Synthesis of fluorescent derivatives of the antibiotic moenomycin A

Article abstract of DOI:10.1002/1099-0690(200204)2002:7<1149::AID-EJOC1149>3.0.CO;2-H

Moenomycin has been highly selectively converted into amino derivative 3b. The primary amino group of this compound can be used to attach various fluorescent labels to moenomycin, through conversion of isothiocyanates into thioureas and squaric acid diesters into diamides. The application of some of the derivatives is outlined. Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002).

Full text of DOI:10.1002/1099-0690(200204)2002:7<1149::AID-EJOC1149>3.0.CO;2-H

FULL PAPER
Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
Andrij Buchynskyy,[a] Uwe Kempin,[a] Stefan Vogel,[a] Lothar Hennig,[a] Matthias Findeisen,[a]
Dietrich Müller,[b] Sabine Giesa,[a] Helmut Knoll,[a] and Peter Welzel*[a]
Dedicated to Professor Lutz Tietze on the occasion of his 60th birthday
Keywords: Antibiotics / Bioconjugate methods / Glycolipids / JappϪKlingemann method / Natural products /
Peptidoglycan
Moenomycin has been highly selectively converted into
amino derivative 3b. The primary amino group of this com-
pound can be used to attach various fluorescent labels to
moenomycin, through conversion of isothiocyanates into
thioureas and squaric acid diesters into diamides. The ap-
plication of some of the derivatives is outlined.
(
Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany,
2002)
Introduction
tional enzymes with two separate active sites: one for trans-
glycosylation and the other for transpeptidation. Each of
these domains can be specifically inhibited by antibiotics.
While β-lactam antibiotics exert their action through cova-
lent binding to an essential serine residue in the transpepti-
dase domain, the transglycosylation step of cell wall assem-
bly can be blocked by a number of antibiotics, including
the moenomycins.[5] Of these antibiotics, however, the mo-
enomycins (see moenomycin A, 1) are the only compounds
known to inhibit the enzyme (Scheme 1).[3] The structure-
activity relationships of the moenomycins have been studied
extensively[6] and a mechanism for their mode of action has
been proposed.[7,8] It is assumed that they are anchored to
the cytoplasmic membrane through the moenocinol lipid
component, followed by highly selective recognition of the
oligosaccharide moiety at a substrate binding site of the
enzyme, most probably the binding site of the growing gly-
can 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 un-
explored. The moenomycins are a unique tool for elucida-
tion of the structure of the enzyme and the detailed mech-
anism of the transglycosylation reaction.
Antibiotic resistance in bacterial pathogens has become
a serious problem for human health and requires both the
development of antiinfectives with novel modes of action
and a deeper mechanistic understanding of already ex-
isting drugs.[1]
The shape of a bacterium is determined by a net-like mul-
tilayer polymer surrounding the cell. This polymer Ϫ pepti-
doglycan Ϫ consists of repeating β-1,4-linked N-acetylglu-
cosaminyl-N-acetylmuramyl units cross-linked through
short peptide side chains.[2] The biosynthesis of peptidogly-
can is an essential 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 thus offer attractive targets for
the development of selective antibacterial agents.
E. coli peptidoglycan biosynthesis, starting from a mem-
brane-bound undecaprenyl-linked disaccharide precursor
(lipid II), is completed by two successive reactions: a trans-
glycosylation reaction producing unbranched glycan
strands[2,3] and a transpeptidation reaction cross-linking the
peptide units of different strands.[2] Both reactions are cata-
lyzed by the major high molecular weight penicillin-binding
proteins (PBPs).[4] PBPs such as PBP 1a and 1b are bifunc-
Research procedures based on fluorescence methods are
of great importance in biochemistry.[9] In the course of in-
vestigations into moenomycin we had already made use of
fluorescence phenomena in studying the interaction of mo-
enomycin with artificial membranes[10] and with moenomy-
cin-binding aptamers selected by an in vitro selection/amp-
lification procedure from a library of approximately 7 ϫ
1014 different modified RNA sequences.[11]
[a]
Universität Leipzig, Fakultät für Chemie und Mineralogie,
Johannisallee 29, 04103 Leipzig, Germany
[b]
Ruhr-Universität Bochum, Institut für Analytische Chemie,
44780 Bochum, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.com or from the author.
Eur. J. Org. Chem. 2002, 1149Ϫ1162
WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1434Ϫ193X/02/0407Ϫ1149 $ 17.50ϩ.50/0
1149

P. Welzel et al.
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Scheme 1
This publication describes the chemistry that can be used cleavage[13] and the thus formed amidrazone cyclizes to give
to attach chromophores to suitable moenomycin derivat- a triazole, the final product of the reaction. The reaction
ives.
occurs at a site that has been found to be of minor import-
ance for the antibiotic activity.[6] By this sequence, the thiol-
substituted compound 3a was prepared and used for the
synthesis of a number of bioconjugates by 1,4-addition to
maleimides[12] and by disulfide formation .[14] Recently, we
reported that the amino compound 3b is also accessible by
this route.[15,16] The heterobifunctional reagent 2 (X ϭ
NH2) was prepared from 5-amino-2-nitrobenzoic acid by
Staab’s procedure.[17,18] The selective formation of the aro-
matic diazonium salt from 2 (X ϭ NH2) is the consequence
Results and Discussion
Treatment of Moenomycin A with Aromatic Diazonium
Salts
We have previously shown that 1 reacts selectively with
diazonium salts at the enolized β-diketone unit A.[12] The
primary coupling product undergoes a JappϪKlingemann
1150
Eur. J. Org. Chem. 2002, 1149Ϫ1162

Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
FULL PAPER
of the different pK values of the amino groups. Under the showed the strong absorption at 2113 cmϪ1 characteristic
conditions of diazonium salt formation, the aliphatic amino of isothiocyanates. Furthermore, two signals appeared in
group is protected by protonation. Compound 3b was ob- the 13C NMR spectrum, at δ ϭ 134.79 and 136.01, corres-
tained in a yield of 78% after careful purification.[19]
ponding to C-4Ј and the isothiocyanato group. The FAB
The formation of moenomycin conjugates by the MS exhibited the expected molecular ion peak.
JappϪKlingemann route seems to be general and can be
Coupling between 5c and 3b was performed in a mixture
used for the synthesis of other conjugates as well. Thus, of DMF and pyridine[27] (to make the reaction solution
under similar conditions the piperazine derivative 3c and homogeneous) at room temperature under argon and in the
the biotin conjugate 3d were prepared. Compound 3d has dark. The reaction mixture was stirred until 3b had been
been employed for the isolation of moenomycin binding ap- completely consumed (TLC analysis). In the H and 13C
1
tamers.[11] Amino compound 3b has turned out to be a NMR spectra of the reaction product 6a, all signals of the
much more flexible ligand than thiol 3a, since more coup- coumarin moiety (Cou), the nitroaromatic ring and the
ling reactions are available for the preparation of bioconju- ethylenediamine-derived components (Ar and DAE), the
gates; they include treatment with activated carboxylic acids triazole ring (TA), the lipid component (I), the carboxypro-
to provide amides,[16] addition to isothiocyanates to give pionyl appendage (A), and the thioureido unit [NHC(S)NH
thioureas, and the attachment to other amines through the at δ ϭ 181.50] were assigned with the aid of 2D NMR ex-
bifunctional squaric acid linker[20,21] as detailed below.
periments (Table 1). The carbon signal of C-1I was covered
by sugar carbon signals and the C-2A carbon signal seemed
to be hidden under the solvent (DMSO) signal (HMQC).
Some sugar proton and carbon signals were assigned, but
most of the sugar part signals could not be identified, due
to overlapping and broadening. The high-resolution ESI
FT ICR MS spectra in both modes confirmed structure 6a.
The fluorescence spectra of 6a and 5c in methanol/water
(1:1) solution were practically identical.
With the aid of 6a, the extent of anchoring of moenomy-
cin to artificial membranes was studied by means of fluores-
cence methods, to yield partition coefficients. Furthermore,
the transfer of 6a between POPC vesicles was investigated
by fluorescence resonance energy transfer (FRET), by mak-
ing use of (4-dimethylamino)azobenzene, which efficiently
quenches the fluorescence of the 7-diethylamino-4-methyl-
coumarin chromophore. From these experiments a rate con-
stant was calculated.[10,28]
Synthesis of Coumarin Conjugate 6a
The synthesis of the coumarin-derived isothiocyanate 5c
started from coumarin 4, the double bond of which was
arylated in the 3-position with the diazonium salt prepared
from N-acetyl-p-phenylenediamine by Meerwein’s arylation
reaction (Scheme 2).[22] Under typical conditions[22] (not
optimized), 5a was obtained in acceptable yield. The ab-
sence of the coumarin 3-H signal in the 1H NMR spectrum
of 5a and the long-range coupling of 2Ј-H, 6Ј-H, and the
CH3-9 protons to C-3 (HMBC) confirmed the proposed
structure. FAB MS displayed the correct molecular ions.
Heating of compound 5a under reflux in 6% hydrochloric
acid in dry ethanol brought about selective removal of the
N-acetyl group. The required amine 5b was obtained in 93%
yield after FC.
The preparation of isothiocyanates from aromatic amines
and thiophosgene can be performed under different condi-
tions: (i) in a heterogeneous mixture of water and chloro-
form[23] with calcium carbonate as the base,[24] (ii) in homo-
Conjugation of 6-Aminotetramethylrhodamine (7a) to 3b
The long-wavelength rhodamine fluorophores are among
geneous organic solutions,[25] or (iii) in water or aqueous the most photostable fluorescent labeling reagents available.
hydrochloric acid.[26] A heterogeneous mixture of water/ Unlike fluorescein, which forms a nonfluorescent spiro
chloroform and calcium carbonate was used for the pre- form at acidic pH, rhodamines exist in a fluorescent qui-
paration of 5c (54% yield after FC). The IR spectrum of 5c none form at neutral and acidic pH values and in a non-
Scheme 2
Eur. J. Org. Chem. 2002, 1149Ϫ1162
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P. Welzel et al.
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Table 1. Structures and fluorescence data of compounds 6aϪ6d
fluorescent spiro form at basic pH values (Scheme 3). Tetra- line of the green He-Ne laser, which is increasingly being
methylrhodamine is readily excited by the intense 546 nm used for analytical purposes such as fluorescence correla-
spectral line of mercury arc lamps or by the 543 nm spectral tion spectroscopy. For conjugation, the rhodamines usually
Scheme 3
1152
Eur. J. Org. Chem. 2002, 1149Ϫ1162

Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
FULL PAPER
have a carboxy or an amino (isothiocyanato) group in posi- ESI FT ICR MS confirmed the structure 6d. Comparison
tion(s) 5 and/or 6 (spiro form).[29] The commercially avail- of the fluorescence spectra of 6d with those of SITS (emis-
able pure tetramethylrhodamine isothiocyanate isomers sion at 421 nm, excitation at 356 nm for SITS) in the same
(Molecular Probes) were not used because of their high solvent system showed red shifts (ϩ9 nm) in the emission
cost, whilst the mixture of the two isomers (Fluka) was and the excitation (ϩ10 nm) maxima.
found to be of unsatisfactory purity. The same problems
Control experiments were performed to test whether the
with the purity of the commercial tetramethylrhodamines isothiocyanates reacted selectively with the amino group of
have been reported previously.[30] A synthesis of the iso- 3b. Thus, moenomycin A (1) was treated with 5c and the
meric 5- and 6-amines (spiro form numbering) has been de- tetramethylrhodamine-derived isothiocyanate 7b under ex-
scribed by Corrie and Craik,[30] and 7a was prepared by actly the same conditions as used for the preparation of 6a
this route. The 13C NMR spectra of amine 7a, with the 13
C
and 6b. After 72 h, no product formation could be observed
signal of the spiro carbon atom at δ ϭ 83.90 (C-9Ј), con- by TLC.
firmed the proposed structure. The corresponding isothi-
ocyanate 7b was prepared as described for 5c, purified and
immediately used for coupling with 3b.
Conjugation of Coumarin 5b and 6-
Aminotetramethylrhodamine (7a) to 3b through the Squaric
Acid Linker
MoenomycinϪtetramethylrhodamine derivative 6b was
characterized, after careful purification, by FAB MS and
ESI FT ICR MS. The mass spectra were consistent with
structure 6b. NMR analysis in this case was not successful
Diethyl squarate as a bifunctional coupling reagent for
bioconjugation was introduced by Tietze and co-
workers.[20,21] The method is based on the fact that, under
neutral conditions, only one of the ethoxy groups is re-
placed by primary or secondary amines whereas under ba-
sic conditions the corresponding symmetrical and unsym-
metrical diamides, respectively, are formed. In the majority
of cases, only aliphatic amines have been conjugated in this
way. We found that diethyl squarate failed to react with 5b
and 7a under the conditions used for the preparation of
aliphatic amide esters (methanolic or ethanolic solution,
room temperature, Scheme 4). However, 5b did form amide
1
for two reasons: (i) the signals were broad both in the H
and in the 13C spectra, and (ii) only a small amount of 6b
was available. In the fluorescence spectrum of 6b, a small
blue shift (compared with 7a) in the excitation (Ϫ3 nm) and
a red shift in emission (ϩ6 nm) maxima were observed.[9]
A derivative of 6b lacking the carbamoyl group in unit F
of the moenomycin part[31] has been used to demonstrate
complex formation between moenomycin and moenomy-
cin-binding aptamers.[11]
Conjugates 6c and 6d
Fluorescein isothiocyanate (FITC, the 5-isomer) belongs
to the most common fluorescent derivatization reagents.
Fluorescein has an excitation maximum that closely
matches the 488 nm spectral line of the argon ion laser, and
at pH Ͼ 7 it has excellent fluorescence properties. However,
it suffers from photobleaching, and the fluorescence is sig-
nificantly reduced at pH Ͻ 7 (pKa ϭ 6.4).[9] We coupled
commercial FITC with 3b under the conditions reported
1
above. The H NMR and the ESI FT ICR mass spectra of
compound 6c were in full accord with the proposed struc-
ture. The fluorescence spectra of 6c were practically ident-
ical with the fluorescence spectra of fluorescein isothiocy-
anate measured in the same solvent system.[9]
4-Acetamido-4Ј-isothiocyanatostilbene-2,2Ј-disulfonic
acid disodium salt (SITS) was conjugated with 3b because
it is soluble in water. Conjugate 6d was obtained in 63%
1
yield. The structure of 6d was elucidated from its H and
13C NMR spectra, with the aid of 2D NMR experiments.
All carbon and proton signals were assigned for the fluores-
cent label (unit SITS), the nitroaromatic ring and the ethyl-
enediamine-derived component (Ar and DAE), the triazole
ring (TA) and the carboxypropionyl residue (A), the lipid
component (I), and the glyceric acid moiety (H). The thiou-
reido carbon signal at δ ϭ 182.28 and the signals of the N-
acetyl, amido, and carbamoyl groups were also observed.
1H and 13C signals characteristic of the sugar part (signals
of all anomeric hydrogen and carbon atoms, CH2-6D, CH2-
6E, C-2C,E) demonstrated that all sugar units were present. Scheme 4
Eur. J. Org. Chem. 2002, 1149Ϫ1162
1153

P. Welzel et al.
FULL PAPER
ester 8 in satisfactory yields when an excess of diethyl sults of ESI FT ICR MS were also in full accord with the
squarate was used and the reaction was performed in meth- structure. The fluorescence spectra of compounds 10a and
anolic or ethanolic solution at room temperature in the 10b were practically identical with those of compounds 6a
presence of triethylamine. For the synthesis of 9 from di- and 6b, respectively (Table 2). For compounds containing
ethyl squarate and 7a, the reaction mixture was heated to the squaric acid linker, an additional maximum at ca.
60 °C. In both cases the reaction stopped at the squaric 351 nm (low intensity) in the excitation spectra was ob-
amide ester stage. Previously, aromatic squaric amide esters served. From the observations reported here, it seems clear
had been briefly described by Maahs and Hegenberg,[32] but that, in cases in which an aromatic and an aliphatic amine
without experimental details.
are to be conjugated through diethyl squarate, the amide
1
The H NMR and FAB mass spectra of both 8 and 9 ester of the aromatic amine has to be formed first, and that
were fully in agreement with the proposed structures. Treat- this may then be treated with the aliphatic amine under
ment of 8 with 3b was performed in methanol/Et3N solu- basic conditions to provide the mixed diamide.
tion, and treatment of 9 in buffer solution (at pH ϭ 9.0)
Synthesis of Rhodamine 6G Conjugate 10c
because of the good solubility of 9 in water. In the case of
9, an excess of 5 was used since only a small amount of the
A major improvement in the synthesis of rhodamine con-
dye was available. Compounds 10a and 10b were carefully jugates was recently reported by Adamczyk and Grote.[33]
purified and were obtained in good yields. For compound These authors showed that rhodamine 2Ј-esters such as
1
10a, good quality H and 13C NMR spectra were obtained rhodamine 6G (11) react selectively with primary amines to
1
in CD3OD/[D6]DMSO solution. The signals in the H and provide rhodamine amides of type 12a, which exist in their
13C NMR spectra of 10a were assigned by comparison with fluorescent open form at low to neutral pH and in the non-
1
1
the H and 13C NMR spectra of 6a. The H NMR spec- fluorescent spirolactam form (shown here) under basic con-
trum of 10b in most solvents (CD3OD and [D6]DMSO or ditions. The method of Adamczyk and Grote avoids the
mixtures of them) displayed very broad signals. This may problem of working with mixture of 5- and 6-functionalized
have been the result of micelle formation and/or the pres- rhodamines as well as the need to synthesize the pure iso-
ence of the quinone form. However, characteristic signals mers (vide supra). Furthermore, the method does not neces-
were found in the 13C NMR spectrum of 10b, and the re- sitate working with fully N-alkylated rhodamines, and so
Table 2. Structures and fluorescence data of compounds 10aϪ10c
1154
Eur. J. Org. Chem. 2002, 1149Ϫ1162

Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
FULL PAPER
Scheme 5
the inexpensive rhodamine 6G (11) can be used as the groups were also identified. Distinction between the carbon
fluorophore. We combined the Adamczyk-Grote approach signals of the AE and the DAE units was difficult because
with the squaric acid linker technology. Thus, treatment of of the similar surroundings of these groups, and the assign-
11 with a large excess of ethylenediamine in DMF at room ments were based on comparison with the 13C NMR spec-
temp. afforded compound 12a in over 90% yield trum of 12b.
(Scheme 5). All the spectra of 12a (spiro carbon signal at
δ ϭ 65.11) were in full agreement with the proposed struc-
ture. Replacement of the ethoxy group in diethyl squarate
Synthesis of Rhodamine 6G Conjugate 15
by the amino group of 12a was performed in methanol/
We recently demonstrated that moenomycin derivatives
chloroform solution at room temp., and furnished amide
lacking the lipid chain nevertheless bind to the enzyme PBP
ester 12b in 79% yield. The proton and carbon signals of
1b through the sugar moiety.[16] Thus, fluorescent derivat-
12b in the 1H and 13C NMR spectra were assigned with the
ives without the lipid chain may be of interest for study of
aid of 2D NMR experiments. Hindered rotation around the
the interaction between moenomycin derivatives and PBP
CϪNH bond in the squaric amide ester[20] resulted in the
1b by fluorescence correlation spectroscopy (FCR). We pre-
appearance of two signals for the CH2-2AE group at δ ϭ
pared one compound of this type from aldehyde 13 by re-
3.38 and 3.44 and two NH (squaric acid part) signals at
ductive amination. Compound 13 is available from moeno-
δ ϭ 5.96 and 6.93 (each corresponding to 0.5 H). The
mycin in high yield by ozonolysis. The amine component
was prepared as follows. Rhodamine 6G was treated with
1
ethoxy group H signals were also doubled. To define the
CH2-1AE and CH2-2AE signals in the H NMR spectrum
1
4,7,10-trioxatridecane-1,13-diamine as described above to
(as well as in the 13C NMR spectrum), HMBC was used.
furnish 14 in 73% yield. Reductive amination of 13 with 14
It was shown that the protons with signals at δ ϭ 3.31 had
provided the desired conjugate 15, albeit in low yield (18%,
long-range couplings to the three carbon atoms C-2AE, C-
Scheme 6). The 1 H, 31P, and 13C NMR and the ESI mass
9ЈR6G, C-3R6G, and thus were the CH2-1AE protons. The
spectra were fully in agreement with structure 15.
other protons, with signals at δ ϭ 3.38, had long-range
couplings to carbon atoms C-1AE and C-3SA and had to
belong to the CH2-2AE group. In the 13C NMR spectrum
of 12b, the spiro carbon signal (at δ ϭ 65.71) and the char-
acteristic double signal of C-2AE were observed. Compound
Antibiotic Properties of Selected Moenomycin Conjugates
The MIC (Minimum Inhibitory Concentration) values
12b was then conjugated with 3b in the usual way to give against seven different Staphylococcus aureus strains
10c. The structure of 10c was elucidated from its high-qual- (ATCC 25923, ATCC 29213, MRSA 1309, SG 511, PEG
1
ity H and 13C NMR spectra with the aid of 2D NMR 18, PEG 5, KNS PEG 5) were determined by a serial
experiments. All carbon and hydrogen signals of the rhoda- double microdilution method on microtiter plates as de-
mine moiety (R6G), the squaric acid unit (SA), the nitro- scribed previously.[10] All compounds displayed antibiotic
aromatic residue (Ar), the triazole ring (TA), the carboxy- activity, although to a much smaller extent than moenomy-
propionyl unit (A), and the lipid component (I) were as- cin A (Table 3). This means that they should be useful in
signed. The characteristic signals of the sugar carbon atoms study by FCS of, for example, the interaction between mo-
(anomeric carbon atoms, C-2C,E, C-4C,E, C-6D,E) and the enomycin analogues and the transglycosylase in competi-
signals of N-acetyl, carbamoyl, amido, and carboxy (H) tion experiments.
Eur. J. Org. Chem. 2002, 1149Ϫ1162
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P. Welzel et al.
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Scheme 6
Experimental Section
Table 3. Minimum inhibitory concentration of moenomycin-NH2
and derivatives against a number of Staph. aureus strains
Gerneral: All O2- 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 aluminum
caps with open top and Teflon-faced septum (Aldrich). ‘‘Usual
workup’’ means partitioning of the reaction mixture between an
aqueous phase and CH2Cl2, drying of the combined organic solu-
tions with Na2SO4, and removal of solvent by distillation with a
rotary evaporator (bath temperature 45 °C). Solvents were purified
by standard techniques. The following materials and methods were
used for chromatographic separations: flash chromatography
(FC):[34] 32Ϫ63 µm silica gel (ICN Biomedicals); medium-pressure
liquid chromatography (MPLC): 40-60 µm silica gel (Grace), Dura-
mat pump (CfG); analytical TLC: Merck precoated 60 F254 silica
gel plates (0.2 mm), spots were identified under a UV lamp (λ ϭ
254 nm, Camag 29 200) and 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)[35] and heating at 140 °C, or an anisaldehyde reagent for
carbohydrates [2 mL of anisaldehyde, 8 mL of conc. H2SO4 in eth-
anol (190 mL)]. Medium-pressure liquid chromatography (MPLC):
LiChroprep RP-18 40Ϫ63 µm material (Merck) was used; the
samples were applied to a precolumn (ca. 2 g of RP-18 material)
and eluted at 1.5Ϫ2.5 bar with a Duramat (CFG) dosage pump.
Analytical HPLC was performed with an HPLC system (Jasco)
consisting of an intelligent PU-980 HPLC pump, a DG-980-05 3-
MIC
µmol/L
µg/mL
1
0.0069
0.15
1.35
0.70
0.58
0.23
1.62
0.029
0.27
2.95
1.63
1.25
0.53
3.75
3b
6a
6b[a]
6c
6d
10c
[a]
Result based on one measurement.
Conclusion
A number of fluorescent moenomycin derivatives have
been prepared. Some of them have already shown their
merits in biophysical studies of moenomycin membrane an-
choring and intervesicle transfer, as well as in fluorescence
correlation spectroscopy (FCS) investigation of the binding
of moenomycin to aptamers.
1156
Eur. J. Org. Chem. 2002, 1149Ϫ1162

Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
FULL PAPER
line degasser, an LG-980-02 ternary gradient unit, and an MD-910 benzamide (270 mg, 98%). 13C NMR (50 MHz, CD3OD): δ ϭ 36.9
multiwavelength detector. As eluent a mixture of buffer (prepared (CH2DAE), 45.2 (CH2PIP), 53.9 (CH2PIP), 57.3 (CH2DAE), 112.4, 113.3
from 0.6 g of KH2PO4, 26.2 g of K2HPO4 ϫ 3H2O, 3.0 g of 1- (C-6Ar, C-4Ar), 127.9 (C-3Ar), 134.0 (C-1Ar), 136.7 (C-2Ar), 155.4
heptanesulfonic acid sodium salt monohydrate, and 1000 mL of
water, pH ϭ 8.0) and acetonitrile (60:40) was used. A 250 ϫ
4.6 mm, nucleosil 300, C18, 5 µm column (Jasco) with a pre-col-
umn was used. Ultrafiltration (UF) was performed using gas-pres-
surized (N2, 3.5 bar), stirred ultrafiltration cells (Amicon, model
8050, 50 mL cell capacity and model 8400, 400 mL capacity) with
YM3 membrane type (Amicon, molecular weight cut-off 3000 Da).
NMR and MS equipment: NMR: UNITY 400 (Varian), DRX 400
(Bruker), DRX 600 (Bruker), GEMINI 200 (Varian), GEMINI
2000 (Varian); MS: FAB MS: VG Autospec (Fisons, matrix: 3-
nitrobenzyl alcohol), ESI MS: FT ICR MS APEX II (Bruker Dal-
tonics, water-methanol). Following the molecular formula, two
masses are always communicated: the first is calculated using the
International Atomic Masses, the second is the monoisotopic mass.
IR: Genesis FTIR (ATI Mattson). For the description of the NMR
spectra the protons and carbon atoms are indexed according to the
indices in the formulae. Fluorescence spectra were recorded with a
Fluoromax-2 (SPEX) in MeOH/H2O (1:1) solution at pH ϭ 6.9.
The fluorescence excitation and emission were corrected according
to the lamp spectrum and according to the photomultiplyer sensit-
ivity, respectively. The maxima of the excitation spectra indicate
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, PEG
18, PEG 5, KNS PEG 5) were determined by a serial double micro-
dilution method on microtiter plates (Iso-Sensitest medium,
Oxoid). A series of decreasing concentrations of the compound un-
der 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 calcu-
lated as the average values from three measurements. For the NMR
spectra of compounds 3c, 3d, 6a, 6c, 6d, 10a, 10b, 10c, and 15 see
Supporting Information (see also the footnote on the first page of
this article).
(C-5Ar), 170.1 (amide CO).
(R)-3-({(5R)-5-[3-(3-Carboxypropionyl)-1-(3-{[2-piperazin-1-yl-
ethyl]carbamoyl}-4-nitrophenyl)-1H-1,2,4-triazol-5-yl]-α-
pyranosyl-(1Ǟ4)-2-acetamido-2, 6-dideoxy-β-
L
-arabino-
-gluco-
D
pyranosyl-(1Ǟ4)-[β-
D-glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-
β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α-D-
D
glucopyranuronamidosyloxy}hydroxyphosphoryloxy)-2-
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-
nonadecatetraenyloxy)propionic Acid (3c): A solution of sodium ni-
trite (4.5 mg, 0.07 mmol) in water (1 mL) was added dropwise at 0
°C to a solution of 5-amino-2-nitro-N-(2-piperazin-1-yl-ethyl)-
benzamide (20.0 mg, 0.07 mmol) in 9% HCl (0.5 mL). After 15 min
at 0 °C, this solution was added dropwise to a solution of 1
(100 mg, 0.06 mmol) and sodium acetate (800 mg) in water (40
mL), and the mixture was stirred at 20 °C. Progress of the reaction
was monitored by RP-HPLC-DAD [buffer/acetonitrile (59:41)].
After 48 h, the intermediate amidrazone (formula not shown) had
been completely converted into triazole 3c. Ultrafiltration followed
by FC [CHCl3/methanol/H2O (18:11:2.7)] furnished 3c (74.8 mg,
63%). Rt ϭ 12.4 min, RP-HPLC [buffer/acetonitrile (59:41), λmax ϭ
279 nm]. C82H124N11O37P (1886.91, 1885.79), ESI FT ICR MS:
m/z ϭ 941.8894 (calcd. 941.8876) [M Ϫ 2 H]2Ϫ, 952.8791 (calcd.
952.8786) [M Ϫ 3 H ϩ Na]2Ϫ
.
5-Amino-2-nitro-N-{2-[5-(2-oxohexahydrothieno[3,4-d]imidazol-6-
yl)pentanoylamino]ethyl}benzamide (Formula not Shown): A solu-
tion of DCC (160 mg) in DMF (10 mL) was added dropwise to a
solution of biotin (200 mg, 0.82 mmol) and N-hydroxysuccinimide
(120 mg) in DMF (20 mL) and the mixture was stirred at 20 °C
overnight. After filtration and solvent evaporation, the residue was
redissolved in DMF (10 mL), a solution of 2 (320 mg of the diacet-
ates, ca. 1 mmol) in DMF (5 mL) was added dropwise, and the
mixture was stirred at 20 °C for 20 h. Solvent evaporation and FC
[(i): CHCl3/methanol (5:1); (ii) CHCl3/methanol/ethyl acetate
(5:10:5)] provided 250 mg (68%, based on biotin) of 5-amino-2-
nitro-N-{2-[5-(2-oxohexahydrothieno[3,4-d]imidazol-6-yl)-
pentanoylamino]ethyl}benzamide. 13C NMR (50 MHz, APT,
[D6]DMSO, two sets of signals): δ ϭ 25.5, 28.3, 28.5, 35.5, (C-
2BTK, C-3BTK, C-1BTK,C-4BTK), 38.3 (C-1DAE, C-2DAE), 55.7, 59.5,
59.6, 61.3, 61.4 (C-4BTR, C-3aBTR, C-6aBTR), 112.2 (C-6Ar), 112.7
(C-4Ar), 127.7 (C-3Ar), 133.2 (C-1Ar), 137.1, 137.2 (C-2Ar), 154.9,
154.9 (C-5Ar), 163.2, 162.3 (COBTR), 167.6, 167.7 (CONHAr sig-
nals), 172.8, 172.8 (CONHBTK). C19H26N6O5S (450.51, 450.17),
FAB MS: m/z ϭ 451.2 [M ϩ H]ϩ, 473.2 [M ϩ Na]ϩ.
(R)-3-({(5R)-5-[3-(3-Carboxypropionyl)-1-(3-{[2-aminoethyl]-
carbamoyl}-4-nitrophenyl)-1H-1,2,4-triazol-5-yl]-α-
pyranosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β- -glucopyranosyl-
(1Ǟ4)-[β- -glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-β- -gluco-
pyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α- -gluco-
L-arabino-
D
D
D
D
pyranuronamidosyloxy}hydroxyphosphoryloxy)-2-[(2Z,6E,13E)-
3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nonadecatetra-
enyloxy]propionic Acid (3b): The preparation and spectroscopic
data of this compound have been described elsewhere.[16] Here we
want to emphasize that the NMR spectra were taken after RP18
chromatography [Kieselgel 60M C18, MachereyϪNagel, H2O, 300
mL, then the compound was eluted with acetonitrile/water (1:2)].
The sample thus obtained contained small amounts of impurities
originating from the column material. These impurities could be
removed by FC [SiO2, nPrOH/H2O (7:3)] to give 269 mg (78%) of
pure compound 3b.
(R)-3-({(5R)-5-[3-(3-Carboxypropionyl)-1-(3-{[2-{[(3aS)-2-oxo-
(3ar,6ac)-hexahydro-1H-thieno[3,4-d]imidazol-4t-yl]pentanoyl-
amino}ethyl]carbamoyl}-4-nitrophenyl)-1H-1,2,4-triazol-5-yl]-
α-
pyranosyl-(1Ǟ4)-[β-
2-deoxyβ- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-
α- -glucopyranuronamidosyloxy}hydroxyphosphoryloxy)-2-
5-Amino-2-nitro-N-(2-piperazin-1-yl-ethyl)benzamide (Formula not [(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-
L
-arabinopyranosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β-
D-gluco-
D
-glucopyranosyl-(1Ǟ6)]-2-acetamido-
D
D
Shown): Carbonyl diimidazole (267 mg, 1.6 mmol) was added to
a solution of 5-amino-2-nitrobenzoic acid (200 mg, 1.1 mmol) in
pyridine (20 mL), and the mixture was stirred at 20 °C for 30 min
nonadecatetraenyloxy]propionic Acid (3d): A solution of sodium
nitrite (9.9 mg, 0.07 mmol) in water (1 mL) was added dropwise at
0 °C to a solution of 5-amino-2-nitro-N-{2-[5-(2-oxohexahydro-
[TLC monitoring: methanol/CHCl3 (1:1)]. A solution of 2-amino- thieno[3,4-d]imidazol-6-yl)pentanoylamino]ethyl}benzamide
ethylpiperazine (350 mg, 2.7 mmol) in pyridine (20 mL) was then (65 mg, 0.14 mmol) in 9% HCl (2 mL, dissolved at elevated temper-
added, and the reaction mixture was stirred at 20 °C for 60 min
and at 80 °C for 90 min. Solvent evaporation and FC [methanol/
CHCl3 (2:1)] provided 5-amino-2-nitro-N-(2-piperazin-1-yl-ethyl)-
ature and then cooled to 0 °C). The mixture was left at 0 °C for
15 min and was then added dropwise to a solution of 1 (170 mg,
0.06 mmol) and sodium acetate (1000 mg) in water (30 mL). The
Eur. J. Org. Chem. 2002, 1149Ϫ1162
1157

P. Welzel et al.
FULL PAPER
resulting mixture was stirred at 20 °C. Progress of the reaction was 8a), 162.9 (C-2). C20H22N2O2 (322.40, 322.17), FAB MS: m/z ϭ
monitored by RP18 HPLC [buffer/acetonitrile (59:41), diode array
detection]. After 48 h, the intermediate amidrazone (formula not
shown) had been completely converted into 3d. Ultrafiltration fol-
lowed by FC [CHCl3/methanol/H2O (18:11:2.7)] furnished 3d
(157.1 mg, 72%) and, as a by-product, a 1-nitrosoimidazole derivat-
ive [RP HPLC, buffer/acetonitrile (63:37), Rt ϭ 22 min]. 13C NMR
signal of the biotin part at δ ϭ 156.8. C88H130N13O40PS (2073.09,
2071.79), FAB MS: m/z ϭ 2095.3 [M ϩ Na]ϩ. Data of 3d: RP18
HPLC [buffer/acetonitrile (63:37) Rt ϭ 16 min, λmax ϭ 275 nm].
C88H131N12O39PS (2044.09, 2042.81), FAB MS: m/z ϭ 2067.0 [M
ϩ Na]ϩ, ESI FT ICR MS: m/z ϭ 1020.3945 (calcd. 1020.3975) [M
323.1 [M ϩ H]ϩ.
7-(Diethylamino)-3-(4-isothiocyanatophenyl)-4-methyl-2H-chromen-
2-one (5c): A solution of amine 5b (112 mg, 0.347 mmol) in chloro-
form was slowly added to a well stirred heterogeneous mixture of
CHCl3/H2O (5:1) (6 mL), CaCO3 (100 mg), and thiophosgene
(45.1 mg, 0.392 mmol). The mixture was stirred at 20 °C and the
reaction was monitored by TLC [CHCl3/methanol (20:1)]. After
2 h, water was added and the mixture was extracted with CHCl3.
The organic extract was dried with sodium sulfate. Solvent was
evaporated under reduced pressure, and the residue was purified
by FC [CHCl3/methanol (20:1)] to provide 68 mg (54%) of pure
crystalline isothiocyanate 5c. Rf ϭ 0.86 [CHCl3/methanol (20:1)].
Ϫ 2H]2Ϫ, 1031.3873 (calcd. 1031.3884) [M Ϫ 3 H ϩ Na]2Ϫ
679.9298 (calcd. 679.9292) [M Ϫ 3 H]3Ϫ
,
.
˜
M.p. 165 °C [CHCl3/methanol (20:1)]. IR (KBr): ν ϭ 3600Ϫ3400
(bs), 2113 (NCS), 1704, 1616, 1584, 1522, 1408, 1353, 1271, 1144,
3-(4-Acetamidophenyl)-7-(diethylamino)-4-methyl-2H-chromen-2-
one (5a): A mixture of 7-diethylamino-4-methylcoumarin (0.5 g,
2 mmol), acetone (11 mL), sodium acetate (2 g), and CuCl2 (80 mg)
was stirred in a two-necked, round-bottomed flask equipped with
a bubbler and a dropping funnel. A cold solution of the diazonium
salt prepared from N-acetyl-p-phenylenediamine (0.45 g, 3 mmol)
was added dropwise to this mixture at such a rate that the nitrogen
evolution was slow (1Ϫ2 bubbles/s). When nitrogen formation was
no longer observed, the mixture was acidified and extracted with
chloroform. The aqueous layer was rendered alkaline and than ex-
tracted with diethyl ether. The ether and chloroform extracts were
collected, washed with water, and dried with sodium sulfate. Solv-
ent evaporation under reduced pressure and purification by FC
[SiO2, CHCl3/toluene/methanol (10:10:1)] gave 207 mg (30%) of 5a.
1075 cmϪ1
.
1H NMR (200 MHz, [D6]DMSO): δ ϭ 1.13 [t, J ϭ
7.1 Hz,
6
H, N(CH2CH3)2], 2.19 (s, 3 H, CH3-9), 3.47
[N(CH2CH3)2, hidden by the water signal], 6.56 (d, J8Ϫ6 ϭ 2.6 Hz,
1 H, 8-H), 6.74 (dd, J6Ϫ8 ϭ 2.6 Hz, J6Ϫ5 ϭ 9.1 Hz, 1 H, 6-H), 7.37
(d, J2Ј-3Ј ϭ 8.4 Hz, 2 H, 2Ј-H, 6Ј-H), 7.49 (d, J3Ј-2Ј ϭ 8.4 Hz, 2 H,
3Ј-H, 5Ј-H), 7.59 (d, J5Ϫ6 ϭ 9.1 Hz, 1 H, 5-H). 13C NMR (50 MHz,
CDCl3): δ ϭ 12.6 [N(CH2CH3)2], 16.4 (C-9), 45.2 [N(CH2CH3)2],
98.0 (C-8), 109.2 (C-6), 109.8 (C-4a), 119.9 (C-3), 125.7 (C-3Ј, C-
5Ј), 126.3 (C-5), 130.7 (C-1Ј), 132.1 (C-2Ј, C-6Ј), 134.8, 136.0 (C-
4Ј, NCS), 148.7 (C-4), 150.4 (C-7), 155.3 (C-8a), 161.7 (C-2).
C21H20N2O2S (364.46, 364.12), FAB MS: m/z ϭ 365.1 [M ϩ H]ϩ.
Fluorescence spectrum [methanol/H2O (1:1), 0.03 mg/5 mL]: ex-
citation (478 nm): λmax ϭ 396 nm, emission (396 nm): λmax
ϭ
478 nm.
˜
Rf ϭ 0.28 [CHCl3/toluene/methanol (10:10:1)]. IR (KBr): ν ϭ 3432,
1685, 1616, 1587, 1525, 1407, 1365, 1313, 1270 cmϪ1 1H NMR
.
6-Amino-3Ј,6Ј-bis(dimethylamino)spiro[1,3-dihydroisobenzofuran-
1,9Ј-xanthen]-3-one (7a): This compound was prepared as described
by Corrie and Craik.[30] 1H NMR (200 MHz, CD3Cl): δ ϭ 2.97 [s,
12 H, 2N(CH3)2], 4.11 (s, 2 H, 6-NH2), 6.26 (d, J7Ϫ5 ϭ 2.0 Hz, 1
H, 7-H), 6.41 (dd, J2Ј-1Ј ϭ 8.7 Hz, J2Ј-4Ј ϭ 2.6 Hz, 2 H, 2Ј-H, 7Ј-
(400 MHz, CDCl3, HMQC, HMBC): δ ϭ 1.23 [t, J ϭ 7.0 Hz, 6 H,
N(CH2CH3)2], 2.16 (s, 3 H, CH3CONH), 2.23 (s, 3 H, CH3-9), 3.44
[q, J ϭ 7.0 Hz, 4 H, N(CH2CH3)2], 6.54 (d, J8Ϫ6 ϭ 2.6 Hz, 1 H, 8-
H), 6.64 (dd, J6Ϫ5 ϭ 8.8 Hz, J6Ϫ8 ϭ 2.6 Hz, 1 H, 6-H), 7.19 (d, J2Ј-
ϭ 8.8 Hz, 2 H, 2Ј-H, 6Ј-H), 7.46 (d, J5Ϫ6 ϭ 8.8 Hz, 1 H, 5-
3Ј
H), 6.46 (d, J4Ј-2Ј ϭ 2.4 Hz, 2 H, 4Ј-H, 5Ј-H), 6.72 (d, J1Ј-2Ј
ϭ
H), 7.47 (d, J3Ј-2Ј ϭ 8.4 Hz, 2 H, 3Ј-H, 5Ј-H), 7.81 (broad s, 1 H,
CH3CONH). 13C NMR (50 MHz, CDCl3, HMQC, HMBC): δ ϭ
12.9 [N(CH2CH3)2], 16.8 (C-9), 24.9 (CH3CONH), 45.2
[N(CH2CH3)2], 97.9 (C-8), 109.2 (C-6), 110.1 (C-4a), 120.3 (C-3Ј,
C-5Ј), 121.4 (C-3), 126.6 (C-5), 131.3 (C-2Ј, C-6Ј), 131.4 (C-1Ј),
138.0 (C-4Ј), 149.3 (C-4), 150.8 (C-7), 155.5 (C-8a), 163.0 (C-2),
169.0 (CH3CONH). C22H24N2O3 (364.44, 364.18), FAB MS: m/z ϭ
365.2 [M ϩ H]ϩ.
8.6 Hz, 2 H, 1Ј-H, 8Ј-H), 6.74 (dd, J5Ϫ7 ϭ 2.0 Hz, J5Ϫ4 ϭ 8.2 Hz,
1 H, 5-H), 7.74 (d, J4Ϫ5 ϭ 8.2 Hz, 1 H, 4-H). 13C NMR (50 MHz,
CD3Cl, APT): δ ϭ 40.9 [N(CH3)2], 83.9 (C-9Ј), 99.2 (C-4Ј, C-5Ј),
106.2 (C-8Јa, C-9Јa), 108.2 (C-3a), 109.3 (C-2Ј, C-7Ј), 116.7 (C-5),
117.6 (C-7a), 120.5 (C-7), 126.9 (C-4), 129.5 (C-1Ј, C-8Ј), 152.6 (C-
3Ј, C-6Ј), 153.2, 153.3 (C-4Јa, C-10Јa), 157.5 (C-6), 170.7 (C-3).
C24H23N3O3 (401.46, 401.17), FAB MS: m/z ϭ 424.2 [M ϩ Na]ϩ,
402.1 [M ϩ H]ϩ. Fluorescence spectrum [methanol/H2O (1:1),
0.0024 mg/5 mL]: excitation (570 nm): λmax ϭ 547 nm, emission
(547 nm): λmax ϭ 571 nm.
3-(4-Aminophenyl)-7-(diethylamino)-4-methyl-2H-chromen-2-one
(5b): A solution of 5a (10 mg, 0.027 mmol) in 6% HCl in ethanol
was stirred at 80 °C. Progress of the reaction was monitored by
TLC [CHCl3/methanol (10:1)]. After 3 h, the mixture was neutral-
ized with potassium carbonate and extracted with CHCl3. The ex-
tract was washed with water and dried with Na2SO4. Solvents were arabinopyranosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β-
evaporated under reduced pressure, and the residue was purified by pyranosyl-(1Ǟ4)-[β-
FC [CHCl3/methanol (10:1)] to give 8 mg (93%) of free amine 5b. β-
(R)-3-({(5R)-5-[3-(3-Carboxypropionyl)-1-(3-{[2-(3-{4-[7-(diethyl-
amino)-4-methyl-2-oxo-2H-chromen-3-yl]phenyl}thioureido)-
ethyl]carbamoyl}-4-nitrophenyl)-1H-1,2,4-triazol-5-yl]-α-
-gluco-
-glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-
-glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α- -gluco-
L-
D
D
D
D
Rf ϭ 0.57 [CHCl3/methanol (10:1)]. IR (KBr): ν˜ ϭ 3428, 1695, pyranuronamidosyloxy}hydroxyphosphoryloxy)-2-[(2Z,6E,13E)-
1616, 1583, 1521, 1467, 1407, 1349, 1301, 1270, 1174, 1143, 1074 3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nonadeca-
cmϪ1 1H NMR (200 MHz, CDCl3): δ ϭ 1.22 [t, J ϭ 7.0 Hz, 6 tetraenyloxy]propionic Acid (6a): The coumarin isothiocyanate 5c
.
H, N(CH2CH3)2], 2.26 (s, 3 H, CH3-9), 3.43 [q, J ϭ 7.0 Hz, 4 H,
N(CH2CH3)2], 6.55 (d, J8Ϫ6 ϭ 2.6 Hz, 1 H, 8-H), 6.62 (dd, J6Ϫ8 ϭ (120 mg, 0.066 mmol) in dry DMF (10 mL) and dry pyridine (3
2.6 Hz, J6Ϫ5 ϭ 9.1 Hz, 1 H, 6-H), 6.74 (d, J3Ј-2Ј ϭ 8.4 Hz, 2 H, 3Ј- mL). After 12 h, the mixture became clear and was stirred under
(48 mg, 2 equiv.) in DMF (2 mL) was added to a solution of 3b
H, 5Ј-H), 7.10 (d, J2Ј-3Ј ϭ 8.4 Hz, 2 H, 2Ј-H, 6Ј-H), 7.44 (d, J5Ϫ6 ϭ argon and in darkness for an additional 12 h. Progress of the reac-
9.1 Hz, 1 H, 5-H). 13C NMR (50 MHz, CDCl3): δ ϭ 12.9 tion was monitored by TLC [CHCl3/methanol/H2O (20:11:2)]. The
[N(CH2CH3)2], 16.8 (C-9), 45.2 [N(CH2CH3)2], 98.0 (C-8), 108.9 solvents were then removed at ambient temperature under reduced
(C-6), 110.3 (C-4a), 115.3 (C-3Ј, C-5Ј), 125.7 (C-3), 126.5 (C-5), pressure, and the residue was purified by FC [SiO2; (i) CHCl3/meth-
131.9 (C-2Ј, C-6Ј), 146.3 (C-4Ј), 148.3 (C-4), 150.5 (C-7), 155.4 (C-
anol (20:11), (2) CHCl3/methanol/H2O (20:11:2)] to give 73 mg
1158
Eur. J. Org. Chem. 2002, 1149Ϫ1162

Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
FULL PAPER
(51%) of pure product 6a. For FRET experiments the substance
was further purified by repeated MPLC [RP18; acetonitrile/H2O
in the dark at 20 °C under argon. Progress of the reaction was
monitored by TLC [nPrOH/methanol/CHCl3/H2O (11:3:3:3)].
(1:1), Rf ϭ 0.40]. C99H137N12O39PS (2182.26, 2180.86), ESI FT After 30 h, no 3b was observed. Solvents were removed under re-
ICR MS: m/z ϭ 1089.4209 (calcd. 1089.4203) [M Ϫ 2 H]2Ϫ
duced pressure, and the residue was purified by FC [nPrOH/meth-
725.9463 (calcd. 725.9442) [M Ϫ 3 H]3Ϫ. Fluorescence spectrum anol/CHCl3/H2O (11:3:3:3)]. Removal of organic solvents under re-
,
[methanol/H2O (1:1), 0.13 mg/5 mL]: excitation (476 nm): λmax
397 nm, emission (397 nm): λmax ϭ 476 nm.
ϭ
duced pressure and water by lyophilization gave 10 mg (26%) 6c.
Rf 0.35 [nPrOH/methanol/CHCl3/H2O (11:3:3:3)].
ϭ
C99H128N11O42PS (2207.18, 2205.77), ESI FT ICR MS: m/z ϭ
1120.8519 (calcd. 1120.8545) [M ϩ K Ϫ 3 H]2Ϫ, 1112.8622 (calcd.
1112.8670) [M ϩ Na Ϫ 3 H]2Ϫ, 1101.8754 (calcd. 1101.8759) [M
Ϫ 2 H]2Ϫ, 746.9038 (calcd. 746.9006) [M ϩ K Ϫ H]3Ϫ, 741.5761
(calcd. 741.5753) [M ϩ Na Ϫ 4 H]3Ϫ, 734.2470 (calcd. 734.2480)
[M Ϫ 3 H]3Ϫ, 550.4358 (calcd. 550.4340) [M Ϫ 4 H]4Ϫ. Fluores-
cence spectrum [methanol/H2O (1:1), 0.14 mg/5 mL]: excitation
(515 nm): λmax ϭ 492 nm, emission (492 nm): λmax ϭ 521 nm.
Control Experiments ؊ Treatment of 1 with 5c and 7b: Compounds
1 (3.2 mg, 0.002 mmol) and 5c (0.73 mg, 0.002 mmol) in DMF (0.3
mL) and pyridine (0.025 mL) were stirred under argon at ambient
temperature. The reaction was monitored by TLC [SiO2; nPrOH/
H2O (7:3); RP18; acetonitrile/H2O (1:1)]. Even after 72 h, no prod-
ucts were observed. The same procedure was performed with 7b.
Again, no reaction product formation was observed. Rf ϭ 0.09
[CHCl3/methanol/H2O (20:11:2)].
(R)-3-[((5R)-5-{1-[3-({2-[3-[((E)-4Ј-Acetamido-2,2Ј-disulfostilben-4-
yl)thioureido]ethyl}carbamoyl)-4-nitrophenyl]-3-(3-carboxy-
(R)-3-({(5R)-5-[1-(3-{[2-(3-{3-[3,6-Bis(dimethylamino)xanthylium-9-
yl]-4-carboxyphenyl}thioureido)ethyl]carbamoyl}-4-nitrophenyl)-3-
propionyl)-1H-1,2,4-triazol-5-yl}-α-
2-acetamido-2,6-dideoxy-β- -glucopyranosyl-(1Ǟ4)-[β-
pyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-β- -glucopyranosyl-(1Ǟ2)-
3-O-carbamoyl-4-C-methyl-α- -glucopyranuronamidosyl-
L
-arabinopyranosyl-(1Ǟ4)-
(3-carboxypropionyl)-1H-1,2,4-triazol-5-yl]-α-
(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- -glucopyranuronamidosyl-
L-arabinopyranosyl-
D
D
-gluco-
D
D-
D
D
D
D
oxy)hydroxyphosphoryloxy]-2-[(2Z,6E,13E)-3,8,8,14,18-
pentamethyl-11-methylene-2,6,13,17-nonadecatetraenyl-
oxy]propionic Acid Disodium Salt (6d): A solution of 4-acetamido-
4Ј-isothiocyanatostilbene-2,2Ј-disulfonic acid disodium salt (SITS)
(11.9 mg, 0.023 mmol) in DMF (0.5 mL) and pyridine (0.1 mL)
was added to a solution of 3b (30 mg, 0.0165 mmol) in DMF (3
mL) and pyridine (0.45 mL). The mixture was stirred in darkness
at 20 °C under argon for 9 h. Progress of the reaction was mon-
itored by TLC [nPrOH/H2O (7:3)]. The solvents were then removed
under reduced pressure, and the residue was diluted with water (100
mL) and lyophilized. Water and Kieselguhr were added, and the
water was lyophilized. The Kieselguhr containing the adsorbed
compound was loaded onto the top of a FC column. Elution with
nPrOH/H2O (7:2) gave 25 mg (63%) of pure product 6d. Rf ϭ 0.53
[nPrOH/H2O (7:3)]. C95H129N12Na2O44PS3 (2316.25, 2314.69), ESI
oxy}hydroxyphosphoryloxy)-2-[(2Z,6E,13E)-3,8,8,14,18-penta-
methyl-11-methylene-2,6,13,17-nonadecatetraenyloxy]-
propionyl Chloride (6b):
A solution of amine 7a (19 mg,
0.047 mmol) was slowly added to a very well stirred suspension of
thiophosgene (5.4 mg, 0.047 mmol) in water:chloroform, 3:1 (3
mL) and CaCO3 (26 mg), and the mixture was stirred at 20 °C for
2 h. The reaction mixture was then neutralized with aq. KOH and
extracted with CHCl3. Solvent evaporation under reduced pressure
and purification by FC [(i) CHCl3, (ii) CHCl3/methanol (20:1, 10:1,
5:1) gave 5.1 mg (21%) of the desired isothiocyanate 7b. A solution
of 7b (5 mg, 0.01 mmol) in DMF (1 mL) and pyridine (0.2 mL)
was added to a solution of 3b (15 mg, 0.008 mmol) in DMF (1
mL). The resulting mixture was stirred in darkness at 20 °C under
argon. Progress of the reaction was monitored by TLC [SiO2;
nPrOH/H2O (7:2)] and [RP18; CHCl3/methanol (1:1)]. After 12 h,
the reaction was complete (no 3b was observed). Solvents were
evaporated under reduced pressure and the crude product was puri-
fied by FC [SiO2; nPrOH/H2O (7:2)] and then several times by
MPLC (RP18) in different solvent systems [acetonitrile/H2O (4:5,
2:3, 1:1)] to give 3.5 mg (18%) of pure 6b. Rf ϭ 0.13 [SiO2; nPrOH/
FT ICR MS: m/z ϭ 1156.3315 (calcd. 1156.3382) [M Ϫ 2 H]2Ϫ
,
1145.3478 (calcd. 1145.3473) [M Ϫ H Ϫ Na]2Ϫ, 770.5556 (calcd.
770.5562) [M Ϫ 3 H]3Ϫ, 763.2281 (calcd. 763.2289) [M Ϫ 2
HϪNa]3Ϫ, 755.9020 (calcd. 755.9016) [M Ϫ 2 Na Ϫ H]3Ϫ, 572.1703
(calcd. 572.1697) [M Ϫ 3 H Ϫ Na]4Ϫ, 566.6751 (calcd. 566.6743)
[M Ϫ 2 H Ϫ 2 Na]4Ϫ. Fluorescence spectrum [methanol/H2O (1:1),
0.21 mg/5 mL]: excitation (436 nm): λmax ϭ 346 nm, emission
(346 nm): λmax ϭ 412 nm.
H2O (7:2)], Rf
ϭ
0.42 [RP18; CHCl3/methanol (1:1)].
C103H138N13O40PS (2261.32, 2259.86), FAB MS: m/z ϭ 2260.6 [M
ϩ H]ϩ. ESI FT ICR MS: m/z ϭ 2260.8743 (calcd. 2260.8700) [M
ϩ H]ϩ, 1149.9212 (calcd. 1149.9169) [M ϩ H ϩ Na]2ϩ, 1141.9105
(calcd. 1141.9338) [M ϩ H ϩ K]2ϩ, 1130.9037 (calcd. 1130.9350)
3-{4-[7-(Diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl]phenyl}-4-
ethoxy-3-cyclobutene-1,2-dione (8):
A solution of 5b (5 mg,
[M ϩ 2 H]2ϩ, 766.9498 (calcd. 766.9472) [M ϩ 2 H ϩ K]3ϩ
,
0.015 mmol) and 3,4-diethoxy-3-cyclobutene-1,2-dione (2.5 mg,
0.013 mmol) in ethanol (0.65 mL) was stirred at room temperature.
Progress of the reaction was monitored by TLC [CHCl3/ethyl acet-
ate (4:1)]. Even after 5 h, however, no product spot was observed.
A few drops of Et3N and more diethyl squarate (10.2 mg,
754.2981 (calcd. 754.2892) [M ϩ 3 H]3ϩ. Fluorescence spectrum
[methanol/H2O (1:1), 0.10 mg/5 mL]: excitation (570 nm): λmax
544 nm, emission (544 nm): λmax ϭ 577 nm.
ϭ
(R)-3-[((5R)-5-{3-(3-Carboxypropionyl)-1-[3-({2-[3-(3Ј,6Ј-dihydroxy-
3-oxospiro[isobenzofuran-1(3H),9Ј-[9H]xanthen]-5-yl)thioureido]- 0.06 mmol) were then added, and the resulting mixture was stirred
ethyl}carbamoyl)-4-nitrophenyl]-1H-1,2,4-triazol-5-yl}-α-
arabinopyranosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β- -gluco-
pyranosyl-(1Ǟ4)-[β-
β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α-
pyranuronamidosyloxy)hydroxyphosphoryloxy]-2-[(2Z,6E,13E)-
L
-
at room temperature. After 4 h, no amine 5b was observed by TLC
[CHCl3/ethyl acetate (4:1)]. Solvent was removed under reduced
D
D
-glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy- pressure and the residue was purified by FC [CHCl3/ethyl acetate
D
D
-gluco-
(4:1)] to give 6 mg (90%) of a specimen of 8 that was not completely
1
free of solvent according to the H NMR. Rf ϭ 0.17 [CHCl3/ethyl
3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nonadeca- acetate (4:1)]. 1H NMR (200 MHz, CDCl3): δ ϭ 1.22 [t, J ϭ
tetraenyloxy)propionic Acid (6c): A solution of fluorescein iso-
7.0 Hz, 6 H, N(CH2CH3)2], 1.49 (t, J ϭ 7.0 Hz, 3 H, OCH2CH3),
thiocyanate (isomer I, Fluka, 9 mg, 0.023 mmol) in dry DMF (2 2.25 (s, 3 H, CH3-9Cou), 3.43 [q, J ϭ 7.0 Hz, 4 H, N(CH2CH3)2],
mL) was added to a solution of 3b (39 mg, 0.021 mmol) in dry 4.88 (q, J ϭ 7.0 Hz, 2 H, OCH2CH3), 6.55 (d, J8Ϫ6 ϭ 2.6 Hz, 1 H,
DMF (2 mL) and dry pyridine (0.5 mL). The mixture was stirred
8Cou-H), 6.64 (dd, J6Ϫ8 ϭ 2.6 Hz, J6Ϫ5 ϭ 9.0 Hz, 1 H, 6Cou-H),
Eur. J. Org. Chem. 2002, 1149Ϫ1162
1159

P. Welzel et al.
FULL PAPER
7.27 (d, J2Ј-3Ј ϭ 8.8 Hz, 2 H, 2ЈCou-H, 6ЈCou-H), 7.34 (d, J3Ј-2Ј
ϭ
pH ϭ 9.03) were stirred under argon at 20 °C. The progress of the
reaction was monitored by TLC [nPrOH/H2O (7:2.5)]. When 9
8.6 Hz, 2 H, 3ЈCou-H, 5ЈCou-H), 7.46 (d, J5Ϫ6 ϭ 9.0 Hz, 1 H, 5Cou
-
H). C26H26N2O5 (446.50, 446.18), FAB MS: m/z ϭ 460.0 [M ϩ could no longer be detected (after 72 h), nPrOH (14 mL) was added
Na]ϩ, 447.5 [M ϩ H]ϩ.
to the reaction mixture and this solution was transferred onto the
top of a FC column. Elution with nPrOH/H2O (7:2.5), solvent
evaporation, and lyophilization gave 28 mg of a crude product.
Further purification by FC [ethyl acetate/ethanol/H2O (5:4:2.5)]
provided 24 mg (58%) of pure product 10b. Rf ϭ 0.20 [nPrOH/H2O
(R)-3-({(5R)-5-[3-(3-Carboxypropionyl)-1-(3-{[2-(2-{4-[7-
(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl]anilino}-3,4-dioxo-
1-cyclobuten-1-yl)ethyl]carbamoyl}-4-nitrophenyl)-1H-1,2,4-triazol-
5-yl]-α-
glucopyranosyl-(1Ǟ4)-[β-
deoxy-β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-methyl-
α- -glucopyranuronamidosyloxy}hydroxyphosphoryloxy)-2-
L
-arabinopyranosyl-(1Ǟ4)-2-acetamido-2,6-dideoxy-β-
D-
1
(7:2.5)]. H NMR {600 MHz, [D6]DMSO and 400 MHz, CD3OD/
D
-glucopyranosyl-(1Ǟ6)]-2-acetamido-2-
[D6]DMSO (2:1)} displayed very broad signals, especially for the
sugar and the aromatic protons. C106H138N13O42P (2297.29,
2295.88), ESI FT ICR MS: m/z ϭ 1157.9266 (calcd. 1157.9231) [M
D
D
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-
nonadecatetraenyloxy)propionic Acid (10a): A solution of 8 (5 mg,
0.011 mmol) in methanol (0.5 mL) and Et3N (10 drops) was added
to an emulsion of 3b (20 mg, 0.011 mmol) in methanol (2 mL). The
whole mixture became homogeneous and was stirred at ambient
temperature under argon for 24 h. Progress of the reaction was
monitored by TLC [CHCl3/methanol/H2O (20:12:3)]. The solvents
were then evaporated under reduced pressure, and the residue was
purified by FC [SiO2; (i) CHCl3/methanol (20:12), (ii) CHCl3/meth-
anol/H2O (20:12:2)]. After solvent evaporation and lyophilization,
13 mg (53%) of pure 10a was obtained. Rf ϭ 0.25 [CHCl3/meth-
anol/H2O (20:12:3)]. C102H137N12O41P (2218.23, 2216.87), ESI FT
Ϫ 3 H ϩ Na]2Ϫ, 1146.9332 (calcd. 1146.9321) [M Ϫ 2 H]2Ϫ
,
764.2851 (calcd. 764.2854) [M Ϫ 3 H]3Ϫ. Fluorescence spectrum
[methanol/H2O (1:1), 0.14 mg/5 mL]: excitation (573 nm): λmax
546 nm, λmax ϭ 354 nm, emission (546 nm): λmax ϭ 575 nm.
ϭ
2-[3Ј,6Ј-Bis(ethylamino)-2Ј,7Ј-dimethyl-3-oxospiro[1H-isoindole-
1,9Ј-[9H]xanthen]-2(3H)-yl]ethylamine (12a): A solution of com-
mercial rhodamine 6G (11, 0.3 g, 0.62 mmol) in DMF (40 mL) was
slowly added to a solution of ethylenediamine (0.6 mL, 14 equiv.)
in DMF (20 mL), and the resulting mixture was stirred at 20 °C
for 22 h. The solvent was then distilled off under reduced pressure
(10Ϫ2 mbar), and the residue was purified by FC [methanol/eth-
anol/CHCl3 (1:1:10)] to give 0.27 g (96%) of product 12a, con-
ICR MS: m/z ϭ 1118.4265 (calcd. 1118.4202) [M Ϫ 3 H ϩ Na]2Ϫ
,
1107.4332 (calcd. 1107.4292) [M Ϫ 2 H]2Ϫ, 737.9519 (calcd.
737.9502) [M Ϫ 3 H]3Ϫ. Fluorescence spectrum [methanol/H2O
(1:1), 0.15 mg/5 mL]: excitation (475 nm): λmax ϭ 396 nm, emission
(396 nm): λmax ϭ 475 nm.
1
taining traces of ethanol and DMF according to H NMR. Rf ϭ
0.38 [methanol/ethanol/CHCl3 (1:1:10)]. 1H NMR (200 MHz,
CDCl3): δ ϭ 1.26 [t, J ϭ 7.0 Hz, 6 H, 2 NH(CH2CH3)], 1.84 (s, 6
H, CH3-11Ј, CH3-12Ј), 2.19 (s, 2 H, NH2), 2.34 (t, J18Ϫ17 ϭ 6.0 Hz,
2 H, CH2-2AE), 3.14 [m, 6 H, CH2-1AE, 2 NH(CH2CH3)], 3.57
[broad t, 2 H, 2 NH(CH2CH3)], 6.18 (s, 2 H, 1Ј-H, 8Ј-H), 6.29 (s,
2 H, 4Ј-H, 5Ј-H), 7.00 (m, 1 H, 7-H), 7.39 (m, 2 H, 5-H, 6-H),
7.85 (m, 1 H, 4-H). 13C NMR (50 MHz, CDCl3, APT): δ ϭ 14.6
[NH(CH2CH3)], 16.6 (C-11Ј, C-12Ј), 38.2 [NH(CH2CH3)], 40.5,
43.3 (C-1AE, C-2AE), 65.1 (C-9Ј), 96.4 (C-4Ј, C-5Ј), 105.8 (C-8Јa,
C-9Јa), 117.9 (C-2Ј, C-7Ј), 122.7 (C-4), 123.7 (C-7), 128.0 (C-5),
128.1 (C-1Ј, C-8Ј), 130.9 (C-3a), 132.4 (C-6), 147.4 (C-3Ј, C-6Ј),
151.6 (C-4Јa, C-10Јa), 153.4 (C-1), 168.6 (C-3). C28H32N4O2
(456.58, 456.25), ESI FT ICR MS: m/z ϭ 457.2593 (calcd.
457.2525) [M ϩ H]ϩ.
9-[2-Carboxy-5-(2-ethoxy-3,4-dioxo-1-cyclobuten-1-ylamino)-
phenyl]-3,6-bis(dimethylamino)xanthylium Chloride (9): Compound
7a (10 mg, 0.025 mmol) and 3,4-diethoxy-3-cyclobutene-1,2-dione
(19 mg, 0.11 mmol) in ethanol (1.5 mL) were stirred at 20 °C for
24 h {progress of the reaction was monitored by TLC [ethyl acetate/
ethanol (5:1)]}. No product formation was observed. Triethylamine
(1.1 mL) was then added, and the reaction mixture was heated
to 60 °C and stirred at this temperature. The formation of a new
compound was observed, and after 14 h, the mixture was allowed
to cool to ambient temperature and Kieselguhr was added. Solv-
ents were evaporated under reduced pressure and the residue was
transferred onto the top of a FC column. Elution with ethyl acet-
ate/ethanol (5:1), gave 9.7 mg (74%) of a product 9, which was not
completely pure according to the 1H NMR. It contained some eth-
anol and a trace of triethylamine. Rf ϭ 0.12 [ethyl acetate/ethanol
3-({2-[3Ј,6Ј-Bis(ethylamino)-2Ј,7Ј-dimethyl-3-oxospiro[1H-isoindole-
1,9Ј-[9H]xanthen]-2(3H)-yl]ethyl}amino)-4-ethoxy-3-cyclobutene-
1,2-dione (12b): A solution of 3,4-diethoxy-3-cyclobutene-1,2-dione
(0.27 g, 1.17 mmol) in methanol (2 mL) was added dropwise to a
solution of 12a (0.24 g, 0.53 mmol) in methanol (4 mL) and chloro-
form (3.5 mL), and the resulting mixture was stirred at 20 °C for
18 h. Progress of the reaction was monitored by TLC [CHCl3/eth-
anol (20:1)]. Solvents were evaporated under reduced pressure and
the residue was purified by FC [CHCl3/ethanol (25:1)]. After re-
moval of the solvents, the crude product was further purified by
FC [(i) CHCl3 (1500 mL), (ii) CHCl3/ethanol (25:1)] to give 0.24 g
(79%) of pure product 12b. Rf ϭ 0.31 [CHCl3/ethanol (20:1)]. IR
1
(5:1)]. H NMR (200 MHz, [D6]DMSO): δ ϭ 1.05 (t, J ϭ 7.0 Hz,
3 H, OCH2CH3), 2.98 (s, 12 H, 2 N(CH3)2TMR), 4.52 (q, J ϭ 7.1 Hz,
2 H, OCH2CH3), 6.61 (broad signal, 6 H, 4ЈTMR-H, 5ЈTMR-H,
2ЈTMR-H, 7ЈTMR-H, 1ЈTMR-H, 8ЈTMR-H), 7.14 (broad signal, 1 H,
6TMR-H), 7.55 (dd, J4Ϫ6 ϭ 1.5 Hz, J4Ϫ3 ϭ 8.4 Hz, 1 H, 4-H), 7.95
(d, J3Ϫ4 ϭ 8.4 Hz, 1 H, 3-H), 11.06 (s, 1 H, COOH). C30H27N3O6
(525.56, 525.19), ESI FT ICR MS: m/z ϭ 548.1785 (calcd.
548.1799) [M ϩ Na]ϩ, 526.1969 (calcd. 526.1979) [M ϩ H]ϩ.
(R)-3-[((5R)-5-{1-[3-({2-[(2-{[3Ј,6Ј-Bis(dimethylamino)-3-oxospiro- (KBr): ν˜ ϭ 3419, 1683, 1608, 1517, 1467, 1444, 1421, 1383, 1348,
[isobenzofuran-1(3 H),9Ј-[9H]xanthen]-6-yl]amino}-3,4- 1268, 1209 cmϪ1. UV (CH3OH): λmax (ε) ϭ 236.0 (66967), 251.5
dioxo-1-cyclobuten-1-yl)amino]ethyl}carbamoyl)-4-nitrophenyl]-3- (S) (49117), 300.5 (S) nm (13589). 1H NMR (400 MHz, CDCl3,
(3-carboxypropionyl)-1H-1,2,4-triazol-5-yl}-α-
(1Ǟ4)-2-acetamido-2,6-dideoxy-β- -glucopyranosyl-(1Ǟ4)-[β-
glucopyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-β-
-glucopyranosyl- (s, 6 H, CH3-11ЈR6G, CH3-12ЈR6G), 3.21 [q, J ϭ 7.2 Hz, 4 H,
(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α-
L
-arabinopyranosyl- 1H,1H COSY, HMQC, HMBC): δ ϭ 1.33 [t, J ϭ 7.2 Hz, 6 H,
D
D
-
NH(CH2CH3)], 1.37/1.44 (two t, J ϭ 7.4 Hz, 3 H, OCH2CH3), 1.89
D
D
-glucopyranuron- NH(CH2CH3)], 3.31 (broad signal, 2 H, CH2-1AE), 3.38/3.44 (two
amidosyloxy)hydroxyphosphoryloxy]-2-[(2Z, 6E,13E)- broad signals, 2 H, CH2-2AE), 4.63/4.70 (two broad q, J ϭ 7.4 Hz,
3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-nonadeca- 2 H, OCH2CH3), 5.96 (broad s, 0.5 H, NH-3SA), 6.18 (s, 2 H,
tetraenyloxy]propionic Acid (10b): Compounds 3b (70 mg,
1ЈR6G-H, 8Ј R6G-H), 6.34 (s, 2 H, 4ЈR6G-H, 5ЈR6G-H), 6.93 (broad
0.038 mmol) and 9 (9.7 mg, 0.018 mmol) in borax buffer (5 mL, signal, 0.5 H, NH-3SA), 7.06 (m, 1 H, 7R6G-H), 7.48 (m, 2 H, 5R6G
-
1160
Eur. J. Org. Chem. 2002, 1149Ϫ1162

Synthesis of Fluorescent Derivatives of the Antibiotic Moenomycin A
H, 6R6G-H), 7.93 (m, 1 H, 4R6G-H). 13C NMR (50 MHz, CDCl3,
FULL PAPER
132.1 (C-6B), 133.9 (C-3B), 149.3 (C-4C, C-4D), 153.2 (C-6C, C-6D),
155.1 (C-1B), 163.7 (?) 169.8 (C-7B). C36H48N4O5 (616.80, 616.36),
FAB MS: m/z ϭ 617.4 [M ϩ H]ϩ.
APT): δ ϭ 14.9 [NH(CH2CH3)], 15.9 (OCH2CH3), 16.9 (C-11ЈR6G
,
C-12ЈR6G), 38.5 [NH(CH2CH3)], 40.9 (C-1AE), 43.2, 45.2 (C-2AE),
65.7 (C-9ЈR6G), 69.5 (OCH2CH3), 96.6 (C-4ЈR6G, C-5ЈR6G), 104.9,
105.4 (C-8ЈaR6G, C-9ЈaR6G), 118.6 (C-2ЈR6G, C-7ЈR6G), 123.0 (C-
4R6G), 124.2 (C-7R6G), 127.9 (?), 128.4 (C-5R6G), 128.5 (C-1ЈR6G, C-
8ЈR6G), 130.9 (C-3aR6G), 133.1 (C-6R6G), 147.9 (C-3ЈR6G, C-6ЈR6G),
151.9 (C-4ЈaR6G, C-10ЈaR6G), 153.7 (C-7aR6G), 170.2 (C-3R6G),
(R)-2-{16-[3Ј,6Ј-Bis(ethylamino)-2Ј,7Ј-dimethyl-3-oxospiro[1H-
isoindole-1,9Ј-[9H]xanthen]-2(3H)-yl]-7,10,13-trioxa-3-azahexa-
decyloxy}-3-({β-
2 , 6 - d i d e ox y - β -
pyranosyl-(1Ǟ6)]-2-acetamido-2-deoxy-β-
(1Ǟ2)-3-O-carbamoyl-4-C-methyl-α- -glucopyranuronamidosyl-
D
-galactopyranuronamidosyl-(1Ǟ4)-2-acetamido-
- gl u c opy ra n o s yl - ( 1Ǟ 4 ) -[ β - - gl u c o -
-glucopyranosyl-
D
D
172.7 (C-3SA), 176.9 (C-4SA), 183.5, 191.0 (C-1SA
,
C-2SA).
D
C34H36N4O5 (580.68, 580.27), ESI FT ICR MS: m/z ϭ 581.2743
(calcd. 581.2765) [M ϩ H]ϩ. Fluorescence spectrum [methanol/
H2O (1:1) (4 mL) ϩ 0.2% TFA (1 mL), 0.045 mg/5 mL]: excitation
(556 nm): λmax ϭ 529 nm, λmax ϭ 351 nm, emission (528 nm):
λmax ϭ 556 nm.
D
oxy}hydroxy-phosphoryloxy)propionic Acid (15): Compounds 13
(80 mg, 67 µmol) and 14 (154.3 mg, 268 µmol), on reductive amin-
ation as described in ref.[36] provided Ϫ after purification [(i) LH-
20, water/methanol (1:4); (ii) Dowex 50Wx8; (iii) FC, ethyl acetate/
2-propanol/water (6:4:2); (iv) LH-20, water/methanol (1:4)] and ly-
ophilization Ϫ 21.3 mg (18%) of pure 15. C77H114N9O37P (1788.76,
1787.70), ESI FT ICR MS: m/z ϭ 892.8440 (892.8454) [M Ϫ 2
(R)-3-{[(5R)-5-(1-{3-[(2-{[2-({2-[3Ј,6Ј-Bis(ethylamino)-2Ј,7Ј-
dimethyl-3-oxospiro[1H-isoindole-1,9Ј-[9H]xanthen]-2(3H)-
yl]ethyl}amino)-3,4-dioxo-1-cyclobuten-1-yl]amino}ethyl)-
carbamoyl]-4-nitrophenyl}-3-(3-carboxypropionyl)-1H-1,2,4-
H]2Ϫ
.
triazol-5-yl)-α-
dideoxy-β- -glucopyranosyl-(1Ǟ4)-[β-
acetamido-2-deoxy-β- -glucopyranosyl-(1Ǟ2)-3-O-carbamoyl-4-C-
methyl-α- -glucopyranuronamidosyloxy]hydroxyphosphoryloxy}-2-
L
-arabinopyranosyl-(1Ǟ4)-2-acetamido-2,6-
D
D
-glucopyranosyl-(1Ǟ6)]-2-
D
D
Acknowledgments
[(2Z,6E,13E)-3,8,8,14,18-pentamethyl-11-methylene-2,6,13,17-
nonadecatetraenyloxy)propionic Acid (10c): A suspension of 12b
(19 mg, 0.032 mmol) in methanol (1 mL) was added to a solution
of 3b (50 mg, 0.027 mmol) in methanol (10 mL) and triethylamine
(0.2 mL). The whole mixture became homogeneous and was stirred
at 20 °C under argon for 6 h. Progress of the reaction was mon-
itored by TLC [nPrOH/H2O (7:2)]. No product formation was ob-
served. More triethylamine (0.5 mL) was added, and stirring was
continued for an additional 44 h (until compound 3b could no
longer be observed by TLC). After removal of the solvents under
reduced pressure, the crude product was purified by FC [nPrOH/
H2O (7:1.5)]. n-Propanol was distilled off under reduced pressure
and water was removed by lyophilization to give 35 mg (54%) of
pure product 10c. Rf ϭ 0.53 [nPrOH/H2O (7:2)]. C110H147N14O41P
(2352.42, 2350.96), ESI FT ICR MS: m/z ϭ 1185.4699 (calcd.
1185.4624) [M ϩ Na Ϫ 3 H]2Ϫ, 1174.4780 (calcd. 1147.4714) [M
Ϫ 2 H]2Ϫ, 782.6458 (calcd. 782.6450) [M Ϫ 3 H]3Ϫ. Fluorescence
spectrum [methanol/H2O (1:1) (4 mL) ϩ 0.2% TFA (1 mL),
0.08 mg/5 mL]: excitation (550 nm): λmax, 528 nm, λmax ϭ 351 nm,
emission (528 nm): λmax ϭ 557 nm.
We wish to thank Dr. K. Stembera and Mrs. R. Herold for the
antibiotic tests. Financial support by the Deutsche Forschungs-
gemeinschaft, BC Biochemie GmbH (Frankfurt), and the Fonds
der Chemischen Industrie is gratefully acknowledged.
[1]
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2.67 mmol) was added slowly to a solution of rhodamine 6G (11,
427 mg, 0.89 mmol) in DMF (50 mL). The mixture was stirred at
60 °C for 24 h. Solvent evaporation and FC [CHCl3/methanol/NH3
(5:1:0.05)] followed by solvent evaporation and lyophilization pro-
vided pure 14 (402 mg, 73%). 1H NMR (400 MHz, D2O, 1H, 1H
COSY): δ ϭ 1.25 (t, CH3-9C, CH3-9D), 1.69Ϫ1.75 (m, CH2-2A),
1.84 (s, CH3-7C, CH3-7D), 1.82Ϫ1.90 (m, CH2-9A), 3.04 (dd, J9A-
10A ϭ 6.1 Hz, CH2-10A), 3.14Ϫ3.20 (m, CH2-8C, CH2-8D), 3.26 (m,
CH2-1A), 3.48 (dd, J2A-3A ϭ 6.0 Hz, CH2-3A), 3.50Ϫ3.57 (m, CH2-
4A, CH2-5A, CH2-6A, CH2-7A), 3.58 Ϫ3.62 (m, CH2-8A), 6.06 (s,
H-5C, H-5D), 6.30 (s, H-2C, H-2D), 6.97Ϫ6.99 (m, H-2B), 7.47Ϫ7.51
(m, H-3B, H-4B), 7.81Ϫ7.83 (m, H-5B). 13C NMR (50 MHz, D2O):
δ ϭ 14.8 (CH3-9C, CH3-9D), 17.2 (CH3-7C, CH3-7D), 27.9 (C-9A),
29.1 (?), 30.2 (C-2A), 36.4 (C-8C, C-8D), 38.5 (?), 39.1 (C-1A), 40.2
(C-10A), 67.2 (C-8A), 69.7, 70.3, 70.5, 70.8, 70.9, 71.1, 71.4 (C-3A
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3D), 123.4 (C-2B), 125.0 (C-5B), 129.0 (C-2C, C-2D), 129.4 (C-4B),
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