M V Uma

P Balaram

Professor P Balaram

R &amp

R Sudha

Spermidine

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M.V. Uma
R. Sudha
P. Balaram
Authors' af®liations:
M.V. Uma and P. Balaram, Molecular Biophysics
Unit, Indian Institute of Science, and Chemical
Biology Unit, Jawaharlal Nehru Center for
Advanced Scienti®c Research, Bangalore, India.
R. Sudha, Chemical Biology Unit, Jawaharlal
Nehru Center for Advanced Scienti®c Research,
Bangalore, India.
Correspondence to:
Professor P. Balaram
Molecular Biophysics Unit
Indian Institute of Science
Bangalore
560012
India
Tel.: 91-80-3602741
Fax: 91-80-3600683/91-80-3600535
E-mail: pb@mbu.iisc.ernet.in
Dates:
Received 20 February 2001
Revised 23 April 2001
Accepted 17 June 2001
To cite this article:
Uma, M.V., Sudha, R. & Balaram, P. Spermidine
as a potential biosynthetic precursor to the
1,5-diazabicyclo[4:3:0]nonene residue in the efrapeptins.
J. Peptide Res., 2001, 58, 375±379.
Copyright Munksgaard International Publishers Ltd, 2001
ISSN 1397±002X
Spermidine as a potential
biosynthetic precursor to the
1,5-diazabicyclo[4:3:0]nonene
residue in the efrapeptins
Key words: efrapeptins; elvapeptins; oxidative cyclization;
spermidine
Abstract: Efrapeptins are a group of microheterogeneous
polypeptide antibiotics produced by the fungus Tolypocladium
niveum, which are potent inhibitors of mitochondrial F1-ATPase.
Efrapeptins contain an unusual 1,5-diazabicyclo[4:3:0]nonene
(DBN) residue at the C-terminus. This study is driven by the
hypothesis that the DBN residue could, in principle, arise by
oxidative cyclization of a spermidine moiety. Electrospray
ionization mass spectrometry of the peptide antibiotics
`elvapeptins' from T. niveum establishes the presence of a
C-terminal spermidine residue. Conversion of elvapeptins to
efrapeptins by CuCl/pyridine demonstrates the transformation
of the spermidine residue to the 1,5-diazabicyclo[4:3:0]nonene
system by oxidative cyclization.
Efrapeptins (1±3) (Fig. 1) are a group of microheterogeneous
polypeptide antibiotics produced by the fungus Tolypocla-
dium niveum, which are potent inhibitors of mitochondrial
F1-ATPase (4±6). The efrapeptins are related to the alamethi-
cin family of peptide antibiotics (7±9), possessing a high
number of a-aminoisobutyryl (Aib) residues (10). The major
difference between the efrapeptins and the peptaibols (11) is
that the former contain an unusual 1,5-diazabicyclo[4:3:0]-
nonene (DBN) residue at the C-terminus (2,3), whereas
the latter possess a C-terminal aminoalcohol (8). Relatively
little is known about the biosynthetic origin of the DBN
moiety of the efrapeptins. In an early study, Bullough et al.
(12) reported the isolation of another microhetero-
geneous mixture of basic polypeptides from the cultures
of T. niveum, which differ in mass from the efrapeptins
by 20 Da and possess the same peptide component as efra-
peptins. Our studies were driven by the hypothesis
375
that the DBN residue could, in principle, arise by oxidative
cyclization of a spermidine moiety (Fig. 2). In this report
we demonstrate by mass spectrometry that the elvapeptins
produced by the fungal cultures contain a C-terminal
spermidine and that conversion of elvapeptins to efrapeptins
can be achieved by oxidative cyclization in the presence of
CuCl and pyridine.
Materials and Methods
Isolation and puri®cation of efrapeptins
The fungus T. niveum (Beauveria nivea) obtained from
ATCC (USA) was maintained by subculturing on agar
slopes containing an oatmeal medium. The slants were
grown for 3 days at 258C and then stored at 48C. The
oatmeal medium used for the maintenance of the fungus
is as follows: 6.00% oatmeal (baby), 0.25% yeast extract,
0.10% K2HPO4, 0.05% KCl, 0.05% MgSO4.7H2O, 0.01%
FeSO4.7H2O and 1.30% agar. The fungus was fermented
for 26 days to obtain efrapeptins. The medium used for
fermentation was 2.5% glucose, 1.0% meat peptone, 0.4%
tryptone and 0.5% MgSO4.7H2O. The seed culture (200 mL)
was grown for 2 days in the fermentation medium and then
subsequently inoculated into 3.2 L of the sane medium.
After fermentation period, the medium was spun at 903g
for 45 min in a Sorvall RC-5B refrigerated superspeed
centrifuge and the supernatant was extracted with chloro-
form. The solvent was evaporated under reduced pressure
and the crude material was puri®ed on a reverse-phase
C4 column. Elvapeptins were isolated after 5 days of
fermentation and separated from efrapeptins by extract-
ing the supernatant at pH 11 and used without further
puri®cation.
Oxidation of elvapeptins to efrapeptins and
acetylation of elvapeptins
The chloroform extract obtained after 5±7 days of fermenta-
tion of the culture medium was rich in a heterogeneous
mixture of elvapeptins. The crude mixture obtained after
solvent evaporation was stirred with CuCl in pyridine at
room temperature, and the oxidation reaction was mon-
itored using electrospray ionization mass spectrometry
(ESI-MS). A small portion of the reaction mixture was
diluted with water, extracted with chloroform and the
extract was successively washed with dilute HCl and water.
The solvent was evaporated under reduced pressure and
the crude product was analyzed by ESI-MS without puri®ca-
tion. Acetylation of elvapeptins was carried out by stirring
the elvapeptins in a 1 : 1 mixture of acetic anhydride and
pyridine at room temperature overnight and the product was
analyzed by ESI-MS.
Mass spectrometry
All electrospray spectra were recorded in the positive ion
detection mode on a Hewlett Packard 1100 MSD model
electrospray mass spectrometer equipped with a single
quadrupole and a conventional electrospray source. Distilled
methanol was used as the running solvent. The solvent
¯ow rate was 30 mL/min. Approximately 100±200 pmol of
sample was injected each time for analysis. The source
temperature was kept at 3008C. Ions were extracted with an
ori®ce potential of 4000 V applied at the nebulizer tip.
Ionization was pneumatically assisted using a constant
¯ow of pure N2 gas at 10 L/min using a Whatman nitrogen
generator. The same gas was used to maintain the nebulizer
pressure at 10 bar. The nozzle skimmer potential was kept
at 20 V. The mass spectrometer was calibrated using ®ve
m/z ions supplied by the manufacturer across the mass
range 118±2500 m/z. The chromatographic peak width for
operation was 0.18 min. The cycle time was 1.90 s/cycle
and the time ®lter was kept on. Data were acquired in
the scan mode for 2 min and averaged over the complete
Figure 1. Sequence of efrapeptins C-G[2,3]. Their corresponding
molecular masses are indicated in parentheses. The underlined
residues indicate the sites of replacement leading to the
microheterogeneity. Aib, a-aminoisobutyric acid; Iva, S-isovaline;
Pip, l-pipecolic acid; b-Ala, b-alanine, X5condensation product
of l-leucinol and 1,5-diazabicyclo[4 : 3 : 0]nonene.
Figure 2. Plausible biosynthetic route to 1,5-diazabicyclo[4:3:0]nonene
from spermidine.
Uma et al . Spermidine as a potential biosynthetic precursor
376 | J. Peptide Res. 58, 2001 / 375±379
chromatogram to obtain the mass spectrum. The charge
states were determined using the isotopic distribution of
the peaks.
Results and Discussion
Figure 3 shows an electrospray ionisation mass spectrum
(ESI-MS) of a mixture of efrapeptins isolated from fungal
cultures (13). As many as seven peptide components
differing in mass from their neighbors by 14 Da can be
seen. The microheterogeneity arises due to the low ®delity
of nonribosomal polypeptide synthesis (14,15), resulting
in the incorporation of Gly, Ala, Aib or Iva residues at
speci®c positions, leading to sequences which differ in
mass from the homologous neighbors by 14 Da. While ®ve
efrapeptins (C±G) have been previously identi®ed (3), two
additional components have been observed in this study.
Figure 4A shows an electrospray mass spectrum of elva-
peptin mixture. The peaks (M+) at 1620 and 1634 Da cor-
respond to a small amount of contaminating efrapeptins.
The elvapeptin masses detected are (MH+) 1641, 1655 and
1669 Da. Two features of the elvapeptins mass spectrum
merit special mention. (i) For elvapeptins MH+ peaks are
observed, in contrast to the efrapeptins in which only M+
peaks are observed, because the DBN group exists as a
positively charged quarternary salt. (ii) The mass spectrum
exhibits peaks at 1653 and 1667, which differ by 2 Da from
that expected for elvapeptins. Presuming a C-terminus sper-
midine residue, it is possible that the presence of oxidized
products (Fig. 2) could lead to species which differ in mass
by 2 Da. Schiff base formation by intramolecular cyclization
could lead to species differing by 1 Da. The apparent
complexity of the mass spectra of the isolated elvapeptins
is a consequence of microheterogeneity, the presence of
peaks due to partially oxidized species superimposed on the
isotopic peaks of the parent peptides.
The ESI mass spectrum of the acetylated elvapeptins
showed clearly that three acetyl groups (DM5126 Da) are
added to the peptide components of the elvapeptin mixture
(1641+12651767 and [M+Na] at 1789, 1655+12651781 and
[M+Na] at 1803, 1669+12651795 and [M+Na] at 1817).
The observation of three sites for acetylation is fully con-
sistent with the presence of spermidine as the C-terminal
residue in elvapeptins.
The conversion of primary amines to aldehydes has been
readily accomplished using CuCl/pyridine (16,17). We there-
fore attempted the in vitro conversion of elvapeptins to
efrapeptins. Figure 4B shows the mass spectrum obtained
after 48 h of oxidation. Clearly at the end of 48 h, trans-
formation is almost complete and only efrapeptins are
detectable (peaks at 1606, 1620, 1634 and 1648). These
results strongly suggest that elvapeptins are biosyntheti-
cally related to the efrapeptins and that the C-terminal
DBN residue is derived from spermidine. Several instances
already exist for spermidine conjugated natural products
Figure 3. ESI mass spectrum of a heterogeneous mixture of
efrapeptins.
Figure 4. ESI mass spectra of a heterogeneous mixture of elvapeptins
(A) and elvapeptins after 48 h of oxidation by CuCl/pyridine (B).
Uma et al . Spermidine as a potential biosynthetic precursor
J. Peptide Res. 58, 2001 / 375±379 | 377
inclusive of steroids like squalamine (18) and antibiotics
like bleomycin-A5 and edeine-A1 (19). Precedence for
conversion of spermidine to 1,5-diazabicyclo[4:3:0]nonane
exists. Weaver et al. (20) used cells of Neisseria per¯ava
grown in spermine rich medium to successfully oxidize
spermidine. Later, after several years, the oxidation product
so obtained (both from enzymatic and chemical oxidation)
was established to be 1,5-diazabicyclo[4:3:0]nonane (21).
The experiments reported here unambiguously con®rm
the chemical and biosynthetic relationship between elva-
peptins and efrapeptins and provide ®rm support for
the assignment of the C-terminal residue of elvapeptins
as spermidine. A plausible mechanistic route for the
conversion of spermidine to 1,5-diazabicyclo[4:3:0]nonene
is shown in Fig. 5.
It must be stressed that although condensation of the
initially formed aldehyde yields 1,5-diazabicyclononane,
further hydrogen abstraction (oxidation) is necessary to
yield the 1,5-diazabicyclo[4:3:0]nonene moiety of the
efrapeptins. Under the conditions used further oxidation is
undoubtedly possible.
Acknowledgments: Financial support from Dr Reddy's Research
Foundation is gratefully acknowledged. The mass spectrometry
facility is supported under the `Drug and Molecular Design'
program of the Department of Biotechnology, India.
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Uma et al . Spermidine as a potential biosynthetic precursor
J. Peptide Res. 58, 2001 / 375±379 | 379


Spermidine as a potential biosynthetic precursor to the 1,5-diazabicyclo[4:3:0]nonene residue in the efrapeptins

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Spermidine as a potential biosynthetic precursor to the 1,5-diazabicyclo[4:3:0]nonene residue in the efrapeptins

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