The structure of hormaomycin and one of its all-peptide aza- analogues in solution: Syntheses and biological activities of new hormaomycin analogues

Article abstract of DOI:10.1002/chem.200400977

Four new aza-analogues of hormaomycin 1, a secondary metabolite with interesting biological activities produced by Streptomyces griseoflavus, were synthesized and subjected to preliminary tests of their antibiotic activity to provide new insights into the

Full text of DOI:10.1002/chem.200400977

FULL PAPER
The Structure of Hormaomycin and One of Its All-Peptide Aza-Analogues in
Solution: Syntheses and Biological Activities of New Hormaomycin
Analogues
Uwe M. Reinscheid,[a] Boris D. Zlatopolskiy,[b] Christian Griesinger,*[a]
Axel Zeeck,*[b] and Armin de Meijere*[b]
Abstract: Four new aza-analogues of
hormaomycin 1, a secondary metabo-
lite with interesting biological activities
produced by Streptomyces griseoflavus,
were synthesized and subjected to pre-
liminary tests of their antibiotic activity
to provide new insights into the struc-
ture–activity relationship studies of this
class of compounds. The solution struc-
tures of hormaomycin 1 and its aza-an-
alogue 2a were determined by NMR
spectroscopy. The data exhibited a rea-
sonably rigid conformation for both
molecules, stabilized by stacking inter-
actions between the aromatic moieties
attached to the ring and the side chain.
According to NMR-spectral data the
aza-analogue epi-2a has a rather differ-
ent conformation and indeed shows no
antibacterial activity whatsoever.
Keywords: amino acids
·
NMR
spectroscopy · peptides · structure
elucidation · synthetic methods
Introduction
acid [Chpca]. The latter three constituents have never been
found in any natural product before. Besides challenging
structural features, hormaomycin 1 possesses quite an inter-
esting spectrum of biological activities, including a marked
influence on the secondary metabolite production of other
streptomycetes, an exceptionally selective antibiotic activity
against coryneform bacteria,[1] and also an antimalaria activ-
ity.[3]
The unique biological properties of 1 prompted feeding
experiments with amino acids, which possibly could replace
(3-Ncp)Ala. This approach yielded several new analogues of
hormaomycin,[4] however, the precursor-directed biosynthe-
sis is apparently limited to such modifications of the build-
ing blocks, which are tolerated by the hormaomycin synthe-
tase. Thus, it was for example impossible to isolate ana-
logues of hormaomycin with a substituted or modified allo-
threonine (a-Thr) moiety.[5] On the other hand, it appeared
to be interesting to study the biological and, in this context,
the conformational properties of hormaomycin and especial-
ly its cyclopeptide analogue 2a with (2R,3R)-diaminobutyric
acid instead of (R)-allo-threonine in the macrocycle. The
more rigid additional amide bond might have a significant
influence on the intramolecular hydrogen bonds and thereby
on the global structure in solution as compared to that of
the peptide lactone. This comparison might provide insights
into the structural requirements for biological activities of
hormaomycin 1 itself and of hormaomycin analogues. Since
Hormaomycin 1 is a secondary metabolite produced by
Streptomyces griseoflavus (strain W-384).[1,2] This peptide
lactone contains (S)-isoleucine [(S)-Ile] as the only proteino-
genic amino acid along with two units of (2S,3R)-3-methyl-
phenylalanine [(bMe)Phe], one of (R)-allo-threonine [a-
Thr] as well as two moieties of (1’R,2’R)-3-(2’-nitrocyclopro-
pyl)alanine [(3-Ncp)Ala; the (2S)-diastereomer in the side
chain and the (2R)-diastereomer in the ring part of the mol-
ecule] as well as one residue of (2S,4R)-4-(Z)-propenylpro-
line [(4-Pe)Pro] (Figure 1). The side chain of 1 is terminated
by an amide-bound 5-chloro-1-hydroxypyrrole-2-carboxylic
[a] Dr. U. M. Reinscheid, Prof. Dr. C. Griesinger
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
Fax : (+49)551-201-2202
E-mail: cigr@nmr.mpibpc.mpg.de
[b] Dr. B. D. Zlatopolskiy, Prof. Dr. A. Zeeck, Prof. Dr. A. de Meijere
Institut fꢀr Organische und Biomolekulare Chemie
der Georg-August-Universitꢁt Gçttingen
Tammannstrasse 2, 37077 Gçttingen (Germany)
Fax : (+49)551-399-475
E-mail: azeeck@gwdg.de
armin.demeijere@chemie.uni-goettingen.de
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 2929 – 2945
DOI: 10.1002/chem.200400977
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1. Structural formulas of hormaomycin 1 and its aza-analogues
2a–c and epi-2a. Analogue epi-2a contains an (R)-a-Ile instead of an Ile
moiety.
amide bonds are usually much more stable towards enzy-
matic cleavage than ester linkages, aza-analogues ought to
have longer half-lives in vivo and thereby provide prolonged
biological activity. Herewith we present the first chemical
syntheses of the hormaomycin analogues 2a–c and epi-2a as
well as a preliminary evaluation of their biological activities
along with a thorough investigation of the three-dimensional
structure of hormaomycin 1 and its aza-analogue 2a in so-
lution by a combination of modern NMR spectroscopic
techniques.
Scheme 1. Syntheses of the suitably protected diamino acids 7a–c.
a) TFA, 208C, 1 h. b) SOCl2, MeOH, ꢀ20 ! 508C, 21 h. c) MeZOSu,
NaHCO3, acetone, H2O, 20 8C, 1.5 h. d) 50% Et2NH in MeCN, 208C,
40 min. e) 5% aq. HF, MeCN, 0 ! 208C, 4 h. f) CH2N2, Et2O/MeOH,
208C, 30 min. g) MsCl, Et3N, CH2Cl2, ꢀ30 ! 208C, 5 h. h) NaN3, DMF,
758C, 15 h. i) Ph3P, THF/H2O, 20 8C, 24 h, then Boc2O, 20 8C, 24 h. j) H2,
10% Pd/C, EtOAc, 208C, 3 h. k) FmocOPfp, HOAt (cat.), TMP, EtOAc,
208C, 15 h. l) 2m HCl, EtOAc, 208C, 3 h. m) MeZOSu, DIEA, TMP,
MeCN, 208C, 16 h. n) Iodobenzene bis(trifluoroacetate), pyridine, DMF/
H2O, 20 8C, 5 h. o) SOCl2, MeOH, ꢀ20 ! 208C, 24 h. MeZOSu=p-meth-
ylbenzyl-N-hydroxysuccinyl carbonate, FmocOPfp=(9-fluorenyl)methyl-
pentafluorophenyl carbonate; HOAt=7-aza-1-hydroxybenzotriazole,
TMP=2,4,6-trimethylpyridine,
Fmoc=(9-fluorenyl)methyloxycarbonyl,
bonyl, TBDMS=tert-butyldimethylsilyl.
DIEA=N,N-diisopropylethylamine,
MeZ=p-methylbenzyloxycar-
Results and Discussion
Synthesis of hormaomycin analogues: At the outset, the ap-
propriate Na-p-methylbenzyloxycarbonyl (MeZ) protected
diamino acid methyl esters 7a–c were synthesized
(Scheme 1). The a-azido tert-butyl ester 4, which was pre-
pared according to a published procedure[6] with a Sharpless
asymmetric aminohydroxylation as a key step followed by
stereoselective azidation, was transformed into the fully pro-
tected (2R,3R)-2,3-diaminobutyric acid (a-Dab) derivative 5
as described by Wen et al.[7] The free acid, after simultane-
ous removal of the N-Boc and O-tert-butyl groups from 5
with trifluoroacetic acid, was esterified with methanol, and
the resulting Nb-protected diamino acid methyl ester was
acetylated with MeZOSu to give 6 in 69% yield over three
steps. Removal of the N-Fmoc group just before the next
step gave the methyl ester 7a.
This transformation was followed by displacement of the
mesylate by an azide group with NaN3 in DMF to give the
azido ester 9, which was further transformed into the Na-
Boc, Nb-Z protected (2R,3R)-3-amino-2-methylaminobutyric
(a-NbMeDab) acid methyl ester, by treatment first with tri-
phenylphosphine and water, and then with Boc2O. Subse-
quent removal of the Z group by hydrogenolysis was fol-
lowed by introduction of the Fmoc group to give the inter-
mediate Na-Boc, Nb-Fmoc protected a-NbMeDab methyl
ester, which, after removal of the Boc group, was finally acy-
lated with MeZOSu to give 10 in 13% yield over ten steps.
The N-Fmoc group in 10 was then removed to give 7b. The
latter was immediately used in the peptide coupling step.
The Na-MeZ protected 2,3-diaminopropionic acid ester 7c
was obtained as a hydrochloride by esterification with meth-
anol of the intermediate 13, which in turn was prepared in
76% yield over three steps starting from (R)-asparagine
(11) by initial acylation with MeZOSu and subsequent oxi-
The fully protected diamino acid 10 was synthesized start-
ing from the known tert-butyl ester 8.[8] After removal of the
tert-butyldimethylsilyl group followed by cleavage of the
tert-butyl ester, the appropriate N-Z-protected isothreonine
was esterified with diazomethane to give an intermediate,
which was further converted to the corresponding mesylate.
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Hormaomycin
FULL PAPER
dation with iodobenzene bis(trifluoroacetate) in close analo-
gy to a published procedure.[9]
The diamino esters 7a–c were coupled with the N-Boc-
protected (2S,3R)-4-(Z)-propenylproline 14[10] to give the in-
termediate methyl esters (Scheme 2).[11] Treatment of the
latter with tetrabutylammonium hydroxide[12] gave the pep-
tide acids 15a–c in 71, 68 and 70% yield over two steps, re-
spectively, which were coupled with the O-dicyclopropyl-
methyl (DCPM) protected tetrapeptide 16[2a] (15a and c) or
with the O-(2-trimethylsilyl)ethyl (TMSE) protected tetra-
peptide 17[13] (15b), after deprotection of their terminal
amino groups, to yield the branched hexapeptides 18a
(80%), 18b (93%), and 18c (59%), respectively.
Scheme 3. Cyclization of the linear precursors 18a–c. a) 2m HCl, EtOAc,
208C, 1 h (for 18a and 18c) or Bu4N+Fꢀ, THF, 20 ! 558C, 2 h then 2m
HCl, in EtOAc, 208C, 1 h (for 18b). b) HATU, DIEA, TMP, CH2Cl2,
0.1 mm, 0 ! 208C, 18–22 h.
as optical rotation values quite different from those of 19a
and 19c indicating distinctions in their solution structures.
The N-MeZ protected cyclohexapeptides were subse-
quently first deprotected and then coupled with N-Teoc-pro-
tected (2S,1’R,2’R)-(3-Ncp)Ala-OH[2a] (Scheme 4). Removal
of the Teoc group and coupling of the intermediates with O-
MOM protected Chpca-OH 20[2a,10] gave the O-MOM pro-
tected hormaomycin aza-analogues. Finally, removal of the
MOM group gave the target compounds 2a–c and epi-2a.
Scheme 2. Syntheses of the linear peptide precursors 18a–c. a) 7a–c,
EDC, HOAt, TMP, CH2Cl2, 0 ! 208C, 16 h. b) 40% aq. Bu4N+OHꢀ,
THF, 08C, 45 min. c) 50% Et2NH in THF, 208C, 40 min. d) 15a–c,
HATU, HOAt, TMP, CH2Cl2, 0 ! 208C, 15 h. EDC=N’-(3-dimethylami-
nopropyl)-N-ethylcarbodiimide hydrochloride, HATU=O-(7-azabenzo-
triazole-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate,
DCPM=dicyclopropylmethyl, TMSE=2-trimethylsilyl)ethyl, Dap=2,3-
diaminopropionic acid.
The acidolytic removal of the Boc and DCPM groups
from the termini of 18a and 18c, as well as the sequential
removal first of the TMSE group with tetrabutylammonium
fluoride, and then the Boc group with acid from the termi-
nus of 18b occurred almost quantitatively, and was succeed-
ed by macrocyclization, by using O-(7-azabenzotriazole-1-
Scheme 4. The final steps in the preparation of aza-analogues 2a–c and
epi-2a. a) Anisole, TFA, 208C, 2 h. b) Teoc-(2S,1’R,2’R)-(3-Ncp)AlaOH,
HATU, HOAt, DIEA, TMP, CH2Cl2, 208C, 15 h. c) TFA, 208C, 1 h.
d) 20, HATU, HOAt, DIEA, TMP, CH2Cl2, 208C, 4 h. e) MgBr2·Et2O,
EtSH, CH2Cl2, 208C, 3–4 h. Teoc=(2-trimethylsilylethyl)oxycarbonyl.
Analogue epi-2a contains an (R)-a-Ile instead of an Ile moiety.
yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
(HATU)[14] in the presence of 7-aza-1-hydroxybenzotriazole
(HOAt)[14] under high-dilution conditions (Scheme 3). The
cyclization of the hexapeptides, containing a-Dab or Dap
residues, caused significant epimerization at the a-carbon of
the Ile residue (Ile ! (R)-a-Ile)[15] and gave, after HPLC
separation, the epimeric macrocycles, 19a (28%) and epi-
19a (19%), as well as 19c (34%) and epi-19c (25%), re-
spectively. In contrast, the cyclization of the a-NbMeDab-
containing peptide (similar to the synthesis of the N-MeZ
protected ring part of hormaomycin 1)[2a] gave almost exclu-
sively the cyclic peptide 19b (44%) along with only traces
(<2%) of the epimer. Not surprisingly, the epimeric prod-
ucts exhibited features in their 1H and 13C NMR spectra
(chemical shifts, coupling constants and line shapes) as well
NMR analysis and conformational modelling of hormaomy-
cin 1 and its all-peptide aza-analogue 2a: The conformation-
al analysis of hormaomycin 1 was performed in CDCl3 so-
lution at 293 K. Spin systems were identified by DQF-
1
COSY, TOCSY and 13C-, H-HMBC experiments. Especially
useful for the assignment of the aromatic components were
the long-range cross peaks between the Hb and the Cipso as
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C. Griesinger, A. Zeeck, A. de Meijere et al.
well as the Haromatic and Cb, because they provided the cor-
rect assignment of the two phenyl rings of (bMe)Phe I and
II.[13,16]
served. Further details about the calculation and NMR
input data are to be found in the Experimental Section.
The average root mean square deviation (RMSD) of the
backbone atoms compared to the average structure was
0.39 ꢃ and for all heavy atoms 0.71 ꢃ.
The c1 angles of each component were determined by the
combined use of coupling constants from P.E. COSY and
13C-HMBC experiments and distance information from
ROESY experiments. A two proton example is the c1 dihe-
dral angle of allo-(R)-threonine. In the HMBC spectrum, a
The propenyl substituent of the (4-Pe)Pro unit of hormao-
mycin 1 is found antiperiplanar relative to the pyrrolidine
nitrogen. It adopts an equatorial position (Figure 2). Allylic
1,3-strain directs the cis-propenyl side chain of (4-Pe)Pro
into one plane with the g-hydrogen of the pyrrolidine ring.
The observation of both NH(i)–NH(i+1) and Ha(i)–NH-
(i+1) ROE values indicates that the peptolide backbone
exists in a tight turn. A strong ROE between Ha [(bMe)Phe
II] and NH [(3-Ncp)Ala I] together with a weak cross peak
between Ha [(3-Ncp)Ala I] and NH [(bMe)Phe I] indicate a
b turn [(bMe)Phe II, (3-Ncp)Ala I, (bMe)Phe I, Ile]. The
CD curves with a positive maximum at 213 nm and a nega-
tive maximum around 240 nm already indicated the pres-
ence of a b turn.[2b]
The general criterium for the presence of a b turn is that
the distance between Ca(i) and Ca(i+3) is less than 7 ꢃ.
Type II and type II’ b turns are further differentiated by
their dihedral angles of the residues i+1 and i+2 (Table 1).
The presence of a CO(i)–HN(i+3) hydrogen bond is possi-
ble, but not necessary for a stabilization of the b turn. The
structure of hormaomycin 1 exhibits a Ca(i)–Ca(i+3) dis-
tance of 7 ꢃ for the components Ile and (bMe)Phe II. These
two constitute the i and i+3 position (i+3 and i position) of
two b turns in the structure of 1. A g turn can be excluded
because the distances between Ha [(bMe)Phe II]–Ha
[(bMe)Phe I] of 6.9 ꢃ and Ha(Ile)–Hb(a-Thr) of 6.8 ꢃ are
too long.
3
strong JCH correlation from CO (d=169.2 ppm) to the b
proton (d=5.44 ppm) is visible. This together with a 3J-
(Ha,Hb) value of 5 Hz is consistent with a gꢀ arrangement of
the two protons.
The chemical shift values of the components are shown in
Table 1 of the Supporting Information. One-dimensional
proton spectra showed one predominant resonance for each
amide NH, suggesting either one dominant isomer or fast
conformational averaging on the NMR time scale in CDCl3.
The coexistence of slowly interconverting conformers could
be ruled out by the absence of exchange cross peaks in the
ROESY spectra and the correct number of resonances in
the 1D-proton spectrum.
The enantiotopic Hb protons of (3-Ncp)Ala I exhibited
Dd values >1.5 ppm indicating a well defined structure.
This agrees with the large chemical shift dispersion within
the set of NH (6.54–9.13 ppm) and Ha (3.51–5.16 ppm)
proton signals. Especially the long-range ROE values be-
tween the aromatic protons of Chpca and (bMe)Phe I indi-
cate a compact conformation.[13]
The presence of strong Ha(i)–NH(i+1) ROE values and
the absence of Ha(i)–Ha(i+1) cross peaks confirmed that all
the amide bonds are in the s-trans conformation.
ꢀ
An s-trans conformation with respect to the Ile Pro pep-
tide bond was assigned according to characteristic ROE
cross peaks between the Ha (Ile) and the Hd [(4-Pe)Pro] as
well as the absence of cross peaks between Ha (Ile) and Ha
[(4-Pe)Pro]. Additionally, the differences in 13C NMR chem-
A type II’ (inverse II) turn is formed with (bMe)Phe II at
position i and with (3-Ncp)Ala I and (bMe)Phe I as the cen-
tral residues (i+1) and (i+2), respectively. The presence of a
Ha(i+1)–HN(i+2) ROE and the absence of other HN–HN
cross peaks differentiates this b turn from the other b turn
of hormaomycin 1 which belongs to type II according to the
corresponding dihedral angles (Table 1). The type II b turn
is formed by Ile at the i position and (4-Pe)Pro and a-Thr as
the central residues. Proline residues are typically found at
the i+1 position of type I and type II b turns.
ꢀ
ical shifts of Cb Cg = ꢀ1.7 ppm [(4-Pe)Pro], are indicative
of trans-peptide bonds.[17] The difference, directly related to
the dihedral angle y(Pro), is usually in the range of 2–
10 ppm for cis-Pro and 0–5 ppm in trans-Pro. In (4-Pe)Pro
residue of hormaomycin 1, the (4R)-substituent further in-
creases the Cg chemical shift value.
Cyclic hexapeptides normally adopt an all-trans-confor-
mation about the peptide bonds and prefer a conformation
with two b turns.[18] The hypothesis that the number of
amino acids in cyclopeptides influences the type of secon-
dary structure adopted was later proved in a modified ver-
sion.[19] However, major influences by the side chains, espe-
cially of non-typical amino acids, have not been taken into
account. This made predictions of the solution structure of
hormaomycin 1 difficult. In fact, hormaomycin 1 combines a
cyclic portion with an extended side chain consisting of two
components. In addition, the ring contains one ester linkage.
From the restrained MD simulations and energy minimi-
zations, one family of low-energy structures was generated,
satisfying the ROE-derived restraints and dihedral angles
(Figure 2). No ROE violation greater than 0.5 ꢃ was ob-
Table 1. Dihedral angles [8] of ideal b turns of type II and II’ and of the
corner components of hormaomycin 1.
f(i+1)
y(i+1)
f(i+2)
y(i+2)
ideal type II
ideal type II’
(4-Pe)Pro, a-Thr
(3-Ncp)Ala I, (bMe)Phe I
ꢀ60
+60
ꢀ61
+69
+120
ꢀ120
+142
ꢀ134
+80
ꢀ80
+90
ꢀ90
0
0
ꢀ77
ꢀ47
Ideal b turns are ten-membered rings when the hydrogen
bond is incorporated. In the case of hormaomycin 1, the
type II b turn is composed of (R)-allo-threonine at the i+2
position and therefore contains the Cb as an additional
atom.
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Hormaomycin
FULL PAPER
Because of the unusual components and the overall struc-
ture that appears to be governed by long-range side-chain
interactions, the b turns deviate from the ideal values. It is
important to underline that the standard distances observed
for turns in peptides and proteins containing only (R)- or
(S)-residues cannot be used here.[20]
In general, distances in oligopeptides are strongly influ-
enced by the configurations of the contained amino acids.
(S)-Xaa-(R)-Yaa and (R)-Xaa-(S)-Yaa dyads have a high
tendency to be in the corner positions of type II and type II’
turns, respectively. Indeed, the type II’ b turn in hormaomy-
cin 1 is formed with the residue of (3-Ncp)Ala I [(R)-amino
acid] in the corner position followed by (bMe)Phe I [(S)-
amino acid].
With the oxygen of the ester linkage in 1 replaced by an
NH in 2a, the f angle (+908) of the i+2 residue (a-Thr) is
almost identical with the ideal f angle of a type II turn
(+808). The dihedral angle defined by O-Ca-Cb-CO (taken
as y(i+2)) is substantially different from an ideal type II. It
is therefore reasonable to refer to it as “type II-like”. The
dihedral angles at the i+1 position agree with the type II
turn (f=ꢀ61 and y=+1428). The twisted nature of the b
turns results in a figure-eight like overall structure for the
macrocyclic ring (Figure 3).
Further corroboration of the structure is derived from the
detailed analysis of the chemical shifts presented in the Ex-
perimental Section and Supporting Information.
The structure of a peptide is not only determined by the
backbone conformation, but also the orientation of the side
chains. Many conformational studies have shown that the
rotamer distribution is the more shifted to a single rotamer,
the more “rigid” the backbone is.[21] Hence, the side-chain
conformation can be taken as an indicator for the rigidity of
the molecule. The two aromatic rings of (bMe)Phe II and
Chpca are stacked in-line with each other (Figure 2). The
compact overall shape of hormaomycin 1 is dictated by
these side-chain interactions which in turn allow only a rigid
macrocyclic structure.
The assignments of proton and carbon resonances of the
aza-analogue 2a are compiled in Table 2 of the Supporting
Information. Due to signal overlap two dihedral angles (Ha/
H3b [(4-Pe)Pro] and Hg/Hda [(4-Pe)Pro] could not be deter-
mined. All the others, which have been determined for hor-
maomycin 1, were also determined for 2a. An identical
range of values was obtained with only one differing di-
hedral angle in the side chain of isoleucin (Ca-Cb-Cg-Cd
=
+1808 for 2a and ꢀ608 for 1). The side chain is therefore
more directed to the solvent. The ROE values measured for
2a differed only slightly, resulting in the same classification
into strong, medium and weak as for hormaomycin 1. An
additional ROESY cross peak was observed between the
HN attached to the Cb atom of the a-Dab unit and the pro-
tons of its methyl group.
The s-trans-conformation of all peptide bonds was con-
firmed by ROE cross peaks as established for hormaomycin
1. Analogously, the ester linkage of a-Thr and (4-Pe)Pro in
the calculated structure of hormaomycin 1 takes an s-trans
orientation which is favored by the anomeric effect.
From the almost identical structural data obtained, one
may conclude that the structure of the macrocyclic ring of
the aza-analogue 2a in solution does not differ from that of
hormaomycin 1 (see Figure 4).
In general, cyclic peptides in which all the peptide bonds
have s-trans-conformations lack internal motions in the
backbone. This agrees with the present findings, that the
modification in the macrocyclic ring from an ester to an
amide linkage does not change the overall structure of the
macrocycle. In the case of 2a, the additional peptide bond
also adopts an s-trans-confor-
mation.
The investigation of the so-
lution structure of the N-
methyl-aza-analogue 2b was
considered to be useless be-
cause of an abundance of
slowly equilibrating conform-
1
ers. As the H NMR spectrum
of the des-methyl-aza-analogue
2c is very similar to those of
hormaomycin 1 and the aza-
analogue 2a, it is quite possi-
ble that this peptide in solution
also adopts approximately the
same overall conformation.
Figure 2. Stereoview of the average structure of hormaomycin 1 in CDCl3.
Antibacterial activity: As an
entry, the antibiotic activity of
the new hormaomycin ana-
logues against Arthrobacter
species was tested (Tables 2,
Figure 3. Stereoview of the macrocyclic ring of the average structure of hormaomycin 1 in CDCl3.
3).[22]
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C. Griesinger, A. Zeeck, A. de Meijere et al.
aza-analogue 2a by solution
NMR experiments. The two
structures turn out to be virtu-
ally identical. Consistent with
this finding, 1 and 2a exhibit
similar antibiotic activities. The
1H NMR spectra of epi-2a sub-
stantially differ from those of 1
and 2a suggesting a different
structure. Consistently, epi-2a
is inactive in the antibiotic
assay indicating that the con-
formation of the whole mole-
cule is important for this bio-
logical activity.
Figure 4. Average structure of the aza-analogous hormaomycin 2a.
Table 2. Relative antibacterial activities of hormaomycin 1, aza-ana-
logues 2a and epi-2a in serial dilution plate diffusion tests against Ar-
throbacter crystallopoites (strain 20117) (%) (estimated relative to the ac-
tivity of hormaomycin at 5ꢄ10ꢀ2 mg per 9ꢄ0.5 mm plate) 288C.
Experimental Section
Compound (mg pro plate)
5ꢄ10ꢀ2
5ꢄ10ꢀ3
5ꢄ10ꢀ4
5ꢄ10ꢀ5
General remarks: Synthesis: 1H NMR spectra: Bruker AM 250
(250 MHz), Varian Unity 300 (300 MHz), Varian Inova 600 (600 MHz).
1H chemical shifts are reported in ppm relative to residual peaks of deu-
terated solvent or tetramethylsilane. Higher order NMR spectra were ap-
proximately interpreted as first-order spectra, if possible. The observed
signal multiplicities are characterized as follows: s=singlet, d=doublet,
t=triplet, q=quartet, quin=quintet, m=multiplet, as well as br=broad,
Ar-H=aryl-H. 13C NMR spectra [additional DEPT (Distortionless En-
hancement by Polarization Transfer) or APT (Attached Proton Test)]:
Bruker AM 250 (62.9 MHz), Varian Unity 300 (75.5 MHz) or Varian
Inova 600 (125.7 MHz) instruments. 13C chemical shifts are reported rela-
tive to peak of solvent or tetramethylsilane. The following abbreviations
were applied: DEPT: +=primary or tertiary (positive signal in DEPT),
ꢀ=secondary (negative signal in DEPT), Cquat =quaternary (no signal in
DEPT); APT: +=primary or tertiary (positive signal in APT), ꢀ=sec-
ondary or quaternary (negative signal in APT); whenever it was necessa-
ry and possible HMBC (Heteronuclear Multiple Bond Connectivity)
and/or HMQC (Heteronuclear Multiple Quantum Coherence) spectra
were also measured. The signals marked with asterisk have been attribut-
ed with uncertain reliability. IR spectra: Bruker IFS 66 (FT-IR) spec-
trometer, samples measured as KBr pellets or oils between KBr plates.
The IR spectra of all synthesized peptides showed a broad NH stretch
band, arising from the amide moieties, between 3500 and 3250 cmꢀ1. MS:
EI-MS: Finnigan MAT 95, 70 eV. High resolution EI-MS spectra with
perfluorokerosene as reference substance; pre-selected ion peak match-
ing at R @ 10000 to be within ꢁ2 ppm of the exact masses. ESI-MS:
Finnigan LCQ. HPLC: pump: Kontron 322 system, detector: Kontron
DAD 440, mixer: Kontron HPLC 360, data system: Kontron Kromasys-
tem 200, columns: Knauer Nucleosil-100 C18 (analytical, 5 mm, 3 mmꢄ
250 mm), preparative: A: Kromasil C18 (7 mm, 20 mmꢄ250 mm), B:
Knauer Nucleosil-100 C18 (5 mm, 8 mmꢄ250 mm). Optical rotations:
Perkin–Elmer 241 digital polarimeter, 1 dm cell; optical rotation values
hormaomycin 1
2a
epi-2a
100
103
0
94
90
0
71
68
–
39
35
–
Table 3. Relative antibacterial activities of several compounds in serial
dilution plate diffusion tests against Arthrobacter oxidans (strain 20119)
(%) (estimated relative to the activity of hormaomycin at 1.5ꢄ10ꢀ2 mg
per 6ꢄ0.65 mm plate) 288C.[23]
Compound (mg per plate)
1.5ꢄ10ꢀ2
1.5ꢄ10ꢀ3
1.5ꢄ10ꢀ4
hormaomycin 1
penicillin G
19a–c, epi-19a
epi-19c
2b
2c
100
78
0
72
0
–
0
72
83
44
0
–
0
42
58
22
94
94
Even these very preliminary biological tests give some in-
formation about structure–activity relationships for hormao-
mycin 1 and its analogues. At least the antibacterial activity
of hormaomycin 1 can neither solely be attributed to its
macrocyclic part nor to its side chain,[4b] but supposedly is
associated with the whole molecule. The weak antibiotic ac-
tivity of the cyclopeptide epi-19c may be due to a mode of
action on bacteria which is different from that of hormao-
mycin 1. The aza-analogues 2a–c displayed spectral and sol-
ubility properties, as well as antibiotic activities very similar
to those of the native compound 1. In contrast, the epi-aza-
are given in 10ꢀ1 degcm2 gꢀ1
; concentrations (c) are given in g per
100 mL. Circular dichroism: Jasco J 500 A. Molar ellipticities (V) are
given in degreecm2 10ꢀ1 molꢀ1. M.p.: Bꢀchi 510 capillary melting point
apparatus, uncorrected values. TLC: Macherey–Nagel precoated sheets,
0.25 mm Sil G/UV254. The chromatograms were viewed under UV light
and/or by treatment with phosphomolybdic acid (10% in ethanol), or
ninhydrin (0.2% in ethanol), or Ehrlichꢅs reagent (freshly prepared so-
lution of 1 g of 4-dimethylamino-benzaldehyde in 25 mL of 36% HCl
and 75 mL methanol). Column chromatography: Merck silica gel, grade
60, 230–400 mesh and Baker silica gel, 40–140 mesh. Preparative TLC:
Macherey–Nagel, silica gel SIL G/UV254, layer thickness 0.25 mm (100ꢄ
200 mm or 200ꢄ200 mm). Elemental analyses: Mikroanalytisches Labo-
ratorium des Instituts fꢀr Organische und Biomolekulare Chemie der
Universitꢁt Gçttingen. Starting materials: Anhydrous solvents were pre-
1
analogue epi-2a, which exhibits CD and H NMR spectra as
well as solubility properties quite different from those of
hormaomycin 1, turned out to be totally inactive within the
used test system.
Conclusion
We have synthesized several analogues of hormaomycin and
investigated the structures of the title compound 1 and its
2934
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 2929 – 2945

Hormaomycin
FULL PAPER
pared according to standard methods by distillation over drying agents
and were stored under argon. All other solvents were distilled before
use. All reactions were carried out with magnetic stirring and, if air or
moisture sensitive, in flame-dried glassware under argon or nitrogen. Or-
ganic extracts were dried with anhydrous MgSO4. tert-Butyl (2R,3R)-2-
azido-3-(benzyloxycarbonylamino)butyrate (4),[6] tert-butyl (2R,3R)-2-
tert-butyloxycarbonylamino-3-(9-fluorenylmethyloxycarbonylamino)buty-
rate (5),[7] tert-butyl (2S,3R)-2-tert-butyldimethylsilyloxy-3-(benzyloxycar-
bonyl-N-methylamino)butyrate (8),[8] (2S,4R)-(N-tert-butyloxycarbonyl)-
4-(Z)-propenylproline (14),[10] 1-hydroxy-7-aza-benzotriazole,[24] tetrapep-
tides 16[2a] and 17,[25] (2S,1’S,2’R)-[N-(2-trimethylsilyl)ethyloxycarbonyl]-
(2’-nitrocyclopropyl)alanine,[2a] 5-chloro-1-methoxymethoxypyrrole-2-car-
boxylic acid (20)[10] were prepared as described elsewhere. Conformation-
al analysis NMR studies: NMR spectra were recorded on Bruker
DRX400 and DRX600 spectrometers. The concentration was 5 mm in
CDCl3 and measurements were run at 293 K. The assignments were car-
ried out with the help of standard DQF-COSY (Double-Quantum Fil-
tered Correlation Spectroscopy), TOCSY (Total Correlation Spectrosco-
py), 13C-HSQC, 15N-HSQC and 13C-HMBC experiments. Typically 2 K
data points in F2 and 512 experiments in F1 were acquired. In some
cases, additional ROESY experiments were used to confirm the assign-
ments made. The spectra were acquired with 16 transients and a relaxa-
tion delay of 2 s except the ROESY experiments with 80 transients. For
ROESY experiments, a spinlock field of 3.1 kHz was used with a mixing
time of 480 ms.[26] The TOCSY experiments were performed with a spin-
lock field of 6.25 kHz by using the MLEV17 sequence with mixing times
of 40 and 80 ms. The data were zero filled and processed as a 4 Kꢄ1 K
matrix. P.E. COSY experiments were processed as an 8 Kꢄ2 K matrix.
To obtain the temperature coefficients of the amide proton chemical
shifts, TOCSY spectra were recorded between +15 and +458C. To de-
termine the c1 torsional angle constraints, the Ha–Hb coupling constants
(3Jab) from the 1D proton and P.E. COSY spectra, the intensity of the in-
Structural validation: The following four interresidual ROEs have not
been used in the calculations for cross validation purposes [Chpca 3-H
and (bMe)Phe I Haromatic, (bMe)Phe II Haromatic and (3-Ncp)Ala I 3’-HA,
(bMe)Phe II Haromatic and (3-Ncp)Ala I 6-HA, (bMe)Phe II Haromatic and
(3-Ncp)Ala I NH]. In the average structure the corresponding distances
are 3.8, 4.6, 5.2, and 3.5 ꢃ, respectively, which reasonably agree with the
measured ROE values. There is an upfield chemical shift of the b-proton
of (bMe)Phe II (3.04 ppm) compared with that of the corresponding
proton in (bMe)Phe I (3.72 ppm), which can be explained by the aniso-
tropy effect of the pyrrole ring. This effect requires a specific folding of
the two-residue side chain. Additional anisotropy effects are seen for the
methyl protons of the Ile residue exerted by the pyrrole ring of Chpca
and for the protons of the (3-Ncp)Ala I side chain by the neighboring ar-
omatic ring of (bMe)Phe I. The large downfield shift of the amide proton
of (3-Ncp)Ala II (8.14 ppm) compared with NH of (3-Ncp)Ala I may be
due to an H-bonding interaction with the oxygen of Chpca. All amide
protons of the macrocyclic ring show low temperature chemical shift
values (all < +/ꢀ1 ppb/8C except NH [(bMe)Phe I]: ꢀ3 ppb per 8C) in-
dicating shielding from solvent or H-bonding. Data have been submitted
to PDB (Protein Data Bank) and BMRB (BioMagResBank).
Biological tests were carried out as described elsewhere.[16b]
Deprotection of N-Fmoc-protected amino acids 7a and 7b, and peptides
16 and 17—General procedure (GP 1): The protected amino acids or
peptides (1 mmol) were taken up with acetonitrile or THF (2 mL), dieth-
ylamine (2 mL) was added, and the resulting mixture left at ambient tem-
perature for 40 min. All volatiles were evaporated under reduced pres-
sure, the residue was taken up with toluene (2ꢄ5 mL), which was evapo-
rated under reduced pressure to remove the last traces of diethylamine.
The obtained crude N-deprotected amino acids or peptides were directly
used in the next condensation step.
Peptide condensation step for the preparation of dipeptide acids 15a–c—
General procedure (GP 2): EDC (1.03 mmol) and HOAt (1.05 mmol)
were added to a cooled (48C) solution of the N-Boc-protected 4-(Z)-pro-
penylproline 14 (1 mmol) in anhydrous CH2Cl2 (3 mL). After 10 min, the
solution of the appropriate crude Nb-deprotected diamino ester
(0.97 mmol) and TMP (3 mmol) in anhydrous CH2Cl2 (1 mL) was added
at the same temperature (in the case of 7c·HCl two additional equiva-
lents of TMP were used). The temperature was allowed to reach 208C,
and stirring was continued for 6 h. Then the reaction mixture was diluted
with Et2O or EtOAc (30 mL), and the mixture washed with 1m KHSO4
(3ꢄ5 mL), water (2ꢄ5 mL), saturated aqueous solution of NaHCO3 (3ꢄ
5 mL), water (3ꢄ5 mL), brine (2ꢄ5 mL), dried and concentrated under
reduced pressure. The residue was purified by column chromatography
and recrystallization to give the respective dipeptide esters.
3
traresidue ROEs (Ha–Hb, NH–Hb) and the intensity of the JCH HMBC
cross peaks were used. Each amino acid residue was classified with re-
3
spect to three rotamers, according to the patterns of the JHH
,
3JCH and
ROE values. The stereospecific assignments were also established for the
b-methylene protons. Assuming that the staggered rotamers are predomi-
nantly populated, qualitative considerations together with homonuclear
coupling constants[13] are often sufficient for the assignment of diastereo-
topic methylene protons (Figure 1). The c1 angle was set at ꢀ608 when
both the 3J(Ha–Hb1) and the 3J(Ha–Hb2) coupling constants are small. If
one strong and one weak coupling is observed, c1 can be either 60 or
1808. To differentiate between these two cases, stereospecific assignments
of the Hb protons are required. This was possible with the help of qualita-
tive heteronuclear J couplings (between 13CO and Hb) and ROE cross-
peak intensities stemming from the different Hb protons. In this way a set
of dihedral angles was obtained and this together with the ROE-derived
Hydrolysis step for the preparation of dipeptide acids 15a–c—General
procedure (GP 3): A 40% aqueous solution of tetra-n-butylammonium
hydroxide (0.15 mmol) was added dropwise to an ice-cold solution of the
respective dipeptide ester (0.10 mmol) in THF (0.91 mL) within 3 min,
and stirring was continued at the same temperature for an additional
45 min (TLC monitoring to detect complete consuming of the starting
material). A 1m aqueous H2SO4 (0.5 mL) was then added, and the mix-
ture was diluted with Et2O (50 mL). The organic layer was separated and
washed with 1m KHSO4 (2ꢄ10 mL), water (5ꢄ10 mL), brine (2ꢄ5 mL),
dried and filtered. The filtrate was concentrated under reduced pressure
to give the crude product which was purified by column chromatography
or/and recrystallization.
distances was the input for
a
molecular modelling (MD) study.[13]
Molecular dynamics: All molecular mechanics/dynamics simulations were
performed with DISCOVER of Insight II (Accelrys) on a Silicon Graph-
ics Octane workstation. The simulations were done using CVFF (Consis-
tent Valence Force Field).[27] A distance-dependent dielectric constant
(e=4.8 r) was used. The molecular structure was first minimized. During
a 100 ps MD run, 100 structures were sampled which represent starting
conformations for the subsequent restrained MD. According to a simulat-
ed annealing approach, the resulting starting molecules were heated to
600 K initially, subsequently cooled and finally subjected to an energy
minimization using both steepest descent and conjugate gradient methods
successively.[28] The final structures were analyzed for similarities by com-
paring the RMSD deviations.
Peptide condensation step for the preparation of the branched hexapepti-
des 18a–c—General procedure (GP 4): Tetrapeptide 16 or 17
(0.21 mmol) was deprotected according to GP 1, taken up with anhydrous
CH2Cl2 (5 mL), the respective dipeptide acid (0.23 mmol), HATU
(0.25 mmol) and HOAt (0.23 mmol) were added, and the reaction mix-
The distance and torsional angle constraints of Tables 3 and 4 in the Sup-
porting Information were used as restraints in the MD runs as well as the
final minimizations. Pseudo-atoms were used for the methyl protons and
aromatic protons. Distance restraints derived from ROE-cross peaks,
classified empirically as strong, medium and weak, were applied as bihar-
monic restraints with lower and upper boundaries of 2.0–2.8, 2.0–3.5, 2.0–
5.0 ꢃ, respectively. The configurations at the stereogenic carbon atoms
were restrained.[13] Likewise, due to the detected trans-conformation of
all peptide bonds, the w dihedral angle was restrained to 1808.
ture was cooled to 48C. After this,
a solution of DIEA (29 mg,
0.22 mmol) and TMP (75 mg, 0.62 mmol) in CH2Cl2 (2 mL) were added
at the same temperature within 5 min. The temperature was allowed to
reach 208C, and stirring was continued for an additional 15 h. The crude
product obtained after aqueous work-up, according to GP 2, was finally
purified by recrystallization and/or column chromatography.
Chem. Eur. J. 2005, 11, 2929 – 2945
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2935

C. Griesinger, A. Zeeck, A. de Meijere et al.
Preparation of N-MeZ-protected cyclohexapeptides 19a–c, epi-19a and
epi-19c—General procedure (GP 5): 2m HCl in EtOAc (2 mL) was
added to the appropriate branched hexapeptide (0.10 mmol); the reac-
tion mixture was stirred at 208C for 1 h in a dark place, and was then
concentrated under reduced pressure at 208C. The residue was triturated
with anhydrous Et2O (2ꢄ5 mL) to give the hydrochloride of the depro-
tected material as a colorless solid, which was taken up with anhydrous
CH2Cl2 (1.0 L). The solution was cooled to 48C (internal temperature),
HATU (0.103 mmol) and HOAt (0.10 mmol) were added, and then a so-
lution of DIEA (0.40 mmol) in CH2Cl2 (50 mL) was added over 30 min.
The cooling bath was removed and stirring continued for an additional
2 h at ambient temperature. Then the reaction mixture was cooled again
to 48C, and a second portion of each, HATU (0.103 mmol) and HOAt
(0.10 mmol), was added, and then a solution of DIEA (0.40 mmol) in
CH2Cl2 (50 mL) was added within 30 min. The temperature was allowed
to reach 208C, and stirring was continued for 15 h. After this, the solvent
was removed under reduced pressure, the residue was taken up with
Et2O, and after the usual aqueous work-up (GP 2) and concentration
under reduced pressure, the crude product was purified first by column
chromatography and then by recrystallization (Et2O/pentane) to give a
mixture of the epimeric cyclopeptides, which was separated by prepara-
tive HPLC to give the respective cyclohexapeptides.
MeZ-a-Dab(Fmoc)-OMe (6): NaHCO3 (0.156 g, 1.85 mmol) and then a
solution of MeZOSu (0.244 g, 0.93 mmol) in acetone (5 mL) were added
to a vigorously stirred solution of H-a-Dab(Fmoc)-OMe·HCl (0.29 g,
max. 0.74 mmol) in water (7 mL), and stirring was continued for 90 min
(if a precipitate formed, acetone and/or water was added to obtain a ho-
mogeneous solution). The mixture was then concentrated under reduced
pressure, diluted with water (40 mL), and the resultant suspension was
filtered. The crude product was washed with Et2O/pentane 1:1 (50 mL),
water (100 mL), 3% NaHCO3 (50 mL), water (20 mL), 1m HCl, water
(50 mL), pentane (50 mL), dried and finally recrystallized from CH2Cl2/
hexane to give 6 (0.272 g, 69% over three steps) as a colorless solid. M.p.
167–1688C; [a]2D0 =8.5 (c=0.40, THF); 1H NMR (250 MHz, CDCl3): d=
1.47 (d, J=6.8 Hz, 3H, 4-H), 2.34 (s, 3H, 1’-H, MeZ), 3.77 (s, 3H, OMe),
4.10–4.51 (m, 4H, 3-H and 9’-H, 1-H, Fmoc), 4.60 (dd, J=8.1, 3.1 Hz,
1H, 2-H), 5.08 (s, 2H, Bzl-H), 5.32 (d, J=8.0 Hz, 1H, NH), 5.67 (d, J=
6.8 Hz, 1H, NH), 7.16 (d, J=7.3 Hz, 2H, Ar-H), 7.21–7.46 (m, 6H, Ar-
H), 7.61 (d, J=6.8 Hz, 2H, Ar-H), 7.77 (d, J=7.3 Hz, 2H, Ar-H);
13C NMR (62.9 MHz, CDCl3): d=16.3 (+, C-4), 21.0 (+, C-1’, MeZ),
47.0 (+, C-3), 48.9 (+, C-9’, Fmoc), 52.5 (+, OMe), 57.8 (+, C-2), 66.8
(ꢀ, Bzl-H, MeZ), 67.1 (ꢀ, C-1, Fmoc), 119.8, 125.0, 126.9, 127.5, 128.3,
129.1 (+, Ar-C), 132.8 (Cquat, Ar-C), 137.9 (Cquat, Ar-C), 141.1 (Cquat, Ar-
C), 143.7, 143.9 (Cquat, Ar-C), 155.8, 156.4 (Cquat, NCO2), 170.7 (Cquat, C-
1); IR (KBr): n˜ =3316, 3067, 2948, 1748, 1691, 1542, 1450, 1338, 1316,
1282, 1231, 1169 cmꢀ1; MS (EI, 70 eV): m/z (%): 502 (1) [M +], 266 (10)
[C14H20NO4+], 178 (100) [C14H10+], 165 (5), 105 (22) [C8H9+], 44 (11)
[CO2+]; HRMS (EI): m/z: calcd for C29H30N2O6: 502.2104, correct mass
found; elemental analysis calcd (%) for C29H30N2O6 (502.6): C 69.31, H
6.02, N 5.57; found C 69.08, H 5.88, N 5.38.
Deprotection of N-MeZ protected cyclohexapeptides 19a–c and epi-
19a—General procedure (GP 6): The N-MeZ-protected cyclopeptides
(18 mmol) were deprotected by treatment with 10% anisole in TFA
(1.1 mL) in the dark at ambient temperature for 2 h. All volatiles were
removed under reduced pressure (0.05 Torr) at 208C. The residues were
triturated with hexane (6ꢄ5 mL) and dried to give the deprotected mate-
rials as trifluoroacetates, which were directly used in the next condensa-
tion step.
Methyl
(2S,3R)-3-(benzyloxycarbonyl-N-methylamino)-2-hydroxybuty-
rate: A solution of the O-TBDMS, NMe-Z protected tert-butyl ester of
(S)-isothreonine 8 (0.73 g, 1.67 mmol) in MeCN (38 mL) was treated with
5% aqueous HF (40 mL) at 48C for 10 min. The mixture was allowed to
warm to 208C, and stirring was continued for an additional 4 h. A satu-
rated aqueous solution of NaHCO3 was then carefully added to adjust
the pH value to about 8, and the mixture was extracted with Et2O (2ꢄ
50 mL). The organic fraction was washed with water (5ꢄ20 mL), brine
(2ꢄ10 mL), dried, filtered and concentrated under reduced pressure. The
resultant crude alcohol was dried at 0.02 Torr for 2 h and then deprotect-
ed by treatment with TFA (6 mL). After 1 h, all volatiles were removed
under reduced pressure, the residue was dissolved in toluene (2ꢄ20 mL),
which was distilled off to remove the last traces of TFA to give the crude
Deprotection of O-MOM protected hormaomycin aza-analogues O-
MOM-2a–c and epi-O-MOM-2a—General procedure (GP 7): The re-
spective O-MOM protected hormaomycin analogue (15 mmol) was de-
protected by treatment with MgBr2·Et2O (0.30 mmol) and EtSH
(0.10 mmol) in CH2Cl2 (10 mL) at ambient temperature for 3 h. The mix-
ture was taken up with Et2O (40 mL) and washed with 1n KHSO4 (3ꢄ
10 mL), water (4ꢄ10 mL), brine (2ꢄ5 mL), dried, filtered and concen-
trated under reduced pressure. The residue was recrystallized from
CH2Cl2/pentane to give the respective hormaomycin analogue, which, if
necessary, was further purified by preparative HPLC. The fraction con-
taining the desired product was collected, and its pH value was carefully
adjusted to 6.9 (pH meter) with diluted aqueous ammonia, and then it
was lyophilized. The residue was dissolved in EtOAc (10 mL), the so-
lution was washed with water (3ꢄ5 mL), dried and filtered. Removal of
the solvent under reduced pressure gave the pure hormaomycin aza-ana-
logue.
(2S,3R)-3-(benzyloxycarbonyl-N-methylamino)-2-hydroxybutyric
acid
(0.42 g, max. 94%). It was dried at 0.02 Torr and ambient temperature
for 2 h, then taken up with Et2O (10 mL; some methanol was added to
obtain a homogeneous solution) and the mixture was treated with an
excess of an ethereal solution of diazomethane untill a yellow coloration
of the reaction mixture persisted. The mixture was then concentrated
under reduced pressure, and the residue was purified by column chroma-
tography to give the title compound (0.361 g, 71% over two steps; Rf =
0.22, EtOAc/hexane 1:3) as a turbid oil, which was directly used for the
next step without any further characterization.
Methyl
(2R,3R)-2-amino-3-(9-fluorenylmethyloxycarbonylamino)buty-
rate hydrochloride: Boc-a-Dab(Fmoc)-OtBu 5 (0.39 g, 0.79 mmol) was
deprotected with TFA (5 mL) for 1 h. All volatiles were removed under
reduced pressure at 208C. The solid residue was taken up with 1m HCl
(5 mL) and methanol (20 mL) and after 10 min the mixture was concen-
trated to give the crude H-a-Dab(Fmoc)-OH·HCl (0.31 g, 100%), which
was dried at 0.02 Torr at ambient temperature for 16 h, and used for the
next step without further purification. SOCl2 (0.60 mL, 8.27 mmol) was
added dropwise to a solution of the crude amino acid hydrochloride
(0.31 g, max 0.79 mmol) in anhydrous methanol (35 mL) at ꢀ208C for
5 min and stirring was continued at the same temperature for an addi-
tional 15 min. The mixture was then allowed to warm to 208C, and, after
stirring at this temperature for 1 h, the reaction flask was sealed, and the
mixture was heated t 508C with stirring for an additional 20 h. The reac-
tion mixture was then concentrated under reduced pressure, and the resi-
due was triturated with Et2O to give the crude title compound (0.29 g,
Methyl (2S,3R)-2-azido-3-(benzyloxycarbonyl-N-methylamino)butyrate
(9): Mesyl chloride (0.14 mL, 1.81 mmol) was added dropwise to a so-
lution of the NMe-Z protected (S)-isothreonine methyl ester (0.36 g,
1.28 mmol) and TEA (0.254 mL, 1.81 mmol) in CH2Cl2 (7 mL) at ꢀ308C
for 3 min, and stirring was continued at the same temperature for 1 h.
The reaction mixture was then allowed to warm to 48C and stirred at this
temperature for an additional 1 h. Finally, the cooling bath was removed,
and stirring was continued for an additional 3 h. Saturated aqueous so-
lution of NaHCO3 (3 mL) was then added, and the mixture was taken up
with Et2O (50 mL). After the usual aqueous work-up (GP 2) the organic
layer was dried, filtered and concentrated under reduced pressure to give
the crude mesylate of NMe-Z protected (S)-isothreonine methyl ester
(0.46 g, 100%; Rf =0.11, EtOAc/hexane 1:6) as a colorless oil. NaN3
(0.086 g, 1.32 mmol) was added to a solution of this compound (0.46 g,
1.28 mmol) in DMF (8 mL), and stirring continued at 708C for 15 h. The
mixture was then cooled, concentrated under reduced pressure, and the
residue was taken up with Et2O (50 mL). After the usual aqueous work-
up (GP 2) the organic layer was dried, filtered and concentrated under
1
max. 94%) as a colorless solid. Rf =0.30 (MeOH/CHCl3 1:100); H NMR
(250 MHz, CD3OD): d=1.36 (d, J=7 Hz, 3H, H-4), 3.97 (s, 3H, OMe),
4.16–4.34 (m, 3H, 2-H and 9’’-H, 1’-Ha), 4.40–4.59 (m, 2H, 3-H, 1’-Hb),
7.27–7.50 (m, 4H, Ar-H), 7.54 (d, J=6.8 Hz, 1H, NH), 7.70 (d, J=
7.3 Hz, 2H, Ar-H), 7.84 (d, J=7.3 Hz, 2H, Ar-H).
2936
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2005, 11, 2929 – 2945

Hormaomycin
FULL PAPER
reduced pressure. The resultant crude product was purified by column
chromatography (EtOAc/hexane 1:6, Rf =0.19) to give 9 (0.191 g, 49%
over two steps) as a mobile colorless oil, which was directly used for the
next step without any further characterization.
137.0 (Cquat, Ar-C), 155.7 (Cquat, NCO2), 170.7 (Cquat, C-1), 173.0 (Cquat, C-
4); IR (KBr): n˜ =3419, 3355, 3214, 3099, 3030, 2989, 2973, 2929, 2827,
2741, 2629, 2533, 1721, 1692, 1645, 1586, 1526, 1346, 1237, 1199, 1183,
1154, 1126 cmꢀ1; MS (EI, 70 eV), m/z (%): 280 (20) [M +], 263 (3) [M +
ꢀOH], 159 (8) [C5H7N2O4+], 122 (46) [C8H10O+], 105 (100) [C8H9+], 87
(16) [C3H7N2O+], 77 (10) [C6H5+], 44 (6) [CO2+]; elemental analysis
calcd (%) for C13H16N2O5 (280.3): C 55.71, H 5.75, N 9.99; found C 55.97,
H 5.73, N 10.08.
Boc-a-NbDab(Fmoc)-OMe: Ph3P (0.262 g, 1.00 mmol) was added to a so-
lution of 9 (0.191 g, 0.62 mmol) in THF/H2O (20:1) (15.8 mL), the resul-
tant mixture was stirred for 24 h. Boc2O (0.272 g, 1.25 mmol) was then
added, and stirring was continued for an additional 24 h. The mixture
was then concentrated, and the residue was purified by column chroma-
tography (twice, hexane 1:4, Rf =0.22) to give the Nb-methylated Boc-a-
Dab(Z)-OMe (0.135 g, 57%) as a viscous colorless oil. This material
(0.135 g, 0.35 mmol) in EtOAc (7 mL) was hydrogenated at ambient pres-
sure of hydrogen over 10% Pd on charcoal (0.07 g) for 3 h. The mixture
was then filtered and concentrated under reduced pressure to give the
crude Boc-a-NbMeDab-OMe (90 mg, 100%), which was immediately
used for the next step. FmocOPfp (0.159 g, 0.39 mmol) was added to a so-
lution of this material, TMP (43 mg, 0.35 mmol), and HOAt (10 mg,
74 mmol) in EtOAc (5 mL) were added and stirring was continued for
15 h. The mixture was then diluted with Et2O (50 mL) and subjected to
the usual aqueous work-up (GP 2). The organic layer was dried, filtered
and concentrated under reduced pressure. The residue was purified by
column chromatography (EtOAc/hexane 1:4, Rf =0.22) to give the Nb-
methylated Boc-a-Dab(Fmoc)-OMe (0.135 g, 81% over two steps) as a
colorless oil, which was directly used for the next step without any fur-
ther characterization.
MeZ-Dap-OH
(13):
Iodobenzene
bis(trifluoroacetate)
(1.46 g,
3.40 mmol) and 12 were suspended by stirring in 50% (v/v) aqueous
DMF (20 mL). After 15 min, pyridine (0.367 g, 4.64 mmol) was added,
and the mixture was stirred for an additional 5 h. The emulsion formed
was evaporated at 40–458C under reduced pressure. The residue was
taken up with water (2ꢄ15 mL), which was evaporated under reduced
pressure. The residual oil was taken up in water (50 mL) and washed
with chloroform (3ꢄ10 mL). The aqueous layer was once more concen-
trated in vacuo, and the residue was dissolved in ethanol (20 mL). The
pH value was adjusted to about 7 with pyridine, and the formed suspen-
sion was left at 48C for 12 h. The precipitate was filtered off and washed
with ether (5ꢄ20 mL) to give, after drying, amino acid 13 (0.51 g, 87%)
as a colorless powder. Rf =0.32 (MeCN/AcOH/H2O 10:1:1); m.p. 210–
2168C (decomp.); [a]2D0 =38.1 (c=0.31, 0.1n HCl); 1H NMR (300 MHz,
DCl in D2O): d=2.28 (s, 3H, 1’-H), 3.28 (dd, J=12.6, 9.6 Hz, 1H, 3-Ha),
3.49 (dd, J=12.6, 4.5 Hz, 1H, 3-Hb), 4.44–4.55 (m, 1H, 2-H), 5.07 (s, 2H,
Bzl-H), 7.22 (d, J=7.5 Hz, 2H, Ar-H), 7.28 (d, J=7.5 Hz, 2H, Ar-H); IR
(KBr): n˜ =3303, 3250–2300, 1695, 1658, 1623, 1592, 1540, 1413, 1273,
1022 cmꢀ1; MS (ESI pos.): m/z: 275 (86) [M+Na+], 253 (12) [M+H+];
neg.: m/z: 251 (10) [MꢀHꢀ]; elemental analysis calcd (%) for
C12H16N2O4 (252.3): C 57.13, H 6.39, N 11.10; found C 56.95, H 6.20, N
10.97.
MeZ-a-NbDab(Fmoc)-OMe (10): Boc-a-NbDab(Fmoc)-OMe (0.135 g,
0.29 mmol) was deprotected with 2m HCl in EtOAc (4 mL) for 3 h. The
mixture was then concentrated under reduced pressure, and the residue
was dissolved in MeCN (4 mL). TMP (45 mg, 0.37 mmol), DIEA (37 mg,
0.29 mmol) and finally MeZOSu (83 mg, 0.32 mmol) were added to this
solution, and it was stirred for 16 h. N,N-Dimethylaminopropylamine
(20 mg, 0.20 mmol) was then added, and after 10 min the mixture was
concentrated under reduced pressure. The residue was taken up with
Et2O and subjected to the usual aqueous work-up (GP 2). The organic
layer was dried, filtered and concentrated under reduced pressure. The
resultant crude product was purified by column chromatography
(EtOAc/hexane 1:3, Rf =0.30) to give 10 (0.122 g, 13% overall yield over
10 steps from 8) as a turbid glass. [a]2D0 =9.2 (c=0.25, CHCl3); 1H NMR
(300 MHz, CDCl3): d=1.16, 1.25 (2 ꢄ d, J=6.9 Hz, 3H, 4-H), 2.33 (s,
3H, 1’-H, MeZ), 2.81 (s, 3H, NMe), 3.66 (s, 3H, OMe), 4.12–4.61 (m,
5H, 2-H, 3-H and 9’-H, 1-H, Fmoc), 5.06 (d, J=5.1 Hz, 2H, Bzl-H), 5.31,
5.60 (2ꢄd, J=7.5 Hz, 1H, NH), 7.15 (d, J=7.8 Hz, 2H, 2-H, MeZ), 7.24
(d, J=7.8 Hz, 2H, 3-H, MeZ), 7.31 (dd, J=7.5, 7.5 Hz, 2H, 3’-H, Fmoc),
7.40 (dd, J=7.5, 7.5 Hz, 2H, 4’-H, Fmoc), 7.58–7.64 (m, 2H, 2’-H, Fmoc),
7.76 (d, J=7.2 Hz, 5’-H, Fmoc); 13C NMR (50.3 MHz, CDCl3): d=14.3
(+, C-4), 21.0 (+, C-1’, MeZ), 28.9 (+, NMe), 47.1 (+, C-3), 52.4 (+, C-
2), 52.9 (+, C-9’, Fmoc), 56.6 (+, OMe), 67.0 (ꢀ, Bzl-H, MeZ), 67.5 (ꢀ,
MeZ-Dap-OMe·HCl (7c·HCl): To
a solution of thionyl chloride
(0.52 mL, 7.26 mmol) in anhydrous MeOH (10 mL) at ꢀ208C was added
with stirring after 10 min the amino acid 13 (0.50 g, 1.98 mmol). The re-
sulting thick suspension was stirred at 208C for 24 h to give a clear so-
lution, which was then left at ꢀ288C for 16 h. Et2O (40 mL) was added
to complete the precipitation, and the solid was filtered off to give
7c·HCl (0.47 g, 78%) as long colorless needles. The mother liquor was
concentrated, and the residue was recrystallized from MeOH/Et2O to
give a second crop of 7c·HCl (26 mg, 83% overall yield). M.p. 159–
1618C; [a]2D0 =32.3 (c=0.86, DMSO); 1H NMR (250 MHz, [D6]DMSO):
d=2.28 (s, 3H, 1’-H), 2.98–3.29 (m, 2H, 3-H), 3.66 (s, 3H, OMe), 4.43
(ddddd, J=4.3 Hz, 1H, 2-H), 5.08 (s, 2H, Bzl-H), 7.17 (d, J=7.9 Hz, 2H,
Ar-H), 7.25 (d, J=7.9 Hz, 2H, Ar-H), 7.52 (d, J=8.3 Hz,1H, CONH),
8.15–8.55 (br, 3H, NH2·HCl); 13C NMR (125.7 MHz, [D6]DMSO): d=
21.0 (+, C-1’), 39.2 (ꢀ, C-3), 52.0 (+, C-2), 52.8 (+, OMe), 66.0 (ꢀ, Bzl-
H), 128.2 (+, Ar-C), 129.1 (+, Ar-C), 133.8 (Cquat, Ar-C), 137.4 (Cquat
,
Ar-C), 156.3 (Cquat, NCO2), 173.6 (Cquat, C-1); IR (KBr): n˜ =3322, 3031,
2884, 2621, 1734, 1690, 1597, 1535, 1307, 1264, 1230, 1015 cmꢀ1; MS (ESI
pos.): m/z: 289 (38) [M+Na+], 267 (93) [M+H+]; elemental analysis
calcd (%) for C13H19N2O4Cl (302.8): C 51.57, H 6.33, N 9.25; found C
51.29, H 6.48, N 9.11.
C-1, Fmoc), 119.8, 124.9, 126.9, 127.5, 128.2, 129.1 (+, Ar-C), 133.0 (Cquat
,
Ar-C), 137.9 (Cquat, Ar-C), 141.2 (Cquat, Ar-C), 143.8, 143.9 (Cquat, Ar-C),
155.9, 156.4 (Cquat, NCO2), 170.9 (Cquat, C-1); IR (KBr): n˜ =2951, 1751,
1725, 1700, 1521, 1451, 1320, 1273, 1242, 1204, 1018 cmꢀ1; MS (ESI pos.):
m/z: 539 (100) [M+Na+].
MeZ-a-Dab[Boc-(4-Pe)Pro]-OMe: Compound 7a (0.191 g, 0.38 mmol)
was deprotected according to GP 1, and the resulting crude Na-protected
diamino ester was coupled with the N-Boc protected 4-(Z)-propenylpro-
line 14 (0.100 g, 0.39 mmol) by treatment with EDC (77 mg, 0.40 mmol),
HOAt (55 mg, 0.41 mmol) and TMP (0.142 g, 1.17 mmol) in CH2Cl2
(4 mL) according to GP 2 for 16 h. The crude product obtained after the
usual aqueous work-up (GP 2) was finally purified by column chromatog-
raphy (EtOAc/hexane 1:1.5, Rf =0.35) to give the title compound
(0.163 g, 83%) as a turbid oil, which solidified during drying at 608C
(0.02 Torr) to a colorless solid. M.p. 94–958C; [a]2D0 =ꢀ41.6 (c=0.32,
MeZ-(R)-Asn-OH (12): NaHCO3 (0.520 g, 6.18 mmol) and then a so-
lution of MeZOSu (0.775 g, 2.97 mmol) in acetone (7 mL) were added to
a vigorously stirred solution of d-aspargine (0.442 g, 2.94 mmol) in water
(10 mL), and stirring was continued for 3 h (if a precipitate formed, ace-
tone and/or water was added to obtain a homogeneous solution). The
mixture was then concentrated under reduced pressure, diluted with
water (40 mL) and washed with CH2Cl2 (3ꢄ10 mL). The pH of the water
fraction was adjusted to 1–2 with solid KHSO4, the resulting precipitate
was filtered off, washed with H2O (5ꢄ20 mL), Et2O (5ꢄ20 mL) and
dried to give 12 (0.75 g, 91%) as a colorless solid. M.p. 181–1838C;
[a]2D0 =6.5 (c=1.00, DMF); 1H NMR (250 MHz, [D6]acetone): d=2.30 (s,
3H, 1’-H), 2.50–3.55 (br, 3H, CO2H, CONH2), 2.65–2.85 (m, 2H, 3-H),
4.39–4.53 (m, 1H, 2-H), 5.03 (s, 2H, Bzl-H), 6.39–6.61 (br, 1H, NH), 7.15
(d, J=8.0 Hz, 2H, Ar-H), 7.26 (d, J=8.0 Hz, 2H, Ar-H); 13C NMR
(125.7 MHz, [D6]DMSO): d=20.7 (+, C-1’), 36.7 (ꢀ, C-3), 50.5 (+, C-2),
65.3 (ꢀ, Bzl-H), 127.8 (+, Ar-C), 128.8 (+, Ar-C), 133.8 (Cquat, Ar-C),
1
CHCl3); H NMR (250 MHz, CDCl3): d=1.04–1.19 (m, 3H, 4-H, a-Dab),
1.41 [s, 9H, C(CH3)3], 1.64 [dd, J=7.0, 1.5 Hz, 3H, 3’-H, (4-Pe)Pro],
1.72–2.00 [m, 1H, 3-Ha, (4-Pe)Pro], 2.34 (s, 3H, 1’-H, MeZ), 2.34–2.54
[m, 1H, 3-Hb, (4-Pe)Pro], 2.92–3.15 [m, 2H, 4-H, 5-Ha, (4-Pe)Pro], 3.76
(s, 3H, OMe), 3.80–3.96 [m, 1H, 5-Hb, (4-Pe)Pro], 4.03–4.22 (m, 1H, 3-
H, a-Dab), 4.35–4.57 (m, 2H, 2-H), 5.01 (d, J=12.3 Hz, 1H, Bzl-Ha), 5.09
(d, J=12.3 Hz, 1H, Bzl-Hb), 5.20–5.37 [m, 1H, 1’-H, (4-Pe)Pro], 5.54 [dq,
Chem. Eur. J. 2005, 11, 2929 – 2945
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2937

C. Griesinger, A. Zeeck, A. de Meijere et al.
J=10.0, 7.0 Hz, 1H, 2’-H, (4-Pe)Pro], 5.59–5.77, 6.20–6.40 (2ꢄm, 1H,
NH), 6.81 (d, J=8.8 Hz, 1H, NH), 7.15 (d, J=7.8 Hz, 2H, Ar-H), 7.25
(d, J=7.8 Hz, 2H, Ar-H); 13C NMR (62.9 MHz, CDCl3): d=13.0 [+, C-
3’, (4-Pe)Pro], 15.3, 16.8 (+, C-4, a-Dab), 21.0 (+, C-1’, MeZ), 28.1 [+,
C(CH3)3], 35.8 [+, C-4, (4-Pe)Pro], 37.8 [ꢀ, C-3, (4-Pe)Pro], 46.6, 47.1
(+, C-3, a-Dab), 52.0 [ꢀ, C-5, (4-Pe)Pro], 52.4 (+, OMe), 57.5 (+, C-2,
a-Dab), 60.9, 61.5 [+, C-2, (4-Pe)Pro], 66.8, 67.2 (ꢀ, Bzl-H, MeZ), 80.2
[Cquat, C(CH3)3], 126.4 [+, C-2’, (4-Pe)Pro], 128.2, 129.0 (+, Ar-C), 129.4
[M+Na+]; elemental analysis calcd (%) for C28H41N3O7 (531.7): C 63.26,
H 7.77, N 7.90; found C 62.95, H 7.70, N 7.70.
MeZ-NbMe-a-Dab[Boc-(4-Pe)Pro]-OH (15b): A solution of the dipep-
tide ester MeZ-NbMe-a-Dab[Boc-(4-Pe)Pro]-OMe (0.128 g, 0.24 mmol)
in THF (2.0 mL) was hydrolyzed according to GP 3 by treatment with
40% aqueous solution of tetra-n-butylammonium hydroxide (0.24 g,
0.36 mmol). The crude product obtained after the usual aqueous work-up
(GP 3) was recrystallized three times from hexane and once from Et2O/
hexane to give 15b (91 mg, 73%) as an extremely viscous turbid oil (Rf =
0.14, acetone/hexane 2:5). [a]2D0 =8.9 (c=0.37, CHCl3); MS (ESI): pos.:
m/z (%): 562 (100) [MꢀH++2Na+], 540 (8) [M+Na+]; neg.: m/z (%):
516 (100) [MꢀHꢀ].
[+, C-1’, (4-Pe)Pro], 132.7, 137.8 (Cquat, Ar-C), 154.2, 156.5 (Cquat,
NCO2), 170.2, 171.0 (Cquat, C-1), 172.0, 172.3 (Cquat, C-1); IR (KBr): n˜ =
3012, 2978, 2929, 2869, 1728, 1703, 1678, 1541, 1519, 1394, 1368, 1259,
1212, 1162 cmꢀ1; MS (EI, 70 eV): m/z (%): 517 (1) [M +], 444 (3)[M +
ꢀC4H9O], 416 (6) [M +ꢀC5H9O2], 281 (52) [C15H24N2O3+], 238 (15)
[C13H20N2O3+], 225 (32) [C11H16N2O3+], 182 (11), 154 (100) [C8H12NO2+],
110 (88) [C7H12N+], 105 (70) [C8H9+], 57 (49) [C4H9+], 44 (68) [CO2+];
HRMS (EI): m/z: calcd for C27H39N3O7: 517.2788, correct mass found;
elemental analysis calcd (%) for C27H39N3O7 (517.6): C 62.65, H 7.59, N
8.12; found C 62.48, H 7.35, N 7.90.
MeZ-Dap[Boc-(4-Pe)Pro]-OMe: Compound 7c (0.127 g, 0.42 mmol) was
coupled with the N-Boc protected (4-propenyl)proline 14 (0.11 g,
0.431 mmol) by treatment with EDC (85 mg, 0.44 mmol), HOAt (60 mg,
0.44 mmol) and TMP (0.314 g, 2.59 mmol) in CH2Cl2 (5 mL) according to
GP 2 for 16 h. The crude product obtained after the usual aqueous work-
up (GP 2) was further purified by column chromatography (acetone/
hexane 1:2.5, Rf =0.13) to give an oily residue which was triturated with
pentane to furnish the title compound (0.14 g, 66%) as a colorless solid.
The mother liquor was cooled to 48C, and the precipitate was filtered off
to give a second crop of the title compound (10 mg, 71% overall yield).
M.p. 160–1628C; [a]2D0 =ꢀ41.4 (c=0.35, CHCl3); 1H NMR (300 MHz,
CDCl3): d=1.40 [s, 9H, C(CH3)3], 1.64 [dd, J=6.9, 1.8 Hz, 3H, 3’-H, (4-
Pe)Pro], 1.78–2.04 [m, 1H, 3-Ha, (4-Pe)Pro], 2.11–2.57 [m, 1H, 3-Hb, (4-
Pe)Pro], 2.34 (s, 3H, 1’-H, MeZ), 2.97–3.15 [m, 1H, 4-H, (4-Pe)Pro], 2.99
[dd, J=9.3 Hz, 5-Ha, (4-Pe)Pro], 3.51–3.92 [m, 3H, 3-H, Dap, 5-Hb, (4-
Pe)Pro], 3.75 (s, 3H, OMe), 4.12 (dd, J=8.1 Hz, 1H, 2-H, Dap), 4.34–
4.51 [m, 1H, 2-H, (4-Pe)Pro], 5.02 (d, J=12.3 Hz, 1H, Bzl-Ha), 5.08 (d,
J=12.3 Hz, 1H, Bzl-Hb), 5.17–5.30 [m, 1H, 1’-H, (4-Pe)Pro], 5.52 [dq,
J=10.5, 6.9 Hz, 1H, 2’-H, (4-Pe)Pro], 5.74–6.17 (br, 1H, NH), 6.43–6.85
(br, 1H, NH), 7.14 (d, J=8.1 Hz, 2H, Ar-H), 7.33 (d, J=8.1 Hz, 2H, Ar-
H); 13C NMR (75.5 MHz, CDCl3): d=13.2 [+, C-3’, (4-Pe)Pro], 21.2 (+,
C-1’, MeZ), 28.3 [+, C(CH3)3], 36.0 [+, C-4, (4-Pe)Pro], 38.1 [ꢀ, C-3,
(4-Pe)Pro], 40.8, 41.5 (ꢀ, C-3, Dap), 52.4 [ꢀ, C-5, (4-Pe)Pro], 52.7 (+,
OMe), 54.3 (+, C-2, Dap), 60.8, 61.4 [+, C-2, (4-Pe)Pro], 67.0 (ꢀ, Bzl-
H, MeZ), 80.7 [Cquat, C(CH3)3], 126.5 [+, C-2’, (4-Pe)Pro], 128.3 (+, Ar-
C), 129.1 (+, Ar-C), 129.4 [+, C-1’, (4-Pe)Pro], 133.2 (Cquat, Ar-C), 137.9
(Cquat, Ar-C), 154.4, 155.1 (Cquat, NCO2), 156.3 (Cquat, NCO2), 170.2, 171.0
(Cquat, C-1), 170.9, 173.0 (Cquat, C-1); IR (KBr): n˜ =3013, 2977, 2953, 2876,
1747, 1728, 1521, 1367, 1259, 1209, 1162, 1118 cmꢀ1; MS (EI, 70 eV): m/z
(%): 503 (4) [M +], 447 (2) [M +ꢀC4H8], 402 (11) [M +ꢀC5H9O2], 210
(15) [C10H14N2O3+], 154 (100) [C8H12NO2+], 110 (84) [C7H12N+], 105
(56) [C8H9+], 57 (38) [C4H9+], 41 (5) [C3H5+]; elemental analysis calcd
(%) for C26H37N3O7 (503.6): C 62.01, H 7.41, N 8.34; found C 62.09, H
7.20, N 8.10.
MeZ-a-Dab[Boc-(4-Pe)Pro]-OH (15a): A solution of the dipeptide ester
MeZ-a-Dab[Boc-(4-Pe)Pro]-OMe (0.145 g, 0.28 mmol) in THF (1.8 mL)
was hydrolyzed according to GP 3 by treatment with 40% aqueous so-
lution of tetra-n-butylammonium hydroxide (0.545 g, 0.84 mmol). The
crude product obtained after the usual aqueous work-up (GP 3) was re-
crystallized from Et2O/hexane to give acid 15a (0.108 g, 76%) as a color-
less solid. The mother liquor was concentrated under reduced pressure,
and the residue was recrystallized twice from Et2O/hexane to give a
second crop of 15a (0.012 g, 85% overall yield). Rf =0.06 (EtOAc/hexane
1:2, 1.5% AcOH); 1H NMR (250 MHz, CDCl3): d=1.13–1.47 (m, 3H, 4-
H, a-Dab), 1.35, 1.39 [2ꢄs, 9H, C(CH3)3], 1.64 [d, J=5.8 Hz, 3H, 3’-H,
(4-Pe)Pro], 1.73–2.00 [m, 1H, 3-Ha, (4-Pe)Pro], 2.33 (s, 3H, 1’-H, MeZ),
2.33–2.57 [m, 1H, 3-Hb, (4-Pe)Pro], 2.87–3.20 [m, 2H, 4-H, 5-Ha, (4-
Pe)Pro], 3.48–3.73, 3.73–3.95 [2ꢄm, 1H, 5-Hb, (4-Pe)Pro], 4.07–4.29 (m,
1H, 3-H, a-Dab), 4.41 (d, J=6.5 Hz, 1H, 2-H), 4.43–4.69 (m, 1H, 2-H),
5.03 (s, 2H, Bzl-H), 5.18–5.33 [m, 1H, 1’-H, (4-Pe)Pro], 5.53 [dq, J=10.8,
7.0 Hz, 1H, 2’-H, (4-Pe)Pro], 5.83–6.02, 6.31–6.48 (2ꢄm, 1H, NH), 6.81
(d, J=8.8 Hz, 1H, NH), 7.14 (d, J=8.0 Hz, 2H, Ar-H), 7.21–7.38 (br,
1H, CO2H), 7.23 (d, J=8.0 Hz, 2H, Ar-H).
MeZ-NbMe-a-Dab[Boc-(4-Pe)Pro]-OMe: Compound 7b (0.108 g,
0.21 mmol) was deprotected according to GP 1, and the resultant crude
monodeprotected diamino ester was coupled with the N-Boc protected 4-
(Z)-propenylproline 14 (64 mg, 0.25 mmol) by using EDC (48 mg,
0.25 mmol), HOAt (34 mg, 0.25 mmol) and TMP (76 mg, 0.63 mmol) in
CH2Cl2 (3 mL) according to GP 2 for 16 h. The crude product, obtained
after the usual aqueous work-up (GP 2), was finally purified by column
chromatography (EtOAc/hexane 1:1.5, Rf =0.35) to give the title com-
pound (0.104 mg, 93%) as a turbid oil. Analytical HPLC: gradient 20 !
90% MeCN in water (0.1% TFA) for 35 min, flow rate=0.5 mLminꢀ1
tR =26.01 min, purity
,
MeZ-Dap[Boc-(4-Pe)Pro]-OH (15c): A solution of the dipeptide ester
MeZ-Dap[Boc-(4-Pe)Pro]-OMe (0.13 g, 0.26 mmol) in THF (2.0 mL) was
hydrolyzed according to GP 3 by treatment with 40% aqueous solution
of tetra-n-butylammonium hydroxide (0.20 g, 0.31 mmol). The crude
product obtained after the usual aqueous work-up (GP 3) was finally pu-
rified by column chromatography [acetone/hexane 4:7 (2% AcOH), Rf =
0.36] to give acid 15c (0.126 g, 99%) as an extremely viscous turbid oil.
1H NMR (250 MHz, CDCl3): d=1.31, 1.41 [2ꢄs, 9H, C(CH3)3], 1.65 [d,
J=6.0 Hz, 3H, 3’-H, (4-Pe)Pro], 1.75–1.98 [m, 1H, 3-Ha, (4-Pe)Pro], 2.33
(s, 3H, 1’-H, MeZ), 2.21–2.53 [m, 1H, 3-Hb, (4-Pe)Pro], 2.93–3.21 [m,
2H, 4-H, 5-Ha, (4-Pe)Pro], 3.44–3.60 (m, 2H, 3-H, Dap), 3.60–4.03 [m,
1H, 5-Hb, (4-Pe)Pro], 4.03–4.19 [m, 1H, 2-H, (4-Pe)Pro], 4.21 (dd, J=
7.5 Hz, 1H, 2-H, Dap), 4.30–4.39, 4.41–4.54 (2ꢄbr, 1H, NH), 5.04 (s, 2H,
Bzl-H), 5.15–5.32 [m, 1H, 1’-H, (4-Pe)Pro], 5.55 [dq, J=10.8, 7.0 Hz, 1H,
2’-H, (4-Pe)Pro], 6.25 (d, J=6.5 Hz, 1H, NH), 7.12 (d, J=7.5 Hz, 2H,
Ar-H), 7.22 (d, J=7.5 Hz, 2H, Ar-H), 7.42–7.65 (br, 1H, CO2H); MS
(ESI): pos.: m/z (%): 534 (100) [MꢀH+2Na+], 512 (45) [M+Na+]; neg.:
m/z (%): 488 (100) [MꢀHꢀ].
>
97%; [a]2D0 =12.0 (c=0.35, CHCl3); 1H NMR
(300 MHz, C2D2Cl4, 373 K): d=1.31 (d, J=7.2 Hz, 3H, 4-H, NMe-a-
Dab), 1.42 [s, 9H, C(CH3)3], 1.50–1.74 [m, 1H, 3-Ha, (4-Pe)Pro], 1.67
[dd, J=7.2, 1.8 Hz, 3H, 3’-H, (4-Pe)Pro], 2.26–2.44 [m, 1H, 3-Hb, (4-
Pe)Pro], 2.36 (s, 3H, 1’-H, MeZ), 2.90 (s, 3H, NMe), 3.01–3.21 [m, 2H, 4-
H, 5-Ha, (4-Pe)Pro], 3.23–3.48 [m, 0.5H, 5-Hb, (4-Pe)Pro], 3.74 (s, 3H,
OMe), 4.47 (dd, J=7.8, 7.8 Hz, 1H, 3-H, NMe-a-Dab), 4.57 (dd, J=7.8,
7.8 Hz, 1H, 2-H, NMe-a-Dab), 4.50–4.95 [m, 1H, C-2, (4-Pe)Pro], 5.08 (s,
2H, Bzl-H), 5.25–5.36 [m, 1H, 1’-H, (4-Pe)Pro], 5.50–5.85 (br, 1H, NH),
5.54 [dq, J=11.4, 7.2 Hz, 1H, 2’-H, (4-Pe)Pro], 7.15 (d, J=7.8 Hz, 2H,
Ar-H), 7.23 (d, J=7.8 Hz, 2H, Ar-H); the signal of OMe overlapped
with the signal of 0.5H, 5-Hb of the (4-Pe)Pro moiety; 13C NMR
(75.5 MHz, C2D2Cl4, 373 K): d=12.7 [+, C-3’, (4-Pe)Pro], 14.5 (+, C-4,
NMe-a-Dab), 20.7 (+, C-1’, MeZ), 28.2 (+, C(CH3)3, NMe), 36.0 [+, C-
4, (4-Pe)Pro], 51.9 [ꢀ, C-5, (4-Pe)Pro], 52.1 (+, C-2, NMe-a-Dab), 57.2
(+, OMe), 66.7 (ꢀ, Bzl-H, MeZ), 79.3 [Cquat, C(CH3)3], 125.8 [+, C-2’,
(4-Pe)Pro], 127.7, 128.8 (+, Ar-C), 130.0 [+, C-1’, (4-Pe)Pro], 133.3,
137.5 (Cquat, Ar-C), 153.5, 155.6 (Cquat, NCO2), 170.6, 173.2 (Cquat, C-1);
the signals of C-2, C-3 of (4-Pe)Pro and C-3 of NMe-a-Dab were unob-
servable because of their low intensity; IR (KBr): n˜ =2977, 1751, 1728,
1700, 1521, 1402, 1281, 1163 cmꢀ1; MS (ESI): pos.: m/z (%): 554 (100)
MeZ-a-Dab[Boc-(4-Pe)Pro]-(bMe)Phe-(R)-(3-Ncp)Ala-(bMe)Phe-Ile-
ODCPM (18a): The tetrapeptide 16 (0.172 g, 0.19 mmol), after removal
of the Fmoc group according to GP 1, was coupled with the dipeptide
acid 15a (0.104 g, 0.21 mmol) by treatment with HATU (79 mg,
2938
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 2929 – 2945

Hormaomycin
FULL PAPER
0.21 mmol), HOAt (30 mg, 0.22 mmol) and TMP (75 mg, 0.62 mmol) in
CH2Cl2 (5 mL) according to GP 4 for 15 h. The mixture was then diluted
with Et2O (50 mL), and subjected to the usual aqueous work-up (GP 2).
The organic layer was dried, filtered and concentrated under reduced
pressure. The residue was recrystallized from hexane, then purified by
column chromatography (EtOAc/hexane 4:3, Rf =0.34) and finally recrys-
tallized from hexane again to give the branched hexapeptide 18a
[s, 9H, Si(CH)3], 0.74 (d, J=6.6 Hz, 3H, 1’-H, Ile), 0.90 (t, J=7.2 Hz,
3H, 5-H, Ile), 1.04 [ddd, J=6.6, 6.6, 6.6 Hz, 1H, 3’-Ha, (3-Ncp)Ala], 1.04
(dd, J=8.4, 8.4 Hz, 2H, TMSE), 1.09–1.23 [m, 2H, 1’-H, (3-Ncp)Ala, 4-
Ha, Ile], 1.26 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe], 1.35 [s, 9H, C(CH3)3],
1.40–1.46 [m, 2H, 3-Hb, 3’-Hb, (3-Ncp)Ala], 1.49 (d, J=7.2 Hz, 3H, 4-H,
a-NbMe-Dab), 1.58 [ddd, J=11.4, 11.4, 11.4 Hz, 1H, 3-Ha, (4-Pe)Pro],
1.68 [dd, J=6.6, 1.2 Hz, 3’-H, (4-Pe)Pro], 1.84–1.93 (m, 1H, 3-H, Ile),
2.28 [ddd, J=11.4, 6.6, 6.6 Hz, 3-Hb, (4-Pe)Pro], 2.32 (s, 3H, 1’-H, MeZ),
2.93 (s, 3H, NMe, a-NbMe-Dab), 3.15 [dd, J=10.5, 10.5 Hz, 1H, 5-Ha, (4-
Pe)Pro], 3.18–3.28 [m, 1H, 4-H, (4-Pe)Pro], 3.31–3.40 [m, 2H, 2ꢄH-3,
(bMe)Phe] 3.71 [dd, J=10.5, 7.8 Hz, 1H, 5-Hb, (4-Pe)Pro], 3.87 [ddd, J=
6.6, 3.0, 3.0 Hz, 1H, 2’-H, (3-Ncp)Ala], 4.12 [dd, J=11.4, 6.6 Hz, 1H, 2-
H, (4-Pe)Pro], 4.19 (d, J=4.8 Hz, 1H, 2-H, a-NbMe-Dab), 4.28 (dd, J=
8.4, 8.4, 1-H, TMSE), 4.37 [dd, J=9.0, 4.5 Hz, 1H, 2-H, (bMe)Phe], 4.53
[ddd, J=9.6, 4.8, 4.8 Hz, 1H, 2-H, (3-Ncp)Ala], 4.65 (dd, J=10.5,
10.5 Hz, 1H, 2-H, Ile), 4.75 [dd, J=9.9, 7.5 Hz, 1H, 2-H, (bMe)Phe], 4.89
(d, J=12.0 Hz, Bzl-Ha), 5.09 (d, J=12.0 Hz, Bzl-Hb), 5.12–5.19 (m, 1H,
3-H, a-NbMe-Dab), 5.22–5.28 [m, 1H, 1’-H, (4-Pe)Pro], 5.55 [dq, J=11.7,
6.6 Hz, 1H, 2’-H, (4-Pe)Pro], 6.92 (d, J=9.0 Hz, 1H, NH), 7.10 (d, J=
8.4 Hz, 2H, Ar-H, MeZ), 7.14–7.28 (m, 11H, Ar-H, NH), 7.22 (d, J=
8.4 Hz, 2H, Ar-H, MeZ), 7.29 (d, J=6.0 Hz, 1H, NH), 7.61 (d, J=9.6 Hz,
1H, NH), 8.02–8.10 (br, 1H, NH); the signal of 4-H of the (bMe)Phe
(1.30 ppm) residue overlapped the signal of 3-Ha of the (3-Ncp)Ala
moiety, and the signal of C(CH3) overlapped the signal of 4-Hb of the Ile
moiety; 13C NMR (150.8 MHz, CDCl3): d=ꢀ1.5 [+, Si(CH)3], 11.7 (+,
C-5, Ile), 13.2 [+, C-3’, (4-Pe)Pro], 15.9 (+, C-1’, Ile), 16.3 (ꢀ, C-2,
TMSE), 18.3 [ꢀ, C-3’, (3-Ncp)Ala], 18.9 [+, C-4, (bMe)Phe], 19.6 [+, C-
4, (bMe)Phe], 21.1 (+, C-1’, MeZ), 21.8 [+, C-1’, (3-Ncp)Ala], 25.2 (ꢀ,
C-4, Ile), 28.3 [+, C(CH3)3], 30.7 (+, NMe, a-NbMe-Dab), 31.3 [ꢀ, C-3,
(3-Ncp)Ala], 35.4 [ꢀ, C-3, (4-Pe)Pro], 36.3 [+, C-4, (4-Pe)Pro], 37.3 (+,
C-3, Ile), 40.0 [+, C-3, (bMe)Phe], 41.9 [+, C-3, (bMe)Phe], 50.3 (+, C-
3, a-NbMe-Dab), 50.5 [+, C-2, (3-Ncp)Ala], 52.3 [ꢀ, C-5, (4-Pe)Pro],
56.2 [+, C-2, (bMe)Phe], 57.5 [+, C-2, (bMe)Phe], 59.6 [+, C-2’, (3-
Ncp)Ala], 61.2 (+, C-2, Ile), 62.6 [+, C-2, (4-Pe)Pro], 63.6 (+, C-2, a-
NbMe-Dab), 63.9 (ꢀ, C-1, TMSE), 66.5 (ꢀ, Bzl-C), 79.8 [Cquat, C(CH3)3],
126.8 [+, C-2’, (4-Pe)Pro], 127.05, 127.08, 127.6, 127.9, 128.5, 128.6,
128.7, 128.9, (+, Ar-C), 129.2 [+, C-1’, (4-Pe)Pro], 133.4, 137.7, 141.2,
142.3 (Cquat, Ar-C), 154.1, 155.9 (Cquat, NCO2), 170.2, 170.73, 170.79,
173.0, 174.0, 175.5, (Cquat, C-1); IR (KBr): n˜ =3059, 2970, 2879, 1660,
1638, 1543, 1400, 1367, 1164 cmꢀ1; MS (ESI): pos.: m/z (%): 1232 (100)
[M+Na+]; neg.: m/z (%): 1207 (20) [MꢀHꢀ]; HRMS (ESI): m/z: calcd
for [C64H82N8O13SiNa+]: 1231.6445; found 1231.6444.
(0.176 g, 80%) as a colorless solid. M.p. 101–1038C (decomp.), [a]D20
=
1
52.8 (c=0.29, THF); H NMR (600 MHz, CDCl3): d=0.34 (dddd, J=4.8,
4.8, 4.8, 4.8 Hz, 1H, 2’-H, DCPM), 0.40 (dddd, J=4.8, 4.8, 4.8, 4.8 Hz,
1H, 2’-H, DCPM), 0.43 (dddd, J=4.8, 4.8, 4.8, 4.8 Hz, 1H, 2’-H, DCPM),
0.45–0.54 (m, 2H, 2’-H, DCPM), 0.54–0.59 (m, 2H, 2’-H, DCPM), 0.66
(ddddd, J=4.2, 4.2, 4.2, 4.2, 4.2 Hz, 1H, 2’-H, DCPM), 0.79 (d, J=
6.6 Hz, 3H, 1’-H, Ile), 0.84–0.92 [m, 1H, 1’-H, (3-Ncp)Ala] 0.90 (t, J=
7.2 Hz, 3H, 5-H, Ile), 1.04 [ddd, J=6.0, 7.2, 7.2 Hz, 1H, 3’-Ha, (3-
Ncp)Ala], 1.06–1.14 (m, 1H, 1’-Ha, DCPM), 1.14–1.23 (m, 1H, 1’-Hb,
DCPM), 1.23 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe], 1.25 [d, J=6.6 Hz, 3H,
4-H, (bMe)Phe], 1.28–1.39 [m, 2H, 3-H, (3-Ncp)Ala], 1.33 (d, J=7.2 Hz,
3H, 4-H, a-Dab), 1.34 [s, 9H, C(CH3)3], 1.45–1.53 [m, 1H, 3’-Hb, (3-
Ncp)Ala], 1.69 [dd, J=6.6, 1.2 Hz, 3’-H, (4-Pe)Pro], 1.81 [ddd, J=12.0,
12.0, 12.0 Hz, 1H, 3-Ha, (4-Pe)Pro], 1.85–1.93 (m, 1H, 3-H, Ile), 2.32 (s,
3H, 1’-H, MeZ), 2.36 [ddd, J=12.0, 6.0, 6.0 Hz, 3-Hb, (4-Pe)Pro], 3.12–
3.23 [m, 2H, 3-H, (bMe)Phe, 5-Ha, (4-Pe)Pro], 3.21–3.30 [m, 2H, 3-H,
(bMe)Phe, 4-H, (4-Pe)Pro], 3.67 [dd, J=9.0, 7.8 Hz, 1H, 5-Hb, (4-
Pe)Pro], 3.89 [ddd, J=7.2, 3.0, 3.0 Hz, 1H, 2’-H, (3-Ncp)Ala], 4.14 (t, J=
7.8 Hz, 1H, 1-H, DCPM), 4.22 [dd, J=4.8, 2.4 Hz, 1H, 2-H, (4-Pe)Pro],
4.24 (dd, J=9.6, 6.6 Hz, 1H, 2-H, a-Dab), 4.30 [dd, J=10.8, 6.0 Hz, 1H,
2-H, (bMe)Phe], 4.34 (dd, J=9.3, 4.5 Hz, 1H, 2-H, Ile), 4.60 [ddd, J=
10.5, 5.4, 5.4 Hz, 1H, 2-H, (3-Ncp)Ala], 4.62–4.70 [m, 2H, 2-H,
(bMe)Phe, 3-H, a-Dab], 5.00 (d, J=12.0 Hz, Bzl-Ha), 5.06 (d, J=12.0 Hz,
Bzl-Hb), 5.26–5.33 [m, 1H, 1’-H, (4-Pe)Pro], 5.56 [dq, J=11.1, 6.6 Hz,
1H, 2’-H, (4-Pe)Pro], 6.61 (d, J=6.6 Hz, 1H, NH), 6.97 (d, J=10.2 Hz,
1H, NH), 7.01 (d, J=9.0 Hz, 1H, NH), 7.10 (d, J=8.4 Hz, 2H, Ar-H),
7.16–7.32 (m, 13H, Ar-H, NH), 7.49 (d, J=9.6 Hz, 1H, NH), 7.60 (d, J=
9.6 Hz, 1H, NH); the signal of 4-H of the (bMe)Phe residue (1.23 ppm)
overlapped the signal of 4-Ha of the Ile fragment, and the signal of C-
(CH3) overlapped the signal of 4-Hb of the Ile moiety; 13C NMR
(150.8 MHz, CDCl3): d=2.5, 2.79, 2.83, 3.0 (ꢀ, C-2’, DCPM), 11.6 (+, C-
5, Ile), 13.2 [+, C-3’, (4-Pe)Pro], 14.1, 14.6 (+, C-1’, DCPM), 15.7 (+, C-
1’, Ile), 18.5 [+, C-4, (bMe)Phe], 18.6 [ꢀ, C-3’, (3-Ncp)Ala], 19.7 [+, C-
4, (bMe)Phe], 19.9 (+, C-4, a-Dab), 21.1 (+, C-1’, MeZ), 21.6 [+, C-1’,
(3-Ncp)Ala], 25.2 (ꢀ, C-4, Ile), 28.3 [+, C(CH3)3], 30.8 [ꢀ, C-3, (3-
Ncp)Ala], 36.2 [ꢀ, C-3, (4-Pe)Pro], 36.5 (+, C-3, Ile), 37.1 [+, C-4, (4-
Pe)Pro], 40.2 [+, C-3, (bMe)Phe], 41.9 [+, C-3, (bMe)Phe], 46.3 (+, C-
3, a-Dab), 50.8 [+, C-2, (3-Ncp)Ala], 52.5 [ꢀ, C-5, (4-Pe)Pro], 56.6 (+,
C-2, Ile), 59.5 [+, C-2’, (3-Ncp)Ala], 60.9 (+, C-2, a-Dab), 61.5 [+, C-2,
(bMe)Phe], 63.3 [+, C-2, (4-Pe)Pro], 63.4 [+, C-2, (bMe)Phe], 66.7 (ꢀ,
Bzl-C), 80.2 [Cquat, C(CH3)3], 83.1 (+, C-1, DCPM), 126.7(+, Ar-C),
127.0 [+, C-2’, (4-Pe)Pro], 127.4, 127.6, 127.7, 128.39, 128.43, 128.85,
128.88 (+, Ar-C), 129.2 [+, C-1’, (4-Pe)Pro], 133.5, 137.7, 141.6, 141.7
(Cquat, Ar-C), 154.4, 155.9 (Cquat, NCO2), 169.7, 170.9, 173.56, 173.59,
174.06, 174.11 (Cquat, C-1); IR (KBr): n˜ =3087, 3010, 2973, 2934, 2876,
1730, 1673, 1545, 1513, 1390, 1368, 1162 cmꢀ1; MS (ESI): pos.: m/z (%):
1212 (100) [M+Na+]; neg.: m/z (%): 1188 (100) [MꢀHꢀ]; elemental
analysis calcd (%) for C65H88N8O13 (1189.5): C 65.64, H 7.46, N 9.42;
found C 65.63, H 7.22, N 9.26.
MeZ-Dap[Boc-(4-Pe)Pro]-(bMe)Phe-(R)-(3-Ncp)Ala-(bMe)Phe-Ile-
ODCPM (18c): The tetrapeptide 16 (0.203 g, 0.22 mmol), after removal
of the Fmoc group according to GP 1, was coupled with the dipeptide
acid 15c (0.120 g, 0.25 mmol) by treatment with HATU (93 mg,
0.25 mmol), HOAt (33 mg, 0.25 mmol) and TMP (0.119 g, 0.98 mmol) in
CH2Cl2 (5 mL) according to GP 4 for 15 h. The mixture was then diluted
with Et2O (50 mL), and subjected to the usual aqueous work-up (GP 2).
The organic layer was dried, filtered and concentrated under reduced
pressure. The oily residue was purified by column chromatography (ace-
tone/hexane 5:2, Rf =0.22, three times) and finally recrystallized twice
from Et2O/hexane to give the branched hexapeptide 18c (0.151 g, 59%)
as
a
colorless solid. M.p. 102–1038C, [a]2D0 =88.9 (c=0.46, CHCl3);
1H NMR (600 MHz, CDCl3): d=0.33 (dddd, J=4.8, 4.8, 4.8, 4.8 Hz, 1H,
2’-H, DCPM), 0.38 (dddd, J=4.8, 4.8, 4.8, 4.8 Hz, 1H, 2’-H, DCPM),
0.41 (dddd, J=4.8, 4.8, 4.8, 4.8 Hz, 1H, 2’-H, DCPM), 0.47–0.61 (m, 2H,
2’-H, DCPM), 0.54–0.59 (m, 2H, 2’-H, DCPM), 0.66 (dddddd, J=4.8, 4.8,
4.8, 4.8, 4.8, 4.8 Hz, 1H, 2’-H, DCPM), 0.81 (d, J=6.6 Hz, 3H, 1’-H, Ile),
0.91 (t, J=7.2 Hz, 3H, 5-H, Ile), 0.92–0.97 [m, 1H, 1’-H, (3-Ncp)Ala] 1.00
[ddd, J=7.2, 7.2, 7.2 Hz, 1H, 3’-Ha, (3-Ncp)Ala], 1.04–1.12 (m, 1H, 1’-Ha,
DCPM), 1.12–1.17 (m, 1H, 1’-Hb, DCPM), 1.17–1.22 (m, 1H, 4-Ha, Ile),
1.23–1.27 [m, 1H, 3-Ha, (3-Ncp)Ala], 1.27 [d, J=6.6 Hz, 3H, 4-H,
(bMe)Phe], 1.34 [s, 9H, C(CH3)3], 1.47–1.54 [m, 1H, 3’-Hb, (3-Ncp)Ala],
1.68 [dd, J=6.6, 1.2 Hz, 3’-H, (4-Pe)Pro], 1.81 [ddd, J=12.0, 12.0,
12.0 Hz, 1H, 3-Ha, (4-Pe)Pro], 1.84–1.92 (m, 1H, 3-H, Ile), 2.33 (s, 3H,
1’-H, MeZ), 2.35 [ddd, J=12.0, 6.0, 6.0 Hz, 3-Hb, (4-Pe)Pro], 3.04 (ddd,
J=13.8, 2.8, 2.8 Hz, 3-Ha, Dap), 3.12–3.21 [m, 2H, 3-H, (bMe)Phe, 5-Ha,
(4-Pe)Pro], 3.21–3.29 [m, 1H, 4-H, (4-Pe)Pro], 3.32 [dq, J=10.2, 6.6 Hz,
MeZ-a-NbMe-Dab[Boc-(4-Pe)Pro]-(bMe)Phe-(R)-(3-Ncp)Ala-
(bMe)Phe-Ile-OTMSE (18b): The tetrapeptide 17 (77 mg, 0.081 mmol),
after removal of the Fmoc group according to GP 1 by treatment with
50% Et2NH in THF (2 mL), was coupled with the dipeptide acid 15b
(0.55 mg, 0.106 mmol) by using HATU (40.4 mg, 0.106 mmol), HOAt
(14.4 mg, 0.106 mmol) and TMP (64 mg, 0.53 mmol) in CH2Cl2 (5 mL) ac-
cording to GP 4 for 15 h. The mixture was then diluted with Et2O
(50 mL), and subjected to the usual aqueous work-up (GP 2). The organ-
ic layer was dried, filtered and concentrated under reduced pressure. The
residue was recrystallized from tBuOMe/hexane, and then purified by
column chromatography (acetone/hexane 1:2, Rf =0.32) to give the
branched hexapeptide 18a (91.0 mg, 93%) as an amorphous colorless
solid. [a]2D0 =10.3 (c=0.31, CHCl3); 1H NMR (600 MHz, CDCl3): d=0.09
Chem. Eur. J. 2005, 11, 2929 – 2945
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2939

C. Griesinger, A. Zeeck, A. de Meijere et al.
1-H, 3-H, (bMe)Phe], 3.67 [dd, J=8.4, 7.2 Hz, 1H, 5-Hb, (4-Pe)Pro], 3.86
[ddd, J=7.2, 3.0, 3.0 Hz, 1H, 2’-H, (3-Ncp)Ala], 4.04 (t, J=7.8 Hz, 1H,
1-H, DCPM), 4.23–4.31 [m, 3H, C-2, (4-Pe)Pro, (bMe)Phe, Dap], 4.32
(ddd, J=13.8, 2.8, 2.8 Hz, 1H, 3-Hb, Dap), 4.37 (dd, J=9.0, 4.2 Hz, 1H,
2-H, Ile), 4.53 [ddd, J=10.8, 6.0, 6.0 Hz, 1H, 2-H, (3-Ncp)Ala], 4.58 [dd,
J=10.2, 10.2 Hz, 1H, 2-H, (bMe)Phe], 5.00 (d, J=12.3 Hz, Bzl-Ha), 5.08
(d, J=12.3 Hz, Bzl-Hb), 5.26–5.33 [m, 1H, 1’-H, (4-Pe)Pro], 5.56 [dq, J=
10.2, 6.6 Hz, 1H, 2’-H, (4-Pe)Pro], 6.59 (d, J=6.0 Hz, 1H, NH), 6.83 (d,
J=9.0 Hz, 1H, NH), 6.2 (d, J=9.6 Hz, 1H, NH), 7.12 (d, J=8.4 Hz, 2H,
Ar-H, MeZ), 7.16–7.20 (m, 2H, Ar-H), 7.21–7.27 (m, 7H, Ar-H), 7.28–
7.37 (m, 3H, Ar-H), 7.50 (d, J=9.0 Hz, 1H, NH), 7.78 (dd, J=10.2,
2.4 Hz, 1H, NH), 8.20 (d, J=6.0 Hz, 1H, NH); the signal of C(CH3)
overlapped the signals of 4-Hb of the Ile moiety, 4-H of (bMe)Phe residue
and 3-Hb of the (3-Ncp)Ala fragment; 13C NMR (150.8 MHz, CDCl3): d=
2.6, 2.87, 2.96, 3.0 (ꢀ, C-2’, DCPM), 11.7 (+, C-5, Ile), 13.2 [+, C-3’, (4-
Pe)Pro], 14.2, 14.6 (+, C-1’, DCPM), 15.6 (+, C-1’, Ile), 18.4 [ꢀ, C-3’, (3-
Ncp)Ala], 18.8 [+, C-4, (bMe)Phe], 19.8 [+, C-4, (bMe)Phe], 21.1 (+,
C-1’, MeZ), 21.8 [+, C-1’, (3-Ncp)Ala], 25.2 (ꢀ, C-4, Ile), 28.3 [+, C-
(CH3)3], 31.1 [ꢀ, C-3, (3-Ncp)Ala], 36.2 [+, C-4, (4-Pe)Pro], 36.4 [ꢀ, C-
3, (4-Pe)Pro], 37.6 (+, C-3, Ile), 40.3 [+, C-3, (bMe)Phe], 40.8 (ꢀ, C-3,
Dap), 41.7 [+, C-3, (bMe)Phe], 51.0 [+, C-2, (3-Ncp)Ala], 52.4 [ꢀ, C-5,
(4-Pe)Pro], 56.5 (+, C-2, Ile), 59.2 (+, C-2, Dap), 59.5 [+, C-2’, (3-
Ncp)Ala], 60.7 [+, C-2, (4-Pe)Pro], 61.0 [+, C-2, (bMe)Phe], 63.4 [+,
C-2, (bMe)Phe], 66.7 (ꢀ, Bzl-C), 80.2 [Cquat, C(CH3)3], 83.5 (+, C-1,
DCPM), 126.7 [+, C-2’, (4-Pe)Pro], 127.0, 127.4, 127.6, 127.7, 128.47,
128.50, 128.93 (? 2) (+, Ar-C), 129.3 [+, C-1’, (4-Pe)Pro], 133.4, 137.7,
141.8, 141.9 (Cquat, Ar-C), 154.4, 156.1 (Cquat, NCO2), 169.7, 170.8, 173.1,
173.4, 174.7, 175.4 (Cquat, C-1); IR (KBr): n˜ =3089, 3062, 3010, 2972, 2933,
2877, 1725, 1667, 1542, 1454, 1416, 1392, 1368, 1258, 1216, 1162 cmꢀ1; MS
(ESI): pos.: m/z (%): 1197 (100) [M+Na+]; neg.: m/z (%): 1173 (100)
[MꢀHꢀ]; elemental analysis calcd (%) for C64H86N8O13 (1175.4): C 65.40,
H 7.37, N 9.53; found C 65.17, H 7.13, N 9.34.
4.60 [m, 2H, 2-H, Ile, 2-H, (bMe)Phe], 4.97 (d, J=12.0 Hz, Bzl-Ha), 5.19
(d, J=12.0 Hz, Bzl-Hb), 5.25 [dd, J=9.6, 9.6 Hz, 1H, 1’-H, (4-Pe)Pro],
5.58 [dq, J=9.6, 7.2 Hz, 1H, 2’-H, (4-Pe)Pro], 5.63–5.80 (br, 1H, NH),
6.04–6.37 (br, 1H, NH), 6.51–6.67 (br, 1H, NH), 6.86–7.02 (br, 1H, NH),
7.02–7.12 (m, 1H, NH), 7.14–7.31 (m, 15H, Ar-H, NH); 13C NMR
(150.8 MHz, CDCl3): d=10.5 (+, C-5, Ile), 13.2 [+, C-3’, (4-Pe)Pro],
13.5 [+, C-4, (bMe)Phe], 14.9 (+, C-1’, Ile), 17.8 [ꢀ, C-3’, (3-Ncp)Ala],
18.0 [+, C-4, (bMe)Phe], 18.7 (+, C-4, a-Dab), 21.0 [+, C-1’, (3-
Ncp)Ala], 21.1 (+, C-1’, MeZ), 24.3 (ꢀ, C-4, Ile), 32.7 [ꢀ, C-3, (3-
Ncp)Ala], 35.2 [ꢀ, C-3, (4-Pe)Pro], 36.6 [+, C-4, (4-Pe)Pro], 37.3 (+, C-
3, Ile), 38.8 [+, C-3, (bMe)Phe], 43.6 [+, C-3, (bMe)Phe], 47.5 (+, C-3,
a-Dab), 52.5 [+, C-2, (3-Ncp)Ala], 52.7 [ꢀ, C-5, (4-Pe)Pro], 54.5 (+, C-
2, Ile), 58.9 [+, C-2’, (3-Ncp)Ala], 59.30 [+, 2 ? C-2, a-Dab, (bMe)Phe],
59.5 [+, C-2, (bMe)Phe], 62.3 [+, C-2, (4-Pe)Pro], 67.0 (ꢀ, Bzl-C),
126.8, 127.1, 127.2, 127.5 (+, Ar-C), 127.7 [C-2’, (4-Pe)Pro], 128.0 (+,
Ar-C), 128.4 [C-1’, (4-Pe)Pro], 128.5, 128.6, 129.1 (+, Ar-C), 133.4, 137.8,
141.4, 142.3 (Cquat, Ar-C), 157.3 (Cquat, NCO2), 169.1, 170.49 (ꢄ 2), 170.90
(ꢄ 2), 172.0 (Cquat, C-1); IR (KBr): n˜ =3060, 3029, 2969, 2936, 2877, 1670,
1634, 1542, 1517, 1452, 1369, 1205 cmꢀ1; MS (ESI): pos.: m/z (%): 1000
(100) [M+Na+]; neg.: m/z (%): 976 (100) [MꢀHꢀ]; HRMS (ESI): m/z:
calcd for [C53H68N8O10Na+]: 999.4951; found 999.4951.
epi-19a: analytical HPLC 1: isocratic, 60% MeCN in H2O (0.1% TFA),
flow rate=0.5 mLminꢀ1, tR =17.25 min, purity
>
97%; [a]2D0 =ꢀ42.68
(c=0.27, CHCl3); 1H NMR (600 MHz, CDCl3): d=0.65–0.73 (m, 3H, 1’-
H, a-Ile), 0.84 (t, J=7.2 Hz, 3H, 5-H, a-Ile), 0.89 [ddd, J=5.4, 5.4, 5.4 Hz,
1H, 3’-Ha, (3-Ncp)Ala], 1.10 [d, J=5.4 Hz, 3H, 4-H, (bMe)Phe], 1.10–
1.15 [m, 1H, 3-Ha*, (3-Ncp)Ala], 1.17–1.26 [m, 4H, 4-H, (bMe)Phe, 4-
Ha*, a-Ile], 1.40–1.48 [m, 1H, 3-Hb*, (3-Ncp)Ala], 1.44 (d, J=7.8 Hz, 3H,
4-H, a-Dab), 1.50–1.59 [m, 1H, 1’-H, (3-Ncp)Ala], 1.59–1.63 [m, 2H, 3’-
Hb, (3-Ncp)Ala, 3-H, a-Ile], 1.63 [d, J=6.6 Hz, 3’-H, (4-Pe)Pro], 2.08–
2.22 [m, 1H, 3-Ha, (4-Pe)Pro], 2.28 (s, 3H, 1’-H, MeZ), 2.25–2.33 [m, 1H,
3-Hb, (4-Pe)Pro], 3.06 [ddddd, J=7.8, 7.8, 7.8, 7.8, 7.8 Hz, 1H, 4-H, (4-
Pe)Pro], 3.17–3.30 [m, 2H, 5-Ha, (4-Pe)Pro, 3-H*, (bMe)Phe], 3.31–3.44
[m, 2H, 2’-H*, (3-Ncp)Ala, 3-H, (bMe)Phe], 3.74 [dd, J=7.8, 7.8 Hz, 1H,
5-Hb, (4-Pe)Pro], 3.74–3.84 (m, 1H, 2-H), 4.08–4.20 (m, 1H, 3-H, a-Dab),
4.31–4.84 (m, 2H, 2-H), 4.48–4.62 (m, 2H, 2-H), 4.73 (dd, J=7.8, 7.8 Hz,
1H, 2-H), 4.98 (d, J=12.0 Hz, Bzl-Ha), 5.09 (d, J=12.0 Hz, Bzl-Hb), 5.29
[dd, J=10.2, 10.2 Hz, 1H, 1’-H, (4-Pe)Pro], 5.52 [dq, J=10.2, 6.6 Hz, 1H,
2’-H, (4-Pe)Pro], 5.92–6.04 (br, 1H, NH), 6.74 (d, J=7.2 Hz, 1H, NH),
7.05–7.12 (br, 1H, NH), 7.07 (d, J=7.8 Hz, 2H, Ar-H), 7.16–7.29 (m,
10H, Ar-H, NH), 7.30–7.36 (m, 4H, Ar-H, NH), 7.44–7.51 (br, 1H, NH);
the absorption of 4-Hb, a-Ile is masked by the signal of 3’-H, (4-Pe)Pro;
13C NMR (150.8 MHz, CDCl3): d=11.7 (+, C-5, a-Ile), 13.2 [+, C-3’, (4-
Pe)Pro], 14.0 (+, C-1’, a-Ile), 17.3 [+, C-4, (bMe)Phe], 17.5 (+, C-4, a-
Dab), 17.6 [ꢀ, C-3’, (3-Ncp)Ala], 17.6 [+, C-4, (bMe)Phe], 21.1 (+, C-1’,
MeZ), 21.9 [+, C-1’, (3-Ncp)Ala], 26.3 (ꢀ, C-4, a-Ile), 31.8 [ꢀ, C-3, (3-
Ncp)Ala], 34.2 [ꢀ, C-3, (4-Pe)Pro], 36.1 [+, C-4, (4-Pe)Pro], 36.9 (+, C-
3, a-Ile), 40.43 [+, 2ꢄC-3, (bMe)Phe], 49.3 (+, C-3, a-Dab), 51.0 (+, C-
2), 52.9 [ꢀ, C-5, (4-Pe)Pro], 54.1 (+, C-2), 58.8 [+, C-2’*, (3-Ncp)Ala],
59.3 (+, C-2*), 59.4 (+, C-2*), 60.34 (+, 2ꢄC-2), 67.2 (ꢀ, Bzl-C), 126.7,
127.0, 127.6, 127.7, 128.2, 128.3, 128.5, 128.7, 129.0, 129.1 [+, Ar-C, C-1’,
MeZ-Protected branched cyclohexapeptide (19a) and its epimer (epi-
19a): The branched hexapeptide 18a (0.188 g, 0.165 mmol) was depro-
tected according to GP 5 by treatment with the freshly prepared 2m HCl
in EtOAc (3 mL) to give the hydrochloride of the deprotected peptide as
a colorless solid [0.145 g; MS (ESI): pos.: m/z (%): 996 (100) [M+H+];
neg.: m/z (%): 994 (100) [MꢀHꢀ], which was taken up with anhydrous
CH2Cl2 (1.5 L) and cyclized by treatment with HATU (2ꢄ61 mg, 2ꢄ
0.160 mmol) and HOAt (2ꢄ18 mg, 2ꢄ0.133 mmol) and solution of
DIEA (2ꢄ55 mg, 2ꢄ0.426 mmol) in CH2Cl2 (2ꢄ20 mL) according to
GP 2 for 18 h. After this, the solvent was removed under reduced pres-
sure, the residue was taken up with Et2O (50 mL), and after the usual
aqueous work-up (GP 5), drying and filtration, the organic layer was con-
centrated under reduced pressure. The residue was purified first by
column chromatography (acetone/hexane 1:1.75, Rf =0.29), and then by
recrystallization (Et2O/pentane) to give a crude product (81.0 mg), which
contained two components according to analytical HPLC. The mixture
was separated by preparative HPLC to give cyclodepsipeptide 19a
(41 mg, 28% over two steps) and its epimer epi-19a (28 mg, 19% over
two steps) as colorless solids. Preparative HPLC: column A, isocratic,
85% MeCN in H2O (0.07% TFA), flow rate 2.5 mLminꢀ1
.
C-2’, (4-Pe)Pro], 132.8, 138.0, 142.0, 142.6 (Cquat, Ar-C), 156.2 (Cquat
,
Compound 19a: analytical HPLC: isocratic, 60% MeCN in H2O (0.1%
TFA), flow rate=0.5 mLminꢀ1, tR =20.18 min, purity > 99%; [a]2D0 =8.6
(c=0.21, CHCl3); 1H NMR (600 MHz, CDCl3): d=0.15–0.31 [m, 1H, 3’-
Ha, (3-Ncp)Ala], 0.32–0.47 [m, 1H, 3-Ha, (3-Ncp)Ala], 0.78 (t, J=7.2 Hz,
3H, 5-H, Ile), 0.84 (d, J=6.6 Hz, 3H, 1’-H, Ile), 0.88–0.97 [m, 1H, 1’-H,
(3-Ncp)Ala], 0.97–1.06 (m, 1H, 4-Ha, Ile), 1.08–1.25 [m, 1H, 3-Hb, (3-
Ncp)Ala], 1.25 [d, J=7.2 Hz, 3H, 4-H, (bMe)Phe], 1.26–1.34 (m, 1H, 4-
Hb, Ile), 1.29 [d, J=7.2 Hz, 3H, 4-H, (bMe)Phe], 1.34–1.44 [m, 1H, 3’-Hb,
(3-Ncp)Ala], 1.40 (d, J=7.2 Hz, 3H, 4-H, a-Dab), 1.63–1.73 (m, 1H, 3-H,
Ile), 1.66 [d, J=6.6 Hz, 3’-H, (4-Pe)Pro], 1.87 [ddd, J=11.4, 11.4, 11.4 Hz,
1H, 3-Ha, (4-Pe)Pro], 2.18 [ddd, J=11.4, 6.0, 6.0 Hz, 1H, 3-Hb, (4-
Pe)Pro], 2.38 (s, 3H, 1’-H, MeZ), 3.01 [dddd, J=1.2, 7.2 Hz, 1H, 3-H,
(bMe)Phe], 3.07–3.19 [m, 1H, 4-H, (4-Pe)Pro], 3.23 [dd, J=9.8, 9.8 Hz,
1H, 5-Ha, (4-Pe)Pro], 3.40–3.51 [m, 1H, 2’-H, (3-Ncp)Ala], 3.64–3.71 [m,
1H, 3-H, (bMe)Phe], 3.71–3.79 [m, 1H, 2-H, (3-Ncp)Ala], 3.78–3.85 [m,
1H, 2-H, (4-Pe)Pro], 3.86–3.94 [m, 1H, 5-Hb, (4-Pe)Pro], 4.28–4.34 [m,
2H, 3-H, a-Dab, 2-H, (bMe)Phe], 4.34–4.50 (m, 1H, 2-H, a-Dab), 4.50–
NCO2), 170.33 (ꢄ 2), 170.82 (ꢄ 2), 170.9, 171.0 (Cquat, C-1); IR (KBr): n˜ =
3061, 3030, 2969, 2934, 2877, 1654, 1540, 1453, 1369, 1270 cmꢀ1; MS
(ESI): pos.: m/z (%): 1000 (100) [M+Na+]; neg.: m/z (%): 976 (100)
[MꢀHꢀ].
MeZ-a-NbMe-Dab[Boc-(4-Pe)Pro]-(bMe)Phe-(R)-(3-Ncp)Ala-
(bMe)Phe-Ile-OH: (Bu)4NꢀF+ (70.0 mg, 0.22 mmol) was added to a stir-
red solution of the ester 18b (88.0 mg, 72.8 mmol) in MeCN (2.0 mL),
and the mixture was stirred at 208C for an additional 1 h. As TLC
showed the presence of the starting material the mixture was carefully
heated at 558C with a heat-gun and then was stirred for another 1 h. 1n
H2SO4 (1 mL) was added, and the reaction mixture was then diluted with
Et2O (40 mL), washed with 1m KHSO4 (3ꢄ10 mL), water (3ꢄ10 mL),
brine (2ꢄ10 mL), dried, filtered and concentrated under reduced pres-
sure. The residue was recrystallized from Et2O/pentane to give the title
compound (79.0 mg, 98%) as a colorless solid which was used for the
next step without additional purification. Rf =0.36, acetone/hexane 4:7
2940
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 2929 – 2945

Hormaomycin
FULL PAPER
(3% AcOH); MS (ESI): pos.: m/z (%): 1153 (78) [MꢀH+2Na+], 1131
(100) [M+Na+]; neg.: m/z (%): 1107 (100) [MꢀHꢀ].
MeZ-Protected branched cyclohexapeptide (19c) and its epimer (epi-
19c): The branched hexapeptide 18c (0.134 g, 0.114 mmol) was depro-
tected according to GP 5 by treatment with a freshly prepared 2m HCl in
EtOAc (2.5 mL) to give the hydrochloride of the deprotected peptide as
a colorless solid, which was taken up with anhydrous CH2Cl2 (1.3 L) and
cyclized by treatment with HATU (2ꢄ44.6 mg, 2ꢄ0.117 mmol), HOAt
(2ꢄ15.9 mg, 2ꢄ0.117 mmol) and a solution of DIEA (2ꢄ59 mg, 2ꢄ
0.456 mmol) in CH2Cl2 (2ꢄ50 mL) according to GP 2 for 18 h. After this,
the solvent was removed under reduced pressure, the residue was taken
up with Et2O (50 mL), and after the usual aqueous work-up (GP 5),
drying and filtration, the organic layer was concentrated under reduced
pressure. The residue was purified by column chromatography (acetone/
hexane 1:1.5, Rf =0.31) to give a crude product (90.0 mg), which con-
tained two components according to analytical HPLC. The mixture was
separated by preparative HPLC to give cyclodepsipeptide 19c (37.7 mg,
34% over two steps) and its epimer epi-19a (27.9 mg, 25% over two
steps) as colorless solids. Preparative HPLC: column B, isocratic, 69%
MeZ-Protected branched cyclohexapeptide (19b): The branched hexa-
peptide
acid
MeZ-a-NbMe-Dab[Boc-(4-Pe)Pro]-(bMe)Phe-(R)-(3-
Ncp)Ala-(bMe)Phe-Ile-OH (79.0 mg, 71.2 mmol) was deprotected accord-
ing to GP 5 by treatment with 2m HCl in EtOAc (2 mL) to give the hy-
drochloride of the deprotected material as a colorless solid (80 mg),
which was taken up with anhydrous CH2Cl2 (1.1 L) and cyclized by treat-
ment with HATU (2ꢄ28.0 mg, 2ꢄ73.3 mmol) and HOAt (2ꢄ9.6 mg, 2ꢄ
73.3 mmol) and solution of DIEA (2ꢄ37 mg, 2ꢄ0.285 mmol) in CH2Cl2
(2ꢄ50 mL) according to GP 2 for 22 h. After this, the solvent was re-
moved under reduced pressure, the residue was taken up with Et2O
(50 mL), and after the usual aqueous work-up (GP 5), drying and filtra-
tion, the organic layer was concentrated under reduced pressure. The res-
idue was purified first by column chromatography (acetone/hexane 1:2,
Rf =0.32) to give a crude product (43.0 mg), which was finally purified by
preparative HPLC to give cyclodepsipeptide 19b (31.6 mg, 44% over
three steps) and a small amount of its epimer epi-19b (1.4 mg, 2% over
three steps) as colorless solids. Preparative HPLC: column B, isocratic,
MeCN in H2O (0.1% TFA), flow rate 2.5 mLminꢀ1
.
Compound 19c: analytical HPLC: isocratic, 70% MeCN in H2O (0.1%
TFA), flow rate=0.5 mLminꢀ1, tR =12.00 min, purity > 99%; [a]2D0 =16.0
(c=0.77, CHCl3); 1H NMR (600 MHz, CDCl3): d=0.40–0.54 [m, 1H, 3-
Ha, (3-Ncp)Ala], 0.58–0.69 [m, 1H, 3’-Ha, (3-Ncp)Ala], 0.76 (t, J=7.2 Hz,
3H, 5-H, Ile), 0.88 (d, J=6.0 Hz, 3H, 1’-H, Ile), 1.00–1.17 [m, 2H, 1’-H,
(3-Ncp)Ala, 1H, 4-Ha, Ile], 1.26 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe],
1.26–1.34 [m, 1H, 3-Hb, (3-Ncp)Ala], 1.34 [d, J=7.2 Hz, 3H, 4-H,
(bMe)Phe], 1.42–1.49 [m, 1H, 3’-Hb, (3-Ncp)Ala], 1.59 [d, J=6.6 Hz, 3’-
H, (4-Pe)Pro], 1.69–1.79 (m, 1H, 3-H, Ile), 1.81 [ddd, J=12.0, 12.0,
12.0 Hz, 1H, 3-Ha, (4-Pe)Pro], 2.16 [ddd, J=12.0, 6.0, 6.0 Hz, 1H, 3-Hb,
(4-Pe)Pro], 2.35 (s, 3H, 1’-H, MeZ), 2.90 [dq, J=6.0, 6.0 Hz, 1H, 3-H,
(bMe)Phe], 3.03–3.15 [m, 1H, 4-H, (4-Pe)Pro], 3.23 [dd, J=9.2, 9.2 Hz,
1H, 5-Ha, (4-Pe)Pro], 3.43–3.58 [m, 2H, 2-H, (3-Ncp)Ala, 3-Ha, Dap],
3.62–3.72 [m, 2H, 3-H, (bMe)Phe, 2’-H, (3-Ncp)Ala], 3.84–3.93 [m, 1H,
3-Hb, Dap], 3.94 [dd, J=9.2, 9.2 Hz, 1H, 5-Hb, (4-Pe)Pro], 4.03–4.12 [m,
1H, 2-H, (4-Pe)Pro], 4.41–4.48 (m, 1H, Dap), 4.50–4.55 [m, 1H, 2-H,
(bMe)Phe], 4.59 (dd, J=8.7, 8.7 Hz, 1H, 2-H, Ile), 4.64 [dd, J=7.8,
7.8 Hz, 1H, 2-H, (bMe)Phe], 5.06 (d, J=12.0 Hz, Bzl-Ha), 5.13 (d, J=
12.0 Hz, Bzl-Hb), 5.22 [dd, J=10.2, 10.2 Hz, 1H, 1’-H, (4-Pe)Pro], 5.57
[dq, J=10.2, 6.6 Hz, 1H, 2’-H, (4-Pe)Pro], 6.06–6.23 (br, 1H, NH), 6.71–
6.90 (br, 2H, 2ꢄNH), 6.98–7.09 (br, 1H, NH), 7.11–7.18 (m, 3H, Ar-H),
7.18–7.22 (m, 2H, Ar-H), 7.22–7.28 (m, 7H, Ar-H), 7.28–7.34 (m, 2H,
Ar-H), 7.34–7.57 (br, 1H, NH), 7.59–7.84 (br, 1H, NH); the signal of 4-H
of the (bMe)Phe residue (1.34 ppm) overlapped the signal of 4-Hb of the
Ile moiety; 13C NMR (150.8 MHz, CDCl3): d=10.6 (+, C-5, Ile), 13.3 [+,
C-3’, (4-Pe)Pro], 13.9 [+, C-4, (bMe)Phe], 15.0 (+, C-1’, Ile), 17.3 [ꢀ, C-
3’, (3-Ncp)Ala], 17.9 [+, C-4, (bMe)Phe], 21.25 (+, C-1’, MeZ), 21.33 [+,
C-1’, (3-Ncp)Ala], 24.6 (ꢀ, C-4, Ile), 32.0 [ꢀ, C-3, (3-Ncp)Ala], 35.2 [ꢀ,
C-3, (4-Pe)Pro], 36.9 [+, C-4, (4-Pe)Pro], 37.3 (+, C-3, Ile), 39.1 [+, C-
3, (bMe)Phe], 40.6 (ꢀ, C-3, Dap), 45.0 [+, C-3, (bMe)Phe], 53.0 [ꢀ, C-5,
(4-Pe)Pro], 53.7 [+, C-2, (3-Ncp)Ala], 54.6 (+, C-2, Ile), 57.8 (+, C-2,
Dap), 58.8 [+, C-2, (bMe)Phe], 59.1 [+, C-2’, (3-Ncp)Ala], 59.5 [+, C-2,
(bMe)Phe], 61.5 [+, C-2, (4-Pe)Pro], 67.2 (ꢀ, Bzl-C), 127.08, 127.12,
127.4, 127.7 [C-2’, (4-Pe)Pro], 128.0 [C-1’, (4-Pe)Pro], 128.3, 128.6, 128.8,
129.17 (ꢄ2) (+, Ar-C), 133.3, 137.9, 141.3, 142.1 (Cquat, Ar-C), 157.3
(Cquat, NCO2), 169.2, 170.9 (ꢄ2), 171.4, 172.0, 174.2 (Cquat, C-1); IR
(KBr): n˜ =3060, 3029, 2972, 2936, 2879, 1725, 1667, 1638, 1543, 1513,
1453, 1369, 1204 cmꢀ1; MS (ESI): pos.: m/z (%): 986 (100) [M+Na+];
neg.: m/z (%): 962 (100) [MꢀHꢀ]; HRMS (ESI): m/z: calcd for
[C52H66N8O10Na+]: 985.4794; found 985.4797.
65% MeCN in H2O (0.1% TFA), flow rate 2.7 mLminꢀ1
.
Compound 19b: analytical HPLC: isocratic, 75% MeCN in H2O (0.1%
TFA), flow rate=0.5 mLminꢀ1, tR =10.64 min, purity > 99%; gradient
55
!
100% MeCN in H2O (0.1% TFA) for 15 min, flow rate=
tR =14.65 min, purity
99%; [a]2D0 =37.8 (c=0.33,
0.5 mLminꢀ1
,
>
CHCl3); 1H NMR (600 MHz, CDCl3): d=0.69–0.77 [m, 1H, 3-Ha, (3-
Ncp)Ala], 0.77 (t, J=7.2 Hz, 3H, 5-H, Ile), 0.79 (d, J=7.2 Hz, 3H, 1’-H,
Ile), 0.85 [ddd, J=7.2, 7.2, 7.2 Hz, 1H, 3’-Ha, (3-Ncp)Ala], 1.11 (ddq, J=
7.8, 6.6, 6.6 Hz, 1H, 4-Ha, Ile), 1.34 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe],
1.32–1.41 [m, 2H, 4-Hb, Ile, 1’-H, (3-Ncp)Ala], 1.42 [d, J=6.6 Hz, 3H, 4-
H, (bMe)Phe], 1.34–1.44 [m, 1H, 3’-Hb, (3-Ncp)Ala], 1.58 (d, J=7.2 Hz,
3H, 4-H, a-NbMe-Dab), 1.59–1.64 [m, 3H, 3-H, Ile, 3-Hb, 3’-Hb, (3-
Ncp)Ala], 1.66 [dd, J=7.2, 1.8 Hz, 3’-H, (4-Pe)Pro], 1.73 [ddd, J=12.0,
12.0, 12.0 Hz, 1H, 3-Ha, (4-Pe)Pro], 2.13 [ddd, J=12.0, 6.0, 6.0 Hz, 1H,
3-Hb, (4-Pe)Pro], 2.35 (s, 3H, 1’-H, MeZ), 2.92 (s, 3H, NMe, a-NbMe-
Dab), 2.99 [dq, J=6.6, 7.5 Hz, 1H, 3-H, (bMe)Phe], 3.17–3.30 [m, 2H, 4-
H, 5-Ha, (4-Pe)Pro], 3.48 [dq, J=6.6, 6.6 Hz, 1H, 3-H, (bMe)Phe], 3.62–
3.72 [m, 1H, 2-H, (3-Ncp)Ala], 3.84 [ddd, J=7.2, 3.0, 3.0 Hz, 1H, 2’-H,
(3-Ncp)Ala], 4.01–4.10 [m, 1H, 5-Hb, (4-Pe)Pro], 4.29 (d, J=7.2 Hz, 1H,
2-H, a-NbMe-Dab), 4.45 [dd, J=10.2, 6.6 Hz, 1H, 2-H, (bMe)Phe], 4.45–
4.54 [m, 2H, 2-H, Ile, 2-H, (4-Pe)Pro], 4.34–4.50 (m, 1H, 2-H, a-Dab),
4.71 [dd, J=7.5, 7.5 Hz, 1H, 2-H, (bMe)Phe], 4.99 (dd, J=7.2, 7.2 Hz, 3-
H, a-NbMe-Dab), 5.09 (s, 2H, Bzl), 5.17–5.27 [dd, J=9.6, 9.6 Hz, 1H, 1’-
H, (4-Pe)Pro], 5.57 [dq, J=9.6, 7.2 Hz, 1H, 2’-H, (4-Pe)Pro], 6.11 (d, J=
6.0 Hz, 1H, NH), 6.23–6.37 (br, 2H, 2ꢄNH), 6.51–6.67 (br, 1H, NH),
7.01 (d, J=6.0 Hz, 1H, NH), 7.09–7.21 (m, 6H, Ar-H), 7.22–7.27 (m, 4H,
Ar-H), 7.28 (dd, J=7.2 Hz, Ar-H), 7.35 (dd, J=7.8, 7.8 Hz, Ar-H), 7.43
(d, J=8.4 Hz, NH); 13C NMR (150.8 MHz, CDCl3): d=10.1 (+, C-5, Ile),
13.4 [+, C-3’, (4-Pe)Pro], 14.7 (+, C-1’, Ile), 15.3 [+, C-4, (bMe)Phe],
17.04 (+, C-4, a-NbMe-Dab), 17.2 [ꢀ, C-3’, (3-Ncp)Ala], 18.2 [+, C-4,
(bMe)Phe], 21.2 (+, C-1’, MeZ), 21.4 [+, C-1’, (3-Ncp)Ala], 24.7 (ꢀ, C-
4, Ile), 31.2 (+, NMe, a-NbMe-Dab), 32.2 [ꢀ, C-3, (3-Ncp)Ala], 35.0 [ꢀ,
C-3, (4-Pe)Pro], 36.3 (+, C-3, Ile), 36.9 [+, C-4, (4-Pe)Pro], 39.6 [+, C-
3, (bMe)Phe], 45.5 [+, C-3, (bMe)Phe], 52.0 (+, C-3, a-NbMe-Dab), 52.6
[ꢀ, C-5, (4-Pe)Pro], 53.8 [+, C-2, (3-Ncp)Ala], 54.6 (+, C-2, Ile), 57.5
[+, C-2, (bMe)Phe], 58.9 [+, C-2, (bMe)Phe], 59.0 [+, C-2’, (3-
Ncp)Ala], 60.3 [+, C-2, (4-Pe)Pro], 61.6 (+, C-2, a-NbMe-Dab), 67.0 (ꢀ,
Bzl-C), 127.1, 127.3, 127.57, 127.62 (+, Ar-C), 127.7 [+, C-2’, (4-Pe)Pro],
128.1 [+, C-1’, (4-Pe)Pro], 128.3, 128.6, 128.8, 129.1 (+, Ar-C), 133.3,
137.9, 140.8, 142.5 (Cquat, Ar-C), 156.6 (Cquat, NCO2), 170.2, 170.4, 170.7,
170.8, 171.0, 174.8 (Cquat, C-1); IR (KBr): n˜ =2971, 2936, 2878, 1720, 1633,
1541, 1506, 1453, 1369, 1209, 1032 cmꢀ1; MS (ESI): pos.: m/z (%): 1013
(100) [M+Na+]; neg.: m/z (%): 989 (100) [MꢀHꢀ]; HRMS (ESI): m/z:
calcd for [C54H71N8O10+]: 991.5288; found 991.5291.
epi-19c: analytical HPLC 1: isocratic, 60% MeCN in H2O (0.1% TFA),
flow rate=0.5 mLminꢀ1, tR =9.44 min, purity > 99%; [a]2D0 =ꢀ43.1 (c=
0.42, CHCl3); 1H NMR (600 MHz, C2D2Cl4, 353.1 K): d=0.67 (d, J=
6.0 Hz, 3H, 1’-H, a-Ile), 0.82 (t, J=7.2 Hz, 3H, 5-H, a-Ile), 0.93 [ddd, J=
6.6, 6.6, 6.6 Hz, 1H, 3’-Ha, (3-Ncp)Ala], 0.93–0.99 (m, 1H, 4-Ha, a-Ile),
1.05–1.15 (m, 1H, 4-Hb, a-Ile), 1.24 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe],
1.37 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe], 1.37–1.45 [m, 1H, 3-Ha, (3-
Ncp)Ala], 1.54–1.63 [m, 2H, 3-Hb, (3-Ncp)Ala, 3-H, a-Ile], 1.66 [d, J=
6.0 Hz, 3’-H, (4-Pe)Pro], 1.67–1.76 [m, 1H, 1’-H, (3-Ncp)Ala], 1.84 [ddd,
J=10.2, 10.2, 10.2 Hz, 1H, 3-Ha, (4-Pe)Pro], 2.34 (s, 3H, 1’-H, MeZ),
2.35–2.44 [m, 1H, 3-Hb, (4-Pe)Pro], 3.07 [m, 1H, 4-H, (4-Pe)Pro], 3.27
epi-19b: analytical HPLC: isocratic, 75% MeCN in H2O (0.1% TFA),
flow rate=0.5 mLminꢀ1, tR =9.61 min, purity
>
95%; gradient 55 !
100% MeCN in H2O (0.1% TFA) for 15 min, flow rate=0.5 mLminꢀ1
,
tR =14.15 min, purity
> 95%; MS (ESI): pos.: m/z (%): 1013 (100)
[M+Na+]; neg.: m/z (%): 989 (100) [MꢀHꢀ].
Chem. Eur. J. 2005, 11, 2929 – 2945
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2941

C. Griesinger, A. Zeeck, A. de Meijere et al.
[dd, J=9.2, 9.2 Hz, 1H, 5-Ha, (4-Pe)Pro], 3.31–3.40 [m, 2H, 2 ? 3-H,
(bMe)Phe], 3.48–3.66 (m, 1H, 3-H, Dap), 3.74 [dd, J=9.2, 9.2 Hz, 1H, 5-
Hb, (4-Pe)Pro], 3.85–3.92 [m, 1H, 2’-H, (3-Ncp)Ala], 4.29–4.40 [m, 3H, 2-
H, a-Ile, 2 ? 2-H, (bMe)Phe], 4.41–4.47 [m, 1H, 2-H, (3-Ncp)Ala], 4.47–
4.54 [m, 1H, 2-H, (4-Pe)Pro], 4.61 (dd, J=8.4, 8.4 Hz, 1H, 2-H, Dap),
5.10 (d, J=12.0 Hz, Bzl-Ha), 5.14 (d, J=12.0 Hz, Bzl-Hb), 5.27–5.35 [dd,
J=10.2, 10.2 Hz, 1H, 1’-H, (4-Pe)Pro], 5.61 [dq, J=10.2, 6.0 Hz, 1H, 2’-
H, (4-Pe)Pro], 6.55–6.69 (br, 2H, NH), 6.89–7.01 (br, 2H, NH), 7.15 (d,
J=7.2 Hz, 2H, Ar-H, MeZ), 7.18–7.22 (m, 1H, Ar-H), 7.22–7.29 (m,
11H, Ar-H), 7.29–7.36 (m, 3H, Ar-H), 7.39–7.48 (br, 2H, NH); the ab-
sorption of 3’-Hb, (3-Ncp)Ala is masked by the signal of 3’-H, (4-Pe)Pro;
13C NMR (150.8 MHz, C2D2Cl4, 353.1 K): d=11.4 (+, C-5, a-Ile), 12.8 [+,
C-3’, (4-Pe)Pro], 13.6 (+, C-1’, a-Ile), 16.9 [+, C-4, (bMe)Phe], 17.2 [+,
C-4, (bMe)Phe], 17.4 [ꢀ, C-3’, (3-Ncp)Ala], 20.8 (+, C-1’, MeZ), 21.9 [+,
C-1’, (3-Ncp)Ala], 26.2 (ꢀ, C-4, a-Ile), 32.1 [ꢀ, C-3, (3-Ncp)Ala], 35.4 [ꢀ,
C-3, (4-Pe)Pro], 36.0 (+, C-3, a-Ile), 36.3 [+, C-4, (4-Pe)Pro], 39.9 [+,
C-3, (bMe)Phe], 41.1 [+, C-3, (bMe)Phe], 42.4 (ꢀ, C-3, Dap), 51.5 [+,
C-2, (3-Ncp)Ala], 52.2 [ꢀ, C-5, (4-Pe)Pro], 54.3 (+, C-2, a-Ile), 55.8 [+,
C-2, (4-Pe)Pro], 58.8 (+, C-2, Dap), 59.3 [+, C-2’, (3-Ncp)Ala], 60.0 [+,
C-2, (bMe)Phe], 60.4 [+, C-2, (bMe)Phe], 67.0 (ꢀ, Bzl-C), 126.5, 126.8
(+, Ar-C), 127.1 [+, C-2’, (4-Pe)Pro], 127.4, 127.5, 128.0, 128.3, 128.4 (+,
Ar-C), 128.6 [+, C-1’, (4-Pe)Pro], 129.0 (+, Ar-C), 132.9, 137.8, 142.3,
142.5 (Cquat, Ar-C), 156.3 (Cquat, NCO2), 170.2, 170.3, 170.6, 170.8, 171.3,
173.2 (Cquat, C-1); IR (KBr): n˜ =3034, 2969, 2871, 1659, 1541, 1453, 1369,
1256, 1206, 1065 cmꢀ1; MS (ESI): pos.: m/z (%): 985 (100) [M+Na+];
neg.: m/z (%): 961 (100) [MꢀHꢀ]; HRMS (ESI): m/z: calcd for
[C52H66N8O10Na+]: 985.4794; found 985.4793.
[m, 1H, 3’-Hb, (3-Ncp)Ala], 1.03 [ddd, J=7.2, 7.2, 7.2 Hz, 1H, 3’-Ha, (3-
Ncp)Ala], 1.07 (d, J=7.2 Hz, 3H, 1’-H, Ile), 1.32 [d, J=7.2 Hz, 3H, 4-H,
(bMe)Phe], 1.35 [d, J=7.2 Hz, 3H, 4-H, (bMe)Phe], 1.41 (d, J=7.8 Hz,
3H, a-Dab), 1.53–1.59 (m, 1H, 4-Hb, Ile), 1.63–1.75 [m, 2H, 3-H, (3-
Ncp)Ala], 1.69 [dd, J=7.2, 1.8 Hz, 3H, 3’-H, (4-Pe)Pro], 1.84–1.95 [m,
3H, 3’-Hb, (3-Ncp)Ala, 3-H, Ile, 3-Ha, (4-Pe)Pro], 1.95–2.01 [m, 1H, 3-Ha,
(4-Pe)Pro], 2.27 [ddd, J=12.0, 6.0, 6.0 Hz, 1H, 4-Hb, (4-Pe)Pro], 2.85
[ddd, J=6.6, 4.8, 4.8 Hz, 1H, 2’-H, (3-Ncp)Ala], 3.02 [dq, J=11.4, 7.2 Hz,
1H, 3-H, (bMe)Phe], 3.22–3.32 [m, 2H, 4-H, 5-Ha, (4-Pe)Pro], 3.46–3.52
[m, 1H, 2-H, (3-Ncp)Ala], 3.70 [dq, J=3.6, 7.2 Hz, 1H, 3-H, (bMe)Phe],
3.92 [dd, J=11.4, 5.4 Hz, 2-H, (4-Pe)Pro], 3.95–3.99 [m, 1H, 5-Hb, (4-
Pe)Pro], 4.03 [ddd, J=7.2, 3.6, 3.6 Hz, 1H, 2’-H, (3-Ncp)Ala], 4.35 (dd,
J=10.8, 10.8 Hz, 1H, 2-H), 4.42–4.48 (m, 1H, 3-H, a-Dab), 4.45 (dd, J=
10.2, 4.2 Hz, 1H, 2-H), 4.50 (dd, J=9.0, 3.0 Hz, 1H, 2-H), 4.66 (dd, J=
9.0, 9.0 Hz, 1H, 2-H), 5.11–5.16 [m, 1H, 2-H, (3-Ncp)Ala], 5.26–5.31 [m,
1H, 1’-H, (4-Pe)Pro], 5.63 [dq, J=10.8, 6.6 Hz, 1H, 2’-H, (4-Pe)Pro],
6.15 (d, J=4.2 Hz, 1H, 4-H, Chpca), 6.43 (d, J=7.8 Hz, 1H, NH), 6.77–
6.86 (br, 1H, NH), 6.85 (d, J=4.2 Hz, 1H, 3-H, Chpca), 7.02–7.06 (m,
2H, Ar-H), 7.12–7.19 (m, 6H, Ar-H, NH), 7.21–7.30 (m, 4H, Ar-H, NH),
7.44 (d, J=10.8 Hz, 1H, NH), 8.87 (d, J=9.0 Hz, 1H, NH), 10.70–11.00
(br, 1H, OH); the signal of 4-Ha, Ile was masked by absorption of 4-H,
(bMe)Phe (1.35 ppm); 13C NMR (150.8 MHz, CDCl3): d=10.4 (+, C-5,
Ile), 13.1 [+, C-3’, (4-Pe)Pro], 13.3 (+, C-4, a-Dab), 14.9 (+, C-1’, Ile),
17.0 [ꢀ, C-3’, (3-Ncp)Ala], 17.2 [ꢀ, C-3’, (3-Ncp)Ala], 17.6 [+, C-4,
(bMe)Phe], 17.7 [+, C-4, (bMe)Phe], 20.0 [+, C-1’, (3-Ncp)Ala], 21.7
[+, C-1’, (3-Ncp)Ala], 25.1 (ꢀ, C-4, Ile), 32.8 [ꢀ, C-3, (3-Ncp)Ala], 35.2
[ꢀ, C-3, (3-Ncp)Ala], 35.9 [ꢀ, C-3, (4-Pe)Pro], 36.4 [+, C-4, (4-Pe)Pro],
38.0 (+, C-3, Ile), 39.2 [+, C-3, (bMe)Phe], 41.2 [+, C-3, (bMe)Phe],
45.3 (+, C-3, a-Dab), 51.0 [+, C-2, (3-Ncp)Ala], 51.8 [+, C-2, (3-
Ncp)Ala], 53.1 [ꢀ, C-5, (4-Pe)Pro], 54.6 (+, C-2), 55.2 (+, C-2), 58.1 [+,
C-2’, (3-Ncp)Ala], 59.3 [+, C-2’, (3-Ncp)Ala], 59.8 (+, C-2), 60.0 (+, C-
2), 63.8 [+, C-2, (4-Pe)Pro], 103.6 (+, C-4, Chpca), 109.9 (+, C-3,
Chpca), 119.9 (Cquat, C-2, Chpca), 121.7 (Cquat, C-5, Chpca), 126.9, 127.3,
127.4, 127.6 (+, Ar-C), 127.87 [+, C-1’, (4-Pe)Pro], 127.92 [+, C-2’, (4-
Pe)Pro], 128.5, 128.7 (+, Ar-C), 141.3, 142.2 (Cquat, Ar-C), 159.3 (Cquat, C-
1, Chpca), 168.3, 169.6, 170.0, 170.2, 171.60, 171.62, 171.9 (Cquat, C-1); IR
(KBr): n˜ =3383, 2968, 2933, 2879, 1747, 1651, 1626, 1548, 1452, 1372,
1321, 1182 cmꢀ1; UV (MeOH): neutral: lmax(e)=277 (1.6ꢄ104) nm; basic:
281 (1.5ꢄ104), 205 (7.0ꢄ104) nm; acidic: 272 (1.4ꢄ104) nm; CD
(MeOH): lmax[V]=280.2 (2.01ꢄ104); 276.5 (2.05ꢄ104), 225.6 ꢀ4.55ꢄ104),
221.5 (ꢀ5.06ꢄ104) nm (c=1.45ꢄ10ꢀ5 m); MS (ESI): pos.: m/z (%): 1151
(100) [M+Na+]; neg.: m/z (%): 1127 (100) [MꢀHꢀ]; HRMS (ESI): m/z:
calcd for [C55H71N11O13Cl+]: 1128.4916; found 1128.4921.
[a-Dab1,a-Ile5]-Hormaomycin (epi-2a): A solution (50 mL) of the CHA
salt of Teoc-(2S,1’R,2’R)-(3-Ncp)AlaOH (27.3 mg, 65.39 mmol) in Et2O
(50 mL) was washed with 1m H2SO4 (3ꢄ5 mL), 1m KHSO4 (2ꢄ5 mL),
water (3ꢄ5 mL), brine (2ꢄ5 mL), dried, filtered and concentrated under
reduced pressure. The resulting N-protected amino acid was dried at
0.02 Torr for 2 h and then coupled with the depsipeptide, obtained after
deprotection of epi-19a (20.0 mg, 20.47 mmol) by treatment with 10%
anisole in TFA (1.1 mL) according to GP 6 for 2 h, applying HATU
(23.4 mg, 61.54 mmol), HOAt (8.3 mg, 61.42 mmol), DIEA (2.64 mg,
20.39 mmol) and TMP (22.36 mg, 183.6 mmol) in CH2Cl2 (3 mL) according
to GP 4 for 15 h. The mixture was then diluted with Et2O (40 mL), and
the crude product obtained after the usual aqueous work-up (GP 2) was
purified by two crystallizations from Et2O/hexane to give the respective
Teoc-(S)-(3-Ncp)Ala-epi-cyclohexapeptide (17.0 mg, 74%; Rf =0.19, ace-
tone/hexane 1:2.5) as a colorless glass which was used for the next step
without any characterization. This substance (17.0 mg, 14.50 mmol) was
deprotected by treatment with TFA (2.0 mL) for 1 h. The mixture was
concentrated under reduced pressure at 208C, and then taken up with
toluene (3ꢄ15 mL) which was distilled off to remove the last traces of
TFA. The resulting deprotected branched peptide was coupled with
Chpca(MOM)-OH 20 (5.5 mg, 26.74 mmol) applying HATU (10.15 mg,
26.69 mmol), DIEA (1.95 mg, 15.09 mmol) and TMP (12.37 mg,
102.08 mmol) in CH2Cl2 (3 mL) according to GP 4 for 5 h. The mixture
was then diluted with Et2O (40 mL), and the crude product obtained
after the usual aqueous work-up (GP 2) was recrystallized twice from
CH2Cl2/hexane to give the O-MOM protected [a-Dab1,a-Ile5]-hormaomy-
[a-Dab1]-Hormaomycin (2a): A solution of the CHA salt of Teoc-
(2S,1’R,2’R)-(3-Ncp)AlaOH (26.6 mg, 63.75 mmol) in Et2O (50 mL) was
washed with 1m H2SO4 (3ꢄ5 mL), 1m KHSO4 (2ꢄ5 mL), water (3ꢄ
5 mL), brine (2ꢄ5 mL), dried, filtered and concentrated under reduced
pressure. The resulting N-protected amino acid was dried at 0.02 Torr for
2 h and then coupled with the depsipeptide, obtained after deprotection
of 19a (19.5 mg, 19.96 mmol) by treatment with 10% anisole in TFA
(1.1 mL) according to GP 6 for 2 h, applying HATU (22.8 mg,
59.96 mmol), HOAt (8.1 mg, 59.94 mmol), DIEA (2.57 mg, 19.88 mmol)
and TMP (21.8 mg, 179.00 mmol) in CH2Cl2 (3 mL) according to GP 4 for
15 h. The mixture was then diluted with Et2O (40 mL), and the crude
product obtained after the usual aqueous work-up (GP 2) was purified by
preparative TLC (200ꢄ200 mm, acetone/hexane 1:2.7) to give the respec-
tive Teoc-(S)-(3-Ncp)Ala-cyclohexapeptide (21.6 mg, 96%; Rf =0.18, ace-
tone/hexane 1:2.5) as a colorless glass which was used for the next step
without any characterization. This substance (21.6 mg, 19.13 mmol) was
deprotected by treatment with TFA (2.0 mL) for 1 h. The mixture was
concentrated under reduced pressure at 208C and then taken up with tol-
uene (3ꢄ15 mL), which was distilled off to remove the last traces of
TFA. The resulting deprotected branched peptide was coupled with
Chpca(MOM)-OH 20 (7.0 mg, 34.04 mmol) by treatment with HATU
(12.9 mg, 33.93 mmol), DIEA (2.47 mg, 19.13 mmol) and TMP (12.37 mg,
102.08 mmol) in CH2Cl2 (3 mL) according to GP 4 for 5 h. The mixture
was then diluted with Et2O (40 mL) and the crude product obtained after
the usual aqueous work-up (GP 2) was purified by preparative TLC
(200ꢄ200 mm, acetone/hexane 1:2.7, two fold development) and finally
by recrystallization from Et2O/hexane to give the O-MOM protected [a-
Dab1]-hormaomycin (20.2 mg, 90%; Rf =0.09, acetone/hexane 1:3) as a
colorless solid which was used for the next step without any characteriza-
tion. MOM-2a (19.1 mg, 16.92 mmol) was deprotected appling MgBr2·
Et2O (164 mg, 633.89 mmol) and EtSH (0.018 mL, 243.07 mmol) in CH2Cl2
(10 mL) according to GP 7 for 3 h. The mixture was diluted with Et2O
(50 mL), and the crude product obtained after the usual aqueous work-
up (GP 7) was recrystallized from Et2O/pentane and then from CH2Cl2/
pentane to give 2a (15.4 mg, 84%, 68% over five steps from 19a) as a
colorless solid. Rf =0.14, acetone/hexane 1:2.5; analytical HPLC: gradient
25 ! 85% MeCN in 0.15% ammonium acetate buffer (pH 5.5) for
25 min, flow rate=0.5 mLminꢀ1, tR =21.72 min, purity > 99%; [a]D20
=
23.0 (c=0.1, CHCl3); 1H NMR (600 MHz, CDCl3): d=ꢀ0.69 [ddd, J=
6.6, 6.6, 6.6 Hz, 1H, 3’-Ha, (3-Ncp)Ala], ꢀ0.17–0.07 [m, 1H, 3-Ha, (3-
Ncp)Ala], 0.20–0.27 [m, 1H, 1’-H, (3-Ncp)Ala], 0.54 [ddd, J=14.4, 4.8,
4.8 Hz, 1H, 3-Hb, (3-Ncp)Ala], 0.90 (t, J=7.2 Hz, 3H, 5-H, Ile), 0.98–1.05
2942
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 2929 – 2945

Hormaomycin
FULL PAPER
cin (14.2 mg, 80%; Rf =0.11, acetone/hexane 1:3) as a colorless solid
which was used for the next step without any characterization. MOM-
epi-2a (14.2 mg, 12.11 mmol) was deprotected applying MgBr2·Et2O
(110 mg, 425.17 mmol) and EtSH (0.018 mL, 243.07 mmol) in CH2Cl2
(10 mL) according to GP 7 for 3 h. The mixture was taken up with Et2O
(50 mL), and the crude product obtained after the usual aqueous work-
up (GP 7) was recrystallized from Et2O/pentane and then from CH2Cl2/
pentane to give the crude product (13.1 mg), which was finally purified
by preparative HPLC to give epi-2a (9.0 mg, 39% over five steps from
epi-19a) as a colorless solid, which was insoluble in CHCl3. Rf =0.14, ace-
tone/hexane 1:2.5; preparative HPLC: column B, 62% MeCN in H2O
obtained after the usual aqueous work-up (GP 2) was purified by crystal-
lization first from CH2Cl2/pentane and then from Et2O/pentane to give
the respective Teoc-(S)-(3-Ncp)Ala-cyclohexapeptide (32.5 mg, 97%;
Rf =0.22, acetone/hexane 4:7) as a colorless solid, which was used for the
next step without any characterization. This substance (32.5 mg,
28.4 mmol) was deprotected by treatment with TFA (2.0 mL) for 1 h. The
mixture was concentrated under reduced pressure at 208C and then
taken up with toluene (3ꢄ15 mL) which was distilled off to remove the
last traces of TFA. The resulting deprotected branched peptide was cou-
pled with Chpca(MOM)-OH 20 (14.6 mg, 71.1 mmol) applying HATU
(25.9 mg, 68.2 mmol), DIEA (3.67 mg, 28.4 mmol) and TMP (26.0 mg,
213.0 mmol) in CH2Cl2 (3 mL) according to GP 4 for 5 h. The mixture
was then diluted with Et2O (40 mL), and the crude product obtained
after the usual aqueous work-up (GP 2) was purified by chromatography
(Rf =0.39, acetone/hexane 1:1.5) to give the O-MOM protected [a-NbMe-
Dab1]-hormaomycin (25.0 mg, 74%) as a colorless solid, which was used
for the next step without any characterization. MOM-2b (25.0 mg,
21.1 mmol) was deprotected by treatment with MgBr2·Et2O (150 mg,
579.7 mmol) and EtSH (0.015 mL, 202.6 mmol) in CH2Cl2 (10 mL) accord-
ing to GP 7 for 3.5 h. The mixture was diluted Et2O (50 mL), and the
crude product obtained after the usual aqueous work-up (GP 7) was re-
crystallized from CH2Cl2/pentane to give a crude product (22.0 mg),
which was finally purified by preparative HPLC to give 2b (16.0 mg,
48% over five steps) as a colorless solid. Preparative HPLC: column B,
69% MeCN in H2O (0.1% TFA), flow rate=2.5 mLminꢀ1; analytical
HPLC: 70% MeCN in H2O (0.1% TFA), flow rate=0.5 mLminꢀ1, tR =
(0.07% TFA), flow rate=2.5 mLminꢀ1
column, the same conditions, tR =17.72 min, purity
;
analytical HPLC: the same
>
99%; 1H NMR
(600 MHz, CD3OD): d=0.69 (t, J=7.2 Hz, 3H, 5-H, a-Ile), 0.72 (d, J=
7.2 Hz, 3H, 1’-H, a-Ile), 0.82–0.92 (m, 1H, 4-Ha, a-Ile), 0.95 (ddd, J=7.2,
7.2, 7.2 Hz, 1H, 4-Hb, Ile), 1.00 [ddd, J=6.0, 6.0, 6.0 Hz, 1H, 3’-Ha, (3-
Ncp)Ala], 1.07 [ddd, J=6.6, 6.6, 6.6 Hz, 1H, 3’-Ha, (3-Ncp)Ala], 1.14 [d,
J=6.6 Hz, 3H, 4-H, (bMe)Phe], 1.25 [d, J=7.2 Hz, 3H, 4-H, (bMe)Phe],
1.31 (d, J=7.2 Hz, 3H, a-Dab), 1.34–1.41 [m, 1H, 3-Ha, (3-Ncp)Ala],
1.44–1.52 [m, 1H, 3-Ha, (3-Ncp)Ala], 1.54–1.60 [m, 1H, 3’-Hb, (3-
Ncp)Ala], 1.65 [dd, J=7.2, 1.8 Hz, 3H, 3’-H, (4-Pe)Pro], 1.82 [ddd, J=
7.8, 7.8, 7.8 Hz, 1H, 3-Ha, (4-Pe)Pro], 1.93–2.01 [m, 2H, 3-Hb, 1’-H, (3-
Ncp)Ala], 2.03–2.15 [m, 2H, 3-Hb, (3-Ncp)Ala, 3-Hb, (4-Pe)Pro], 2.98
[dd, J=10.8, 10.8 Hz, 1H, 5-Ha, (4-Pe)Pro], 3.07 [m, 1H, 4-H, (4-
Pe)Pro], 3.13–3.24 [m, 2H, 2ꢄ3-H, (bMe)Phe], 3.69 [dd, J=10.8, 7.2 Hz,
1H, 5-Hb, (4-Pe)Pro], 4.08 [ddd, J=6.6, 3.0, 3.0 Hz, 1H, 2’-H, (3-
Ncp)Ala], 4.10–4.14 (m, 1H, 2-H, a-Ile), 4.19–4.29 [m, 3H, 3-H, a-Dab, 2-
H, 2’-H, (3-Ncp)Ala], 4.40 [d, J=10.8 Hz, 2-H, (bMe)Phe], 4.49 (d, J=
3.6 Hz, 1H, 2-H, a-Dab), 4.51 [dd, J=7.8, 7.8 Hz, 1H, 2-H, (4-Pe)Pro],
4.77 [d, J=11.4 Hz, 1H, 2-H, (bMe)Phe], 4.79–4.82 [m, 1H, 2-H, (3-
Ncp)Ala], 5.38–5.44 [m, 1H, 1’-H, (4-Pe)Pro], 5.59 [dq, J=10.2, 7.2 Hz,
1H, 2’-H, (4-Pe)Pro], 6.00 (d, J=4.5 Hz, 1H, 4-H, Chpca), 6.73 (d, J=
4.5 Hz, 1H, 3-H, Chpca), 7.01–7.15 (m, 1H, Ar-H), 7.17–7.25 (m, 3H, Ar-
H), 7.25–7.31 (m, 6H, Ar-H), 7.41–7.49 (br, 1H, NH), 7.80–7.84 (br, 1H,
NH); the signal of 1’-H, (3-Ncp)Ala was masked by absorption of 4-H, a-
Dab and the signals of 3-H, a-Ile and 3’-Hb, (3-Ncp)Ala were masked by
absorption of 3’-H, (4-Pe)Pro; 13C NMR (150.8 MHz, CD3OD): d=12.2
(+, C-5, a-Ile), 13.3 [+, C-3’, (4-Pe)Pro], 14.6 (+, C-1’, a-Ile), 17.9 [ꢀ, C-
3’, (3-Ncp)Ala], 18.0 [+, C-4, (bMe)Phe], 18.9 [ꢀ, C-3’, (3-Ncp)Ala],
19.1 (+, C-4, a-Dab), 19.4 [+, C-4, (bMe)Phe], 23.6 [+, C-1’, (3-
Ncp)Ala], 23.8 [+, C-1’, (3-Ncp)Ala], 27.3 (ꢀ, C-4, a-Ile), 34.06 [ꢀ, C-3,
(3-Ncp)Ala], 34.15 [ꢀ, C-3, (3-Ncp)Ala], 34.18 [ꢀ, C-3, (4-Pe)Pro], 36.5
(+, C-3, a-Ile), 37.7 [+, C-4, (4-Pe)Pro], 41.6 [+, C-3, (bMe)Phe], 43.4
[+, C-3, (bMe)Phe], 48.9 (+, C-3, a-Dab), 51.0 [+, C-2, (3-Ncp)Ala],
53.2 [ꢀ, C-5, (4-Pe)Pro], 53.3 [+, C-2’, (3-Ncp)Ala], 53.9 [+, C-2, (3-
Ncp)Ala], 55.9 (+, C-2, a-Ile), 58.4 (+, C-2, a-Dab), 59.5 [+, C-2,
(bMe)Phe], 60.2 [+, C-2, (3-Ncp)Ala], 60.3 [+, C-2’, (3-Ncp)Ala], 61.3
[+, C-2, (4-Pe)Pro], 62.0 [+, C-2, (bMe)Phe], 104.0 (+, C-4, Chpca),
10.00 min, purity
>
99%; Rf =0.24, acetone/hexane 1:1.5; [a]2D0 =75.0
(c=0.15, MeOH); 1H NMR (600 MHz, CDCl3): d=ꢀ0.01–0.16 [m, 2H,
3-Ha, 3’-Ha, (3-Ncp)Ala], 0.64–0.76 [m, 1H, 1’-H, (3-Ncp)Ala], 0.81 (t,
J=7.2 Hz, 3H, 5-H, Ile), 1.03 (d, J=6.6 Hz, 3H, 1’-H, Ile), 1.15–1.25 (m,
1H, 4-Ha, Ile), 1.26 [d, J=6.6 Hz, 3H, 4-H, (bMe)Phe], 1.37 [d, J=
7.2 Hz, 3H, 4-H, (bMe)Phe], 1.45–1.53 (m, 1H, 4-Hb, Ile), 1.55 (d, J=
7.2 Hz, 3H, a-NbMe-Dab), 1.66 [dd, J=6.6, 1.2 Hz, 3H, 3’-H, (4-Pe)Pro],
1.70–1.79 [m, 3H, 3-H, (3-Ncp)Ala, 3-Ha, (4-Pe)Pro], 1.83–1.88 [m, 1H,
3’-Hb, (3-Ncp)Ala], 1.94–2.02 [m, 1H, 3-H, Ile], 2.03–2.10 [m, 1H, 1’-H,
(3-Ncp)Ala], 2.17–2.23 [m, 1H, 4-Hb, (4-Pe)Pro], 2.85 [dq, J=9.6, 6.6 Hz,
1H, 3-H, (bMe)Phe], 3.10 (s, 3H, NMe, a-NbMe-Dab), 3.20–3.34 [m, 3H,
4-H, 5-Ha, (4-Pe)Pro, 2’-H, (3-Ncp)Ala], 3.54–3.60 [m, 1H, 2-H, (3-
Ncp)Ala], 3.70 [dq, J=5.2, 7.2 Hz, 1H, 3-H, (bMe)Phe], 4.02 [dd, J=7.2,
7.2 Hz, 1H, 5-Hb, (4-Pe)Pro], 4.19–4.24 [m, 1H, 2’-H, (3-Ncp)Ala], 4.50
[dd, J=9.6 Hz, 1H, 2-H, (bMe)Phe], 4.52 [dd, J=12.0, 5.2 Hz, 1H, 2-H,
(bMe)Phe], 4.59–4.64 [m, 4H, 2-H, Ile, 2-H, 3-H, a-NbMe-Dab, 2-H, (4-
Pe)Pro], 4.98–5.02 [m, 1H, 2-H, (3-Ncp)Ala], 5.24–5.29 [m, 1H, 1’-H, (4-
Pe)Pro], 5.60 [dq, J=11.2, 6.6 Hz, 1H, 2’-H, (4-Pe)Pro], 5.98–6.01 (m,
1H, 4-H, Chpca), 6.60–6.67 (m, 1H, 3-H, Chpca), 6.64–6.71 (br, 1H,
NH), 7.02–7.08 (m, 2H, Ar-H), 7.10–7.19 (m, 4H, Ar-H, NH), 7.16–7.20
(m, 2H, Ar-H), 7.22–7.28 (m, 4H, Ar-H, NH), 7.34–7.40 (br, 1H, NH),
7.40–7.47 (br, 1H, NH), 7.98–8.10 (br, 1H, NH), 8.52–8.62 (br, 1H, NH),
12.0–13.2 (br, 1H, OH); the signals of 3-Hb, (3-Ncp)Ala and 3’-Hb, (3-
Ncp)Ala was masked by absorption of 1’-H, Ile and the signal of 3’-Hb,
(3-Ncp)Ala by absorption of 4-H, (bMe)Phe (1.26 ppm); 13C NMR
(150.8 MHz, CDCl3): d=10.4 (+, C-5, Ile), 13.26 [+, C-4, (bMe)Phe],
13.31 [+, C-3’, (4-Pe)Pro], 15.4 (+, C-1’, Ile), 16.6 (+, C-4, a-NbMe-
Dab), 17.0 [ꢀ, C-3’, (3-Ncp)Ala], 17.2 [ꢀ, C-3’, (3-Ncp)Ala], 18.0 [+, C-
4, (bMe)Phe], 20.7 [+, C-1’, (3-Ncp)Ala], 21.9 [+, C-1’, (3-Ncp)Ala],
24.7 (ꢀ, C-4, Ile), 32.4 (+, NMe, a-NbMe-Dab), 32.6 [ꢀ, C-3, (3-
Ncp)Ala], 33.5 [ꢀ, C-3, (3-Ncp)Ala], 34.8 [ꢀ, C-3, (4-Pe)Pro], 36.6 (+,
C-3, Ile), 36.8 [+, C-4, (4-Pe)Pro], 39.1 [+, C-3, (bMe)Phe], 43.7 [+, C-
3, (bMe)Phe], 50.6 [+, C-2, (3-Ncp)Ala], 52.3 (+, C-3, a-NbMe-Dab),
52.60 [+, C-2, (3-Ncp)Ala], 52.65 [ꢀ, C-5, (4-Pe)Pro], 54.9 (+, C-2, Ile),
58.6 [+, C-2’, (3-Ncp)Ala], 58.8 (+, C-2, a-NbMe-Dab), 58.9 [+, C-2,
(bMe)Phe], 59.4 [+, C-2’, (3-Ncp)Ala], 59.5 [+, C-2, (bMe)Phe], 59.6
[+, C-2, (4-Pe)Pro], 103.1 (+, C-4, Chpca), 108.5 (+, C-3, Chpca), 117.7
(Cquat, C-2, Chpca), 119.0 (Cquat, C-5, Chpca), 126.8, 127.0, 127.3, 127.6 (+,
Ar-C), 127.8 [+, C-1’, (4-Pe)Pro], 128.0 [+, C-2’, (4-Pe)Pro], 128.44 (ꢄ
2) (+, Ar-C), 141.9, 142.3 (Cquat, Ar-C), 160.1 (Cquat, C-1, Chpca), 169.8,
170.0, 170.2, 170.7, 170.8, 171.2, 174.2 (Cquat, C-1); IR (KBr): n˜ =3383,
2968, 2935, 2877, 1634, 1543, 1440, 1368, 1311, 1263, 1212, 1129 cmꢀ1; UV
(MeOH): neutral: lmax(e)=277 (1.5ꢄ104) nm; basic: 281 (1.3ꢄ104), 205
111.1 (+, C-3, Chpca), 119.2 (Cquat, C-2, Chpca), 122.1 (Cquat, C-5,
Chpca), 127.5 [+, C-2’, (4-Pe)Pro], 127.8, 128.2, 128.95, 128.99, 129.59,
129.67 (+, Ar-C), 130.8 [+, C-1’, (4-Pe)Pro], 143.6, 144.5 (Cquat, Ar-C),
161.7 (Cquat, C-1, Chpca), 171.3, 172.0, 172.2, 172.7, 172.87, 172.90, 174.3
(Cquat, C-1); IR (KBr): n˜ =3445, 2926, 2850, 1653, 1558, 1543, 1458, 1383,
1321, 1020 cmꢀ1; UV (MeOH): neutral: lmax(e)=279 (8.3ꢄ103) nm; basic:
283 (8.0ꢄ103), 209 (2.3ꢄ104) nm; acidic: 271 (8.7ꢄ103) nm; CD
(MeOH): lmax[V]=279.6 (1.15ꢄ104); 275.7 (1.08ꢄ104), 225.3 (ꢀ3.85ꢄ104)
nm (c=1.26ꢄ10ꢀ5 m); MS (ESI): pos.: m/z (%): 1151 (100) [M+Na+],
1129 (52) [M+H+]; neg.: m/z (%): 1127 (100) [MꢀHꢀ].
[a-NbMe-Dab1]-Hormaomycin (2b): A solution of the CHA salt of Teoc-
(2S,1’R,2’R)-(3-Ncp)AlaOH (40.3 mg, 96.5 mmol) in Et2O (50 mL) was
washed with 1m H2SO4 (3ꢄ5 mL), 1m KHSO4 (2ꢄ5 mL), water (3ꢄ
5 mL), brine (2ꢄ5 mL), dried, filtered and concentrated under reduced
pressure. The resulting N-protected amino acid was dried at 0.02 Torr for
2 h and then coupled with the peptolide, obtained after deprotection of
19b (29.0 mg, 29.3 mmol) by treatment with 10% anisole in TFA
(1.5 mL) according to GP 6 for 2 h, applying HATU (33.3 mg,
87.8 mmol), HOAt (13.0 mg, 96.5 mmol), DIEA (3.78 mg, 29.3 mmol) and
TMP (31.9 mg, 263.3 mmol) in CH2Cl2 (3 mL) according to GP 4 for 15 h.
The mixture was then diluted with Et2O (40 mL), and the crude product
Chem. Eur. J. 2005, 11, 2929 – 2945
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2943

C. Griesinger, A. Zeeck, A. de Meijere et al.
(7.0ꢄ104) nm; acidic: 273 (1.6ꢄ104) nm; CD (MeOH): lmax[V]=278.8
(3.4ꢄ104); 229.0 (ꢀ2.54ꢄ104) nm (c=2.1ꢄ10ꢀ5 m); MS (ESI): pos.: m/z
(%): 1165 (100) [M+Na+]; neg.: m/z (%): 1141 (100) [MꢀHꢀ]; HRMS
(ESI): m/z: calcd for [C56H73N11O13Cl+]: 1142.5072; found 1142.5072.
Ile), 13.2 [+, C-4, (bMe)Phe], 13.3 [+, C-3’, (4-Pe)Pro], 14.8 (+, C-1’,
Ile), 16.9 [ꢀ, C-3’, (3-Ncp)Ala], 17.1 [ꢀ, C-3’, (3-Ncp)Ala], 17.5 [+, C-4,
(bMe)Phe], 20.0 [+, C-1’, (3-Ncp)Ala], 21.6 [+, C-1’, (3-Ncp)Ala], 25.1
(ꢀ, C-4, Ile), 32.9 [ꢀ, C-3, (3-Ncp)Ala], 35.0 [ꢀ, C-3, (3-Ncp)Ala], 35.7
[ꢀ, C-3, (4-Pe)Pro], 36.3 [+, C-4, (4-Pe)Pro], 37.8 (+, C-3, Ile), 38.0 (ꢀ,
C-3, Dap), 39.1 [+, C-3, (bMe)Phe], 41.6 [+, C-3, (bMe)Phe], 50.9 [+,
C-2, (3-Ncp)Ala], 51.8 (+, C-2, Dap), 52.0 [+, C-2, (3-Ncp)Ala], 53.0 [ꢀ,
C-5, (4-Pe)Pro], 54.5 (+, C-2, Ile), 58.0 [+, C-2’, (3-Ncp)Ala], 59.2 [+,
C-2’, (3-Ncp)Ala], 60.0 [+, C-2, (bMe)Phe], 60.3 [+, C-2, (bMe)Phe],
63.5 [+, C-2, (4-Pe)Pro], 103.6 (+, C-4, Chpca), 109.8 (+, C-3, Chpca),
119.9 (Cquat, C-2, Chpca), 121.6 (Cquat, C-5, Chpca), 126.9, 127.3, 127.4,
127.6 (+, Ar-C), 127.8 [+, C-1’, (4-Pe)Pro], 127.9 [+, C-2’, (4-Pe)Pro],
128.5, 128.6 (+, Ar-C), 141.3, 142.1 (Cquat, Ar-C), 159.2 (Cquat, C-1,
Chpca), 168.4, 169.5, 170.3, 170.8, 171.7, 172.4, 172.5 (Cquat, C-1); IR
(KBr): n˜ =3347, 2968, 2933, 2877, 1625, 1544, 1428, 1368, 1260,
1210 cmꢀ1; UV (MeOH): neutral: lmax(e)=277 (1.6ꢄ104), 209 (5.6ꢄ104)
nm; basic: 280 (1.7ꢄ104), 211 (5.6ꢄ104) nm; acidic: 273 (1.6ꢄ104), 208
(5.6ꢄ104) nm; CD (MeOH): lmax[V]=276.0 (3.31ꢄ104); 222.6 (ꢀ3.47ꢄ
104), 210.8 (ꢀ5.67ꢄ103) nm (c=2.9ꢄ10ꢀ5 m); MS (ESI): pos.: m/z (%):
1137 (100) [M+Na+], 1115 (32) [M+H+]; neg.: m/z (%): 1113 (72)
[MꢀHꢀ]; HRMS (ESI): m/z: calcd for [C54H69N11O13Cl+]: 1114.4759;
found 1114.4760.
[Dap1]-Hormaomycin (2c):
A solution of the CHA salt of Teoc-
(2S,1’R,2’R)-(3-Ncp)AlaOH (40.6 mg, 97.3 mmol) in Et2O (50 mL) was
washed with 1m H2SO4 (3ꢄ5 mL), 1m KHSO4 (2ꢄ5 mL), water (3ꢄ
5 mL), brine (2ꢄ5 mL), dried, filtered and concentrated under reduced
pressure. The resulting N-protected amino acid was dried at 0.02 Torr for
2 h and then coupled with the peptolide, obtained after deprotection of
19c (28.4 mg, 29.5 mmol) by treatment with 10% anisole in TFA (1.5 mL)
according to GP 6 for 2 h, applying HATU (33.6 mg, 88.5 mmol), HOAt
(13.2 mg, 97.3 mmol), DIEA (3.81 mg, 29.5 mmol) and TMP (32.2 mg,
265.4 mmol) in CH2Cl2 (3 mL) according to GP 4 for 15 h. The mixture
was then diluted with Et2O (40 mL), and the crude product obtained
after the usual aqueous work-up (GP 2) was purified by crystallization
first from CH2Cl2/pentane and then from Et2O/pentane to give the re-
spective Teoc-(S)-(3-Ncp)Ala-cyclohexapeptide (30.2 mg, 92%; Rf =0.25,
acetone/hexane 4:7) as a colorless solid, which was used for the next step
without any characterization. This substance (30.2 mg, 27.1 mmol) was de-
protected by treatment with TFA (2.0 mL) for 1 h. The mixture was con-
centrated under reduced pressure at 208C and then taken up with tolu-
ene (3ꢄ15 mL), which was distilled off to remove the last traces of TFA.
The resultant deprotected branched peptide was coupled with Chpca-
(MOM)-OH 20 (13.9 mg, 67.7 mmol) applying HATU (23.7 mg,
62.3 mmol), DIEA (3.50 mg, 27.1 mmol) and TMP (25.0 mg, 203.1 mmol)
in CH2Cl2 (3 mL) according to GP 4 for 5 h. The mixture was then dilut-
ed with Et2O (40 mL), and the crude product obtained after the usual
aqueous work-up (GP 2) was purified by chromatography (acetone/
hexane 1:1.5) to give the O-MOM protected [Dap1]-hormaomycin
(22.0 mg, 70%; Rf =0.10, acetone/hexane 4:7) as a colorless solid, which
was used for the next step without any characterization. MOM-2c
(22.0 mg, 21.1 mmol) was deprotected by treatment with MgBr2·Et2O
(150 mg, 579.7 mmol) and EtSH (0.015 mL, 202.6 mmol) in CH2Cl2
(10 mL) according to GP 7 for 3.5 h. The mixture was diluted Et2O
(50 mL), and the crude product obtained after the usual aqueous work-
up (GP 7) was recrystallized from CH2Cl2/pentane to give a crude prod-
uct (21.5 mg), which was finally purified by preparative HPLC to give 2c
(14.2 mg, 43% over five steps) as a colorless solid. Preparative HPLC:
column B, 70% MeCN in H2O (0.1% TFA), flow rate=2.5 mLminꢀ1; an-
alytical HPLC: 70% MeCN in H2O (0.1% TFA), flow rate=
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (SFB-
416, Projects A3, B5 and ZS), the Max-Planck-Gesellschaft and the
Fonds der Chemischen Industrie. The authors are indebted to Dr. H.
Frauendorf (Gçttingen) for performing the HPLC/MS experiments, to
Mr. H.-P. Kroll (Gçttingen) for excellent technical assistance and to Dr.
B. Knieriem (Gçttingen) for his careful proofreading of the final manu-
script.
[1] a) N. Andres, H. Wolf, H. Zꢁhner, E. Rçssner, A. Zeeck, W. A.
Kçnig, V. Sinnwell, Helv. Chim. Acta 1989, 72, 426–437; b) E. Rçss-
ner, A. Zeeck, W. A. Kçnig, Angew. Chem. 1990, 102, 84–85;
Angew. Chem. Int. Ed. Engl. 1990, 29, 64–65.
0.5 mLminꢀ1, tR =9.27 min, purity
> 99%; Rf =0.10 (acetone/hexane
4:7); [a]2D0 =61.0 (c=0.10, MeOH); 1H NMR (600 MHz, CDCl3): d=
ꢀ0.60 [ddd, J=6.6, 6.6, 6.6 Hz, 1H, 3’-Ha, (3-Ncp)Ala], ꢀ0.20–0.02 [m,
1H, 3-Ha, (3-Ncp)Ala], 0.25–0.31 [m, 1H, 1’-H, (3-Ncp)Ala], 0.52 [ddd,
J=13.8, 4.8, 4.8 Hz, 1H, 3-Hb, (3-Ncp)Ala], 0.89 (t, J=7.2 Hz, 3H, 5-H,
Ile), 0.98–1.05 [m, 2H, 3’-Ha, 3’-Hb, (3-Ncp)Ala], 1.07 (d, J=7.2 Hz, 3H,
1’-H, Ile), 1.30 [d, J=7.2 Hz, 3H, 4-H, (bMe)Phe], 1.40 [d, J=7.2 Hz,
3H, 4-H, (bMe)Phe], 1.54–1.60 (m, 1H, 4-Hb, Ile), 1.67 [dd, J=6.6 Hz,
3H, 3’-H, (4-Pe)Pro], 1.67–1.75 [m, 2H, 3-H, (3-Ncp)Ala], 1.84–1.93 [m,
3H, 3’-Hb, (3-Ncp)Ala, 3-H, Ile, 3-Ha, (4-Pe)Pro], 1.95–2.01 [m, 1H, 3-Ha,
(4-Pe)Pro], 2.23 [ddd, J=12.0, 6.0, 6.0 Hz, 1H, 4-Hb, (4-Pe)Pro], 2.87
[ddd, J=6.6, 3.0, 3.0 Hz, 1H, 2’-H, (3-Ncp)Ala], 3.04 [dq, J=10.5, 7.2 Hz,
1H, 3-H, (bMe)Phe], 3.18–3.30 [m, 2H, 4-H, 5-Ha, (4-Pe)Pro], 3.33 (d,
J=13.8 Hz, 1H, 3-Ha, Dap), 3.49 [ddd, J=7.2, 3.6 Hz, 1H, 2-H, (3-
Ncp)Ala], 3.68 [dq, J=4.8, 7.2 Hz, 1H, 3-H, (bMe)Phe], 3.93 [dd, J=
12.0, 5.4 Hz, 2-H, (4-Pe)Pro], 3.94–3.98 [m, 1H, 5-Hb, (4-Pe)Pro], 4.04
[ddd, J=7.2, 3.6, 3.6 Hz, 1H, 2’-H, (3-Ncp)Ala], 4.16 (dddd, J=13.8,
10.8, 3.0, 3.0 Hz, 1H, 3-Hb, Dap), 4.33 [dd, J=10.5, 10.5 Hz, 1H, 2-H,
(bMe)Phe], 4.47 [dd, J=9.6, 4.8 Hz, 1H, 2-H, (bMe)Phe], 4.50 (dd, J=
9.0, 3.0 Hz, 1H, 2-H), 4.60–4.68 (m, 2H, 2-H, Ile, 2-H, Dap), 5.14–5.20
[m, 1H, 2-H, (3-Ncp)Ala], 5.24–5.30 [m, 1H, 1’-H, (4-Pe)Pro], 5.61 [dq,
J=10.8, 6.6 Hz, 1H, 2’-H, (4-Pe)Pro], 6.15 (d, J=4.8 Hz, 1H, 4-H,
Chpca), 6.46 (d, J=6.6 Hz, 1H, NH), 6.78–6.83 (br, 1H, NH), 6.83 (d, J=
4.8 Hz, 1H, 3-H, Chpca), 7.02–7.06 (m, 2H, Ar-H), 7.10–7.19 (m, 6H, Ar-
H, NH), 7.20–7.24 (m, 5H, Ar-H, NH), 7.32 (d, J=9.0 Hz, 1H, NH), 8.17
(d, J=7.8 Hz, 1H, NH), 8.75 (d, J=8.4 Hz, 1H, NH), 10.75–11.15 (br,
1H, OH); The signal of 4-Ha, Ile was masked by absorption of C-4,
(bMe)Phe (1.30 ppm); 13C NMR (150.8 MHz, CDCl3): d=10.3 (+, C-5,
[2] For the final elucidation of the structure of hormaomycin 1, see:
a) B. D. Zlatopolskiy, K. Loscha, P. Alvermann, S. I. Kozhushkov,
S. V. Nikolaev, A. Zeeck, A. de Meijere, Chem. Eur. J. 2004, 10,
4708–4717; for its total synthesis see: b) B. D. Zlatopolskiy, A. de
Meijere, Chem. Eur. J. 2004, 10, 4718–4727. The formula 1 as pre-
sented here depicts the correct absolute configuration in the (S)-iso-
leucine (Ile) moiety. The formulas in our recently published corri-
gendum in Chem. Eur. J. 2004, 10, 5568, were indeed erroneous in
this respect.
[3] K. Otoguro, H. Ui, A. Ishiyama, N. Arai, M. Kobayashi, Y. Takaha-
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56, 322–324.
[4] a) M. Brandl, S. I. Kozhushkov, B. D. Zlatopolskiy, B. Geers, P. Al-
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125; b) S. I. Kozhushkov, B. D. Zlatopolskiy, M. Brandl, M. Radzom,
B. Geers, P. Alvermann, A. Zeeck, A. de Meijere, Eur. J. Org.
Chem. 2005, in press; c) B. D. Zlatopolskiy, M. Radzom, A. Zeeck,
A. de Meijere, unpublished results.
[5] P. Alvermann, PhD Thesis, Universitꢁt Gçttingen, 2001.
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[8] D. D. Hennings, R. M. Williams, Synthesis 2000, 1310–1314.
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Org. Chem. 2004, 4492–4502.
2944
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Chem. Eur. J. 2005, 11, 2929 – 2945

Hormaomycin
FULL PAPER
[11] It is noteworthy that the NMR spectra measured in CDCl3 and
C2D2Cl4 of the acid 15b (as well as of the corresponding methyl
ester) showed the presence of at least four conformers instead of
the usual two as in the case of the peptides 15a, 15c, MeZ-a-Thr-
[Boc-(4-Pe)Pro-OH] (and of the corresponding methyl esters) due
to the hindered rotation around the tertiary urethane bond of Boc-
(4-Pe)Pro. This is presumed to be attributable to the additionally
hindered rotation around the tertiary amide bond between (4-
Pe)Pro and a-NbMe-Dab fragments. A complicated temperature de-
pendence for the spectra measured in C2D2Cl4 was also observed.
On heating up to 1008C, the coalescence point was almost reached,
but spectra measured at 1258C again showed the presence of several
conformers; as already at this temperature the compound started to
decompose, no attempts were made to measure spectra at higher
temperatures.
[18] a) R. Schwyzer, J. P. Garriꢆn, B. Gorup, H. Nolting, A. Tun-Kyi,
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[22] Some hormaomycin analogues show interesting shifts of their antibi-
otic activities.[4c] Consequently, the antibiotic activities of all synthe-
sized hormaomycin analogues against E. coli, B. subtilis, S. aureus,
C. albicans were also tested. Analogue 2c was more active against S.
aureus species than hormaomycin 1. Nevertheless, its activity was
lower than that of penicillin G.
[23] Cyclopeptides obtained after deprotection of 19a–c, epi-19a and
epi-19c as well as the N-MeZ protected ring part of hormaomycin 1
were also tested (as trifluoroacetates), but were all totally inactive.
[24] L. A. Carpino, Patent US5580981, 1996.
[12] See: A. F. Abdel-Magid, J. H. Cohen, C. A. Maryanoff, R. D. Shah,
F. J. Villani, F. Zhang, Tetrahedron Lett. 1998, 39, 3391–3394.
[13] See Supporting Information for details.
[14] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397–4398.
[15] Determined according to the advanced Marfey method (cf.: K.
Fujii, Y. Ikai, T. Mayumi, H. Oka, M. Suzuki, K. Harada, Anal.
Chem. 1997, 69, 3346–3352; Y. Suzuki, M. Ojika, Y. Sakagami, K.
Kaida, R. Fudou, T. Kameyama, J. Antibiot. 2001, 54, 22–28) after
acidic hydrolysis of the appropriate peptides. See Supporting Infor-
mation for details.
[25] a) B. D. Zlatopolski, Dissertation, Universitꢁt Gçttingen, 2003;
b) Supporting Information.
[26] T. L. Hwang, A. J. Shaka, J. Am. Chem. Soc. 1992, 114, 3157–3159.
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Genest, A. T. Hagler, Proteins Struct. Funct. Genet. 1988, 4, 31–47.
[28] M. Nilges, G. M. Clore, A. M. Gronenborn, FEBS Lett. 1988, 229,
317–324.
[16] For preliminary assignments of 1H and 13C NMR spectra of 1, see:
a) P. Henne, Dissertation, Universitꢁt Gçttingen, 1994; b) B. Geers,
Dissertation, Universitꢁt Gçttingen, 1998.
Received: September 24, 2004
[17] D. E. Dorman, F. A. Bovey, J. Org. Chem. 1973, 38, 2379–2383.
Published online: March 7, 2005
Chem. Eur. J. 2005, 11, 2929 – 2945
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2945