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Elucidation of the Structure of Epidermin a Ribosomally Synthesized Tetracyclic Heterodetic Polypeptide Antibiotic.

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5) Addition of acrylamide to the electrolyte (0.5 M) leads
to a decrease o f j l l mto 40% of the original value. The formation of polyacrylamide in the oxidation with chromic
acid has been regarded as proof for the occurrence of radical
In the oxidation at the Ti/Cr20,
anode, however, the possibility that layers of polymers on
the electrodes leads to a decrease in current cannot be
ruled out.
Summarizing, it has been established that 2-propanol is
oxidized to acetone at a Ti/Cr,03 anode via a mechanism
analogous to that postulated by Westheimer for homogeneous oxidation with chromic acid. The lifetimes of the electrodes are still too short for use on an industrial scale, but
we have already been able to achieve substantial improvements by using Sb20, as additional oxide component.[*]
Received: July 4, 1985:
revised: August 28, 1985 [ Z 1376 IE]
German version: Angew. Chem. 97 (1985) 1047
meso-Lanthionine
3-Methyllanthionine
$HI
CHz-
S-
i"'
(5lCH
I
~
S
rz
-
1
2
1
L
5
6
9 10
12
13
H-lle-Ala-NH-CH-CO-Lys-Phe-lle-NH-CH-CO-NH-CH-CO-Pro-Gly-HN-CH-CO-Ala-Lys(S1
iR1
(51
(Rl
CH3
I
6"
r'
meso-Lanthtontne
15
17
18
IS1
20
-HN-C-CO-Gly-HN-CH-CO-Phe-Asn-HN-CH-CO-Tyr-HN-CH-CO-NH-CH
IZI
IS1
I
iR)
CH, -5-CH
(Zl
I/
5-(2-A~tnovtnyll-o-cystelne
Fig. 1. Sequence of the tetracyclic heterodetic 22-peptide antibiotic epidermin. Trypsin cleaves behind Lys" into the fragments PI and P2 (cf. Fig. 2).
All chiral amino acids without indicated configurations have the L-configuration, i.e. they belong to the (S)-series (exceptions are sulfur-containing amino acids such as cysteine: L-CYSis (R)-configurated).
[ I ] K. B. Wiberg: Oxidation in Organic Chemistry, Part A , Academic Press,
New York 1965.
[2] R. L. Augustine (Ed.): Oxidation. Vol. I, 2 , Marcel Dekker, New York
1969 and 1971.
[ 3 ] M. Kappel, Chem. Ing. Techn. 35 (1963) 386.
[4] a) F. H. Westheimer, A. Novick, J. Chem. Phys. I1 (1943) 506; b) F. H.
Westheimer, N. Nicolaides, J . Am. Chem. Soc. 71 (1949) 25; c) F. H.
Westheimer, Chem. Rev. 45 (1949) 419.
[5] P. Miiller, Chimia 31 (1977) 209.
[6] H. B. Beer, DDR-Pat. 55223 (Priority: 12. 5. 1965), Belg. Pat. 710551
(2167).
[7] F. Beck, H. Schulz, Ber. Bunsenges. Phys. Chem. 88 (1984) 155.
[S] F. Beck, H. Schulz, Electrochim. Acta 29 (1984) 1569.
191 C. P. Andrieux, J. M. Dumas-Bouchiat, J. M. Saveant, J. Electroanal.
Chem. 123 (1981) 171.
[ 101 F. Beck, H. Schulz, B. Jansen, Electrochim. Acta. in press.
[ I l l K. B. Wiberg, Chem. Rev. 55 (1955) 713.
1121 K. 8. Wiherg, H. Schafer, 1.Am. Chem. SOC.91 (1969) 933.
[I31 We thank Prof. J. Koryta for a helpful remark made at the HeyrovskyDiscussion-Meeting in Liblice. CSSR, in May 1985.
[I41 P. Miiller, J.-C. Perlberger, Helu. Chim. Acta 57(1974) 1943.
[IS] M. Fleischmann, K. Korinek, D. Pletcher, J. Chem. SOC.Perkin Trans. 2
1972. 1396.
[I61 K. Elbs, 0. Brunner, 2. Elektrochem. 6 (1900) 609.
[I71 W. Watanabe, F. H. Westheimer, J. Chem. Phys. 17 (1949) 61.
[IS] M. Rahman, Jan RoEek, J . Am. Chem. Soc. 93 (1971) 5462.
The acidic total hydrolyzate of epidermin contained
thirteen protein amino acids, two lanthionines, and one 3methyllanthionine. The meso- and (2S,3S,6R)-configurations of lanthionine and 3-methyllanthionine, respectively,
were determined by gas chromatography on chiral stationary phases."] In the nuclear resonance spectra of the antibiotic, two unsaturated amino acids could be detected;
these were completely destroyed upon acidic total hydrolysis (Table 1).
Elucidation of the Structure of Epidermin, a
Ribosomally Synthesized,
Tetracyclic Heterodetic Polypeptide Antibiotic
Starting point of the sequencing of the complex basic
polypeptide was the tryptic cleavage into an N-terminal
fragment P1 and a C-terminal fragment P2. These fragments could be separated from each other, since P2 precipitated upon enzymatic cleavage of epidermin. C-terminal lysine could be cleaved from the tryptic fragment P1 by
reaction with carboxypeptidase B (Fig. 2). A subsequent
desulfurization with Raney nickel['] afforded a bridge-free
dodecapeptide 1-12, which was sequenced by FAB-MS
and Edman degradation in a gas-phase sequencer. A further fragmentation of the dodecapeptide into the peptides
1-4 and 5-12 was achieved by further tryptic cleavage. The
positions of the sulfur bridges in P1 could be determined
by reactions with carboxypeptidase A and B, trypsin, and
endoproteinase Lys-C, in combination with Edman degradation and FAB-MS (Fig. 2).
The tryptic fragment P2, which is insoluble in water, was
N-terminally blocked with 2-oxobutyric acid and C-terminally blocked by the &-amino group of the amino acid S(2-aminovinyl)-~-cysteine.Tryptic cleavage furnished the
2-oxobutyryl residue, in which the dehydroaminobutyric
acid (Dhb) after LysI3 was converted.[31S-(2-Aminovinyl)D-CySteine (Cys(Avi)), which is likewise labile under the
conditions of acidic total hydrolysis, could be converted by
By Hermann Allgaier, Giinther Jung,* Rolf G. Werner,
Ursula Schneider, and Hans Zahner
From the culture filtrate of Staphylococcus epidermidis
Tii 3298, we have isolated a novel active compound which
is highly effective, especially against the pathogen Propzonibacterium acnes occurring in acne disease and against staphylococci and streptococci (Fig. 1). After adsorption o n
Amberlite XAD-8 and chromatography on Sephadex LH20 the antibiotic could be isolated in pure form via two
purification steps, by multiplicative countercurrent distribution.
[*I Prof. Dr. G. Jung, Dr. H. Allgaier
lnstitut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-7400 Tiibingen (FRG)
Priv.-Doz. Dr. R. G. Werner
Abteilung Biotechnische Verfahren der Dr. Karl Thomae GmbH
D-7950 Biberach (FRG)
Prof. Dr. H. Zahner, Dr. U. Schneider
Institut fur Biologie, Mikrobiologie I der Universitat
Auf der Morgenstelle 28, D-7400 Tuhingen (FRG)
Angew. Chrm. I n t . Ed. Engl. 24 (1985) No. 12
Table I . Characterization of epidermin. Dhb =dehydroaminobutyric acid (2amino-2,3-didehydrobutyricacid).
Amino acid composition: L-Asn (I), L-Pro ( I ) , Gly (2), L-Ala ( 2 ) , ~ - I l e(2),
L-Tyr (I), L-Phe (2), L-LYS(2), Dhb ( I ) , meso-lanthionine (2), (2S,3S,6R)-3methyllanthionine (I), S-(2-aminovinyl)-~-cysteine
(I)
Thin-layer chromatography (silica gel 60 FZ54plates, Merck): chloroform/
methanol/ 17% ammonia (2 : 2 : I), RF= 0.73: I-butanol/glacial acetic acid/
water (4: 1 : I), RF=0.05
Mass spectra: Fragment PI [ M + H ] + : m/z 1302 (FAB-MS), fragment P2
[ M s N a ] ' : m/z904(FD-MS)
UV spectrum (c=O.15 mg/mL, water, pH =7): 1,,,=267 nm, E,,, = I I000
0 V C H Verlagsgesellschaft mbH. 0-6940 Weinheim. 1985
0570-0833/85/1212-1051 $ 02.50/0
1051
I
N-terminal fragment P1
N-terminal fragment P1
Carboxypsptidase B
Carboxyppptidase A
Raney nickel
Endoproteirase Lys-C
9
Oodecapeptide 1-12
,
Peptide P12-0
,
Q e m m o p e p t ! d a s e
C-terminal fragment P2
a-Chymotrypsin
a-Chymotrypsin
Peptide P21
HCI. H20
I
I
H-o- Ala-Phe-OH
H-Gly-o-Ala- Phe-OH
H-lle- Ala-o-Ala-Lys-Phe
Fragment P1
-1le-Ala-o- Abu-Pro-Gly-Afa-Ala- Lys-OH
r-S-CH=Y
Fragment P2
C2H5-CO-CO-Gly-o-Afa-Phe-Asn-o-Ala-Tyr-Ala-NH
I
,
I
s
] l
Peptide P12
H-lle- Ala-o-Ala-Lys- Phe-lle- Ala-o-Abu-Pro-Gly-Ala- Ala-OH
Peptide P12-0
H-lle-Ala-o-Ala-Lys-OH
H-Phe-lle-Afa-o-Abu-Pro-Gly-Afa-Ala-OH
Peptide P12-2
H-lle-Ala-o-Afa-Lys-OH
H-lle-Afa-o-Abu-Pro-Gly-Ala-Ala-OH
I
s
1
L
S
-s---J
Peptlde PZI
C2H,-CO-CO-Gly-o-Ala-Phe-Asn-o-Ala-Tyr-Ala-NHEt
Peptide P22
C2H5-CO-CO-Gly-D-Afaa-Phe-Asn-o-Ala-Tyr-Afa-NHEt
I
I
S
&
I
s
IsI
-
Peptide P21(16-20) C,H,-CO-CO-Gly-D-Ala-Phe- Asn-o-Ala-Tyr-OH
Peptide P22-0
C,H,-CO-CO-Gly-o-Ala-Phe-Asn-D-Ala-Tyr-OH
LsI
I
s
)
I
H-lle-Ala-o-Ala-Lys-Phe-lle-Ala-o-Abu-Pro-Gly-Als-OH
Peptide P12-1
I
s
JsI
Peptide P22-1
Oodecapeptide 1-12 H-lle-Ala-o-Ala-LysTetrapaptide 1-6
S
H-o-Ala-Phe-OH
H-o-Ala-Tyr-Ala-NHEt
Phe- Ile- Ala-o- Abu-Pro-GIy-Ala- Ala-OH
Peptide P22-2
H-lle- Ala-o-Ala-Lys-OH
H-Gly-D-Afa-Phe-OH
j
Octrpcptide 5-12
H-Ala-NHEt
-
H-o-Ala-Tyr-Aid-NHEt
s
-
H-Phe-lle- Ala-o-Abu-Pro-Gly-Ala-Ala-OH
Fig. 2. Elucidation of the structure of epidermin. The halves of the thioether amino acids nearest to the N-termini are each o-configurated at C , (cf. Fig. I).
Without this excessive fragmentation of the molecule, an assignment of the thioether bridges is impossible. D-Ala Ala: meso-lanthionine; D-Abu Ala:
(2.S,3S,6R)-3-methylianthionine;D-Ala CH=CH- NH2: S-(2-aminovinyl)-o-cysteine).
LSJ
LSJ
LS--l
hydrogenation of the native antibiotic into the stable
amino acid S-(2-aminoethyl)-~-cysteine(Cys(Aet)), which
was detectable by amino acid analysis and by mass spectrometry.
The key reaction for the elucidation of the structure of
P2 was desulfurization with Raney nickel.”] S(2-Aminoviny1)-D-cysteine was thereby completely converted into Dalanine and C-terminal N-ethylamide. Under the chosen
reaction conditions, meso-lanthionine could only be partly
desulfurized, so that the bridge-free peptide P21 and the
peptide P22 resulted, which still contained one meso-lanthionine bridge (Fig. 2). The sequencing of the C- and Nterminally blocked peptide P2 1 was accomplished by
acidic partial hydrolysis. The assignment of the sulfur
bridges in P2 was achieved by a combination of enzymatic
and acidolytic cleavages with FAB-MS.
The 22-peptide (docosapeptide) epidermin, like nisini4]
and subtilin,’’’ belongs to the polycyclic heterodetic class
of peptide antibiotics. It differs from the latter mentioned
peptides, however, not only in the primary structure but
also by its extremely rigid conformation, as shown by comparative CD investigations. The recently described poly1052
0 VCH Verlugsgesellschufr mbH. 0-6940 Weinheim, 1985
cyclic heterodetic peptide ancovenini6]is not antibiotically
active, but it inhibits the angiotensin I-converting enzyme
ACE.
1
2
3 L
5 6
R‘-lle-Ala-Ser-Lys-Phe-Ile-
7 a 9 10 11
12 13 I L 15 16 17 la 19 20 21 22
CyS-Thr-Pro-Gly-Cys-All-Lys-lhr-Gly-Ser-Phe-Asn-Ser-Tyr-Cys-Cys-R‘
1
2
3 L
5 6 7 8
9 10 11 12 13
H-lle-Ala-A.la-Lys-Phe-lle-Al~-Abe-Pro-Gly-Aia-Ala-Lys-
I
1L
15
16
17
l8
T
S1
20 21 22
19
Dhb-Gly-Ala-Phe-Asn-Aia-Tyr-Ala-Ala-0
oLp
sL
l sI
oLsLA
EnzymeskCO2 ;H21
1
2
3
L
5 6 7
8
9 10 11 12 13 1L 15 16
H-lle-Ala-A l a - L y s - P h e - l l e - A / ~ - A b ~ - P l o - G l y - A l ~ - A l a - L y-~D h b - G l y - A l s O
L
-
1
DLsA
17
18
22
rS-CH=CH
19 20 21
DLs2
Fig. 3. Possible biosynthesis of epidermin from a postulated ribosomal precursor. The sequence of the steps-dehydration of serine and threonine, addition of the SH groups of cysteine with formation of the sulfur bridges,
cleavage of COz and Hz. thereby C-terminal formation of S-(2-aminovinyl)o-cysteine-is presently being tested by isolation of the respective intermediates (see text). R ’ and R2 have not been defined; meso-lanthionine and
3-methyllanthionine are abbreviated as in Figure 2 ; however, the configuration symbol D has been placed below “Ala”.
0570-0833/85/1212-1052 $ 02.50/0
I
Phe-Asn-Ala-lyr-Als-NH
Angew. Chem. Int. Ed. Engl. 24 (1985) No. 12
The addition of inhibitors of the protein synthesis or
RNA synthesis (erythromycin, rifampicin) to cultures of
Staphylococcus epidermidis completely prevented the production of antibiotics. Epidermin thus belongs to the few
antibiotics that are formed from a ribosomally synthesized
precursor (Fig. 3) by posttranslational, enzymatic modification.[’] Dehydration of Ser3, Thr’, ThrI4, SerI6, and Ser”
leads thereby to formation of dehydroalanine (Dha) and
dehydroaminobutyric acid (Dhb). The subsequent o r
synchronous addition of thiol groups of the cysteine residues in the positions 7, 11, 21, and 22 to the CC double
bonds of Dha and Dhb leads, with formation of sulfide
bridges to meso-lanthionine and (2S,3S,6R)-3-methyllanthionine. The new C-terminal amino acid S-(2-aminoviny1)-D-cysteine is presumably formed by enzymatic oxidative decarboxylation, whereas the central dehydroaminobutyric acid remains unsaturated. We are at present attempting to detect and isolate precursor proteins with the
aid of antibodies against synthetic segments of the postulated prosequence in order to prove this biosynthesis experimentally.
The unsaturated character of 2 is exhibited in a number
of addition reactions (cf. Experimental Procedure). With
water, 2 forms the addition product 4 (the product of insertion of 2 into an OH bond of H20), which, in turn, can
react with 2 to form 5 . With propene, the ene reaction
product 6 is formed, with methyl vinyl ether, the [2 21 cycloadduct 7. Dinitrogen oxide also reacts with 2 ; the primary product is probably the [2 + 31 cycloadduct 8, which,
however, is unstable and decomposes with formation of
tri-tert-butylsilylazide and further products of tBu2Si0,
which have not yet been identified.
+
3- Ye0
L
tB%Si-NSitB%
%C-CH(OMe)
I
I
Received: July 29, 1985 [Z 1403 IE]
German version: Angew. Chem. 97 (1985) 1052
[I] E. Kiisters, H. Allgaier, G. Jung, E. Bayer, Chromatographia 18 (1984)
287, and references cited therein.
[2] M. T. Perlstein, M. Z. Atassi, S. G. Cheng, Biochim. Biophys. Acfa 236
(1971) 174.
[3] E. Nebelin, E. Gross, Hoppe-Seyler’s 2. Physiol. Chem. 254 (1973) 807.
[4] E. Gross, J. L. Morell, J. Am. Chem. Soc. 93 (1971) 4634.
(51 E. Gross, H. H. Kiltz, E. Nebelin, Hoppe-Seyler’s Z. Physiol. Chem. 354
(1973) 810.
[6] T. Wakamiya, Y. Ueki, T. Shiba, Y. Kido, Y. Motoki, Tetrahedron Lett. 26
(1985) 665.
[7] L. G. Ingram, Biochim. Biophys. Acfa 184 (1969) 216.
Isolation of the Stable Silaketimine
tBu2Si=N -Si?Bu3* *
By Nils Wiberg,* Klaus Schurz, and Gerd Fischer
Dedicated to Professor Max Schmidt on the occasion of
his 60th birthday
We were recently able to isolate the silaethene 1, which
is stable at room temperature.[’I We have now succeeded in
preparing the first stable silaketimine 2.
Me\
/
Si=C
/
S i e t B u 2 tBu,
\
SiMe3
Me
1
A
Si=N-SitB%
tBu/
/ SitB%
I
t-- t Bu-Si-N
- ucl
I
I
c1 Li
2
9
2, like 1 , can be obtained by “thermal elimination of
salt.” The tetrahydrofuran (THF) adduct of compound 31z1
serves as the precursor, which slowly eliminates LiCl upon
heating.I3l In the presence of CF3S03SiMe3, 3 is transformed rapidly, even at room temperature, into the unsaturated system 2, which can be crystallized from pentane at
-78”C.151The silaketimine 2 gives the expected ‘H-NMR,
29Si-NMR, and mass spectra (see Experimental Procedure).
[*] Prof. Dr. N. Wiberg, K. Schurz, Dr. G. Fischer
[**I
Institut fur Anorganische Chemie der Universitat
Meiserstrasse 1, D-8000 Munchen 2 (FRG)
Unsaturated Silicon Compounds, Part 10; Compounds of Silicon, Part
61. This work was supported by the Deutsche Forschungsgemein%haft.-Parts 9 and 60: [l].
Angew. Chem Int. Ed. Engl. 24 (1985) No. 12
J/ \
+ O=N=N
\
+
~
H
t-------2
7
6
t &Si-NSit
I
I
tBu2Si-NHSitBu,
+ 2
Bu,
3-
OH H
6
I
tB%Si-
4
NHSitBu3
5
Experimental Procedure
Preparation of 2 : MeLi (15.99mmol) in 1 7 m L of T H F was added to
tBu2SiCI-NH(SitBu,) 9 (6.27 g, 15.98 mmol) dissolved in 50 mL of T H F at
- 78°C. The mixture was allowed to react at - 60°C for 70 h (quantitative
conversion according to ‘H-NMR spectroscopy). After removal of all volatiles in vacuum at room temperature, the residue was dissolved in 100 mL of
pentane and the resulting solution treated with 2.9 mL (16 mmol) of
CF3S03SiMe3.After 1 d, the solution was filtered to remove all precipitate
formed (CF,SO,Li) and concentrated to 50 mL: THF-containing 2 crystal,
298 ( M +-rBu,
lized out at -78°C (80%). MS (15 eV): m / z 355 ( M +0.1%),
lOO%), 256, 214, 172, 130 (298+-nxpropene, 3, 14, 7, 25). ‘H-NMR (THF,
28°C): S= 1.178 (tBu2Si), 1.107 (SitBu,). 29Si-NMR (THF, 28°C): 6 = -2.555
(tBu2Si), - 16.571 (tBu,Si).
Reactions of 2: 2 (0.427 g, 1 mmol), dissolved in benzene, was allowed to
react at room temperature with 0.5 or 1 mmol of H 2 0 and at 60°C with
10 mmol of propene or 10 mmol of methyl vinyl ether. After removal of all
volatiles in vacuum at room temperature, the analytically pure products 4-7
were obtained. The following melting points and ‘H-NMR signals (C,D,)
were obtained for the samples characterized by elemental analysis and mass
spectroscopy. 4: m.p.= 13OOC; 6 = 1.099 (s, SifBu?), 1.216 (s, SitBu,), 1.443 (s,
OH).-5: m.p.=344’C (decomp.); S= 1.279 (s, SitBu,), 1.380 (s, SitBu2).-6:
m.p.= 121 “ C ;S= 1.176 (s, SitBu,), 1.192 (s, SitBu,), 2.074 (m, SiCH2), 5.129
(m, CHZ), 6.070 (m, CH).-7: m.p.=114”C (decomp.): 6=1.148, 1.214 (s,
SitBu~),1.290 (s, SitBu,), 2.907 (s, CH,), 4.934 (m, CH).
Received: August 5 , 1985:
supplemented: September 18, 1985 [Z 1415 IE]
German version: Angew. Chem. 97 (1985) 1058
CAS-Registry-Numbers :
2, 99112-70-6: 4, 99112-71-7; 5, 99112-72-8; 6, 99112-73-9; 7, 99112-74-0;
t-Bu2SiCI-NH(Si-t-Bu,), 991 12-69-3.
(11 N. Wiberg, G. Wagner, G. Miiller, Angew. Chem. 97 (1985) 220: Angew.
Chem. Int. Ed. Engl. 24 (1985) 229.
[21 3 was synthesized in collaboration with Dr. P. Karampatses. An important intermediate is the compound 9 , which reacts with MeLi to give 3
(cf. Experimental Procedure). Synthesis of 9:
tBu2SiC1-N=N=N 2
~
~
~+N2+NaCl+NMe3
~ ~ ~ , +
9
(cf. N. Wiberg, G. Fischer, P. Karampatses, Angew. Chem. 96 (1984) 5 8 :
Angew. Chem. Int. Ed. Engl. 23 (1984) 59).
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1053
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structure, epidermis, antibiotics, synthesizers, ribosomal, polypeptide, elucidation, tetracycline, heterodetic
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