.3, 13.8 Hz, 1 H, 3-H),
1.65 (dd, J ϭ 6.5, 13.8 Hz, 1 H, 3-H), 2.58 (t, J ϭ 6.8 Hz, 2 H, 6-
H), 3.99 (dd, J ϭ 6.5, 7.3 Hz, 1 H, 2-H), 5.15 (br. s, 1 H, NH),
5.30 (br. s, 2 H, NH2) ppm. 13C NMR: δ ϭ 21.6 (quint, JCD
ϭ
1
cmϪ1, 2671, 1734, 1694, 1367, 1123, 733. H NMR: δ ϭ 1.48 [s, 18
20.8 Hz, C-4), 27.8, 28.1 [ϩ, C(CH3)3], 32.3 (Ϫ, C-3), 33.1 (Ϫ, C-
5), 41.7 (Ϫ, C-6), 53.7 (ϩ, C-2), 79.2, 81.3 [Cquat, C(CH3)3], 155.2
(Cquat, NCϭO), 171.8 (Cquat, CϭO) ppm.
H, 2 C(CH3)3], 1.56Ϫ1.77 (m, 10 H, 4Ј-H, 5Ј-H, 3Ј-H, 2-H, 3-H),
3.39Ϫ3.49 (m, 2 H, 4-H), 3.70Ϫ3.84 (m, 2 H, 6Ј-H), 4.55 (br. s, 1
H, 2Ј-H) ppm. 13C NMR: δ ϭ 19.5 (Ϫ, C-4Ј), 25.4 (Ϫ, C-5Ј), 27.0
(Ϫ, C-3), 28.0 [ϩ, 2 C(CH3)3], 30.5 (Ϫ, C-2), 30.7 (Ϫ, C-3Ј), 62.2
tert-Butyl 6-Amino-2-(tert-butoxycarbonylamino)-5,5-dideuteriohex-
anoate (21d): Compound 21d (2.89 g, 100%) was obtained from (Ϫ, C-4), 67.1 (Ϫ, C-6Ј), 82.2 [Cquat, 2 C(CH3)3], 98.7 (ϩ, C-2Ј),
20d (2.86 g, 9.52 mmol) according to GP5 (reaction time 15 h) as 152.6 (Cquat, 2 NCϭO) ppm. The intensity of the CD2 carbon sig-
a slightly yellow oil. 1H NMR: δ ϭ 1.23Ϫ1.38 (m, 2 H, 4-H), 1.40, nal was too low. MS (CI): m/z (%) ϭ 768 (85) [2M ϩ NH4ϩ], 712
Eur. J. Org. Chem. 2005, 123Ϫ135
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
131

A. Zeeck, A. de Meijere et al.
FULL PAPER
(40) [2M Ϫ C4H8 ϩ NH4ϩ], 393 (100) [M ϩ NH4ϩ], 337 (100) [M into ice-cold 10% aqueous HCl solution (200 mL), the aqueous
Ϫ C4H8 ϩ NH4ϩ], 253 (50).
phase was extracted with Et2O (2 ϫ 80 mL), and the combined
organic layers were washed with H2O (200 mL), and dried and con-
centrated under reduced pressure. Column chromatography (200 g
of silica gel, 6 ϫ 20 cm column, hexane/Et2O, 1:1) gave the starting
material 30 (3.42 g, 48%, Rf ϭ 0.42) and the product 31 (3.46 g,
42%, Rf ϭ 0.16) as a 2:1 mixture of diastereomers 31a and 31b. 1H
NMR (31a,b): δ ϭ 1.45, 1.48 [s, 9 H, C(CH3)3, 31a,b] 1.65Ϫ1.89
(m, 1 H, 3-H, 31a,b), 1.98 (ddd, J ϭ 3.0, 11.1, 12.5 Hz, 1 H, 3-H,
31a), 2.12 (ddd, J ϭ 3.0, 10.0, 13.5 Hz, 1 H, 3-H, 31b), 3.49 (br. s,
1 H, OH, 31a,b), 3.95 (dd, J ϭ 4.0, 10.1 Hz, 1 H, 4-H, 31a), 4.11
(dd, J ϭ 3.0, 10.0 Hz, 1 H, 4-H, 31b), 4.18 (ddd, J ϭ 4.0, 7.4,
7.5 Hz, 1 H, 2-H, 31b), 4.37 (ddd, J ϭ 4.0, 6.7, 11.1 Hz, 1 H, 2-H,
31a), 4.85 (d, J ϭ 4.0 Hz, 1 H, NH, 31b), 5.42 (d, J ϭ 6.7 Hz, 1
H, NH, 31a) ppm. 13C NMR (31a): δ ϭ 27.8, 28.1 [ϩ, C(CH3)3],
N,N-Bis(tert-butoxycarbonyl)-1,1-dideuterio-4-iodobutylamine (26):
Column chromatography (200 g of silica gel, 5 ϫ 30 cm column,
hexane/Et2O, 5:1) of a reaction mixture obtained from the pro-
tected amino alcohol 25 (6.67 g, 17.8 mmol), dppe (8.53 g,
21.4 mmol) and I2 (5.45 g, 21.5 mmol) according to GP2b gave the
diprotected aminoiodide 26 (3.65 g, 51%) and N-tert-butoxycar-
bonyl-1,1-dideuterio-4-iodobutylamine (27) (2.01 g, 38%). 26: Rf ϭ
1
0.34. H NMR: δ ϭ 1.45 [s, 18 H, 2 C(CH3)3], 1.61 (t, J ϭ 8.2 Hz,
2 H, 2-H), 1.75Ϫ1.81 (m, 2 H, 3-H), 3.14 (t, J ϭ 6.8 Hz, 2 H, 4-
H) ppm. 13C NMR: δ ϭ 6.0 (Ϫ, C-4), 27.9 [ϩ, 2 C(CH3)3], 29.6
(Ϫ, C-3), 30.6 (Ϫ, C-2), 44.4 (quint, J ϭ 19.0 Hz, C-1), 82.1 [Cquat
2 C(CH3)3], 152.4 (Cquat, 2 NCϭO) ppm. 27: Rf ϭ 0.18.
,
40.8 (Ϫ, C-3), 50.7 (ϩ, C-2), 63.2 (ϩ, C-4), 80.8, 82.9 [Cquat
C(CH3)3], 117.4 (Cquat, CN), 156.4 (Cquat, NCϭO), 171.1 (Cquat
,
,
tert-Butyl 6-[N,N-Bis(tert-butoxycarbonyl)]-6,6-dideuterio-2-(N-di-
phenylmethyleneamino)hexanoate (28): Compound 28 (5.16 g,
100%) was obtained from the iodide 26 (3.65 g, 9.1 mmol), tert-
butyl N-(diphenylmethylene)glycinate (7) (2.96 g, 10.0 mmol) and
nBuLi (10.1 mmol, 6.1 mL of a 1.65 n solution in hexane) accord-
ing to GP3 as a colorless oil after column chromatography (150 g
of silica gel, 4.5 ϫ 25 cm column, hexane/Et2O, 3:1, Rf ϭ 0.36). 1H
NMR: δ ϭ 1.21Ϫ1.36 (m, 4 H, 3-H, 4-H), 1.42, 1.44, 1.45 [s, 9 H,
C(CH3)3], 1.82Ϫ1.96 (m, 2 H, 5-H), 3.83 (t, J ϭ 7.4 Hz, 1 H, 2-H),
7.15Ϫ7.19 (m, 2 H, Ph-H), 7.29Ϫ7.38 (m, 3 H, Ph-H), 7.44Ϫ7.46
(m, 3 H, Ph-H), 7.63Ϫ7.67 (m, 2 H, Ph-H) ppm. 13C NMR: δ ϭ
23.4 (Ϫ, C-4), 28.0 [ϩ, 3 C(CH3)3], 28.7 (Ϫ, C-5), 33.4 (Ϫ, C-3),
66.0 (ϩ, C-2), 80.8 [Cquat, C(CH3)3], 81.9 [Cquat, 2 C(CH3)3], 127.8,
127.9, 128.4, 128.7, 130.1 (ϩ, Ph-C), 136.7, 139.6 (Cquat, Ph-C),
152.6 (Cquat, 2 NCϭO), 170.0 (Cquat, CϭN), 170.4 (Cquat, CϭO)
ppm. The intensity of CD2 carbon signals was too low. MS (CI):
m/z (%) ϭ 569 (23) [M ϩ Hϩ], 297 (72), 296 (100).
CϭO) ppm. 13C NMR (31b): δ ϭ 27.7, 28.0 [ϩ, C(CH3)3], 39.3 (Ϫ,
C-3), 51.6 (ϩ, C-2), 64.93 (ϩ, C-4), 80.2, 82.6 [Cquat, C(CH3)3],
117.35 (Cquat, CN), 155.6 (Cquat, NCϭO), 170.9 (Cquat, CϭO) ppm.
The intensity of CD2 carbon signals was too low.
tert-Butyl 2-(tert-Butoxycarbonylamino)-5-cyano-5,5-dideuterio-4-
(tetrahydropyran-2-yloxy)pentanoate (32): A solution of alcohol 31
(1.48 g, 4.68 mmol) and 3,4-dihydro-2H-pyran (DHP) (789 mg,
0.85 mL, 9.38 mmol) in anhydrous CH2Cl2 (50 mL) was stirred
with PPTS (118 mg, 0.47 mmol) at ambient temperature for 24 h.
After this, the reaction mixture was diluted with Et2O (50 mL),
washed with half-saturated sodium chloride solution (50 mL), and
dried and concentrated under reduced pressure. Column chroma-
tography of the residue (60 g of silica gel, 4 ϫ 15 cm column, hex-
ane/Et2O, 1:1, Rf ϭ 0.38) gave the cyano ester 32 (1.34 g, 71%) as
a slightly yellow oil. 1H NMR: δ ϭ 1.37, 1.40 [s, 9 H, C(CH3)3],
1.48Ϫ1.65 (m, 4 H, 4Ј-H, 5Ј-H), 1.66Ϫ1.76 (m, 2 H, 3Ј-H), 1.90
(ddd, J ϭ 3.5, 8.0, 14.2 Hz, 1 H, 3-H), 2.10 (ddd, J ϭ 4.2, 8.8,
14.2 Hz, 1 H, 3-H), 3.39Ϫ3.46 (m, 1 H, 6Ј-H), 3.78Ϫ3.82 (m, 1 H,
6Ј-H), 4.04 (dd, J ϭ 3.5, 8.8 Hz, 1 H, 4-H), 4.10Ϫ4.23 (m, 1 H, 2-
H), 4.68 (br. s, 1 H, 2Ј-H), 5.53 (d, J ϭ 8.5 Hz, 1 H, NH) ppm.
13C NMR: δ ϭ 19.5 (Ϫ, C-4Ј), 24.9 (Ϫ, C-5Ј), 27.8, 28.1 [ϩ,
C(CH3)3], 30.6 (Ϫ, C-3Ј), 36.5 (Ϫ, C-3), 51.4 (ϩ, C-4), 63.2 (Ϫ, C-
6Ј), 68.2 (ϩ, C-2), 79.3, 81.1 [Cquat, C(CH3)3], 97.3 (ϩ, C-2Ј), 116.8
(Cquat, CN), 155.3 (Cquat, NCϭO), 171.0 (Cquat, CϭO) ppm. The
intensity of CD2 carbon signals was too low. MS (CI): m/z (%) ϭ
818 (5) [2M ϩ NH4ϩ], 418 (100) [M ϩ NH4ϩ], 334 (60).
rac-2,6-Diamino-6,6-dideuteriohexanoic Acid (rac-6,6-Dideuteriolys-
ine) Dihydrochloride (6e·2 HCl): The protected amino acid 28
(5.80 g, 10.2 mmol) was taken up with diethyl ether (100 mL), 1 n
aqueous HCl solution (150 mL) was added, and the resulting mix-
ture was vigorously stirred at ambient temperature for two days.
The aqueous phase was washed with Et2O (2 ϫ 80 mL), and the
solvents evaporated in vacuo. The residue was washed with Et2O
(30 mL) and dried in a desiccator over P2O5 under reduced pressure
to give amino acid dihydrochloride 6e·2 HCl (1.79 g, 79%) as a
1
colorless powder, m.p. 168Ϫ170 °C. H NMR (D2O): δ ϭ 1.34 (tt,
J ϭ 6.8, 9.0 Hz, 2 H, 4-H), 1.54 (t, J ϭ 7.1 Hz, 2 H, 5-H),
1.78Ϫ1.85 (m, 2 H, 3-H), 3.92 (dd, J ϭ 6.1, 6.1 Hz, 1 H, 2-H) ppm.
13C NMR (D2O): δ ϭ 23.8 (Ϫ, C-4), 28.5 (Ϫ, C-3), 31.7 (Ϫ, C-5),
41.3 (quint, JCD ϭ 32.7 Hz, C-6), 55.1 (ϩ, C-2), 174.5 (Cquat, Cϭ
O) ppm. MS (CI): m/z (%) ϭ 166 (32) [M Ϫ 2HCl ϩ NH4ϩ], 149
(100) [Mϩ Ϫ 2HCl ϩ H].
tert-Butyl 2-(tert-Butoxycarbonylamino)-6-(tert-butoxycarbonylam-
ino)-5,5-dideuterio-4-(tetrahydropyran-2-yloxy)hexanoate (33): The
residue obtained from the nitrile 32 (1.10 g, 2.75 mmol) after hydro-
genation under the conditions described in GP5 [MeOH (20 mL),
a 3.5 n ammonia solution in MeOH (5 mL), Raney Ni (1.0 g), 3 h]
was stirred in MeOH (50 mL) with Boc2O (830 mg, 3.8 mmol) at
ambient temperature for 9 h. Workup according to GP4 and col-
umn chromatography (50 g of silica gel, 3 ϫ 15 cm column, hexane/
Et2O, 1:1, Rf ϭ 0.24) gave 33 as a colorless oil which was immedi-
ately deprotected. MS (CI): m/z (%) ϭ 522 (100) [M ϩ NH4ϩ], 505
(8) [M ϩ Hϩ].
tert-Butyl 2-(tert-Butoxycarbonylamino)-5-cyano-5,5-dideuterio-4-
hydroxypentanoate (31): A mixture of anhydrous CeCl3 (6.36 g,
25.8 mmol, dried at 140 °C/0.01 mbar for 2 h) and anhydrous THF
(200 mL) was vigorously stirred under Ar at ambient temperature
for 2 h. A solution of tert-butyl 2-(tert-butoxycarbonylamino)-4-
oxobutyrate (30) (7.05 g, 25.8 mmol) in anhydrous THF (30 mL)
was then added in one portion, the stirring was continued for an
additional 1 h, and the reaction mixture was then cooled to Ϫ78
°C. After this, a cold suspension of LiCD2CN Ϫ prepared from
3-Amino-5-(2-amino-1,1-dideuterioethyl)-4,5-dihydrofuran-2-one Di-
hydrochloride (34·2 HCl): The residue from the previous prep-
aration was taken up with MeOH (40 mL), and the solution vigor-
[D3]acetonitrile (CD3CN) (2.27 g, 2.70 mL, 51.5 mmol) and nBuLi ously stirred with 1 n aqueous HCl solution (40 mL) at ambient
(51.5 mmol, 20.6 mL of a 2.5 n solution in hexane) in anhydrous
THF (100 mL) at Ϫ78 °C as described above for the preparation
of compound 14c Ϫ was cannulated to the reaction mixture. After
additional stirring at Ϫ78 °C for 20 min, the mixture was poured
temperature for two days. Methanol was evaporated under reduced
pressure, the residual aqueous solution was washed with Et2O (2
ϫ 30 mL) and concentrated in vacuo to give 357 mg of a yellow oil,
crystallization of which from aqueous EtOH gave lactone 34·2HCl
132
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 123Ϫ135

Biosynthesis of a Constituent of the Signal Metabolite Hormaomycin
FULL PAPER
(196 mg, 33% over three steps, a 1.1:1 mixture of diastereomers 34a
and 34b) as a colorless powder, m.p. 176Ϫ181 °C. 1H NMR (D2O,
ppm. 13C NMR: decreased: δ ϭ 33.0 [C-3, (3-Ncp)Ala I], 35.1 [C-
3, (3-Ncp)Ala II]; duplicated (β-shift): δ ϭ 20.3 and 20.2 ppm[C-
34a,b): δ ϭ 2.43Ϫ2.50 (m, 2 H, 4-H), 2.98Ϫ3.10 (m, 2 H, 2Ј-H), 1Ј, (3-Ncp)Ala I], 21.9 and 21.6 [C-1Ј, (3-Ncp)Ala II], 51.1 and
4.35Ϫ4.49 (m, 1 H, 3-H) 4.81Ϫ4.90 (m, 1 H, 5-H) ppm. 13C NMR 51.0 [C-2, (3-Ncp)Ala II], 52.1 and 51.8 [C-2, (3-Ncp)Ala I] ppm.
(D2O, 34a): δ ϭ 32.9 (Ϫ, C-4), 33.5 (quint, J ϭ 20.2 Hz, C-1Ј), 38.8 MS (ESI), positive m/z (%) ϭ 1155/1153/1151 [M ϩ Naϩ].
(Ϫ, C-2Ј), 51.8 (ϩ, C-3), 79.9 (ϩ, C-5), 175.8 (Cquat, CϭO) ppm.
Feeding Experiment with 6c·2 HCl: This was carried out according
13C NMR (D2O, 34b): δ ϭ 34.8 (Ϫ, C-4), 35.3 (quint, J ϭ 20.2 Hz,
to GP 7 with 6c·2 HCl (460 mg, 2.08 mmol) to give hormaomycin
C-1Ј), 38.9 (Ϫ, C-2Ј), 51.5 (ϩ, C-3), 79.8 (ϩ, C-5), 175.8 (Cquat
,
1c (7.6 mg). 1H NMR: decreased: δ ϭ 0.33 [1Ј-H, (3-Ncp)Ala I]
ppm. 13C NMR: decreased: δ ϭ 20.0 [C-1Ј, (3-Ncp)Ala I], 21.6 [C-
1Ј, (3-Ncp)Ala II] ppm. MS (ESI), positive m/z (%) ϭ 1153/1152/
1151 [M ϩ Naϩ], 1131/1130/1129 [M ϩ Hϩ].
CϭO) ppm. MS (CI): m/z (%) ϭ 164 (100) [M Ϫ 2HCl ϩ NH4ϩ],
147 (100) [M Ϫ 2HCl ϩ Hϩ].
Feeding Experiments. General Procedure (GP) 7: Streptomyces gri-
seoflavus (strain W-384) was obtained from Prof. H. Wolf
(Stuttgart). An M2Ca agar in a culture tube was inoculated with
the spores of the strain W-384 and the tube was incubated at 28
°C until the sporulation was complete (grey colored aerial mycelia,
orange colored agar, incubation up to 3 weeks). The tubes were
stored at 4 °C.
Aliquots of this material (2 ϫ 2 mL, not older than 2 months) were
used to inoculate medium M6 (2 ϫ 50 mL) in two 250 mL flasks,
and the flasks were incubated in a shaking incubator at 27 °C and
120 rpm for 31 h. The seed culture was transferred into 900 mL of
medium M10 in a 1 L fermenter. Just before inoculation, 1 mL of
the vitamin solution and 5 drops of olive oil (to prevent foaming)
were added.
The production culture was incubated at 27 °C, with a stirring rate
of 700 rpm and an air flow rate of 1.6 vvm over 20 h. The pH was
maintained at 6.5 Ϯ 1.0 automatically by addition of 2 m citric
acid. The temperature was then decreased to 20 °C. After 24 h, the
solution of the respective synthetic precursor substance in 50 mL
of distilled, sterilized water (the pH of this solution was adjusted
to 7.0 with 0.5 m NaOH or 0.5 m HCl) was added over a period of
10 h. After 66 h, the production culture was harvested by filtration.
The separated mycelium was lyophilized, homogenized and ex-
tracted with ethyl acetate (2 ϫ 250 mL) by applying sonification
for 15 min. The collected organic extracts were filtered and concen-
trated under reduced pressure. The resulting crude material was
purified twice by column chromatography [acetone/hexane (2:3),
Rf ϭ 0.33 and CHCl3/CH3OH (9:1), Rf ϭ 0.63]. The thus prepared
labeled hormaomycins had purities of about 90% or higher, as de-
termined by HPLC analysis. tR ϭ 14.25 min [column: Nucleosil®-
100C18, 5 µm, 250 ϫ 3.0 mm; eluent: 0.1% TFA in water (A), 0.1%
TFA in acetonitrile (B); gradient elution: 40 Ǟ 100% B for 15 min,
flow rate Ϫ0.5 mL/min; detection: 220 or 270 nm].
Feeding Experiment with 6d·2 HCl: This was carried out according
to GP 7 with 6d·2 HCl (460 mg, 2.08 mmol) to give hormaomycin
1d (5.8 mg). 1H NMR: decreased: δ ϭ Ϫ0.60 ppm [3Ј-Ha, (3-
Ncp)Ala I], 1.01 [3Ј-Hb, (3-Ncp)Ala I, 3Ј-Ha, (3-Ncp)Ala II], 1.90
[3Ј-Hb, (3-Ncp)Ala II]. D NMR: δ ϭ Ϫ0.06, 1.00, 1.92 ppm. 13C
NMR: decreased: δ ϭ 17.4 [C-3Ј, (3-Ncp)Ala I and C-3Ј, (3-
Ncp)Ala II]; duplicated (β-shift): δ ϭ 20.0 [C-1Ј, (3-Ncp)Ala I],
21.6 [C-1Ј, (3-Ncp)Ala II], 58.1 [C-2Ј, (3-Ncp)Ala I], 59.1 [C-2Ј, (3-
Ncp)Ala II] ppm. MS (ESI), positive m/z (%) ϭ 1155/1153/1151
[M ϩ Naϩ].
Feeding Experiment with 6e·2 HCl: This was carried out according
to GP 7 with 6e·2 HCl (460 mg, 2.08 mmol) to give the unlabeled
hormaomycin 1a (18.0 mg).
Feeding Experiment with 34·2 HCl: This was carried out according
to GP 7 with 34·2 HCl (100 mg, 0.49 mmol) to give hormaomycin
1d (1.1 mg).
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB-416, Projects A3 and B5) and the Fonds der Chemischen In-
dustrie. The authors are indebted to Mrs. M. Klingebiel and Mr.
H.-P. Kroll (Göttingen) for their excellent technical assistance and
to Dr. B. Knieriem (Göttingen) for his careful proofreading of the
final manuscript.
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Feeding Experiment with (RS)-[1؊13C]-Lysine: This was carried out
For the total synthesis of Hormaomycin (1) see: [2a] B. D. Zlato-
[2]
according to GP
7
with (RS)-[1-13C]-lysine dihydrochloride
polskiy, A. de Meijere, Chem. Eur. J. 2004, 10, 4718Ϫ4727. For
the final elucidation of its structure see also:
(560 mg, 2.54 mmol) to give the 13C-labeled hormaomycin
(10.2 mg). The isotopic enrichment was 10.4% for C-1 (δ ϭ 168.4
ppm) of (3-Ncp)Ala I and 45.7% for C-1 of (3-Ncp)Ala II (δ ϭ
171.).[8,47,48]
[2b]
B. Zlatopol-
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4708Ϫ4717. The formula of 1a as presented here and in these
previous publications do depict the correct absolute configura-
tion in the (S)-isoleucine (Ile) moiety. The formulas in our re-
cently published “Corrigendum” (see Chem. Eur. J. 2004, 10,
5568) were indeed erroneous in this respect.
Feeding Experiment with (RS)-[6؊15N]-Lysine: This was carried out
according to GP
7
with (R,S)-[6-15N]-lysine dihydrochloride
(500 mg, 2.27 mmol) to give the 15N-labeled hormaomycin
(9.3 mg). 13C NMR: duplicated: δ ϭ 58.0 ([15N-13C]-1J ϭ 14.1 Hz)
and 58.2 [C-2Ј, (3-Ncp)Ala I], 59.1 ([15N-13C]-1J ϭ 14.1 Hz) and
59.2 [C-2Ј, (3-Ncp)Ala II] ppm. MS (FAB): 1131/1130/1129 [M ϩ
Hϩ].
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Feeding Experiment with 6b·2 HCl: This was carried out according
to GP 7 with 6b·2 HCl (460 mg, 2.08 mmol) to give hormaomycin
1b (4.6 mg). 1H NMR: decreased: δ ϭ Ϫ0.07 ppm [3-Ha, (3-
Ncp)Ala I], 0.58 [3-Hb, (3-Ncp)Ala I], 1.60 [3-Ha, (3-Ncp)Ala II];
1.81 [3-Hb, (3-Ncp)Ala II]. D NMR: δ ϭ 0.00, 0.50, 1.60 (1:1:2)
[4]
Eur. J. Org. Chem. 2005, 123Ϫ135
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
133

A. Zeeck, A. de Meijere et al.
FULL PAPER
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,
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[11a]
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M. J. OЈDonnell, R. L. Polt, J. Org. Chem. 1982, 47,
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[32b]
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J. A. Long, N. J. Harris, K. Lam-
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S. P. Schmidt, D. W. Brooks, Tetrahedron Lett. 1987, 28,
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134
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2005, 123Ϫ135

Biosynthesis of a Constituent of the Signal Metabolite Hormaomycin
FULL PAPER
cause of the additional strain in an azamethylenecyclopropane
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ory] of for nitrosocyclopropane and cyclopropanone oxime to
be Ϫ28.20 and Ϫ26.72 kcal·molϪ1, only 1.5 kcal·molϪ1 apart
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Although (2S)-35b should be by ca. 16 kcal·molϪ1 thermo-
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Estimated by means of 13C NMR spectroscopy as follows: %
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trum of the labeled hormaomycin)/(intensity of the signal in
the spectrum of the unlabeled hormaomycin) Ϫ1.1%.
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Received July 13, 2004
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
135