Secondary metabolites by chemical screening, 39: Acyl α-L- rhamnopyranosides, a novel family of secondary metabolites from streptomyces sp.: Isolation and biosynthesis

Article abstract of DOI:10.1002/(sici)1099-0690(200003)2000:6<929::aid-ejoc929>3.0.co;2-u

In the course of our chemical screening program, the novel acyl α-L- rhamnopyranosides (1-6) were detected as metabolites from five different strains of Streptomycetes. The structures of all these compounds were elucidated by chemical and spectroscopic methods. The biosynthesis of 1 and 3 was established by feeding ¹³C-labelled acetate, glycerol, and d-glucose to Streptomyces griseoviridis (strain Tu 3634), and resulted in a complete labelling pattern of the 2,4-dimethyl-3-furanylcarbonyl and benzoyl residues, as well as the rhamnose moiety. These results reveal biosynthetic pathways of general importance and give an insight into the generation of the hexose phosphates, from which deoxysugars are formed. The acyl rhamnosides are members of a novel family of microbial metabolites and are considered as rhamnoconjugates from Streptomycetes.

Full text of DOI:10.1002/(sici)1099-0690(200003)2000:6<929::aid-ejoc929>3.0.co;2-u

FULL PAPER
Secondary Metabolites by Chemical Screening, 39[‡]
Acyl α--Rhamnopyranosides, a Novel Family of Secondary Metabolites from
Streptomyces sp.: Isolation and Biosynthesis
Stephanie Grond,[a] Hans-Jörg Langer,[a] Petra Henne,[a] Isabel Sattler,[b] Ralf Thiericke,[b]
Susanne Grabley,[b] Hans Zähner,[c] and Axel Zeeck*[a]
Keywords: α-L-Rhamnopyranosides / Secondary metabolites / Streptomyces / Biosynthesis / 2,4-Dimethylfuran-3-
carboxylic acid
In the course of our chemical screening program, the novel
acyl α- -rhamnopyranosides (1–6) were detected as metabol- nose moiety. These results reveal biosynthetic pathways of
ites from five different strains of Streptomycetes. The struc- general importance and give an insight into the generation
tures of all these compounds were elucidated by chemical of the hexose phosphates, from which deoxysugars are
3-furanylcarbonyl and benzoyl residues, as well as the rham-
L
and spectroscopic methods. The biosynthesis of 1 and 3 was
formed. The acyl rhamnosides are members of a novel family
of microbial metabolites and are considered as rhamnoconju-
gates from Streptomycetes.
established by feeding 13C-labelled acetate, glycerol, and
d-
glucose to Streptomyces griseoviridis (strain Tü 3634), and
resulted in a complete labelling pattern of the 2,4-dimethyl-
Introduction
strictly glycosides, we use the term here for the acylated
sugars described in this paper. Biosynthetic studies from
feeding experiments with 13C-labelled sodium acetate, gly-
cerol, and -glucose, respectively, gave interesting and unex-
pected results on the respective pathways and intermediates
for the aglyca and the sugar assembly.
Deoxyhexoses are typical building blocks of microbial
secondary metabolites either as individual moieties or as
components of oligosaccharides.[2,3] Usually they are linked
to an aglycone as O-glycosides or to another sugar moiety
and modulate the biological activity of a large number of
antibiotics[4] e.g. anthracyclines, angucyclines, macrolides,
enediynes, and the aureolic acid group.[5] Whilst the divers-
ity of biological activity is tremendous and depends on the
number and substituents present on the sugar, the mode of
action is seldom known, e.g. interaction of sugars with spe-
cific DNA regions.[6,7] The O-glycosidic linkage is formed
in the late biosynthesis by glycosyltransferases, which utilize
deoxysugars activated at the anomeric C-atom and free ali-
phatic or phenolic hydroxy groups of the aglycone.[8] Using
the well established chemical screening method for the de-
tection of new secondary metabolites,[9,10] we have disco-
vered a novel family of glycosides due to their remarkable
yellow to green coloration on silica gel TLC plates when
stained with anisaldehyde–sulfuric acid. This paper deals
with the isolation and structure elucidation of six new acyl
α--rhamnopyranosides (1–6) and related compounds (7–9)
from different strains of Streptomycetes. Although not
Results
Producing Organisms and Isolation
During an extensive screening program we found five
strains, which produce varying ratios of an unusual type of
glycoside with different co-metabolites. Streptomyces griseo-
viridis (strain Tü 3634), isolated from a soil sample from
Nepal, produces compound 1 and 3 in an oatmeal medium,
2 after addition of ammonium hydrogenphosphate, 2 and 4
in a malt/yeast extract/glycerol medium and 6 in a malt/
yeast extract medium. Strain FH-S 2087 produces 3, 4, and
5 in a glycerol/casein peptone medium, which yields only
small amounts of compound 1 with strains Tü 3634, FH-S
1071 (Streptomyces fragilis), FH-S 1512, and FH-S 1298.[11]
In addition different amounts of butanolides, e.g. acetomy-
cin (7) and its derivatives 8 and 9 can be detected as by-
products.[12–14] In the screening procedure, cultivations were
run in 300-mL Erlenmeyer flasks containing 100 mL of the
different media for 3–4 days at 28–30 °C. Using the isola-
tion procedures described below,[15] we obtained crude ex-
tracts of the mycelium and the culture filtrate. Analysis by
chromatography on silica gel TLC plates indicated that the
glycosides were predominantly present in the culture fil-
[‡]
Part 38: Ref.[1]
Institut für Organische Chemie, Universität Göttingen,
[a]
Tammannstrasse 2, D-37077 Göttingen, Germany
Fax: (internat.) ϩ49 (0)551/39-9660
E-mail: azeeck@gwdg.de
[b]
Hans-Knöll-Institut für Naturstoff-Forschung e. V.,
Beutenbergstrasse 11, D-07745 Jena, Germany
Kistlerweg 7, CH-3006 Bern, Switzerland
[c]
Supporting Information for this article is available on the
WWW under http://www.wiley-vch.de/home/eurjoc or from the
author.
Eur. J. Org. Chem. 2000, 929Ϫ937
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0306Ϫ0929 $ 17.50ϩ.50/0
929

A. Zeeck et al.
FULL PAPER
trate. Their Rf-values and color reactions with different
staining reagents are given in Table 1.
Structure Elucidation
The pure acyl rhamnosides were spectroscopically char-
acterized and, in some cases, derivatives were prepared by
nopyranosides 1–6 and some of their triacetates on silica gel TLC methanolysis and acetylation.
Table 1. Rf values and discoloration of the natural acyl α--rham-
plates
2,4-Dimethyl-3-furanylcarbonyl α--Rhamnopyranoside (1)
Compound
A[a]
B
C
I
II
The molecular formula C13H18O7 of 1 was established
from the high resolution EI mass spectrum (Mϩ ϭ 286).
The elemental analysis gave no reproducible values due to
the inclusion of varying amounts of water and other solv-
ents. The fragmentation peak at m/z ϭ 140 originates from
the loss of C6H10O4, indicating a deoxy sugar moiety. UV
maxima (MeOH) at 201 and 254 nm characterize the chro-
mophore and the IR spectrum exhibits a strong band at
1710 (sh) indicating a conjugated ester.
1
0.39
0.82
0.21
0.31
0.81
0.32
0.83
0.20
0.76
0.09
0.64
0.77
0.62
0.65
0.76
0.65
0.80
0.61
0.79
0.75
0.19
0.63
0.11
0.18
0.62
0.20
0.64
0.14
0.58
0.15
bright green
yellow
pink
pink
red violet
–
1a
2
bright yellow
yellow
3
3a
4
yellow
–
yellow
pink
yellow
violet
violet
–
4a
5
yellow
light brown
yellow
5a
6
yellow
[a] Solvent systems: (A) CHCl3/MeOH ϭ 9:1; (B) acetic acid/1-butyl
alcohol/water ϭ 1:4:5 (upper layer); (C) ethyl acetate/n-hexane
The 1H-NMR spectrum (CDCl3) reveals 18 protons,
(4:1). – Staining reagents: (I) anisaldehyde/sulfuric acid, (II) three of which were exchangeable in [D4]methanol. The
EhrlichЈs reagent; after spraying the plates were heated at 120 °C
for 5 min.
presence of one methyl singlet (δ ϭ 2.54), two methyl doub-
lets [δ ϭ 1.26 (J ϭ 6.0 Hz), 2.15 (J ϭ 1.3 Hz)], four aliphatic
protons as multiplets (δ ϭ 3.48–3.91), the anomeric proton
as a doublet [δ ϭ 6.16 (J ϭ 1.9 Hz)] and an aromatic me-
thine proton [δ ϭ 7.18 (J ϭ 1.3 Hz)] were observed.
In order to isolate reasonable quantities of the glycosides,
strains Tü 3634 and FH-S 2087 were cultivated in 1 L and
10 L fermentors, respectively, with different culture media.
Acetylation gave the triacetate 1a, thus indicating the pres-
ence of three hydroxyl groups. The sugar moiety of 1 was
After harvesting, the culture filtrate was separated by filtra-
determined as rhamnose by comparing the chemical shifts
tion and applied to Amberlite XAD-2 or MCI-gel HP-20.
with literature data and by an analysis of the coupling pat-
Elution with methanol or a methanol/water gradient gave a
crude extract, which was separated by column chromato-
graphy on Sephadex LH-20 (MeOH) and on silica gel
terns of 1Ј-H (δH ϭ 6.16) to 6Ј-H (δH ϭ 1.26).[17] From the
1
13C chemical shift (δ ϭ 95.2) and the JC–H coupling con-
stant (J ϭ 172.5 Hz) at C-1Ј, an α-glycosidic linkage was
(CH2Cl2/MeOH, 4:1) according to analysis by TLC
deduced.[18] In order to assign the absolute configuration of
(Table 1). Whilst 1, 2, 4, and 6 could be obtained by this
procedure, further purification on silica gel with acetone/
hexane (2:1) or CHCl3/MeOH (9:1) as eluents were neces-
the deoxyhexose, the glycoside was cleaved with methanol/
HCl and gave the methyl rhamnopyranosides in both an-
omeric forms (α-anomer: [α]2D9 ϭ Ϫ65, β-anomer: [α]2D9
ϭ
sary to obtain pure samples of 3 and 5.[16] Typical fermenta-
tions (on 1-L and 10-L scale) followed by the purification
procedure yielded 11 mg/L of acyl α--rhamnopyranoside
1, 3Ϫ5 mg/L of 2, 6 mg/L of 3, 23 mg/L of 4, 0.8 mg/L of
5 and 0.4 mg/L of 6. All the glycosides are colorless solids.
ϩ86).[19] A comparison of the optical rotation values with
those in the literature allowed the -configuration to be as-
signed for the rhamnose moiety in 1 ([α]2D0 ϭ Ϫ42). The α-
configuration of -deoxyhexoses is a characteristic struc-
tural feature in microbial secondary metabolites.
Thirteen signals were observed in the 13C-NMR spec-
trum and APT experiment (CD3OD), six of which con-
firmed the rhamnose moiety. The remaining seven signals
originate from two methyl groups (δC ϭ 14.7, 10.6), one
ester carbonyl (δC ϭ 164.0) and four furan ring carbons
(δC ϭ 162.4, 139.6 d, 122.2, 113.9), one of which carries a
hydrogen atom (δH ϭ 7.18). These structural elements form
the aglycone, the structure of which was confirmed by 2D
NMR methods and by comparison with synthetic 2,4-di-
methylfuran-3-carboxylic acid (11). The synthesis of 11 was
readily achieved by the dropwise addition of bromine to an
aqueous suspension of isodehydracetic acid. Ether extrac-
tion followed by chromatography on silica gel (CH2Cl2/
MeOH, 7:1) gave the pure aglycone 11 (95%).[20] A natural
sample of 11 and synthetic 11 had identical properties
(NMR and MS). The upfield shift of the anomeric carbon
(C-1Ј, δC ϭ 95.2) of 1 and the long-range correlation of the
ester carbonyl C-7 at δC ϭ 164.0 with the anomeric proton
930
Eur. J. Org. Chem. 2000, 929Ϫ937

Secondary Metabolites by Chemical Screening, 39
FULL PAPER
Phenylacetyl α--Rhamnopyranoside (4)
1Ј-H at δH ϭ 6.16 in a COLOC-experiment account for an
ester glycosidic linkage.[17] The appearance of only one set
of NMR signals clearly indicates that 1 is a diastereomer-
ically pure natural product. The structure of the new rham-
nopyranoside was assigned as indicated in formula 1.
Compound 4 was isolated from the culture broth of
strains Tü 3634 and FH-S 2087 in amounts up to 23 mg/L.
The results of the DCI-MS spectra (m/z ϭ 300, M ϩ NHϩ4 )
indicate one more methylene group than 3. The IR spec-
trum reveals an ester absorption at 1745 cm–1. In the H-
1
and 13C-NMR spectra (CD3OD) all the signals due to the
rhamnose moiety are identical to those in 1–3. This also
suggests the α--configuration of the deoxysugar.[16,19]
Again, this was confirmed by conversion into the triacetate
4a and from isolation of the anomeric methyl rhamnosides
obtained on methanolysis. Focussing on the aglycone: the
methylene signals (δH ϭ 3.68, δC ϭ 42.1) and the presence
of aromatic signals allow the identification of the benzyl
group, which subsequently leads to structure 4.
2,4-Dimethyl-3-pyrrolylcarbonyl α--Rhamnopyranoside (2)
The analogue of 1, containing nitrogen was produced (3
to 5 mg/L) in medium A enriched with ammonium hydro-
genphosphate as nitrogen source or in medium C. The spec-
troscopic data of 2 are similar to those obtained for 1. From
HREI-mass spectral data (m/z ϭ 285, Mϩ, C13H19NO6),
component 2 exhibits one O-atom less than 1 and an addi-
tional NH. In the IR spectrum the band for the ester car-
bonyl appears at 1683 cm–1. A comparison of the 1H-NMR
and 13C-NMR spectra (CD3OD) revealed the same number
of signals with different chemical shifts for the aglycone
moiety only. The upfield shift of C-2 and C-5 of 2 indicates
the exchange of oxygen by NH in the five-membered ring.
Thus, structure 2 was deduced, which gave an insight into
alternative routes in the biosynthesis of the aglycone start-
ing from a common C7 precursor.
2-Pyrrolylcarbonyl α--Rhamnopyranoside (5)
Compound 5 was only isolated (0.8 mg/L) from the ex-
tracts of strain FH-S 2087. The DCI-MS [m/z ϭ 258 (M ϩ
Hϩ), 275 (M ϩ NH4ϩ)] confirms the molecular weight in
agreement with the molecular formula C11H15NO6 as estab-
lished from the spectral data and from the elemental ana-
lysis of the triacetate 5a. The IR spectrum shows the char-
acteristic ester carbonyl at 1696 cm–1. In the upfield region
of the 1H-NMR spectrum (CD3OD) all signals are again in
agreement with those of an α--rhamnopyranosyl moiety.
The optical rotation ([α]2D0 ϭ Ϫ39) of 5 again points to the
α--configuration of the rhamnose in 5. Three doublets of
doublets in the lowfield region (δH ϭ 6.21, 6.91, 7.01, J ϭ
1.5, 2.5, 3.5 Hz) correspond to a 2-pyrrolylcarbonyl agly-
cone in 5, which was confirmed from the 13C-NMR spec-
troscopic data. In addition to the four 13C-NMR signals of
the pyrrole nucleus (δC ϭ 125.7, 122.7, 117.5, 111.0), the
ester carbon appears as a single signal at δC ϭ 160.6.
Benzoyl α--Rhamnopyranoside (3)
Compound 3 was formed in culture broths of strain Tü
3634 and in smaller amounts from strain FH-S 2087. The
DCI-MS of 3 shows the molecular ion at m/z ϭ 286 (M ϩ
NH4ϩ). The presence of a benzoyl group was indicated by
characteristic UV absorption maxima in methanol and
from the ester carbonyl IR absorption at 1726 cm–1. The
1
upfield region of the H- and 13C-NMR (CD3OD) spectra
were attributed to an ester linked rhamnose moiety, which
was confirmed by facile conversion into the triacetate 3a.
Signals in the low field range of the NMR spectra indicate
the aglycone to be a benzoyl residue. The optical rotation
value ([α]2D0 ϭ Ϫ52) again supports the stereochemical as-
signment of a α--rhamnopyranoside. All these data con-
firm the structure as 3.
4-Hydroxymethyl-2-methyl-3-furanylcarbonyl α--
Rhamnopyranoside (6)
Compound 6 was isolated (0.4 mg/L) from a culture of
strain Tü 3634 in a 10 L airlift-loop fermentor with a high
Table 2. 13C-NMR analysis of enriched 1 with stable isotope precursors; *as estimated by spin simulation
1
Specific
JC–C [Hz]
Specific
incorporation
JC–C [Hz]
incorporation (JC–C [Hz])[a]
Carbon
δc
[1-13C]acetate
[1,2-13C2]acetate
[2-13C]glycerol
[U-13C3]glycerol
52.7
2
162.4
113.9
122.2
139.9
14.7
164.0
10.6
95.2
71.4
72.4
73.4
72.7
18.1
21.5 (8.0)
52.3
1.8
–
3
–
86.4
86. 0
4
–
–
–
1.7
–
48.0, 72.0
72.0, 6.0
52.7
5
–
6
–
52.3
–
7
22.2 (8.0)
86.4
–
[0.9]
–
86.0
8
–
–
–
–
–
–
–
48.0, 6.0
47, 3*
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
–
–
–
1.6
–
47, 37,
37, 3*
–
–
8.0
–
40, 1
–
40, 40
–
–
40, 1
[a]
Statistical coupling.
Eur. J. Org. Chem. 2000, 929Ϫ937
931

A. Zeeck et al.
FULL PAPER
Table 3. 13C-NMR analysis of 1 and 3 enriched with stable isotope precursors; *as estimated by spin simulation
3
Sugar moiety of 1
Specific
incorporation
JC–C [Hz]
JC–C [Hz]
Carbon
δc
δc
[2-13C]glycerol
[U-13C3]glycerol
75*, 64
56*, 64*, 2*
52, 56
[U-13C6]glucose
1
130.8
130.7
129.8
134.8
166.0
96.0
71.3
72.3
73.5
72.6
18.1
3.44
–
–
2/6
3/5
4
–
2.93
–
–
52
–
7
–
–
75*, 2*
47, 3*
–
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
95.2
71.4
72.4
73.4
72.7
18.1
47, 3*
47, 37
37*, 38*, 3*
38, 40, 1
40, 40
40, 1
1.6
–
47, 37,
37, 3*
8.0
–
40, 1
40, 40
–
40, 1
aeration rate. The DCI-MS [m/z ϭ 320 (M ϩ NH4ϩ), 302 3. The results of 13C-NMR spectral analysis are presented
(Mϩ)] confirms the molecular weight as C13H18O8. Com- in Table 2 and Table 3 and in Scheme 1.
parison with the 1H-NMR spectrum (CD3OD) of 1 showed
that the methyl group of 1 (C-8) is replaced by a methylene
group as indicated by a doublet (J ϭ 1.0 Hz) at δH ϭ 4.64.
The 13C-NMR spectrum of 6 exhibits all thirteen signals
with the methylene group (δC ϭ 57.1) in place of the methyl
group of 1. From all spectroscopic data, the metabolite cle-
arly has structure 6 and is probably produced by the oxida-
tion of 1.
Biosynthetic Studies
A working hypothesis for the biosynthesis of the novel
Feeding of sodium [1-13C]acetate resulted in enhanced
acyl rhamnoside 1 envisaged a C3ϩC4 pathway for the agly-
13C-NMR signals for C-2 and C-7 of the aglycone only. The
cone (11), while -rhamnose should be derived from the
13C-NMR spectrum of 1, obtained from feeding of sodium
usual carbohydrate pathway.[4] We postulated that 11 is as-
[1,2-13C2]acetate, revealed two pairs of signals assigned by
sembled from a triketide and a C1 unit or a diketide and a
the coupling constants as C-6/C-2 and C-3/C-7. Thus, a di-
C3 unit (e.g. glycerol) as in acetomycin (7), A-factor, vir-
ketide precursor was assumed for C-6/C-2/C-3/C-7. While
ginae butanolides or acaterin (10).[12,14,21] Consequently, we
feeding experiments with -[methyl-13C]methionine gave no
carried out feeding experiments with Streptomyces griseovir-
enrichments whatsoever, experiments with [U-13C3]glycerol
idis (strain Tü 3634) adding 13C-labelled acetate, methion-
led to an intact incorporation at C-5/C-4/C-8 as indicated
ine, glycerol, and glucose, respectively, to the growing cul-
1
3
by the JC–C and JC–C coupling constants. Due to scram-
bling from glycerol into acetate, intact C2-units were also
detected for the diketide. Likewise [2-13C]glycerol gave en-
richments of C-2, C-7, and C-4 (Table 2). Thus, the assem-
bly of the aglycone requires a diketide and a C3-unit derived
from glycerol. The C7-intermediate (conformation A,
Scheme 2) is converted into 11 by formation of a cyclic
hemiacetal and subsequently into 1 in the predominant
pathway. Remarkably, strains Tü 3634, FH-S 1071, and
FH-S 1512 also produce butanolides of the acetomycin
type, e.g. 8 and 9, as minor components (see ref.[10–12]). For
these, we propose the same C7-intermediate (conformation
B) which is converted by lactonization and further trans-
formations (Scheme 2).
tures.
Once the production of 1 had commenced (TLC analysis,
16 hours after inoculation), precursors were continuously
added to the culture over a period of 10 hours. The cultures
were harvested after 60 hours. Isolation by the above de-
scribed procedure gave varying yields of 1 and, additionally,
In the preceding feeding experiments with strain Tü 3634,
no incorporation of labelled acetate, methionine and glu-
cose was observed for the aglycone of 3. Whereas [2-13C]gly-
cerol labelled C-1 and C-3/C-5 (chemical equivalent nuclei)
and [U-13C3]glycerol labelled C-4/C-5/C-6 and C-2/C-1/C-7
(Table 3). These findings were in accord with the biosyn-
Scheme 1. Labelling pattern of 2,4-dimethyl-3-furanylcarbonyl α--
rhamnopyranoside (1) derived from 13C-labelled precursors
932
Eur. J. Org. Chem. 2000, 929Ϫ937

Secondary Metabolites by Chemical Screening, 39
FULL PAPER
order spin coupling system, which consequently makes
quantitative determinations impossible as the eligible coup-
ling partners C-1 (δ ϭ 130.84) and C-2/C-6 (δ ϭ 130.72)
have similar chemical shifts. Therefore further evidence for
an intact C-7/C-1/C-2-unit is not directly available by ob-
serving C-1 and C-2. Spin simulation experiments for C-7/
C-1/C-2 gave a nearly corresponding coupling pattern for
the C-7 signal, and from the spin simulations for C4/C-5/
C-6, the signal patterns at δ ϭ 130.2–131.2 were found to be
comparable.[24] A strong doublet at δ ϭ 134.8 (J ϭ 52 Hz)
accounts for C-4 as an outer carbon of a glycerol unit. Spin-
spin coupling of the overlapping signals for C-3/C-5 (δ ϭ
129.8, J ϭ 52, 56 Hz) point to the incorporation of at least
one intact C3-unit. The incorporation pattern for the agly-
cone of 3 (Scheme 3) results from an examination of the
coupling constants of 52 (C-4/C-5) and 56 Hz (C-5/C-6),
the assumption of a singlet for C-3 and spin simulations,
and is in accord with that of shikimate.[22] Feeding of unla-
belled -phenylalanine (3.3 m, 7.3 m) under the condi-
tions of normal incorporation experiments did not raise the
yields of 3. Thus the biogenesis of an unsubstituted benzoyl
moiety from a microbial source is described for the first
time.[25]
Scheme 2. Proposed biosynthetic pathways for acyl rhamnosides 1,
6 and acetomycin 7 starting with a C7 unit
Additionally, feeding experiments with 13C-labelled gly-
cerol and -glucose led to interesting incorporation patterns
in the rhamnose residues of 1 and 3. Glucose feeding was
followed by a decrease in the amount of products and 3
could be isolated in only small yield. Both C-2Ј (weak) and
C-5Ј (strong) were unevenly enriched with [2-13C]glycerol.
[U-13C3]glycerol gave intact incorporation which led to vari-
able intensities for the ‘‘top’’ half (C-1Ј/C-2Ј/C-3Ј, weak)
and ‘‘bottom’’ half (C-4Ј/C-5Ј/C-6Ј, very strong). Moreover,
an additional strong doublet within the C-2Ј-coupling pat-
tern also indicated the incorporation of an intact C2-unit
(C-1Ј/C-2Ј). Feeding [U-13C6]glucose to strain Tü 3634 re-
sulted in the 13C-NMR spectra of a highly enriched rham-
nose moiety in 3 which showed further additional complex
coupling patterns as discussed above. This was especially
the case for C-4Ј, which could be clearly analyzed as a
doublet of doublets (J ϭ 38, 40 Hz) thus indicating an in-
tact incorporation of glucose (Scheme 1). It is well-known
that deoxyhexoses e.g. rhamnose arise intact from glucose
phosphate. Its labeling pattern reflects the labelling of the
hexose phosphate pool, which is formed by different path-
ways: The ‘‘top’’ and the ‘‘bottom’’ C3-unit are formed
from triose phosphate pools and since these are not equilib-
rating, the addition of [U-13C3]glycerol via phosphoglyeral-
dehyde and dihydroxyacetone phosphate leads to uneven
pulse-labelling as previously described in the literature.[26]
The separate C2-unit (C-1Ј/C-2Ј) derived from a clear coup-
ling pattern of C-2Ј (d, 1JC-1/C-2 ϭ 47 Hz) indicates, that the
pentose phosphate pathway also feeds the hexose phosphate
pool, since transketolases transfer C2-units and thus labels
the final product hexose. Although much is known about
the biosynthesis of deoxysugars from glucose, detailed in-
vestigations of the hexoses as sources have been rare, and a
separate C-1/C-2-coupling has been observed only in the
case of polyketomycin.[27]
thesis of 3 via a shikimate pathway from phosphoenolpy-
ruvate and erythrose-4-phosphate.[22] Analysis of the extens-
ive labelling pattern of 3, obtained from the complex 13C-
NMR spectra with higher order couplings after feeding
with [U-13C3]glycerol, was accompanied by spin simula-
tions. Based on these results the biosynthetic pathway is
assumed to start directly from shikimate and thus rules out
a pathway via phenylalanine (Scheme 3).[23] A decisive fact
here is, that instead of a strong singlet indicating a biosyn-
thetic pathway via phenylalanine, a broad multiplet for C-7
3(δ ϭ 166.0) is observed. Thus C-7 must be part of a higher
Scheme 3. Labelling pattern and proposed biosynthetic pathway to
benzoic acid, the aglycone of 3, ruling out a pathway via phenylal-
anine
Eur. J. Org. Chem. 2000, 929Ϫ937
933

A. Zeeck et al.
FULL PAPER
extract 10 g/L, yeast extract 4 g/L, glucose 4 g/L, pH ϭ 7.0 prior
to sterilization; medium C: malt 10 g/L, yeast extract 4 g/L, glycerol
20 g/L, CaCO3 20 mg/L, pH ϭ 7.0 prior to sterilization; medium
D: meat meal 20 g/L, malt extract 100 g/L, CaCO3 10 g/L, pH ϭ
7.2 prior to sterilization; medium E: glycerol 30 g/L, casein peptone
2 g/L, K2HPO4 1 g/L, NaCl 1 g/L, MgSO4 7H2O 0.5 g/L, 5 mL
trace element solution, pH ϭ 7.3 prior to sterilization; trace ele-
ment solution: CaCl2 2H2O 3 g/L, FeIII-citrate 1 g/L, MnSO4 0.2 g/
L, ZnCl2 0.1 g/L, CuSO4 5 H2O 25 mg/L, Na2B4O7 10 H2O
0.02 g/L, CoCl2 4 mg/L, Na2MoO4 2 H2O 0.01 g/L.
Discussion
The acyl rhamnopyranosides (1–6) represent a novel fam-
ily of natural products produced by Streptomyces strains
and were discovered by the chemical screening method.
Glycosyltransferases use carboxylic acids as acceptor, the
resulting ester linked rhamnose moiety is very uncommon
in microbial secondary metabolites. The phenazine derivat-
ives aestivophoenins and phenazoviridins are the only ex-
amples for acyl glycosides known so far.[17] In its biosyn-
thesis, the aglycone of 1 is formed from a diketide and a
glycerol unit (Scheme 1). In the presence of an increased
amount of a nitrogen source the corresponding pyrrole de-
Fermentation: a) Strain Tü 3634 (Streptomyces griseoviridis) was
maintained as a stock culture on agar slants containing medium C
stored at 4 °C no longer than 14 d. A 1-cm2 piece of agar from 7
d old cultures was used to inoculate 100 mL of medium A in 1000-
rivative 2 is formed, presumably by a new pyrrole biosyn- mL Erlenmeyer flasks with flow spoilers. These cultures were in-
thetic pathway.[28] Higher aeration during the fermentation
cubated on a rotary shaker (250 rpm) at 28 °C for 48 h as standard
conditions. In order to isolate 1 and 4 100 mL of the culture broth
led to 6 by a hydroxylation of 1. The benzoyl residue of 3
were used to inoculate a stirred vessel (1 L working volume, me-
is derived from the shikimate pathway by dehydration and
dium A, 700 rpm, 28 °C, aeration 1.6 vvm). During the fermenta-
tion the pH decreased from 6.5 to 5.3 and after 12 h of fermenta-
tion the pH was maintained between 6.7 and 5.3 automatically.
Acyl rhamnosides could be detected after 16 h and the culture was
harvested after 67 h. For the isolation of 2, strain Tü 3634 was
cultivated in a 1-L fermentor under standard conditions (see above)
for 3 d in flasks each containing 100 mL of medium C. For the
reduction of the precursor. Investigations of the biosyn-
thesis of the α--rhamnosyl moiety with [U-13C3]glycerol
and [U-13C6]glucose revealed striking differences in the
amount of incorporation in the ‘‘top’’ and ‘‘bottom’’ halves
of the deoxyhexose and a characteristic labelling pattern
which indicated the pentose phosphate pathway as one ad-
ditional source for the hexose phosphate pool used for - isolation of 3, medium A was used. Compound 6 was isolated from
the culture filtrate of Tü 3634 in medium B (airlift-loop fermentor,
10 L, 28 °C, 72 h, aeration 4 vvm).
rhamnose.
One important result of the work presented here is the
observation that different aglyca are combined with the α-
-rhamnopyranosyl residue in a regio- and stereoselective
manner. The best interpretation is, that the rhamnosyl-
transferase is specific for -rhamnosyl nucleotide phos-
phates but much less specific for carboxylic acids as the ac-
cepting aglycone. Experiments to study this observation are
in progress.
b) Strains FH-S 1071 (Streptomyces fragilis, DSM 4200), FH-S
1512 (DSM 4355), FH-S 2087, and FH-S 1298 (Streptomyces sp.,
DSM 4211) were maintained as stock cultures on agar slants con-
taining medium B. A 72 h old submerged culture, cultivated on a
rotary shaker, 180 rpm at 30 °C for 72 h, medium A or medium D,
was used to inoculate (3%) a fermentor (10 L working volume,
medium A or D, 200 rpm, 30 °C, 0.5 vvm). All cultures were har-
vested after 96 h.
Isolation and Purification: a) For 1 to 4 and 6 similar procedures
were applied. The culture broths of strain Tü 3634 (Streptomyces
griseoviridis) were separated from the mycelium by filtration. The
mycelium was discarded. The light-red solutions obtained were
passed through Amberlite XAD-2 columns and impurities washed
out with deionized water and the metabolites then eluted with
methanol. Evaporation yielded crude extracts (approx. 180 mg/L).
Experimental Section
General: Melting points: Reichert hot-stage microscope (not cor-
rected). – IR spectra: Perkin–Elmer 298 spectrometer. – UV spec-
tra: Kontron Uvikon 860 spectrophotometer. – Optical rotation:
Perkin–Elmer 241. – EI-MS: Varian 311 A, 70 eV, direct insert,
high resolution with perfluorokerosine as standard. – DCI-MS:
Finnigan MAT 95, 200 eV, reaction gas: NH3. – 1H- and 13C-NMR
spectra: Varian VXR 500, U300, Bruker AMX 300. Chemical shifts
are expressed in δ values, TMS was used as internal standard. –
TLC: silica gel 60 (Macherey–Nagel, SIL G/UV 254ϩ366,
0.25 mm), Polygram (Macherey–Nagel, Alox N/UV254 0.20 mm);
Rf values were determined on 20 ϫ 20 cm plates; the evaluation
length was 10 cm. – Column chromatography: silica gel Ͻ 0.08 mm
(Macherey–Nagel), Sephadex LH-20 (Pharmacia). – Staining re-
agents: Anisaldehyde/sulfuric acid: 1.0 mL of anisaldehyde in
85 mL of methanol, 5 mL of conc. sulfuric acid and 10 mL of acetic
acid; vanillin/sulfuric acid: 1 g of vanillin in 100 mL of sulfuric
acid; Ehrlich’s reagent: 1 g of 4-dimethylamino benzaldehyde,
25 mL conc. hydrochloric acid, 75 mL of methanol – Fermentation:
Biostat E (10 L), Biostat M (1 L), Biostat U (50 L), all Braun-
Diessel (Melsungen, Germany), Airlift-loop fermentor (10 L).
b) For the isolation of 1 and 3 strain Tü 3634 was cultivated in
medium A. To obtain 6 cultivation was carried out in medium B.
The crude extract obtained (180 mg/L) was chromatographed on
Sephadex LH-20 (column: 100 ϫ 2.5 cm, methanol) and the main
fractions (detection by TLC) further purified on silica gel (column:
30 ϫ 2.5 cm, CH2Cl2/MeOH 4:1) to yield 11 mg/L of pure 1, 6 mg/
L of 3 and 0.4 mg/L of 6.
c) Rhamnoside 2 was isolated from the culture broth of strain Tü
3634, either obtained from medium C in 1-L flasks or medium A
with additional 2.9 g (21.9 m) ammonium hydrogenphosphate in
a 1-L fermentor. The crude extract was chromatographed on Se-
phadex LH-20 (column: 100 ϫ 2.5 cm, MeOH) and silica gel (col-
umn: 25 ϫ 1.5 cm, CH2Cl2/MeOH, 4:1). Compound 2, 5.3 mg/L
(medium C) or 2.7 mg/L [medium A/(NH4)2HPO4] was obtained
along with 1 (8.0 mg/L) as white powders.
Nutrient Solutions: Medium A: oatmeal 20 g/L, 2.5 mL trace ele-
ment solution, pH ϭ 6.8 prior to sterilization; medium B: malt
d) Rhamnoside 4 was isolated from the culture broth of strain Tü
3634 (medium C, 1-L fermentor). The crude extract was chromato-
934
Eur. J. Org. Chem. 2000, 929Ϫ937

Secondary Metabolites by Chemical Screening, 39
FULL PAPER
graphed on Sephadex LH-20 (column: 100 ϫ 2.5 cm, MeOH) and
5Ј-H), 3.83 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 3.97 (dd, J ϭ 3.5,
silica gel (column: 25 ϫ 1.5 cm, CH2Cl2/MeOH 4:1). Compound 4 1.9 Hz, 1 H, 2Ј-H), 6.18 (d, J ϭ 1.9 Hz, 1 H, 1Ј-H), 7.50 (dd, Jϭ
(23 mg/L) was obtained as a white powder. Other acyl rhamnosides
were not detected.
7.5, 7.5, 2 H, 3-H, 5-H), 7.64 (tt, J ϭ 7.5, 1.5 Hz, 1 H, 4-H), 8.03
(dd, J ϭ 7.5, 1.5 Hz, 2 H, 2-H, 6-H). Ϫ 13C NMR (125.7 MHz,
CD3OD): δ ϭ 18.1 (q, C-6Ј), 71.2 (d, C-2Ј), 72.3 (d, C-3Ј), 72.6 (d,
C-5Ј), 73.5 (d, C-4Ј), 95.9 (d, C-1Ј), 129.8 (d, C-3, C-5), 130.5 (s,
C-1), 130. 7 (d, C-2, C-6), 134.8 (d, C-4), 166.0 (s, C-7). Ϫ DCI-
MS: m/z (%) ϭ 303 (100) [M ϩ NH4ϩ ϩ NH3], 286 (33) [M ϩ
NH4ϩ]. – C13H16O6 (268.27).
e) Rhamnosides 3, 4, and 5 were obtained by separation of the
culture filtrate of strain FH-S 2087 (cultivated in a 10-L fermentor,
medium E) from the mycelium by filtration or centrifugation. The
aqueous solution was applied to MCI-Gel HP-20, followed by elu-
tion with a 50–100% MeOH gradient in H2O to yield 6 fractions
of crude material. The isolation of the rhamnosides 3 and 5 from
strain FH-S 2087 was accomplished by chromatography on silica
gel (column: 40 ϫ 5 cm, acetone/hexane 2:1, acetone/hexane/H2O
2:1:1), on Sephadex LH-20 (column: 100 ϫ 2.5 cm, MeOH) and
on silica gel (column: 35 ϫ 2.5 cm, CHCl3/MeOH 9:1) to yield
1 mg/L of pure 3 and 0.8 mg/L of pure 5 as white powders. For the
isolation of 4, the main fraction of crude material was purified
by chromatography on Sephadex LH-20 (column: 100 ϫ 2.5 cm,
MeOH) and on silica gel (column: 35 ϫ 2.5 cm, CH2Cl2/MeOH
3:1) and yielded 3.1 mg/L of 4.
Phenylacetyl α-L-Rhamnopyranoside (4): m.p. 85 °C (dec.). –
[α]2D0 ϭ –52 (c ϭ 1.0 in MeOH). – IR (KBr): ν˜ ϭ 3400 cm–1, 2980,
2930, 1745, 1500, 1455, 1250, 1130, 1060, 970. – UV (MeOH): λmax
(log ε) ϭ 206 (3.48), 258 (2.58); (MeOH/HCl): λmax (log ε) ϭ 206
1
(3.78); (MeOH/NaOH): λmax (log ε) ϭ 206 (3.88), 258 (2.55). – H
NMR (500 MHz, CD3OD): δ ϭ 1.18 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3),
3.38 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 3.48 (dq, J ϭ 6.5, 9.5 Hz, 1
H, 5Ј-H), 3.57 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 3.68 (s, 2 H, 8-H2),
3.75 (dd, J ϭ 3.5, 1.9 Hz, 1 H, 2Ј-H), 5.93 (d, J ϭ 1.9 Hz, 1 H, 1Ј-
H), 7.28 (m, 5 H, C6H5). – 13C NMR (75.5 MHz, CD3OD): δ ϭ
17.9 (q, C-6Ј), 42.1 (t, C-8), 71.1 (d, C-2Ј), 72.0 (d, C-3Ј), 72.3 (d,
C-5Ј), 73.3 (d, C-4Ј), 95.7 (d, C-1Ј), 128.2 (d, C-4), 129.6 (2 d, C-
3, C-5), 130.3 (2 d, C-2, C-6), 135.3 (s, C-1), 171.4 (s, C-7). – DCI-
MS: m/z (%) ϭ 317 (35) [M ϩ NH4ϩ ϩ NH3], 300 (100) [M ϩ
NH4ϩ]. – C14H18O6 (282.3).
All glycosides (1–6) are readily soluble in MeOH, ethyl acetate,
DMSO, slightly soluble in CHCl3 and water, and insoluble in n-
hexane. They are unstable under acidic (pH Ͻ 4) or alkaline
(pH Ͼ 9) conditions, as well as on heating above 40 °C.
2,4-Dimethyl-3-furanylcarbonyl α-L-Rhamnopyranoside (1): m.p. 87
2-Pyrrolylcarbonyl α-L-Rhamnopyranoside (5): m.p. 89 °C (dec.). –
°C (dec.). – [α]2D0 ϭ Ϫ42 (c ϭ 0.05 in MeOH). Ϫ IR (KBr): ν˜ ϭ
3400 cm–1, 2980, 2930, 1710, 1610, 1560. Ϫ UV (MeOH): λmax (log
[α]2D0 ϭ –39 (c ϭ 0.56 in MeOH). – IR (KBr): ν˜ ϭ 3396 cm–1, 2980,
2931, 2362, 1696, 1553, 1448, 1410, 1306. – UV (MeOH): λmax (log
1
ε) ϭ 201 (3.94), 254 (3.30). Ϫ H NMR (500 MHz, CD3OD): δ ϭ
1
ε) ϭ 210 (3.37), 267 (4.23). – H NMR (500 MHz, CD3OD): δ ϭ
1.26 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 2.15 (d, J ϭ 1.3 Hz, 3 H, 8-H3),
2.54 (s, 3 H, 6-H3), 3.48 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 3.65 (dq,
J ϭ 6.5, 9.5 Hz, 1 H, 5Ј-H), 3.77 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H),
3.91 (dd, J ϭ 3.5, 1.9 Hz, 1 H, 2Ј-H), 6.16 (d, J ϭ 1.9 Hz, 1 H, 1Ј-
H), 7.18 (q, J ϭ 1.3 Hz, 1 H, 5-H). Ϫ 13C- NMR (125.7 MHz,
CD3OD): δ ϭ 10.6 (q, C-8), 14.7 (q, C-6), 18.1 (q, C-6Ј), 71.4 (d,
C-2Ј), 72.4 (d, C-3Ј), 72.7 (d, C-5Ј), 73.4 (d, C-4Ј), 95.2 (d, C-1Ј),
113.9 (s, C-3), 122.2 (s, C-4), 139.6 (d, C-5), 162.4 (s, C-2), 164.0
(s, C-7). Ϫ HREI-MS: calcd. for C13H18O7: 286.1052, found:
1.26 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 3.47 (dd, J ϭ 9.5, 9.5 Hz, 1 H,
4Ј-H), 3.74 (dq, J ϭ 6.5, 9.5 Hz, 1 H, 5Ј-H), 3.83 (dd, J ϭ 9.5,
3.5 Hz, 1 H, 3Ј-H), 3.90 (dd, J ϭ 3.5, 2.0 Hz, 1 H, 2Ј-H), 6.09 (d,
J ϭ 2.0 Hz, 1 H, 1Ј-H), 6.21 (dd, J ϭ 2.5, 3.5 Hz, 1 H, 4-H), 6.91
(dd, J ϭ 1.5, 3.5 Hz, 1 H, 3-H), 7.01 (dd, J ϭ 1.5, 2.5 Hz, 1 H, 5-
H). – 13C NMR (125.7 MHz, CD3OD): δ ϭ18.0 (q, C-6Ј), 71.4 (d,
C-2Ј), 72.2 (d, C-3Ј), 72.3 (d, C-5Ј), 73.6 (d, C-4Ј), 95.1 (d, C-1Ј),
111.0 (d, C-4), 117.5 (d, C-3), 122.7 (s, C-2), 125.7 (d, C-5), 160.6
(s, C-6). – DCI-MS: m/z (%) ϭ 258.3 (20) [M ϩ Hϩ], 275 (100) [M
ϩ NH4ϩ]. – C11H15NO6 (257.24).
286.1058. Ϫ EI MS: m/z (%) ϭ 286 (9) [Mϩ], 140 (53) [Mϩ
C6H10O4], 123 (100) [Mϩ – C6H11O5]. Ϫ C13H18O7 (286.3).
–
4-Hydroxymethyl-2-methyl-3-furanylcarbonyl α-L-Rhamnopyrano-
2,4-Dimethyl-3-pyrrolylcarbonyl α-L-Rhamnopyranoside (2): m.p. 90
side (6): m.p. 119 °C. – [α]2D0 ϭ –21 (c ϭ 0.09 in MeOH). – IR
(KBr): ν˜ ϭ 3425 cm–1, 2926, 2363, 1632, 1383, 1062. – UV
(MeOH): λmax (log ε) ϭ 201 (3.88), 248 (3.35). – 1H NMR
(500 MHz, CD3OD): δ ϭ 1.28 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 2.56 (s,
3 H, 6-H3), 3.48 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 3.69 (dq, J ϭ
6.5, 9.5 Hz, 1 H, 5Ј-H), 3.76 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 3.91
(dd, J ϭ 3.5, 2.0 Hz, 1 H, 2Ј-H), 4.64 (d, J ϭ 1.0 Hz, 2 H, 8-H2),
6.15 (d, J ϭ 2.0 Hz, 1 H, 1Ј-H), 7.37 (t, J ϭ 1.0 Hz, 1 H, 5-H). –
13C NMR (125.7 MHz, CD3OD): δ ϭ14.6 (q, C-6), 18.1 (q, C-6Ј),
57.1 (t, C-8), 71.3 (d, C-2Ј), 72.4 (d, C-3Ј), 72.8 (d, C-5Ј), 73.4 (d,
C-4Ј), 95.6 (d, C-1Ј), 112.6 (s, C-3), 127.8 (s, C-4), 140.2 (d, C-5),
162.7 (s, C-2), 163.8 (s, C-7). – DCI-MS: m/z (%) ϭ 320 (100) [M
ϩ NH4ϩ], 302 (10) [Mϩ]. – C13H18O8 (302.28).
°C (dec.). Ϫ [α]2D0 ϭ Ϫ22 (c ϭ 0.1 in MeOH). Ϫ IR (KBr): ν˜ ϭ
3406 cm–1, 2924, 2363, 1683, 1582, 1441, 1383, 1259. Ϫ UV
(MeOH): λmax (log ε) ϭ 205 (4.10), 221 (3.81), 233 (3.88), 252
1
(3.62), 262 (3.64). Ϫ H NMR (500 MHz, CD3OD): δ ϭ 1.27 (d,
J ϭ 6.0 Hz, 3 H, 6Ј-H3), 2.20 (d, J ϭ 1.3 Hz, 3 H, 8-H3), 2.44 (s,
3 H, 6-H3), 3.48 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 3.70 (dq, J ϭ
6.5, 9.5 Hz, 1 H, 5Ј-H), 3.81 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 3.89
(dd, J ϭ 3.5, 1.9 Hz, 1 H, 2Ј-H), 6.13 (d, J ϭ 1.9 Hz, 1 H, 1Ј-H),
6.35 (q, J ϭ 1.3 Hz, 1 H, 5-H). Ϫ 13C NMR (75.5 MHz, CD3OD):
δ ϭ 13.5 (q, C-8), 14.3 (q, C-6), 18.1 (q, C-6Ј), 71.7 (d, C-2Ј), 72.3
(d, C-3Ј), 72.5 (d, C-5Ј), 73.5 (d, C-4Ј), 94.3 (d, C-1Ј), 110.1 (s, C-
3), 116.1 (d, C-5), 122.1 (s, C-4), 138.6 (s, C-2), 165.9 (s, C-7). Ϫ
EI-MS: m/z (%) ϭ 285 (20) [Mϩ], 139 (100) [MϩϪC6H10O4], 122
(98) [C7H8NOϩ], 94 (10) [C6H8Nϩ]; DCI-MS: m/z (%) ϭ 303 (30)
[M ϩ NH4ϩ], 286 (100) [M ϩ Hϩ]. Ϫ HREI-MS: found as calcd.
for C13H19NO6: 285.1212. – C13H19NO6 (285.3).
Acetylation of 1, 3, 4, and 5: The respective rhamnoside (0.04
mmol) was dissolved in acetic anhydride (2 mL) at 0 °C. Pyridine
(0.75 mL) was added, the solution stirred for 16 h at room temper-
ature and the reaction stopped by hydrolysis with ice water (10 mL)
Benzoyl α-L ϭ
-Rhamnopyranoside (3): m.p. 145 °C (dec.). Ϫ [α]2D0
Ϫ52 (c ϭ 0.35 in MeOH). Ϫ IR (KBr): ν˜ ϭ 3384 cm–1, 2979, 2916, for 2 h. The reaction product was extracted twice with ethyl acetate
1726, 1601, 1451, 1320, 1271. Ϫ UV (MeOH): λmax (log ε) ϭ 201
(4.02), 209 (3.51), 230 (4.11), 258 (2.83), 274 (2.98). Ϫ 1H NMR removed by evaporation with toluene in vacuo. Flash chromato-
(500 MHz, CD3OD): δ ϭ 1.28 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 3.50 graphy on silica gel (column: 20 ϫ 1, CHCl3/MeOH 98:2) yielded
(dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 3.76 (dq, J ϭ 6.5, 9.5 Hz, 1 H, 14.8 mg (0.03 mmol, 75%) of 1a, 18.8 mg (0.05 mmol, 90%) of 3a,
or chloroform, the organic phases dried with Na2SO4, and pyridine
Eur. J. Org. Chem. 2000, 929Ϫ937
935

A. Zeeck et al.
6.0 mg (0.015 mmol, 30%) of 4a and 21 mg (0.05 mmol, 100%) of H, 1Ј-H), 6.29 (dd, J ϭ 2.5, 3.8 Hz, 1 H, 4-H), 7.02 (m, 2 H, 3-H,
FULL PAPER
5a.
5-H). – 13C NMR (125.7 MHz, CD3OD): δ ϭ17.8 (q, C-6Ј), 20.6
(q, CH3CO), 20.6 (q, CH3CO), 20.7 (q, CH3CO), 69.9 (d, C-2Ј),
70.2 (d, C-5Ј), 70.6 (d, C-3Ј), 71.7 (d, C-4Ј), 91.8 (d, C-1Ј), 111.3
(d, C-4), 118.2 (d, C-3), 122.0 (s, C-2), 126.2 (d, C-5), 159.5 (s, C-
6), 171.4 (s, CH3CO), 171.6 (s, CH3CO), 171.6 (s, CH3CO). – DCI-
MS: m/z (%) ϭ 258.3 (20) [M ϩ Hϩ], 275 (100) [M ϩ NH4ϩ]. –
C17H21NO9 (383.4): calcd. C 53.26, H 5.52; found C 53.49, 5.63.
2,4-Dimethyl-3-furanylcarbonyl 2,3,4-Tri-O-acetyl-α-L-rhamnopyr-
anoside (1a): Colorless oil. – [α]2D0 ϭ –65 (c ϭ 0.2 in MeOH). – IR
(KBr): ν˜ ϭ 3431 cm–1, 2986, 1752, 1610, 1562. – UV (MeOH): λmax
1
(log ε) ϭ 203 (4.02), 256 (3.54). – H NMR (300 MHz, CD3OD):
δ ϭ 1.20 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 1.97 (s, 3 H, CH3CO), 2.06
(s, 3 H, CH3CO), 2.17 (s, 3 H, CH3CO), 2.18 (d, J ϭ 1.5 Hz, 3 H,
8-H3), 2.57 (s, 3 H, 6-H3), 3.98 (dq, J ϭ 6.0, 9.5 Hz, 1 H, 5Ј-H),
5.11 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 5.33 (m, 1 H, 2Ј-H), 5.35
(dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 6.18 (d, J ϭ 2.0 Hz, 1 H, 1Ј-H),
7.20 (q, J ϭ 1.5 Hz, 1 H, 7-H). – 13C NMR (75.5 MHz, CD3OD):
δ ϭ 10.6 (q, C-8), 14.8 (q, C-6), 17.9 (q, C-6Ј), 20.5 (q, CH3CO),
20.6 (q, CH3CO), 20.6 (q, CH3CO), 70.1 (d, C-2Ј), 70.4 (d, C-5Ј),
70.6 (d, C-3Ј), 71.4 (d, C-4Ј), 91.7 (d, C-1Ј), 113.5 (s, C-3), 122.2
(s, C-4), 139.8 (d, C-5), 163.0 (s, C-2 or C-7), 163.1 (s, C-2 or C-
7), 171.4 (s, CH3CO), 171.5 (s, CH3CO), 171.7 (s, CH3CO). – EI-
MS: m/z (%) ϭ 412 (10) [Mϩ], 273 (50) [Mϩ – C7H7O3], 123.1 (100)
[C7H7O2]. – C19H24O10 (412.4).
Methanolysis of 1 and 4: a) Compound 1 (49 mg, 0.171 mmol) was
stirred with MeOH/HCl (3 mL, 0.1 ) for 24 h at room temper-
ature. Solvent and HCl were removed in vacuo and the residue chro-
matographed on silica gel (column 28 ϫ 2.5, gradient CHCl3/
MeOH 95:5 to 8:1) to yield 14 mg (0.1 mmol, 58%) of aglycone 10,
5 mg (0.03 mmol, 16%) of methyl α--rhamnopyranoside and 4 mg
(0.02 mmol, 13%) of methyl β--rhamnopyranoside. – b) Com-
pound 4 (40 mg, 0.14 mmol) was mixed with MeOH/HCl (2 mL,
0.1 ) and stirred for 16 h at room temperature. An identical isola-
tion procedure yielded 13 mg (0.088 mmol, 62%) of phenyl acetic
acid methyl ester, 9.0 mg (0.05 mmol, 72%) of methyl α--rhamno-
pyranoside and 6 mg (0.03 mmol, 48%) of methyl β--rhamnopyr-
anoside.
Benzoyl 2,3,4-Tri-O-acetyl-α-L-rhamnopyranoside (3a): Colorless
oil. – [α]2D0 ϭ –74 (c ϭ 0.5 in MeOH). – IR (KBr): ν˜ ϭ 3450 cm–1,
2980, 2920, 1750, 1600, 1580. – UV (MeOH): λmax (log ε) ϭ 202
(2.66), 228 (3.79), 273 (2.78). – 1H NMR (500 MHz, CD3OD): δ ϭ
1.24 (d, J ϭ 6.0 Hz, 3 H, 6Ј-H3), 2.00 (s, 3 H, CH3CO), 2.06 (s, 3
H, CH3CO), 2.19 (s, 3 H, CH3CO), 4.02 (dq, J ϭ 6.0, 9.5 Hz, 1 H,
5Ј-H), 5.17 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 5.40 (dd, J ϭ 3.5,
2.0 Hz, 1 H, 2Ј-H), 5.43 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 6.25 (d,
J ϭ 2.0 Hz, 1 H, 1Ј-H), 7.47 (dd, J ϭ 7.5, 8.0 Hz, 2 H, 3-H, 5-H),
7.61 (tt, J ϭ 1.5, 7.5 Hz, 1 H, 4-H), 8.08 (dd, J ϭ 1.5, 8.0 Hz, 2 H,
2-H, 6-H). – 13C NMR (75.5 MHz, CD3OD): δ ϭ 17.8 (q, C-6Ј),
20.6 (q, CH3CO), 20.6 (q, CH3CO), 20.6 (q, CH3CO), 69.9 (d, C-
2Ј), 70.3 (d, C-5Ј), 70.6 (d, C-3Ј), 71.5 (d, C-4Ј), 92.7 (d, C-1Ј),
129.9 (2 d, C-3, C-5), 130.2 (s, C-1), 130.9 (2 d, C-2, C-6), 135.1
(d, C-4), 165.2 (s, C-7), 171.4 (s, CH3CO), 171.6 (s, CH3CO), 171.7
(s, CH3CO). – DCI-MS: m/z (%) ϭ 412 (100) [M ϩ NH4ϩ]. – HREI-
MS: calcd. for C19H22O9: 394.1264, found: 394.1263. – C19H22O9
(394.4).
Methyl α-L-Rhamnopyranoside: Colorless oil. – Rf ϭ 0.20, CHCl3/
MeOH, 85:15. – [α]2D0 ϭ –65 (c ϭ 0.33 in MeOH). – 1H NMR
(500 MHz, CD3OD): δ ϭ 1.26 (d, J ϭ 6.0 Hz, 3 H, 6-H3), 3.33 (s,
1-OCH3), 3.35 (dd, J ϭ 9.5, 9.5, 1 H, 4-H), 3.52 (dq, J ϭ 9.5,
6.0 Hz, 1 H, 5-H), 3.58 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3-H), 3.77 (dd,
J ϭ 3.5, 2.0 Hz, 1 H, 2-H), 4.54 (d, J ϭ 2.0 Hz, 1 H, 1-H). – DCI-
MS: m/z (%) ϭ 213 (24) [M ϩ NH4ϩ ϩ NH3], 196 (100) [M ϩ
NH4ϩ). – C7H14O5 (178.2).
Methyl β-L-Rhamnopyranoside: Colorless oil. – Rf ϭ 0.17 CHCl3/
1
MeOH, 85:15. – [α]2D0 ϭ ϩ85.8 (c ϭ 0.33 in MeOH). – H NMR
(500 MHz, CD3OD): δ ϭ 1.31 (d, J ϭ 6.0 Hz, 3 H, 6-H3), 3.22 (dq,
J ϭ 9.5, 6.0 Hz, 1 H, 5-H), 3.32 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4-H),
3.38 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3-H), 3.49 (s, 3 H, 1-OCH3), 3.84
(dd, J ϭ 3.5, 2.0 Hz, 1 H, 2-H), 4.36 (d, J ϭ 2.0 Hz, 1 H, 1-H). –
DCI-MS: m/z (%) ϭ 196 (100) [M ϩ NH4ϩ]. – C7H14O5 (178.2).
2,4-Dimethylfuran-3-carboxylic Acid (11): White amorphous pow-
der, m.p. 132 °C. – Rf ϭ 0.54 (CHCl3/MeOH 85:15). – IR (KBr):
ν˜ ϭ 3400 cm–1, 3000, 2620, 1670, 1605, 1560. – UV (MeOH): λmax
(log ε) ϭ 202 (3.89), 243 (3.43). – 1H NMR (300 MHz, CDCl3/
CD3OD): δ ϭ 2.16 (d, J ϭ 1.3 Hz, 3 H, 8-H3), 2.58 (s, 3 H, 6-H3),
7.06 (d, J ϭ 1.3, 1 H, 5-H). – 13C NMR (50.3 MHz, CDCl3): δ ϭ
9.9 (t, C-8), 14.6 (t, C-6), 112.8 (s, C-3), 121.5 (s, C-4), 137.9 (d, C-
5), 162.0 (s, C-2), 170.4 (s, C-7). – EI-MS: m/z (%) ϭ 140 (100)
[Mϩ], 122 (48) [Mϩ – H2O], 44 (100) [CO2]. – HREI-MS: found as
calcd. for C7H8O3: 140.0473.
Phenylacetyl 2,3,4-Tri-O-acetyl-α-
less oil. – [α]2D0 ϭ –35 (c ϭ 1.15 in MeOH). – IR (KBr): ν˜ ϭ
2980 cm–1, 2930, 1750, 1495, 1450. 1H NMR (500 MHz,
L-rhamnopyranoside (4a): Color-
–
CD3OD): δ ϭ 1.08 (d, J ϭ 6 Hz, 3 H, 6Ј-H3), 1.98 (s, 3 H, CH3CO),
2.06 (s, 3 H, CH3CO), 2.15 (s, 3 H, CH3OH), 3.64 (dq, J ϭ 6.0,
9.5 Hz, 1 H, 5Ј-H), 3.75 (d, J ϭ 14.5 Hz, 1 H, 8-H), 3.79 (d, J ϭ
14.5 Hz, 1 H, 8-H), 5.02 (dd, J ϭ 9.5, 9.5 Hz, 1 H, 4Ј-H), 5.16 (dd,
J ϭ 3.5, 9.5 Hz, 1 H, 3Ј-H), 5.25 (dd, J ϭ 2.0, 3.5 Hz, 1 H, 2Ј-H),
5.98 (d, J ϭ 2.0 Hz, 1-H, 1Ј-H), 7.29 (m, 1 H, 4-H), 7.34 (m, 4 H,
2-H, 3-H, 5-H, 6-H). – 13C NMR (75.5 MHz, CD3OD): δ ϭ 17.7
(q, C-6Ј), 20.5 (2q, 2 CH3CO), 20.6 (q, CH3CO), 41.2 (t, C-2), 69.8
(d, C-2Ј), 69.8 (d, C-5Ј), 70.4 (d, C-3Ј), 71.4 (d, C-4Ј), 92.2 (d, C-
1Ј), 128.3 (d, C-4), 129.8 (2 d, C-3, C-5), 130.4 (2 d, C-2, C-6),
135.1 (s, C-1), 170.6 (s, C-7), 171.5 (s, CH3CO), 171.6 (2s, 2
CH3CO). – DCI-MS: m/z (%) ϭ 426 (100) [M ϩ NH4ϩ]. –
C20H24O9 (408.4).
Phenylacetic Acid Methyl Ester: 1H NMR (500 MHz, CD3OD):
δ ϭ3.63 (s, 2 H, CH2), 3.66 (s, 3 H, CH3O), 7.27 (m, 5 H, C6H6). –
EI-MS: m/z (%) ϭ 150 (50) [Mϩ], 91 (100) (C7H7). – HREI-MS:
calcd. for C9H10O2: 150.0681, found: 150.0680).
Incorporation Experiments: Feedings were carried out in standard
cultivations (1-L fermentor) by continuous addition of labelled pre-
2-Pyrrolylcarbonyl 2,3,4-Tri-O-acetyl-α-
L
-rhamnopyranoside (5a): cursors in aqueous solutions (7 mL/h) for 10 h by a low rate pump.
Addition was started 16 h after inoculation. Precursors were dis-
Light-yellow oil. – [α]2D0 ϭ –89 (c ϭ 0.2 in MeOH). – IR (KBr):
ν˜ ϭ 3426 cm–1, 2926, 1743, 1653, 1635, 1410. – UV (MeOH): λmax solved in 100 mL of deionized water, adjusted to pH ϭ 6.5 with
(log ε) ϭ 267 (4.20). – 1H NMR (500 MHz, CD3OD): δ ϭ 1.20 (d, 2  citric acid and sterilized at 120 °C for 30 min. The following
J ϭ 6.0 Hz, 3 H, 6Ј-H3), 1.99 (s, 3 H, CDCl3), 2.06 (s, 3 H, quantities of labelled compounds (all obtained from Campro Sci-
CH3CO), 2.17 (s, 3 H, CH3CO), 4.00 (dq, J ϭ 9.5, 6.0, 1 H, 5Ј-H),
5.14 (dd, J ϭ 9.5, 9.5, 1 H, 4Ј-H), 5.36 (dd, J ϭ 3.5, 2.0, 1 H, 2Ј-
entific or Cambridge Isotope Laboratories) were used: 9.1 m of
sodium [1-13C]acetate (99.0% 13C), 5.5 m of sodium [1,2-13C2]a-
H), 5.39 (dd, J ϭ 9.5, 3.5 Hz, 1 H, 3Ј-H), 6.17 (d, J ϭ 2.0 Hz, 1 cetate (99.0% 13C), 0.7 m of -[methyl-13C]methionine (99.0%
936
Eur. J. Org. Chem. 2000, 929Ϫ937

Secondary Metabolites by Chemical Screening, 39
FULL PAPER
13C), 5.4 m of [2-13C]glycerol (99.0% 13C), 5.4 m of [U-13C3]gly-
cerol (99.0% 13C) and 2.8 m of [U-13C6]glucose. Labelled products
were obtained in the following amounts: 7.5 mg 1, 2.0 mg 3 ([1-
13C]acetate); 10.2 mg 1, 2.0 mg 3 ([1,2-13C2]acetate); 11.6 mg 1,
2.0 mg 3 ([2-13C]glycerol); 5.0 mg 1, 1.0 mg 3 ([U-13C3]glycerol);
1.0 mg 1, 1.0 mg 3 ([U-13C6]glucose). Specific incorporations, sum-
marized in Table 2 and Table 3, were calculated according Scott
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We wish to thank I. Papastavrou for valuable contributions. We are
grateful to the Hoechst AG (Frankfurt) for providing us with the
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Received July 9, 1999
[O99413]
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