Attempted introduction of a fourth amide NH into the carboxylate-binding pocket of glycopeptide antibiotics

Article abstract of DOI:10.1039/a906502d

We report the synthesis of a novel derivative of the glycopeptide antibiotic vancomycin, modified at the N-terminus. This incorporates a fourth amide NH into the antibiotic, at the position which was formerly the N-terminus of the antibiotic-binding pocke

Full text of DOI:10.1039/a906502d

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Attempted introduction of a fourth amide NH into the
carboxylate-binding pocket of glycopeptide antibiotics
Jochen Görlitzer, Thomas F. Gale and Dudley H. Williams*
Cambridge Centre for Molecular Recognition, University Chemical Laboratory,
Lensfield Road, Cambridge, UK CB2 1EW. E-mail: dhw1@cus.cam.ac.uk
Received (in Cambridge, UK) 10th August 1999, Accepted 15th September 1999
We report the synthesis of a novel derivative of the glycopeptide antibiotic vancomycin, modified at the N-terminus.
This incorporates a fourth amide NH into the antibiotic, at the position which was formerly the N-terminus of the
antibiotic-binding pocket. Although this modification gives the potential to form an extra hydrogen bond to the
carboxylate anion of the bacterial cell-wall precursor analogue (N,N)-diacetyl--lysyl--alanyl--alanine, the modified
antibiotic does not show enhanced binding to this precursor. This lack of enhanced binding is associated with an
unfavourable conformational change (relative to the desired conformation) in the antibiotic.
Introduction
The glycopeptide antibiotics vancomycin and teicoplanin find
widespread clinical use against infections due to multi-resistant
bacteria such as strains of Staphylococcus aureus (MRSA) and
1,2
are often the antibiotics of last resort. Vancomycin-group
antibiotics bind to bacterial cell-wall mucopeptide precursors
2
terminating in the sequence -lysyl--alanyl--alanine, thereby
3,4
inhibiting the crosslinking enzymes. The resulting inter-
ference with peptidoglycan synthesis causes a loss of mechan-
ical strength of the cell wall and leads ultimately to cell lysis.
In the binding of bacterial cell-wall analogues terminating in
5

-Ala--Ala to vancomycin (2) and ristocetin A, the main
source of the binding energy is to be found in the binding of the
carboxylate of the terminal -Ala residue into a pocket of three
adjacent NHs of the antibiotic (the NHs of residues 1, 2, and 3,
6
Fig. 1a). We reasoned that if this pocket could be extended to
one of four adjacent NHs (Fig. 1b), then a dramatic increase
in the binding affinity might occur. The covalent connectivity
which can allow, in principle, such an extension does not simply
involve the acylation (e.g., N-acetylation) of the N-terminal
amino group (residue 1) of, for example N-demethylvanco-
mycin (3) (Scheme 1). This is because the -stereochemistry at
this centre causes the N-terminal ammonium ion to point out
of the carboxylate anion-binding pocket, and the α-CH of resi-
due 1 to point into this pocket. Thus an inversion of the stereo-
chemistry of residue 1 is required, prior to its acetylation,
to give N-acetyl-N-demethyl--leucyl-vancomycin (7). In
principle, the required modifications can be achieved either
by inverting the stereochemistry of the existing N-terminal
Fig. 1 The cooperative array of hydrogen bonds is responsible for
ligand binding.

-leucine of N-demethylvancomycin (3) and subsequently
acetylating this material, or alternatively by removing this resi-
due, and then replacing it by N-acetyl--leucine. Both methods
were attempted.
Results and discussion
Initial attempts to obtain 7 involved introduction of sp -
2
hybridisation (in the form of a ketone) at the centre which
7
originally constituted the α-CH centre of -leucine. In the case
of ristocetin aglycone, there is precedence for reduction of such
resulted in very low yields of the desired product, despite a
broad variation of the reaction conditions. Therefore, the alter-
native strategy involving the removal of the N-methyl--leucine
a ketone (1), using ammonium acetate and cyanoborohydride,
8
to give a product with the required stereochemistry. However,
9
the application of this method to the vancomycin ketone (10)
from vancomycin by an Edman degradation and subsequent
J. Chem. Soc., Perkin Trans. 1, 1999, 3253–3257
This journal is © The Royal Society of Chemistry 1999
3253

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Scheme 1 Scheme for the synthesis of vancomycin derivatives. Reagents and conditions: a, 1 M HCl, reflux; b, PhNCS, pyridine–water; then
TFA, 50 ЊC.
Table 1 Binding constants for compounds 1–13
Ϫ1
K /M
lig
Derivative
(N,N)Ac KAA
2
6
5
1
5.6 × 10
8.0 × 10
1.7 × 10
4.6 × 10
5
5
2
5
6
3
9
5
4
5
5
a1.5 × 10
10
6
7
8
9
0
1
2
3
4
3.6 × 10
1.0 × 10
5.0 × 10
a4.2 × 10
4
9
5
3
3
1
1
1
1
a
4
2.0 × 10
a
No binding observed.
10
attachment of N-acetyl--leucine was employed. Since the
vancomycin sugar residues do not contribute largely to the
binding of cell-wall precursors in in vitro experiments, we
decided to remove them in order to avoid problems with the
regioselectivity in the coupling step.
Scheme 2 Scheme for the synthesis of the N-acetyldemethylvanco-
mycin aglycone 8 and its epi-compound 7.
Vancomycin (2) and N-demethylvancomycin (3) were trans-
formed into their corresponding aglycones (4 and 5) in good
11
yields, by deglycosylation with aq. HCl. Edman degradation
with phenyl isothiocyanate, and subsequent cleavage with
TFA, gave the des-leucylvancomycin aglycone (6) in 49%
the extra NH proton of such derivatives might be suitably
located for interaction with the carboxylate residue of the lig-
and. The ketone 10 was condensed, in turn, with acetyl-
hydrazine, semicarbazide hydrochloride and tosylhydrazine in
pyridine to give the corresponding hydrazones 11, 12 and 13,
respectively, in yields between 49 and 64% (Scheme 3).
7
12
yield. Compound 6 was coupled with N-acetyl--leucine
in DMF using o-(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyl-
1
3
uronium hexafluorophosphate (HBTU)
as a coupling
14
reagent. The coupling occurred with racemisation at the leu-
cine asymmetric centre, and the two resulting diastereoisomers
were separated by HPLC. However, the use of (TDBTU)
The binding of the cell-wall precursor analogue Ac --Lys--
2
Ala--Ala to N-demethylvancomycin aglycone (5) and the
newly synthesised derivatives 7, 8 and 11–13 were measured by
UV difference spectrophotometry (Table 1). The affinity of lig-
ands for vancomycin aglycone 4 was, within experimental
15
efficiently suppressed racemisation, and the desired product 7
featuring the -stereochemistry was obtained in 41% yield.
The other diastereoisomer 8, featuring the -stereochemistry,
11
error, in agreement with previous studies, and N-demethyl-
vancomycin aglycone 5 showed a comparable binding constant
was prepared independently by acetylation of 5 with Ac O in
2
5
Ϫ1
MeOH in 78% yield (Scheme 2). This was done in order to
support the assigned stereochemistry at the C-α centre of
leucine in compound 7 by reference to its isomer 8.
of 1.5 × 10 M .
4
Ϫ1
The binding constant obtained for 7 (Klig = 3.6 × 10 M )
strongly suggests that a fourth hydrogen bond does not form
A second approach to the incorporation of an extra amide
bond involved the reaction of acetylated or sulfonylated hydra-
zines with vancomycin ketone (10). CPK models suggested that
to the carboxylate anion of Ac --Lys--Ala--Ala (which
requires the binding pocket conformation shown in Fig. 2a).
It seems possible that its formation might be precluded by 7
2
3
254
J. Chem. Soc., Perkin Trans. 1, 1999, 3253–3257

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Scheme 3 Scheme for the synthesis of derivatives of N-demethylvancomycin.
undergoing ≈120Њ rotation about the α-CH-to-carbonyl bond
Experimental
All chemicals used were purchased from Aldrich and used
without further purification. N-Demethylvancomycin was
obtained from Dr Yan Husheng of Nankai University. Ac --
of the leucine residue (Fig. 2b). This conclusion is supported
1
NMR data; a H NOESY spectrum shows cross-peaks between
the methyl protons of the N-acetyl group at residue 1 and the
NH protons of the asparagine side-chain. This observation is
consistent with the proposed 120Њ rotation, since an X-ray study
2
Lys--Ala--Ala was synthesised according to standard liter-
18
16
ature methods.
of vancomycin shows that the asparagine NH group occu-
2
1
H NMR spectra were recorded on a Bruker DRX-500
spectrometer at 300 K; J-values are given in Hz, and the
letter code used for proton assignment is as previously
pies the ‘lower face of the vancomycin structure’, a conform-
ation which would bring this NH close to the N-acetyl moiety
2
17
(
Fig. 2b). Most convincingly, a strong NOE cross-peak
19
described. The descriptor G refers to protons in the glucose
and V refers to protons of the vancosamine sugar. High-
resolution mass spectra were obtained on a Fourier transform
ion cyclotron resonance mass spectrometer equipped with a
between x (the α-CH of the -leucine residue) and w (the NH
1
2
of the β-hydroxy-chloro-tyrosine residue) can be observed
Fig. 2b). We note that such a conformation might be favoured
(
because it avoids the additional electrostatic repulsion of bring-
ing a fourth positive end of an NH-CO dipole parallel to the
existing three parallel dipoles (constituted from the NHs of
residues 2, 3, and 4). We further note that the tolerance of these
latter three parallel dipoles in vancomycin (despite their
intrinsic electrostatic repulsion) seems to be occasioned by the
cross-linking of the side-chains of residues 2 and 4.
4
.7 T superconducting magnet and an external electrospray
ionisation source (Analytica of Branford, Branford, USA). In
reversed-phase HPLC, solvent A was water with 0.1% TFA
modifier, and solvent B was acetonitrile with 0.1% TFA
modifier. The gradient used with analytical HPLC (Phenom-
enex Jupiter column 5 µ, C-18; 300 Å; 250 × 4.6 mm) was
0
min, 0% B; 15 min, 30% B; 23 min, 30% B; 25 min, 80% B;
30 min 80% B; 32 min 0% B. A Phenomenex Primesphere
bead size 5 µ, C-18-MC; pore size 300 Å; 150 × 21.2 mm)
The acetylated derivative 8 displayed only weak binding
Klig = 1 × 10 M ). This is comparable to binding affinities
4
Ϫ1
(
(
of compound 9, derived from vancomycin, reported previ-
11
column was used for preparative HPLC.
ously. Therefore the substitution of an NH proton in 8 for
a methyl group in 9 does not make a significant difference to
binding.
Measurement of binding constants
No measurable binding was observed for the derivatives 11
and 12, which suggests that the additional NH is not pointing
into the binding pocket. The tosyl hydrazone 13 showed mod-
erate binding (Klig = 2 × 10 M ), and significantly enhanced
solubility of this derivative was observed.
Binding constants for the association between the vancomycin
derivatives and Ac --Lys--Ala--Ala were determined by UV
2
difference spectrophotometry. All binding measurements were
performed using a double-beam UVIKON 940 spectro-
photometer equipped with a thermocirculator to maintain the
temperature at 300 K. The light path was 1 cm. All antibiotic
solutions were 50 mM, using KH PO (50 mM, buffered to pH
4
Ϫ1
Conclusions
2
4
We observe only weak binding between the cell-wall precursor
analogue (N,N)-diacetyl--lysyl--alanyl--alanine and vanco-
mycin derivatives which carry a fourth amide proton at the
N-terminus. There is at least a 10-fold decrease of binding com-
pared with vancomycin aglycone. Unfavourable conformational
changes relative to the geometry required for enhanced binding
appear likely to be the cause of this behaviour.
7) as the diluent. The tripeptide ligand was dissolved in this
antibiotic solution to maintain a constant concentration of
antibiotic. Appropriate increments (10–40 µl) of the ligand
solution (typically 5 mM) were added to antibiotic solution in
the titration cell, until the antibiotic was ≈95% bound. In all
cases the large change in absorbance at ≈293 nm was monitored
by obtaining 2A293 Ϫ (A283 ϩ A303) throughout the titration
J. Chem. Soc., Perkin Trans. 1, 1999, 3253–3257
3255

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Fig. 2 CPK models showing (a) the desired conformation of a carboxylate-binding pocket containing four amide NHs, (b) the conformation which
is indicated experimentally by the observation of proton NOEs. Protons, white; nitrogens, blue; carbons, black; oxygens, red (see text for proton
code).
(
A is the difference in absorbance at x nm between the titration
x
L-N-Acetyldemethylvancomycin aglycone 7
cell and the reference cell). The quoted wavelengths varied by
several nm between derivatives. The large change in absorb-
ance near 242 nm was not used in these determinations, to
avoid overlap with peaks due to the free tripeptide. Associ-
ation constants were obtained using a least-squares curve-
fitting programme in Kaleidagraph 3.0.5 (Adelbeck Software)
by plotting 2A293 Ϫ (A283 ϩ A303) against ligand concentration
for each titration. Titrations were repeated to ensure repro-
ducibility, and cases of large error were the result of small
absorbance changes on formation of the complex. All com-
pounds 2 and 4–6, 8, 10–13 were indicated to be pure by the
criterion of analytical HPLC after initial isolation by pre-
parative HPLC.
To an ice-cold solution of N-acetyl--leucine (27 mg, 152
mmol) in DMF (1 ml) were added TDBTU (105 mg, 0.3
mmol) and diisopropylethylamine (DIEA) (33 µl, 195 mmol)
and the mixture was stirred for 15 min at this temperature.
Des-leucylaglycovancomycin 6 (40 mg, 40 mmol) in DMF (1 ml)
was cooled to 0 ЊC, added to the other solution, and the mixture
was stirred for 1 h at 0 ЊC and for 5 h at ambient temperature,
and evaporated to dryness. The residue was brought to pH 10
with aq. NH and incubated at 37 ЊC for 1 h. Preparative HPLC
3
followed by lyophilisation yielded -N-acetyldemethylaglyco-
vancomycin 7 (19 mg, 41%) as a white solid, tR 21.5 min; δH
(
500 MHz; DMSO-d ) 0.93 (3H, d, J 6.4, 1c), 1.03 (3H, d, J 6.8,
6
1
6
cЈ), 1.49 (1H, dd, J 13.2 and 6.6, 1a), 1.65 (1H, dd, J 13.2 and
.4, 1aЈ), 1.77 (3H, s, Ac), 1.82 (1H, hept, J 6.4, 1b), 2.20 (1H,
N-Demethylaglycovancomycin 5
dd, J 14.4 and 7.0, 3a), 2.42–2.50 (1H, m, 3aЈ), 4.08–4.13 (1H, m,
x ), 4.17 (1H, d, J 12.0, x ), 4.38 (1H, d, J 4.7, x ), 4.45 (1H, d,
N-Demethylvancomycin 3 (1.0 g, 0.96 mmol) was dissolved in
1
6
5
2
5 ml of 1 M HCl and heated in a boiling water-bath for 4 min.
J 5.5, x ), 4.48–4.54 (1H, m, x ), 4.90 (1H, dd, J 8.5 and 4.7, x ),
7
3
2
The mixture was cooled and filtered. Preparative HPLC fol-
lowed by lyophilisation yielded N-demethylaglycovancomycin 5
5.09 (1H, br s, z ), 5.19–5.23 (1H, m, z ), 5.41 (2H, br s, 4b, z ),
6 2 2
5.61–5.65 (1H, br s, 4f), 5.68 (1H, d, J 7.7, x ), 5.92–5.97 (1H,
4
(
0.79 g, 66%) as a white solid, tR 16.2 min; δH (500 MHz;
m, z OH), 6.25 (1H, s, 7f), 6.40 (1H, s, 7d), 6.68 (1H, d, J 8.2,
6
DMSO-d ) 0.92 (3H, d, J 6.2, 1c), 0.93 (3H, d, J 6.2, 1cЈ), 1.54–
Asn NH), 6.73 (1H, d, J 8.8, 5e), 6.79 (1H, d, J 8.5, 5f), 7.01–
6
d
1
.64 (2H, m, 1a, 1aЈ), 1.70 (1H, ap. sext, J 6.6, 1b), 2.14 (1H, dd,
7.04 (1H, m, Asn NH), 7.07–7.11 (1H, m, w ), 7.18 (1H, s,
d
3
J 16.4 and 8.5, 3a), 2.68–2.74 (1H, m, 3aЈ), 4.07 (1H, br s, x ),
5b), 7.25 (1H, d, J 8.5, 6e), 7.29 (1H, d, J 8.1, 2e), 7.45–7.49
1
4
.13–4.21 (2H, m, x , x ), 4.44–4.47 (2H, m, x , x ), 4.82 (1H,
(1H, m, 2f, 6f), 7.73 (1H, d, J 9.4, w ), 7.84 (1H, s, 6b),
3
6
5
7
2
dd, J 7.2 and 3.6, x ), 5.12 (1H, br d, J 7.2, z ), 5.14–5.16 (1H,
7.87–7.90 (1H, m, w ), 8.29 (1H, d, J 5.1, w ), 8.43 (1H, br d,
2
2
4
1
m, z ), 5.67 (1H, br s, z OH), 5.76 (1H, d, J 8.1, x ), 5.85 (1H, br
J 5.5, w ), 8.48 (1H, br s, w ), 12.74 (1H, br s, COOH); m/z
7 5
ϩ
6
6
4
s, x ), 6.27 (1H, d, J 1.9, 7d), 6.41 (1H, d, J 1.5, 7f), 6.45 (1H, br
(ESI) (C H Cl N O m/z 1171.2511. [M ϩ H] requires
54 53 2 8 18
2
s, w ), 6.70 (1H, d, J 1.3, w ), 6.72 (1H, d, J 8.5, 5e), 6.79 (1H,
m/z 1171.2545).
3
6
dd, J 8.8 and 1.3, 5f), 7.09–7.12 (1H, br s, Asn NH), 7.14 (1H,
d
br s, 5b), 7.25 (1H, d, J 8.3, 6e), 7.47 (1H, dd, J 8.3 and 1.3,
N-Acetyldemethylvancomycin aglycone 8
Asn NH), 7.50–7.53 (1H, m, 2f), 7.86 (1H, d, J 1.3, 6b), 8.21
d
(
1H, br s, w ), 8.25 (1H, br s, w ), 8.53 (1H, br s, w ), 8.61 (1H,
N-Demethylaglycovancomycin 5 (100 mg, 80 mmol) was dis-
1
4
2
d, J 5.5, w ), 8.71 (1H, d, J 4.6, w ), 12.75 (1H, br s, COOH);
solved in a mixture of MeOH–Ac O (4:1; 5 ml) and the solution
was stirred at ambient temperature for 4 h and evaporated to
dryness. The residue was brought to pH 10 with aq. NH3
7
5
2
ϩ
m/z (ESI) (C H Cl N O m/z 1129.2785. [M ϩ H] requires
52
57
2
8
17
m/z 1129.2748).
3
256 J. Chem. Soc., Perkin Trans. 1, 1999, 3253–3257

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and incubated at 37 ЊC for 1 h. Preparative HPLC followed
by lyophilisation yielded N-acetyldemethylaglycovancomycin 8
Vancomycin ketone tosylhydrazone 13
To a solution of the ketone 10 (40 mg, 27 µmol) in pyridine
1 ml) were added tosylhydrazine (52 mg, 280 µmol) and acetic
(
73 mg, 78%) as a white solid, tR 18.0 min; δH (500 MHz;
(
DMSO-d ) 0.83 (3H, d, J 6.4, 1c), 0.93 (3H, d, J 6.4, 1cЈ), 1.46–
6
acid (35 µl, 0.6 mmol), and the solution was stirred for 18 h at
ambient temperature. The residual pyridine was azeotropically
removed with toluene (3 × 10 ml). Preparative HPLC followed
by lyophilisation yielded the tosylhydrazone 13 (21 mg, 49%) as
a white solid, t 17.8 min; δ (600 MHz; DMSO-d ) 0.76 (3H, d,
1
.58 (2H, m, 1a/1aЈ), 1.71 (1H, dhept, J 7.2 and 6.4, 1b), 1.71
(
3H, s, Ac), 2.12 (1H, dd, J 15.7 and 5.5, 3a), 2.18–2.24 (1H, m,
3
4
4
aЈ), 4.18 (1H, d, J 11.9, x ), 4.24 (1H, dd, J 14.0 and 6.4, x ),
6
2
.49 (1H, d, J 5.5, x ), 4.45 (1H, d, J 6.0, x ), 4.54 (1H, br s, x ),
5
7
3
R
H
6
.68 (1H, d, J 8.5 and 4.7, x ), 5.11 (1H, br s, z ), 5.20 (1H, br s,
2
6
J 6.4, 1c), 0.87 (3H, d, J 6.4, 1cЈ), 1.11 (3H, d, J 6.0, V ), 1.30
6
z ), 5.23 (1H, s, 4f), 5.54 (1H, s, 4b), 5.71 (1H, br s, z OH), 5.73
2
2
(
3H, s, V ), 1.75 (1H, d, J 12.8, V ), 1.83 (2H, m, 1b, V ), 2.10–
7 2a 2e
(
1H, br s, x ), 5.94 (1H, br s, z OH), 6.26 (1H, s, 7f), 6.41 (1H, s,
4
6
2
.15 (2H, m, 3a, 3aЈ), 2.30–2.44 (1H, m, 1a), 2.38 (3H, s,
7
d), 6.71 (1H, d, J 13.2, Asn NH), 6.73 (1H, d, J 8.5, 5e), 6.78
d
C H Me), 2.50–2.54 (1H, m, 1aЈ), 3.17 (1H, d, J 7.3, V ), 3.45
6
4
4
(
(
1H, d, J 8.5, 5f), 6.89 (1H, br s, w ), 7.01 (1H, br s, w ), 7.15
3
2
(
1H, dd, J 14.3 and 6.0, G ), 3.55–3.60 (2H, m, G , G ), 3.65–
3
2
6
1H, s, 5b), 7.22 (1H, d, J 8.5, 2e), 7.23 (1H, d, J 13.2, Asn NH),
d
3
.70 (1H, m, G ), 3.92 (1H, t , J 5.5, G ), 4.45 (1H, d, J 5.5,
6 app 5
Ј
7
.28 (1H, s, 2b), 7.28–7.30 (1H, m, 6e), 7.47 (1H, d, J 8.1, 6f),
x ), 4.49–4.51 (1H, m, x ), 4.52–4.59 (2H, m, x , x ), 4.68–4.75
6
7
5
3
7
.68 (1H, d, J 8.5, 2f), 7.84 (1H, s, 6b), 7.99 (1H, br s, w ), 8.46
4
(
1H, m, x , V ), 5.12 (1H, d, J 6.8, z ), 5.20–5.26 (3H, m, G , V ,
2 5 6 1 1
(
1H, d, J 6.0, w ), 8.53 (1H, d, J 5.6, w ), 8.64 (1H, d, J 5.5, w ),
1
7
5
z ), 5.34 (1H, d, J 5.1, G OH), 5.43 (1H, d, J 6.8, V OH), 5.53
2
3
4
1
1
2.76 (1H, br s, COOH); m/z (ESI) (C H Cl N O m/z
54 53 2 8 18
ϩ
(
1H, s, 4b), 5.75–5.78 (1H, m, x ), 5.95 (1H, d, J 6.4, z OH),
4
6
171.2553. [M ϩ H] requires m/z 1171.2545).
6
.26 (1H, d, J 1.3, 7f), 6.48 (1H, d, J 1.3, 7d), 6.64–6.72 (2H, m,
w , Ans NH), 6.74 (1H, d, J 8.1, 5e), 6.79 (1H, d, J 8.1, 5f),
6
d
Vancomycin ketone acetylhydrazone 11
7
.09–7.14 (2H, m, w , Ans NH), 7.22 (1H, s, 5b), 7.28–7.33
3 d
To a solution of the ketone 10 (65 mg, 45 mmol) in pyridine (1
ml) were added acetylhydrazine (37 mg, 0.5 mmol) and glacial
acetic acid (40 ml, 0.7 mmol), and the solution was stirred for
(1H, m, 2e), 7.39–7.42 (3H, m, 6e, 3,5-ArH), 7.47–7.52 (2H, m,
6f, 2b), 7.62 (1H, m, w ), 7.81 (1H, d, J 8.1, 2f), 7.83 (2H, d,
2
J 8.1, 2,6-ArH), 7.86 (1H, s, 6b), 8.19 (1H, br s, w ), 8.45–8.48
4
1
8 h at ambient temperature. The residual pyridine was azeo-
(1H, m, w ), 8.65–8.67 (1H, m, w ), 11.22 (1H, s, hydrazineNH);
5
7
ϩ
tropically removed with toluene (3 × 10 ml). Preparative HPLC
followed by lyophilisation yielded the acetylhydrazone 11 (43
mg, 64%) as a white solid, t 14.3 min; δ (500 MHz; DMSO-
m/z (ESI) (C H Cl N O S m/z 801.2086. [M ϩ H] requires
72 79 2 10 26
m/z 801.2088).
R
H
d6) 0.83 (3H, d, J 6.4, 1c), 0.89 (3H, d, J 6.4, 1cЈ), 1.12 (3H, d,
J 6.8, V ), 1.34 (3H, s, V ), 1.74–1.79 (1H, m, V ), 1.89–1.94
References
1 H. C. Neu, Science, 1992, 257, 1064.
6
7
2e
(
2H, m, V , 1b), 2.08 (3H, s, NAc), 2.07–2.12 (2H, m, 3a/3aЈ),
2e
2
D. H. Williams and B. Bardsley, Angew. Chem., Int. Ed., 1999, 38,
172.
P. E. Reynolds, Eur. J. Clin. Microbiol. Inf. Dis., 1989, 8, 943.
3
.16–3.20 (1H, m, V ), 3.44–3.50 (1H, m, G ), 3.55–3.61 (2H, m,
4
3
1
G , G ), 3.66–3.71 (1H, m, G ), 3.94–3.99 (1H, m, G ), 4.20–
2
6
6Ј
5
3
4
4
.24 (1H, m, x ), 4.45 (1H, d, J 6.0, x ), 4.49–4.51 (1H, m, x ),
6
7
5
4 M. Ge, Z. Chen, H. R. Onishi, J. Kohler, L. S. Silver, R. Kerns,
S. Fukuzawa, C. Thompson and D. Kahne, Science, 1999, 284, 507.
5 J. P. Waltho and D. H. Williams, J. Am. Chem. Soc., 1989, 111, 2475.
6 D. H. Williams, M. S. Searle, M. S. Westwell, U. Gerhard and
S. E. Holroyd, Philos. Trans. R. Soc. London, Ser. A, 1993, 345, 11.
.54–4.58 (1H, m, x ), 4.70 (1H, dd, J 6.8 and 5.5, V ), 4.78–4.82
3
5
(
1H, m, x ), 5.23–5.30 (4H, m, G , V , 4f, z ), 5.71–5.74 (2H, m,
2 1 1 2
x , z OH), 6.25 (1H, s, 7d), 6.41 (1H, s, 7f), 6.67–6.70 (1H, m,
4
2
w ), 7.66–7.69 (1H, m, w ), 8.23 (1H, br s, w ), 8.47 (1H, br s,
6
2
4
7
T. F. Gale, J. Görlitzer, S. W. O’Brien and D. H. Williams, J. Chem.
Soc., Perkin Trans. 1, in print.
w ), 8.61 (1H, br s, w ), 10.80 (1H, br s, hydrazoneNH), 12.74
4
4
(
1H, br s, COOH); m/z (ESI) (C H Cl N O m/z 1489.3978.
67 75 2 10 25
ϩ
8 T. R. Herrin, A. M. Thomas, T. J. Perrun, J. C. Mao and S. W. Fesik,
[M ϩ H] requires m/z 1489.4281).
J. Med. Chem., 1985, 28, 1371.
P. Edman, Acta Chem. Scand., 1950, 4, 277.
9
Vancomycin ketone semicarbazone 12
10 M. F. Cristofaro, D. A. Beauregard, H. Yan, N. J. Osborn and
D. H. Williams, J. Antibiot., 1995, 48, 805.
11 R. Kannan, C. M. Harris, T. M. Harris, J. P. Waltho, N. J. Skelton
and D. H. Williams, J. Am. Chem. Soc., 1988, 110, 2946.
To a solution of the ketone 10 (50 mg, 35 µmol) in pyridine (1
ml) was added semicarbazide hydrochloride (40 mg, 358 µmol),
and the solution was stirred for 18 h at ambient temperature.
The residual pyridine was azeotropically removed with toluene
1
2 P. M. Booth and D. H. Williams, J. Chem. Soc., Perkin Trans. 1,
989, 2355.
1
1
3 V. Dourtoglou, B. Gross, V. Lambropoulou and C. Zioudrou,
Synthesis, 1984, 572.
(
3 × 10 ml). Preparative HPLC followed by lyophilisation yield-
ed the semicarbazone 12 (26 mg, 50%) as a white solid, t 14.6
R
14 W. König and R. Geiger, Chem. Ber., 1970, 103, 2024.
15 R. Knorr, A. Treciak, W. Bannwarth and D. Gillessen, Tetrahedron
Lett., 1989, 30, 1927.
min; δ (500 MHz; DMSO-d ) 0.83 (3H, d, J 6.4, 1c), 0.84 (3H,
H
6
d, J 6.4, 1cЈ), 1.11 (3H, d, J 6.8, V ), 1.31 (3H, s, V ), 1.75–1.79
6
7
1
6 M. Schäfer, T. R. Schneider and G. M. Sheldrick, Structure, 1996, 4,
509.
(
1H, d, J 13.6, V ), 1.87–1.95 (2H, m, V , 1b), 2.10–2.15 (1H,
2e 2a
1
m, 3a), 2.25 (1H, br s, 3aЈ), 2.41–2.47 (1H, m, 1a), 2.51–2.55
1
7 D. H. Williams, M. P. Williamson, D. W. Butcher and S. J.
Hammond, J. Am. Chem. Soc., 1983, 105, 1332.
(
1H, m, 1aЈ), 4.21 (1H, d, J 11.5, x ), 4.45 (1H, d, J 6.0, x ),
6
7
4
.47–4.49 (2H, m, x , x ), 4.69 (1H, d, J 6.8, V ), 4.81 (1H, d,
3 5 5
18 M. Bodansky and A. Bodansky, The Practice of Peptide Synthesis,
Springer-Verlag, Berlin, 1994.
J 3.8, x ), 5.22–5.29 (3H, m, G , V , z ), 5.57 (1H, m, z OH),
2
1
1
2
2
5
7
.75 (1H, d, J 7.7, x ), 6.26 (1H, d, J 1.7, 7f), 6.41 (1H, d, J 1.7,
19 J. P. Mackay, U. Gerhard, D. A. Beauregard, R. A. Maplestone and
D. H. Williams, J. Am. Chem. Soc., 1994, 116, 4573.
4
d), 6.73 (1H, d, J 8.5, w ), 7.63 (1H, br s, w ), 8.27 (1H, br s,
6
2
w ), 8.48 (1H, br s, w ), 8.60 (1H, br s, w ), 9.88 (1H, br s,
4
7
5
hydrazoneNH); m/z (ESI) (C H Cl N O m/z 745.7126.
6
6
74
2
11 25
ϩ
[
M ϩ H] requires m/z 745.7151).
Paper 9/06502D
J. Chem. Soc., Perkin Trans. 1, 1999, 3253–3257
3257