A method for the introduction of reporter groups into moenomycin A, based on thiouronium salt chemistry

Article abstract of DOI:10.1002/1099-0690(200204)2002:7<1163::AID-EJOC1163>3.0.CO;2-C

p-Alkoxy-substituted cinnamylthiouronium salts 7, 13, 21a, and 21b reacted selectively with the 2-acylamino-1,3-cyclopentanedione unit A of moenomycin A (1) to give the corresponding 2-alkylated products 14a, 14b, 22a, and 22b. These products, depending on the pH of the solution, were either stable under the reaction conditions or they underwent retro-Claisen-type reactions. The method can be used for the attachment of reporter groups to moenomycin A for the synthesis of, for example, 25. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002).

Full text of DOI:10.1002/1099-0690(200204)2002:7<1163::AID-EJOC1163>3.0.CO;2-C

FULL PAPER
A Method for the Introduction of Reporter Groups into Moenomycin A,
Based on Thiouronium Salt Chemistry
Andrij Buchynskyy,[a] Katherina Stembera,[a] Lothar Hennig,[a] Matthias Findeisen,[a]
Sabine Giesa,[a] and Peter Welzel*[a]
Keywords: Antibiotics / Bioconjugate methods / Glycolipids / Peptidoglycan / Transglycosylase
p-Alkoxy-substituted cinnamylthiouronium salts 7, 13, 21a,
and 21b reacted selectively with the 2-acylamino-1,3-cyclo-
Claisen-type reactions. The method can be used for the at-
tachment of reporter groups to moenomycin A for the syn-
pentanedione unit A of moenomycin A (1) to give the corres- thesis of, for example, 25.
ponding 2-alkylated products 14a, 14b, 22a, and 22b. These
products, depending on the pH of the solution, were either
stable under the reaction conditions or they underwent retro-
(
Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
mode of action has been proposed.[6,7] It is assumed that
they are anchored to the cytoplasmic membrane through
the C25 lipid component, followed by highly selective recog-
nition of the oligosaccharide moiety at a substrate binding
site of the enzyme, most probably the binding site of the
growing glycan chain (the glycosyl donor). Whereas the
mechanism of the transpeptidation reaction is reasonably
well understood, the active site of the transglycosylase is
still unknown and the mechanism of the transglycosylation
step is largely unexplored. The moenomycins represent a
unique tool for elucidating the structure of the enzyme and
the detailed mechanism of the transglycosylation reaction.
One array of methods that can be used for this purpose is
based on the attachment of moenomycin to solid supports
(affinity chromatography for isolating PBP1b[8] or moeno-
mycin aptamers[9]), reporter groups (such as fluorescent la-
bels for binding studies,[10,11] photoactive groups for affinity
labeling,[12] etc.) or proteins (raising of antibodies[13]). For
moenomycin A, there are a number of essentials that have
to be taken into account:
The shape of bacteria is determined by a net-like multi-
layer polymer surrounding the cell. The polymer Ϫ peptido-
glycan Ϫ consists of repeating β-1,4-linked N-acetylglucosa-
minyl-N-acetylmuramyl units cross-linked by short peptide
side chains.[1] The biosynthesis of peptidoglycan is an essen-
tial pathway for bacteria and has no direct counterpart in
eukaryotic cells. Defects or disruption of peptidoglycan or
inhibition of its biosynthesis result in cell lysis caused by
the osmotic pressure. The distinct stages of peptidoglycan
biosynthesis offer attractive targets for the development of
selective antibacterial agents.
The biosynthesis of peptidoglycan in E. coli, starting
from a membrane-bound undecaprenyl-linked disaccharide
precursor (lipid II), is completed by two successive reac-
tions, a transglycosylation reaction producing unbranched
glycan strands[2] and a transpeptidation reaction cross-link-
ing the peptide units of different strands.[1] Both reactions
are catalyzed by the major high molecular weight penicillin-
binding proteins (PBPs).[3] PBPs such as PBP1a and PBP1b
are bifunctional enzymes with two separate active sites, one
for transglycosylation and the other for transpeptidation.
Each of these domains can be specifically inhibited by anti-
biotics. While β-lactam antibiotics exert their action
through covalent binding to an essential serine residue in
the transpeptidase domain, the transglycosylation step of
cell wall assembly can be blocked by a number of antibi-
otics, including the moenomycins.[4] Of these, the moeno-
mycins (see moenomycin A, 1, Figure 1) are the only com-
pounds known to inhibit the enzyme.[2]
(i) A selective reaction has to be used in order to be sure
that a structurally well-defined conjugate is formed.
(ii) The conjugate must bind efficiently to the moenomy-
cin binding site at the enzyme (it should be antibiotically
active). This means that the conjugation reaction should
not occur at units C-E-F-G-H-I (see 1, Figure 1), be-
cause they have been shown to be prerequisites of antibi-
otic activity.[5]
(iii) The reaction has to be performed in highly polar
media, preferably in aqueous solution, for solubility
reasons.
The structure-activity relationships of the moenomycins
have been studied extensively[5] and a mechanism for their
Some time ago we reported that the enolized 1,3-diketone
unit A of moenomycin A reacts selectively with p-acceptor-
substituted aromatic diazonium salts Ϫ that is, with soft
[a]
Universität Leipzig, Fakultät für Chemie und Mineralogie,
Johannisallee 29, 04103 Leipzig, Germany
Eur. J. Org. Chem. 2002, 1163Ϫ1174
WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0407Ϫ1163 $ 17.50ϩ.50/0
1163

A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S. Giesa, P. Welzel
FULL PAPER
Figure 1. Moenomycin A
electrophiles
Ϫ
with
a
well-balanced reactivity (see aqueous solution, provided secosteroid 5 in 80Ϫ85% yield
Scheme 1) to give the corresponding azo derivatives. These (Scheme 2).[15] The method was later used by others.[16,17]
were not stable under the reaction conditions, and under-
went JappϪKlingemann cleavage to form the correspond-
ing amidrazones, which slowly cyclized to yield triazoles.[14]
We described the synthesis of some triazoles in which the
functional groups R (SH, NH2) were orthogonal in their
reactivities to all other functional groups in 2a or 2b and
could be used for
a large variety of conjugation
reactions.[8Ϫ10,12,13] Here we wish to outline experiments
with another class of soft electrophiles, which eventually re-
sulted in a new conjugation method.
Scheme 2
Repeating work by Schick and Hilgetag,[16] we obtained
8 from 4 and thiouronium acetate 7 in 80% yield. Under
the same conditions, moenomycin model compound 9 was
converted into alkylation product 10 in 71% yield. Thio-
uronium acetate 7 was prepared from p-methoxyaceto-
phenone (6) (see Scheme 3) as reported previously.[16]
Since the olefinic methyl group (see Formula 7) was not
essential for our purposes, and since we had found in the
alkylation experiments that, instead of the thiouronium
acetates used in all previous reports, the corresponding
bromides could also be employed, we prepared thiouronium
Scheme 1
Results and Discussion
In the course of synthetic work in the steroid field, Kuo, bromide 13 from (E)-p-methoxycinnamic acid (11) as sum-
Taub, and Wendler reported in 1965 that thiouronium salt marized in Scheme 4. The yields in the individual steps were
3, on treatment with 2-methylcyclopentane-1,3-dione (4) in very satisfying and in our hands appreciably higher than
1164
Eur. J. Org. Chem. 2002, 1163Ϫ1174

Introduction of Reporter Groups into Moenomycin A
FULL PAPER
Scheme 5
The structure of 14b was determined with the aid of 2D
NMR experiments. Thus, the diastereotopic aliphatic CH2-
1Ј protons (broad multiplet at δ ϭ 2.60Ϫ2.69) were as-
1
signed by their correlation with 2Ј-H (1H, H COSY). The
corresponding carbon C-1Ј signal at δ ϭ 36.89 was assigned
on the basis of long-range couplings with 2Ј-H and 3Ј-H
(HMBC). The CH2-1Ј protons were found to have long-
range couplings to C-1A, C-2Ј, C-3Ј, and to the two car-
bonyl carbon atoms C-2A and C-5A. The ESI ICR, FAB,
and MALDI TOF mass spectra displayed the expected mo-
lecular ion peaks. The structure of 14a was accordingly de-
duced.
In order to find out ways in which the thiouronium salts
could be modified to allow the attachment of reporter
groups, we prepared thiouronium salts 16, 17b, and 18b as
depicted in Scheme 6. None of these salts reacted with mo-
enomycin A in aqueous, acetate-buffered solution at 60 °C.
The p-methoxy-substituted benzylthiouronium chloride 17d
did react with 1 to give the expected conjugate (formula
not shown), but only very slowly (144 h at 75 °C) and with
insufficient yields (7%). This implied that the conjugated
olefinic double bond and also an alkoxy substituent in the
aromatic ring should be present in thiouronium salt re-
agents suitable for conjugation reactions with 1.
Scheme 3. a) refs.[16,19]; b) water/toluene, 60 °C, 80%; c) water/tolu-
ene, 60 °C, 71%
those found in the synthesis of 7. Furthermore, bromide 13
crystallized very nicely from acetonitrile solution.
Scheme 4. a) refs.[18,19]; b) PBr3, Ϫ10 °C; c) SC(NH2)2, acetonitrile
Compounds 7 and 13 both reacted readily with moeno-
mycin A in aqueous solution buffered with sodium acetate
to give alkylation products 14a and 14b, respectively, in
reasonable yields (57% and 55%, Scheme 5).[20]
Scheme 6. a) refs.[10,17]; b) SC(NH2)2, EtOH, 70%; c) SOCl2, THF;
d) SC(NH2)2, acetonitrile; e) SC(NH2)2, acetonitrile, 88%
Eur. J. Org. Chem. 2002, 1163Ϫ1174
1165

A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S. Giesa, P. Welzel
1
FULL PAPER
We therefore prepared the aminoethoxy-functionalized CD3OD) had been stored in a refrigerator for 10 d, the H
cinnamyl thiouronium bromide 21b via 21a, as summarized NMR spectrum showed the presence of a mixture of two
in Scheme 7. The only remarkable step in this sequence was compounds, consisting of 14b and a new product with sig-
the reduction of 20a to give 20b, which worked well under nals at δ ϭ 4.15 and 4.40 and somewhat different signals
the conditions described by Sakaitani and Ohfune.[21]
for the aromatic protons 2Ј-H and 3Ј-H. The CH2-3A and
CH2-4A signals of 14b (at δ ϭ 2.78Ϫ2.88) were absent be-
cause of H/D exchange. We speculated that the cyclopen-
tanedione ring in the second compound had been cleaved
by a retro-Claisen-type reaction. This assumption was
shown to be correct by performing the treatment of thio-
uronium salt 21b with 1 at different pH values. At a pH of
approximately 6.4, formation only of 22b was observed by
TLC (RP18, Rf ϭ 0.26). After adjustment of the pH to 7.7
and further stirring at 60 °C for 25 h, however, a new prod-
uct Ϫ 23b (TLC, RP18, Rf ϭ 0.37) Ϫ was obtained in 32%
yield (based on 1 consumed). NMR and MS analysis of
this product showed that the cyclopentanedione ring had
been cleaved under the reaction conditions (retro-Claisen-
type reaction) and a propoxycarbonyl group had been
formed (Scheme 8). Similar results were obtained with 21a
(see Exp. Sect.). On cleavage of the cyclopentanedione ring,
a new stereogenic center at C-1Ј in 23a and 23b is formed
(the change in the numbering of the carbon atoms in these
compounds follows the IUPAC rules). Hence, compounds
23a and 23b exist as mixtures of two diastereomers that we
were not able to separate by flash chromatography (FC).
Scheme 7. a) EtOH/Hϩ; b) BrCH2CH2NHBoc, K2CO3, DMF; c)
BF3·Et2O, DIBAL-H, CH2Cl2, Ϫ78 °C, 72%; d) PBr3, CH2Cl2,
Ϫ10 °C; e) SC(NH2)2, MeCN; f) HCl/EtOH, 20 °C, 82%
1
However, in the H NMR spectra of 23a and 23b the well-
separated signals of 3Ј-H and 4Ј-H of each diastereomer
(1:1 ratio) and the signals of the CH2-2Ј protons (long-
range coupling to C-3Ј and C-4Ј) and of CH2-2A and CH2-
3A (long-range coupling to C-1A, COOHA) of each diaster-
eoisomer could be assigned for each diastereoisomer with
Treatment both of 21a and of 21b with moenomycin A
(1) in buffered aqueous solution at pH ϭ 5.7 gave the de-
sired conjugates 22a and 22b in yields of 45 and 81%, re-
spectively, based on 1 consumed (Scheme 8). The structures
were confirmed by NMR and MS as described for 14b.
1
the aid of H,1H COSY and HMBC experiments. In the
13C NMR spectra, instead of the two carbonyl carbon (C-
2A, C-5A) signals of 22a and 22b, only one carbonyl carbon
signal (C-1A) was observed in each case for compounds 23a
and 23b, together with the carboxylic group carbon signal
(COOHA). The 1Ј-H signal (broad signal in the region of
δ ϭ 4.50Ϫ4.69) could be identified because of the coupling
of this proton with 2Ј-H (1H,1H COSY). The corresponding
C-1Ј signal was identified by its coupling with 1Ј-H
(HMQC). The proton signals of the CH2-2Ј group were
1
identified by H,1H COSY (coupling with 3Ј-H), while C-
2Ј in each diastereomer showed long-range couplings with
3Ј-H and 4Ј-H (HMBC). With the help of HMQC and
HMBC experiments, practically all the carbon signals (ex-
cept some overlapping sugar carbon and C-3H, C-1AE, and
C-2A signals) in 23a and 23b were assigned. Some corres-
ponding carbon signals of each diastereomer had slightly
different chemical shifts (see Exp. Sect.).
Thus, through control of the pH of the reaction mixture
of thiouronium salt 21b and moenomycin A (1), either 22b
Ϫ with the intact cyclopentanedione ring (pH ϭ 5.74) Ϫ or
23b Ϫ with the carboxypropionyl substituent (pH Ͼ 7.0) Ϫ
could be obtained, in 81% and 37% yield, respectively
(based on 1 consumed). The same behavior on treatment
Scheme 8
In the course of the structure elucidation of 14b, we made with 1 was observed with the Boc-protected aminothio-
an interesting observation. After the NMR sample (in uronium salt 21a.
1166
Eur. J. Org. Chem. 2002, 1163Ϫ1174

Introduction of Reporter Groups into Moenomycin A
FULL PAPER
In order to demonstrate that the new amine 22b could enomycin derivative 22b was almost the same as for 2b. The
be used for the preparation of moenomycin conjugates, the Boc protection of the amino function in the moenomycin
coupling of 22b to 24[22] was studied (Scheme 9). The reac- derivatives 22a and 23a had practically no influence on the
tion was performed in methanol containing a small amount antibiotic activity.
of ethanol (to make the solution homogeneous) in the pres-
ence of Et3N at 20 °C. It was observed by TLC analysis
that two products with very similar Rf values were formed,
and these were isolated in 56% yield. As then shown by
NMR analysis, a mixture of two diastereoisomers had been
Table 1. Minimum inhibitory concentrations (MICs) of the moeno-
mycin derivatives obtained from moenomycin A by the thiouron-
ium salt method
obtained. The formation of these diastereomers can be ex-
plained as described above. In the 1H and 13C NMR spectra
of 25, almost all hydrogen and carbon signals (with the ex-
ception of some broad and overlapping carbon and proton
signals of the sugar part and units AE and DAE, COOHH)
were assigned with the aid of 2D NMR experiments, as
Compound
MIC
µmol/L
µg/mL
1[10]
2b[10]
14a
0.0069
0.15
0.69
0.28
0.27
0.58
0.13
0.15
1.50
0.029
0.27
1.21
0.49
0.50
1.08
0.24
0.28
3.5
14b
described for 23a and 23b. The CH3OA groups (two diaster- 23a
1
22a
eomers) gave rise to a broad unresolved H NMR signal
23b
(δ ϭ 3.67), while two OCH3 signals (51.48, δ ϭ 51.42, as-
22b
signed by HMQC) appeared in the 13C NMR spectrum.
25[a]
[a]
Based on one measurement.
Conclusions
New amino derivatives of moenomycin A have been pre-
pared by making use of thiouronium salt 21b. Depending
on the pH value of the reaction mixture, the conjugates had
structures of type 22b or of type 23b. In one example it was
demonstrated that a fluorescent reporter group could be in-
troduced selectively at the amino group through a squaric
acid linker. An appreciable degree of antibiotic activity was
retained in the conjugates.
Experimental Section
General: All air- or moisture-sensitive reactions were performed in
oven-dried glassware under a positive pressure of argon. Liquids
and solutions were transferred by syringe. Small-scale reactions
were performed in Wheaton serum bottles sealed with aluminium
caps with open top and Teflon-faced septum (Aldrich). ‘‘Usual
workup’’ means partitioning the reaction mixture between an aque-
ous phase and CH2Cl2, drying the combined organic solutions with
Na2SO4, and removal of solvent by distillation in a rotary evapor-
ator (bath temperature 35 °C). Solvents were purified by standard
techniques. The following materials and methods were used for
chromatographic separations: flash chromatography (FC):[23]
32Ϫ63 µm silica gel (ICN Biomedicals) and LiChroprep 40Ϫ63 µm
RP-18 material (Merck), Kieselguhr (Merck); analytical TLC:
Merck precoated 60 F254 silica gel and RP-18 F254s plates (0.2 mm),
spots were identified under a UV lamp (λ ϭ 254 nm, Camag 29
200), with a 2.22 mol/L H2SO4 solution containing Ce(SO4)2·4H2O
(10 g/L) and H3[PO4(Mo3O9)4]·H2O (25 g/L)[24] and heating at 140
°C, or with an anisaldehyde reagent for carbohydrates [2 mL of
anisaldehyde, 8 mL of conc. H2SO4 in ethanol (190 mL)].Analytical
HPLC was performed with a HPLC system (Jasco) consisting of
an intelligent PU-980 HPLC pump, a DG-980Ϫ053 line degasser,
an LG-980Ϫ02 ternary gradient unit, and an MD-910 multiwave-
length detector. As eluent, a mixture of buffer (prepared from
Scheme 9
Antibiotic Properties of the New Moenomycin Derivatives in
Comparison with 1 and 2b
All the new derivatives were antibiotically less active than
moenomycin A itself, but still showed high activities, with
MIC values in the range of 0.13 (23b) to 1.50 (25) µmol/L
(Table 1). It therefore seemed justified to use them for the
conjugation methods indicated above. The largest decrease
in antibiotic activity (ca. twentyfold) was observed on con-
version of the enolized cyclopentane-1,3-dione unit A of
moenomycin A (1) into the derivatives alkylated in the 2-
position (Table 1). The MIC value for the new amino-mo- 0.6 g KH2PO4, 26.2 g K2HPO4 ϫ 3H2O, 3.0 g 1-heptanesulfonic
Eur. J. Org. Chem. 2002, 1163Ϫ1174 1167

A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S. Giesa, P. Welzel
FULL PAPER
°
acid sodium salt monohydrate and 1000 mL of water, pH ϭ 8.0)
(0.5 mL) and toluene (0.3 mL) were stirred at 60 C for 6 h. The
and acetonitrile (60:40) was used. A 250 ϫ 4.6 mm, Nucleosil 300, solvents were removed under reduced pressure and the residue was
C18, 5 µm column (Jasco) with a pre-column was used. Ultrafiltra- purified by FC (toluene/ethyl acetate/methanol, 10:5:2). The crude
tion (UF) was performed in gas-pressurized (N2, 3.5 bar) stirred product was further purified by FC (toluene/ethyl acetate, 2:1, Rf ϭ
ultrafiltration cells (Amicon, model 8050, 50-mL cell capacity and 0.19) to give 22 mg (71%) of pure 10 as an oil. Rf ϭ 0.58 (toluene/
˜
model 8400, 400-mL capacity) with YM3 membrane type (Amicon, ethyl acetate/methanol, 10:5:2). IR (KBr): ν ϭ 3432, 3274, 1729,
molecular weight cut-off 3000 Da). NMR and MS equipment:
1629, 1511, 1288, 1245 cmϪ1. UV (methanol): λmax (ε) ϭ 256 nm
NMR: DRX 400 (Bruker), DRX 600 (Bruker), GEMINI 200 (15294). 1H NMR (200 MHz, CD3OD): δ ϭ 1.93 (d, J4Ј-2Ј
(Varian), GEMINI 2000 (Varian); for the description of the NMR 0.9 Hz, 3 H, CH3-4Ј), 1.95 (s, 3 H, CH3CONH), 2.62 (d, J1Ј-2Ј
ϭ
ϭ
spectra, the protons and carbon atoms are indexed according to
the indices in the formulae. Mass spectrometry: FAB MS: VG CH3O), 5.59 (t, J2Ј-1Ј ϭ 7.7 Hz, 1 H, 2Ј-H), 6.86 (d, J3-2 ϭ 7.9 Hz,
Autospec (Fisons, matrix: 3-nitrobenzyl alcohol), ESI MS: FT ICR
2 H, 3Ph-H, 5Ph-H), 7.29 (d, J2-3 ϭ 7.9 Hz, 2 H, 2Ph-H, 6Ph-H). 13
7.7 Hz, 2 H, CH2-1Ј), 2.75 (s, 4 H, CH2-4, CH2-5), 3.77 (s, 3 H,
C
MS ApeX II [Bruker Daltonics, solvent: water/acetonitrile (1:1) or NMR (50 MHz, CD3OD): δ ϭ 16.2 (C-4Ј), 20.5 (CH3CONH), 32.5
water/methanol (1:1)], MALDI TOF MS: VOYAGERTM spectro-
meter [PerSeptive Biosystems, matrix: 2,4,6-trihydroxyaceto-
phenone from water/acetonitrile (1:1) solution]. Two masses are al-
(C-1Ј), 35.7 (C-4, C-5), 55.7 (CH3O), 67.1 (C-2), 114.7 (C-3Ph, C-
5Ph), 116.8 (C-2Ј), 127.8 (C-2Ph, C-6Ph), 136.5 (C-1Ph), 141.5 (C-3Ј),
160.7 (C-4Ph), 172.6 (CH3CONH), 214.0 (C-1, C-3). C18H21NO4
ways communicated after the molecular formula; the first is that (315.36, 315.15), FAB MS: m/z ϭ 316.1 [M ϩ H]ϩ.
calculated with the International Atomic Masses, the second the
(R)-3-[(N-{1-[(E)-3-(4-Methoxyphenyl)-2-butenyl]-2,5-dioxocyclo-
pentyl}-β- -galactopyranuronamidosyl-(1Ǟ4)-2-acetamido-2,6-
dideoxy-β- -glucopyranosyl-(1Ǟ4)-[β- -glucopyranosyl-(1Ǟ6)]-2-
acetamido-2-deoxy-β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-
methyl-α- -glucopyranuronamidosyloxy)hydroxyphosphoryloxy]-2-
mono-isotopic mass. IR: Genesis FTIR (ATI Mattson). UV: DU-
650 (Beckman). Fluorescence spectra were recorded with a Fluoro-
max-2 (SPEX). The fluorescence excitation and emission were cor-
rected according to the lamp spectrum and the photomultiplier
sensitivity, respectively. The maxima of the excitation spectra indic-
ate the UV/Vis absorption maxima. The MIC (minimum inhibitory
concentration) values against seven different Staphylococcus aureus
strains (ATCC 25923, ATCC 29213, MRSA 1309, SG 511, petro-
leum etherG 18, petroleum etherG 5, KNS petroleum etherG 5)
were determined by a serial double microdilution method on micro-
titer plates (Iso-Sensitest medium, Oxoid). A series of decreasing
concentrations of the compound under investigation was prepared
in the medium. For inoculations, 1 ϫ 105 cfu mLϪ1 were used. The
MICs were determined (absence of visible turbidity) after 24 h at
37 °C. The MIC values were calculated as the average values from
three measurements.
D
D
D
D
D
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nona-
decatetraenyloxy)]propionic Acid (14a): Moenomycin A (1) (99 mg,
0.06 mmol) and 7 (28 mg, 0.09 mmol) were stirred under argon in
water (1.5 mL) at 60 °C. Progress of the reaction was monitored by
TLC (1-propanol/H2O, 7:2). After 8 h, the TLC spot of 14a ap-
peared. Even after 36 h, however, the reaction was not complete.
More thiouronium salt 7 (18 mg, total 46 mg, 0.15 mmol) and
water (1 mL, total 2.5 mL) were added, and heating and stirring
were continued for 49 h. Even then, moenomycin A was not com-
pletely consumed. Heating was stopped and the reaction mixture
was allowed to cool to ambient temperature. The excess of thiou-
ronium salt and thiourea formed in the reaction were then removed
by ultrafiltration (4 ϫ 50 mL). After freeze-drying, 108 mg of a
solid product was obtained, and this was purified twice by FC (1-
propanol/H2O, 5:1) to give 57 mg (55%) of pure 14a, with 17 mg
of 1 recovered. Rf ϭ 0.57 (1-propanol/H2O, 7:2). UV: λmax ϭ 257
(ε ϭ 10815, methanol), 256 (methanol ϩ HCl), 253 (methanol ϩ
NaOH), for 1: λmax 258 nm (ε ϭ 17305, methanol), 242 (methanol
ϩ HCl), 258 (methanol ϩ NaOH).[25] 1H NMR (600 MHz,
CD3OD, HMQC): δ ϭ 0.99 (s, 6 H, CH3-23I, CH3-24I), 1.26 (s, 3
H, CH3F), 1.39 (m, 2 H, CH2-9I), 1.42 (d, 3 H, CH3-6C, J ϭ
4.7 Hz), 1.61 (s, 3 H, CH3-20I), 1.62 (s, 3 H, CH3-21I), 1.68 (s, 3
H, CH3-19I), 1.78 (s, 3 H, CH3-25I), 1.92 (m, 2 H, CH2-10I), 1.96
(s, 6 H, CH3-4Ј, CH3CONHE or CH3CONHC), 2.00Ϫ2.20 (m, 11
H, CH2-4I, CH2-5I, CH2-15I, CH2-16I and 2.03 (s, CH3CONHC or
CH3CONHE), 2.63Ϫ2.86 (m, 8 H, CH2-1Ј, CH2-3A, CH2-4A and
2.70 (d, J12-13 ϭ 7.3 Hz, CH2-12I)], 3.20Ϫ4.60 [broad signals of the
sugar protons with 3.80 (s, 3 H, OCH3), 4.09 (5B-H, 4B-H), 4.46
(d, J ϭ 6.8 Hz, 1D-H)], 4.68 (d, J ϭ 6.8 Hz, 2 H, CH-22I), 5.11 (bt,
J17-16 ϭ 6.3 Hz, 2 H, 17I-H, and 3F-H), 5.15 (t, J13-12 ϭ 7.3 Hz, 1
H, 13I-H), 5.33 (broad signal, 1 H, 6I-H), 5.39 (d, J7-6 ϭ 15.7 Hz,
1 H, 7I-H), 5.44 (broad signal, 1 H, 2I-H), 5.65 (t, J2Ј-1Ј ϭ 7.9 Hz,
1 H, 2Ј-H), 5.91 (broad signal, 1 H, 1F-H), 6.88 (d, J3-2 ϭ 8.4 Hz,
2-[(E)-3-(4-Methoxyphenyl)-2-butenyl]thiouronium Acetate (7):
Compound 7 was prepared as described in refs.[16,19]
. M.p.
141Ϫ142 °C (ref.[16] 145Ϫ147 °C). IR (KBr): ν˜ ϭ 3550, 1633, 1604,
1569, 1511, 1409, 1249, 1178, 829 cmϪ1. UV (methanol): λmax (ε) ϭ
240 (39312), 308 nm (6960). 1H NMR (200 MHz, CD3OD): δ ϭ
1.91 (s, 3 H, CH3COOϪ), 2.14 (s, long range coupling, J4Ј-1Ј
ϭ
0.4 Hz, J4Ј-2Ј ϭ 1.3 Hz, 3 H, CH3-4Ј), 3.80 (s, 3 H, CH3O), 4.04 (d,
J1Ј-2Ј ϭ 7.7 Hz, 2 H, CH2-1Ј), 5.81 (tq, J2Ј-1Ј ϭ 7.7 Hz, J2Ј-4Ј
ϭ
1.3 Hz, 1 H, 2Ј-H), 6.89 (J3-2 ϭ 9.2 Hz, 2 H, 3Ph-H, 5Ph-H), 7.37
(J2-3 ϭ 9.2 Hz, 2 H, 2Ph-H, 6Ph-H). 13C NMR (50 MHz, CD3OD):
δ ϭ 16.4 (C-4Ј), 31.3 (C-1Ј), 56.0 (CH3O), 115.1 (C-3Ph, C-5Ph),
117.5 (C-2Ј), 128.3 (C-2Ph, C-6Ph), 136.01 (C-1Ph), 143.7 (C-3Ј),
161.3 (C-4Ph). C12H16N2OS (base, 236.33, 236.09), FAB MS: m/z ϭ
237.1 [M ϩ H]ϩ, 161.1 [M Ϫ SC(NH2)2 ϩ H]ϩ.
2-[(E)-3-(4-Methoxyphenyl)but-2-enyl]-2-methylcyclopentane-1,3-
dione (8):[16,19] Rf ϭ 0.93 (toluene/ethyl acetate/methanol, 10:5:2).
1H NMR (200 MHz, CD3OD): δ ϭ 1.13 (s, 3 H, 2-CH3), 1.96 (d,
J4Ј-2Ј ϭ 1.2 Hz, 3 H, CH3-4Ј), 2.50 (d, J1Ј-2Ј ϭ 8.0 Hz, 2 H, CH2-
1Ј), 2.74 (m, 4 H, CH2-4, CH2-5), 3.78 (s, 3 H, CH3O), 5.49 (tq,
J2Ј-1Ј ϭ 8.0 Hz, J2Ј-4Ј ϭ 1.2 Hz, 1 H, 2Ј-H), 6.85 (J3-2 ϭ 8.8 Hz, 2
H, 3Ph-H, 5Ph-H), 7.26 (J2-3 ϭ 8.8 Hz, 2 H, 2Ph-H, 6Ph-H). 13C
NMR (50 MHz, CD3OD): δ ϭ 16.2 (2-CH3), 18.4 (C-4Ј), 36.2 (C-
4, C-5), 36.3 (C-1Ј), 55.7 (CH3O), 58.1 (C-2), 114.6 (C-3Ph, C-5Ph),
120.2 (C-2Ј), 127.8 (C-2Ph, C-6Ph), 137.1 (C-1Ph), 139.8 (C-3Ј), 160.4
(C-4Ph), 218.7 (C-1, C-3).
2 H, 3Ph-H, 5Ph-H), 7.35 (d, J2-3 ϭ 8.4 Hz, 2 H, 2Ph-H, 6Ph-H). 13
C
NMR (150 MHz, CD3OD, HMQC): δ ϭ 16.2 (C-21I), 16.3 (C-4Ј),
16.5 (CH3F), 17.8 (C-6C, C-20I), 23.4 (CH3CONHC and
CH3CONHE), 24.2 (C-25I), 25.9 (C-19I), 27.7 (C-16I), 27.9 (C-23I,
C-24I), 32.4 (C-10I), 32.8 (C-5I), 33.2 (C-1Ј), 33.5 (C-4I), 35.8, 35.9
2-Acetamido-2-[(E)-3-(4-Methoxyphenyl)but-2-enyl]cyclopentane- (C-3A, C-4A), 35.9 (C-12I), 36.5 (C-8I), 40.9 (C-15I), 42.9 (C-9I),
1,3-dione (10): 2-Acetaminocyclopentane-1,3-dione (9)[14] (15.5 mg, 55.8 (OCH3), 56.2 (C-2E), 56.7 (C-2C), 62.9 (C-6D), 67.1 (C-1I or
0.1 mmol) and thiouronium salt 7 (29.6 mg, 0.1 mmol) in water
C-1A), 69.9 (broad, C-3 H), 70.5 (C-4B), [72.0, 72.3, 72.6, 73.6, 73.7,
1168
Eur. J. Org. Chem. 2002, 1163Ϫ1174

Introduction of Reporter Groups into Moenomycin A
FULL PAPER
74.1, 74.2, 74.3, 75.1, 75.3 (C-5B), 76.3 (broad), 78.0, 78.8 (broad),
extraction with diethyl ether. The ethereal solution was washed
82.5 (broad), (unassigned signals of the sugar carbon atoms)], 84.7 with water and dried with sodium sulfate. After filtration and solv-
(C-4C), 95.9 (C-1F), 103.4, 103.9, 104.4, 104.6 (C-1C, C-1E, C-1D, ent removal, 0.45 g of a white solid was obtained. The solid was
C-1B), 109.3 (C-22I), 114.7 (C-3Ph, C-5Ph), 116.9 (C-2Ј), 123.5 (C- dissolved in acetonitrile (2 mL), and a solution of thiourea (0.1 g,
13I), 125.4 (C-17I), 126.9 (C-6I), 127.9 (C-2Ph, C-6Ph), 132.2 (C- 1.3 mmol) in acetonitrile (2 mL) was added. After 1Ϫ2 min, a solid
18I), 136.6 (C-3Ј), 137.3 (C-14I), 141.5 (C-3I), 141.6 (C-7I), 151.1 precipitated from the clear solution. The precipitate was collected
(C-11I), 159.2 (OCONH2F), 160.6 (C-4Ph), 170.5 (CONHB), 173.7 by filtration, washed with acetonitrile and dried in vacuum to pro-
(CONH2F), 174.1, 174.3 (CH3CONHC and CH3CONHE), 212.9, vide 0.338 g (85% based on thiourea) of pure 12. M.p. 148 °C (de-
213.8 (C-2A, C-5A). C80H120N5O35P (1742.81, 1741.75), ESI ICR comp., acetonitrile). IR (KBr): ν ϭ 3600Ϫ3200 (broad, NH, NH2),
˜
MS: m/z ϭ 1740.74642 (calcd. 1740.74225) [M Ϫ H]Ϫ, 869.86951
(calcd. 869.86780) [M Ϫ 2 H]2Ϫ
3200Ϫ3000 (C-HAr), 1637, 1511, 1438, 1250, 1175, 609 cmϪ1. UV
(methanol): λmax (ε) ϭ 230 (20424), 274 (2763), 283 (s) nm (2218).
1H NMR (200 MHz, CD3OD): δ ϭ 3.78 (s, 3 H, OCH3), 4.00 (dd,
J1Ј-2Ј ϭ 7.3 Hz, J1Ј-3Ј ϭ 1.1 Hz, 2 H, 1Ј-H) 6.12 (dt, J2Ј-3Ј ϭ 15.8 Hz,
J2Ј-1Ј ϭ 7.3 Hz, 1 H, 2Ј-H), 6.70 (d, J3Ј-2Ј ϭ 15.8 Hz, 1 H, 3Ј-H),
6.89 (J3-2 ϭ 8.8 Hz, 2 H, 3Ph-H, 5Ph-H), 7.35 (J2-3 ϭ 8.8 Hz, 2 H,
2Ph-H, 6Ph-H). 13C NMR (50 Hz, CD3OD, APT): δ ϭ 35.0 (C-1Ј),
55.8 (OCH3), 115.2 (C-3Ph, C-5Ph), 119.7 (C-2Ј), 129.1 (C-2Ph, C-
6Ph), 130.0 (C-1Ph), 136.5 (C-3Ј), 161.5 (C-4Ph), 172.4
[SC(NH)NH2). C11H14N2OS (base, 222.30, 222.08), FAB MS:
m/z ϭ 223.1 [M ϩ H]ϩ, 147.1 [M Ϫ SC(NH2)2 ϩH]ϩ.
.
2-[(E)-Cinnamyl]thiouronium Bromide (18b):[26,19] M.p. 195Ϫ197 °C
(ref.[26] 196Ϫ197 °C). IR (KBr): ν ϭ 3320Ϫ3370, 3120Ϫ3270, 1636,
˜
1440, 986, 747, 688, 622, 469 cmϪ1. UV (methanol): λmax (ε) ϭ
1
254 nm (24756). H NMR (200 MHz, [D6]DMSO/D2O, 10:1): δ ϭ
3.98 (d, J1Ј-2Ј ϭ 7.0, Hz 2 H, CH2-1Ј), 6.25 (dt, J2Ј-1Ј ϭ 7.0 Hz,
J2Ј-3Ј ϭ 15.7 Hz, 1 H, 2Ј-H), 6.69 (d, J3Ј-2Ј ϭ 15.7 Hz, 1 H, 3Ј-H),
7.23Ϫ7.44 (m, 5 H, 2-H, 3-H, 4-H, 5-H, 6-H). 13C NMR (50 MHz,
[D6]DMSO/D2O, 10:1): δ ϭ 33.7 (C-1Ј), 123.1 (C-2Ј), 127.4 (C-2,
C-6), 129.3 (C-4), 129.8 (C-3, C-5), 135.2, 136.6 (C-1, C-3Ј), 170.1
[SC(NH)NH2). C10H12N2S (base, 192.27, 192.07), FAB MS: m/z ϭ
193.0 [M ϩ H]ϩ, 117.1 [M Ϫ SC(NH2)2 ϩ H]ϩ.
(R)-3-[(N-{1-[(E)-3-(4-Methoxyphenyl)propenyl]-2,5-dioxocyclo-
pentyl}-β-
dideoxy-β-
acetamido-2-deoxy-β-
methyl-α- -glucopyranuronamidosyloxy)hydroxyphosphoryloxy]-2-
D
-galactopyranuronamidosyl-(1Ǟ4)-2-acetamido-2,6-
-glucopyranosyl-(1Ǟ4)-[β- -glucopyranosyl-(1Ǟ6)]-2-
-glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-
D
D
2-(Benzyl)thiouronium Chloride (17b):[27,19] M.p. 145 °C (aq. HCl)
(ref.[27] 148 °C). IR (KBr): ν˜ ϭ 3400 (broad), 3070Ϫ3200, 1640,
1420, 760, 703, 677, 568 cmϪ1. UV (methanol): λmax (ε) ϭ 209 nm
D
D
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nona-
decatetraenyloxy]propionic Acid (14b): A solution of moenomycin
A (1, 50 mg, 0.032 mmol), sodium acetate (9 mg), and thiouronium
bromide (13, 14.3 mg, 0.048 mmol) in water (1.5 mL) was stirred
under argon at 60 °C for 30 h. Progress of the reaction was mon-
itored by TLC (1-propanol/H2O, 7:2) and HPLC (Rt ϭ 29.15 min,
Rtmoenomycin A ϭ 13.49 min, flow 0.5 mL/min). Since the reaction
was not complete, more 13 (9.6 mg, 0.032 mmol), sodium acetate
(7 mg), and water (0.5 mL) were added and the reaction mixture
was stirred at 60 °C for an additional 30 h (together 60 h). Water
was then removed by lyophilization, and the crude product was
purified twice by FC (1-propanol/H2O, 6:1). Solvent evaporation
under reduced pressure and water lyophilization gave 31.5 mg
(57%) of pure (HPLC) 14b. Rf ϭ 0.50 (1-propanol/H2O, 7:2). UV
(methanol): λmax (ε) ϭ 264 nm (27970). 1H NMR (600 MHz,
1
(14864). H NMR (200 MHz, D2O): δ ϭ 4.35 (s, 2 H, -CH2ϪS-),
7.33Ϫ7.44 (m, 5 H, 2-H, 3-H, 4-H, 5-H, 6-H). 13C NMR (50 MHz,
D2O): δ ϭ 35.5 (ϪCH2ϪS), 128.9 (C-4), 129.4, 129.6 (C-3 and C-5,
C-2 and C-6), 134.5 (C-1), 171.0 [SC(NH)NH2). C8H10N2S (base,
166.24, 166.06), FAB MS: m/z
ϭ 167.0 [M ϩ
H]ϩ, 91.1
[C6H5CH2]ϩ.
2-[(E)-3-Phenyl-2-butenyl]thiouronium Acetate (16): Compound 16
was prepared from acetophenone (15) as described for 7. M.p.
163Ϫ164 °C (water). IR (KBr): ν˜ ϭ 3385Ϫ3275, 3177Ϫ3100, 1613,
1470, 1414, 1084, 729 cmϪ1. UV (methanol): λmax (ε) ϭ 241 nm
(29303). 1H NMR (200 MHz, [D6]DMSO): δ ϭ 1.79 (s, 3 H,
CH3COOϪ), 2.05 (s, 3 H, CH3-4Ј), 3.77 (d, J1Ј-2Ј ϭ 8.0 Hz, 2 H,
CH2-1Ј), 5.85 (t, J2Ј-1Ј ϭ 8.0 Hz, 1 H, 2Ј-H), 7.05 (broad s, NH,
NH2), 7.20Ϫ7.40 (m, 5 H, 2Ph-H, 3Ph-H, 4Ph-H, 5Ph-H, 6Ph-H). H
1
NMR (200 MHz, CD3OD): δ ϭ 1.91 (s, 3 H, CH3COOϪ), 2.17
(long range coupling, J4Ј-2Ј ϭ 1.4 Hz, J4Ј-1Ј ϭ 0.7 Hz, 3 H, CH3-
4Ј), 4.06 (d, J1Ј-2Ј ϭ 7.8 Hz, 2 H, CH2-1Ј), 5.87 (td, J2Ј-1Ј ϭ 7.8 Hz,
J1Ј-4Ј ϭ 0.7 Hz, 1 H, 2Ј-H), 7.13 (broad s, NH, NH2), 7.29Ϫ7.45
(m, 5 H, 2Ph-H, 3Ph-H, 4Ph-H, 5Ph-H, 6Ph-H). 13C NMR (50 MHz,
1
CD3OD, H,1H COSY, HMQC, HMBC): δ ϭ 0.98 (s, 6 H, CH3-
23I, CH3-24I), 1.27 (s, 3 H, CH3F), 1.38 (m, 2 H, CH2-9I), 1.42 (d,
J ϭ 5.8 Hz, 3 H, CH3-6C), 1.61 (s, 3 H, CH3-20I), 1.62 (s, 3 H,
CH3-21I), 1.68 (s, 3 H, CH3-19I), 1.77 (s, 3 H, CH3-25I), 1.91 (s),
1.91 (m, 2 H, CH2-10I), 1.94 (s, 3 H, CH3CONHC or CH3CONHE),
2.00Ϫ2.20 (m, 11 H, CH2-4I, CH2-5I, CH2-15I, 16I and 2.07 (s,
CH3CONHE or CH3CONHC), 2.60Ϫ2.69 (m, 2 H, CH2-1Ј), 2.70
(d, J12-13 ϭ 7.3 Hz, 2 H, CH2-12I), 2.78Ϫ2.88 (m, 2 H, CH2-3A,
CH2-4A), 3.40Ϫ4.65 (broad signals of the sugar protons with 3.81
(s, 3 H, OCH3), 4.68 (d, J ϭ 8.9 Hz, CH2-22I), 5.11 (broad t,
1
CD3OD, H,13C COSY): δ ϭ 16.3 (C-4Ј), 24.1 (CH3COOϪ), 30.9
(C-1Ј), 119.1 (C-2Ј), 126.8 (C-3Ph, C-5Ph), 128.8 (C-1Ph), 129.4 (C-
2Ph, C-6Ph), 143.3, 143.9 (C-3Ј, C-4Ph), 172.4 [SC(NH)NH2), 185.2
(CH3COOϪ). C11H14N2S (base, 206.30, 206.09), FAB MS: m/z ϭ
230.0 [M ϩ Na]ϩ, 207.1 [M ϩ H]ϩ.
J
17-16 ϭ 5.5 Hz, 2 H, 17I-H and 5.12 (3F-H), 5.14 (t, J13-12 ϭ 7.3 Hz,
Treatment of Moenomycin A with 16 and 17b: Mixtures of moeno-
mycin A (30 mg, 0.019 mmol) and thiouronium salts 16 and 17b
(each 2.5 equiv.) in water (5 mL) were stirred under argon at 60
°C . TLC monitoring (1-propanol/H2O, 7:2) indicated no product
formation even after 24 h.
1 H, 13I-H), 5.31 (dt, J6-7 ϭ 15.2 Hz, J6-5 ϭ 6.7 Hz, 1 H, 6I-H),
5.39 (d, J7-6 ϭ 15.2 Hz, 1 H, 7I-H), 5.45 (broad signal, 1 H, 2I-H),
5.89 (broad signal, 1 H, 1F-H), 6.04 (dt, J2Ј-3Ј ϭ 15.7 Hz, J2Ј-1Ј
ϭ
7.6 Hz, 1 H, 2Ј-H), 6.46 (d, J3Ј-2Ј ϭ 15.7 Hz, 1 H, 3Ј-H), 6.89 (d,
J
3-2 ϭ 8.9 Hz, 2 H, 3Ph-H, 5Ph-H), 7.35 (d, J2-3 ϭ 8.9 Hz, 2 H, 2Ph
-
2-[(E)-4-Methoxycinnamyl]thiouronium Bromide (13): A solution of
PBr3 (0.218 g, 0.075 mL, 0.8 mmol) in dry CH2Cl2 (1 mL) was
H, 6Ph-H). 13C NMR (150 MHz, CD3OD, APT, HMQC, HMBC):
δ ϭ 16.2 (C-21I), 16.4 (CH3F), 17.8 (C-6C), 17.9 (C-20I), 23.5
slowly added under an argon and at Ϫ10 °C to a stirred solution (CH3CONHC, CH3CONHE), 24.1 (C-25I), 26.0 (C-19I), 27.7 (C-
of 4-methoxycinnamyl alcohol[18] (0.36 g, 2.2 mmol) in dry CH2Cl2
(4 mL). The reaction mixture was then stirred for an additional (broad, C-3A, C-4A), 35.9 (C-12I), 36.5 (C-8I), 36.9 (C-1Ј), 40.8 (C-
20 min and quenched with a saturated aqueous solution of
15I), 42.9 (C-9I), 55.9 (OCH3), 56.1, 56.7 (C-2E, C-2C), 62.0*, 62.7
NaHCO3. The product was separated from the aqueous layer by (OCH2D), 66.9* (C-1I), 68.2 (C-1A), 70.4, 71.3*, 71.6, 72.3, 72.5 (C-
16I), 27.9 (C-23I, C-24I), 32.3 (C-10I), 32.7 (C-5I), 33.5 (C-4I), 34.8
Eur. J. Org. Chem. 2002, 1163Ϫ1174
1169

A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S. Giesa, P. Welzel
FULL PAPER
5C), 73.5*, 73.8, 73.9, 74.1, 74.2*, 74.3 (C-5F), 75.0, 75.3, 76.1 (C-
J ϭ 7.8 Hz, 2 H, CH2-22I), 5.11 (t, J17-16 ϭ 7.1 Hz, 1 H, 17I-H),
3F), 77.8, 78.0, 78.6, 80.9 (broad, C-2 H), 82.2, 84.7 (C-4C, C-4E), 5.16 (t, J13-12 ϭ 7.3 Hz, 1 H, 13I-H), 5.33 (m, 1 H, 6I-H), 5.39 (d,
95.8 (C-1F), 103.3 (C-1C), 103.9 (C-1E), 104.3, 104.5 (C-1D, C-1B), J7-6 ϭ 15.1 Hz, 7I-H), 5.45 (broad signal, 1 H, 2I-H), 5.89 (broad
109.3 (C-22I), 115.0 (C-3Ph, C-5Ph), 118.9 (C-2Ј), 123.1 (broad, C-
2I), 123.5 (C-17I), 125.3 (C-13I), 126.9 (C-6I), 128.6, 128.8 (C-2Ph
signal, 1 H, 1F-H), 6.88 (d, J3-2 ϭ 8.9 Hz, 2 H, 3Bz-H, 5Bz-H), 7.02
(d, J2-3 ϭ 8.9 Hz, 2 H, 2Bz-H, 6Bz-H). 13C NMR (600 MHz,
,
C-6Ph), 130.8 (C-1Ph), 132.3 (C-18I), 136.5 (C-3Ј), 137.4 (C-14I), CD3OD, APT, HMQC): δ ϭ 16.2 (C-21I), 16.5 (CH3F), 17.8 (C-
141.6 (C-7I), 151.1 (C-11I), 159.2 (OCONH2F), 160.9 (C-4Ph), 170.5 6C)*, 17.8 (C-20I), 23.4, 23.4 (CH3CONHC, CH3CONHE)*, 24.1
(CONHB), 173.9, 173.9 (CH3CONHC or CH3CONHE, CONH2F),
(C-25I)*, 25.9 (C-19I), 27.7 (C-16I), 27.9 (C-23I, C-24I), 32.4 (C-
174.4 (CH3CONHE or CH3CONHC), 177.8 (CO2HH), 180.6, 212.8, 10I), 32.8, 33.6 (C-4I, C-5I), 33.0 (C-3A, C-4A), 35.9 (C-12I), 36.5
213.7 (C-2A, C-5A). * Signals of secondary or quaternary carbon (C-8I), 40.1 (CH2Bz)*, 40.9 (C-15I), 42.9 (C-9I), 55.7 (OCH3), 62.1
atoms (APT) in the δ ϭ 60Ϫ80 range. C79H118N5O35P (1728.79, (C-6D), 67.6 (C-1I or C-3 H), 70.0Ϫ80.0 (broad signals of the sugar
1727.73), ESI ICR MS: m/z ϭ 1748.72412 (calcd. 1748.70870) [M
carbon atoms), 109.3 (C-22I), 115.3 (C-3Bz, C-5Bz), 123.5 (C-13I),
125.4 (C-17I), 126.9 (C-6I), 132.2 (C-18I), 132.4 (C-2Bz, C-6Bz),
ϩ Na Ϫ 2H]Ϫ, 1726.74254 (calcd. 1726.72660) [M Ϫ H]Ϫ,
862.85829 (calcd. 862.85935) [M Ϫ 2H]2Ϫ. FAB MS: m/z ϭ 1788.5 137.4 (C-14I), 141.6 (C-7I), 151.1 (C-11I), 160.9 (OCONH2F). *
[M ϩ Na ϩ K Ϫ H]ϩ, 1772.5 [M ϩ 2Na Ϫ H]ϩ, 1766.5 [M ϩ Chemical shifts and assignments were obtained from the HMQC
K]ϩ, 1750.5 [M ϩ Na]ϩ. MALDI TOF MS: m/z ϭ 1748.3 [M Ϫ 2 spectrum. C77H116N5O35P (1702.75, 1701.72), ESI ICR MS: m/z ϭ
H ϩ Na]Ϫ, 1726.4 [M Ϫ H]Ϫ.
1700.73109 (calcd. 1700.71095) [M Ϫ H]Ϫ, 849.85112 (calcd.
849.85152) [M Ϫ 2H]2Ϫ. FAB MS: m/z ϭ 1740.6 [M ϩ K]ϩ, 1724.6
[M ϩ Na]ϩ.
2-(4-Methoxybenzyl)thiouronium Chloride (17d): The crude benzyl
chloride 17c[28] was converted into 17d as described for 13. Drying
in vacuum gave 0.674 g (67% based on thiourea) of pure 17d. M.p.
Ethyl (E)-4-Hydroxycinnamate:[19,30] Rf ϭ 0.22 (CHCl3). IR (KBr):
165Ϫ166 °C (acetonitrile) (ref.[29] 161Ϫ163 °C). IR (KBr): ν ϭ ν ϭ 3286, 1677, 1631, 1598, 1513, 1442, 1371, 1319, 1276, 1195,
3247, 3077, 2967, 1646, 1511, 1430, 1303, 1245, 1180, 1025, 840, 1033, 977, 831, 518 cmϪ1. UV (methanol): λmax (ε) ϭ 227 (32090),
˜
˜
1
728, 703 cmϪ1. UV (methanol): λmax (ε) ϭ 230 (18471). H NMR 301 (s) (52769), 310 nm (56851). 1H NMR (200 MHz, CDCl3): δ ϭ
(200 MHz, CD3OD): δ ϭ 3.78 (s, 3 H, OCH3), 4.40 (s, 2 H, 1.34 (t, J ϭ 7.1 Hz, 3 H, OCH2CH3), 4.26 (q, J ϭ 7.1 Hz, 2 H,
ϪCH2ϪSϪ), 6.91 (J3-2 ϭ 8.8 Hz, 2 H, 3-H, 5-H), 7.34 (J2-3
ϭ
OCH2CH), 6.29 (d, J2Ј-3Ј ϭ 15.9 Hz, 1 H, 2Ј-H), 6.86 (J3-2 ϭ
8.8 Hz, 2 H, 2-H, 6-H). 13C NMR (50 MHz, CD3OD): δ ϭ 36.0 8.7 Hz, 2 H, 3-H, 5-H), 7.40 (J2-3 ϭ 8.7 Hz, 2 H, 2-H, 6-H,), 7.63
(ϪCH2ϪSϪ), 55.8 (OCH3), 115.5 (C-3, C-5), 126.7 (C-1), 131.6 (d, J3Ј-2Ј ϭ 15.9 Hz, 1 H, 3Ј-H). 13C NMR (50 MHz, CDCl3): δ ϭ
(C-2, C-6), 161.4 (C-4), 172.4 [SC(NH)NH2]. C9H12N2OS (base, 14.4 (OCH2CH3), 60.8 (OCH2CH3), 115.3 (C-2Ј), 116.1 (C-3, C-5),
196.26, 196.07), FAB MS: m/z ϭ 197.1 [M ϩ H]ϩ, 121.1 [M Ϫ 126.9 (C-1), 130.1 (C-2, C-6), 145.0 (C-3Ј), 158.3 (C-4), 168.2 (C-
SC(NH2)2 ϩ H]ϩ.
1Ј). C11H12O3 (192.21, 192.08), FAB MS: m/z ϭ 193.1 [M ϩ H]ϩ.
(R)-3-({N-[1-(4-Methoxybenzyl)-2,5-dioxocyclopentyl]-β-
D
-galacto-
-gluco-
-glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy- 5.2 mmol) in DMF (10 mL) was added over 25 min to a suspension
Ethyl (E)-4-[2-(tert-Butoxycarbonylamino)ethoxy]cinnamate (20a):
pyranuronamidosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β-
pyranosyl-(1Ǟ4)-[β-
β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α-
ranuronamidosyloxy}hydroxyphosphoryloxy)-2-[(2Z,6E,13E)-
D
A
solution of tert-butyl 2-bromoethylcarbamate[31] (1.17 g,
D
D
D
-glucopy-
of ethyl 4-hydroxycinnamate (1.00 g, 5.2 mmol) and K2CO3 (0.72 g,
5.2 mmol) in DMF (10 mL). The mixture was stirred at 20 °C for
3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nona- 17 h, but no product formation was observed [TLC, CHCl3/petro-
decatetraenyloxy]propionic Acid (Formula Not Shown): A solution
of moenomycin A (1, 50 mg, 0.032 mmol), sodium acetate (8 mg),
and thiouronium salt 17d (12.8 mg, 0.054 mmol) in water (1.5 mL)
was stirred under argon at 60 °C for 22 h. The reaction was mon-
leum ether (light)/2-propanol, 10:10:1]. More K2CO3 (0.87 mg,
6.3 mmol) was then added, and the mixture was stirred at 20 °C
for 3 d. TLC [CHCl3/petroleum ether (light)/2-propanol, 10:10:1]
indicated formation of 20a, but the reaction was still not complete.
itored by TLC (1-propanol/H2O, 7:2). No product formation was Stirring was then continued at 60 °C for an additional 90 min (until
observed. The temperature was then increased to 75 °C and the starting material could no longer be observed). The reaction mix-
mixture was stirred at this temperature for an additional 52 h. TLC ture was poured into water (170 mL) and extracted with ethyl acet-
(1-propanol/H2O, 7:2) indicated product formation only to a minor ate (3 ϫ 70 mL). The solvent was removed from the combined or-
extent. further portion of 17d (15 mg 0.064 mmol, total ganic extracts under reduced pressure, and the residue was purified
0.118 mmol, 3.7equiv.), sodium acetate (12 mg), and water (0.5 mL) by FC [CHCl3/petroleum ether (light)/2-propanol, 10:10:1] to give
were added, and the reaction mixture was stirred at 75 °C for an- a crude product that was further purified by FC (CHCl3/petroleum
other 70 h. According to TLC, moenomycin A was still present. ether/2-propanol, 10:15:1) to provide 1.7 g (97%) of pure 20a. Rf ϭ
A
The solvent was removed by lyophilization and the residue was
purified by FC (1-propanol/H2O, 7:2) to provide 4 mg (7.3%) of
0.37 [CHCl3/petroleum ether (light)/2-propanol, 10:10:1]. M.p. 59
˜
°C (after FC). IR (KBr): ν ϭ 3280, 2975, 2929, 1708, 1693, 1631,
the pure conjugate (formula not shown). Rf ϭ 0.51 (1-propanol/
1600, 1542, 1511, 1363, 1251, 1172, 829 cmϪ1. UV (methanol): λmax
1
1
H2O, 7:2). Only characteristic signals in H NMR and 13C NMR (ε) ϭ 225 (13221), 308 nm (26070). H NMR (200 MHz, CDCl3):
could be assigned, due to the lack of sufficient amounts of the
δ ϭ 1.32 (t, J ϭ 7.1 Hz, 3 H, OCH2CH3), 1.45 [s, 9 H, (CH3)3C],
compound. 1H NMR (600 MHz, CD3OD, H,1H COSY): δ ϭ 3.53 (dt, J2-1 ϭ 5.2 Hz, J2-NH ϭ 5.6 Hz, 2 H, CH2-2AE), 4.04 (t,
0.99 (s, 6 H, CH3-23I, CH3-24I), 1.27 (s, 3 H, CH3F), 1.39 (m, CH2- J1-2 ϭ 5.2 Hz, 2 H, CH2-1AE), 4.25 (q, J ϭ 7.1 Hz, 2 H,
9I), 1.44 (d, J ϭ 5.7 Hz, 3 H, CH3-6C), 1.62 (s, 3 H, CH3-20I), 1.63 OCH2CH3), 4.98 (broad s, 1 H, NH), 6.29 (d, J2Ј-3Ј ϭ 15.9 Hz, 1
(s, 3 H, CH3-21I), 1.68 (s, 3 H, CH3-19I), 1.77 (s, 3 H, CH3-25I), H, 2Ј-H), 6.88 (J3-2 ϭ 8.8 Hz, 2 H, 3Ph-H, 5Ph-H), 7.46 (J2-3
1.92 (m, CH2-10I), 1.97 (broad s, H, CH3CONHC or 8.8 Hz, 2 H, 2Ph-H, 6Ph-H), 7.63 (d, J3Ј-2Ј ϭ 15.9 Hz, 1 H, 3Ј-H).
CH3CONHE), 2.00Ϫ2.15 (m, 11 H, CH2-4I, CH2-5I, CH2-15I, 13C NMR (50 MHz, CDCl3):
14.4 (OCH2CH3), 28.5
CH2-16I, and 2.07 (broad s, CH3CONHE or CH3CONHC), 2.71 [(CH3)3C], 40.2 (C-2AE), 60.4 (OCH2CH3), 67.4 (C-1AE), 79.8
(d, J12-13 ϭ 7.3 Hz, 2 H, CH2-12I), 3.04 (s, 2 H, CH2Bz), 3.25Ϫ4.60 [(CH3)3C], 115.0 (C-3Ph, C-5Ph), 116.3 (C-2Ј), 127.8 (C-1Ph), 129.9
[broad signals of the sugar protons with 3.78 (s, OCH3)], 4.68 (d, (C-2Ph, C-6Ph), 144.3 (C-3Ј), 156.1 [OC(O)NH], 160.6 (C-4), 167.5
1
ϭ
3
δ
ϭ
1170
Eur. J. Org. Chem. 2002, 1163Ϫ1174

Introduction of Reporter Groups into Moenomycin A
FULL PAPER
(C-1Ј). C18H25NO5 (335.40, 335.17), ESI ICR MS: m/z
ϭ
(C-4Ph), 172.4 [SC(NH)NH2]. C17H25N3O3S (base, 351.46, 351.16),
693.33723 (calcd. 693.33554) [2 M ϩ Na]ϩ, 671.35511 (calcd. FAB MS: m/z ϭ 352.1 [M ϩ H]ϩ, 276.1 [M Ϫ SC(NH2)2 ϩ H]ϩ.
671.35434) [2 M ϩ H]ϩ, 571.30328 (calcd. 571.30214) [2 M Ϫ
2-[(E)-4-(2-Ammonioethoxy)cinnamyl]thiouronium Bromide Chloride
(CH3)2CCH2 Ϫ CO2 ϩ H]ϩ, 336.18102 (calcd. 336.18107) [M ϩ
(21b): A mixture of 21a (0.50 g, 1.16 mmol) in dry ethanol (15 mL)
H]ϩ, 280.11835 (calcd. 280.09127) [M Ϫ (CH3)2CCH2 Ϫ CO2 Ϫ H
and HCl in ethanol (22%, 8 mL) was stirred at 20 °C for 2 h. The
ϩ 2 Na]ϩ.
mixture was then poured into diethyl ether and the white precipit-
ate that formed was collected, washed with ether, and dried in va-
(E)-4-[2-(tert-Butoxycarbonylamino)ethoxy]cinnamyl Alcohol (20b):
˜
cuo to give 0.35 g (82%) of 21b. M.p. 194Ϫ196 °C. IR (KBr): ν ϭ
A solution of BF3·Et2O (0.2 mL, 1.6 mmol) was added over 20 min
to a cold (Ϫ78 °C) solution of 20a (0.5 g, 1.5 mmol) in dry CH2Cl2
(7 mL). A solution of DIBAL-H (1 mol/L, 4.5 mL, 4.5 mmol) in
CH2Cl2 (4.5 mL) was added at Ϫ78 °C, and the mixture was stirred
at Ϫ78 °C for 2 h. Acetic acid (0.8 mL) and water (0.6 mL) were
added to the reaction mixture. The temperature was allowed to
increase to 20 °C, and the reaction mixture was extracted with di-
ethyl ether. The ethereal extract was washed with aqueous
NaHCO3 and water, dried with Na2SO4, and filtered. The solvent
was evaporated under reduced pressure to give 0.4 g of a white
solid. Further purification by FC (cyclohexane/CH2Cl2/methanol,
10:10:1) gave 0.3 g (72%) of pure 20b. Rf ϭ 0.30 (cyclohexane/
3600Ϫ3100, 1649, 1603, 1509, 1248, 1016, 711 cmϪ1. UV (meth-
1
anol): λmax (ε) ϭ 265 nm (21583). H NMR (200 MHz, CD3OD):
δ ϭ 3.38 (t, J2-1 ϭ 4.9 Hz, 2 H, CH2-2AE), 4.02 (dd, J1Ј-2Ј ϭ 7.1 Hz,
J1Ј-3Ј ϭ 1.1 Hz, 2 H, CH2-1Ј), 4.25 (t, J1-2 ϭ 4.9 Hz, 2 H, CH2-
1AE), 6.17 (dt, J2Ј-3Ј ϭ 15.8 Hz, J2Ј-1Ј ϭ 7.1 Hz, 1 H, 2Ј-H), 6.73 (d,
J3Ј-2Ј ϭ 15.8 Hz, 1 H, 3Ј-H), 7.00 (J3-2 ϭ 8.8 Hz, 2 H, 3Ph-H, 5Ph
-
H), 7.41 (J2-3 ϭ 8.8 Hz, 2 H, 2Ph-H, 6Ph-H). 13C NMR (50 MHz,
CD3OD): δ ϭ 34.9 (C-1Ј), 40.3 (C-2AE), 65.3 (C-1AE), 115.9 (C-
3Ph, C-5Ph), 120.4 (C-2Ј), 129.1 (C-2Ph, C-6Ph), 131.1 (C-1Ph), 136.1
(C-3Ј), 159.6 (C-4Ph), 172.3 [SC(NH)NH2]. C12H17N3OS (base,
251.34, 251.11), FAB MS: m/z ϭ 252.1 [M ϩ H]ϩ, 176.1 [M Ϫ
SC(NH2)2 ϩ H]ϩ.
˜
ν ϭ
CH2Cl2/methanol, 10:10:1). M.p. 108 °C. IR (KBr):
3565Ϫ3300, 2977, 2927, 1716, 1687, 1602, 1542, 1509, 1461, 1388,
1363, 1251, 1174, 1108, 1064, 971 cmϪ1. UV (methanol): λmax (ε) ϭ
260 nm (19130). 1H NMR (200 MHz, CDCl3): δ ϭ 1.45 [s, 9 H,
(CH3)3C], 3.52 (dt, J2-1 ϭ 5.1 Hz, J2-NH ϭ 5.3 Hz, 2 H, CH2-2AE),
4.00 (t, J1-2 ϭ 5.1 Hz, 2 H, CH2-1AE), 4.28 (dd, J1Ј-2Ј ϭ 5.8 Hz,
J1Ј-3Ј ϭ 1.4 Hz, 2 H, CH2-1Ј), 5.02 (broad s, 1 H, NH), 6.23 (dt,
Treatment of
1 with 21a: A) Moenomycin A (1, 30 mg,
0.019 mmol), thiouronium salt 21a (11.7 mg, 0.027 mmol), and so-
dium acetate (20 mg) in water (3 mL) were stirred under argon at
60 °C for 8 h. Progress of the reaction was monitored by TLC
(RP18, acetonitrile/H2O, 7:10). No new product was observed. The
mixture was then cooled to 20 °C, a further portion of thiouronium
salt 21a (11 mg, 0.019 mmol) and water (1 mL) were added, and
stirring was continued at 60 °C for an additional 20 h. Formation
of only one product was observed by TLC (RP18, acetonitrile/H2O,
7:10). The cold reaction mixture was directly transferred to the top
of a FC (RP18) column. The inorganic salts were removed by elu-
tion with water (50 mL) and the product was then eluted with ace-
tonitrile/H2O (7:10) to give 10 mg (45% yield, based on consumed
1) of pure 22a, with 11 mg of 1 recovered. B) Moenomycin A (1,
100 mg, 0.063 mmol), sodium acetate (50 mg), and thiouronium
salt 21a (85 mg, 0.23 mmol) in water (10 mL) were stirred under
argon at 60 °C for 8 h. The mixture was then allowed to cool to 20
°C, and the pH was measured (pH ϭ 5.38). The pH was adjusted
to 7.25 by addition of sat. aqueous Na2CO3, and the reaction mix-
ture was stirred at 60 °C for an additional 13 h. Progress of the
reaction was monitored by TLC (RP18, acetonitrile/H2O, 6:10).
Formation of two products (23a and 22a) was observed. Kieselguhr
was then added to the cold reaction solution, and water was lyo-
philized. The residue was transferred to the top of a FC (RP18)
column and the products were separated by elution with acetonitr-
ile/H2O, 6:10. 37.6 mg (37%) of 1, 14.0 mg (12%) of 23a and
17.4 mg (15%) of 22a were obtained.
J2Ј-3Ј ϭ 15.8 Hz, J2Ј-1Ј ϭ 5.8 Hz, 1 H, 2Ј-H), 6.54 (d, J3Ј-2Ј
ϭ
15.8 Hz, 1 H, 3Ј-H), 6.83 (J3-2 ϭ 8.8 Hz, 2 H, 3Ph-H, 5Ph-H,), 7.30
(J2-3 ϭ 8.8 Hz, 2 H, 2Ph-H, 6Ph-H). 13C NMR (50 MHz, CDCl3):
δ ϭ 28.5 [(CH3)3C], 40.3 (C-2AE), 63.9 (C-1Ј), 67.3 (C-1AE), 79.7
[(CH3)3C], 114.7 (C-3Ph, C-5Ph), 126.7 (C-2Ј), 127.8 (C-2Ph, C-6Ph),
130.0 (C-1Ph), 130.8 (C-3Ј), 156.0 [OC(O)NH], 158.4 (C-4Ph).
C16H23NO4 (293.36, 293.16), ESI ICR MS: m/z ϭ 316.15195
(calcd. 316.15193) [M ϩ Na]ϩ, 609.31464 (calcd. 609.31464) [2 M
ϩ Na]ϩ.
2-{(E)-4-[2-(tert-Butoxycarbonylamino)ethoxy]cinnamyl}-
thiouronium Bromide (21a): A solution of PBr3 (34 mg, 12 µL,
0.125 mmol) in dry CH2Cl2 (0.5 mL) was added over 15 min to a
cooled (Ϫ10 °C) solution of 20b (100 mg, 0.34 mmol) in dry
CH2Cl2 (1.3 mL), and the reaction mixture was stirred at Ϫ10 °C
for 25 min. Sat. aqueous NaHCO3 (2 mL) was then added, the
cooling bath was removed, and the mixture was extracted with di-
ethyl ether. The ethereal extract was washed with water, dried with
sodium sulfate, and filtered. The solvent was evaporated under re-
duced pressure and the crude product was dissolved in acetonitrile
(2 mL). A solution of thiourea (23 mg, 0.27 mmol) in acetonitrile
(0.5 mL) was then added to this solution. After a few minutes, a
white precipitate formed in the clear reaction solution. The mixture
(R)-3-{[N-(1-{(E)-4-[2-(tert-Butoxycarbonylamino)ethoxy]-
was stirred for an additional 20 min. The product was then isolated cinnamyl}-2,5-dioxocyclopentyl)-β-
by filtration and washed with acetonitrile. Drying in vacuum gave (1Ǟ4)-2-acetamido-2,6-dideoxy-β-
93 mg (63% overall yield) of pure 21a. IR (KBr): ν ϭ 3600Ϫ3100 glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-β-
(broad, NH, NH2), 1691, 1646, 1509, 1245, 1172 cmϪ1. UV (water):
(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α- -glucopyranuron-
D
-galactopyranuronamidosyl-
-glucopyranosyl-(1Ǟ4)-[β-
-glucopyranosyl-
D
D-
˜
D
D
λmax (ε) ϭ 266 nm (17297). UV (methanol): λmax (ε) ϭ 271 nm amidosyloxy]hydroxyphosphoryloxy}-2-[(2Z,6E,13E)-3,8,8,14,18-
(27195). 1H NMR (200 MHz, CD3OD): δ ϭ 1.44 [s, 9 H, (CH3)3C], pentamethyl-11-methylene-2,6,13,17-nonadecatetraenyloxy)-
3.42 (t, J2-1 ϭ 5.8 Hz, 2 H, CH2-2AE), 4.00 (dd, J1Ј-2Ј ϭ 7.4 Hz, propionic Acid (22a): Rf ϭ 0.28 (RP18, acetonitrile/H2O, 7:10). UV
J1Ј-3Ј ϭ 1.1 Hz, 2 H, CH2-1Ј), 4.01 (t, J1-2 ϭ 5.8 Hz, 2 H, CH2-
(methanol): λmax (ε) ϭ 264 nm (13959). 1H NMR (600 MHz,
1
1AE), 6.14 (dt, J2Ј-3Ј ϭ 15.7 Hz, J2Ј-1Ј ϭ 7.4 Hz, 1 H, 2Ј-H), 6.70 (d, CD3OD, H,1H COSY, HMQC, HMBC): δ ϭ 0.98 (s, 6 H, CH3-
J3Ј-2Ј ϭ 15.7 Hz, 1 H, 3Ј-H), 6.90 (J3-2 ϭ 8.8 Hz, 2 H, 3Ph-H, 5Ph
H), 7.35 (J2-3 ϭ 8.8 Hz, 2 H, 2Ph-H, 6Ph-H). 13C NMR (50 MHz, J ϭ 5.7 Hz, 3 H, CH3-6C), 1.46 (s, 9 H, (CH3)3C), 1.61 (s, 3 H,
CD3OD): δ ϭ 28.7 [(CH3)3C], 35.1 (C-1Ј), 41.0 (C-2AE), 68.0 (C- CH3-20I), 1.63 (s, 3 H, CH3-21I), 1.68 (s, 3 H, CH3-19I), 1.76 (s, 3
-
23I, CH3-24I), 1.26 (s, 3 H, CH3F), 1.39 (m, 2 H, CH2-9I), 1.43 (d,
1AE), 80.3 [(CH3)3C], 115.8 (C-3Ph, C-5Ph), 119.8 (C-2Ј), 128.9 (C- H, CH3-25I), 1.92 (m, 2 H, CH2-10I), 1.95 (s, 3 H, CH3CONHC or
2Ph, C-6Ph), 130.2 (C-1Ph), 136.3 (C-3Ј), 158.5 [OC(O)NH], 160.5
CH3CONHE), 2.01Ϫ2.19 (m, 11 H, CH2-4I, CH2-5I, CH2-15I,
Eur. J. Org. Chem. 2002, 1163Ϫ1174
1171

A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S. Giesa, P. Welzel
FULL PAPER
CH2-16I, and 2.04 (s, CH3CONHE or CH3CONHC], 2.64 (m, 2 H,
CH2-1Ј), 2.71 (d, J12-13 ϭ 7.3 Hz, 2 H, CH2-12I), 2.79 (broad m, 1 24.0 (C-25I), 25.9 (C-19I), 27.7 (C-16I), 27.8 (C-23I, C-24I), 28.8
H), 3.25Ϫ4.40 [broad signals of the sugar protons: 3.44 (t, J2-1
[(CH3)3C), 32.3 (C-10I), 32.5 (C-3A), 32.7 (C-5I), 33.5 (C-4I), 34.8,
17.8, 17.9 (C-20I, C-6C), 23.4, 23.5 (CH3CONHC, CH3CONHE),
ϭ
5.2 Hz, CH2-2AE), 4.02 (bt, J1-2 ϭ 5.2 Hz, CH2-1AE)] , 4.47, 4.57 35.0 (C-2Ј)*, 35.9 (C-12I), 36.4 (C-8I), 40.9 (C-15I), 41.0 (C-2AE),
(d, d, J1-2 ϭ 7.3 Hz, 1 H, 1 H, 1C-H, 1E-H), 4.49 (d, J1-2 ϭ 7.3 Hz, 42.9 (C-9I), 55.9, 55.9 (C-2E)*, 56.9, 57.1 (C-2C)*, 59.9, 60.3 (C-
1 H, 1D-H), 4.68 (d, J ϭ 8.4 Hz, 2 H, CH2-22I), 5.09Ϫ5.14 (m, 2 1Ј)*, 62.7 (C-6D), 66.9 (C-1I), 67.9 (C-1AE), 69.2, 69.8, 70.0, 70.1,
H, 3F-H, 17I-H), 5.15 (t, J13-12 ϭ 7.3 Hz, 1 H, 13I-H), 5.31 (dt, 70.8, 70.9, 71.7, 72.1, 72.2, 72.6, 73.8 (C-5F), 74.1, 74.4, 75.0, 75.1,
J6-7 ϭ 15.7 Hz, J6-5 ϭ 6.8 Hz, 1 H, 6I-H), 5.39 (d, J7-6 ϭ 15.7 Hz,
76.2 (C-3F), 77.9, 78.1, 79.1 (C-2F), 80.2 (C-13Ar), 81.0 (C-2 H),
1 H, 7I-H), 5.45 (broad signal, 1 H, 2I-H), 5.95 (broad signal, 1 H, 82.4 (C-4E), 84.5, 84.8 (C-4C)*, 95.9 (C-1F), 103.1, 103.3 (C-1C)*,
1F-H), 6.03 (dt, J2Ј-3Ј ϭ 15.7 Hz, J2Ј-1Ј ϭ 7.7 Hz, 1 H, 2Ј-H), 6.46 104.2, 104.4 (C-1E, C-1D), 104.6, 104.7 (C-1B)*, 109.3 (C-22I), 115.6
(d, J3Ј-2Ј ϭ 15.7 Hz, 1 H, 3Ј-H), 6.89 (d, J3-2 ϭ 8.4 Hz, 2 H, 3Ph
-
(C-3Ar, C-5Ar), 123.5 (C-13I, C-2I), 123.9, 124.1 (C-3Ј)*, 125.4 (C-
H, 5Ph-H), 7.35 (d, J2-3 ϭ 8.4 Hz, 2 H, 2Ph-H, 6Ph-H). 13C NMR 17I), 126.9 (C-6I), 128.6 (C-2Ar, C-6Ar), 131.8 (C-1Ar), 132.2 (C-
(150 MHz, CD3OD, APT, HMQC, HMBC): δ ϭ 16.1 (C-21I), 16.4 18I), 133.8, 133.9 (C-4Ј)*, 137.3 (C-14I), 140.8 (C-3I), 141.5 (C-7I),
(CH3F), 17.7 (C-6C), 17.8 (C-20I), 23.4, 23.5 (CH3CONHC, 151.1 (C-11I), 158.5 (NHC(O)O), 159.3 (OCONH2F), 159.6 (C-
CH3CONHE), 23.9 (C-25I), 25.9 (C-19I), 27.7 (C-16I), 27.9 (C-23I, 4Ar), 170.7, 171.1 (CONHB)*, 173.9, 174.2 (CH3CONHC,
C-24I), 28.8 [(CH3)3C], 32.3 (C-10I), 32.7 (C-5I), 33.6 (C-4I), 34.7 CH3CONHE), 174.3 (CONH2F), 177.7 (C-1H), 180.8 (CO2HA),
(broad signal), 35.9 (C-12I), 36.4 (C-8I), 36.9 (C-1Ј), 40.9 (C-15I), 210.1 (C-1A).
41.0 (C-2AE), 42.9 (C-9I), 56.1 (C-2C), 56.8 (C-2E), 62.8 (C-6D), 67.0 C85H131N6O38P (1875.96, 1874.82), ESI ICR MS: m/z ϭ 947.39030
(C-1I), 68.0 (C-1AE, C-1A), 69.2, 70.0, 70.5, 70.7, 71.8, 72.2, 72.3, (calcd. 947.39517) [M H]2Ϫ
Na 936.40002 (calcd.
936.40412) [M Ϫ 2 H]2Ϫ, 623.93157 (calcd. 623.93345) [M Ϫ 3
H]3Ϫ
*
Signals from the second diastereomer.
ϩ
Ϫ
3
,
72.6, 73.7, 74.1, 74.2, 74.3, 74.9, 75.1, 75.4, 76.1, 76.3 (C-3F), 78.0,
78.1, 79.0 [(CH3)3C], 80.3, 81.2, 82.6 (C-4E), 84.6 (C-4C), 95.9 (C-
1F), 103.4 (C-1C), 104.1, 104.5 (C-1D, C-1E), 104.7 (C-1B), 109.3
(C-22I), 115.7 (C-3Ph, C-5Ph), 119.1 (C-2Ј), 123.5 (C-13I), 123.6 (C-
2I), 125.4 (C-17I), 126.9 (C-6I), 128.6, 128.8 (C-2Ph, C-6Ph), 131.1
(C-1Ph), 131.8 (same HMBC as C-1Ph), 132.2 (C-18I), 133.9 (same
HMBC as C-3Ј), 136.4 (C-3Ј), 137.3 (C-14I), 140.7 (C-3I), 141.5
(C-7I), 151.1 (C-11I), 158.5 [OC(O)NH), 159.3 (OCONH2F), 159.6
(same HMBC as C-4Ph), 160.1 (C-4Ph), 170.4 (C-6B), 170.9, 171.2,
173.8, 174.3 (CH3CONHC and CH3CONHE, C-6F), 177.7 (CO-
OHH), 212.4, 213.2 (C-2A, C-5A). C85H129N6O37P (1857.95,
1856.81), ESI ICR MS: m/z ϭ 1855.83150 (calcd. 1855.80558) [M
.
Treatment of
1 with 21b: A) Moenomycin A (1, 100 mg,
0.063 mmol), sodium acetate (100 mg), and thiouronium salt 21b
(42 mg, 0.114 mmol) in water (3 mL) were stirred under argon at
60 °C for 20 h. More thiouronium salt 21b (30 mg, 0.081 mmol),
sodium acetate (230 mg), and water (0.8 mL) were then added to
the reaction mixture, and stirring and heating were continued for
an additional 25 h. A further amount of compound 21b (30 mg,
0.081 mmol, total 102 mg, 0.276 mmol), sodium acetate (100 mg,
total 430 mg), and water (0.4 mL, total 4.2 mL) were added, and
the mixture was stirred at 60 °C for 21 h (altogether 66 h). Progress
of the reaction was continuously monitored by TLC (RP18, aceto-
Ϫ H]Ϫ, 927.40576 (calcd. 927.39884) [M Ϫ 2H]2Ϫ
.
(R)-3-[(N-{(E)-4-[4-(2-tert-Butoxycarbonylamino)ethoxyphenyl]-1- nitrile/H2O, 7:10) and it was found that two products had been
(3-carboxypropionyl)-3-butenyl}-β-
(1Ǟ4)-2-acetamido-2,6-dideoxy-β-
glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-β-
(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α-
amidosyloxy)hydroxyphosphoryloxy]-2-[(2Z,6E,13E)-3,8,8,14,18-
D
-galactopyranuronamidosyl-
-glucopyranosyl-(1Ǟ4)-[β-
-glucopyranosyl-
formed but the reaction was still not complete. The reaction mix-
ture was allowed to cool to ambient temperature. Inorganic salts
were removed by ultrafiltration (4 ϫ 50 mL water). Water was lyo-
D
D-
D
D
-glucopyranuron- philized, and the crude products were purified twice by FC (RP18,
acetonitrile/H2O, 7:10). After acetonitrile evaporation under re-
pentamethyl-11-methylene-2,6,13,17-nonadecatetraenyloxy)- duced pressure and lyophilization, 34 mg (15%) of 22b and 18 mg
propionic Acid (23a): Rf ϭ 0.38 (RP18, acetonitrile/H2O, 6:10). UV (8%) of 23b were obtained. B) A solution of moenomycin A (1,
(methanol): λmax (ε) ϭ 262 nm (14886). 1H NMR (600 MHz,
150 mg, 0.095 mmol), sodium acetate (50 mg, 0.609 mmol), and
1
CD3OD, H,1H COSY, HMQC, HMBC): δ ϭ 0.98 (s, 6 H, CH3- thiouronium salt 21b (49 mg, 0.133 mmol) in water (15 mL, pH ϭ
23I, CH3-24I), 1.26 (s, 3 H, CH3F), 1.39 (m, 5 H, CH2-9I, CH3-6C), 5.74) was stirred under argon at 60 °C for 47 h and then at 20 °C
1.46 (s, 9 H, (CH3)3C), 1.61 (s, 3 H, CH3-20I), 1.63 (s, 3 H, CH3-
overnight. Progress of the reaction was monitored by TLC (RP18,
21I), 1.68 (s, 3 H, CH3-19I), 1.76 (s, 3 H, CH3-25I), 1.92 (m, 2 H, acetonitrile/H2O, 7:10, Rf ϭ 0.23, Rfmoenomycin A ϭ 0.47) (22b could
CH2-10I), 2.00Ϫ2.20 (m, 16 H, CH2-4I, CH2-5I, CH2-15I, CH2-16I, be not detected and separated by normal phase chromatography)
CH3CONHC, CH3CONHE), 2.39Ϫ2.49 (m, 2 H, CH2-3A, CH2*- and MALDI TOF MS. Kieselguhr was then added to the cold
3A), 2.49Ϫ2.56 (m, 0.5 H, 2Ј-Ha), 2.57Ϫ2.65 (m, 0.5 H, 2Ј-Ha*), reaction mixture, and water was lyophilized. The residue was trans-
2.71 (d, 2 H, CH2-12I, J12-13 ϭ 7.3 Hz), 2.73Ϫ2.84 (m, 2 H, 2A- ferred to the top of a FC column (RP18). Elution with acetonitrile/
Ha, 2A-Ha*, 2Ј-Hb, 2Ј-Hb*), 2.84Ϫ2.95 (m, 1 H, 2A-Hb, 2A-Hb*),
3.22Ϫ4.52 [broad signals of the sugar protons: 3.43 (broad, CH2-
H2O (6:10) gave (after lyophilization of solvents) 71 mg (43%, 81%
based on consumed 1) of pure 22b, 121 mg of 1, and inorganic
2AE), 3.99 (broad, CH2-1AE), 3.66 (2F-H), 4.12 (1I-Hb), 4.23 (1I- salts. C) A solution of moenomycin A (1) (100 mg, 0.063 mmol),
Ha), 4.51 (1D-H)], 4.54Ϫ4.63 (broad, 1Ј-H), 4.68 (d, J ϭ 8.4 Hz, 2 sodium acetate (80 mg), and thiouronium salt 21b (73 mg,
H, CH2-22I), 5.07Ϫ5.13 (m, 2 H, 3F-H, 17I-H), 5.15 (t, J13-12
ϭ
0.197 mmol) in water (10 mL, pH ϭ 7.30) was stirred under argon
at 60 °C for 7 h. Progress of the reaction was monitored by TLC
(RP18, acetonitrile/H2O, 6:10) and formation only of product 22b
was observed (Rf ϭ 0.26). After the reaction mixture had cooled,
7.3 Hz, 1 H, 13I-H), 5.31 (dt, J6-7 ϭ 15.1 Hz, J6-5 ϭ 6.4 Hz, 1 H,
6I-H,), 5.38 (d, J7-6 ϭ 15.1 Hz, 1 H, 7I-H), 5.45 (bt, 1 H, 2I-H),
5.96 (broad signal, 1 H, 1F-H), 6.04 (dt, J3Ј-4Ј ϭ 15.7 Hz, J3Ј-2Ј
ϭ
7.7 Hz, 0.5 H, 3Ј-H*), 6.11 (dt, J3Ј-4Ј ϭ 15.7 Hz, J3Ј-2Ј ϭ 7.7 Hz, the pH was measured (pH ϭ 5.56). The pH of the reaction mixture
0.5 H, 3Ј-H), 6.44 (d, J4Ј-3Ј ϭ 15.2 Hz, 0.5 H, 4Ј-H), 6.52 (d, was then adjusted to 7.71 by addition of sat. aq. Na2CO3 and the
J4Ј-3Ј ϭ 15.2 Hz, 0.5 H, 4Ј-H*), 6.87 (d, J3-2 ϭ 7.8 Hz, 2 H, 3Ar-H,
mixture was stirred at 60 °C for an additional 25 h. After this
period, TLC showed only one product (23b), which differed from
5Ar-H, 3Ar-H*, 5Ar-H*), [2 H, 7.33 (d, J2-3 ϭ 7.8 Hz, 2Ar-H*, 6Ar
-
H*), 7.35 (d, J2-3 ϭ 7.8 Hz, 2Ar-H, 6Ar-H)]. 13C NMR (150 MHz, that first detected (Rf ϭ 0.37). The cold reaction mixture was
CD3OD, APT, HMQC, HMBC): δ ϭ 16.1 (C-21I), 16.3 (CH3F), poured into an ultrafiltration cell (50 mL) and inorganic salts were
1172
Eur. J. Org. Chem. 2002, 1163Ϫ1174

Introduction of Reporter Groups into Moenomycin A
FULL PAPER
removed by elution with water (120 mL). Kieselguhr was then ad-
ded to the residue, and the solvent was lyophilized. The Kieselguhr
1.63 (s, 3 H, CH3-21I), 1.69 (s, 3 H, CH3-19I), 1.75 (s, 3 H, CH3-
25I), 1.92 (broad signal, 5 H, CH3CONHC or CH3CONHE and
with the adsorbed substances was transferred to the top of a FC CH2-10I), 2.00Ϫ2.07 (m,
5
H, Ha-15I, Ha-16I and 2.03 (s,
column (RP18). Elution with acetonitrile/H2O (6:10) gave 42 mg of CH3CONHE or CH3CONHC)], 2.08Ϫ2.20 (m, 6 H, CH2-4I, CH2-
crude 23b, with 33 mg of 1 recovered. Repeated FC (RP18, aceto-
nitrile/H2O, 6:10) provided 24 mg (22%) of pure 23b.
5I, 15I-Hb, 16I-Hb), 2.58Ϫ2.68 (broad signal, hidden by DMSO sig-
nal, CH2-1Ј), 2.71 (d, J12-13 ϭ 7.1 Hz, 2 H, CH2-12I), 2.80 (broad
d, 2 H, CH2-3A or CH2-4A), 2.90 (broad d, 2 H, CH2-4A or CH2-
3A), 3.26 (broad signal, 1 H, 2D-H), 3.25Ϫ4.60 [broad overlapping
signals of the sugar protons: 3.33 (4D-H), 3.43 (broad signal, CH2-
2AE), 3.66 (2F-H, 5D-H), 3.89 (6D-Ha), 3.97 (broad signal, 1 H, 2H-
H), 4.03Ϫ4.19 (broad signal, 3 H, 1I-Ha, 6D-Hb, 3H-Ha), 4.21
(broad signal, 1 H, 1I-Hb), 4.30 (broad signal, 3H-Hb, CH2-1AE),
4.46 (broad signal, 3 H, 1B-H, 1C-H, 1E-H)], 5.07 (d, J3-2 ϭ 10.0 Hz,
(R)-3-[(N-{(E)-4-[4-(2-Aminoethoxy)phenyl]-1-(3-carboxypropionyl)-
3-butenyl}-β-
dideoxy-β- -glucopyranosyl-(1Ǟ4)-[β-
acetamido-2-deoxy-β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-
methyl-α- -glucopyranuronamidosyloxy)hydroxyphosphoryloxy]-2-
D
-galactopyranuronamidosyl-(1Ǟ4)-2-acetamido-2,6-
D
D
-glucopyranosyl-(1Ǟ6)]-2-
D
D
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nona-
decatetraenyloxy)propionic Acid (23b): Rf ϭ 0.33 (RP18, acetonitr-
ile/H2O, 7:10). UV (methanol): λmax (ε) ϭ 262 nm (14704). 1H
1 H, 3F-H), 5.12 (t, J17-16 ϭ 6.9 Hz, 1 H, 17I-H), 5.16 (t, J13-12
ϭ
7.1 Hz, 1 H, 13I-H), 5.30 (dt, J6-7 ϭ 15.2 Hz, J6-5 ϭ 5.7 Hz, 1 H,
6I-H), 5.38 (d, J7-6 ϭ 15.2 Hz, 1 H, 7I-H), 5.45 (broad signal, 1 H,
2I-H), 5.90 (broad signal, 1 H, 1F-H), 6.10 (m, 1 H, 2Ј-H), 6.47 (d,
J3Ј-2Ј ϭ 14.8 Hz, 1 H, 3Ј-H), 7.04 (d, J3-2 ϭ 8.1 Hz, 2 H, 3Ar-H,
5Ar-H), 7.40 (d, J2-3 ϭ 8.1 Hz, 2 H, 2Ar-H, 6Ar-H). 13C NMR
(150 MHz, CD3OD/[D6]DMSO, 3:1, HMQC, HMBC): δ ϭ 13.3
(impurities from RP18 material), 15.4 (CH3-21I), 15.9 (CH3F), 17.1
(CH3-20I), 17.2 (C-6C), 19.89 (impurities from RP18 material), 22.8
(CH3CONHC), 22.9 (CH3CONHE), 23.3 (C-25I), 25.3 (C-19I), 26.8
(C-16I), 27.1 (C-23I, C-24I), 29.7 (impurities from RP18 material),
31.4 (C-10I), 31.8 (C-5I), 32.7 (C-4I), 34.5 (broad, C-3A, C-4A), 35.1
(C-12I), 35.6 (C-8I, C-1Ј (HMBC 2Ј, 3Ј-H), 39.5 (C-2AE), 39.9 (C-
15I), 41.9 (C-9I), 55.5 (C-2E), 55.9 (C-2C), 61.8 (C-6D), 64.9 (C-
1AE), 66.1 (C-1I), 66.9, 68.2, 68.8, 69.5 (C-3 H), 70.7, 71.2, 71.6,
72.8 (C-2F), 73.1, 73.3, 73.4, 74.1, 74.6, 75.0 (C-3F), 77.3, 77.9 (C-
2F), 80.3 (C-2H), 81.6 (C-4E.), 83.8 (C-4C), 94.9 (C-1F), 102.4, 102.8,
1
NMR (600 MHz, CD3OD, H,1H COSY, HMBC, HMQC): δ ϭ
0.96 (s, 6 H, CH3-23I, CH3-24I), 1.25 (s, 3 H, CH3F), 1.29 (d, J ϭ
6.3 Hz, 3 H, CH3-6C), 1.35 (m, 2 H, CH2-9I), 1.59 (s, 3 H, CH3-
20I), 1.61 (s, 3 H, CH3-21I), 1.67 (s, 3 H, CH3-19I), 1.74 (s, 3 H,
CH3-25I) 1.89 (m, 2 H, CH2-10I), 2.00Ϫ2.20 (m, 14 H, CH2-4I,
CH2-5I, CH2-15I, CH2-16I, and 2.06, 2.07 (s, s, CH3CONHC,
CH3CONHE), 2.40Ϫ2.48 (m, 2 H, CH2-3A, CH2*-3A), 2.60Ϫ2.66
(m, 0.5 H, 2Ј-H*a), 2.69 (d, J12-13 ϭ 7.3 Hz, 2 H, CH2-12I),
2.72Ϫ2.77 (m, 1 H, 2Ј-Ha,b), 2.79Ϫ2.94 (m, 2.5 H, 2A-Ha,b, 2A-
Ha,b*, 2Ј-Hb*), 3.20Ϫ4.80 [broad signals of the sugar protons: 3.43
(CH2-2AE), 4.28 (CH2-1AE), 4.50, 4.69* (CH-1Ј)], 5.07Ϫ5.14 (m,
13I-H, 17I-H), 5.27 (dt, J6-7 ϭ 15.5 Hz, J6-5 ϭ 6.4 Hz, 1 H, 6I-H),
5.34 (d, J7-6 ϭ 15.5 Hz, 1 H, 7I-H), 5.42 (broad signal, 1 H, 2I-H),
5.88 (m, 1 H, 1F-H), 5.96 (dt, J3Ј-4Ј ϭ 15.5 Hz, J3Ј-2Ј ϭ 7.6 Hz, 0.5
H, 3Ј-H*), 6.09 (dt, J3Ј-4Ј ϭ 15.5 Hz, J3Ј-2Ј ϭ 7.6 Hz, 0.5 H, 3Ј-H),
6.46 (d, J4Ј-3Ј ϭ 15.5 Hz, 0.5 H, 4Ј-H), 6.48 (d, J4Ј-3Ј ϭ 15.5 Hz, 0.5
H, 4Ј-H*), 6.99 (d, J3-2 ϭ 8.9 Hz, 1 H, 3Ar-H, 5Ar-H), 7.01 (d,
J3-2 ϭ 8.9 Hz, 1 H, 3Ar-H*, 5Ar-H*), 7.34 (d, J2-3 ϭ 8.9 Hz, 1 H,
2Ar-H, 6Ar-H), 7.39 (d, J2-3 ϭ 8.9 Hz, 1 H, 2Ar-H*, 6Ar-H*). 13C
NMR (150 MHz, CD3OD, HMBC, HMQC): δ ϭ 16.1 (C-21I),
16.3 (CH3F), 17.8 (C-20I), 18.0, 18.2 (C-6C)*, 23.5 (CH3CONHC,
CH3CONHE), 23.9 (C-25I), 25.9 (C-19I), 27.4 (C-16I), 27.8 (C-23I,
C-24I), 32.0 (C-10I), 32.2 (C-3A), 32.5 (C-5I), 33.3 (C-4I), 34.2, 34.6
(C-2Ј)*, 35.9 (C-12I), 36.3 (C-8I), 40.3 (C-2AE), 40.6 (C-15I), 42.7
(C-9I), 56.7 (C-2C, C-2E), 59.6, 59.9 (C-1Ј)*, 62.4 (C-6D), 65.6 (C-
1AE), 66.9Ϫ81.1 (broad overlapping signals of the sugar carbon
atoms), 95.7 (C-1F), 102.7Ϫ103.4 (broad signals, C-1C, C-1B, C-1E,
C-1D), 109.4 (C-22I), 115.8, 116.1 (C-3Ar, C-5Ar)*, 122.9 (C-2I),
123.4 (C-13I), 123.6 (C-3Ј), 125.2 (C-17I), 126.9 (C-6I), 128.6, 128.7
(C-2Ar, C-6Ar)*, 132.2 (C-1Ar), 132.7 (C-8I), 134.1 (C-4Ј), 137.3 (C-
14I), 141.4 (C-7I, C-3I), 151.2 (C-11I), 158.8 (C-4Ar), 159.2
(OCONH2F), 170.1, 170.8 (CONHB)*, 174.3 (CONH2F), 174.4,
174.5 (CH3CONHC, CH3CONHE), 177.6 (C-1 H), 181.1 (CO2HA),
201.4 (C-1A). * Signals of the second diastereomer. C80H123N6O36P
103.6, 103.6 (C-1C, C-1E, C-1B, C-1D), 108.6 (C-22I), 115.3 (C-3Ar
,
C-5Ar), 119.1 (C-2Ј), 122.6 (C-13I), 122.9 (C-2I), 124.5 (C-17I),
126.1 (C-6I), 128.1 (C-2, 6Ar), 130.7 (C-1Ar), 131.3 (C-18I), 135.1
(C-3Ј), 136.5 (C-14I), 139.52 (C-3I), 140.5 (C-7I), 150.2 (C-11I),
157.9 (OCONH2F), 158.5 (C-4Ar), 169.4 (CONHB), 172.3, 172.6
(CH3CONHC, CH3CONHE), 173.3 (CONH2 F), 176.0 (small sig-
nal), 176.3 (C-1 H), 210.9, 211.5 (C-2A, C-5A). C80H121N6O35P
(1757.83, 1756.76), ESI ICR MS: m/z ϭ 1755.73669 (calcd.
1755.75377) [M Ϫ H]Ϫ, 877.37116 (calcd. 877.37325) [M Ϫ 2 H]2Ϫ
.
(R)-3-[(N-{(E)-4-[4-(2-{[2-({2-[3Ј,6Ј-Bis(ethylamino)-2Ј,7Ј-dimethyl-
3-oxospiro[1H-isoindole-1,9Ј-[9H]xanthen]-2(3 H)-yl]ethyl}amino)-
3,4-dioxo-1-cyclobuten-1-yl]amino}ethoxy)phenyl]-1-[3-
( m e t h o x y c a r b o n y l ) p r o p i o n y l ] - 3 - b u t e n y l } - β -
galactopyranuronamidosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β-
glucopyranosyl-(1Ǟ4)-[β-
deoxy-β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α-
D
-
-
D
D
-glucopyranosyl-(1Ǟ6)]-2-acetamido-2-
D
D
-
glucopyranuronamidosyloxy)hydroxyphosphoryloxy]-2-
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nona-
decatetraenyloxy)propionic Acid (25): A solution of 24 (15 mg,
0.025 mmol) in EtOH (0.5 mL) was added to a solution of 22b
(30 mg, 0.017 mmol) in methanol (7 mL) and Et3N (1 mL). The
heterogeneous mixture was stirred at 20 °C. After 18 h, the mixture
(1775.84, 1774.77), ESI ICR MS: m/z
886.37791) [M Ϫ 2 H]2Ϫ, 590.58070 (calcd. 590.58264) [M Ϫ 3
H]3Ϫ
ϭ 886.37477 (calcd.
.
(R)-3-{[N-(1-{(E)-4-[2-Aminoethoxy]cinnamyl}-2,5-dioxo- became homogeneous, and it was stirred at 20 °C for an additional
cyclopentyl)-β-
dideoxy-β- -glucopyranosyl-(1Ǟ4)-[β-
acetamido-2-deoxy-β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-
methyl-α-
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nona- was transferred to the top of a FC column. Elution with 1-pro-
panol/H2O (7:1.5), solvent evaporation, and lyophilization pro-
D
-galactopyranuronamidosyl-(1Ǟ4)-2-acetamido-2,6-
72 h. Progress of the reaction was continuously monitored by TLC
(1-propanol/H2O, 7:2). TLC indicated the formation of two diaster-
eoisomers of 25. Kieselguhr was added to the cooled solution and
D
D
-glucopyranosyl-(1Ǟ6)]-2-
D
D
-glucopyranuronamidosyloxy]hydroxyphosphoryloxy}-2- the solvents were evaporated under reduced pressure. The residue
decatetraenyloxy)propionic Acid (22b): Rf ϭ 0.23 (RP18, acetonitr-
ile/H2O, 7:10). UV (methanol): λmax (ε) ϭ 263 nm (27344). 1H vided 22 mg (56%) of pure 25 (two diastereomers). Rf * ϭ 0.62,
NMR (600 MHz, CD3OD/[D6]DMSO, 3:1, 1 H,1H COSY, HMQC, Rf ϭ 0.58 (1-propanol/H2O, 7:2). 1H NMR (600 MHz, CD3OD/
HMBC): δ ϭ 0.98 (s, 6 H, CH3-23I, CH3-24I), 1.24 (s, 3 H, CH3F), [D6]DMSO, 5:1, 1H,1H COSY, HMQC, HMBC): δ ϭ 1.01 (s, 6 H,
1.40 (broad signal, 5 H, CH3-6C, CH2-9I), 1.62 (s, 3 H, CH3-20I), CH3-23I, CH3-24I), 1.26 (s, 3 H, CH3F), 1.32, 1.33 (broad signals,
Eur. J. Org. Chem. 2002, 1163Ϫ1174
1173

A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S. Giesa, P. Welzel
FULL PAPER
[1]
6 H, 2 NHCH2CH3R6G), 1.37Ϫ1.47 (broad signal, 5 H, CH2-9I,
CH3-6C), 1.64 (s, 6 H, CH3-20I, CH3-21I), 1.71 (s, 3 H, CH3-19I),
1.79 (s, 3 H, CH3-25I), 1.92, 1.93 (s, 6 H, 2ЈR6G-CH3, 7ЈR6G-CH3),
1.92Ϫ1.98 (broad signal, 2 H, CH2-10I), 2.02Ϫ2.10 (broad signals,
8 H, CH3CONHC, CH3CONHE, 15I-Ha, 16I-Ha), 2.11Ϫ2.17
(broad signal, 6 H, CH2-4I, CH2-5I, 15I-Hb, 16I-Hb), 2.18Ϫ2.25
(acetone), 2.49Ϫ2.64 (broad m, 3 H, 2Ј-Ha, CH2*-3A, CH2-3A, 2Ј-
H*a), 2.70Ϫ2.80 (broad signal, 3 H, CH2-12I, 2Ј-Hb, 2Ј-H*b),
2.90Ϫ3.02 (broad signal, 2 H, CH2-2A, CH2*-2A), 3.20Ϫ4.60
[broad overlapping signals of the sugar protons with 3.24 (broad
signal, 2 NHCH2CH3R6G), 3.67 (broad s, OCH3A), 4.48 (broad sig-
nal, 1Ј-H)], 5.14 (broad m, 2 H, 17I-H, 3F-H), 5.18 (broad signal,
Review: T. D. H. Bugg in Comprehensive Natural Products
Chemistry, vol. 3 (Ed.: D. Barton, K. Nakanishi), Elsevier, Am-
sterdam, 1999, p. 241Ϫ294.
Review: J. van Heijenoort, Glycobiology 2001, 13, 25RϪ36R.
C. Goffin, J.-M. Ghuysen, Microbiol. Mol. Biol. Rev. 1998,
62, 1079Ϫ1093.
[2]
[3]
[4]
[5]
R. C. Goldman, D. Gange, Curr. Med. Chem. 2000, 7,
801Ϫ820.
N. El-Abadla, M. Lampilas, L. Hennig, M. Findeisen, P. Wel-
zel, D. Müller, A. Markus, J. van Heijenoort, Tetrahedron 1999,
55, 699Ϫ722.
O. Ritzeler, L. Hennig, M. Findeisen, P. Welzel, D. Müller, A.
Markus, G. Lemoine, M. Lampilas, J. van Heijenoort, Tetra-
hedron 1997, 53, 1675Ϫ1694.
[6]
[7]
1 H, 13I-H), 5.36 (broad signal, 1 H, 6I-H), 5.43 (d, 7I-H, J7-6
ϭ
M. Terrak, T. K. Ghosh, J. van Heijenoort, J. Van Beeumen,
M. Lampilas, J. Aszodi, J. A. Ayala, J.-M. Ghuysen, M.
17.8 Hz), 5.46 (broad signal, 1 H, 2I-H), 5.95 (broad signal, 1 H,
1F-H), 6.05 (broad signal, 0.5 H, 3Ј-H), 6.14 (broad signal, 0.5 H,
3Ј-H*), 6.18 (s, 2 H, 1ЈR6G-H, 8ЈR6G-H), 6.37 (s, 2 H, 4ЈR6G-H,
`
Nguyen-Desteche, Mol. Microbiol. 1999, 34, 350Ϫ364.
[8]
[9]
K. Stembera, A. Buchynskyy, S. Vogel, D. Knoll, A. A. Osman,
J. A. Ayala, P. Welzel, ChemBioChem, in press.
H. Schürer, K. Stembera, D. Knoll, G. Mayer, M. Blind, H.-
H. Förster, M. Famulok, P. Welzel, U. Hahn, Bioorg. Med.
Chem. 2001, 9, 2557Ϫ2563.
5ЈR6G-H), 6.44 (d, J4Ј-3Ј ϭ 15.5 Hz, 0.5 H, 4Ј-H*), 6.55 (d, J4Ј- 3Ј
. ϭ
15.5 Hz, 0.5 H, 4Ј-H), 6.93 (broad signal, 2 H, 3Ph-H, 5Ph-H), 7.07
(broad signals, 1 H, 7R6G-H), 7.36 (broad signal, 2 H, 2Ph-H, 6Ph
-
H), 7.58 (broad signal, 2 H, 5R6G-H, 6R6G-H), 7.92 (broad signal,
1 H, 4R6G-H). 13C NMR (150 MHz, CD3OD/[D6]DMSO, 5:1,
HMQC, HMBC): δ ϭ 14.1 (NHCH2CH3R6G), 15.5 (C-21I), 15.9
(CH3F), 16.6 (2Ј-CH3R6G, 7Ј-CH3R6G), 17.2 (C-20I, C-6C), 22.9
(CH3CONHC, CH3CONHE), 23.4 (C-25I), 25.3 (C-19I), 26.8 (C-
16I), 27.2 (C-23I, C-24I), 27.7, 27.7 (C-3A)*, 30.1 (acetone), 31.5
(C-10I), 31.9 (C-5I, C-2Ј (HMBC), 32.6 (C-4I), 34.0, 34.1 (C-2A)*,
34.3 (impurities), 35.1 (C-12I), 35.6 (C-8I), 38.2 (NHCH2CH3R6G),
39.9 (C-15I), 41.8 (C-2AE), 41.9 (C-9I), 43.8 (C-1DAE), 51.4, 51.5
(OCH3A)*, 55.2 (C-2E), 56.0 (C-2C), 58.9, 59.2 (C-1Ј)*, 61.9 (C-
6D), 66.3 (C-9ЈR6G), 68.0, 69.0 (broad signal, C-3 H), C-70.1, 70.9,
71.1, 71.2, 71.6, 71.7, 72.8, 73.1, 73.3, 73.6, 74.2, 75.2, 75.3, 77.2,
77.3 (broad unassigned signals of the sugar carbon atoms), 77.9
(broad C-2F), 81.8 (C-4E), 83.7, 83.9 (C-2 H, C-4C), 94.9 (C-1F),
96.5 (C-4ЈR6G, C-5ЈR6G), 102.4, 103.1, 103.7 (broad, C-1C, C-1B, C-
[10]
A. Anikin, A. Buchynskyy, U. Kempin, K. Stembera, P. Welzel,
G. Lantzsch, Angew. Chem. 1999, 111, 3931Ϫ3935; Angew.
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H. Schürer, A. Buchynskyy, K. Korn, M. Famulok, P. Welzel,
U. Hahn, Biol. Chem. 2001, 382, 479Ϫ481.
For preliminary experiments, see: T. Rühl, L. Hennig, Y. Hat-
anaka, K. Burger, P. Welzel, Tetrahedron Lett. 2000, 41,
4555Ϫ4558.
[11]
[12]
[13]
[14]
Results to be published.
U. Kempin, L. Hennig, D. Knoll, P. Welzel, Tetrahedron 1997,
53, 17669Ϫ17690, and references therein.
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C. H. Kuo, D. Taub, N. L. Wendler, Angew. Chem. 1965, 77,
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A. Buchynskyy, Dissertation, Universität Leipzig, 2001.
For a preliminary communication, see: S. Vogel, A. Buchyn-
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M. Sakaitani, Y. Ohfune, J. Am. Chem. Soc. 1990, 112,
1150Ϫ1158.
A. Buchynskyy, K. Stembera, L. Hennig, M. Findeisen, S.
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2923Ϫ2925.
D. Kritchevsky, M. R. Kirk, Arch. Biochem. Biophys. 1952,
35, 346Ϫ351.
R. Tschesche, J. Blumbach, P. Welzel, Justus Liebigs Ann.
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Prepared as described by: N. Cohen, B. Banner, J. Heterocycl.
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Asymmetry 1996, 7, 2863Ϫ2870.
[16]
[17]
[18]
1E, C-1D), 104.9 (C-8ЈaR6G, C-9ЈaR6G), 108.6 (C-22I), 115.1 (C-3Ph
,
C-5Ph), 119.1 (C-2ЈR6G, C-7ЈR6G), 122.6 (C-13I), 122.9 (C-4R6G),
123.4, 123.4 (C-2I), 124.3 (C-7R6G), 124.6 (C-17I), 126.1 (C-6I),
127.9 (C-2Ph, C-6Ph), 128.1 (C-13I), 128.8 (C-5R6G), 131.1 (C-
3aR6G), 131.4 (C-18I), 132.7 (C-1Ph), 133.1 (C-4Ј), 133.4 (C-6R6G),
136.5 (C-14I), 140.6 (C-7I), 148.5 (C-3ЈR6G, C-6ЈR6G), 150.1 (C-11I),
152.3 (C-4ЈaR6G, C-10ЈaR6G), 154.4 (C-7aR6G), 157.9 (OCONH2F),
158.5 (C-4Ph), 168.3, 168.6 (CONHB)*, 169.6 (CONHR6G), 169.8,
170.1 (C-1SA, C-2SA), 172.3 (CH3CONHC, CH3CONHE), 173.2
(CONHF), 173.8 (CO2HA), 182.8, 183.3 (C-3SA, C-4SA), 207.8
(acetone), 208.4, 208.5 (C-1A)*. * Signals of the second diastere-
omer. C113H155N10O40P (2324.49, 2323.01), ESI ICR MS: m/z ϭ
1160.50236 (calcd. 1160.49971) [M Ϫ 2H]2Ϫ, 773.33291 (calcd.
773.33071) [M Ϫ 3 H]3Ϫ. Fluorescence spectrum [methanol/H2O,
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
1:1 (4 mL)
ϩ 0.2% TFA (1 mL), 0.22 mg/5 mL]: excitation
(550 nm): λmax ϭ 527 nm, λmax ϭ 350 nm, emission (527 nm):
λmax ϭ 555 nm.
Acknowledgments
We are grateful to Mrs. R. Herold for technical assistance and to
Dr. H. Knoll for the fluorescence spectrum of 25. Financial sup-
port by the Deutsche Forschungsgemeinschaft (Innovationskolleg
‘‘Chemisches Signal und biologische Antwort’’), BC Biochemie
GmbH, and the Fonds der Chemischen Industrie is also grate-
fully acknowledged.
E-M. Ehrenstorfer-Schaefers, N. Steiner, J. Altman, Z. Natur-
forsch., Teil B 1990, 45, 817Ϫ827.
Received October 16, 2001
[O01496]
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Eur. J. Org. Chem. 2002, 1163Ϫ1174