The first low molecular weight antibiotic from lactic acid bacteria: Reutericyclin, a new tetramic acid

Article abstract of DOI:10.1002/1521-3773(20000804)39:15<2766::AID-ANIE2766>3.0.CO;2-G

The first chemical proof is available to show that lactic acid bacteria, generally known for their limited metabolite spectrum, are able to produce antibiotics with a broad inhibitory spectrum. The new tetramic acid, reutericyclin (1), opens a new dimension in the discussion regarding the application of lactic acid bacteria in food preservation or for positively influencing the human intestinal microflora.

Full text of DOI:10.1002/1521-3773(20000804)39:15<2766::AID-ANIE2766>3.0.CO;2-G

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Table 1. 1H and 13C NMR chemical shifts for compound 1, as well as
observed HMBC correlations ([D3]acetonitrile, 298 K). Due to keto ± enol
tautomerism two signal sets were detected for several nuclei (1a to 60%,
1b/1c up to 40%).
The First Low Molecular Weight Antibiotic
from Lactic Acid Bacteria: Reutericyclin, a
New Tetramic Acid
d(13C)
d(1H)
1b/1c
HMBC
correlations
Alexandra Höltzel, Michael G. Gänzle,
Graeme J. Nicholson, Walter P. Hammes, and
Günther Jung*
1a
1b/1c
1a
2
3
4
5
167.3
106.1
199.2
60.2
174.8
103.6
194.5
64.1
±
±
±
4.68
1.81
±
11-H
5-H, 6-H
6-H
Based on a screening of lactic acid bacteria from cereal
fermentations, an inhibitory effect of Lactobacillus reuteri
LTH2584 against Gram-positive bacteria has been demon-
strated.[1] This strain had been isolated as one of the strains
dominating the flora of a sourdough which is prepared for the
production of a commercial baking aid.[2] Here, we describe
for the first time a low molecular weight antibiotic, reuter-
icyclin (1), which is produced by lactic acid bacteria.
4.41
1.76
6
39.8
5-H, 7-H, 8-H, 9-H
725.2
1.82
5-H, 6-H, 8-H, 9-H
8
9
10
11
12
13
14
15
16
23.8
22.6
195.6
22.8
165.9
124.2
150.7
33.3
0.88
0.93
±
2.51
±
7.29
7.06
2.28
1.50
9-H
8-H
11-H
±
13-H, 14-H
15-H
15-H, 16-H
13-H, 14-H, 16-H
14-H, 15-H
189.3
20.2
165.5
7.22
7.10
2.29
151.8
33.2
Compound 1 was purified as a yellow-brown oil from cell
extracts and culture filtrate of L. reuteri LTH2584 with a yield
of approximately 1 mgLÀ1.[3] The elemental formula of 1 was
determined by high-resolution ESI-FT-ICR mass spectrome-
28.8
1729.8
18
19
20
21
1.34
15-H, 16-H, 18-H
29.8
32.5
23.3
14.3
1.34
1.30
1.32
0.90
16-H, 17-H
20-H, 21-H
19-H, 21-H
20-H
‡
try (ICR ˆ ion cyclotron resonance; [M ‡ H ] for C20H31NO4:
calculated m/z 350.23257, experimental m/z 350.23210). The
UV/Vis spectrum (acetonitrile) showed absorption maxima at
238 and 286 nm. An intense absorption at 1726 cmÀ1, as well as
several strong bands between 1657cm À1 and 1617cm À1, in the
IR spectrum (film) pointed to the presence of several
carbonyl groups in the molecule. The structure of 1 was
elucidated by NMR spectroscopy and GC-MS analysis of
methanolysis products.
vicinal connectivity of methyl group and keto function.
HMBC connectivities were not observed between the three
structural fragments established so far or to the carbonyl
carbon C-2.
The acid methanolyzate of 1 (formed by treatment with
CH3OH/1.5n HCl, 1108C, 15 min) was, after addition of
water, extracted with n-pentane (Scheme 1). The MeOH/H2O
fraction was derivatized with trifluoroacetic anhydride. The
GC-MS of the pentane fraction showed two major peaks. The
peak at m/z 184 was identified as 2-decenoic acid methyl ester
(2) while the second peak at m/z 297was assigned to N-(2-
decenoyl)leucine methyl ester (3), on the basis of character-
The HSQC spectrum showed that 1 consists of four methyl
and seven methylene groups as well as two aliphatic and two
olefinic methine groups. Signals for five quarternary carbons
were identified in the 13C NMR spectrum, two of which were
classified as carbonyl carbons of acid, ester, or amide
functionalities, and two as ketone carbons (Table 1). TOCSY
spectra (acquired with short and long mixing times) exhibited
signals for two distinct spin systems, the first one including
5-H to 9-H and the second one 13-H to 21-H. The E
configuration of the double bond between C-13 and C-14 was
3
reflected by the vicinal coupling constant J(13-H, 14-H) ˆ
15.4 Hz. Connectivities observed in the HMBC spectrum
(Table 1) confirmed the assignments within the spin systems
and enabled the addition of the quarternary carbons C-4 and
C-12 to the respective structural fragments. A third fragment
was established from the HMBC connectivities of the methyl
protons 11-H to the quarternary carbons C-3 and C-10; the
1H NMR signal for 11-H appeared at d ˆ 2.51, suggesting the
[*] Prof. Dr. G. Jung, Dr. A. Höltzel, G. J. Nicholson
Institut für Organische Chemie der Universität Tübingen
Auf der Morgenstelle 18, 72076 Tübingen (Germany)
Fax : (‡49)7071-29-5560
E-mail: guenther.jung@uni-tuebingen.de
Dr. M. G. Gänzle,[‡] Prof. Dr. W. P. Hammes
Institut für Lebensmitteltechnologie
der Universität Hohenheim (Germany)
‡
[ ] Current Address:
Lehrstuhl für Technische Mikrobiologie
Institut für Brauereitechnologie und Mikrobiologie
TU München in Freising-Weihenstephan (Germany)
Scheme 1. Methanolysis products of reutericyclin, detected by GC-MS
analysis at m/z 184 (2), 297( 3), 265 (4), and 241 (5).
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istic fragmentations. The identification of 3 in the acid
methanolyzate of 1 suggested that C-5 and C-12 are connected
by N-1 in the native molecule. The observed 13C NMR signals
left but one possibility for the arrangement of the third
fragment and the carbonyl group at C-2 in the molecule. The
formulation of 1 as 3-acyltetramic acid was confirmed by the
identification of compound 4 as the main component of the
MeOH/H2O fraction of the acid methanolyzate. The deacy-
lation of 3-acyltetramic acids under acidic conditions has long
been known,[4] and similarly the O-alkylation of tetramic
acids.[5] The MeOH/H2O fraction also contained N-trifluoro-
acetylleucine methyl ester 5. From the absolute configuration
of 5, as determined by GC-MS analysis with a Chirasil-Val
column,[6] the (5R)-configuration of 1 was concluded. The
methanolysis products 3 and 5, the identification of which was
essential for the structure elucidation of 1, are unusual in that
unprecedented N-1 substitution with an a,b-unsaturated fatty
acid. For a possible application of 1 as an antibiotic, it is
noteworthy that L. reuteri is utilized in food fermentations[2]
and has been described as a stable constituent of the intestinal
microflora of both humans and animals.[11] Investigations on
the inhibitory spectrum of 1 and its production by L. reuteri
LTH2584 support the conclusion that inhibitory concentra-
tions of the amphiphilic compound are present in sourdough
fermentations with L. reuteri.[1, 12] Lactic acid bacteria produce
a multitude of unspecific low molecular weight compounds,
mainly side products of carbohydrate metabolism, which
contribute to the inhibitory effect of lactic acid.[13] However,
all antibiotics from lactic acid bacteria known to date are
bacteriocins. The inhibitory effect of bacteriocins is restricted
to closely related species and their potential for application to
foods is therefore limited.[14] In recent years, a considerable
amount of research has focused on the positive effect of lactic
acid bacteria on human health (probiotics). In animal tests, a
protective effect of antimicrobial substances from lactobacilli
against Helicobacter and Salmonella infections has been
demonstrated,[15] but an active substance has not been chemi-
cally characterized to date. As 1 differs in its chemical and
biological properties from all other active substances pro-
duced by lactic acid bacteria, the structure elucidation of this
natural compound adds a new dimension to the discussion
regarding the application of lactic acid bacteria and their
antimicrobial metabolites for food preservation or for pos-
itively influencing the human intestinal microflora.
À
their formation requires the cleavage of a C C bond. Up to
now the release of an amino acid from 3-acyltetramic acids
has only been reported upon treatment with oxidative
reagents.[4] The structure and the results of the acid meth-
anolysis of 1 were validated by the synthesis of racemic 1.[7]
The formulation of 1 as (5R)-3-acyl-1-(2-decenoyl)-2-hy-
droxy-5-isobutyl-D2-pyrroline-4-one refers to tautomer 1a
(Scheme 2), which analysis of the 13C NMR signals (Table 1)
revealed to be the major tautomer (60%) in acetonitrile
Received: February 11, 2000 [Z14692]
[1] M. G. Gänzle, C. Hertel, W. P. Hammes in Beijerinck Centennial.
Microbial physiology and gene regulation (Eds.: W. A. Scheffers, J. P.
van Dijken), Delft University Press, 1995, pp. 380 ± 381.
[2] G. Böcker, P. Stolz, W. P. Hammes, Getreide Mehl Brot 1995, 49, 37 0 ±
374.
[3] The isolation of 1 from cell extracts involved extraction with a 70/30
mixture of phosphate buffer (50mm, pH 6.5) and isopropanol, gel
filtration on Superdex S30 (Pharmacia, 75/25 mixture of triethylamine
buffer (50mm, pH 7.2) and isopropanol), and medium-pressure
reversed-phase chromatography (Pharmacia, 15 mm, eluent A: H2O/
0.1% trifluoroacetic acid (TFA) solution, eluent B: isopropanol/0.1%
TFA solution, gradient: from 25% to 50% B in 20 min). The isolation
of 1 from culture filtrate followed a slightly modified protocol. In both
cases, HPLC (Advanced Separation Technologies, C18 column, 5 mm,
eluent A: acetonitrile/0.1% TFA solution, eluent B: H2O/0.1% TFA
solution, A/B ˆ 85/15) was used as a final purification step.
[4] C. E. Stickings, Biochem. J. 1959, 72, 332 ± 340.
Scheme 2. Reutericyclin is subjected to keto ± enol tautomerism in sol-
ution. In [D3]acetonitrile 1a is present to 60% and 1b and 1c in up to 20%
each.
[5] a) H.-D. Stachel, K. K. Harigel, H. Poschenrieder, H. Burghard, J.
Heterocycl. Chem. 1980, 17, 1195 ± 1199; b) K. Inami, T. Shiba,
Tetrahedron Lett. 1984, 25, 2009 ± 2012. Both, the 4-methoxy- and
the 2-methoxy derivatives have been reported as the methylation
products of tetramic acids. The formulation of 2-methoxy-D2-pyrro-
line-4-one as the methanolysis product of 1 is based on the mass
spectrum of 4 in which the base peak at m/z 209 originates from a
McLafferty rearrangement which would require the carbonyl group at
position 4.
[6] H. Frank, G. J. Nicholson, E. Bayer, J. Chromatogr. 1978, 167, 187±
196.
[7] U. Marquardt, D. Schmid, G. Jung, Synlett, in press.
[8] The naturally occurring 3-acyltetramic acids known to date areÐwith
the exception of magnesidins A and B-±either unsubstituted or
alkylated at N-1. For these compounds the dominance of the exocyclic
pyrrolidine-2,4-dione form, in which the amide carbonyl group acts as
solution. The 13C NMR data for the minor signal set indicate
equal contributions (20% each) from the internal tautomers
1b and 1c. In this respect, 1 differs from all other naturally
occurring 3-acyltetramic acids. These prefer almost exclusive-
ly the pyrrolidine-2,4-dione form in solution.[8, 9].The excep-
tional preference for the D2-pyrroline-4-one form in 1 is
brought about by the 2-decenoyl substituent at N-1, which not
only reduces the hydrogen bridge acceptor ability of the
carbonyl group at C-2, but also stabilizes the endocyclic
double bond by conjugation.
Among the naturally occurring tetramic acids,[9, 10] which
display a remarkable spectrum of biological activity, the
structure of 1 is chemically interesting because of the
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a hydrogen bond acceptor, has been deduced from a comprehensive
analysis of the 13C NMR spectra: a) P. S. Steyn, P. L. Wessels,
Tetrahedron Lett. 1978, 47, 4707 ± 4710; b) M. J. Nolte, P. S. Steyn,
P. L. Wessels, J. Chem. Soc. Perkin Trans. 1 1980, 1057± 1065.
[9] The major tautomers of magnesidins A and B in solution are the D3-
pyrroline-2-one forms: N. Imamura, K. Adachi, H. Sano, J. Antibiot.
1994, 47, 257± 261.
[10] B. J. L. Royles, Chem. Rev. 1995, 95, 1981 ± 2001. So far, the naturally
occurring N-acyltetramic acids known are only 4-methoxy-D3-pyrro-
line-2-ones (with the exception of magnesidins A and B).
[11] I. A. Casas, W. J. Dobrogosz, Microecol. Ther. 1997, 26, 221 ± 231.
[12] a) The optimal conditions with respect to temperature, pH, and
substrate supply correspond to the conditions during sourdough
fermentations: M. G. Gänzle, PhD. thesis, Universität Hohenheim,
1998; b) additionally, it has been shown that reutericyclin-sensitive
strains are inhibited in their growth or killed during cofermentation in
sourdough containing L. reuteri LTH2584: W. P. Hammes, J. Walter,
unpublished results; M. G. Gänzle, R. Vogel, unpublished results.
[13] a) P. A. Vandenbergh, FEMS Microbiol. Rev. 1993, 12, 221 ± 337;
b) M.-L. Niku-Paavola, A. Laitila, T. Mattila-Sandholm, A. Haikara,
J. Appl. Microbiol. 1999, 86, 29 ± 35.
[14] a) R. W. Jack, J. R. Tagg, B. Ray, Microbiol. Rev. 1995, 59, 171 ± 200;
b) R. W. Jack, F. Götz, G. Jung in Biotechnology, Vol. 7, 2nd edition
(Eds.: H. Kleinkauf, H. von Döhren), VCH, Weinheim, 1997, pp. 325 ±
368.
Quantum-chemical calculations predict a linear arangement
‡
‡ [6]
of atoms (Dh) for the cations PO2 and PS2 . For these
cations a bis-donor adduct has been obtained recently.[7] Here
we report on the first synthesis and crystal structure analysis
of a bis-donor adduct of the imine analogue of II as well as on
ab initio calculations on model compounds that support the
findings.
Â
[15] a) M.-H. Coconnier, V. Lievin, E. Hemery, A. L. Servin, Appl.
Environ. Microbiol. 1998, 64, 4573 ± 4580; b) M.-F. Bernet-Camard,
Â
V. Lievin, D. Brassart, J.-R. Neeser, A. L. Servia, S. Hudault, Appl.
Environ. Microbiol 1997, 63, 2747 ± 2753.
Treatment of the bromobis(arylimino)phosphorane 1 (R ˆ
Mes* ˆ 2,4,6-tBu3C6H2)[8] with one equivalent of p-dimethyl-
aminopyridine (DMAP ˆ D) in CH2Cl2 furnished the donor/
acceptor adduct 2 in good yield (75%) in the form of air- and
moisture-sensitive light yellow crystals. Subsequent reaction
of 2 with a second equivalent of DMAP proceeded by
halogen/nucleophile exchange to give the cation 3, whose
bromide was isolated at low temperature as yellow crystals
from a little CH2Cl2.
Synthesis of the Adduct
ˆ
DMAP ´ BrP( N-Mes*)2 and of the Salt
‡
ˆ
[(DMAP)2P( NMes*)2] Br**
Markus Blättner, Martin Nieger, Alexander Ruban,
Wolfgang W. Schoeller, and Edgar Niecke*
Dedicated to Professor Reinhard Schmutzler
on the occasion of his 65th birthday
Allenes I are fundamental compounds with a linear
structure (D2d symmetry). This implies sp hybridization at
the central carbon atom.[1] The hypothetical 2-phosphonio-
allenic cation IIa is isovalent to I, but its central phosphorus
atom is not sp hybridized.[2] The s orbital at the phosphorus
atom becomes stereochemically active under formation of a
ªbentº 2-phosphaallyl cation IIb.[3] According to ab initio
calculations[5] this geometry is favored over that of IIa.
Initial information concerning the constitution of 2 and 3 ´
BrÀ was obtained from multinuclear NMR investigations. The
1H and 13C NMR data clearly indicated the presence of Mes*
substituents at the nitrogen atoms as well as the attachment of
one (2) or two donor molecules (DMAP) (3) to the
phosphorus atom. As expected the 31P NMR signals appeared
at higher fields (d ˆ À93.2 (2), À57.2 (3)) than those of
bis(imino)phosphoranes,[9] indicating an increase of coordi-
nation number at the phosphorus atom.
[*] Prof. Dr. E. Niecke, Dr. M. Blättner, Dr. M. Nieger, Dr. A. Ruban
Anorganisch-Chemisches Institut der Universität
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
Fax : (‡49)228-73-5327
E-mail: e.niecke@uni-bonn.de
Crystal structure analyses of 2 and 3 ´ BrÀ gave evidence for
the formation as a monoadduct of the bromobis(imino)phos-
phorane and as a bisadduct of the bis(imino)phosphonium
cation (Figures 1 and 2).[10] In both compounds the phospho-
rus atoms adopt a strongly distorted tetrahedral geometry.
The N-P-N bond angle at the phosphorus atom (N1-P1-N2
Prof. Dr. W. W. Schoeller
Fakultät für Chemie der Universität Bielefeld (Germany)
[**] This work was supported by the Fonds der Chemischen Industrie and
the Deutsche Forschungsgemeinschaft (SFB 334). Part of this work
was reported at the International Conference on Phosphorus Chem-
istry, Cincinnati, USA, 1998. DMAP ˆ p-dimethylaminopyridine;
Mes* ˆ 2,4,6-tBu3C6H2.
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Angew. Chem. Int. Ed. 2000, 39, No. 15