Synthesis of isotope-labeled aminoacids contained in servamycin antibiotic

Article abstract of DOI:10.1023/A:1012417532363

Approach was developed to a preparative synthesis of isotope-labeled aminoacids contained in servamycin IIB antibiotic. Glutamines labeled with ¹⁵N, ¹³C, and ²H were prepared in 70-80% yield starting with the corresponding labeled glutamic acids under catalysis with the glutamine synthetase enzyme. ¹⁵N-2-aminoisohutanoic acid and ¹⁵N-isovaline were obtained by Strecker method in 65 and 31% yields respectively. All compounds synthesized were identified and characterized by NMR spectroscopy.

Full text of DOI:10.1023/A:1012417532363

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Russian Journal of Organic Chemistry, Vol. 37, No. 4, 2001, pp. 475 479. Translated from Zhurnal Organicheskoi Khimii, Vol. 37, No. 4, 2001,
pp. 510 514.
Original Russian Text Copyright
,
2001 by Ogrel , Rimavi, Raap, Shvets.
Synthesis of Isotope-labeled Aminoacids Contained
in Servamycin Antibiotic
,
1
A. Ogrel , V. Rimavi1, J. Raap2, and V. Shvets1
1Moscow State Academy of Fine Chemical Processing, Moscow, 117571 Russia
2J.Raap, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Germany
Received April 17, 2000
Abstract Approach was developed to a preparative synthesis of isotope-labeled aminoacids contained in
2
servamycin IIB antibiotic. Glutamines labeled with 15N, 13C, and H were prepared in 70 80% yield starting
with the corresponding labeled glutamic acids under catalysis with the glutamine synthetase enzyme.
15N-2-aminoisobutanoic acid and 15N-isovaline were obtained by Strecker method in 65 and 31% yields
respectively. All compounds synthesized were identified and characterized by NMR spectroscopy.
The study of peptide antibiotics labeled with stable
isotopes with the use of isotope-sensitive methods is
among the most informative procedures for investiga-
tion of interaction of these compounds with phospho-
lipide membranes [1]. Therefore the preparation of
isotope-labeled aminoacids and introducing them into
the molecules of peptide antibiotics is indispensable
step of such studies. Here we report on the developed
approach to the preparative synthesis of aminoacids
[L-glutamine, 2-aminoisobutanoic acid (Aib), and
(4,4-2H2)-Glu (I) was obtained from nonlabeled Glu
via reaction of the chemical isotope exchange in 20%
2
2
solution of HCl in H2O (Scheme 2). The reaction
was carried out at 100 C for 4 days. The completely
labeled glutamic acid (I) was obtained in 75% yield.
The glutamine synthetase is known to be the
catalyst of ATP-dependent conversion of glutamic
acid into glutamine. However the ADP arising in the
process operates as competitive inhibitor for the
enzyme. To avoid this complication an alternative
process may be used: ADP-dependent transformation
2
D-isovaline (D-Ival)] labeled with isotopes H, 15N,
and 13C in different positions aiming to introduce
them into servamycin IIB molecule [2]. The
servamycin IIB is a 16-membered peptide antibiotic
efficient against both gram-positive and gram-
negative bacteria that is capable to form ionic
channels in the membranes of bacteria [3]. However
the mechanism of formation and operation of these
channels is not completely understood as yet.
Scheme 1.
Introduction of isotopes into the rests of Aib and
Ival of servamycin provides a possibility to study the
antibiotic directly in the membrane phase and also to
determine the part played by the above aminoacids
in formation and operation of the ionic channels. The
selection of Gln for isotope labelling was done under
assumption that this rest plays a decisive role in
opening and closing of the ionic channels formed by
servamycin molecules [4].
Scheme 2.
In the synthesis of labeled glutamines II IX we
applied a chemical enzymatic approach. The base of
the method consisted in enzymatic conversion of
labeled glutamic acids into the corresponding glut-
amines with the use of glutamine synthetase (GS)
(Scheme 1).
1070-4280/01/3704-0475$25.00 2001 MAIK Nauka/Interperiodica

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476
OGREL, et al.
multiplet at 2.12 ppm in the spectrum of compound
III and also singlets from the protons H2 and H4
indicate the replacement by deuterium of the proton
H3. In the spectrum of compound IV is lacking the
signal at 3.74 ppm corresponding to H2 proton.
1
The H NMR spectra of the prepared (13C)-glut-
amines are presented on Fig. 1. As seen, the signal
of H2 proton at 3.76 ppm appears as a multiplet
instead of a triplet due to the coupling with the 13C
isotope. Moreover, the presence of the 13C in position
3 (or) 4 provides the splitting of the geminal protons
H3 and H4 (J 128 Hz). The rest coupling constants in
13
2
the (3,4- C )-Gln molecule are listed in Table 2.
Quite a number of procedures in known nowadays
for the preparation of , -dialkylated amino acids.
Most of the methods are based on intermediate
synthesis of -aminonitriles by Strecker reaction [6].
In this study we used this approach for preparative
synthesis of (15n)-D-Ival (XII) and Aib (XIII)
proceeding from mixtures of the respective ketones,
15N-ammonium chloride, and sodium cyanide.
1
Fig. 1. H NMR spectra of (13C)-glutamines.
of phosphoenol pyruvate into pyruvate catalyzed by
pyruvate kinase. However we believe this path to
be economically unfeasible. Therefore to suppress
the inhibition by ADP we used ATP in 3-fold excess
with respect to the amino acid. The catalytic activity
of the glutamine synthetase appears in the presence
of bivalent metal cations in the reaction medium.
As such we used magnesium cations (Mg2+ ) [5].
In the synthesis of (5-15N)-Gln (VI) as the nitrogen
source was used 15NH4Cl. The reactions were
carried out in 80 mM imidazole buffer (pH 7.1) at
38 C for 24 h. The isolation of pure labeled
glutamines was carried out by ion-exchange
chromatography on a cation-exchanger AG 5OW-X8
(form NH+4 ) to remove all positive ions, and then
on an anion-exchanger AG1-X8 (form Ac ) to remove
all negative ions. Thus in the course of this study
we succeeded in developing conditions for prepara-
tion of fully labeled glutamines in 70 80% yield
(Table 1).
The synthesis of D-Ival (XII) consists of three stages.
In the first stage ( -15N)-D,L-aminoisovaleronitrile
(X) was obtained in the quantitative yield from
2-butanone and water solution of NaCN and 15N4Cl
taken at the molar ratio 2: 2: 1. The prepared amino-
nitrile X was subjected to mild hydrolysis with formic
acid saturated with HCl in the presence of the equi-
molar amount of water. As a result arose ( -15N)-
D,L-aminoisovalerylamide (XI). In the second stage
amide (XI) was subjected to stereoselective hydro-
lysis effected by bacteria Mycobacterium neoarum
ATTC 25795 [7] that contained L-specific amino-
peptidase. As a result formed the L-isomer of the
labeled Ival whereas the ( -15N)-D-aminoisovaleryl-
1
The H NMR spectra of (2H)-Gln are presented in
Table 1. The analysis of the spectrum of compound I
as compared with that of unlabeled glutamic acid
demonstrates the lack of a multiplet in the 2.5 ppm
region and appearance of a doublet from H3 proton;
these facts evidence the replacement of H4 proton by
deuterium. Similar features are observed in the
spectrum of compound II. The disappearance of the
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 37 No. 4 2001

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SYNTHESIS OF ISOTOPE-LABELED AMINOACIDS
477
amide remained intact. Then the cells were removed
by centrifugation, and the mixture of (15N)-L-Ival and
( -15N)-D-aminoisovalerylamide was separated on
cation-exchanger Amberlite CG 50. At the last stage
the optically pure D-amide was hydrolyzed with
conc. HCl to obtain compound XII in overall yield
31%. The stereoselectivity of the procedure according
to the data of HPLC was over 99.5%.
ether 36%, with ethyl acetate 40%, and with dichloro-
methane 65%.
The synthesized compounds labeled with 15N were
1
characterized with H NMR spectra (Figs. 2, 3). The
presence of 15N was proved by 15N NMR spectra, and
the degree of isotope enrichment was confirmed by
mass spectrometry. As seen from Fig. 2 and 3, in the
region of 1.57 ppm instead of a singlet appeared a
doublet due to the coupling between the protons of
the -methyl groups with 15N [J(CH3, 15N) was
2.8 Hz in Ival (XII) and 2.6 Hz in Aib (XIII)]. In the
spectrum of compound XII two multiplets should be
indicated at 2.00 and 1.90 ppm belonging to the
methylene protons. This splitting of signal cor-
responds to nonequivalence of the protons in the
methylene group. This fact is an indirect evidence of
the conformational rigidity of Ival molecule.
Under the same conditions compound XIII was
obtained from acetone in low yield (20%) and with
partial loss of the label (isotope enrichment did not
exceed 75%) (Fig. 2b). It seemed unreasonable
since the chemical difference between acetone and
2-butanone is slight. However acetone is miscible
with water whereas the 2-butanone forms a separate
phase in the reaction mixture. Therefore we presumed
that the presence of the second phase is important for
this reaction. Actually, when the reaction was carried
out in the presence of a solvent unmiscible with
water the resulting product XIII was fully labeled
(Fig. 2). We also observed that the yield of the
product depended on the polarity of solvent forming
the second phase. For instance, at the use of hexane
the yield of compound XIII was 32%, with ethyl
Thus as a result of this study applying the modern
enzymatic and chemical-enzymatic approach were
obtained at a preparative scale and characterized
various aminoacids labeled with stable isotopes (Gln,
Aib, Ival) that were further successfully introduced
into the molecule of servamycin IIB.
Fig. 2. H NMR spectra of(15N)-Aib, prepared (a) in two-
phase and (b) in single-phase systems, and (c) of unlabeled
Aib.
1
Fig. 3. 1H NMR spectra of unlabeled and labeled (15N)-
isovaline.
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478
OGREL, et al.
Table 1. Yields and data of 1H NMR spectra of glutamines labeled with 2H, 15N, and 13
C
Compound
no.
Yield,
%
mp,
(water)
C
Chemical shift, , ppm
Name of compound
H2
H3
H4
L-Glutamic acid
226 [8]
225 227
185 [8]
184 186
183 185
186 188
184 186
182 184
185 187
183 185
184 186
3.94 t
3.93 t
3.74 t
3.76 t
3.74 s
2.06 m
2.02 m
2.12 m
2.10 d
2.50 m
2.42 m
I
(4,4-2H2)-L-Glutamic acid
L-Glutamine
75
II
(4,4-2H2)-L-Glutamine
(3,3-2H2)-L-Glutamine
(2-2H)-L-Glutamine
( -15N)-L-Glutamine
(5-15N)-L-Glutamine
(3-13C)-L-Glutamine
(4-13C)-L-Glutamine
(3,4-13C2)-L-Glutamine
75
74
79
71
80
76
74
71
III
IV
V
VI
VII
VIII
IX
2.43 s
2.44 m
2.43 m
2.45 m
2.43 m
2.12 m
2.17 m
2.15 m
3.79 t
3.77 t
3.73 m
3.75 m
3.76
2.10 a
m
2.11 m
2.40a
2.43 a
m
m
2.12 a
m
a
For coupling constants see Table 2.
Table 2. Spin-spin coupling constants (JA,X, Hz) in
molecules of (3-13C-) (VII), (4-13C-)- (VIII) and
(3,4-13C2)-Gln (IX)
methanol concentration from 15% in the mixture
methanol sodium acetate (15: 85) to 100%. Ion-ex-
change chromatography was carried out on columns
50 80 mm and 100 25 mm packed with ion ex-
changers AG 50 W-X8 (NH+4 form) and AG-1X8
(Ac form) respectively (Bio-Rad Laboratories).
The 1H NMR spectra were registered on Bruker
WM-300 spectrometer operating in Fourrier-trans-
form pulse mode at 300 MHz, internal reference
TMS ( 0.00 ppm) in D2O or CD3OD, and Ival
spectrum was recorded in CD3OD on a spectrometer
with the operating frequency 600 MHz. The
15N NMR spectra were registered on Bruker DMX-
600 instrument at 30.4 MHz, internal standard am-
monium nitrate-15N ( 19.0 ppm).
L-glutamic-4,4-2H2 acid (I). Into an ampule was
charged 1 g (6.8 mmol) of L-glutamic acid and 10 ml
of 20% DCl solution in D2O. The ampule was cooled
in liquid nitrogen and sealed in a vacuum. The
reaction was carried out for 4 days at 100 C. Then
the reaction mixture was cooled, and 25 ml of
ethanol was added thereto. The separated pre-
cipitate was filtered off and washed with ethanol.
Yield of aminoacid I 0.75 g (75%), mp 225 227 C
(226 C [8]), colorless crystals Rf 0.42 (A). The data
on 1H NMR spectrum are given in Table 1.
X
H2
H3
4.7a
H4
13C3
13C4
H2
H3
H4
13C3
1XC4
1.3
4.5b 128
2.0
4.7
1.3
2.0
128
4.5
128
2.0
2.0
128
EXPERIMENTAL
In the study were used 3,3-2H2-, 2-2H-, 3-13C-,
4-13C-, 3,4-13C2- and -15N-glutamic acids kindly
provided by Leiden University (the Netherlands),
15NH4Cl (Cambridge Isotope Laboratories), 2H2O
(Aldrich), and also Russian reagents and solvents of
high purity. Enzymatic processes were carried out at
pH 7.1 and 38 C with the use of enzyme glutamine
synthetase (GS, E.C.6.3.1.2.) Escherichia coli from
Sigma Co (USA). The monitoring of reactions was
performed by thin-layer chromatography of the plates
DC-Alufolien, Kieselgel Go, F 254 (Merck) in the
Synthesis of labeled glutamines II IX (general
following solvent systems: 2-propanol H2O HCOOH, procedure). In 50 ml of 80 mM imidazole buffer
20: 5: 1 (A); 2-propanol ammonia, 7: 3 (B). Develop-
ment was performed with ninhydrin (0.5% solution
in acetone). HPLC was carried out on Knauer instru-
ment (Germany) using a column 4 250 mm, Spheri-
sorb ODS-2 (C18), flow rate 0.4 ml/min, gradient of
(pH 7.1) were dissolved 0.5 mmol of labeled glutamic
acid, 1.5 mmol of ATP, 1 mmol of 15NH4Cl, and 4
mmol of MgCl2 6 H2O; then was added thereto
2.5 mg (70 units) of glutamine synthetase, and the
mixture was maintained for 24 h at 38 C. The solu-
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 37 No. 4 2001