New derivatives of eremomycin containing 15N or F atoms for NMR study

Article abstract of DOI:10.1134/S1068162008060162

New semisynthetic derivatives of eremomycin containing ¹⁵N or F atoms were obtained for studying the antibiotic-target interaction in intact cells of Gram-positive bacteria by REDOR NMR method. Interaction of the terminal carboxyl group of amino acid 7 (AA7) of eremomycin with amines in the presence of PyBOP and TBTU reagents resulted in the corresponding [¹⁵N]-amide, p-fluorobenzylamide, p-fluorophenylpiperazide, and 6-N-(p-fluorobenzyl) aminohexylamide. A selective method of [¹⁵N]-amidation of carboxyl group of amino acid 3 (AA3) of carboxyeremomycin was developed, and the amide of eremomycin containing [¹⁵N] in AA3 amide group near the antibiotic binding pocket was obtained. Carboxyeremomycin bisamides substituted at AA3 and AA7 and containing two atoms of [¹⁵N] or F were obtained from carboxyeremomycin and [¹⁵N]NH₄Cl or the corresponding p-fluorobenzylamine hydrochloride in the presence of PyBOP at pH ～8. The Edman degradation of eremomycin p-fluorobenzylamide gave de-(D-MeLeu)-eremomycin p-fluorobenzylamide, a hexapeptide derivative incapable of the antibiotic binding with-D-Ala-D-Ala fragment of growing cell wall peptidoglycan. Among the compounds studied, carboxyeremomycin bis-p-fluorobenzylamide showed the best activity against both the glycopeptides-sensitive and glycopeptides-resistant strains of staphylococci and enterococci.

Full text of DOI:10.1134/S1068162008060162

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 6, pp. 747–754. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © S.E. Solov’eva, S.S. Printsevskaya, E.N. Olsuf’eva, G. Batta, M.N. Preobrazhenskaya, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34,
No. 6, pp. 831–839.
New Derivatives of Eremomycin Containing 15N
or F Atoms for NMR Study
S. E. Solov’evaa, S. S. Printsevskayaa, E. N. Olsuf’evaa, 1,
G. Battab, and M. N. Preobrazhenskayaa
a Gause Institute of New Antibiotics, Russian Academy of Medical Sciences,
B. Pirogovskaya ul. 11, Moscow, 119021 Russia
b Department of Biochemistry, University of Debrecen, Egyetem tér 1, Debrecen, H-4010 Hungary
Received December 17, 2007; in final form, February 11, 2008
Abstract—New semisynthetic derivatives of eremomycin containing 15N or F atoms were obtained for study-
ing the antibiotic–target interaction in intact cells of Gram-positive bacteria by REDOR NMR method. Inter-
action of the terminal carboxyl group of amino acid 7 (AA7) of eremomycin with amines in the presence of
PyBOP and TBTU reagents resulted in the corresponding [15N]-amide, p-fluorobenzylamide, p-fluorophe-
nylpiperazide, and 6-N-(p-fluorobenzyl)aminohexylamide. A selective method of [15N]-amidation of carboxyl
group of amino acid 3 (AA3) of carboxyeremomycin was developed, and the amide of eremomycin containing
[15N] in AA3 amide group near the antibiotic binding pocket was obtained. Carboxyeremomycin bisamides
substituted at AA3 and AA7 and containing two atoms of [15N] or F were obtained from carboxyeremomycin
and [15N]NH4Cl or the corresponding p-fluorobenzylamine hydrochloride in the presence of PyBOP at pH ~8.
The Edman degradation of eremomycin p-fluorobenzylamide gave de-(D-MeLeu)-eremomycin p-fluorobenzy-
lamide, a hexapeptide derivative incapable of the antibiotic binding with -D-Ala-D-Ala fragment of growing
cell wall peptidoglycan. Among the compounds studied, carboxyeremomycin bis-p-fluorobenzylamide showed
the best activity against both the glycopeptides-sensitive and glycopeptides-resistant strains of staphylococci
and enterococci.
Key words: eremomycin chemical modification, mechanism of action, NMR spectroscopy
DOI: 10.1134/S1068162008060162
infections caused by Gram-positive pathogens and fre-
quently resulting in lethal outcome [1, p. 88]. In many
countries of the world, increasingly distributed become
the infections caused by GRS and GRE [1, p. 38],
against which natural glycopeptides are inactive. Such
infections are treated by only few new drugs; however,
a fast appearance of isolates resistant to them is
observed.
INTRODUCTION
Currently, the problem of polyresistant bacterial
infections is extremely topical2. Vancomycin and teico-
planin, belonging to the group of macrocyclic glyco-
peptides, are drugs of last choice at treatment of the
1
Corresponding author; phone: +7 (499) 245-3753; fax: +7 (499)
245-0295; e-mail: eolsufeva@list.ru.
Abbreviations: AA, amino acid residues; Bzl, benzyl; CM, car-
2
boxymethylcellulose; ESI, electrospray ionization; GISA, sta-
phylococci (Staphylococcus aureus) with medium resistance to
glycopeptides; GRE, glycopeptide-resistant enterococci; GRS,
glycopeptide-resistant staphylococci; GSE, glycopeptide sensi-
tive enterococci; iGln, isoglutamine; MIC, minimal inhibitory
concentration of antibiotic (µg/ml); MS, mass spectometry;
One of directions of search for new medicines active
toward GRE and GRS is the chemical modification of
glycopeptide antibiotics. Study of the mechanism of
antibacterial action of already known glycopeptides
derivatives that are active toward the glycopeptide-
resistant bacteria is necessary for the purposeful syn-
thesis of new derivatives.
PyBOP,
(benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate; REDOR, Rotation Echo Double Reso-
nance, an NMR method similar to Overhauser decoupling (in
solid state); TBTU, (benzotriazol-1-yl)-1,1,3,3-bis(tetramethyl-
ene)uronium tetrafluoroborate.
747

748
SOLOV’EVA et al.
Transglycosylation,
transpeptidation
NH2
Transpeptidation
OH
A fragment of linear structure
of peptidoglycan
OH
Transglycosylation
Transglycosylation,
transpeptidation
lipid-II-pentapeptide
OH
OH
O
P
O
P
O
P
O
P
–
–
O
O
O
O
O
O
–
–
O
O
O
O
cell membrane
A fragment of three-dimensional structure of peptidoglycan
D-Ala
D-Ala
D-Ala
Gly
D-Ala
D-Ala
Gly
Gly
Gly
Lys
iGln
Ala
Gly Lys
iGln
Lys
=
iGln
Ala
Ala
–GlcNAc-MurNAc–
–GlcNAc-MurNAc–
–GlcNAc-MurNAc–
= – Gly5 –
Fig. 1. The scheme of biosynthesis of peptidoglycan of the wall in Gram-positive bacteria. In a frame, the dashed line indicates the
peptidoglycan fragment, a binding site for antibiotic.
Glycopeptide antibiotics inhibit the growth of
Gram-positive bacteria by inhibition of the biosynthetic
stage of growing peptidoglycan of their cell wall. The
peptidoglycan biosynthesis in bacteria includes the pro-
cesses of transglycosylation (the formation of linear
structure from the fragments of lipid II pentapeptide)
and transpeptidation. They result in the formation of
three-dimensional structure of peptidoglycan. There
are some models of the organization of such a three-
dimensional structure [2]. A simplified variant of one of
the models is given in Fig. 1. In bacteria sensitive to
glycopeptides, there is an interaction of fragment
D-Ala-D-Ala of the built up peptidoglycan with the
peptide core of an antibiotic (a binding pocket) with the
formation of five hydrogen bonds between amide
groups of the amino acid residues AA2, AA3, AA4,
AA6, and AA7 of an antibiotic and the corresponding
carboxyl and amide groups of the glycopeptide target,
N-acyl-D-Ala-D-Ala [3, p. 47]. In this case, side radi-
cals of the AA1 and AA3 residues form the walls of the
binding pocket.
GRE bacteria use for the construction of the bacte-
rial wall the peptidoglycan that contains depsipeptide -
D-Ala-D-Lac instead of the -D-Ala-D-Ala fragment [3,
p. 151]. This allows the formation of only four hydro-
gen bonds and there is repulsion between carbonyl
group of theAA4 residue of an antibiotic and oxygen of
the depsipeptide ester group arises. Such complex is
unstable, which leads to a sharp decrease in the antibac-
terial activity of antibiotic.
It was shown in various laboratories abroad and
also in our laboratory that the introduction of hydro-
phobic substituents into antibiotics of the macrocy-
clic glycopeptide group can lead to the activity
toward the vancomycin-resistant strains [4, 5]. As
starting scaffolds, in addition to vancomycin, teico-
planin, and some other glycopeptides, our laboratory
widely used the domestic antibiotic of the same
group, eremomycin (I). It is 2 to 5 times more active
than vancomycin and teicoplanin toward the Gram-
positive bacteria; however, like these, is just inactive
to GRE.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

NEW DERIVATIVES OF EREMOMYCIN CONTAINING 15N
749
OH
HO
H3C
CH3
HO
H2N
OH
O
O
OH
H3C
O
CH3
O
O
Cl
H2N
O
O
O
6
4
2
OH
O
CH3
NH
O
O
H
N
H
H
N
N
HO
HO
N
N
N
H
H
H
O
O
O
O
CH3
CH3
3
7
5
1
X
OH
HO
Eremomycin (I): X = NH2
Carboxyeremomycin (II): X = OH
The mechanisms of antibacterial action of hydro-
Similar results were obtained [9] at the study by the
phobic derivatives of glycopeptides differ from those of same method of peptidoglycan interactions with ere-
starting natural antibiotics and are still insufficiently momycin amides we obtained: p-fluorobenzylamide
investigated. The major factor determining their activ- (Ib) and p-fluorophenylpiperazide (Ic) (Fig. 2); they
ity was shown to be the presence of hydrophobic ali- contain hydrophobic substituents and a fluorine atom in
phatic substituent of ~ë9–ë15 size or an aromatic sub- other position of molecule than in N'-p-(p-fluorophe-
stituent at the periphery of molecule [4–6]. The con- nyl)benzylchloroeremomycin. These data allowed us to
crete position of introduction of the hydrophobic come to the conclusion that the arrangement of sites of
substituent plays no particular role in this case [7]. It molecules of the investigated antibiotic derivatives rel-
was established that the hydrophobic derivatives of ative to peptidoglycan fragments has a similar charac-
vancomycin and eremomycin inhibit the peptidoglycan ter. The antibiotic is located close to the peptidoglycan
biosynthesis by another manner than the natural antibi- disaccharide fragment, and the stability of the antibiotic
otics, their activity being caused neither by interaction bond with target is amplified by a noncovalent interac-
with fragments -D-Ala-D-Ala nor depsipeptide -D-Ala- tion of the hydrophobic substituent to an antibiotic with
D-Lac [6, 7].
Ala-iGln fragment of the binding site on peptidoglycan
(see Fig. 1). The formation of such a complex can pre-
vent the peptidoglycan growth, interfering with the
activity of enzymes involved in its biosynthesis.
The task of search for antibiotics of the new genera-
tion overcoming the resistance to Gram-positive bacte-
ria is closely connected to the determination of exact
targets of action onto a bacterial cell.
REDOR, a solid phase NMR method, allows the
determination of distances between certain atoms in an
antibiotic molecule and atoms of peptidoglycan in
intact cells of S. aureus at their interaction [8]. On the
basis of the data obtained, a model of antibiotic–target
complex was constructed. The fragment Ala-iGln-Lys
RESULTS AND DISCUSSION
This work is devoted to synthesis and study of anti-
bacterial activity of new derivatives of eremomycin
containing 15N and F atoms in the differentAA residues
of antibiotic molecule. The compounds are intended for
a through study of their interaction with targets intact
cells of S. aureus with the use of REDOR NMR.
Derivatives of the following types (Fig. 2) were
obtained: (1) AA7-amide of carboxyeremomycin, i.e.,
the eremomycin isomer (iso-I); (2) AA7-amide of ere-
momycin (III) and AA7-amides of eremomycin (Ia),
(IIa), and (IIIa) containing 15Nç4Cl group; (3) AA7-
amides of eremomycin containing a fluorine atom in
the hydrophobic substituent (Ib)–(Id); (4) AA3,AA7-
bisamide of carboxyeremomycin (IIIb) with two fluo-
(
Gly5
D-Ala)-D-Ala-D-Ala-) (see also Fig. 1)
was shown to serve as the binding site for N'-p-(p-fluo-
rophenyl)benzylchloroeremomycin (a derivative con-
taining a hydrophobic fragment at N'-amino group of
the eremosamine residue of the disaccharide branch of
chloroeremosamine) on the peptidoglycan of intact cell
of S. aureus. In this site, it is possible to isolate two key
fragments: dipeptide -D-Ala-D-Ala and nonapeptide -
Ala-iGln-Lys(Gly5D-Ala), including a pentaglycine
bridge folded in a helix.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

750
SOLOV’EVA et al.
Edman’s degradation
ROC
ROC
7 6 5 4 3 2 1 NHMe
7 6 5 4 3 2 NH2
CONH2
(only for (Ib))
a) or b) or c)
at pH 8.5, PyBOP
CONH2
Eremomycin AA7-amide
De-D-MeLeu-eremomycin
AA7-(p-fluorobenzyl)amide
(IVb) R = NHCH2C6H4F-p
(Ia) R = 15NH2
(Ib) R = NHCH2C6H4F-p
(Ic) R = N(CH2CH2)2NPh-F-p
(Id) R = NH(CH2)6NHCH2C6H4F-p
Saturated solution of Ba(OH)2, 37ºC, 4 h
HOOC
7 6 5 4 3 2 1 NHMe
HOOC
7 6 5 4 3 2 1 NHMe
CONH2
COOH
NH4Cl, pH 8, TBTU
column chromatography
on CM-32
Eremomycin (I)
Carboxyeremomycin (II)
b) pH 8.5, PyBOP
Excess of NH4Cl,
pH 8, TBTU
a) pH 8.5, PyBOP
H2NOC
+ (I) + (III)
7 6 5 4 3 2 1 NHMe
COOH
(Iso-I) carboxyeremomycin
AA7-amide
H2NOC
7 6 5 4 3 2 1 NHMe
CONH2
15NH4Cl
pH 8.5, PyBOP
H215NOC
7 6 5 4 3 2 1 NHMe
CONHCH2C6H4F-p
p-FC6H4H2CHNOC
7 6 5 4 3 2 1
NHMe
CO15NH2
(III) Eremomycin AA7-amide
(IIIb)
Carboxyeremomycin AA3,AA7-bisamide
(IIIa)
H2NOC
7 6 5 4 3 2 1 NHMe
CO15NH2
(IIa) AA7-amide [15N]AA3-eremomycin
a) 15NH4Cl; b) NCl×NH2CH2C6H4F-p c) HCl×HN
d) NCl×NH2(CH2)6NHCH2C6H4F-p
N-C6H4-F-p
;
;
Fig. 2. The scheme of synthesis of eremomycin and carboxyeremomycin amides.
rine-containing hydrophobic substituents; and (5) de- considerably weakened could lead to the revealing of
(D-MeLeu)-eremomycin p-fluorobenzylamide (IVb), a an additional binding site of the antibiotic with target.
hydrophobic derivative with the destructed binding
pocket that contains a fluorine atom.
Such an additional binding site was found at study-
ing of analogue (IVb) with destructed binding pocket,
N'-p-(p-fluorophenylbenzyl)-de-(D-MeLeu)-chloroer-
emomycin with the use of REDOR method [8]. It was
shown that, despite the lack of antibiotic interaction
with a -D-Ala-D-Ala fragment of S. aureus, the binding
of antibiotic with peptidoglycan is realized by means of
fragment -Ala-iGln-Lys(Gly5D-Ala) (see Fig. 1). This
antibiotic derivative shows an activity toward both sen-
sitive and glycopeptide-resistant bacteria. Thus, the
absence of interaction with a -D-Ala-D-Ala fragment in
hydrophobic derivatives of such type is compensated
by the interaction with an -Ala-iGln-Lys(Gly5D-Ala)
fragment and does not lead to a sharp loss of activity.
Derivatives of glycopeptide antibiotics containing
labels near to the binding pocket (i.e., at AA3 (IIa),
(IIIa), and (IIIb)) were obtained for the first time. The
presence of labels near to the binding pocket will allow
a detailed study of the arrangement of antibiotic frag-
ments relative to sites of peptidoglycan bound to it in an
antibiotic–target complex.
Compound (IVb) is also of interest because the
binding pocket is destructed in it. We had earlier inves-
tigated the structure–activity relationship in a series of
hydrophobic derivatives of eremomycin, vancomycin,
and teicoplanin with a different destruction degree of
molecule [10]. The removal of the first amino acid res-
idue from hydrophobic derivatives led to a slight
decrease in activity toward sensitive bacteria and GRE.
It was presumed that the hydrophobic glycopeptide
derivatives have the mechanism of activity that is not
due to the interaction with -D-Ala-D-Ala [7, 10]. Prob-
ably, it is caused by existence of an additional binding
site of the antibiotic with target. The study of the mech-
The AA7-amide derivative (IVb) we synthesized
with the destructed binding pocket contains a hydro-
phobic substituent in inother position of molecule than
in N'-p-(p-fluorophenylbenzyl)-de-(D-MeLeu)-chloro-
eremomycin.
Obtaining of Eremomycin Derivatives
The substituents containing 15N or F nuclei were
anism of action for the derivatives with the destructed entered into amino acid residues AA3 and/or AA7 of
binding pocket whose interaction with -D-Ala-D-Ala is the peptides chain of eremomycin (Fig. 2). The AA7
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

NEW DERIVATIVES OF EREMOMYCIN CONTAINING 15N
751
monoamides of eremomycin were obtained using the AA7 residues (7.80, 7.30, and 110.25 ppm) and AA3
earlier developed method [11] by the eremomycin (7.35, 6.60, and 113.45 ppm).
interaction with the corresponding amines in the pres-
ence of PyBOP: [15N]-amide (Ia), p-fluorobenzylamide
(Ib), N-(p-fluorophanyl)piperazinamide (Ic), and 6-N-
Antibacterial Activity of Eremomycin Derivatives
(p-fluorobenzyl)aminohexylamide (Id). Carboxyere-
momycin (II) in which the residue of asparagin (AA3)
was replaced by the aspartic acid residue was prepared
by the method earlier described (the treatment with bar-
ium hydroxide at 37°ë (4 h) [12]. Selective amidation
of the terminal carboxyl group in (II) with ammonium
chloride at pH ~8 in the presence of TBTU led to ere-
momycin isomer, AA7-monoamide (iso-I). The not
reacted carboxyeremomycin (II) (~1/3 from the start-
ing amount) and eremomycin (I), as an insignificant
impurity, were also isolated from the reaction mixture.
However, the use of increased quantity of ammonium
chloride and/or TBTU, and also a prolonged reaction
time led to a by-product, a significant amount of car-
boxyeremomycin bisamide (AA7-amide of eremomy-
cin) (III), which had earlier been obtained by another
method and described in [13]. The presence of eremo-
mycin (I) in reaction mixture can also be explained by
the amidation of AA3 residue of the carboxyeremomy-
cin (II) molecule.
Replacement of the amide group of the AA3 residue
close to the binding pocket of antibiotic by the carboxyl
group [compounds (II) and (iso-I)] leads to a decrease
in the activity toward the sensitive staphylococcus and
enterococcus strains (MIC ~0.5–4 and ~0.5–2 µg/ml,
respectively) and toward two GISA strains (MIC ~ 4–
8 µg/ml for (iso-I) (Table 3). Eremomycin amide (III)
has the same as eremomycin (I) activity toward sensi-
tive strains of Gram-positive bacteria (MIC ~0.06–
0.25 µg/ml), but is more active toward clinical GISA
strains (MIC ~0.25–0.5 instead of ~8 µg/ml), typical of
which is a thickening of cellular wall. Thus, amide
group on the terminal AA7 improves the action of anti-
biotic on the GISA strains.
The derivatives (iso-I), (II), and (III) do not exhibit
activity toward GRE strains. Introduction of radicals p-
FC6H4CH2NH- (Ib) and p-FPh-N(CH2CH2)2N- (Ic) in
eremomycin molecule led to a slight decrease in the
activity toward sensitive strains and to increase in activ-
ity toward GISA (MIC 1 µg/ml) and GRE (MIC
32 µg/ml).
Introduction
of
the
substituent
AA7 amide of [15N] AA3-eremomycin (IIa) was
obtained by amidation of (iso-I) by [15N]NH4Cl in the
presence of PyBOP. Exhaustive amidation of carboxy-
eremomycin (II) under the earlier described conditions
[12] by [15N]NH4Cl or a p-fluorobenzylamine hydro-
chloride at pH ~8.5 led to the corresponding bisamides
(IIIa) and (IIIb) with two identical substituents on
AA3 and AA7. The removal of AA1 from the peptide
chain of (Ib) by the Edman degradation under the con-
ditions described in [14] led to p-fluorobenzylamide of
de-(D-MeLeu)-eremomycin (IVb) with the destructed
binding pocket.
p-FC6H4CH2NH(CH2)6NH- (Id) resulted in the loss of
activity toward both sensitive and resistant strains.
The splitting of terminal residue D-MeLeu (AA1)
from (Ib) [hexapeptide (IVb)] significantly reduced the
activity toward sensitive strains (MIC 8–32 µg/ml), loss
of activity toward GISA, and the preservation of activ-
ity toward GRE at the former level (MIC = 32 µg/ml).
Introduction of two substituents p-FC6H4CH2NH-
in a carboxyeremomycin molecule (II) [compound
(IIIb)] led to a decrease in the activity to sensitive
strains (MIC 2–4 µg/ml) compared with both carboxy-
eremomycin (II) and with the monosubstituted deriva-
tive (Ib), but to appearance of the expressed activity
toward GRE (MIC 4–8 µg/ml).
We are planning to study the interaction of (IIa),
(IIIa), (IIIb), and (IVb) with intact staphylococcus and
enterococcus cells with the use of REDOR. Compound
(IVb) is also interesting because it possesses approxi-
mately equal activity toward both sensitive and resis-
tant enterococcus strains. It probably inhibits growth of
both sensitive and resistant strains by the same mecha-
nism. Such data can form a basis for understanding the
action mechanism of glycopeptide antibiotics, includ-
ing their hydrophobic derivatives possessing activity
toward resistant strains.
The physicochemical properties of new eremomy-
cin derivatives (iso-I), (Ia)–(Id), (IIa), (IIIa), (IIIb),
and (IVb) in comparison with eremomycin (I), carbox-
yeremomycin (II), and eremomycin AA7-amide (III)
are given in Table 1. Structures of new eremomycin
derivatives were proved by mass spectrometry of high
resolution; the purity of preparations was >90% (by
HPLC).
The introduction of 15N in different positions of
antibiotic molecule with the formation of isomeric
amides (Ia) and (IIa) and also simultaneously two 15N
atoms with the formation of bisamide (IIIa) were con-
firmed by NMR spectra (Table 2). The values of chem-
ical shifts of two protons and 15N in amide group of
AA7 for (Ia) (7.81, 7.37, and 110.3 ppm, respectively)
strictly differ from the corresponding values in the
amide group of AA3 of (IIa) (7.36, 6.66, and
113.4 ppm). At the NMR spectrum of labeled bisamide
EXPERIMENTAL
We used in the work eremomycin sulfate (I)
obtained on the pilot plant of the Gause Institute of New
(IIIa), there are two sets of signals proving the presence Antibiotics, Russian Academy of Medical Sciences,
of 15N-atom simultaneously in both amide groups of and carboxyeremomycin (II) and eremomycin amide
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

752
SOLOV’EVA et al.
Table 1. Physicochemical properties of new eremomycin derivatives in comparison with eremomycin (I), carboxyeremomy-
cin (II), and eremomycin amode (III)
ESI-MSa)
Num-
ber
*
Rf
Compound
RT*
Empirical formula
calculated,
found,
[M]
[M + H]+1
(I)
(II)
(iso-I) Carboxyeremomycin AA7-amide
Eremomycin
0.24 (A) 15.61 (1) C73H89ClN10O26
0.23 (A) 14.03 (1) C73H88ClN9O27
0.28 (A) 16.79 (1) C73H89ClN10O26
0.34 (A) 17.80 (1) C73H90ClN11O25
0.34 (A) 17.81 (1) C73H90ClN1015NO25
0.21 (B) 15.79 (2) C80H95ClFN11O25
1556.56
779.20b)
780.30b)
1557.55
1556.22
1557.60
1664.43
1719.32
1763.40
1557.61
779.80b)
886.80b)
769.30b)
(1557.99)
Carboxyeremomycin
1557.55
(1558.98)
1556.56
(1557.99)
(III)
(Ia)
(Ib)
(Ic)
Eremomycin AA7-amide
1555.58
(1557.01)
[15N]Eremomycin AA7-amide
1556.58
(1558.00)
Eremomycin AA7-(p-fluoroben-
zyl)amide
1663.62
(1665.12)
Eremomycin AA7-(p-fluorophe-
nyl)piperazinamide
0.51 (B)
9.71 (4) C83H100ClFN12O25
1718.66
(1720.20)
(Id)
(IIa)
Eremomycin AA7-N-(p-fluoroben-
0.48 (C) 13.89 (2) C86H108ClFN12O25
0.34 (A) 17.80 (1) C73H90ClN1015NO25
0.34 (A) 17.81 (1) C73H90ClN915N2O25
0.28 (B) 15.75 (3) C87H100ClF2N11O25
1762.72
(1764.29)
zyl)-6-aminohexylamide
[15N]AA3-Eremomycin AA7-amide
1556.58
(1558.00)
(IIIa) Carboxyeremomycin AA3,AA7-
1557.57
(1558.99)
bis[15N]amide
(IIIb) Carboxyeremomycin AA3,AA7-
bis(p-fluorobenzyl)amide
1771.65
(1773.23)
(IVb) De-(D-MeLeu)-eremomycin AA7-(p- 0.19 (B) 25.93 (1) C73H82ClFN10O24
1536.52
(1537.94)
fluorobenzyl)amide
Note: * For TLC and HPLC systems, see the Experimental section
a
For glycopeptides, peaks in ESI-MS have a form of complex clusters due to the sets of isotope peaks corresponding to monomer
or dimer forms of the ions formed (for details, see [15]).
b
+2
[M + 2H]
.
(III) earlier described in [12] and [11], respectively. All 7 : 7 : 9; (B) 17 : 10 : 10; and (C) 1 : 1 : 1 EtOAc–PrOH–
25% ammonia. Substance spots on chromatograms
were visualized under UV light. Analytical HPLC was
carried out on a LC-10 chromatograph (Shimadzu,
Japan) with the use of an UV detector at a wavelength
of 280 nm. The following systems and columns were
used: (1) 1% HCOONH4, pH 7.8 and MeCN 8–15% for
15 min, 15–40% for 15 min, 40–90% for 10 min (col-
umn Kromasil C-18, 4.0 × 250 mm, flow rate of
1.0 ml/min); (2) 1% H2PO4NH4 and MeCN 5–45 % for
25 min (column N18 Diaspher 4.0 × 250 mm, flow rate
of 0.9 ml/min); (3) 1% H2PO4NH4 and MeCN 15–45%
for 20 min, 45–45% for 10 min (column Diaspher-110
N18, flow rate of 1.0 ml/min); and (4) 0.01M H3PO4
and MeCN 10–90% for 30 min (column N18 Diaspher
4.0 × 250 mm, flow rate of 0.9 ml/mines).
reagents and solvents were from Aldrich (United
States), Fluka (Switzerland), and Merck (Germany).
The reaction course and completeness, processes of
purification and isolation, and also the purity of the
compounds obtained were monitored by the TLC and
HPLC methods. TLC was carried out on silica gel
60F254 plates (Merck, Germany) in solvent systems: (A)
Table 2. Data of NMR spectra for eremomycin amides con-
taining 15NH2 (Ia)–(IIIa)
Compound
15N, ppm H; H, ppm
(Ia) (15NH2 in AA7)
(IIa) (15NH2 in AA3)
110.3*
113.4
7.81; 7.37
7.36; 6.66
7.80; 7.30
7.35; 6.60
Preparative isolation and purification of compounds
were carried out on the columns packed with car-
boxymethylcellulose CM-32 (Whatman, UK),
silanized Kieselgel 60 (0.063-0.200 mm, Merck, Ger-
many), and sulfocation exchanger SDV-3 [H+]-form,
(IIIa) (15NH2 in AA7 and AA3) 110.25
113.45
Note: * The values correspond to the signal of maximal intensity.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

NEW DERIVATIVES OF EREMOMYCIN CONTAINING 15N
753
Table 3. Antibacterial activity* (MIC, µg/ml) of eremomycin amides (Ia)–(Id), (iso-I), and (III)** in comparison with ere-
momycin (I) and carboxyeremomycin (II)***
Strains
Compound
533 S. epider- 602 S. haemo- 3797 S. au- 3798 S. au- 568 E. faeci- 559 E. faeca- 569 E. faeci- 560 E. faeca-
midis
lyticus
reus (GISA) reus (GISA) um (GSE)
lis (GSE)
um (GRE)
lis (GRE)
(I)
(iso-I)
(Ib)
(Ic)
(Id)
0.13 [0.13]
0.5
0.13 [0.13]
8 [Nd]
4
8 [Nd]
8
0.25 [0.13] 0.25 [0.5] >128 [>128] >128 [>128]
1
0.5
1
2
2
>64
32
>64
32
0.25
0.5
1
1
0.13
0.5
>64
[Nd]
0.13
4
0.13
0.5
>64
[4]
0.25
2
1
1
32
32
>32
[2]
>32
[4]
0.13
2
>32
[Nd]
0.25
4
>32
[Nd]
0.5
4
>64
[>128]
>64
4
>64
[>128]
>64
8
(II)
(III)
(IIIb)
(IVb)
0.06
2
16
8
>32
>32
64
32
32
32
Notes: *S., Staphylococcus; E., Enterococcus
15
** Data for eremomycin amides labeled with N are not shown because their activity does not differ from that of unlabeled amide
(III).
*** Data on the antibacterial activity of (II) in comparison with eremomycin (I) are given in brackets (see [12]).
Russia, an analogue of Dowex 50 × 2) with detection by of the AA7 amide group of compound (Ia). The split-
LKB Uvicord 2138 and Recorder 6520. Mass spectra tings observable in NMR spectra probably correspond
were measured at electrospray ionization (ESI-MS) on to conformational isomers of amide groups.
TheAA7 amides of eremomycin: [15N]amide (Ia),
p-fluorobenzylamide (Ib), N-(p-fluorophenyl)piper-
azide (Ic), and 6-N-(p-fluorobenzyl)aminohexylamide
(Id), were obtained by the method described in [7] from
eremomycin sulfate and a hydrochloride of the corre-
sponding amine. Yields were approximately 50–70%.
Constants are given in Table 1.
Mono-AA7-amide of carboxyeremomycin, or
isoeremomycin (iso-I). A solution of carboxyeremo-
mycin (II) (250 mg, 0.16 mmol) in DMSO (3 ml) was
treated under stirring at room temperature (~23°C) with
ammonium chloride (8.6 mg, 0.16 mmol), triethy-
lamine up to pH 8, and three portions (at an interval of
5 min) of condensing reagent TBTU (25.7 mg, (0.08
mmol), when maintaining pH of reaction mixture equal
to 8 by addition of triethylamine. After addition of the
last TBTU portion, the reaction mixture was stirred at
room temperature for 15 min. Then additional ammo-
nium chloride was added (2.15 mg, 0.04 mmol) and
TBTU (12.8 mg, 0.04 mmol) in portions. The reaction
was stirred at room temperature for 10 min, monitoring
a Finnigan MAT 900S device (Germany).
NMR spectroscopy. Spectra of ç- and 15N NMR
1
were registered on an NMR spectrometer Bruker DRX-
500 with working frequency of 500.13 and 50.68 MHz
for 1H and 15N nuclei, respectively.
1
For registration of signals H/15N, a multinuclear
sensing element with a pulse gradient of field along the
Z axis was used. 15N NMR spectra were registered by a
technique of indirect observation 2D-HSQC (heteronu-
clear one-quantum correlation), 90-angle pulses for
1H/15N being 14.5/23 µs, respectively. Relaxation times
and spin-coupling constants (1JNH ≅ 90 Hz) for all com-
pounds were practically identical.
Samples were dissolved in 50 mM acetate buffer in
a mixture of H2O/D2O (95 : 5, 500 µl) at temperature
298 K. Concentration for each antibiotic was no less
than 6 mM. In most time-consuming experiments for
each of 64 increments, 400 scans were accumulated in
the domain 15N with a delay of 1.3 s between scans; as
a result, such an experiment was about 11 h long.
In experiments with a very high resolution degree, the completeness of reaction course by TLC in system
three signals at 113.4, 112.9, and 112.6 ppm [15N] cor- A. The reaction product was precipitated by acetone;
respond to the amide group of AA3 of AA7-amide of the precipitate was filtered, washed on filter with ace-
[15N] AA3-eremomycin (IIa) with the distribution of tone, and dried in a vacuum. It was purified on a column
intensities of 79, 18, and 3%. A similar splitting of sig- with carboxymethylcellulose (CMC-32) (2 × 13 cm),
nal from the amide group of asparagine (AA3) can be preliminarily equilibrated with buffer system of 0.05 M
observed for the side radical of the residue in a spec- ëH3COONH4–EtOH (9 : 1). Elution was carried out by
trum of [15N]eremomycin (practically all atoms of the gradient from 0.05 M ëH3COONH4–EtOH (9 : 1) to
nitrogen of the antibiotic were replaced by 15N [16]). It 0.2 M ëH3COONH4–EtOH (9 : 1). Target compound
is interesting that two signals at 110.3 and 109.8 ppm (iso-I) was isolated from fractions by sorption from the
and the intensity ratio of 90 : 10% correspond to [15N] buffer solution (acidified by 2 N INl to pH 3) on sulfo-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008

754
SOLOV’EVA et al.
cation exchanger SDV-3 (H+) with the subsequent des-
orption with 0.25 N ammonia. The fractions containing
small amounts (HPLC) of the starting (II) and a by-
product of eremomycin (I) were rejected. Eluate was
evaporated to a small volume and precipitated with ace-
tone. The precipitate was filtered, washed with acetone,
and dried in a vacuum; yield of (iso-I) 92 mg (37%);
Rf 0.28 (A), RT 16.79 (1).
REFERENCES
1. Practical Handbook On Antiinfection Chemotherapy, L.S.
Strachunskii, Yu.B. Belousov, and S.N. Kozlov, eds., Ios-
cow: Borges, 2002.
2. Dmitriev, B.A., Toukach, F. V., Holst, O., Rietschel, E.T.,
Ehlers, S., J. Bacteriol., 2004, vol. 186, pp. 7141–7148.
3. Walsh, C., Antibiotics: Actions, Origins, Resistance,
Washington: ASM, 2003.
Eremomycin AA7-amide (III). Under the condi-
tions of the synthesis similar to theose for (iso-I), ere-
momycin AA7-amide (III) was obtained from carbox-
yeremomycin (II) and fivefold excess of ammonium
chloride and/or twofold excess of TBTU; reaction time
was 1 h. All constants of this compound coincided with
those for eremomycin amide described in [13].
4. Olsuf’eva, E.N. and Preobrazhenskaya, M.N., Bioorg.
Khim., 2006, vol. 32, pp. 339–359; Rus. J. Bioorg.
Chem., 2006, vol. 32, pp. 303–322.
5. Allen, N.E. and Nicas, T., FEBS Microbiol. Rev., 2003,
vol. 26, pp. 511–532.
6. Leimkuhler, C., Chen, L., Barret, D., Panzone, G., Sun, B.,
Falcone, B., Oberthur, M., Donadio, S., Walker, S., and
Kahne, D., J. Am. Chem. Soc., 2005, vol. 127, pp. 3250–
3251.
Bis[15N] amide (IIIa) and bis(p-fluorobenzyla-
mide) of carboxyeremomycin (IIIb) were obtained by
exhaustive amidation of carboxyeremomycin (II)
under the conditions described in [11], using
[15N]ammonium chloride or p-fluorobenzylamine at
pH ~8.5. Yields were ~60–70%; constants are given in
Table 1.
7. Printsevskaya, S.S., Pavlov, A.Y., Olsufyeva, E.N.,
Mirchink, E.P., Isakova, E.B., Reznikova, M.I., Gold-
man, R.C., Brandstrom,A.A., Baizman, E.R., Longley, C.B.,
Sztaricskai, F., Batta, G., and Preobrazhenskaya, M.N.,
J. Med. Chem., 2002, vol. 45, pp. 1340–1345.
AA-7 p-fluorobenzylamide of de-(D-MeLeu)-ere-
momycin (IVb) was obtained by the removal of AA1
from the peptyide chain of (Ib) by the Edman degrada-
tion under the conditions described in [14]; yield 60%.
Constants are given in Table 1.
8. Kim, S.J., Dietrich, E., Tanaka, K.S.E., Rafai, Far, and
Schaefer J., 47 Intern. Conf. on Antimicrobial Agents
and Chemotherapy, Chicago, Illinois (United States),
2007. Sept. 17–20. C1–1473.
9. Kim, S.J., Cegelski, L., Preobrazhenskaya, M., and
Schaefer, J. Biochemistry, 2006, vol. 45, pp. 5235–5250.
Determination of antibacterial activity was car-
ried out in a Mueller Hinton medium as recommended
in the procedure of National Committee for Clinical
Laboratory Standard. MIC (µg/ ml) is the minimal
inhibiting concentration of antibiotic. All the strains
were kind gifts of the Biosearch Italia SpA (Gerenzano,
Italy). The resistant strains with the confirmed geno-
type for vancomycin-resistant enterococci 533 Staphy-
lococcus epidermidis and 602 S. haemoliticus were
clinical isolates and were identical to strains described
in [17]. The strains also were used with an intermediate
level of resistance to glycopeptides: 3797 Staphylococ-
cus aureus (GISA HIP-5836 New Jersey) and 3798
S. aureus (GISA HIP-5827 Michigan). Results were
well reproduced; they were at least two times repeated.
10. Printsevskaya, S.S., Pavlov, A.Y., Olsufyeva, E.N.,
Mirchink, E.P., and Preobrazhenskaya, M.N., J. Med.
Chem., 2003, vol. 46, pp. 1204–1209.
11. Miroshnikova, O.V., Printsevskaya, S.S., Olsufyeva, E.N.,
Pavlov, A.Y., Nilius, A., Hensey-Rudloff, D., and Preo-
brazhenskaya, M.N., J. Antibiot., 2001, vol. 54, pp. 455–
459.
12. Olsufyeva, E.N., Berdnikova, T.F., Miroshnikova, O.V.,
Reznikova, M.I., and Preobrazhenskaya, M.N., J. Anti-
biot., 1999, vol. 52, pp. 319–324.
13. Pavlov, A.Y., Berdnikova, T.F., Olsufyeva, E.N., Miroshni-
kova, O.V., Filipposyanz, S.T., Preobrazhenskaya, M.N.,
Sottani, C., Colombo, L., and Goldstein, B.P., J. Antibiot.,
1996, vol. 49, pp. 194–198.
14. Printsevskaya, S.S., Olsuf’yeva, E.N., Lazhko, E.I., Pre-
obrazhenskaya, M.N., Bioorg. Khim., 2002, vol. 28.
pp. 79-88; Russ. J. Bioorg. Chem., 2002, vol. 28. pp. 65–73.
ACKNOWLEDGMENTS
15. Mirgorodskaya, O.A., Olsufyeva, E.N., Kolume, D.E.,
Joergensen, T.J.D., Roepstorff, P., Pavlov, A.Yu.,
Miroshnikova, O.V., and Preobrazhenskaya, M.N.,
Bioorg. Khim., 2000, vol. 26, pp. 631–640; Rus. J.
Bioorg. Chem., 2000, vol. 26, pp. 566–574.
We thank E.P. Mirchink and M.I. Reznikov (Gause
Institute of NewAntibiotics, RussianAcademy of Med-
ical Sciences) for determination of antibacterial activity
and HPLC analysis.
Work of S.S. Printsevskaya was supported by the 16. Batta, G., Sztaricskai, F., Makarova, M.O., Gladkikh, E.G.,
Pogozheva, V.V., and Berdnikova, T.F., Chem. Commun.,
grant of the President of the Russian Federation for the
state support of young scientists no. 02.120.11.24179,
2007–2008; and also by Regional Foundation for Sup-
port of Domestic Medicine of the Presidium of Russian
Academy of Medical Sciences, 2006–2007.
2001, pp. 501–502.
17. Pavlov, A.Y., Preobrazhenskaya, M.N., Malabarba, A.,
Ciabatti, R., and Colombo, L., J. Antibiot., 1998, vol. 51,
pp. 73–81.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 6 2008