Site-selective epimerization of a fungal cyclodepsipeptide via a 5- aminooxazole intermediate

Article abstract of DOI:10.1055/s-1999-3103

The epimerization of the cyclic depsiheptapeptide 1 at a single amino acid (Leu) was achieved in four steps. After regio-selective thionation of 1 at one of its six amide-bonds, subsequent S-benzylation of the thioamide 2 led to the thioimidate 3, which w

Full text of DOI:10.1055/s-1999-3103

LETTER
893
Site-Selective Epimerization of a Fungal Cyclodepsipeptide via a
5
-Aminooxazole Intermediate
Berndt Oberhauser,* Karl Baumann, Brigitte Grohmann, Hildegard Sperner
Novartis Forschungsinstitut GmbH, Brunner Str. 59, A-1235 Wien, Austria
Fax +43(1)86634354; E-mail: berndt.oberhauser@pharma.novartis.com
Received 1 February 1999
Dedicated to Professor Albert Eschenmoser for his brilliant contributions to the understanding of the molecules of life
lardoides Tassi. The compound potently inhibits the ex-
Abstract: The epimerization of the cyclic depsiheptapeptide 1 at a
single amino acid (Leu) was achieved in four steps. After regio-se-
lective thionation of 1 at one of its six amide-bonds, subsequent
pression of the adhesion molecules VCAM-1 and
2
ICAM-1 on activated human endothelial cells. Despite its
S-benzylation of the thioamide 2 led to the thioimidate 3, which was peptidic character, the compound has only one unmodi-
converted to the peptide-bridged 5-aminooxazole 5 by an unusual fied natural amino acid (Leu). All other constituents are
mercury promoted intramolecular cyclization. Re-opening of the either N-methylated (MeAla and MeLeu) or further mod-
oxazole ring by acid hydrolysis regenerated the parent cyclopep-
tolide framework 6, but preferentially with inverted configuration at
MeOMeTrp, δ-propylleucine PrLeu2 and PrLeu6). The lac-
leucine.
ified amino acids (N1’-methoxy-N-methyltryptophan
tone linkage is formed between MeAla and the D-configured
Key words: natural products, cyclodepsipeptide, epimerization,
α-hydroxy acid D-4-cyano-2-hydroxybutyric acid (CHBA).
5
-aminooxazole
Selective transformation of identical functional groups in
natural products is usually attributed to the selective
power of enzymatic reactions. In many cases, the 3D
structure of the natural products per se poses a remarkable
differentiation on otherwise chemically identical groups.
One elegant example for the use of this differentiation
through conformational organization in chemical synthe-
sis is the selective coupling of the nucleotide component
in a biomimetic synthesis of Vitamin B by the
1
2
1
Eschenmoser group. There, the nucleotide component
selectively reacts with one out of seven otherwise almost
identical ester groups of cobyrinic acid heptakis(cyano-
methylester). In our paper we describe the selective
functionalization of a single amide bond in a cyclic
depsipeptide similarly exploiting conformational differ-
entiation.
The constitution and 3-dimensional structure of the depsi-
heptapeptide has been determined by 2D-NMR methods
3
and X-ray diffraction analysis (Figure 2). The conforma-
tion is characterized by two of the three methylated amide
bonds (PrLeu2-MeLeu and Leu-MeOMeTrp) adopting a
cis-conformation, allowing the necessary turns to accom-
modate the cyclic structure. The ring is further stabilized
by two trans-annular hydrogen bonds and a close hydro-
phobic contact of the sidechains of the hydroxy acid CH-
BA, MeOMeTrp and Leu on one face of the macrocycle.
This tight spatial arrangement is reflected in a remarkably
strong magnetic shielding effect of the aromatic ring on
one of the Leu β-protons (δ = -0.36 ppm). The two in-
tramolecular hydrogen bonds leave only one NH group
Figure 1: The cyclic depsiheptapeptide HUN-7293 1
During screening of fermentation broths for inhibitors of
induced expression of adhesion molecules, a novel cyclic
(
Leu NH) free to interact with the solvent as seen by the
formation of an H-bond to a DMSO molecule in the crys-
tal structure.
depsiheptapeptide
(cyclopeptolide
HUN-7293) 1
(Figure 1) was isolated from the fungus Bartalinia robil-
Synlett 1999, S1, 893–896 ISSN 0936-5214 © Thieme Stuttgart · New York

8
94
B. Oberhauser et al.
LETTER
The structural differentiation of the three unmethylated Despite the potentially labile lactone moiety, S-alkylation
amide bonds could be exploited chemically for a regiose- of the mono-thioamide 2 with benzyl bromide under
lective thionation of the most accessible amide between phase-transfer conditions (aq. NaOH, dichloromethane,
1
2
MeLeu and Leu. This was easily achieved with 0.5 ultrasound) proceeded smoothly to the S-benzyl
equivalents Lawesson’s reagent in xylene at 130°C.4 In thioimidate 3. Taken together, this two-step sequence
contrast to the thionation of cyclosporin A, where the re- resulted in the selective transformation of the most acces-
action in homogenous solution leads to parallel formation sible amide into a reactive thioimidate intermediate which
,5
13
4
of three mono-thioamides , the only detectable by-prod- allows further modification of the peptidic backbone
ucts were the double thioamide (additional thionation at (Scheme 1):
MeOMeTrp-PrLeu6) and a triple thioamide (thionation at
1
) Aqueous acidic hydrolysis of the imide-bond (hydro-
all unmethylated amides). The absence of other mono-
thionated products suggests, that thionation of the other
amides is dependent on a conformational change, result-
ing from the introduction of the first thioamide group. In-
deed, the NMR spectrum of the mono-thioamide 2 lacks
the characteristic shielding effect on the Leu β-protons in-
dicative for the cis-amide between Leu-MeOMeTrp. This
quite dramatic effect on the ring-conformation (NOE ex-
periments indicate an all-cis conformation of the amides)
by the isosteric replacement of a single amide bond can be
explained by the weaker H-bond acceptor strength of the
C=S bond which results in the loss of this crucially stabi-
lizing H-bond.
chloric acid) of the S-benzyl thioimidate 3, with an appro-
priate co-solvent (t-BuOH) to increase solubility, led to
quantitative formation of the ring-opened depsipeptide 4
without affecting the ester linkage. The S-benzyl
thioester, which is formed by this hydrolysis, can serve as
a C-terminal protecting group of the linear depsipeptide
during further manipulation of the N-terminus since it is
stable towards standard reaction conditions in peptide
chemistry.
14
2
) Instead of ring cleavage via protonation of the imide-
nitrogen with aqueous acid, a selective activation of sulfur
with mercury- or silver ions in aqueous solvents led to a
Synlett 1999, S1, 893–896 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Site-Selective Epimerization of a Fungal Cyclodepsipeptide
895
1
5
regeneration of the starting material 1 through hydroly-
sis of the thioimidate C-S bond.
(5) Eberle, M. K.; Jutzi-Eme, A.-M.; Nuninger, F. J. Org. Chem.
994, 59, 7249.
1
(
6) Lipshutz, B. H.; Huff, B. E.; McCarthy, K. E.; Mukarram, S.
M. J.; Siahaan, T. J.; Vaccaro, W. D.; Webb, H.; Falick, A. M..
Miller, T. A. J. Am. Chem. Soc. 1990, 112, 7032.
3
) In the absence of external nucleophiles, activation of
thioimidate 3 with silver- or mercury salts (i.e. formation
of a leaving group at sulfur) in dry acetonitrile resulted in
the intramolecular attack of the n+1 amide oxygen
(7) Lipshutz, B. H.; Hungate, R. W.; McCarthy, K. E.
Isr. J. Chem. 1986, 27, 49.
(
(
8) Lipshutz, B. H.; Hungate, R. W.; McCarthy, K. E.
Tetrahedron Lett. 1983, 24, 5155.
9) Lipshutz, B. H.; Hungate, R. W.; McCarthy, K. E.
J. Am. Chem. Soc. 1983, 105, 7703.
(
Leu-MeOMeTrp amide). This led to the quite unusual
16
formation of a peptide-bridged 5-aminooxazole 5 with
destruction of chirality at Leu by aromatization. In con-
trast to other oxazole-containing peptidic natural prod-
ucts, no sidechain atoms are involved in the ring-closure
of this rather exotic type of a rigid dipeptide mimetic.6
(
10) Engelfried, C.; Ludwig, K.; Jaenichen, H.; Koenig, W. A.
Liebigs Ann. Chem. 1979, 973.
-10
(11) Simulated annealing with ROESY distance constrains: Sybyl,
00 cycles 700K 1000fs, 200K 1000fs, energy minimization:
1
Tripos forcefield, Gasteiger charges, no violations of distance
constraints greater than 20 pm.
(12) A solution of 1.17 g 1 and 0.36 g of Lawesson's reagent in
50 mL of xylene was heated to 130°C for 30 min. The mixture
was evaporated in vacuo, filtered over silicagel (toluene/
MeOH 95:5) and purified by chromatography (SiO , gradient
2
toluene / 0.5-4% MeOH) to give 393 mg (33%) of the
thioamide 2 (colorless solid foam) and 110 mg (9%) of the
bis-thioamide (colorless solid foam) and recovered starting
1
material. Selected analytical data for 2: H NMR (CDCl , 500
3
MHz): δ = 9.38 (d br, 1H; Leu NH), 8.02 (d br, J=9Hz, 1H;
2
6
PrLeu NH), 7.49 (d, J=9.5Hz, 1H; PrLeu NH), 5.32 (ddd,
6
1
H; PrLeu a-H), 5.16 (ddd, 1H; Leu a-H), 5.13 (dd, 1H;
2
CHBA a-H), 5.02 (ddd, 1H; PrLeu a-H), 4.19 (dd, 1H; Me-
Leu a-H), 3.72 (q, J=7Hz, 1H; MeAla a-H), 3.47 (s, 3H;
MeAla NMe), 3.45 (m, 1H; MeOMeTrp a-H), 3.29 (s, 3H;
1
3
MeLeu NMe), 2.37 (s, 3H; MeOMeTrp NMe). C NMR
CDCl , 125 MHz): δ = 197.4 (C=S).
(
3
(
13) A mixture of aqueous NaOH (30%, 1 mL) and a CH Cl solu-
2 2
tion (3 mL) of 37.8 mg 2 and 100 µL benzyl bromide (5 mL)
was stirred for 30 min at r.t. with occasional sonication. The
mixture was neutralized with 4N HCl and extracted with AcO-
Et. The organic phase was dried over Na SO , evaporated and
Utilizing the sensitivity of 5-aminooxazoles towards acid
hydrolysis, the two masked amide bonds could easily be
regenerated by treatment with TFA in aqueous t-BuOH.
In contrast to the direct silver promoted hydrolysis of the
thioimidate, which led to the exclusive regeneration of the
parent compound HUN-7293 1, this two-step procedure
via the achiral oxazole intermediate resulted in the prefer-
ential formation of the D-Leu epimer 6 together with 1 in
a 62:38 ratio as determined by HPLC. The excess of one
diastereomer probably reflects the rigid structure of the
peptide-bridged oxazole, as also indicated by a predomi-
nant conformation (2D-ROESY: all-trans conformation
of the amides). The configuration of 6 was established by
2
4
®
the residue was subjected to gel chromatography (Sephadex
LH-20, MeOH/AcOEt 1:1) to yield 38 mg 3 (94%) as color-
1
less solid foam. Selected analytical data for 3: H NMR
6
(CDCl , 500 MHz, 330 K): δ = 7.93 (d br, 1H; PrLeu NH),
7
3
2
.81 (d br, J=8Hz, 1H; PrLeu NH), 5.27 (dd, J=6.8, 3.4Hz,
1
H; Leu a-H), 5.23 (dd, J=11.1, 2.2Hz, 1H; CHBA a-H), 5.16
6
2
1
7
(ddd, 1H; PrLeu a-H), 4.74 (ddd, 1H; PrLeu a-H), 4.48 (dd,
J=9.4, 4.5Hz, 1H; MeLeu a-H), 4.26 (d, J=13.5Hz, 1H;
S-CH a), 4.02 (s, 3H; MTO N1’-OMe), 3.83 (d, J=13.5Hz, 1H;
2
S-CH b), 3.59 (q, J=7Hz, 1H; MeAla a-H), 3.12, 3.03, 2.94
2
1
3
(3s; 3xNMe). C NMR (CDCl , 125 MHz, 330 K): δ = 174.6
(PrLEU CO), 171.8, 171.2, 169.5, 168.7 (4xCO), 138.0 (Ph),
3
2
7
2.6 (CHBA C-a), 66.0 (MeOMeTrp C-a), 59.8 (MeAla C-a),
2
59.2 (MeLeu C-a), 56.6 (Leu C-a), 49.2 (PrLeu C-a), 47.1
2
D-NMR methods (Figure 3) and independently proven
6
(PrLeu C-a), 31.1 (SCH ).
2
-
by partial synthesis.
(
14) A solution of 26 mg benzyl thioimidate 3 in 2 mL t-BuOH and
00 µL conc. HCl was heated to 55°C for 20 min. The reaction
2
mixture was diluted with AcOEt and evaporated in vacuo to
References and Notes
give 30 mg 4 as a colorless solid. Selected analytical data for
1
(1) Eschenmoser A. Angew. Chem. 1988, 100, 5; Angew. Chem.,
Int. Ed. Engl. 1988, 27, 5.
4: H NMR (CDCl , 500 MHz, main conformer): δ = 7.96 (d
3
br, J=9Hz; NH), 7.3-7.2 (m; Ph), 5.55 (ddd; a H), 5.24 (dd,
6
(
2) Foster, C. A.; Dreyfuss, M.; Mandak, B.; Meingassner, J. G.;
Naegeli, H. U.; Nussbaumer, A.; Oberer, L.; Scheel, G.; Swo-
boda, E.-M. J. Dermatol. 1994, 21, 847.
1H; CHBA a-H), 5.00 (m, 1H; PrLeu a-H), 4.78 (ddd, 1H;
2
PrLeu a-H), 4.33 (m; a-H), 4.16 (d, J=13.8Hz, 1H; Ph-CH ),
2
4.01 (s, 3H; MeOMeTrp N1'-OMe), 3.99 (d, J=13.8Hz, 1H;
(
3) Hommel, U.; Weber, H.-P.; Oberer, L.; Naegeli, H. U.; Ober-
hauser, B.; Foster, C. A. FEBS Letters 1995, 379, 69.
4) Seebach, D.; Ko, S. Y.; Kessler, H.; Köck, M.; Reggelin, M.;
Schmieder, P.; Walkinshaw, M. D.; Bölsterli, J. J.; Bevec, D.
Helv. Chim. Acta 1991, 74, 1953.
Ph-CH ), 3.79 (q, J=7Hz, 1H; MeAla a-H), 3.08 (s, 6H;
2
2xNMe), 2.93 (s, 3H; MeOMeTrp NMe), 0.63 (d, J=6.5Hz,
1
3
(
3H; MeLeu d1-Me). C NMR (CDCl , 125 MHz): δ = 199.8
3
(COS), 174.7, 171.6, 169.9, 169.7, 169.4, 168.6 (6xCO).
Synlett 1999, S1, 893–896 ISSN 0936-5214 © Thieme Stuttgart · New York

8
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B. Oberhauser et al.
LETTER
2
6
(
15) A solution of 50 mg 3 in 2 mL t-BuOH/H O 9:1 was stirred
C-a), 48.3* (PrLeu C-a), 47.4* (PrLeu C-a), 36.3, 35.8
(2xNMe), 33.8* (Leu C-b), 33.8* (NMe), 27.5 (Leu C-g),
21.8* (Leu C-d1).
2
with 38 mg AgNO at r.t. for 2.5 h. The precipitated silver salt
3
was removed by filtration and after dilution with AcOEt, the
reaction mixture was extracted with brine. After filtration the
organic phase was dried over Na SO , evaporated in vacuo
(17) A solution of 17 mg 5 in 4 mL t-BuOH/H O 9:1 was stirred
2
with 400 µL TFA at r.t. for 15 h. The reaction mixture was
2
4
and purified by chromatography (SiO , gradient toluene/
neutralized with aq. NaHCO and extracted with AcOEt. The
2
3
MeOH 0.5-1.5%) to yield 15 mg HUN-7293 1 (35%).
organic phase was dried over Na SO , evaporated and the re-
2
4
(
16) A solution of 110 mg 3 in 2 mL dry CH CN was stirred with
sidue was subjected to silicagel chromatography (gradient to-
luene/MeOH 0.5-2%) to yield 6 mg 6 (35%) as a colorless
solid foam. The ratio of the epimeric compounds 6:1 (62:38)
3
1
31 mg HgCl and 90 mg CaCO at r.t. for two hours. The re-
2
3
action mixture was diluted with 1N HCl and extracted with
AcOEt. The organic phase was dried over Na SO , evaporated
was determined by RP-HPLC. Selected analytical data for 6:
2
4
1
and the residue was subjected to gel-chromatography (Sepha-
H NMR (CDCl , 500 MHz): δ = 8.04 (d, J=9.2Hz, 1H;
3
®
2
6
dex LH-20, MeOH/AcOEt 1:1) and silicagel chromatogra-
PrLeu NH), 7.65 (d, J=9.5Hz, 1H; PrLeu NH), 6.79 (d,
phy (gradient toluene/MeOH 0.5-2%) to yield 42 mg 5 (45%)
J=10.2Hz, 1H; Leu NH), 5.41 (t, J=7.4Hz, 1H; MeOMeTrp a-
1
6
as colorless solid foam. Selected analytical data for 5: H
H), 5.23 (ddd, 1H; PrLeu a-H), 5.07 (dd, J=8.5, 3.7Hz, 1H;
2
NMR (CDCl , 500 MHz): δ = 7.67 (d, J=9.3Hz, 1H; PrLeu
HCBA a-H), 4.99 (ddd, 1H; PrLeu2 a-H), 4.97 (ddd, 1H; Leu
a-H), 4.03 (s, 3H; MeOMeTrp N1'-OMe), 3.91 (dd, J=9.2,
4.3Hz, 1H; MeLeu a-H), 3.63 (q, J=7Hz, 1H; MeAla a-H),
3.53 (dd, J=15, 7Hz, 1H; MeOMeTrp b1-H), 3.36, 3.23, 3.16
(3s, 3xNMe ), 2.89 (dd, J=15, 8Hz, 1H; MeOMeTrp b2-H),
1.46 (d, J=7Hz, 1H; MeAla b-H), 0.73 (d, J=6.6Hz, 3H; Leu
d1-Me), 0.67 (d, J=6.6Hz, 3H; Leu d2-Me).
3
6
NH), 7.52 (d, J=9.7Hz, 1H; PrLeu NH), 5.20 (dd, J=6.8,
.7Hz, 1H; CHBA a-H), 4.84 (ddd, 1H; PrLeu a-H), 4.30
dd, J=8.2, 7Hz, 1H; MeLeu a-H), 4.03 (s, 3H; MeOMeTrp
N1’-OMe), 3.68 (q, J=7Hz, 1H; MeAla a-H), 3.62 (t, J=7.6Hz,
H; MeOMeTrp a-H), 3.08 (s, 3H; MeLeu NMe), 2.87 (s, 3H;
MeAla NMe), 2.78 (s, 3H; MeOMeTrp NMe), 1.41 (d, J=7Hz,
2
3
(
1
1
H; MeAla b-H), 0.70 (d, J=6.4Hz, 3H; Leu d1-Me), 0.67 (d,
1
3
J=6.3Hz, 3H; Leu d2-Me). C NMR (CDCl , 125 MHz):
δ = 172.0, 171.4, 169.9, 169.1, 167.3 (5xC=O), 156.9 (oxazo-
3
Article Identifier:
1437-2096,E;1999,0,S1,0893,0896,ftx,en;W04999ST.pdf
le C-2), 152.0 (oxazole C-5), 132.3 (MeOMeTrp C-7a’), 125.5
(
6
oxazole C-4), 72.5 (CHBA C-a), 69.0 (MeOMeTrp C-a),
5.6 (MeOMeTrp N1’-OMe), 58.8 (MeAla C-a), 57.6 (MeLeu
Synlett 1999, S1, 893–896 ISSN 0936-5214 © Thieme Stuttgart · New York