On Penicillin-Binding Protein 1b Affinity-Labeling Reagents

Article abstract of DOI:10.1002/hlca.200390346

The synthesis of some 3-aryl-3-(trifluoromethyl)3H-diazirine and benzophenone-based photoaffinity labels is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an indicator unit (biotin) and the bioacti

Full text of DOI:10.1002/hlca.200390346

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Helvetica Chimica Acta Vol. 86 (2003)
On Penicillin-Binding Protein 1b Affinity-Labeling Reagents
by Daniela Volke, Mohammed Daghish, Lothar Hennig, Matthias Findeisen, Sabine Giesa, Ramona Oehme,
and Peter Welzel*
Universit‰t Leipzig, Fakult‰t f¸r Chemie und Mineralogie, Johannisallee 29, D-04103 Leipzig
(e-mail: welzel@chemie.uni-leipzig.de)
Dedicated to Professor Duilio Arigoni on the occasion of his 75th birthday
The synthesis of some 3-aryl-3-(trifluoromethyl)3H-diazirine and benzophenone-based photoaffinity labels
is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an
indicator unit (biotin) and the bioactive ligand can be attached orthogonally. To three of the labels, moenomycin
was conjugated with the aim to provide tools for the identification of the moenomycin binding site within the
transglycosylase domain of the enzyme PBP 1b. Some preliminary photoaffinity-labeling experiments were
carried out.
Introduction. Peptidoglycan is an essential cell-wall constituent of almost all
eubacteria. It is a heteropolymer consisting of glycan strands cross-linked via short
peptide chains. The final events of peptidoglycan biosynthesis at the outside of the
cytoplasmic membrane are the formation of the macromolecular architecture by two
major types of reaction, i) formation of the polysaccharide strands (transglycosylation)
and ii) formation of peptide cross-linkages between the glycan chains (transpeptida-
tion) (for review, see [1]). A representation of the transition state of the trans-
glycosylation reaction is shown in Fig. 1,a [2]. Both the glycosyl donor (the growing
peptidoglycan chain) and the glycosyl acceptor (the disaccharide-derived lipid II) (see
[1]) are attached to the outer face of the cytoplasmic membrane via a C55 anchor. The
transglycosylation reaction is catalyzed by a number of multimodular bifunctional
polymerases (that catalyze also the transpeptidation reaction) designated as class-A
high-molecular-mass penicillin-binding proteins (PBPs). Of these, PBP 1b from
Escherichia coli has been studied in great detail [3]. It comprises 844 amino acid
residues and contains a short cytosolic N-terminus, a membrane span, the D198-G435
glycosyl-transferase module, and the Q447-N844 acyl-transferase module. Within the
glycosyl-transferase module, Glu233 has been shown to be central to the trans-
glycosylation, and, in the active site of the acyl transferase, Ser510 is essential, the
acylation of which forms the basis of the antibiotic properties of the b-lactams.
Recently, the soluble extracellular region of PBP 1b from Streptococcus pneumoniae
has been expressed and characterized biochemically [4]. From the most-recent kinetic
characterization of the E. coli PBP 1b, it was deduced that a doubly charged metal ion
bound to the diphosphate group assists departure of the leaving group, whereas the
essential Glu233 acts as a base, removing the proton from the 4-OH group of the
glycosyl acceptor (see Fig. 1,a) [5].

Helvetica Chimica Acta Vol. 86 (2003)
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Fig. 1. a) Putative mechanism of the transglycosylation reaction; b) SAR results of moenomycin A. Arrows
indicate indispensible pharmacophoric groups.
We are interested mainly in the interactions of the antibiotic moenomycin A with
PBP 1b. The moenomycin antibiotics are known to interfere with the transglycosylation
reaction [6] (for a recent review on transglycosylase inhibition, see [7]). They belong to
the rare group of compounds known with certainty to inhibit the transglycosylase [8]
and are, thus, important tools for localizing the transglycosylase domain on PBP 1b and
for unraveling the mechanism of the transglycosylation reaction. Furthermore, they are
very interesting lead compounds for the development of medicinally valuable new
antibiotics [9]. Moenomycin A (2) has been demonstrated to bind reversibly to E. coli
PBP 1b [1]. From structure-activity relationships [10][11], it has been concluded that
the moenomycins, after unselective anchoring to the cytoplasmic membrane via their
lipid moiety (for a discussion, see [12]), bind highly selectively via their carbohydrate
part to the donor-binding subsites of the enzyme. The structural features that are
known to be responsible for the antibiotic activity are indicated by arrows in 1 (see
Fig. 1,b) [10][11]. It is assumed that units C and E of 1 and 2 compete with the second
and the third sugar unit of the growing peptidoglycan chain for the binding subsites at
the enzyme. The different binding of the first two sugar units (1 ! 4 in the growing
peptidoglycan strand vs. 1 ! 2 in 1 and 2) has been suggested to be responsible for the
inhibition of the enzyme (for a detailed discussion, see [2][10]). The conclusions from

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Helvetica Chimica Acta Vol. 86 (2003)
structure-activity-relationship (SAR) studies are corroborated by NMR results,
especially from saturation-transfer-difference (STD) NMR experiments [13][14] from
which it has been inferred that the N-acetyl groups of units C and E are in contact with
the enzyme in the ligand/protein complex [15] (see also [16])1). The generation of a
soluble and functional extracellular form of PBP 1b [4] in Vernet×s laboratory was
undertaken with the goal to determine eventually a crystal structure. Even the crystal
structure of the complex with moenomycin or a suitable moenomycin derivative with
this PBP 1b construct may become available. But some care should be taken. There are
differences in the in vivo (Staphylococcus aureus MIC values) and the in vitro SAR
results which have been taken as a hint that the access to the donor and acceptor
binding sites of the transglycosylase differs in the enzyme preparations from those in
the intact bacterium [1][11]. With this in mind, we started some time ago to develop
moenomycin-derived tools that will allow us to perform affinity-labeling studies [18
20]. It is hoped that they will bring about independent information on the mode of
binding of the antibiotic to the transglycosylase domain of PBP 1b (for some reviews on
photoaffinity labeling, see [21]).
We describe here the synthesis of three moenomycin-derived photoaffinity labels as
well as some preliminary photoaffinity-labeling studies.
Results2). Syntheses. All new photoactivatable probes3) are based on our concept
that allows the attachment of the photophore, the biotin tag, and the ligand
orthogonally to a trifunctional carrier [18][23]. In 4d, 8, 10e, and 11, the trifunctional
scaffold is isoserine (ˆ 3-amino-2-hydroxypropanoic acid), and in 4e, 9, and 10f, it is an
amine N-atom. The probes, 4d, 4e, 4f, 8, 9 contain furthermore an alkyl 3-nitrophenyl
ether unit, i.e., an element with a cleavable bond, which is broken by a photo-
substitution reaction to yield the corresponding 3-nitrophenol [24]. As suggested by
Nakanishi and co-workers [25], the latter reaction can be used at the end of the
1
)
The conclusions drawn from some recent kinetic studies need to be rediscussed in view of our previous
results [17].
Some results have been published in a preliminary communication [20].
For a comparison of photoaffinity-labeling probes, see [22].
2
3
)
)

Helvetica Chimica Acta Vol. 86 (2003)
4217
photoaffinity-labeling procedure to remove both the ligand and the biotin tag from
the labeled peptide prior to MS sequencing to diminish complications in the MS
analysis.
The synthesis of 3a followed a route developed by Nakanishi and co-workers [25].
Amine 3a has previously been oxidized to the corresponding nitro compound 3b with
dimethyldioxirane [25]. The reported yield was 66%. We found the reaction to be
rather capricious. In our hands, the method of Krohn and co-workers [26], i.e.,
Zr(tBuO)4-mediated oxidation with tert-butyl hydroperoxide, gave far more-reliable
results. The yields of 3b were uniformly in the range of 80%. On oxidative degradation
(OsO4, then NaIO4, immobilized on silica gel [27]) 3b provided aldehyde 4a (yield
74%), and reduction of the latter with NaBH4 furnished primary alcohol 4b in 84%
yield.
Isocyanates 5 (both enantiomers) were obtained from (R)- and (S)-malic acid,
respectively, as described previously [28]. Coupling of 5a with primary alcohol 4b gave
urethane 4c in 88% yield. Reaction of 4c with the biotin derivative 6b (obtained from
the corresponding commercial diamine and 1,1'-carbonylbis[1H-imidazole] (CDI)-
activated biotin in pyridine) in 1,2-dimethoxyethane/H2O 1:4 gave 4d by amide
formation and concomitant loss of the protecting group (65% yield after lyophilization
and flash chromatography). The 1H- and 13C-NMR spectra of 4d were fully assigned by
using 1H,1H-COSY and 13C,1H-COSY experiments. The 19F-NMR signal was found at d
12.65 (CDCl3 solution, d (CF3COOH) 0). The ESI mass spectrum showed the expected
quasi-molecular-ion peaks. Isoserine derivative 4d is a broadly applicable biotin-tagged
photoaffinity label to which any suitably functionalized ligand can be attached.
We coupled 4d to the moenomycin derivative 7b (obtained from moenomycin A via
the azo coupling/JappÀKlingemann route as described previously [29]). The bifunc-
tional linker 4-thiocyanatobenzoyl chloride was used for the conjugation reaction. First
4d was treated with the acid chloride in dry pyridine in the presence of Steglich×s base to
provide the corresponding ester. Due to its hydrolytic sensitivity, the ester could not be
isolated and was immediately treated with 7b. Wasteful chromatographic separations
were necessary to obtain pure 8, which was, thus, isolated in a poor yield of 10%.
Compound 8 could be detected on TLC by anisaldehyde and 4-(dimethylamino)benz-
aldehyde dipping reagents that suggested the presence of a sugar and the biotin part,
respectively. The structure of 8 was confirmed by 1H- and 13C-NMR and mass
spectrometry.
A second broadly applicable photoaffinity reagent based on a 3-nitro-alkoxyphenyl
substituted 3-(trifluoromethyl)-3H-diazirine was obtained from aldehyde 4a and amino
compound 6a by reductive amination to give secondary amine 4e in 60% yield. Like 4d,
4e can be attached to any ligand armed with a suitable functional group. We coupled 4e
to the moenomycin-derived amine 7a [29] via the squaric acid linker [30 32]. Thus,
reaction of 4e with diethyl squarate provided −squaric acid ester amide× 4f (a 3-amino-
4-ethoxycyclobut-3-ene-1,2-dione) in 91% yield. As usual for −squaric acid amides×
[30], two sets of 13C-NMR signals appeared for many C-atoms, indicating the presence
of two conformers [29]. Accordingly, there were two 19F-NMR signals. On reaction
with the moenomycin derivative 7a in H2O/MeOH 1:1 at pH 9, 4f furnished compound
9 (32% yield), which was characterized by NMR and mass spectrometry. The relevant
photochemistry of both 8 and 9 has been discussed elsewhere [20].

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Helvetica Chimica Acta Vol. 86 (2003)
The synthesis of the benzophenone-based photoprobes 10e and 10f started with 4-
benzoylbenzoic acid, which was converted to the allyl [33] and the 3-hydroxypropyl
esters 10a and 10c, respectively, making use of Staab×s 1,1'-carbonylbis[[1H]-imidazole]

Helvetica Chimica Acta Vol. 86 (2003)
4219
method [34]. Ozonolysis followed by reduction converted allyl ester 10a to the 2-
oxoethyl-ester 10b.
Reaction of 10a with Burger×s isocyanate 5b [28] furnished urethane 10d, which on
reaction with the biotin-derived amine 6a, provided the isoserine derivative 10e to
which again any ligand can be attached by suitable methods. We prepared 11 from 10e
and the moenomycin-derived amine 7b as described above for 8. The structure of 11 is
in accord with the 1H- and 13C-NMR and mass spectra. Finally, amine 10f was prepared
in 43% yield by reductive amination from aldehyde 10b and the biotin-derived amine
6a.
Antibiotic Properties of Selected Moenomycin Conjugates. The minimum inhibitory
concentration (MIC) values against seven different Staph. aureus strains (ATCC 25923,
ATCC 29213, MRSA 1309, SG 511, PEG 18, PEG 5, and KNS PEG 5) were deter-
mined by a serial twofold micro-dilution method on microtiter plates as described
previously [12]. All compounds displayed strong antibiotic activity, albeit weaker than
that of moenomycin A itself [35] (Table 1).
Table 1. Minimum Inhibitory Concentration (MIC) of Moenomycin Derivatives against a Number of Staph.
aureus Strains
MIC
[mmol/l]
[mg/ml]
2
0.00690.029
0.15
0.038
0.053
0.32
7a
7b
7c
8
0.27
0.013
0.11
0.81
0.14
0.16
9
11
0.48
0.058
Preliminary Photoaffinity-Labeling Experiments. PBP 1b was obtained and purified
by ampicillin affinity chromatography as described previously [11]. Cross-linking with
affinity label 9 was achieved by irradiation at 350 nm (Rayonet reactor). The
Coomassie-Blue staining after SDS-PAGE (sodium dodecyl sulfate polyacrylamide

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Helvetica Chimica Acta Vol. 86 (2003)
electrophoresis) revealed the expected pattern (not shown, see [36]). Evidence for a
selective affinity labeling was obtained from the Western-blot analysis. Visualization of
the transferred band by the streptavidineÀalkaline-phosphatase-conjugate method
clearly demonstrated a biotin-containing, i.e., labeled component in the band (Fig. 2,
Lane 3). Two control experiments were carried out. In the first, all experimental steps
were performed as described above, with the exception that the enzyme was incubated
both with 9 and an excess of moenomycin A (2). After SDS-PAGE and Western
blotting, the visualization by the streptavidinÀalkaline-phosphatese-conjugate method
showed a band with greatly diminished intensity (Fig. 2, Lane 1) indicating the
competition of 9 and 2 for the binding site at the enzyme. In the second control
experiment, again all steps were executed as described for the labeling experiment,
with the exception that irradiation was omitted. After blotting, the PBP 1b band gave
no color reaction on applying the streptavidinÀalkaline-phosphatase-conjugate
method (Fig. 2, Lane 2).
We were interested to further localize the labeling site on the enzyme. An
endoproteinase Lys-C digest seemed ideally suited since, as Table 2 shows, theoretically

Helvetica Chimica Acta Vol. 86 (2003)
4221
only two large fragments are expected from such a digest, one comprising a large part
(109amino acid residues) of the PBP 1b transglycosylase domain and a second one
from the C-terminal part of the enzyme. After submitting PBP 1b to an endoproteinase
Lys-C digest, the SDS-tricine-PAGE (Coomassie-Blue staining) displayed many bands
(not shown). However, when the affinity-labeled specimen of PBP 1b was submitted to
the endoproteinase Lys-C digest, only one major band in the expected molecular-mass
region (Fig. 3) could be identified after SDS-tricine-PAGE followed by Western
blotting and visualization with the streptavidinÀalkaline-phosphatese conjugate
method. We take this result as a hint that indeed the transglycosylase domain of PBP
1b was labeled. As Fig. 3 indicates, there is still a large portion of uncleaved and only
partially degraded PBP 1b is present. We did not attempt to optimize the digest since
the amount of material in the desired mass-region band was obviously sufficient for
mass-spectrometric sequencing. This work has yet to be performed.
We would like to express our gratitude to Renate Herold (this department) for determining the MIC values
and to Dr. J. A. Ayala (Madrid) for providing us with the PBP 1b overproducing E. coli strain JM109/pJP13.
Financial support by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and Sandoz
Industrial Products GmbH (formerly BC Biochemie GmbH) is gratefully acknowledged.

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Helvetica Chimica Acta Vol. 86 (2003)
Experimental Part
General. All O2- or moisture-sensitive reactions were performed in oven-dried glassware under a positive
pressure of Ar. Liquids and solns. were transferred by syringe. Small-scale reactions were performed in Wheaton
serum bottles sealed with aluminium caps with open top and Teflon-faced septa (Aldrich). Usual workup means
partitioning the reaction mixture between an aq. phase and CH2Cl2, drying the combined org. soln. (Na2SO4),
and evaporation (bath temp. 458). All light-sensitive compounds were handled under dimmed light and in
brown glassware. Solvents were purified by standard techniques. TLC: pre-coated aluminium plates (silica gel
60 F254, Merck); unless stated otherwise, visualization with phosphormolybdate/ceric sulfate reagent [37]. Flash
column chromatography (FC): silica gel 60 (Merck; 40 63 mm). UV Spectra: lmax (e) in nm. IR Spectra: FT-IR
1
spectrometer (ATI Mattson, Genesis series); in cmÀ1. H- and 13C-NMR Spectra: Varian Gemini-200, Gemini-
2000, Gemini-300, Bruker DRX-400, and DRX-600 spectrometers; CDCl3 as a solvent unless otherwise stated; d
in ppm, with the signals of CHCl3 (d 7.26) and of CDCl3 (d 77.16) as internal references, J values in Hz; signal
assignments by means of 1H,1H-COSY, HMQC, HMBC, and, in some complicated cases, partly by analogy with
the fully analyzed moenomycin spectra [38]; H- and C-atoms are characterized according to Formulae 2 and 8,
bi ˆ biotin, is ˆ isoserine, sp ˆ spacer, sa ˆ squaric acid unit. MS: VG ZAB-HSQ (VG Analytics) with 3-
nitrobenzyl alcohol as a matrix (FAB), Finnigan MAT 212 (EI) and FT ICR (MS 7 T APEX II, Bruker
Daltonics, H2O/MeOH) in the pos. or neg. mode (ESI); in m/z; following the molecular formula, two calc.

Helvetica Chimica Acta Vol. 86 (2003)
4223
masses are given in parentheses, the first one being calculated with the International Atomic Masses, the second
one being the mono-isotopic mass.
3-[3-Nitro-5-(prop-2-enyloxy)phenyl)-3-(trifluoromethyl)-3H-diazirine (3b). To a soln. of 3-(prop-2-
enyloxy)-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzenamine [25] (3a; 970.0 mg, 3.77 mmol) in dry CH2Cl2
(200 ml) containing freshly activated 3ä molecular sieves (5.0 g) at 208, zirconium tetra(tert-butoxide) (0.15 ml,
0.4 mmol) was added, and the mixture was stirred for 2 h. Then, within 10 min tert-butyl hydroperoxide (2 ml,
11 mmol) was added. After 24 h (TLC monitoring (cyclohexane/AcOEt 8 :2), visualization with tin(II)
chloride, hydrochloric acid, 4-(dimethylamino)benzaldehyde), H2O (5 ml) was added. Usual workup (CH2Cl2)
and FC (MeOH/CHCl3 0.7:9.3, and AcOEt/cyclohexane 2 :8) furnished 3b (900.0 mg, 83%). Yellow highly
viscous oil, which, according to the spectra, was slightly impure. 1H-NMR (200 MHz, CDCl3): 6.40
(m, 1 arom. H); 6.19( m, 2 arom. H); 6.12 5.96 (m, CH2ˆCHCH2O); 5.49 5.42 (1 H, C H2ˆCHCH2O);
5.34 5.29(1 H, C H2ˆCHCH2O); 4.60 4.56 (m, CH2ˆCHCH2O). 13C-NMR (50 Hz, CDCl3): 159.66 (C(5'));
149.65 (C(3')); 132.04 (C(1')); 131.56 (CH2ˆCHCH2O); ca. 121.00 (CF3); 119.93 (arom. CH); 119.29
(CH2ˆCHCH2O); 113.89, 110.45 (2 arom. CH); 69.92 (CH2ˆCHCH2O); 28.31 (q, 2J(C,F) ˆ 41.2 (C(3)). 19F-
.
‡
NMR (188 Hz, CDCl3): 11.35 (s, CF3). EI-MS (C11H8F3N3O3; 287.20, 287.052): 286.9(10,
C11H8NO3F3 ), 212.9(35, C 11H8OF3 ), 40.8 (100, C3H5 ).
M ), 258.9(25,
‡
‡
‡
2-{3-Nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenoxy}ethanol (4b). To a soln. of 2-{3-nitro-5-[3-
(trifluoromethyl)-3H-diazirin-3-yl]phenoxy]acetaldehyde [25] (4a; 419.0 mg, 1.50 mmol) in dry THF/MeOH
4 :1 (50 ml) at À28, NaBH4 (55 mg, 0.89mmol) was added, and the mixture was allowed to warm to 5 8. After 1 h
(TLC monitoring (AcOEt/petroleum ether/toluene 3 :3 :4)), the reaction was stopped by addition of AcOH
(1 ml). Evaporation and FC (AcOEt/petroleum ether/toluene 0.4 :1:1) provided 4b (354 mg, 84%). Yellow

4224
Helvetica Chimica Acta Vol. 86 (2003)
Fig. 2. Western-blot analysis of the photoaffinity labeling of PBP
1b with 9. Photolabeling, SDS-PAGE, and Western blotting
were performed as described in the Exper. Part. For staining, the
streptavidineÀalkaline-phosphatase-conjugate method was em-
ployed. Lane 1: control experiment, i.e., PBP 1b was incubated
with 9 and moenomycin A (1000-fold excess) prior to the
photolabeling procedure; Lane 2: control experiment, i.e., PBP
1b ‡ 9, not irradiated; Lane 3: photolabeling of PBP 1b with 9.
Fig. 3. Western-blot analysis of the endo-
proteinase Lys-C digested photolabeled
PBP 1b. For staining, the streptavidine-
Àalkaline-phosphatase-conjugate method
was used. For exper. conditions and details,
see text.
solid. UV (CHCl3): 241.50 (5777), 263.50 (3599), 324.50 (1992). IR (KBr): 1622, 1587, 1547, 1444, 1349, 1294,
1263, 1164, 1082, 1061, 957, 905, 870, 750, 687. 1H-NMR (300 MHz, CDCl3): 7.85 7.83 (m, 1 arom. H); 7.67 7.66
(m, 1 arom. H); 7.16 7.14 (m, 1 arom. H); 4.24 4.21 (m, OCH2CH2OH); 4.08 4.06 (m, OCH2CH2OH); 2.10
(br. s, OH). 13C-NMR (75 MHz, CDCl3): 159.74 (C(1')); 149.52 (C(3')); 132.09(C(5 ')); 121.67 (q, ˆ 1J(C,F) ˆ
274.8, CF3); 119.76, 114.04, 110.02 (3 arom. CH); 70.49 (OCH2CH2OH); 61.10 (OCH2CH2OH); 28.32
(q 2J(C,F) ˆ 40.8, CF3-CN2). 19F-NMR (282 MHz, CDCl3): 12.63 (s, CF3). EI-MS (C10H8F3N3O4; 291.19,
.
.
.
‡
‡
‡
‡
291.047): 291.0 (10, M ), 263.0 (25, C10H8F3NO4 ), 217.0 (25, C10H8F3O2 ), 172.9(100, C H3F3O ), 44.9(75,
C2H5O ).
8
‡
1,2-O-(Hexafluoroisopropylidine)-N-{{2-{3-nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenoxy}ethox-
y}carbonyl}-d-isoserine (ˆ2-{3-Nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenoxy}ethyl {[(4R)-2,2-Bis(tri-
fluoromethyl)-5-oxo-1,3-dioxolan-4-yl]methyl}carbamate; 4c). To a soln. of 4b (220.0 mg, 0.76 mmol) in dry
CHCl3 (40 ml), 5a [28] (316.0 mg, 1134 mmol) was added, and the mixture was stirred at 208 for 5 d (TLC
control (petroleum ether/AcOEt 1:1)). Evaporation and removal of the excess isocyanate by bulb-to-bulb

Helvetica Chimica Acta Vol. 86 (2003)
4225
Table 2. Fragments Expected from Endoproteinase Lys-C Digest of PBP 1b
Cleavage Resulting peptide sequence
Peptide
Peptide
site
length [ä] mass [Da]
12
14
20
22
MAGNDREPIGRK
GK
PTRPVK
QK
12
2
6
1342.678
203.127
696.428
274.164
2
49VSRRRYEDDDDYDDYDDYEDEEPMPRK
27
3485.423
51
53
55
58
62
GK
GK
GK
GRK
2
2
2
3
4
203.127
203.127
203.127
359.228
456.281
PRGK
71
RGWLWLLLK
91183.723
90
98
LAIVFAVLIAIYGVYLDQK
IRSRIDGK
19
8
2108.228
943.556
121
126
138
165
274
VWQLPAAVYGRMVNLEPDMTISK
NEMVK
LLEATQYRQVSK
23
5
12
27
2617.340
619.300
1434.783
3168.548
MTRPGEFTVQANSIEMIRRPFDFPDSK
EGQVRARLTFDGDHLATIVNMENNRQFGFFRLDPRLITMISSPN- 7.1300991219
GEQRLFVPRSGFPDLLVDTLLATEDRHFYEHDGISLYSIGR-
AVLANLTAGRTVQGASTLTQQLVK
287
303
355
NLFLSSERSYWRK
ANEAYMALIMDARYSK
DRILELYMNEVYLGQSGDNEIRGFPLASLYYFGRPVEE-
LSLDQQALLVGMVK
13
16
52
1684.869
1845.875
5948.017
367
425
GASIYNPWRNPK
LALERRNLVLRLLQQQQIIDQELYDMLSARPLGVQPR-
GGVISPQPAFMQLVRQELQAK
12
58
1401.715
6663.674
429LGDK
431
437
452
462
463
466
513
4
431.238
VK
DLSGVK
IFTTFDSVAQDAAEK
AAVEGIPALK
K
QRK
2
6
15
10
1
245.174
617.338
1641.789
967.570
146.106
430.265
5038.587
3
47
LSDLETAIVVVDRFSGEVRAMVGGSEPQFAGYNRAMQARR-
SIGSLAK
525
595
PATYLTALSQPK
12
70
1288.703
7864.056
IYRLNTWIADAPIALRQPNGQVWSPQNDDRRYSESGRVMLV-
DALTRSMNVPTVNLGMALGLPAVTETWIK
LGVPK
600
5
512.332
649DQLHPVPAMLLGALNLTPIEVAQAFQTIASGGNRAPLSA-
LRSVIAEDGK
.707495049
689VLYQSFPQAERAVPAQAAYLTLWTMQQVVQRGTGRQLGAK
40
4460.354
698
732
828
YPNLHLAGK
9
34
96
1011.550
3679.750
10609.909
TGTTNNNVDTWFAGIDGSTVTITWVGRDNNQPTK
LYGASGAMSIYQRYLANQTPTPLNLVPPEDIADMGVDYD-
GNFVCSGGMRILPVWTSDPQSLCQQSEMQQQPSGNPFDQSS-
QPQQQPQQQPAQQEQK
DSDGVAGWIK
DMFGSN
838
844
10
6
1046.503
669.243

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Helvetica Chimica Acta Vol. 86 (2003)
distillation at 2 ¥ 10À2 mbar followed by FC (cyclohexane/AcOEt 2 :1) furnished 4c (385.0 mg, 88%). Colorless
oil. UV (MeOH): 233.5 (49959), 255.0 (sh, 26153). IR (KBr): 1840, 1707, 1541, 1325, 1271, 1240, 1200, 1136, 984.
1H-NMR (300 MHz, CDCl3): 7.80 7.78 (t, J ˆ 1.4, 1 arom. H); 7.65 (br. s, 1 arom. H); 7.09(br. s, 1 arom. H);
5.15 5.11 (m, NH); 4.83 4.78 (m, CHCH2NH); 4.52 4.48 (t, J ˆ 4.8, COOCH2); 4.29 4.24 ( t, J ˆ 4.4,
ArOCH2); 3.90 3.64 (m, CHCH2NH). 13C-NMR (75.5 MHz, CDCl3): 166.08 (CH(O)C(ˆO)O); 159.55
(arom. C(1')); 155.87 (NHCOO); 149.64 (arom. C(3')); 132.25 (arom. C(5')); 121.73 (q, 1J(C,F) ˆ 274.8,
CF3ÀCN2), 119.80 (arom. CH); 119.66 (q, J ˆ 288.57, 1 CF3); 118.85 (q, 1J(C,F) ˆ 286.9, 1 CF3); 114.29
(arom. CH); 110.03 (arom. CH); 97.77 (sept. 1J(C,F) ˆ 36.6, (CF3)2C); 74.44 (CHCH2NH); 67.32 (COOCH2);
63.46 (ArOCH2); 61.85 (not assigned); 41.30 (CHCH2NH); 27.52 (q, 2J(C,F) ˆ 27.4, CF3ÀC); 27.51 (not
assigned). 19F-NMR (282 MHz, CDCl3): 12.45 (s, CF3ÀCN2); À2.98 to À3.09( m, CF3); À3.27 to À3.38
‡
(m, CF3). FAB-MS (C17H11F9N4O8; 570.28, 570.043): 570.97 ([M ‡ H] ).
(5BR)-5B-{1-[3-(10-Amino-1-oxo-5,8-dioxa-2-azadecyl)-4-nitrophenyl]-3-(3-carboxy-1-oxopropyl)-1H-
1,2,4-triazol-5-yl}-5B-de[(2-hydroxy-5-oxocyclopent-1-en-1-yl)carbamoyl]moenomycin A (7b). To a soln. of 5-
amino-N-(8-amino-3,6-dioxaoctyl)-2-nitrobenzamide (prepared as described for 5-amino-N-(2-aminoethyl)-2-
nitrobenzamide [29]; 73.0 mg, 233.7 mmol) in 6% HCl soln. (0.7 ml) at 38, a soln. of NaNO2 (14.4 mg,
208.7 mmol) in H2O (1 ml) was added, and the mixture was stirred at 38 for 15 min. The resulting soln. was added
slowly to a soln. of 2 (300.0 mg, 189.6 mmol) and NaOAc (600 mg) in H2O (50 ml) kept at 208. The mixture was
stirred at 208 for 24 h and then filtered through a column of RP18 with MeCN/H2O 3 :7. The solvent of the
fractions containing 7b was removed by distillation, the residue taken up in H2O (100 ml), and the soln.
lyophilized. FC (H2O/PrOH 2 :7) followed by lyophilization provided pure 7b (250.0 mg, 55%). Pale yellow
1
hygroscopic powder. H-NMR (600 MHz, CD3OD; only signals that could be assigned): 8.47 8.45 (d, J ˆ 8.4,
1 arom. H); 8.10 (d, J ˆ 8.4, 1 arom. H); 8.05 (s, 1 arom. H); 5.85 (br. s, HÀC(1F)); 5.44 (m, HÀC(2I)); 5.36
5.33 (d, J ˆ 15.2, HÀC(7I)); 5.29( m, HÀC(6I)); 5.13 5.10 (t, J ˆ 6.8, HÀC(17I)); 4.67 4.66 (2s, CH2(22I),
overlapped by H2O); 3.00 2.98 (dd, J ꢀ 7.8, HÀC(2D)); 2.66 2.65 (d, J ˆ 6.6, CH2(12I)); 2.06 (br. s, 6 H,
MeCONHCE); 1.88 (m, CH2(10I)); 1.68 1.65 (s, Me(25I)); 1.58, 1.57 (2s, Me(20I), Me(21I)); 1.35 (Me(6C)); 1.25
(s, Me(4F)); 0.95 (br. s, Me(23I), Me(24I)). 13C-NMR (150, 100 MHz; HMBC, HMQC; CD3OD/(D6)DMSO
4 :1; only signals that could be assigned): 95.11, 180.45, 176.80, 173.95, 173.19, 167.32, 159.21 (7 CO); 158.43,
154.32 (2 CTA ); 149.87 (C(11I)); 146.69(arom. C); 141.54 (arom. C); 140.63 (C(7 I); 136.36 (C(3I)); 133.77 (arom.
C); 131.41 (C(14I)); 128.42 (arom. C); 127.01 (arom. C); 126.06 (C(6I)); 125.59(C(17 I)); 122.02 (C(13I)); 121.42
(C(2I)); 108.98 (C(22I)); 104.61, 102.73, 101.61 (C(1B,C,D)); 94.94 (C(1F)); 83.47 (C(4C)); 80.87, 80.04 (C(2H),
C(4E)); 77.30, 76.72, 76.59, 75.19, 73.82, 73.47, 73.16, 72.44, 72.25, 71.17, 70.21, 70.40, 69.83, 67.80, 69.16
(carbohydrate Cs, CH2Osp); 66.98 (C(1I)); 66.25 (C(6E)); 61.43 (C(6D)); 55.53 (C(2E,C)); 42.00 (C(9I)); 40.10
(C(15I)); 39.67, 39.64 (CH2NH2, CONHCH2sp), 35.63 (C(12I)); 35.21 (C(8I)); 32.52, 31.84, 31.53, 31.42 (C(4I),
C(5I), C(10I), C(8I)); 29.46 (CH2A); 27.34, 27.31 (C(23I), C(24I)); 26.86 (C(19I)); 25.66 (C(25I)); 23.53, 22.82
(MeCONHC,E); 17.58 (C(20I)); 17.15 (C(6C)); 15.85 (C(21I)); 15.48 (Me(4F)). ESI-MS (C82H125N10O39P; 1905.91,
1904.7843): 951.38600 ([M À 2H]2À; calc. 951.38487), 633.91943 ([M À 3H]3À); calc. 633.92082).
(5BR)-5B-{1-[3-(15-Amino-1-oxo-6,9,12-trioxa-2-azapentadecyl)-4-nitrophenyl]-3-(3-carboxy-1-oxoprop-
yl)-1H-1,2,4-triazol-5-yl}-5B-de[(2-hydroxy-5-oxocyclopent-1-en-1-yl)carbamoyl]moenomycin A (7c). As de-
scribed for 7b, from 5-amino-N-(13-amino-3,6,9-trioxadecyl)-2-nitrobenzamide (prepared as described for 5-
amino-N-(2-aminoethyl)-2-nitrobenzamide [29]; 31.7 mg, 77 mmol) and moenomycin A (118.0 mg, 74.6 mmol):
1
7c (100.0 mg, 68%). Pale yellow hygroscopic powder. H-NMR (600 MHz, CD3OD/(D6)DMSO 0.5 :0.4; only
signals that could be assigned): 8.14 8.12 (d, J ˆ 8.4, 1 arom. H); 7.98 (d, J ˆ 8.4, 1 arom. H); 7.91 (br. s,
1 arom. H); 5.69(br. s, HÀC(1F)); 5.24 (br. s, HÀC(2I)); 5.21 5.18 (d, J ˆ 15.8, HÀC(7I)); 5.13 5.10 (d, J ˆ
15.8, HÀC(6I)); 4.97 4.95 (t, J ˆ 7.8, HÀC(17I)); 4.88 4.86 (d, J ˆ 10.5, HÀC(3F)); 2.52 2.51 (d, J ˆ 7.0,
CH2(12I)); 1.86 1.82 (br. s, 8 H, CH2(15I), MeCOC,E); 1.57 (s, Me(25I)); 1.49( s, Me(19I)); 1.43 (s, Me(21I)); 1.42
(s, Me(20I)); 1.21 1.11 (CH2(9I), Me(6C)); 1.05 (s, Me(4F)); 0.79( s, Me(23I), Me(24I)). 13C-NMR (100,
150 MHz, CD3OD/(D6)DMSO 0.5 :0.4; only signals that could be assigned): 195.97, 181.53, 174.85, 173.19,
167.65, 159.65, 159.02 (7 CO); 154.74 (CTA ); 150.10 (C(11I)); 147.15 (arom. CNO2); 142.00, 141.50, 141.10, 136.78
(4CI); 134.37 (arom. C); 131.68 (CI); 126.69(arom. C); 125.23 (C I); 123.10 (CI); 109.66 (C(22I)); 104.29, 103.56
(C(1B,C,D,E)); 77.12, 74.03, 70.60, 69.35 (br., carbohydrate Cs, Csp); 61.92 (CH2); 56.03 (C(2E,C)); 42.56, 40.56
(2CI); 38.68, 38.10 (2 Csp); 36.19, 35.71 (CI); 33.11, 32.32, 31.92 (3CI); 29.28 (CH2); 28.01, 27.67, 27.51 (C(23I),
C(24I), C(16I)); 26.40 (C(19I)); 24.24 (C(25I)); 23.44 (MeCONHC,E); 18.33 (C(6C)); 17.68 (Me(4F)); 16.62
(C(20I)); 16.00 (C(21I)). 31P-NMR (80 MHz, CD3OD): À1.18. ESI-MS (C86H133N10O40P; 1978.02, 1976.8418):
987.41356 ([M À 2 H]2À; calc. 987.41363), 657.94185 ([M À 3 H]3À; calc. 657.9400).
(5BR)-5B-{1-{3-{13-{4-{(3R)-18-Biotinamido-3-{{{{2-{3-nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phen-
oxy}ethoxy}carbonyl}amino}methyl}-1,4-dioxo-2,9,12,15-tetraoxa-5-azaoctadecyl}phenyl}-1-oxo-12-thioxo-5,8-

Helvetica Chimica Acta Vol. 86 (2003)
4227
dioxa-2,11,13-triazatridecyl}-4-nitrophenyl}-3-(3-carboxy-1-oxopropyl)-1H-1,2,4-triazol-5-yl}-5B-de[(2-hy-
droxy-5-oxocyclopent-1-en-1-yl)carbamoyl]moenomycin A (8). A soln. of 4d (80.0 mg, 94 mmol) and N,N-
dimethylpyridin-4-amine (DMAP; 1.0 mg, 8.2 mmol) in dry pyridine (5 ml) was stirred at 08 for 10 min (Ar,
dimmed light). Upon addition of 4-isothiocyanatobenzoyl chloride (18.5 mg, 94 mmol), the soln. turned brown.
The mixture was stirred at 08 for 2 h, then allowed to warm to 58 and stirred at this temp. for 13 min (TLC
control (MeOH/CHCl3 2 :8), 4-(dimethylamino)cinnamaldehyde dipping reagent). A suspension of 7b
(150.0 mg, 79 mmol) in H2O (5 ml) was added. The mixture was stirred at 58 for 30 h. H2O (30 ml) was added,
and the mixture was evaporated. The residue was taken up in H2O (30 ml) and lyophilized. Chromatography
(RP18 MeCN/H2O 1:1, Rf 0.9), solvent evaporation, lyophilization, repeated FC (MeOH/CHCl3 2 :8, Rf 0.1;
PrOH/H2O 3 :7, Rf 0.7), and chromatography (RP18, MeCN/H2O 3 :7) provided 8 (30.0 mg, 10%). Pale yellow
hygroscopic powder. 1H-NMR (600 MHz, CD3OD/(D6)DMSO 4 :1; only signals that could be assigned): 8.20
8.19( d, J ˆ 8.8, 1 arom. H); 8.07 8.06 (d, J ˆ 8.8, 1 arom. H); 7.91 7.90 (m, 3 arom. H); 7.78 (br. s, 2 arom. H);
7.79 7.67 ( d, J ˆ 8.8, 2 arom. H); 7.04 (br. s, 1 arom. H); 5.66 (br. s, HÀC(1F)); 5.32 (m, HÀC(2I)); 5.31 5.28
(d, J ˆ 15.9, HÀC(7I)); 5.25 5.18 (m, HÀC(6I)); 5.12 5.10 (t, J ˆ 4.7, HÀC(13I)); 5.09 5.03 ( t, J ˆ 7.6,
HÀC(17I)); 4.95 4.93 (d, J ˆ 10.6, HÀC(3F)); 4.58, 4.57 (2s, CH2(22I)); 4.17 4.15 (m, SCHCHbi); 2.82 2.79
(dd, 2JAB ˆ 12.9, JBX ˆ 4.7, 1 H, SCH2CHbi), 2.60 2.58 (2JAB ˆ 12.9, 1 H, SCH2CHbi); 2.47 (s, CH2A); 2.07 1.98
3
(m, CH2(4I), CH2(5I), CH2(15I)); 1.94 1.91 (m, CH2(16I)); 1.88 (s, 6 H, MeCONHC,D), 1.82 1.79(CH (10I));
2
1.66 (s, Me(25I)); 1.63 1.60 (m, 4 H, CH2bi); 1.57 (s, Me(19I)); 1.51 (s, Me(21I)); 1.50 (s, Me(20I)); 1.40 1.26
(m, Me(6C), CH2(9I), 2 CH2sp); 1.11 (s, Me(4F)); 0.87 (s, Me(23I), Me(24I)). 13C-NMR (150, 100 MHz; HMBC,
HMQC; CD3OD/(D6)DMSO 4 :1; only signals that could be assigned): 174.27, 173.46, 171.88, 168.96, 166.28,
165.64, 164.52, 160.26 (9CO); 159.50 (arom. OC); 157.38 (C TA ); 157.23 (CO); 154.75 (CO); 150.08 (C(11I));
146.88 (arom. CNO2); 145.41 (arom. CNO2); 141.55 (arom. C); 140.55 (C(3I), C(7I)); 136.45 (C(14I)); 134.25
(arom. C); 131.31 (C(18I)); 130.82 (arom. C); 127.97 (arom. C); 126.02 (arom. C); 124.51 (C(17I)); 122.47
(C(13I)); 121.19(br. s, arom. C, CF3); 119.69 (arom. C); 113.97 (arom. C); 111.01 (arom. C); 108.61 (C(22I));
104.27, 103.72, 102.53, 101.91 (C(1B,C,D,E)); 94.83 (C(1F)); 77.20, 74.09, 68.11 (carbohydrate Cs, CH2Osp); 62.97,
60.39(C(6 D), biotin signals); 56.07 (SCHCH2bi); 55.81, 55.97 (C(2E), C(2C)); 44.02 (CH2is); 42.51 (C(9I)); 41.82
(C(15I)); 36.86, 36.70 (CHsp); 35.87, 35.60 (CH2A, CH2bi); 34.99 (C(8I)); 32.54 (CH2A); 31.72 (CH2sp); 31.40
(C(10I)); 29.64, 29.44, 28.84, 28.59 (2 CH2bi, C(5I), C(16I), CH2A); 27.13 (C(23I), C(24I)); 26.76 (C(25I)); 25.92
(CH2bi); 25.32 (MeCONHC,D); 23.32, 22.80 (C(6C)); 17.20 (C(19I), C(22I)); 16.13 (C(21I)); 15.48 (Me(4F)). 19F-
NMR (188 MHz, CDCl3): À67.08 (s, CF3). ESI-MS (C124H178F3N19O25PS2; 2918.96, 2917.100): 971.02420 ([M À
3 H]3À; calc. 971.02343).
N-{11-{3-Nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenoxy}-3,6-dioxa-9-azaundecyl}biotinamide
(4e). Commercially available N-[2-(2-ethoxyethoxy)ethyl]biotinamide (199.0 mg, 0.53 mmol), which we
prepared using the method of Braun et al. [39]; and 4a (99.0 mg, 0.34 mmol) were dissolved in MeOH
(10 ml). At 208, the pH was adjusted to 6.5 by addition of 0.25m CF3COOH in MeOH. Sodium
cyanoborohydride (23.0 mg, 0.37 mmol) was added, and the mixture was stirred for 30 h at 208 (TLC control
(CHCl3/MeOH 8 :2), 4-(dimethylamino)cinnamaldehyde dipping reagent). AcOH (0.2 ml) was added, and
solvents were evaporated. FC (CHCl3/MeOH 9:1) provided 4e (133.0 mg, 60%). Colorless oil. UV (CH2Cl2):
1
229(13484), 263 (4924), 325 (2463). 1H-NMR (200 MHz, CH3OD; H,1H-COSY): 7.95 7.93 (m, 1 arom. H);
7.72 (br. s, 1 arom. H); 7.21 (br. s, 1 arom. H); 4.53 4.48 (m, SCH2CHbi); 4.34 4.24 (m, SCHCH, CH2OAr);
3.69 3.65 ( m, 3 CH2O); 3.58 3.52 (m, CH2O); 3.39 3.33 ( m, CH2CONH, overlapped by MeOH); 3.25 3.18
(m, SCHCH); 3.16 3.10 (m, CH2NH); 2.98 2.93 (dd, 2JAB ˆ 12.5, 1 H, SCH2CH); 2.95 2.89 (m, CH2NH);
2.74 2.67 (d, 2JAB ˆ 12.5, 1 H, SCH2CHbi); 2.25 2.18 (t, J ˆ 7.0, CH2CONHbi); 1.83 1.26 (m, 3 CH2bi).
13C-NMR (75 MHz, CDCl3): 176.09(CONH bi); 165.96 (HNCONHbi); 161.41 (CH2OAr); 151.10 (arom. CNO2);
132.67 (arom. CÀCN2); 120.48, 114.80, 111.83 (arom. CH); 71.39, 71.28 (2 CH2O); 70.64, 70.56
(OCH2CH2NHCH2CH2OAr); 69.20 (CONHCH2CH2O); 63.39( CHCHSbi); 61.67 (SCH2CHbi); 56.93
(SCHCHbi); 41.01 (HNCH2CH2OAr); 40.30 (SCH2CHbi); 36.75 (CH2CONHbi); 29.71, 29.49, 26.80 (3 CH2bi).
19F-NMR (282 MHz, CDCl3): 2.65 (s, CF3); 2.03 (s, CF3COO). ESI-MS (C26H36F3N7O7S; 647.67, 647.24):
‡
648.24211 ([M ‡ H] ; calc. 648.2418).
N-{9-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)-11-{3-nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenoxy}-
3,6-dioxa-9-azaundecyl}biotinamide (4f). To a soln. of 4e (120.0 mg, 0.185 mmol) in EtOH (7 ml), 3,4-
diethoxycyclobut-3-ene-1,2-dione (75 ml, 0.51 mmol) was added, and the mixture was stirred at 208 for 19h
(TLC control (CHCl3/MeOH 8 :2), 4-(dimethylamino)cinnamaldehyde dipping reagent). Evaporation and FC
(CHCl3/MeOH 10 :1) provided 4f (130.0 mg, 91%). Colorless oil. 1H-NMR (200 MHz, CDCl3; 1H,1H-COSY):
7.79 7.77 ( m, 1 arom. H); 7.67 (m, 1 arom. H); 7.01 (m, 1 arom. H); 6.62 (w1/2 ˆ 3.92, 0.5 H, NHCH2); 6.46
(w1/2 ˆ 5.0, 0.5 H, NHCH2); 6.37 (w1/2 ˆ 5.74, 1 H, HNCONH); 5.47 (w1/2 ˆ 5.62, 1 H, HNCONH); 4.84 4.72

4228
Helvetica Chimica Acta Vol. 86 (2003)
(m, CH3CH2O); 4.53 4.46 (m, SCHCHbi); 4.39 4.20 ( m, SCH2CHbi, ArOCH2, CH2N); 3.97 3.95 (CH2N);
3.75 3.71 (m, CH2O); 3.60 3.50 (m, 3 CH2O); 3.43 3.38 (m, CONHCH2); 3.18 3.05 (w1/2 ˆ 5.56,
3
SCHCH2bi); 2.94 2.85 (dd, 2JAB ˆ 13.2, JBX ˆ 4.8, 1 H, SCH2); 2.74 2.68 (d, 2JAB ˆ 13.2, 1 H, SCH2); 2.29
2.18 (m, CH2CONHbi); 1.71 1.61 (2 CH2bi); 1.49 1.24 ( m, MeCH2O, CH2bi). 13C-NMR (50 MHz, CDCl3):
189.06, 188.76 (COsa); 183.13, 183.03 (COsa); 177.58, 177.18 and 173.16, 172.84 (CˆCsa); 173.71, 173.63
(CH2CONH); 164.14 (CObi); 159.49, 159.41 (arom. OC); 149.78 (arom. CNO2); 132.52, 132.38 (arom. CCN2);
130.08, 121.85 (2q, each with 1J(C,F) ˆ 273, CF3); 119.91, 119.60 (arom. CH); 114.69 (arom. CH); 110.44, 109.96
(arom. CH); 70.93 69.77 (MeCH2Osa, CH2O); 68.22 (CH2O); 67.17 (CH2O); 62.03 (CHCHSbi); 60.44
(CHCH2Sbi); 55.89(S CHCH2bi); 50.99 (2 CH2N); 40.86 (SCH2CHbi); 39.40 (CH2NCO); 36.21 (CH2CONH);
28.43 (2J(C,F) ˆ 42.0, CCF3); 28.44, 28.39(2 CH bi); 25.90 (CH2bi); 16.25 (MeCH2O). 19F-NMR (188 MHz,
2
‡
CDCl3): 11.36, 11.34 (CF3). ESI-MS (C32H40F3N7O10S; 771.77, 771.251): 772.25759([ M ‡ H] ; calc. 772.25095),
‡
‡
794.23993 ([M ‡ Na] , calc. 794.24017), 1543.51046 ([2M ‡ H] ; calc. 1543.50972).
(5BR)-5B-{1-{3-{{{2-{{2-{(8-Biotinamido-3,6-dioxaoctyl){2-{3-nitro-5-[3-(trifluoromethyl)-3H-diazirin-3-
yl]phenoxy}ethyl}amino}-3,4-dioxocyclobut-1-en-1-yl}amino}ethyl}amino}carbonyl}-4-nitrophenyl}-3-(3-car-
boxy-1-oxopropyl)-1H-1,2,4-triazol-5-yl}-5B-de[(2-hydroxy-5-oxocyclopent-1-en-1-yl)carbamoyl]moenomycin
A (9). To a suspension of 7a (67.0 mg, 36 mmol) in MeOH/H2O 1:1 (8 ml), 0.2m Et3N in MeOH was added until
a pH value of 9was reached (dimmed light). At 20 8, a soln. of squaric acid ester amide 4f (28.0 mg, 36 mmol) in
MeOH (5 ml) was added. The mixture was stirred at 208 (TLC control PrOH/H2O 7:3), anisaldehyde and 4-
(dimethylamino)cinnamaldehye dipping reagents). After 72 h, the mixture was evaporated, the residue taken
up in H2O (20 ml) and separated by chromatography (RP18, MeCN/H2O 3 :7). The product fractions were
evaporated. The residue was redissolved in H2O (30 ml) and the soln. adsorbed onto −Kieselguhr× (1.0 g) by
lyophilization. FC (PrOH/H2O 7.5 :2.5) provided 9 (30.0 mg, 33%). Hygroscopic pale yellow powder. 1H-NMR
1
(600 MHz; H,1H-COSY, HMBC; CD3OD/(D6)DMSO 4 :1; only signals that could be assigned): 8.17 (d, J ˆ
8.4, 1 arom. H); 8.00 7.97 (m, 2 arom. H); 7.81 (br. s, 1 arom. HBPP), 7.60 (s, 1 arom. HBPP); 7.03 (br. s,
1 arom. HBPP); 5.69(br. s, HÀC(1F)); 5.31 (m, HÀC(2I)); 5.27 5.25 (d, J ˆ 15.2, HÀC(7I)); 5.21 5.17
(m, HÀC(6I)); 5.03 5.01 (t, J ˆ 7.0, HÀC(17I)); 4.99 4.97 (m, CH2OAr); 4.94 4.93 (br., carbohydrate Hs);
4.46 (m, HÀC(1C)); 4.35 4.33 (m, SCH2CHbi); 4.12 (s, O(CH2)2Osp); 4.17 4.15 (m, SCHCHbi); 4.10 3.73 (br.,
carbohydrate Hs); 3.60 (s, HN(CH2)2CONH); 3.53 3.41 (br., carbohydrate Hs); 3.38 3.35 (m, N(CH2CH2O)2sa);
3
3.07 3.04 (m, SCHCHbi); 2.90 (m, HÀC(4C)); 2.80 2.77 (dd, 2JAB ˆ 12.5, JBX ˆ 4.7, 1 H, SCH2CHbi); 2.58
2.56 (d, 3JBX ˆ 12.5, 3 H, SCH2CHbi, COCH2A); 2.44 (s, 2 H, NCH2CH2Osa); 2.07 2.05 (t, J ˆ 7.3, 1 CH2); 2.01
1.96 (m, CH2(4I), CH2(15I), CH2(16I)); 1.94 1.80 (m, COCH2CH2COOHA, MeCONHE, CH2bi); 1.86 (s, Me-
CONHC); 1.79 1.74 ( m, CH2(10I)); 1.63 (s, Me(25I)); 1.55 (s, Me(19I)); 1.49( s, Me(21I)); 1.48 (s, Me(20I));
1.49 1.39 ( m, 6 H, 2 CH2bi, partly overlapped); 1.30 1.23 (m, 7 H, CH2bi, CH2(9I), NCH2CH2Osa); 1.10
(Me(4F)); 0.85 (Me(23I), Me(24I)). 13C-NMR (150, 100 MHz; HMBC, HMQC; CD3OD/(D6)DMSO 4 :1; only
signals that could be assigned): 194.35, 183.79, 183.47, 179.63, 175.41, 174.03, 172.88, 170.21, 167.35, 165.38 (8 CO,
CˆCsa); 160.80 (CTA ); 160.07 (arom. CO); 158.41, 155.29(2 CO); 150.66 (C(11 I)); 147.52 (arom. C); 147.25
(arom. CBPPÀNO2); 142.14 (arom. C); 141.21 (C(7I)); 137.08 (C(3I)); 134.87 (arom. C); 132.16 (CCN2CF3);
131.94 (C(17I)); 128.50 (arom. C); 126.83 (arom. C); 126.66 (C(6I)); 126.23 (arom. C); 125.13 (C(13I)); 124.20
(arom. C); 123.16 (C(2I)); ca. 121.5 (CF3); 120.28 (CBPP); 114.75 (CBPP); 111.87 (CBPP); 109.15 (C(22I)); 105.01,
104.45, 103.76, 102.56 (C(1B,C,D,E)); 85.23 (C(4C)); 81.78 (C(4E)); 77.82, 75.73, 74.73, 73.87, 73.58, 72.18
(carbohydrate Cs, OCH2); 62.89, 62.48, 61.16 (C(6D), SCH2CHbi); 56.71 (CHCHSbi); 56.39, 55.59 (C(2E,C));
36.43 (CH2N); 36.22 (C(8I)); 35.64 (C(12I)); 33.23 (CH2bi); 32.41, 32.14 (C(4I), C(4I)); 32.05 (C(10I)); 30.39
(CH2A); 29.43, 29.22 (CH2bi); 27.71, 27.42 (C(23I), C(24I)); 26.55 (CH2bi); 25.86 (C(25I)); 23.90, 23.36 (MeCOC,
MeCOE); 17.72 (C(20I), C(6c)); 16.50 (C(21I)); 16.02 (Me(4F)). 19F-NMR (282 MHz, CDCl3): 11.79( s, CF3).
31P-NMR (282 MHz, CDCl3): À1.84. ESI-MS (C108H151F3N17O46PS; 2543.50, 2541.940): 1269.96424 ([M ‡ H]2À
;
calc. 1269.96320), 864.30686 ([M À 3 H]3À; calc. 864.30637).
Prop-2-enyl 4-Benzoylbenzoate (10a). A mixture of 4-benzoylbenzoic acid (2.5 g, 11 mmol), N,N'-
diisopropylcarbodiimide (2.5 ml, 16 mmol), DMAP (0.1 g, 0.8 mmol), and dry CH2Cl2 (25 ml) was stirred for
2 h at 208 (dimmed light). Allyl alcohol (10 ml) was added, and the mixture stirred at 208 for 24 h (TLC control
(petroleum ether/AcOEt 1 :1), potassium permanganate dipping reagent). The mixture was evaporated, the
residue taken up in CH2Cl2 (ca. 300 ml), the soln. washed with sat. aq. NaHCO3 soln. (2 Â 300 ml) and H2O
(600 ml), dried, and evaporated, and the residue purified by FC (petroleum ether/AcOEt 9:2): 10a (2.5 g,
85%). Colorless oil. UV (MeOH): 256.50 (27952). IR (KBr): 1722, 1662, 1267, 1107, 930, 710. 1H-NMR
(200 MHz, CDCl3): 8.20 8.14 (m, 2 arom. H); 7.87 7.78 (m, 4 arom. H); 7.66 7.57 (m, 1 arom. H); 7.54 7.45
(m, 2 arom. H); 6.16 5.96 (m, CH2ˆCHCH2); 5.49 5.38 ( ddd, J ˆ 17.2, 2.9, 1.0, 1 H, CH2ˆCHCH2); 5.35
5.28 (dd, J ˆ 10.1, 2.2, 1.0, 1 H, CH2ˆCHCH2); 4.89 4.84 ( dt, 3J ˆ 5.3, 4J ˆ 1.0, CH2ˆCHCH2). 13C-NMR

Helvetica Chimica Acta Vol. 86 (2003)
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(50 Hz, CDCl3): 196.08 (CO2); 165.60 (CˆO); 141.55 (C(4)); 137.12 (C(1')); 133.42 (C(4')); 133.11
(CH2ˆCHCH2); 133.09 (C(1)); 130.28 (C(2)); 129.94, 129.73 (C(3), C(2')); 128.63 (C(3')); 118.38
‡
(CH2ˆCHCH2); 66.19(CH ˆCHCH2). FAB-MS (C17H14O3; 266.30, 266.0943): 267.1 ([M ‡ H] ).
2
2-Oxoethyl 4-Benzoylbenzoate (10b). Into a soln. of 10a (2 g, 7.51 mmol) in CH2Cl2/MeOH 4 :1 (50 ml) at
À738 (stirring), ozone (50 l hÀ1, 2.5 g hÀ1) was introduced for 10 min (TLC control (every 2 min; petroleum
ether/AcOEt 8 :2), fuchsin dipping reagent). Purging with N2, addition of Me2S (2 ml, 44 mmol), stirring at 208
for 3 h, evaporation, and FC (petroleum ether/AcOEt 10 :4) provided 10b (1.80 g, 90%). Colorless oil. UV
(MeOH): 256.00 (33929). IR (KBr): 3431, 1718, 1649, 1273. 1H-NMR (CDCl3, 200 MHz): 9.74 (s, CHO); 8.24
8.18 (m, 2 arom. H); 7.89 7.78 ( m, 4 arom. H); 7.67 7.58 (m, 1 arom. H); 7.54 7.46 (m, 2 arom. H); 4.96
(s, CH2). 13C-NMR (75 MHz, CDCl3, APT): 195.93 (‡, CO); 195.25 (À, HCO); 165.22 (‡, COOR); 142.02 (‡,
C(4)); 136.87 (‡, C(1')); 133.16 (À, C(4')); 131.97 (‡, C(1)); 130.19( À, C(2)); 129.94, 129.79 (À, C(3), C(2'));
‡
128.59( À, C(3')); 69.38 (‡, CH2). FAB-MS (C16H12O4; 268.27, 268.073559): 269.0 ([ M ‡ H] ).
3-Hydroxypropyl 4-Benzoylbenzoate (10c). A mixture of 4-benzoylbenzoic acid (2.1 g, 9.3 mmol), dry
pyridine (70 ml), 1,1'-carbonylbis[1H-imidazole] (2.3 g, 14 mmol) was stirred at 208 for 2 h. Propane-1,3-diol
(5 ml, 66 mmol) was added, and the mixture was stirred at 208 for 24 h. Pyridine was evaporated and CH2Cl2
(200 ml) was added. Usual workup followed by FC (CH2Cl2) furnished 10c (2.6 g, 91%). Colorless oil. UV
(MeOH): 256.0 (25209). IR (KBr): 3282, 1716, 1650, 1317, 1280, 1111, 1059, 708. 1H-NMR (300 MHz, CDCl3):
8.16 8.12 (m, 2 arom. H); 7.85 8.78 (m, 4 arom. H); 7.62 7.46 (m, 3 arom. H); 4.56 (t, J ˆ 6.2, CH2CO2); 3.80
(t, J ˆ 6.2, CH2OH); 2.27 (br. s, OH); 2.07 2.01 (quint., J ˆ 6.2, CH2CH2CH2). 13C-NMR (50 Hz, CDCl3,
APT): 196.10 (‡, CO); 166.20 (‡, COO); 141.48 (‡, C(4)); 136.98 (‡, C(1')); 133.30 (C(1)); 133.06 (C(4'));
130.18 (À, C(2)); 129.85, 129.59 (À, C(3), C(2')); 128.55 (À, C(3')); 62.42 (‡, CH2CO2); 59.21 (‡, CH2OH);
‡
‡
31.93 (‡, CH2CH2CH2). ESI-MS (C17H16O4; 284.3, 284.105): 285.0 ([M ‡ H] ), 306.9([ M ‡ Na] ).
N-{{3-[(4-Benzoylbenzoyl)oxy]propoxy}carbonyl}-1,2-O-(hexafluoroisopropylidene)-l-isoserine (ˆ 3-
{{{{[(4S)-2,2-Bis(trifluoromethyl)-5-oxo-1,3-dioxolan-4-yl]methyl}amino}carbonyl}oxy}propyl 4-Benzoylben-
zoate; 10d). As described for 4c from 10a and 5b. UV (MeOH): 256 (2414). IR (KBr): 3350, 1734, 1659,
1527, 1319, 1281, 1268, 1228, 1130, 982, 721. 1H-NMR (300 MHz, CDCl3): 8.16 8.12 (m, 2 arom. H); 7.86 7.78
(m, 4 arom. H); 7.67 7.58 (m, 1 arom. H); 7.54 7.45 (m, 2 arom. H); 5.22 5.16 (m, CH2NHCOO); 4.83 4.78
(t, J ˆ 4.9, CHCH2NH); 4.48 4.41 (t, J ˆ 6.2, CO2CH2); 4.32 4.26 (t, J ˆ 6.2, CO2CH2); 3.91 3.59
(m, CH2NH); 2.20 1.95 (quint., CH2CH2CH2). 13C-NMR (50 and 75 MHz, CDCl3): 196.42 (PhCO); 166.25
(CO); 166.00 (CO); 156.41 (HNCO); 141.58 (C(4)); 136.94 (C(1)); 133.25 (C(1')); 133.18 (C(4')); 130.30 (C(2));
129.98 (C(2), C(3')); 129.66 (C(3)); 128.63 (C(2')); 119.62 (q, 1J(C,F) ˆ 289.1, CF3); 118.84 (d, 1J(C,F) ˆ 286.9,
CF3); 74.40 (OCHCH2is); 62.48 (NHCO2CH2CH2); 62.00 (ArCO2CH2); 41.23 (CHCH2NH); 28.34
(CH2CH2CH2). 19F-NMR (282.3 MHz, CDCl3): À2.81 to À2.87 (m, CF3); À3.10 to À3.16 (m, CF3); À4.80
‡
(s, unassigned). ESI-MS (C24H19F6NO8; 563.41, 563.101); 586.2 ([M ‡ Na] ).
(8S)-18-Biotinamido-8-hydroxy-5,9-dioxo-4,13,16-trioxa-6,10-diazaoctadecyl 4-Benzoylbenzoate (10e). To
a soln. of 10d (700.0 mg, 1.24 mmol) in 1,2-dimethoxyethane (15 ml), a soln. of 6a (600.0 mg, 1.60 mmol) in 1,2-
dimethoxyethane (10 ml) was added dropwise at 08 (dimmed light). The mixture was stirred at 08 for 2.5 h and
then allowed to warm to r.t. H2O (10 ml) was added, the mixture evaporated, and the residue taken up in H2O
(30 ml) and freeze-dried. FC (MeOH/CHCl3 2.5 :7.5) provided 10e (475.0 mg, 62%). Colorless oil. UV
(MeOH): 256 (22063). IR (KBr): 3290, 3282, 1695, 1645, 1547, 1319, 1275, 1111, 721. 1H-NMR (600 MHz,
CDCl3; 1H,1H-COSY): 8.08 8.06 (d, J ˆ 8.4, 2 arom. H); 7.85 7.76 (d, J ˆ 8.4, 2 arom. H); 7.74 7.73 (d, J ˆ 6.8,
2 arom. H); 7.59 7.55 ( m, 1 arom. H); 7.55 (w1/2 ˆ 11, CONH); 7.45 7.43 (m, 2 arom. H); 6.10 (m, w1/2 ˆ 19,
0.5 H, CONH); 4.52 (m, SCH2CHbi); 4.38 4.36 (m, SCHCHbi, 1 COOCH2); 4.19 4.18 ( m, 1 COOCH2,
CHOHis); 3.56 3.52 (m, 10 H, 3 CH2O, CH2CHOH, CONHCH2); 3.42 3.40 (m, 5 H, CH2O, CONHCH2,
3
OH); 3.11 (m, SCHCHbi); 2.87 2.85 (dd, 2JAB ˆ 13.1, JBX ꢀ 4, 1 H, SCH2CHbi); 2.76 2.74 (d, 2JAB ˆ 13.1, 1 H,
SCH2CHbi); 2.26 2.24 (m (ˆ−t×), J ˆ 6.3, CH2CONHbi); 2.07 2.05 (m (ˆ−t×), J ˆ 5.8, ArCOOCH2CH2); 1.70
1.40 (m, 3 CH2bi). 13C-NMR (50 Hz, CDCl3; HETCOR): 198.00 (PhCO); 175.70 (CONHCH2); 174.61
(CONHCH2); 167.75 (ArCOOCH2); 166.21 (HNCOOCH2); 160.17 (HNCONH); 143.42, 138.86, 135.13
(arom. C(1), C(1'), and C(4)); 135.02 (arom. C(4')); 132.11, 131.79, 131.50, 130.49 (6 arom. CH); 74.22
(CHOHis); 72.16 (2 CH2O); 71.88, 71.51 (2 CH2O); 64.08, 64.00 (OCH2CH2CH2O); 63.80 (SCHCH); 62.35
(SCH2CH); 57.75 (SCHCH); 46.87 (HOCHCH2NHis); 42.50 (SCH2CHbi); 41.32, 40.85 (2 CONHCH2sp); 37.84
(CH2CONHbi); 30.48 (ArCOOCH2CH2); 30.30, 30.09, 27.65 (3 CH2bi). ESI-MS (C37H49N5O11S; 771.88, 771.315):
‡
‡
‡
772.32286 ([M ‡ H] ; calc. 772.32220), 794.30583 ([M ‡ Na] ; calc. 794.30470), 810.27953 ([M ‡ K] ; calc.
810.27864).
(5BR)-5B-{{3-{13-{4-{(3S)-3-{{{{3-[(4-Benzoylbenzoyl)oxy]propoxy}carbonyl}amino}methyl}-13-biotin-
amido-1,4-dioxo-2,8,11-trioxa-5-azatridecyl}phenyl}-1-oxo-12-thioxo-5,8-dioxa-2,11,13-triazatridecyl}-4-nitro-

4230
Helvetica Chimica Acta Vol. 86 (2003)
phenyl}-3-(3-carboxy-1-oxopropyl)-1H-1,2,4-triazol-5-yl}-5B-de[(2-hydroxy-5-oxocyclopent-1-en-1-yl)car-
bamoyl]moenomycin A (11). As described for 8, from 10e (170.0 mg, 221 mmol), 4-isothiocyanatobenzoyl
chloride (45.0 mg, 227 mmol), and 7a (170.0 mg, 94 mmol): 11 (33.0 mg, 13%). Pale yellow hygroscopic powder.
1H-NMR (600 MHz, CD3OD/(D6)DMSO 4 :1; only signals that could be assigned): 8.22 7.35 (m, 14 arom. H);
5.68 (br. s, HÀC(1F)); 5.33 (m, HÀC(2I)); 5.32 5.29( d, J ˆ 16.2, HÀC(7I)); 5.16 5.13 (m, HÀC(6I)); 5.10
5.00 (t, J ˆ 6.8, HÀC(17I)); 5.59, 5.58 (2s, CH2(22I)); 1.94 1.90 (m, 6 H, MeCONHC,E); 1.67 (s, Me(25I)); 1.58
(s, Me(19I)); 1.52 (s, Me(21I)); 1.51 (s, Me(20I)); 1.31 1.17 (m, 10 H, CH2bi, Me(6C), CH2(9I)); 1.11 (s, Me(4F));
0.88 (Me(23I), Me(24I)). 13C-NMR (150, 100 MHz; HMBC, HMQC; CD3OD/(D6)DMSO 4 :1; only signals that
could be assigned): 172.46, 159.42 (2 CO); 157.64, 154.8 (2 CTA ); 150.19(C(10 I)); 146.96 (arom. CNO2); 141.84,
140.67 (arom. C); 137.32, 136.53, 136.29, 135.95 (C(3I), arom. C); 133.50 (C(14I)); 131.38, 130.42, 130.41, 130.32,
130.12, 130.08, 129.78, 129.77, 128.99, 128.43, 128.32 (arom. C); 126.15 (C(6I)); 124.56 (C(13I)); 122.57 (C(2I));
108.60 (C(22I)); 103.73, 102.61, 101.98 (C(1B,C,D)); 95.02 (C(1F)); 81.43 (C(4H)); 77.31 69.74 (carbohydrate Cs,
CH2Osp); 62.63, 62.28, 61.91, 60.53 (C(6D), CO2CH2, biotin Cs); 56.15, 55.26, 55.03, 54.32 (C(2C), C(2E), CHbi);
41.92, 35.67, 35.05 (C(15I), 2 CH2sp); 32.54 (C(12I)); 32.12, 31.48 (CH2); 31.83 (C(10I)); 29.82, 29.43, 28.65, 27.13,
26.82 (2 CI, 3 CH2bi); 25.96 (CH2bi); 25.31 (C(25I)); 23.36 (MeCONC,E); 20.39(C(20 I)); 19.97 (C(6C)); 16.07
(C(21I)); 16.00 (Me(4F)). ESI-MS (C123H169N16O49PS2; 2750.87, 2749.040): 1373.50848 ([M À 2 H]2À; calc.
1373.51289), 915.33825 ([M À 3 H]3À; calc. 915.33950).
2-{{2-[2-(2-Biotinamidoethoxy)ethoxy]ethyl}amino}ethyl 4-Benzoylbenzoate (10f). A soln. of 6a (600.0 mg,
1.6 mmol) and aldehyde 10b (100.0 mg, 0.37 mmol) in dry MeOH (40 ml; stirring under dimmed light) was
adjusted to pH 6.5 with AcOH. At 208, sodium cyanoborohydride (23 mg, 0.84 mmol) was added, and the
mixture was stirred at 208 for 48 h (TLC control (MeOH/CHCl3/iPrNH2 2 :10 :0.1), 4-(dimethylamino)cin-
namaldehyde dipping reagent). Evaporation and FC (MeOH/CHCl3/iPrOH 0.75 :10 :0.1) furnished 10f
(100.0 mg, 43%). Colorless oil. UV (MeOH): 206.0 (20783), 257 (16030). IR (KBr): 3388, 3327, 2931, 1691, 1652,
1275, 756. 1H-NMR (CDCl3, 200 MHz): 7.85 7.76 (m, 4 arom. H); 7.70 7.57 (m, 3 arom. H); 7.54 7.40
(m, 2 arom. H); 7.04 (br. s, 0.5 H, CONH); 6.20 (br. s, 0.5 H, HNCONH); 5.65 (br. s, 0.5 H, HNCONH); 4.55
4.42 (m, SCH2CHbi); 4.29 4.23 ( m, SCHCHbi); 4.15 3.90 (m, ArCOOCH2, CONHCH2); 3.74 3.30 (m, 12 H,
3
4 CH2O, 2 CH2NH); 3.15 3.06 (m, SCHbi); 2.90 2.80 (dd, 2JAB ˆ 12.8, JBX ˆ 4.4, 1 H, SCH2CHbi); 2.72 2.65
(d, 2JAB ˆ 12.8, 1 H, SCH2CHbi); 2.21 2.13 (t, J ˆ 7.2, CH2CONHbi); 1.68 1.33 (m, 4 H, 3 CH2bi, overlapped by
iPrNH2). 13C-NMR (50 MHz, CDCl3): 196.21 (PhCO); 173.62 (CONH); 172.14 (ArCOO); 163.90 (HNCONHbi);
140.34, 138.48, 137.13, 132.96, 130.17, 129.85, 128.56, 127.51, 127.36, 127.20 (10 C, arom. CH, arom. C); 70.56,
70.47 (OCH2CH2Osp); 69.80 (CH2CO2Ar); 68.82 (2 CH2Osp); 61.97, 60.28 (SCH2CHbi, SCHCHbi); 59.76
(SCHbi); 55.62 (SCH2bi); 53.61, 50.68 (CH2NHCH2); 47.20 (CONHCH2sp); 43.85 (SCH2CHbi); 39.27 (iPrNH2);
37.82 (CONHCH2sp); 35.85 (CH2CONHbi); 28.36, 28.18, 25.74 (3 CH2bi). ESI-MS (C32H42N4O7S; 626.77,
‡
626.277): 627.28528 ([M ‡ H] , calc. 627.28470).
MIC Values. The MIC values against seven different Staph. aureus strains (ATCC 25923, ATCC 29213,
MRSA 1309, SG 511, PEG 18, PEG 5, and KNS PEG 5) were determined by a serial twofold micro dilution
method on microtiter plates (Iso-Sensitest medium, Oxoid). A series of decreasing concentrations of the
compound under investigation was prepared in the medium. For inoculations, 1 ¥ 105 cfu mlÀ1 were used. The
MICs were determined (absence of visible turbidity) after 24 h at 378. The MIC values were calculated as the
average values from three measurements.
Photoaffinity Labeling of PBP 1b with Affinity Label 9. PBP 1b was obtained and purified by ampicillin
affinity chromatography as described previously [36]. PBP 1b (74.25 mg) from a dialyzed ampicillin-affinity
chromatography fraction (150 mg/ml in 50 mm Tris ¥ HCl (pH 7.6) containing 0.1 mm MgCl2 and 1 mm 2-
mercaptoethanol) was mixed in the dark with photoaffinity label X (5 mg) in an Eppendorf tube. The mixture
was gently swirled for 90 min in the dark to facilitate complex formation. The soln. was then transferred into a
glass tube (5-mm diameter) and irradiated at 350 nm for 20 min in a Rayonet reactor which was cooled. The SDS
PAGE on a 8.5% gel was performed by the Laemmli method as described previously (BioRad Mini-Protean 3,
0.75 mm). Proteins were detected after separation by staining with Coomassie Brilliant Blue G250 (Serva) and
by Western blotting. For Western-blot analysis, the semi-dry method was used in a discontinuous buffer system.
The proteins were transferred within 1 h at 25 Vonto a Millipore PVDF membrane (pore size 0.45 mm) using the
Trans-Blot unit of BioRad. For the detection of labeled protein, the streptavidineÀalkaline-phosphatase
conjugate was employed. In a control experiment, the Western blotting and visualization was also performed
with botinylated bovine serum albumin. Two control experiments were carried out: i) Two Eppendorf tubes
were charged with PBP 1b from a dialyzed ampicillin-affinity-chromatography fraction, each with 74.25 mg. ii)
Two Eppendorf tubes were charged with PBP 1b (each 74.25 mg from a dialyzed ampicillin-affinity
chromatography fraction), photoaffinity label 9 (5 mg), and moenomycin A (5 mg). The samples were treated

Helvetica Chimica Acta Vol. 86 (2003)
4231
as described above, with the exception that one sample of each series was not irradiated. All samples were
analyzed by SDS PAGE and Western blot as described. For the endoproteinase digest, a sample containing
labeled PBP 1b was precipitated with 80% acetone. The pellet was solubilized in 50 ml of digest buffer (25 mm
Tris ¥ HCl (pH 8.5), 1 mm EDTA, 0.1% (w/v) SDS) in Eppendorf tubes and mixed with endoproteinase Lys-C
(100 mg/ml in digest buffer) at a ratio of 1:20. The digest was incubated for 18 h at 378. For control, an aliquot of
the endoproteinase-Lys-C-containing soln. was incubated with melittin, whereupon the typical four cleavage
products were formed. After the endoproteinase digest, the sample was analyzed by SDS-tricine PAGE, which
was performed in a vertical apparatus (BioRad Mini-Protean 3, 0.75 mm) as described by Sch‰gger and von
Jagow [40]. Routinely, two gels were prepared, one for Coomassie Brilliant Blue G250 (Serva) staining and one
for the Western-blot analysis. For Western blotting, the semi-dry method was used in a discontinuous buffer
system. The peptides were transferred onto a PVDF membrane (Millipore, pore size 0.2 mm) by using the
BioRad-Trans-Blot unit (1 h at 25 V). For the detection, again the streptavidineÀalkaline-phosphatase-
conjugate method was used (control with biotinylated bovine serum albumine).
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Received August 22, 2003