Synthesis and conformational analysis of stevastelin C3 analogues and their activity against the dual-specific vaccina H1-related phosphatase

Article abstract of DOI:10.1002/chem.200800692

The biological activity of macrocyclic natural products depends on their conformational properties. For both the elucidation of enzyme binding affinities as well as the development of selective drugs, rigid macrocyclic scaffolds carry high potential. In this study, 13-membered cyclodepsipeptides based on the structure of naturally occurring stevastelins were studied in detail. Six diastereomeric stevastelin C3 analogues and four phosphorylated derivatives were synthesized. The synthesis of linear precursors was achieved on solid support by starting from stereoisomerically pure 2-methyl-3-hydroxy acids. Subsequent macro-lactamization gave the cyclic depsipeptides in very good yields (36-62%). The conformational space of these stevastelin C3 analogues was computationally investigated. On the basis of NMR spectroscopic data, homogeneous conformations were determined for each benzylated depsipeptide and the influence of phosphorylation on the overall conformation was investigated. Importantly, phosphorylation was found to significantly weaken the conformational preferences of the 13-membered depsipeptides. Finally, the cyclic depsipeptides were tested for activity against phosphatases. Inhibitory activity on vaccina H1-related phosphatase was observed depending on the derivatization of the cycles. The activity profiles are discussed in the light of the structural data.

Full text of DOI:10.1002/chem.200800692

FULL PAPER
DOI: 10.1002/chem.200800692
Synthesis and Conformational Analysis of Stevastelin C3 Analogues and
Their Activity Against the Dual-Specific Vaccina H1-Related Phosphatase
Nicola Bisek,[a, b] Stefan Wetzel,[a, b] Hans-Dieter Arndt,*[a, b] and Herbert Waldmann*[a, b]
Abstract: The biological activity of
macrocyclic natural products depends
on their conformational properties. For
both the elucidation of enzyme binding
affinities as well as the development of
selective drugs, rigid macrocyclic scaf-
folds carry high potential. In this study,
13-membered cyclodepsipeptides based
on the structure of naturally occurring
stevastelins were studied in detail. Six
diastereomeric stevastelin C3 ana-
logues and four phosphorylated deriva-
tives were synthesized. The synthesis of
linear precursors was achieved on solid
support by starting from stereoisomeri-
cally pure 2-methyl-3-hydroxy acids.
Subsequent macro-lactamization gave
the cyclic depsipeptides in very good
yields (36–62%). The conformational
space of these stevastelin C3 analogues
was computationally investigated. On
the basis of NMR spectroscopic data,
homogeneous conformations were de-
termined for each benzylated depsipep-
tide and the influence of phosphoryla-
tion on the overall conformation was
investigated. Importantly, phosphoryla-
tion was found to significantly weaken
the conformational preferences of the
13-membered depsipeptides. Finally,
the cyclic depsipeptides were tested for
activity against phosphatases. Inhibito-
ry activity on vaccina H1-related phos-
phatase was observed depending on
the derivatization of the cycles. The ac-
tivity profiles are discussed in the light
of the structural data.
Keywords: biological activity
conformation analysis cyclo-
depsipeptides · macrocyclization ·
phosphorylation
·
·
AHCTUNGTRENNUNG
Introduction
cursors of membrane lipoproteins in bacterial cell walls and
thereby promotes cell death. In further biological studies, it
has been found to be a specific inhibitor of lipoprotein
signal peptidase II.[3]
Naturally occurring cyclic depsipeptides provide a rich
source of structurally interesting compounds endowed with
various biological activities. Prominent representatives even
entered clinical trials. Among them kahalalide F (1)[1] has
been shown to be a potent anticancer agent acting on cell ly-
sosomes and is currently undergoing phase II clinical trials.
Another relevant example is globomycin (2).[2] This cyclic
lipodepsipeptide antibiotic leads to the accumulation of pre-
However, within numerous cyclic depsipeptides only 39
13-membered rings can be found in the dictionary of natural
products, representing seven different compound classes.[4]
Lydiamycin A (3) has shown antibacterial activity against a
series of mycobacteria, including Mycobacterium tuberculo-
sis.[5] The beauveriolides I and III (4, 5) have an antiathero-
sclerotic effect in vivo and show promise as potential lead
compounds for therapeutic agents.[6] Apart from antibacteri-
al and antitumoral activities, some cyclic depsipeptides have
shown immunosuppressive properties. Among them, the ste-
vastelins (6–12; stevastelin A (6), B (7), A3 (8), B3 (9), C3
(10), D3 (11), E3 (12)) were identified, which have been iso-
lated from a culture broth of Penicillium.[7] These depsipep-
tides have not only shown effects on the immune response
but also inhibition of phosphatases.[8]
[a] Dipl.-Chem. N. Bisek, Dipl.-Chem. S. Wetzel, Dr. H.-D. Arndt,
Prof. Dr. H. Waldmann
Abteilung Chemische Biologie
Max-Planck-Institut fꢀr Molekulare Physiologie
Otto-Hahn-Straße 11, 44227 Dortmund (Germany)
Fax : (+49)231-133-2499
E-mail: hans-dieter.arndt@mpi-dortmund.mpg.de
herbert.waldmann@mpi-dortmund.mpg.de
[b] Dipl.-Chem. N. Bisek, Dipl.-Chem. S. Wetzel, Dr. H.-D. Arndt,
Prof. Dr. H. Waldmann
On the molecular level, cyclic peptides often display in-
creased bioactivity and stability when compared to their
linear epitopes.[9–11] Furthermore, cyclization of peptide
chains has been used extensively as a method for introduc-
ing conformational constraints in peptide backbones and to
Fakultꢁt Chemie, Technische Universitꢁt Dortmund
Otto-Hahn-Straße 6, 44227 Dortmund (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800692.
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8847

present diverse functionality in a defined and predictable
manner.[12–14] Such conformational rigidification has not only
been used for mimicking protein turns, but also for the
design of orally available therapeutics.[15] Even slight
changes in the macrocyclic backbone can influence biologi-
cal activities dramatically.[16–18]
vastelin C3 analogues with variations of selected stereocen-
ters in the backbone of the cyclic depsipeptide (13, 14). Due
to the strained macrocyclic system, we presumed low confor-
Therefore, a rigid macrocyclic scaffold for orientating po-
tential enzyme binding groups in space would be highly val-
uable. Such a template could serve as powerful tool for the
elucidation of enzyme binding affinities or the development
of selective drugs, filling an enzyme pocket by arranging
functional groups on the rigid template in a defined way.
In this regard, 13-membered cyclic depsipeptides are es-
pecially interesting as 12-membered tetrapeptides possess
unfavorable ring strain resulting in instability and difficulties
in synthesis, which often leads to the necessity for turn-
inducing elements like prolines or d-amino acids.[19,20] Fur-
thermore, many cyclic tetrapeptides show multiple confor-
mations in water or DMSO.[21,22] Hence, the 13-membered
cyclic depsipeptides characterized by one ester linkage
belong to the smallest cyclic peptide scaffolds that can be
accessed efficiently without the need of particular turn
mational flexibility. It was planned to compare the low-
energy conformations of the diastereomeric macrocycles by
using 2D NMR spectroscopic experiments and computation-
al analysis of the conformational space and to investigate
the influence of topological changes on the inhibitory activi-
ty against the dual-specific phosphatase VHR.
ACHTUNGTRENNUNG
These results should lead to valuable insights into the po-
tential of 13-membered cyclodepsipeptide scaffolds to serve
the need for rigid templates for the elucidation of enzyme
binding affinity and selectivity, in particular for phosphatase
targeting.
bered stevastelin analogues display interesting biological ac-
tivities on the dual-specific vaccina H1-related (VHR) phos-
phatase.[8,23] The inhibitory activity has been shown to
depend on the configuration of the stereocenters in the b-
hydroxy acid.
To systematically investigate the influence of stereochemi-
cal alterations in the backbone, we decided to develop a
straightforward synthesis for a series of 13-membered ste-
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Stevastelin C3 Analogues
FULL PAPER
Results and Discussion
The syn-aldol adducts were prepared in 80% yield and
with a diastereomeric ratio of 92:8. To this end, tetradecanal
was added to the boron-enolate derived from Evans imides
17 or ent-17 and nBu2BOTf in the presence of DIPEA.
Cleavage of the auxiliary with aqueous LiOH and H2O2
yielded acids 19 and 21, respectively.
For a flexible and straightforward synthesis, we focused
on a solid-phase synthesis with a 2-chlorotrityl chloride resin
(22) that tolerates the reaction conditions and can be
cleaved in one step with Boc and tBu protecting groups. The
synthesis of linear depsipeptide (2S,3R)-27 is exemplified in
Scheme 2.
Stevastelin C3 analogue synthesis: Our synthetic approach
towards stevastelin C3 analogues (2R,3S)-13, (2R,3R)-13,
(2S,3R)-13, and (2S,3S)-13 involved solid-phase synthesis of
the linear peptide esters 27 from building blocks 18–21 and
the three amino acids, l-Ser, l-Thr, and l-Val. Subsequent
macrolactamization in solution was planned to yield the
cyclic depsipeptides (2R,3S)-13, (2R,3R)-13, (2S,3R)-13, and
(2S,3S)-13.
To sample the stereocenters in the macrocyclic backbone,
four different b-hydroxy acids were synthesized as outlined
in Scheme 1. anti-Configured acids 18 and 20 were accessed
Scheme 1. Asymmetric synthesis of b-hydroxy acid building blocks:
a) cHex2BOTf, NEt3, CH2Cl2, ꢀ788C to RT, 3 h, d.r. 96:4, 71%; b) 30%
H2O2, nBu4NOH, THF, 08C to RT, then Na2SO3, 86%; c) nBu2BOTf,
DIPEA, CH2Cl2, ꢀ788C to 08C, 2 h, d.r. 92:8, 80%; d) 30% H2O2,
LiOH, THF/H2O, 0 8C, 2 h then Na2SO3, 77%. b-Hydroxy acids 20 and
21 have been synthesized accordingly by using the enantiomeric auxilia-
ries. DIPEA: diisopropyl ethyl amine. Tf: trifluoromethanesulfonyl.
Scheme 2. Solid-phase synthesis of the linear cyclization precursors:
a) DIPEA, CH2Cl2, 2.5 h, 99%; b) FmocSer
CH2Cl2/DMF, 08C, 3 h, d.r. 8:1, 98%; c) 2ꢃ15 min 20% piperidine in
DMF, 99%; d) FmocThr(OtBu)OH, DIC, HOBt, CH2Cl2/DMF, 2.5 h,
ACHTUNGTREN(NUGN OBn)OH, DIC, DMAP,
AHCTUNGTRENNUNG
99%; e) BocValOH, DIC, HOBt, CH2Cl2/DMF, 2.5 h, 99%; f) 2ꢃ30 min
TFA/CH2Cl2 (1:1), 88% overall yield, d.r. 8:1. Boc: tert-butyloxycarbon-
yl; DIC:diisopropylcarbodiimide; DMAP: 4-dimethylaminopyridine;
Fmoc: fluorenylmethyloxycarbonyl; HOBt: hydroxybenzotriazole; TFA:
trifluoroacetic acid.
by using the asymmetric aldol reaction developed by Abiko
and Masamune,[24] whereas syn-configured acids 19 and 21
were prepared by using Evans aldol methodology.[25,26] The
acylated auxiliaries 15 and 17 and their enantiomers were
prepared by following known procedures.[27–29] Tetradecanal
(16) was derived from its alcohol by oxidation with o-io-
A
ACHTUNGTRENNUNG
benzoic acid (IBX).[30]
The hydroxy acid 19 was chemoselectively attached to
resin 22 in the presence of DIPEA in CH2Cl2. Initial esterifi-
cation attempts of FmocSerACTHNUTRGENUGN(OBn)OH and the immobilized
adding tetradecanal 16 to the boron-enolate derived from
norephedrin ester 15 or ent-15 and cHex2BOTf and yielded
the aldol adducts in a 71% yield and with diastereomeric
ratio of 96:4. After separation of the stereoisomers by
column chromatography, the chiral auxiliary was cleaved by
hydrolysis with aqueous nBu4NOH and H2O2 to give pure 2-
methyl-3-hydroxy acids 18 and 20 in 86% yield. For repro-
ducible results, it was crucial to use freshly distilled alde-
hyde 16.
b-hydroxy acid 23 showed epimerization of the serine in a
ratio of 5:1. By lowering the temperature to 08C and the
amount of DMAP to 0.2 equivalents, the reaction could be
improved to a diastereomeric ratio of 8:1. Alternatively,
AHCTUNGTRENNUNG
Yamaguchi conditions[31] (2,4,6-trichlorobenzoyl chloride,
NEt3) could be applied, but delivered comparable yields and
stereoisomeric purities.
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8849

H. Waldmann, H.-D. Arndt et al.
The Fmoc group of resin-attached ester 24 was removed
with 20% piperidine in DMF and subsequent coupling of
FmocThrACHTUNGTRENNUNG(OtBu)OH with DIC and HOBt yielded immobi-
lized depsipeptide 25. Elongation of the sequence by Fmoc
deprotection and coupling of BocValOH resulted in 26. Fi-
nally, cleavage from the solid support with simultaneous de-
protection of the Boc and tBu groups with 50% TFA in
CH2Cl2 released the desired cyclization precursor (2S,3R)-27
together with its a-serine epimer. As separation of epimers
at this point of synthesis proved to be difficult, the mixtures
were cyclized and the isomers separated afterwards. The dia-
stereomers ((2R,3R)-27, (2R,3S)-27, (2S,3S)-27) of the linear
precursor were prepared in the same manner in excellent
overall yields (80–90%).
The key step of our synthesis was the ring closure of the
strained 13-membered depsipeptide. Macrolactamization of
(2S,3R)-27 with HBTU and DIPEA under high dilution con-
ditions and separation of diastereomers by preparative
HPLC afforded stevastelin C3 analogue (2S,3R)-13 in 46%
yield and epimer (2S,3R)-30 in 7% yield (Scheme 3). The
Scheme 3. Macrolactamization and derivatization of stevastelin C3 ana-
logues: a) HBTU, DIPEA, CH2Cl2/DMF, 1.5 mm, RT, o/n, separation of
diastereomers, 46% with l-Ser, 7% with d-Ser; b) 10% Pd/C, H2, HCl,
EtOH, o/n, 91%; c) tetrazole, (BnO)2PNACTHNUTRGNEU(NG iPr)2, CH2Cl2, RT, then 08C,
tBuOOH, 77%; d) 10% Pd/C, H2, EtOH, 4 h, 99%. HBTU: o-benzotri-
azol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate; o/n: over-
night.
In summary, a new straightforward synthesis of stevastelin
C3 analogues has been developed. The esterification on
solid support was found to be particularly challenging. The
key steps towards stevastelin analogues 13 and 14 have been
the macrolactamization of the linear depsipeptide and sub-
sequent phosphorylation of the macrocycle. These reactions
were optimized successfully to yield the analogues in very
good yields. Due to the high yielding and flexible building-
block approach, this synthesis needs only a few purification
steps and can also be applied for the incorporation of other
building blocks, such as modified b-amino acids, instead of
the fatty acid or d-amino acids for further variation of the
stereocenters in the peptidic backbone.
formation of cyclic dimers could be suppressed by slowly
adding the open-chain precursor to a solution of coupling
reagents with the help of a syringe pump. Following this
strategy, three additional diastereomeric macrocycles
((2R,3R)-13 in 36%, (2S,3R)-13 in 55%, (2S,3S)-13 in 62%
yield) and two of the minor epimers could be isolated
((2R,3S)-30 in 7%, (2S,3R)-30 in 8% yield). Pd/C-catalyzed
benzyl ether hydrogenolysis of cyclic depsipeptide (2S,3R)-
13 delivered stevastelin C3 analogue 29 in 77% yield.
Earlier studies had suggested that neutral stevastelins
become phosphorylated or sulfated in vivo to reach their
full activity.[8] Therefore, macrocycles (2S,3R)-13, (2R,3R)-
13, (2R,3S)-13, and (2S,3S)-13 were functionalized further
(Scheme 3). Derivatization was achieved in three steps.
Phosphitylation of the threonine residue in macrocycle
(2S,3R)-13 with O,O-dibenzyl(N,N-diisopropyl)phosphor-
Conformational analysis: To investigate the ability of the
diastereomeric macrocycles (2S,3R)-13, (2R,3R)-13, (2R,3S)-
13, and (2S,3S)-13 as well as d-serine-containing macrocycles
(2S,3R)-30 and (2R,3S)-30 to serve as 3D templates for de-
fined and predictable orientation of residues in space, these
compounds were studied in detail.
1
A
In H NMR spectroscopic experiments, a single set of res-
ed macrocycle (2S,3R)-28. To avoid phosphonate formation,
it was crucial to work under rigorously dry conditions. Fur-
thermore, the very mild oxidation reagent tBuOOH was
found to be optimal to suppress acid-mediated side reac-
tions. Finally, macrocycle (2S,3R)-28 was debenzylated quan-
titatively by Pd/C-catalyzed hydrogenolysis to give the phos-
phorylated analogue (2S,3R)-14. Diastereomeric analogues
(2R,3R)-14, (2R,3S)-14, and (2S,3S)-14 have been synthe-
sized accordingly in excellent yields.
onances without line broadening was found for all mole-
cules, which indicates just one stable conformation or fast
averaging. Only macrocycle (2R,3R)-13 showed line broad-
ening for valine signals suggesting some intermediate flexi-
bility at this position. TOCSY, gHSQC, and gHMBC experi-
ments were then performed for complete signal assignment.
Comparison of H NMR spectroscopic experiments of dif-
ferent stereoisomers in [D6]DMSO already provided strong
indications of different conformations, as dramatic differen-
1
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Stevastelin C3 Analogues
FULL PAPER
ces in chemical shifts and coupling constants for both the
CHa- and NH-protons were observed. In proteins and linear
peptides an upfield shift of NH and CHa is known to corre-
late with helical structures, whereas a downfield shift is re-
lated to b-sheet conformations.[33] As the conformations of
small and constrained peptide rings cannot be matched
easily with proteins, one can only conclude that different
stereoisomers induce distinct conformations within the mac-
rocyclic ring. For instance, the comparison between the epi-
Figure 2. Selected ROESY contacts of macrocycle (2S,3R)-13.
(Figure 2). 33 to 40 proton–
proton distances have been as-
signed and qualified for each
diastereomer. These distances
were classified into approxi-
mately 24 interresidual, 12 se-
quential, and one medium-
range contact. Only distinctly
assigned protons have been
used in further studies. Diaste-
reotopic protons and torsion
angles have been used for struc-
ture validations. More details
can be found in the Supporting
Information.
1
Figure 1. Comparison of H NMR spectra in [D6]DMSO of the two epimers of 13. The spectra show significant
shifts in the NH and CHa region. A) (2S,3R)-13; B) (2S,3S)-13.
Based on NMR-derived dis-
tance constraints, a conforma-
mers (2S,3R)-13 and (2S,3S)-13 (Figure 1) demonstrates
drastic differences in chemical shifts of Ser-NH and Thr-NH
protons of Dd=0.79 and 0.43 ppm, respectively. Further-
more, the a-protons and Val-Hb are significantly affected as
well.
tional search was performed by using the low-mode search
(LMOD) algorithm that has been especially developed for
calculations of protein loops and cyclic peptides.[36] The low-
energy conformations of the cyclic depsipeptides were com-
puted with Macromodel 9.1[37] within Maestro. A total of
35,000 starting structures were generated, energy-minimized
(OPLS2005, GB/SA solvation model), and compared.
By using the LMOD method and conformational energy
calculations, we found stable low-energy structures for each
analogue without dramatic violations of experimentally vali-
dated restraints (Figure 3). Comparing the diastereomeric
stevastelin C3 analogues 13, the lowest global energy could
be found for (2S,3S)-13 (ꢀ258.3 kJmolꢀ1) followed by the
syn-configured analogues (2S,3R)-13 and (2R,3S)-13 (ꢀ239.4
and ꢀ237.0 kJmolꢀ1). Macrocycle (2R,3R)-13 exhibited the
highest global energy (ꢀ229.3 kJmolꢀ1) with a difference of
29 kJmolꢀ1 relative to (2S,3S)-13.
In these calculated conformations, the orientation of the
amino acid side chains is comparable for all the analogues,
as they are all pointing to one side of the macrocycle. How-
ever, the long alkyl chain of the fatty acid can be oriented
by setting the stereocenter in C-3 of the b-hydroxy acid.
In detail, we find that macrocyles (2S,3S)-13 and (2R,3S)-
13 possess nearly identical backbone conformations. They
only differ in the orientation of the methyl group at the al-
tered stereocenter. From this result, we can conclude that
the orientation of the methyl group does not change the
topology of the rigid template that seems to be set by the
peptidic section of the cycle and the S configuration at C-3
of the b-hydroxy acid.
An all-trans peptide conformation was evident for all ste-
reoisomers from NOE crosspeaks of NH protons and dihe-
3
dral angle calculations from J
G
(>8.2 Hz) by using the Karplus equation (Table 1).[34,35] Due
Table 1. Shifts and coupling constants of NH protons in [D6]DMSO.
SNH
TNH
VNH
d [ppm] J [Hz][a] d [ppm] J [Hz][a] d [ppm] J [Hz][a]
A
7.086
8.33
8.98
8.71
9.31
6.52
8.36
7.796
7.937
7.369
7.966
7.622
7.366
9.30
10.09
9.74
9.55
9.25
9.29
8.007
7.599
8.048
7.685
8.230
7.805
8.21
nd[b]
10.11
9.23
9.84
7.46
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
[a] 3J(NHCHa). [b] Not detected, one broad signal.
to the significant differences in 1D NMR experiments when
altering the stereocenters in the macrocyclic backbone, we
were encouraged to investigate the conformations in more
detail. Therefore, 2D ROESY experiments were performed
and used to identify and quantify intramolecular proton–
proton contacts. The calculated proton distances along the
depsipeptide backbone and selected interresidual correla-
tions are shown exemplarily for macrocycle (2S,3R)-13
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H. Waldmann, H.-D. Arndt et al.
Figure 3. Ten lowest-energy conformations of stevastelin C3 analogues: A) (2S,3S)-13; B) (2S,3R)-13; C) (2R,3R)-13; D) (2R,3S)-13; E) d-Ser-containing
(2S,3R)-30; F) d-Ser-containing (2R,3S)-30; grey=carbon, blue=nitrogen, red=oxygen.
In contrast to this result, macrocyles with an R configura-
tion at C-3 behave completely differently. For diastereomers
(2R,3R)-13 and (2S,3R)-13, significantly different geometries
of the backbone can be observed, which shows the strong in-
fluence of alteration in C-3 stereochemistry. Compound
(2R,3R)-13 has the same configuration as the natural stevas-
telin C3. Here, the R configuration of C-3 forces the macro-
cycle in a twisted form in which the threonine residue is
bent downwards relative to backbone conformations of
(2S,3S)-13 and (2R,3S)-13, whereas the serine residue and
carbonyl are bent upwards out of the plane of the cycle. In
stevastelin analogue (2S,3R)-13, the R configuration of C-3
leads to an even more dramatic change in the orientation of
the serine carbonyl, which is now pointing to the opposite
side of the macrocyclic plane. In return, the threonine resi-
due keeps the same geometry as in depsipeptides (2S,3S)-13
and (2R,3S)-13.
Interestingly, an incorporated d-Ser did not lead to less-
strained conformations as is known for cyclic tetrapeptides,
but induced the opposite effect. Macrocycles (2S,3R)-30 and
(2R,3S)-30 are energetically much less favored. Especially,
analogue (2S,3R)-30 (ꢀ147.5 kJmolꢀ1) is 92 kJmolꢀ1 higher
in energy than the its epimer (2S,3R)-13 (ꢀ239.4 kJmolꢀ1).
Besides the obvious effect on the orientation of the serine
residue in cyclic analogues 30, the valine carbonyl is point-
ing to the opposite side of the macrocycle relative to mole-
cules 13. In contrast to depsipeptide (2S,3R)-13, its epimer
(2S,3R)-30 shows completely different possibilities for the
formation of hydrogen bonds and hydrophobic interactions.
Furthermore, the conformations of phosphorylated and
debenzylated macrocycles 14 were analyzed. In a physiologi-
cal surrounding phosphorylation is used as a key regulator
of signal transduction by activation or deactivation of enzy-
matic functions. These effects are often caused by a change
in protein conformation, due to the introduced steric hin-
drance, charge, and new possible hydrogen bonds after
phosphorylation.[38–41] Normally, threonine phosphorylation
leads to a coordination of the phosphate to the peptide
backbone and thereby to a downfield shift of the coordinat-
ed NH.[42] However, comparing the synthesized macrocycles
13 and their phosphorylated and debenzylated analogues 14,
all amide protons are shifted downfield (Figure 4).
Additionally, phosphorylated macrocycles 14 show much
1
broader signals in the H NMR spectra than the benzylated
cycles. As these findings can result from high flexibility in
macrocycles 14, a conformational search without constraints
was performed and compared to unrestrained calculations
of unphosphorylated macrocycles 13. Indeed, it became evi-
dent that phosphorylation influences the backbone confor-
mation and induces enormous flexibility. In an energy
window of 50 kJmolꢀ1 147 conformation clusters (RMSD<
0.1 kJmolꢀ1) can be found for phosphorylated cycle (2S,3R)-
14, whereas only 14 clusters were found for unphosphorylat-
ed but benzylated cycle (2S,3R)-13 (Table 2).
The phosphorylation-induced flexibility and signal broad-
ening in the proton NMR can be explained by the calculated
conformations of the phosphorylated cycles (Figure 5). In
the strained macrocycle all amide protons are pointing to-
wards the center of the cycle and the threonine residue
places the phosphate just above the macrocyclic plane,
which enables hydrogen bonding to any amide proton and
the debenzylated serine residue.
In conclusion, we found conformationally homogeneous
scaffolds for the benzylated molecules, which are deter-
mined by the stereocenters at C-2 and C-3 of the b-hydroxy
acid part of the backbone and the a-Ser position. Altera-
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Stevastelin C3 Analogues
FULL PAPER
13-Membered cyclic tetrapep-
tides with incorporated b-amino
acids instead of b-hydroxy acids
have been shown to serve as
rigid templates and are used as
b- or g-turn mimics.[14,43] But
not all 13-membered peptides
possess rigid conformations. It
was reported, that the position
of b-amino acid introduction is
important to stabilize a single
conformation.[14] Alternatively,
15-membered
pentapeptides
have frequently been used as
conformationally rigid tem-
plates.[44–46] Furthermore, d-
amino acids are known to
induce bII’-turns[47,48] and the
introduction of N-methylated
amino acids lowers the energy
Figure 4. Differences in chemical shifts between unphosphorylated and phosphorylated macrocycles (13, 14).
Table 2. Number of conformation clusters for each synthesized macrocy-
cle within an energy window of 50 kJmolꢀ1
.
Inhibitor
(2R,3S)-13
(2R,3R)-13
(2S,3R)-13[c]
(2S,3S)-13
(2R,3S)-14
(2R,3R)-14
(2S,3R)-14
(2S,3S)-14
Cluster[a]
Cluster[b]
R1
R2
G
32
37
14
38
246
115
147
106
2
21
4
19
–
–
Bn
Bn
Bn
Bn
H
H
H
H
H
H
H
H
PO3H2
PO3H2
PO3H2
PO3H2
E
N
R
N
G
N
–
–
A
[a] Calculated without NMR-derived constraints. [b] Calculated with
NMR-derived constraints. [c] Only 50% more unique structures were cal-
culated for the debenzylated analogue (2S,3R)-29.
Figure 5. Low-energy conformations of phosphorylated stevastelin C3 an-
alogues: A) (2R,3R)-14; B) (2R,3S)-14; C) (2S,3R)-14; D) (2S,3S)-14;
grey=carbon, blue=nitrogen, red=oxygen, orange=phosphorus.
tions in these stereocenters do affect the macrocycle geome-
try. The orientation of the long alkyl chain seems to be
more important than variation at C-2, which leads not only
to different steric hindrance due to its orientation, but also
populates a different conformation of the macrocyclic ring.
In particular, the configuration at the a-Ser position deter-
mines the overall geometry of the stevastelin analogues, ar-
ranging possible enzyme binding groups in a completely dif-
ferent way. However, phosphorylation and subsequent de-
benzylation of the macrocyles leads to much more flexible
structures, confirming the power of phosphorylation as a
biological tool for switching conformational properties in
peptides or proteins.
barrier between cis and trans peptide bonds and leads to the
loss of a hydrogen donor in the backbone generating new
rigid templates.[12,48]
Application of such rigid scaffolds can be found in drug
design. Kessler et al. have realized spatial screening with
cyclic peptides, in which the conformation-inducing effect of
special amino acids is utilized to generate rigid templates
that are screened for their activity against selected en-
zymes.[13,49]
Hence, the synthesis of 13-membered depsipeptides
serves as a new tool for the design of rigid macrocyles as
biologically active compounds, which could be used to study
Chem. Eur. J. 2008, 14, 8847 – 8860
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8853

H. Waldmann, H.-D. Arndt et al.
Table 3. Inhibition of dual-specific phosphatase VHR in vitro.
the affinity and selectivity of enzymes to give insights in
their biological targets and functions.
Biochemical investigations: Due to the conclusive results
from the conformational analysis in which different stable
conformations for the synthesized stevastelin C3 analogues
have been found, we were interested in the consequences on
enzyme inhibition. As stevastelins have been reported to in-
hibit the dual-specific phosphatase VHR,[8] we investigated
the inhibitory activity of the synthesized analogues against
this phosphatase.
The 21 kDa dual-specific phosphatase VHR is one of the
smallest known phosphatases.[50] It plays an essential role in
MAP kinase regulation of cell cycle progression and the
immune response in activated T-cells due to its activation by
tyrosine kinase ZAP-70 and negative regulation of the mito-
gen-activated protein kinases, extracellular regulated kinases
(ERK) and c-Jun N-terminal kinases (JNK).[51–53] To date,
very few inhibitors of VHR are known.[8,54,55] New inhibitors
or more detailed investigation of known inhibitors could
give better insights into the physiological function of VHR
and serve as lead structures for the development of new
drug candidates.
Inhibitor
(2R,3S)-13
(2R,3R)-13
(2S,3R)-13
(2S,3S)-13
(2R,3S)-30
(2S,3R)-30
(2S,3R)-29
(2R,3S)-14
(2R,3R)-14
(2S,3R)-14
(2S,3S)-14
IC
(VHR)[b]
2[a]
3[a]
4[a]
R1
R2
G
>100 mm
>100 mm
>100 mm
>100 mm
>100 mm
>100 mm
>100 mm
R
R
S
S
R
S
R
R
R
S
S
S
S
S
S
R
R
S
S
S
S
S
Bn
Bn
Bn
Bn
Bn
Bn
H
H
H
H
H
H
H
H
H
H
H
H
PO3H2
PO3H2
PO3H2
PO3H2
E
R
R
S
S
R
S
E
R
R
E
G
R
T
S
E
N
R
R
S
T
R
A
A
S
[a] Configuration of the stereocenter. [b] Mean of three independent ex-
periments.
VHR was assayed by using para-nitrophenol phosphate
(pNPP) as a substrate. The reaction buffer contained 1 mm
DTE to ensure an active state of the enzyme, as Cys-124 is
involved in the catalytic enzyme mechanism.[56] Further-
more, 0.025% detergent NP-40 was found crucial to sup-
press unspecific binding. The assay was performed at an op-
timal pH of 6.5 and 378C.
Initially, the inhibitory activities of the synthesized com-
pounds were tested at 100 mm. From active compounds, the
IC50 values were determined. Only phosphorylated macrocy-
cles inhibited the dual-specific phosphatase VHR in vitro,
whereas unphosphorylated but benzylated stevastelin C3 an-
alogues were inactive (Figure 6). Interestingly, only a slight
dependence of inhibitory activity on orientation of the long
alkyl chain could be observed (Table 3). An S configuration
at position 2 of the b-hydroxy acid seems to be slightly fa-
vored, whereas an R configuration at position 3 gives better
inhibition relative to its epimer.
These results stand in contrast to data reported earlier in
the literature.[23] During the course of our studies, we found
that VHR apparently seems to be very sensitive to changes
in assay conditions, which makes these data difficult to com-
pare. In the current investigations, a clear dependence on
threonine derivatization has been found.
The minor differences in biochemical activity among the
phosphorylated analogues (2S,3R)-14, (2R,3R)-14, (2R,3S)-
14, and (2S,3S)-14 are supported by our results from confor-
mational analysis, in which these derivatives have shown
higher flexibility than the unphosphorylated macrocycles.
Furthermore, these new results support previous observa-
tions in which the natural stevastelins that are sulfated or
phosphorylated are more active against VHR in vitro.[8]
However, in living cells the reverse effect has been ob-
served. Thus, the hypothesis was put forward that uncharged
stevastelins penetrate the membrane and become phos-
phorylated or sulfated in the cell, thus generating the active
form of the inhibitor in vivo. Our results suggest that this
hypothesis may be valid for VHR. Recently, it has been
shown, that VHR inhibition can lead to highly activated
ERK and JNK resulting in a cell cycle arrest at the G1–S
transition.[57] Therefore, further cellular studies of stevastelin
C3 analogues 13 in HeLa cells are ongoing.
On the other hand, the phosphorylated stevastelin C3 an-
alogues could also serve as substrates for the dual-specific
phosphatase and become dephosphorylated. The natural
substrate for VHR is a double phosphorylated protein, with
phosphorylated Thr and Tyr separated by one amino acid in
the peptide sequence.[58] It has been shown that VHR turns
over phosphotyrosine very efficiently, whereas phospho-
threonine and pNPP are converted rather slowly. To moni-
Figure 6. VHR inhibition at 100 mm inhibitor concentration. nI: control
with no inhibitor; PTP inhibitor IV: control.
8854
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Chem. Eur. J. 2008, 14, 8847 – 8860

Stevastelin C3 Analogues
FULL PAPER
tor VHR-mediated phosphate release from the stevastelin
analogues, a malachite green control experiment[59] was per-
formed. The result shows that macrocyle (2S,3S)-14 indeed
serves as substrate, but gets dephosphorylated very slowly.
After 45 min, 15% dephosphorylation could be observed.
Deactivation of the active inhibitor (2S,3S)-14 by VHR-
mediated dephosphorylation could, in principle, influence
our assay results. However, in the performed setup the con-
centration of the substrate pNPP is much higher than the in-
hibitor concentration ((2S,3S)-14). Furthermore, with VHR
a significantly higher kcat./Km was reported in the literature
for pNPP in comparison to pThr,[58] which is present in
(2S,3S)-14. Thus, from the equation for substrate specificity
[Eq.: (1)], which describes the preferred enzymatic reaction
in the presence of two potentially competing substrates, it
follows that in our in vitro assay, competition should be neg-
ligible. Therefore, phosphorylated stevastelin analogues 14
are acting as inhibitors, not as substrates during our VHR
assay, and IC50 values are not affected by dephosphoryla-
tion.
studies are necessary to further explore the potential of the
stevastelins as phosphatase inhibitors.
Experimental Section
General procedure: Immobilization of 2-methyl-3-hydroxyhexadecanoic
acids: In a syringe reactor, 2-chlorotrityl chloride PS resin (1.4 mmolgꢀ1
,
1.85 g, 2.59 mmol) was swollen in dry CH2Cl2 for 10 min before 2-methyl-
3-hydroxytetradecanoic acid (965 mg, 3.37 mmol) and DIPEA (2.23 mL,
13.5 mmol) in dry CH2Cl2 (10 mL) were added. The mixture was shaken
for 2.5 h at RT. Then the resin was washed with CH2Cl2 (3ꢃ), CH2Cl2/
MeOH/DIPEA (17:2:1; 3ꢃ), CH2Cl2 (3ꢃ), MeOH (3ꢃ), and CH2Cl2
(3ꢃ) and dried in high vacuum overnight. A test cleavage with 5% TFA
in CH2Cl2 showed quantitative loading.
General procedure: Esterification of the immobilized 2-methyl-3-hy-
AHCTUNGERTGdNNUN roxyhexadecanoic acids: In a flask, 2-methyl-3-hydroxytetradecanoic
acid (2.3 g, 2.4 mmol) loaded resin (23) was swollen in dry CH2Cl2
(10 mL) for 10 min. At 08C, a solution of DIC (2.04 mL, 13.2 mmol) and
FmocSerACTHNUGTREN(UNG OBn)OH (5.00 g, 12.0 mmol) in DMF/CH2Cl2 (1:1, 15 mL) was
added, followed by DMAP (88 mg, 0.72 mmol) in DMF/CH2Cl2 (1:1,
5 mL) and the mixture was stirred at 08C for 3 h. After washing the resin
with CH2Cl2 (3ꢃ), DMF (3ꢃ), MeOH (3ꢃ), and CH2Cl2 (3ꢃ), the resin
was dried at high vacuum overnight. An 86–96% yield was concluded
from loading determinations. A test cleavage with TFA/CH2Cl2 (1:1) and
subsequent HPLC analysis (chiral OD-H, 17% iPrOH in nhexane)
showed a diastereomeric ratio of 89:11 to 82:18.
½nI ꢂ ½kcat:=Kmꢂ ½Iꢂ
½nSꢂ ½kcat:=KmꢂSI½Sꢂ
ð1Þ
¼
General procedure: Solid-phase peptide synthesis: For Fmoc deprotec-
tion, the resin was treated twice with 20% piperidine in DMF for 15 min
each in a syringe reactor and washed with DMF and CH2Cl2. Then a solu-
However, regarding their activity in cells, the phosphory-
lated stevastelins and analogues may become dephosphory-
lated by other endogeneous phosphatases. Therefore, either
phosphorylation of the inhibitors must be competitive with
phosphatase-mediated deactivation or the active molecule is
not a phosphorylated stevastelin but another derivative, for
example the sulfate.
tion of FmocThrACHTUNTRGNEUNG(OtBu)OH (5 equiv), HOBt (5 equiv), and DIC
(5 equiv) in CH2Cl2/DMF (2:1) was added. The mixture was agitated for
3 h at RT and then washed with DMF, MeOH, and CH2Cl2 (3ꢃ each).
The Fmoc deprotection and an amino acid coupling were repeated with
BocValOH. Finally the resin was treated with TFA/CH2Cl2 (1:1) for
30 min twice. The resin was thoroughly washed with CH2Cl2 and the com-
bined filtrates were coevaporated with toluene. After preparative HPLC,
the product together with its Sera-epimer was obtained as a colorless
solid. For the solid-phase synthesis of linear depsipeptides 27 an overall
yield of 80–88% was calculated.
Conclusion
l-Valyl-l-threonyl-3-O-benzyl-l-seryl-(2R,3S)-2-methyl-3-hydroxyhexa-
decanoic acid ((2R,3S)-27): b-Hydroxy acid (2-methyl-3-hydroxyhexade-
canoic acid) is represented as BHA throughout. Yield: 220 mg
(331 mmol), 88% (d.r. 8:1); major isomer: 1H NMR (400 MHz, CDCl3):
d=8.51 (d, J=6.99 Hz, 1H; NH), 7.93 (d, J=7.7 Hz, 1H; NH), 7.29–7.11
(m, 6H; aromat. H; NH), 5.26–5.19 (m, 1H; BHA3-H), 4.61–4.55 (m,
1H; Ser-Ha), 4.48 (d, J=11.9 Hz, 1H; Ser-CH2Ph), 4.44 (d, J=11.9 Hz,
1H; Ser-CH2Ph), 4.39 (d, J=4.6 Hz, 1H; Thr-Ha), 4.07–3.97 (m, 1H; Thr-
Hb), 3.90 (d, J=6.0 Hz, 1H; Val-Ha), 3.82 (dd, J=9.5, 4.1 Hz, 1H; Ser-
Hba), 3.65 (dd, J=9.2, 3.2 Hz, 1H; Ser-Hbb), 2.59 (dq, J=6.9, 2.6 Hz,
1H; BHA2-H), 2.15 (qd, J=13.5, 6.8 Hz, 1H; Val-Hb), 1.60–1.47 (m, 1H;
BHA4a-H2), 1.45–1.33 (m, 1H; BHA4b-H2), 1.26–1.09 (m, 23H; BHA),
1.13 (d, J=6.3 Hz, 3H; Thr-Hg), 1.09 (d, J=7.1 Hz, 3H; BHA2’-H3), 0.95
(d, J=6.8 Hz, 6H; Val-Hg), 0.86 ppm (t, J=6.9 Hz, 3H; BHA16-H3);
13C NMR (101 MHz, CDCl3): d=177.3 (BHA-1), 170.5 (Thr-1), 169.9
(Ser-1), 169.6 (Val-1), 137.1 (aromat. C), 128.3 (aromat. C), 127.8
(aromat. C), 127.6 (aromat. C), 75.9 (BHA-3), 73.4 (Ser-CH2Ph), 69.0
(Ser-b), 67.2 (Thr-b), 59.4 (Thr-a), 58.7 (Val-a), 52.8 (Ser-a), 42.3 (BHA-
2), 31.9 (BHA), 31.7 (BHA), 30.2 (Val-b), 29.7 (BHA), 29.6 (BHA), 29.4
(BHA), 29.3 (BHA), 25.5 (BHA), 22.6 (BHA), 18.8 (Thr-g), 18.1 (Val-
ga), 17.3 (Val-gb), 14.0 (BHA-16), 9.9 ppm (BHA-2’); IR (neat): n˜ =
3306, 3072, 2921, 2853, 1740, 1666, 1639, 1537, 1467 cmꢀ1; HPLC-ESIMS
(C4–10; tR =8.32 min): m/z: 664.19 [M+H]+, 1326.84 [2M+H]+; HRMS
(ESI): m/z: calcd for C36H62N3O8: 664.4531 [M+H]+; found: 664.4528.
Small macrocycles that are restrained in conformational
flexibility can serve as 3D topological templates, which ori-
entate potential enzyme binding groups in space. A series of
13-membered stevastelin C3 analogues was successfully syn-
thesized based on solid-phase synthesis and macrocycliza-
tion in solution. Conformational studies on different stereo-
isomers revealed rigid conformations and defined orienta-
tion of functional groups attached to the macrocyclic back-
bone. Thus, we demonstrated that the introduction of a b-
hydroxy acid into a small cyclic peptide can serve as a new
tool for the generation of rigid macrocycles. These templates
can be utilized for spatial screening against various enzymes
to give insights into enzyme affinity and selectivity.
Furthermore, it could be shown that phosphorylation of
the threonine moiety is essential for inhibitory activity
against VHR in vitro. In phosphorylated and debenzylated
analogues, the alteration of the backbone stereochemistry
only moderately influenced the inhibitory activity on the
dual-specific phosphatase VHR, which is in line with an in-
creased level of flexibility of the cyclic depsipeptides after
phosphorylation. As phosphorylated stevastelins were found
to be substrates for VHR themselves, further biological
l-Valyl-l-threonyl-3-O-benzyl-l-seryl-(2S,3R)-2-methyl-3-hydroxyhexa-
decanoic acid ((2S,3R)-27): Yield: 267 mg (402 mmol), 87% (d.r. 8:1);
major isomer: 1H NMR (400 MHz, CDCl3/MeOD 4:1): d=8.13 (d, J=
Chem. Eur. J. 2008, 14, 8847 – 8860
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8855

H. Waldmann, H.-D. Arndt et al.
8.2 Hz, 1H; NH), 7.73 (d, J=8.2 Hz, 1H; NH), 7.24–7.09 (m, 5H;
aromat. H), 5.12 (td, J=8.34, 4.96 Hz, 1H; BHA3-H), 4.55 (t, J=3.5 Hz,
1H; Ser-Ha), 4.38 (s, 2H; Ser-CH2Ph), 4.34 (d, J=4.6 Hz, 1H; Thr-Ha),
4.01 (dq, J=6.4, 4.6 Hz, 1H; Thr-Hb), 3.79 (dd, J=9.6, 3.7 Hz, 1H; Ser-
Hba), 3.66 (d, J=6.1 Hz, 1H; Val-Ha), 3.57 (dd, J=9.6, 3.4 Hz, 1H; Ser-
Hbb), 2.52 (dq, J=7.0, 5.2 Hz, 1H; BHA2-H), 2.05 (qd, J=13.6, 6.9 Hz,
1H; Val-Hb), 1.56–1.35 (m, 2H; BHA4-H2), 1.23–1.11 (m, 22H; BHA),
1.11 (d, J=6.5 Hz, 3H; Thr-Hg), 1.03 (d, J=7.1 Hz, 3H; BHA2’-H3), 0.89
(d, J=6.9 Hz, 3H; Val-Hga), 0.88 (d, J=6.9 Hz, 3H; Val-Hgb), 0.75 ppm
(t, J=6.9 Hz, 3H; BHA16-H3); 13C NMR (101 MHz, CDCl3): d=175.9
(BHA-1), 169.9 (Thr-1), 169.4 (Ser-1), 168.4 (Val-1), 137.0 (aromat. C),
128.2 (aromat. C), 127.7 (aromat. C), 127.4 (aromat. C), 75.9 (BHA-3),
73.2 (Ser-CH2Ph), 69.2 (Ser-b), 67.2 (Thr-b), 58.4 (Thr-a), 58.3 (Val-a),
52.6 (Ser-a), 42.6 (BHA-2), 31.7 (BHA-4), 30.0 (Val-b), 29.4 (BHA), 29.2
(BHA), 29.1 (BHA), 25.1 (BHA), 22.4 (BHA), 18.3 (Thr-g), 17.9 (Val-
ga), 17.2 (Val-gb), 13.7 (BHA-16), 11.3 ppm (BHA-2’); IR (neat): n˜ =
3292, 3066, 2922, 2852, 1740, 1688, 1666, 1658, 1641, 1544, 1536, 1468,
1454 cmꢀ1; HPLC-ESIMS (C4–10; tR =8.39 min): m/z: 664.13 [M+H]+,
1326.84 [2M+H]+; HRMS (ESI): m/z: calcd for C36H62N3O8: 664.4531
[M+H]+; found: 664.4528.
removed. The crude product was purified by preparative HPLC, which
provided both the title compound 13 and the minor epimer 30 resulting
from partial epimerization of Ser-a as colorless powders.
1,3.4-Anhydro[l-valyl-l-threonyl-3-O-benzyl-l-seryl-(2R,3S)-2-methyl-3-
hydroxyhexadecanoic acid] ((2R,3S)-13): Yield: 53.5 mg (82.8 mmol),
46%; [a]2D0 =ꢀ45.9 (c=1 in CHCl3); m.p.: 1978C; 1H NMR (600 MHz,
[D6]DMSO): d=7.97 (d, J=9.6 Hz, 1H; Thr-NH), 7.68 (d, J=9.2 Hz,
1H; Val-NH), 7.35–7.24 (m, 5H; aromat. H), 6.78 (d, J=9.3 Hz, 1H; Ser-
NH), 5.28 (d, J=4.4 Hz, 1H; Thr-OH), 4.88 (ddd, J=8.4, 5.9, 2.3 Hz,
1H; BHA3-H), 4.55 (d, J=12.2 Hz, 1H; Ser-CH2Ph), 4.47 (dt, J=9.1,
5.4 Hz, 1H; Ser-Ha), 4.43 (d, J=12.2 Hz, 1H; Ser-CH2Ph), 4.12–4.07 (m,
1H; Thr-Hb), 4.05 (dd, J=9.6, 2.4 Hz, 1H; Thr-Ha), 3.86 (dd, J=11.1,
9.3 Hz, 1H; Val-Ha), 3.52 (dd, J=9.2, 5.4 Hz, 1H; Ser-Hba), 3.47 (dd, J=
9.1, 9.1 Hz, 1H; Ser-Hbb), 2.57 (dq, J=7.2, 2.1 Hz, 1H; BHA2-H), 2.03–
1.95 (m, 1H; Val-Hb), 1.71–1.62 (m, 1H; BHA4a-H2), 1.47–1.38 (m, 1H;
BHA4b-H2), 1.26–1.19 (m, 22H; BHA5–15-H2), 1.17 (d, J=7.3 Hz, 3H;
BHA2’-H3), 1.02 (d, J=6.3 Hz, 3H; Thr-Hg), 0.89 (d, J=6.5 Hz, 3H; Val-
Hga), 0.87 (d, J=6.7 Hz, 3H; Val-Hgb), 0.83 ppm (t, J=7.0 Hz, 3H;
BHA16-H3); 13C NMR (151 MHz, [D6]DMSO): d=171.8 (BHA-1), 171.2
(Val-1), 170.2 (Thr-1), 167.4 (Ser-1), 137.6 (aromat. C), 128.1 (aromat. C),
127.5 (aromat. C), 127.4 (aromat. C), 76.4 (BHA-3), 72.1 (Ser-CH2Ph),
68.8 (Ser-b), 64.8 (Thr-b), 62.0 (Val-a), 59.0 (Thr-a), 53.0 (Ser-a), 42.8
(BHA-2), 31.2 (BHA), 30.1 (BHA), 29.0 (Val-b), 28.9 (BHA), 28.7
(BHA), 28.6 (BHA), 24.5 (BHA), 22.0 (BHA), 20.7 (Thr-g), 19.3 (Val-
ga), 19.0 (Val-gb), 13.9 (BHA-16), 8.3 ppm (BHA-2’); IR (neat): n˜ =
3424, 3283, 2923, 2853, 1745, 1671, 1656, 1536, 1468 cmꢀ1; HPLC-ESIMS
(C4–70; tR =5.13 min): m/z: 646.3 [M+H]+, 668.5 [M+Na]+; HRMS
(ESI): m/z: calcd for C36H60N3O7: 646.4426 [M+H]+; found: 646.4426.
l-Valyl-l-threonyl-3-O-benzyl-l-seryl-(2S,3S)-2-methyl-3-hydroxyhexade-
canoic acid ((2S,3S)-27): Yield: 252 mg (380 mmol), 80% (d.r. 8:1); major
isomer: 1H NMR (400 MHz, CDCl3): d=7.24–7.07 (m, 5H; aromat. H),
5.07–4.97 (m, 1H; BHA3-H), 4.57 (t, J=3.3 Hz, 1H; Ser-Ha), 4.40–4.36
(m, 2H; Ser-CH2Ph), 4.31 (d, J=4.5 Hz, 1H; Thr-Ha), 4.07–3.98 (m, 1H;
Thr-Hb), 3.79 (dd, J=9.5, 3.6 Hz, 1H; Ser-Hba), 3.67 (dd, J=10.1, 6.0 Hz,
1H; Val-Ha), 3.56 (dd, J=9.5, 3.3 Hz, 1H; Ser-Hbb), 2.66–2.55 (m, 1H;
BHA2-H), 2.11–1.97 (m, 1H; Val-Hb), 1.53–1.41 (m, 2H; BHA-4), 1.21–
1.07 (m, 22H; BHA), 1.05 (d, J=6.5 Hz, 3H; Thr-Hg), 1.03–0.97 (m, 3H;
BHA2’-H3), 0.91–0.84 (m, 6H; Val-g), 0.75 ppm (t, J=6.9 Hz, 3H;
BHA16-H3); 13C NMR (101 MHz, CDCl3): d=175.5 (BHA-1), 169.7
(Thr-1), 168.7 (Ser-1), 168.4 (Val-1), 136.9 (aromat. C), 128.1 (aromat. C),
127.6 (aromat. C), 127.3 (aromat. C), 76.6 (BHA-3), 73.1 (Ser-CH2Ph),
69.3 (Ser-b), 66.9 (Thr-b), 58.6 (Thr-a), 58.3 (Val-a), 52.6 (Ser-a), 42.7
(BHA-2), 31.6 (BHA), 31.0 (BHA), 30.8 (BHA), 30.0 (Val-b), 29.4
(BHA), 29.2 (BHA), 29.1 (BHA), 24.9 (BHA), 24.7 (BHA), 22.4 (BHA),
18.3 (Thr-g), 17.9 (Val-ga), 17.0 (Val-gb), 13.7 (BHA-16), 12.7 ppm
(BHA-2’); IR (neat): n˜ =3277, 3066, 2924, 2853, 1742, 1659, 1525,
1465 cmꢀ1; HPLC-ESIMS (C4–10; tR =8.44 min): m/z: 664.15 [M+H]+,
1326.89 [2M+H]+; HRMS (ESI): m/z: calcd for C36H62N3O8: 664.4531
[M+H]+; found: 664.4528.
1,3.4-Anhydro[l-valyl-l-threonyl-3-O-benzyl-d-seryl-(2R,3S)-2-methyl-3-
hydroxyhexadecanoic acid] ((2R,3S)-30): Yield: 8.1 mg (12.6 mmol), 7%;
m.p.: 1788C; 1H NMR (600 MHz, [D6]DMSO): d=7.81 (d, J=7.5 Hz,
1H; Val-NH), 7.60 (d, J=8.4 Hz, 1H; Ser-NH), 7.37 (d, J=9.3 Hz, 1H;
Thr-NH), 7.33–7.21 (m, 5H; aromat. H), 5.19 (dt, J=7.3, 2.4 Hz, 1H;
BHA3-H), 4.78 (d, J=4.5 Hz, 1H; Thr-OH), 4.52 (ddd, J=8.1, 7.1,
5.4 Hz, 1H; Ser-Ha), 4.48–4.42 (m, 2H; Ser-CH2Ph), 4.11–4.04 (m, 2H;
Thr-Hb, Thr-Ha), 3.73 (dd, J=9.8, 7.5 Hz, 1H; Val-Ha), 3.68 (dd, J=9.7,
7.1 Hz, 1H; Ser-Hba), 3.50 (dd, J=9.7, 5.3 Hz, 1H; Ser-Hbb), 2.55 (dq,
J=7.2, 2.3 Hz, 1H; BHA2-H), 2.18–2.08 (m, 1H; Val-Hb), 1.64 (qd, J=
14.0, 7.0 Hz, 1H; BHA4a-H2), 1.48–1.40 (m, 1H; BHA4b-H2), 1.26–1.19
(m, 22H; BHA5–15-H2), 1.18 (d, J=7.3 Hz, 3H; BHA2’-H3), 0.98 (d, J=
6.1 Hz, 3H; Thr-Hg), 0.90 (d, J=3.3 Hz, 3H; Val-Hga), 0.88 (d, J=3.4 Hz,
3H; Val-Hgb), 0.83 ppm (t, J=7.0 Hz, 3H; BHA16-H3); 13C NMR
(151 MHz, [D6]DMSO): d=173.3 (BHA-1), 171.8 (Val-1), 169.8 (Thr-1),
169.0 (Ser-1), 138.2 (aromat. H), 128.1 (aromat. H), 127.2 (aromat. H),
75.8 (BHA-3), 72.1 (Ser-CH2Ph), 67.5 (Ser-b), 64.3 (Thr-b), 62.9 (Val-a),
57.0 (Thr-a), 51.0 (Ser-a), 41.6 (BHA-2), 31.2 (BHA), 30.4 (BHA), 29.0
(BHA), 28.9 (BHA), 28.8 (BHA), 28.6 (BHA), 28.0 (Val-b), 24.9 (BHA),
22.0 (BHA), 20.1 (Thr-g), 19.8 (Val-ga), 19.2 (Val-gb), 13.9 (BHA-16),
9.3 ppm (BHA-2’); IR (neat): n˜ =3394, 3299, 3062, 2959, 2921, 2852,
1741, 1651, 1537, 1463 cmꢀ1; HRMS (ESI): m/z: calcd for C36H60N3O7:
[M+H]+: 646.4426; found: 646.4425.
l-Valyl-l-threonyl-3-O-benzyl-l-seryl-(2R,3R)-2-methyl-3-hydroxyhexa-
decanoic acid ((2R,3R)-27): Yield: 133 mg (200 mmol), 82% (d.r. 8:1);
major isomer: 1H NMR (400 MHz, CDCl3): d=7.24–7.06 (m, 5H;
aromat. H), 5.05–4.97 (m, 1H; BHA3-H), 4.51 (t, J=3.5 Hz, 1H; Ser-
Ha), 4.35 (s, 2H; Ser-CH2Ph), 4.34–4.29 (m, 1H; Thr-Ha), 4.04–3.95 (m,
1H; Thr-Hb), 3.77 (dd, J=9.6, 3.7 Hz, 1H; Ser-Hba), 3.65 (d, J=5.9 Hz,
1H; Val-Ha), 3.54 (dd, J=9.6, 3.4 Hz, 1H; Ser-Hbb), 2.64–2.52 (m, 1H;
BHA2-H), 2.02 (qd, J=13.6, 6.9 Hz, 1H; Val-Hb), 1.52–1.37 (m, 2H;
BHA-4), 1.18–1.03 (m, 25H; BHA, Thr-g), 1.00 (d, J=7.1 Hz, 3H;
BHA2’-H3), 0.88–0.82 (m, 6H; Val-g), 0.72 ppm (t, J=6.9 Hz, 3H;
BHA16-H3); 13C NMR (101 MHz, CDCl3): d=175.9 (BHA-1), 169.9
(Thr-1), 169.2 (Ser-1), 168.4 (Val-1), 136.9 (aromat. C), 128.1 (aromat. C),
127.5 (aromat. C), 127.2 (aromat. C), 76.4 (BHA-3), 73.1 (Ser-CH2Ph),
69.2 (Ser-b), 67.2 (Thr-b), 58.3 (Thr-a), 58.2 (Val-a), 52.6 (Ser-a), 42.9
(BHA-2), 31.6 (BHA), 30.8 (BHA), 29.9 (Val-b), 29.3 (BHA), 29.2
(BHA), 29.0 (BHA), 24.6 (BHA), 22.3 (BHA), 18.3 (Thr-g), 17.8 (Val-
ga), 17.0 (Val-gb), 13.6 (BHA-16), 12.5 ppm (BHA-2’); IR (neat): n˜ =
3291, 3066, 2923, 2853, 1735, 1652, 1533, 1465 cmꢀ1; HPLC-ESIMS (C4–
10; tR =8.46 min): m/z: 664.16 [M+H]+, 1326.71 [2M+H]+; HRMS
(ESI): m/z: calcd for C36H62N3O8: 664.4531 [M+H]+; found: 664.4529.
1,3.4-Anhydro[l-valyl-l-threonyl-3-O-benzyl-l-seryl-(2S,3R)-2-methyl-3-
hydroxyhexadecanoic acid] ((2S,3R)-13): Yield: 59.3 mg (91.8 mmol),
51%; [a]2D0 =ꢀ37.8 (c=1 in CHCl3); m.p.: 2268C; 1H NMR (600 MHz,
[D6]DMSO): d=8.05 (d, J=10.1 Hz, 1H; Val-NH), 7.88 (d, J=8.7 Hz,
1H; Ser-NH), 7.37 (d, J=9.7 Hz, 1H; Thr-NH), 7.35–7.23 (m, 5H;
aromat. H), 5.14 (d, J=5.1 Hz, 1H; Thr-OH), 4.83 (ddd, J=10.0, 3.8,
3.0 Hz, 1H; BHA3-H), 4.63 (td, J=8.5, 4.2 Hz, 1H; Ser-Ha), 4.53 (d, J=
12.6 Hz, 1H; Ser-CH2Ph), 4.50 (d, J=12.6 Hz, 1H; Ser-CH2Ph), 4.08 (dd,
J=9.7, 3.4 Hz, 1H; Thr-Ha), 4.01–3.96 (m, 1H; Thr-Hb), 3.97–3.91 (m,
1H; Val-Ha), 3.71 (dd, J=9.8, 4.0 Hz, 1H; Ser-Hba), 3.57 (dd, J=9.8,
4.4 Hz, 1H; Ser-Hbb), 2.47–2.44 (m, 1H; BHA2-H), 1.63–1.54 (m, 1H;
BHA4a-H2), 1.92–1.82 (m, 1H; Val-Hb), 1.45–1.36 (m, 1H; BHA4b-H2),
1.26–1.14 (m, 22H; BHA5–15-H2), 1.06 (d, J=6.3 Hz, 3H; Thr-Hg), 1.02
(d, J=7.1 Hz, 3H; BHA2’-H3), 0.90 (d, J=6.6 Hz, 1H; Val-Hga), 0.87 (d,
J=6.5 Hz, 3H; Val-Hgb), 0.82 ppm (t, J=7.0 Hz, 3H; BHA16-H3);
13C NMR (151 MHz, [D6]DMSO): d=170.9 (BHA-1, Val-1), 170.3 (Thr-
1), 168.7 (Ser-1), 138.0 (aromat. C), 128.2 (aromat. C), 127.4 (aromat. C),
General procedure: Macrolactamization: A solution of open chain pre-
cursor 27 (119 mg, 0.180 mmol) in dry CH2Cl2/DMF (15:1, 4 mL) was
added slowly with a syringe pump to a solution of HBTU (137 mg,
0.360 mmol) and DIPEA (59 mL, 0.360 mmol) in dry CH2Cl2/DMF (15:1,
124 mL). The mixture was stirred for 14 h. After removal of the solvents,
the residue was dissolved in ethyl acetate and washed with water and
brine. The organic layer was dried with MgSO4 and the ethyl acetate was
8856
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ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8847 – 8860

Stevastelin C3 Analogues
FULL PAPER
76.6 (BHA-3), 72.2 (Ser-CH2Ph), 68.7 (Ser-b), 65.3 (Thr-b), 61.3 (Val-a),
59.7 (Thr-a), 51.4 (Ser-a), 43.2 (BHA-2), 31.2 (BHA), 29.4 (Val-b), 29.0
(BHA), 28.9 (BHA), 28.9 (BHA), 28.8 (BHA), 28.6 (BHA), 28.4 (BHA),
25.7 (BHA), 22.0 (BHA), 20.9 (Thr-g), 19.1 (Val-ga), 19.0 (Val-gb), 14.0
(BHA-2’), 13.9 ppm (BHA-16); IR: n˜ =3311, 2963, 2919, 2852, 1726,
1673, 1642, 1556, 1508, 1469 cmꢀ1; HPLC-ESIMS (C4–70; tR =5.00 min):
m/z: 646.41 [M+H]+, 668.51 [M+Na]+; HRMS (ESI): m/z: calcd for
C36H60N3O7: 646.4426 [M+H]+; found: 646.4424.
BHA5–15-H2), 1.13 (d, J=7.3 Hz, 3H; BHA2’-H3), 1.05 (d, J=6.3 Hz,
3H; Thr-Hg), 0.90 (d, J=6.6 Hz, 3H; Val-Hga), 0.85 (d, J=6.5 Hz, 3H;
Val-Hgb), 0.83 ppm (t, J=7.0 Hz, 3H; BHA16-H3); 13C NMR (151 MHz,
[D6]DMSO): d=171.0 (BHA-1), 170.8 (Val-1), 170.3 (Thr-1), 168.9 (Ser-
1), 137.9 (aromat. C), 129.8 (aromat. C), 128.1 (aromat. C), 127.3
(aromat. C), 77.9 (BHA-3), 72.4 (Ser-CH2Ph), 70.0 (Ser-b), 65.2 (Thr-b),
61.3 (Val-a), 59.1 (Thr-a), 52.5 (Ser-a), 52.4, 42.8 (BHA-2), 40.0 (BHA),
31.7 (BHA), 31.2 (BHA), 29.0 (BHA), 29.1 (Val-b), 28.9 (BHA), 28.8
(BHA), 28.7 (BHA), 28.6 (BHA), 24.8 (BHA), 22.0 (BHA), 20.9 (Thr-g),
19.2 (Val-ga), 18.9 (Val-gb), 13.9 ppm (BHA-16, BHA-2’); IR (neat): n˜ =
3285, 2923, 2853, 1725, 1684, 1637, 1544, 1455 cmꢀ1; HRMS (ESI): m/z:
calcd for C36H60N3O7: 646.4426 [M+H]+; found: 646.4425.
1,3.4-Anhydro[l-valyl-l-threonyl-3-O-benzyl-d-seryl-(2S,3R)-2-methyl-3-
hydroxyhexadecanoic acid] ((2S,3R)-30): Yield: 9.3 mg (14.4 mmol), 8%;
m.p.: 2128C; 1H NMR (600 MHz, [D6]DMSO): d=8.23 (d, J=9.8 Hz,
1H; Val-NH), 7.62 (d, J=9.3 Hz, 1H; Thr-NH), 7.35–7.17 (m, 5H;
aromat. H), 7.09 (d, J=6.5 Hz, 1H; Ser-NH), 4.96 (ddd, J=10.4, 4.8,
2.6 Hz, 1H; BHA3-H), 4.87 (d, J=4.5 Hz, 1H; Thr-OH), 4.44 (s, 2H;
Ser-CH2Ph), 4.40 (td, J=6.2, 4.6 Hz, 1H; Ser-Ha), 4.30–4.24 (m, 1H; Thr-
Hb), 4.15 (dd, J=9.2, 2.9 Hz, 1H; Thr-Ha), 4.00 (t, J=10.0 Hz, 1H; Val-
Ha), 3.71 (dd, J=9.5, 4.4 Hz, 1H; Ser-Hba), 3.58 (dd, J=9.5, 6.1 Hz, 1H;
Ser-Hbb), 2.60–2.53 (m, 1H; BHA2-H), 1.88–1.74 (m, 2H; Val-Hb,
BHA4a-H2), 1.62–1.51 (m, 1H; BHA4b-H2), 1.29–1.07 (m, 22H; BHA5–
15-H2), 1.04 (d, J=7.1 Hz, 3H; BHA2’-H3), 0.99 (d, J=6.4 Hz, 3H; Thr-
Hg), 0.89 (d, J=6.7 Hz, 3H; Val-Hga), 0.86 (d, J=6.5 Hz, 3H; Val-Hgb),
0.82 ppm (t, J=7.0 Hz, 3H; BHA16-H3); 13C NMR (151 MHz,
[D6]DMSO): d=171.8 (Val-1), 171.5 (BHA-1), 169.5 (Thr-1), 167.4 (Ser-
1), 137.6 (aromat. H), 128.0 (aromat. H), 127.4 (aromat. H), 127.2
(aromat. H), 76.5 (BHA-3), 72.2 (Ser-CH2Ph), 68.9 (Ser-b), 63.9 (Thr-b),
60.8 (Val-a), 56.9 (Thr-a), 54.3 (Ser-a), 42.8 (BHA-2), 31.2 (BHA), 29.7
(Val-b), 29.0 (BHA), 28.9 (BHA), 28.8 (BHA), 28.6 (BHA), 28.1 (BHA),
25.4 (BHA), 22.0 (BHA), 20.5 (Thr-g), 19.1 (Val-ga), 19.0 (Val-gb),
13.9 ppm (BHA-16, BHA-2’); IR (neat): n˜ =3313, 2964, 2918, 2852, 1720,
1672, 1638, 1541, 1507, 1468 cmꢀ1; HPLC-ESIMS (C4–70; tR =5.57 min):
m/z: 646.41, 668.53; HRMS (ESI): m/z: calcd for C36H60N3O7: 646.4426
[M+H]+; found: 646.4425.
General procedure: Phosphorylation: Macrocyle 13 (20.5 mg, 31.7 mmol)
and tetrazole (0.45m in CH3CN, 353 mL, 159 mmol) were dissolved in dry
CH2Cl2 (634 mL) and stirred for 10 min at RT. Dibenzylisopropyl phos-
phoramidite[32] (626 mL, 190 mmol) was added and after 3 h, the mixture
was cooled to 08C, and a tBuOOH solution (5.5m in hexane, 35 mL,
190 mmol) was added. The mixture was stirred for 15 min and quenched
with 10% Na2S2O3 (3 mL) at 08C. The aqueous layer was extracted with
ethyl acetate (4ꢃ3 mL) and the combined organic layers were dried with
MgSO4. After removal of the solvent, the crude product was purified by
preparative HPLC and lyophilized to give a colorless powder. The ob-
tained products were always found contaminated with minor amounts of
monodebenzylated compound (1–5%), which were apparent in the
31P NMR at ca. d=ꢀ6 ppm.
1,3.4-Anhydro[l-valyl-3-O-dibenzylphospho-l-threonyl-3-O-benzyl-l-
seryl-(2S,3R)-2-methyl-3-hydroxyhexadecanoic acid] ((2S,3R)-28): Yield:
20.1 mg (22.2 mmol), 70%; 1H NMR (400 MHz, CDCl3): d=7.63 (d, J=
6.2 Hz, 1H; Ser-NH), 7.35–7.15 (m, 15H; aromat. H), 7.00 (d, J=7.1 Hz,
1H; Thr-NH), 6.46 (brs, 1H; Val-NH), 5.41–5.30 (m, 1H; Thr-Hb), 4.98–
4.89 (m, 5H; POCH2Ph, BHA3-H), 4.50 (td, J=7.1, 2.0 Hz, 1H; Ser-Ha),
4.32 (d, J=10.8 Hz, 1H; Ser-CH2Ph), 4.24 (d, J=10.8 Hz, 1H; Ser-
CH2Ph), 4.23–4.18 (m, 1H; Thr-Ha), 4.15 (t, J=10.5 Hz, 1H; Val-Ha),
3.88 (dd, J=9.2, 2.1 Hz, 1H; Ser-Hba), 3.70 (dd, J=9.2, 2.3 Hz, 1H; Ser-
Hbb), 2.24–2.15 (m, 1H; BHA2-H), 1.73–1.60 (m, 2H; Val-Hb, BHA4a-
H2), 1.59–1.49 (m, 1H; BHA4b-H2), 1.32 (d, J=6.5 Hz, 3H; Thr-Hg),
1.25–1.13 (m, 25H; BHA), 1.05 (d, J=7.1 Hz, 3H; BHA2’-H3), 0.88 (d,
J=6.6 Hz, 3H; Val-Hga), 0.81 (t, J=7.0 Hz, 3H; BHA16-H3), 0.79 ppm
(d, J=6.5 Hz, 3H; Val-Hgb); 31P NMR (162.1 MHz, CDCl3): d=
ꢀ1.26 ppm.
1,3.4-Anhydro[l-valyl-l-threonyl-3-O-benzyl-l-seryl-(2S,3S)-2-methyl-3-
hydroxyhexadecanoic acid] ((2S,3S)-13): Yield: 72.1 mg (0.112 mmol),
62%; [a]2D0 =ꢀ58.2 (c=1 in CHCl3); m.p.: 2358C; 1H NMR (600 MHz,
[D6]DMSO): d=8.01 (d, J=8.2 Hz, 1H; Val-NH), 7.80 (d, J=9.3 Hz,
1H; Thr-NH), 7.33–7.20 (m, 5H; aromat. H), 7.09 (d, J=8.3 Hz, 1H;
Ser-NH), 5.16 (d, J=5.2 Hz, 1H; Thr-OH), 4.75 (ddd, J=10.5, 5.0,
3.9 Hz, 1H; BHA3-H), 4.48 (d, J=12.3 Hz, 1H; Ser-CH2Ph), 4.46 (d, J=
12.3 Hz, 1H; Ser-CH2Ph), 4.39 (ddd, J=8.2, 6.9, 4.7 Hz, 1H; Ser-Ha),
4.09–4.02 (m, 2H; Thr-Hb, Thr-Ha), 3.73 (t, J=8.6 Hz, 1H; Val-Ha), 3.62
(dd, J=9.5, 4.7 Hz, 1H; Ser-Hba), 3.46 (dd, J=9.5, 6.9 Hz, 1H; Ser-Hbb),
2.72–2.61 (m, 1H; BHA2-H), 1.77–1.69 (m, 1H; BHA4a-H2), 1.94–1.84
(m, 1H; Val-Hb), 1.50–1.41 (m, 1H; BHA4b-H2), 1.28–1.08 (m, 22H;
BHA5–15-H3), 1.02 (d, J=6.2 Hz, 3H; Thr-Hg), 0.99 (d, J=6.9 Hz, 3H;
BHA2’-H3), 0.93 (d, J=6.6 Hz, 3H; Val-Hga), 0.88 (d, J=6.8 Hz, 3H;
Val-Hgb), 0.82 ppm (t, J=7.1 Hz, 3H; BHA16-H3); 13C NMR (151 MHz,
[D6]DMSO): d=173.3 (BHA-1), 171.6 (Val-1), 170.2 (Thr-1), 167.2 (Ser-
1), 137.6 (ipso-aromat. C), 128.1 (2x m-aromat. C), 127.4 (p-aromat. C),
127.1 (2x o-aromat. C), 76.6 (BHA-3), 72.1 (Ser-CH2Ph), 69.6 (Ser-b),
64.9 (Thr-b), 61.6 (Val-a), 59.5 (Thr-a), 53.3 (Ser-a), 42.8 (BHA-2), 31.2
(BHA), 29.9 (BHA), 29.1 (Val-b), 28.9 (7x BHA), 28.6 (BHA), 22.8
(BHA), 22.0 (BHA), 20.8 (Thr-g), 19.3 (Val-ga), 19.0 (Val-gb), 14.7
(BHA-2’), 13.9 ppm (BHA-16); IR (neat): n˜ =3333, 2958, 2921, 2852,
1,3.4-Anhydro[l-valyl-3-O-dibenzylphospho-l-threonyl-3-O-benzyl-l-
seryl-(2R,3R)-2-methyl-3-hydroxyhexadecanoic
acid]
((2R,3R)-28):
Yield: 22.1 mg (24.4 mmol), 80%; 1H NMR (400 MHz, CDCl3): d=7.67
(d, J=6.8 Hz, 1H; Ser-NH), 7.24 (m, 15H; aromat. H), 7.08 (d, J=
7.0 Hz, 1H; Thr-NH), 6.04 (d, J=9.1 Hz, 1H; Val-NH), 5.44–5.32 (m,
1H; Thr-Hb), 4.99–4.92 (m, 4H; POCH2Ph), 4.71 (t, J=6.7 Hz, 1H;
BHA3-H), 4.47 (td, J=7.3, 2.2 Hz, 1H; Ser-Ha), 4.36 (d, J=10.7 Hz, 1H;
Ser-CH2Ph), 4.25 (d, J=10.7 Hz, 1H; Ser-CH2Ph), 4.18–4.01 (m, 2H;
Thr-Ha, Val-Ha), 3.86 (dd, J=9.1, 2.2 Hz, 1H; Ser-Hba), 3.65 (dd, J=9.1,
2.5 Hz, 1H; Ser-Hbb), 2.56 (dq, J=7.1, 0.55 Hz, 1H; BHA2-H), 1.95–1.82
(m, 1H; Val-Hb), 1.76–1.55 (m, 2H; BHA4-H2), 1.31 (d, J=6.5 Hz, 3H;
Thr-Hg), 1.26–1.09 (m, 25H; BHA, BHA2’-H3), 0.91 (d, J=6.6 Hz, 3H;
Val-Hga), 0.82 (d, J=6.0 Hz, 3H; Val-Hga), 0.81 ppm (t, J=6.5 Hz, 3H;
BHA16-H3); 31P NMR (162.1 MHz, CDCl3): d=ꢀ1.10 ppm.
1739, 1660, 1530, 1503, 1469 cmꢀ1
; HRMS (ESI): m/z: calcd for
C36H60N3O7: 646.4426 [M+H]+; found: 646.4425.
1,3.4-Anhydro[l-valyl-3-O-dibenzylphospho-l-threonyl-3-O-benzyl-l-
seryl-(2S,3S)-2-methyl-3-hydroxyhexadecanoic acid] ((2S,3S)-28): Yield:
23.8 mg (26.3 mmol), 84%; 1H NMR (400 MHz, CDCl3): d=7.70 (brs,
1H; Thr-NH), 7.37–7.02 (m, 15H; aromat. H), 6.87 (bs, 1H; Ser-NH),
6.30 (brs, 1H; Val-NH), 5.02 (ddd, J=14.4, 6.6, 3.9 Hz, 1H; Thr-Hb),
4.96–4.87 (m, 5H; BHA3-H, POCH2Ph), 4.51 (dd, J=9.0, 3.4 Hz, 1H;
Thr-Ha), 4.44 (dd, J=9.8, 5.4 Hz, 1H; Ser-Ha), 4.26 (d, J=1.7 Hz, 2H;
Ser-CH2Ph), 4.02–3.94 (m, 1H; Val-Ha), 3.70 (dd, J=9.5, 4.0 Hz, 1H;
Ser-Hba), 3.60 (dd, J=9.5, 5.6 Hz, 1H; Ser-Hbb), 2.59 (qd, J=13.9,
6.9 Hz, 1H; BHA2-H), 2.04–1.77 (m, 2H; Val-Hb, BHA4a-H2), 1.44 (ddd,
J=14.9, 10.3, 5.1 Hz, 1H; BHA4b-H2), 1.31 (d, J=6.6 Hz, 3H; Thr-Hg),
1.28–1.12 (m, 22H; BHA), 1.11 (d, J=6.9 Hz, 3H; BHA2’-H3), 0.97 (d,
J=6.6 Hz, 3H; Val-Hga), 0.93 (d, J=6.7 Hz, 3H; Val-Hgb), 0.81 ppm (t,
1,3.4-Anhydro[l-valyl-l-threonyl-3-O-benzyl-l-seryl-(2R,3R)-2-methyl-3-
hydroxyhexadecanoic acid] ((2R,3R)-13): Yield: 41.8 mg (64.8 mmol),
36%; [a]2D0 =ꢀ49.3 (c=0.5 in CHCl3); m.p.: 2348C; 1H NMR (600 MHz,
[D6]DMSO): d=7.94 (d, J=10.1 Hz, 1H; Thr-NH), 7.72 (d, J=9.0 Hz,
1H; Ser-NH), 7.60 (b, 1H; Val-NH), 7.40–7.20 (m, 5H; aromat. H), 5.21
(d, J=4.6 Hz, 1H; Thr-OH), 4.77–4.73 (m, 1H; Ser-Ha), 4.70 (ddd, J=
8.0, 6.2, 2.0 Hz, 1H; BHA3-H), 4.54 (d, J=12.4 Hz, 1H; Ser-CH2Ph),
4.47 (d, J=12.4 Hz, 1H; Ser-CH2Ph), 4.11 (dd, J=10.1, 2.6 Hz, 1H; Thr-
Ha), 4.06 (m, 1H; Thr-Hb), 4.06–4.01 (m, 1H; Val-Ha), 3.84 (dd, J=9.0,
2.6 Hz, 1H; Ser-Hba), 3.59 (dd, J=9.6, 3.0 Hz, 1H; Ser-Hbb), 2.58 (dq,
J=7.3, 1.90 Hz, 1H; BHA2-H), 2.09–1.95 (m, 1H; Val-Hb), 1.61–1.52 (m,
1H; BHA4a-H2), 1.48–1.40 (m, 1H; BHA4b-H2), 1.33–1.17 (m, 22H;
Chem. Eur. J. 2008, 14, 8847 – 8860
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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8857

H. Waldmann, H.-D. Arndt et al.
J=6.9 Hz, 3H; BHA16-H3); 31P NMR (162.1 MHz, CDCl3): d=
ꢀ1.11 ppm.
25H; BHA, Thr-Hg), 0.97 (d, J=6.9 Hz, 1H; BHA2’-H3), 0.92 (d, J=
6.5 Hz, 3H; Val-Hga), 0.88 (d, J=6.5 Hz, 3H; Val-Hgb), 0.85 ppm (t, J=
6.7 Hz, 3H; BHA16-H3); 13C NMR (151 MHz, [D6]DMSO): d=173.3
(BHA-1), 171.8 (Val-1), 169.4 (Thr-1), 168.1 (Ser-1), 76.5 (BHA-3), 69.5
(Thr-b), 62.0 (Ser-b), 61.8 (Val-a), 59.6 (Thr-a), 55.7 (Ser-a), 42.9 (BHA-
2), 31.2 (BHA), 30.0 (BHA-4), 29.1 (BHA), 29.0 (Val-b), 28.8 (BHA),
28.7 (BHA), 22.6 (BHA), 22.0 (BHA), 19.5 (Val-ga), 19.4 (Thr-g), 19.1
(Val-gb), 14.5 (BHA-2’), 13.9 ppm (BHA-16); 31P NMR (162.1 MHz,
CDCl3): d=ꢀ0.37; HPLC-ESIMS (C4–50, tR =5.52 min): m/z: 636.12
[M+H]+, 658.35 [M+Na]+; HRMS (ESI): m/z: calcd for C29H55N3O10P:
636.3620 [M+H]+; found: 636.3619.
1,3.4-Anhydro[l-valyl-3-O-dibenzylphospho-l-threonyl-3-O-benzyl-l-
seryl-(2R,3S)-2-methyl-3-hydroxyhexadecanoic acid] ((2R,3S)-28): Yield:
21.8 mg (24.1 mmol), 76%; 1H NMR (400 MHz, CDCl3): d=8.14 (brs,
1H; Thr-NH), 7.42–6.99 (m, 15H; aromat. H), 6.80 (brs, 1H; Ser-NH),
6.43 (brs, 1H; Val-NH), 5.24–5.15 (m, 1H; BHA3-H), 5.02 (ddd, J=16.7,
6.4, 3.4 Hz, 1H; Thr-Hb), 4.93–4.81 (m, 4H; POCH2Ph), 4.53–4.44 (m,
2H; Thr-Ha, Ser-Ha), 4.29 (d, J=12.0 Hz, 1H; Ser-CH2Ph), 4.22 (d, J=
12.0 Hz, 1H; Ser-CH2Ph), 4.00 (t, J=8.4 Hz, 1H; Val-Ha), 3.65 (dd, J=
9.5, 4.4 Hz, 1H; Ser-Hba), 3.55 (dd, J=8.8, 6.9 Hz, 1H; Ser-Hbb), 2.76–
2.59 (m, 1H; BHA2-H), 2.09–1.97 (m, 1H; Val-Hb), 1.98–1.69 (m, 2H;
BHA4-H2), 1.32 (d, J=6.8 Hz, 3H; Thr-Hg), 1.21 (d, J=7.4 Hz, 3H;
BHA2’-H3), 1.18 (s, 22H; BHA), 1.00–0.90 (m, 6H; Val-Hg), 0.81 ppm (t,
J=6.7 Hz, 3H; BHA16-H3); 31P NMR (162.1 MHz, CDCl3): d=
ꢀ1.24 ppm.
1,3.4-Anhydro[l-valyl-phospho-l-threonyl-l-seryl-(2R,3R)-2-methyl-3-hy-
droxyhexadecanoic acid) ((2R,3R)-14): Yield: 12.8 mg (23.1 mmol), 95%;
1H NMR (600 MHz, [D6]DMSO): d=8.79–8.41 (m, 2H; Ser-NH, Thr-
NH) 8.26 (brs, 1H; Val-NH), 4.64 (t, J=6.6 Hz, 1H; BHA3-H), 4.59–
4.46 (m, 2H; Ser-Ha, Thr-Hb), 4.15 (d, J=9.5 Hz, 1H; Thr-Ha), 4.01 (dd,
J=10.8, 10.8 Hz, 1H; Val-Ha), 3.75 (dd, J=11.2, 6.0 Hz, 1H; Ser-Hba),
3.61 (dd, J=10.9, 2.3 Hz, 1H; Ser-Hbb), 2.53–2.51 (m, 1H; BHA2-H),
2.22–2.13 (m, 1H; Val-Hb), 1.56–1.44 (m, 1H; BHA4a-H2), 1.42–1.32 (m,
1H; BHA4b-H2), 1.32–1.08 (m, 28H; BHA2’-H3, BHA, Thr-Hg), 0.89 (d,
J=6.5 Hz, 3H; Val-Hga), 0.85 (t, J=6.8 Hz, 3H; BHA16-H3), 0.84 ppm
(d, J=6.2 Hz, 3H; Val-Hgb); 13C NMR (151 MHz, [D6]DMSO): d=171.2
(Val-1), 170.2 (BHA-1), 170.0 (Thr-1), 169.7 (Ser-1), 76.9 (BHA-3), 70.0
(Thr-b), 61.6 (Val-a), 61.5 (Ser-b), 59.1 (Thr-a), 54.5 (Ser-a), 43.5 (BHA-
2), 32.0 (BHA-4), 31.2 (BHA), 29.0 (BHA), 28.9 (BHA), 28.7 (BHA),
28.5 (Val-b), 25.0 (BHA), 22.0 (BHA), 20.0 (Thr-g), 19.2 (Val-ga), 19.0
(Val-gb), 14.1 (BHA-2’), 13.9 ppm (BHA-16); 31P NMR (162.1 MHz,
[D6]DMSO): d=ꢀ1.26 ppm; HPLC-ESIMS (C4–50; tR =6.98 min): m/z:
636.16 [M+H]+, 658.35 [M+Na]+; HRMS (ESI): m/z: calcd for
C29H55N3O10P: 636.3620 [M+H]+; found: 636.3618.
General procedure: Debenzylation of phosphorylated depsipeptides:
Phosphotriester 28 (19.0 mg, 20.97 mmol) was dissolved in EtOH (3 mL).
10% Pd/C (7 mg) was added and the mixture was hydrogenated (H2 bal-
loon) for 4 h. Filtration through a pad of Celite and evaporation of the
solvent gave product 14 as a colorless oil.
1,3.4-Anhydro[l-valyl-phospho-l-threonyl-l-seryl-(2S,3R)-2-methyl-3-hy-
droxyhexadecanoic acid] ((2S,3R)-14): Yield: 8.2 mg (14.7 mmol), 87%;
1H NMR (600 MHz, [D6]DMSO): d=8.15 (d, J=8.6 Hz, 1H; Val-NH),
8.01 (d, J=7.9 Hz, 1H; Ser-NH), 7.61 (d, J=8.9 Hz, 1H; Thr-NH), 4.85–
4.77 (m, 1H; BHA3-H), 4.53–4.47 (m, 1H; Thr-Hb), 4.45 (dt, J=8.7,
4.3 Hz, 1H; Ser-Ha) 4.19 (dd, J=9.0, 4.6 Hz, 1H; Thr-Ha), 3.97 (dd, J=
10.7, 10.7 Hz, 1H; Val-Ha), 3.72 (dd, J=11.3, 4.5 Hz, 1H; Ser-Hba), 3.62
(dd, J=11.3, 4.1 Hz, 1H; Ser-Hbb), 2.47–2.43 (m, 1H; BHA2-H), 1.96–
1.80 (m, 1H; Val-Hb), 1.63–1.51 (m, 1H; BHA4a-H2), 1.42–1.29 (m, 1H;
BHA4b-H2), 1.30–1.12 (m, 25H; Thr-Hg, BHA), 1.03 (d, J=7.0 Hz, 1H;
BHA2’-H3), 0.91 (d, J=6.6 Hz, 3H; Val-Hga), 0.88 (d, J=6.6 Hz, 3H;
Val-Hgb), 0.85 ppm (t, J=6.9 Hz, 3H; BHA16-H3); 13C NMR (151 MHz,
[D6]DMSO): d=170.8 (BHA-1, Val-1), 169.0 (Ser-1), 168.8 (Thr-1), 76.3
(BHA-3), 71.1 (Thr-b), 61.3 (Val-a), 60.3 (Ser-b), 59.3 (Thr-a), 53.4 (Ser-
a), 43.3 (BHA-2), 31.3 (BHA), 29.3 (Val-b), 29.0 (BHA), 28.9 (BHA),
28.8 (BHA), 28.7 (BHA), 28.5 (BHA), 25.7 (BHA), 22.1 (BHA), 19.1
(Val-g), 18.9 (Thr-g), 13.9 ppm (BHA-2’, BHA-16); 31P NMR
(162.1 MHz, CDCl3): d=ꢀ0.46; HPLC-ESIMS (C4–50, tR =6.35 min):
m/z: 636.12 [M+H]+, 658.33 [M+Na]+; HRMS (ESI): m/z: calcd for
C29H55N3O10P: 636.3620 [M+H]+; found: 636.3619.
1,3.4-Anhydro[l-valyl-l-threonyl-l-seryl-(2S,3R)-2-methyl-3-hydroxyhex-
adecanoic acid] ((2S,3R)-29): Benzylated cylodepsipeptide 13 (15.8 mg,
24.6 mmol) was dissolved in EtOH (2.5 mL). 10% Pd/C (7 mg) and HCl
(25 mL, 1n) were added and the mixture was hydrogenated (H2 balloon)
for 4 h. Filtration through a pad of Celite and evaporation of the solvent
gave product 29 as a colorless solid. Yield: 12.4 mg (22.4 mmol), 91%; Rf:
0.27 (CH2Cl2/MeOH 10:1, v/v); [a]2D0 =ꢀ30.0 (c=0.5 in 1:1 MeOH/
CHCl3); 1H NMR (600 MHz, [D6]DMSO): d=8.12 (d, J=10.1 Hz, 1H;
Val-NH), 7.83 (d, J=8.7 Hz, 1H; Ser-NH), 7.46 (d, J=9.8 Hz, 1H; Thr-
NH), 5.10 (d, J=5.1 Hz, 1H; Thr-OH), 4.88–4.77 (m, 2H; Ser-OH,
BHA3-H), 4.49–4.42 (m, 1H; Ser-Ha), 4.10 (dd, J=9.8, 3.0 Hz, 1H; Thr-
Ha), 4.06–3.94 (m, 2H; Thr-Hb, Val-Ha), 3.78 (ddd, J=10.2, 5.5, 3.6 Hz,
1H; Ser-Hba), 3.57 (dd, J=10.8, 4.5 Hz, 1H; Ser-Hbb), 2.48–2.43 (m, 1H;
BHA2-H), 1.91 (dq, J=13.0, 6.5 Hz, 1H; Val-Hb), 1.67–1.56 (m, 1H;
BHA4a-H2), 1.46–1.33 (m, 1H; BHA4b-H2), 1.33–1.11 (m, 22H; BHA),
1.07 (d, J=6.3 Hz, 3H; Thr-Hg), 1.04 (d, J=7.1 Hz, 3H; BHA2’-H3), 0.92
(d, J=8.8 Hz, 3H; Val-Hga), 0.90 (d, J=8.6 Hz, 3H; Val-Hgb), 0.85 ppm
(t, J=6.7 Hz, 3H; BHA16-H3); 13C NMR (151 MHz, [D6]DMSO): d=
170.9 (BHA-1), 170.8 (Val-1), 170.1 (Thr-1), 169.0 (Ser-1), 76.3 (BHA-3),
65.3 (Thr-b), 61.5 (Thr-a), 61.1 (Ser-b), 60.2 (Val-a), 53.5 (Ser-a), 43.1
(BHA-2), 39.7 (Val-b), 31.2 (BHA), 28.9 (BHA), 28.9 (BHA), 28.9
(BHA), 28.8 (BHA), 28.6 (BHA), 25.7 (BHA), 22.0 (BHA), 20.7 (Thr-g),
19.1 (Val-ga), 19.0 (Val-gb), 13.9 ppm (BHA-16; BHA-2’); IR (neat): n˜ =
3444, 3333, 3085, 2922, 2852, 1729, 1656, 1550, 1466, 1367 cmꢀ1; HRMS
(ESI): m/z: calcd for C29H54N3O7: 556.3956 [M+H]+; found: 556.3950.
1,3.4-Anhydro[l-valyl-phospho-l-threonyl-l-seryl-(2R,3S)-2-methyl-3-hy-
droxyhexadecanoic acid] ((2R,3S)-14): Yield: 13.1 mg (20.3 mmol), 97%;
1H NMR (600 MHz, [D6]DMSO): d=9.01 (d, J=6.3 Hz, 1H; Thr-NH),
8.38 (brs, 1H; Val-NH), 7.69 (brs, 1H; Ser-NH), 4.75 (ddd, J=7.5, 4.8,
1.7 Hz, 1H; BHA3-H), 4.58–4.47 (m, 1H; Thr-Hb), 4.32 (td, J=9.8,
6.1 Hz, 1H; Ser-Ha), 4.05 (dd, J=9.0, 2.2 Hz, 1H; Thr-Ha), 3.87 (dd, J=
10.8, 9.8 Hz, 1H; Val-Ha), 3.59–3.45 (m, 2H; Ser-Hb), 2.50 (m, 1H;
BHA2-H), 2.24–2.13 (m, 1H; Val-Hb), 1.77–1.63 (m, 1H; BHA4a-H2),
1.47–1.35 (m, 1H; BHA4b-H2), 1.32–1.12 (m, 28H; BHA2’-H3, BHA,
Thr-Hg), 0.93–0.73 ppm (m, 9H; Val-Hg, BHA16-H3); 13C NMR
(151 MHz, [D6]DMSO): d=172.2 (BHA-1), 171.6 (Val-1), 170.1 (Thr-1),
168.8 (Ser-1), 76.1 (BHA-3), 69.2 (Thr-b), 62.0 (Ser-b, Val-a), 59.6 (Thr-
a), 55.8 (Ser-a), 43.0 (BHA-2), 31.3 (BHA), 29.8 (BHA), 29.0 (BHA),
28.9 (BHA), 28.8 (BHA), 28.7 (Val-b), 24.4 (BHA), 22.1 (BHA), 19.6
(Thr-g), 19.1 (Val-g), 13.9 (BHA-16), 8.8 ppm (BHA-2’); 31P NMR
(162.1 MHz, CDCl3): d=ꢀ0.87 ppm; HPLC-ESIMS (C4–50, tR =
5.98 min): m/z: 636.15 [M+H]+, 658.35 [M+Na]+; HRMS (ESI): m/z:
calcd for C29H55N3O10P: 636.3620 [M+H]+; found: 636.3619.
NMR spectroscopic experiments: All spectra were recorded at RT on a
Varian Mercury-Vx 400 MHz or a Varian Unity Inova 600 MHz NMR
spectrometer. For signal assignment 1H-, APT, TOCSY, gHSQC, and
gHMBC spectra were recorded. ROESY experiments have been per-
formed with 6 mg depsipeptide in 0.5 mL degassed [D6]DMSO at a
mixing time of 300 ms. Integration of the signals was performed with the
program Aurelia from Bruker and the integrated volumes were offset
corrected.[60] Proton–proton distances were calculated and ranked by
using two methylene protons as reference (1.8 ꢄ).
1,3.4-Anhydro[l-valyl-phospho-l-threonyl-l-seryl-(2S,3S)-2-methyl-3-hy-
droxyhexadecanoic acid] ((2S,3S)-14): Yield: 13.3 mg (24.0 mmol), 95%;
1H NMR (600 MHz, [D6]DMSO): d=8.59 (d, J=6.0 Hz, 1H; Val-NH),
8.37 (d, J=8.6 Hz, 1H; Thr-NH), 7.33 (d, J=8.7 Hz, 1H; Ser-NH), 4.66
(td, J=9.3, 4.4 Hz, 1H; BHA3-H), 4.58–4.51 (m, 1H; Thr-Hb), 4.25 (td,
J=9.4, 5.5 Hz, 1H; Ser-Ha), 4.11 (dd, J=8.8, 4.1 Hz, 1H; Thr-Ha), 3.72
(dd, J=9.3, 9.3 Hz, 1H; Val-Ha), 3.52 (d, J=5.5 Hz, 2H; Ser-Hb), 2.76
(qd, J=13.7, 6.7 Hz, 1H; BHA2-H), 2.07–1.95 (m, 1H; Val-Hb), 1.78–
1.67 (m, 1H; BHA4a-H2), 1.50–1.39 (m, 1H; BHA4b-H2), 1.32–1.14 (m,
Computational methods: The low-energy conformations of the cyclic
depsipeptides were calculated by using the programs Macromodel 9.1[37]
and Maestro. A total of 35,000 starting structures was generated for each
molecule by using the LMOD method with low-frequency eigenvector
8858
www.chemeurj.org
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8847 – 8860

Stevastelin C3 Analogues
FULL PAPER
sampling.[36] All ring atoms as well as the first atoms of the side chains
were used for the identification of new unique structures applying a root-
mean-square deviation (RMSD) threshold of 0.25 ꢄ. All conformers
within an energy window of 50 kJmolꢀ1 were used. Subsequently, energy
minimization with the truncated Newton conjugated gradient (TNCG)[61]
was performed on each conformer by using the OPLS 2005 force field[62]
with implemented GB/SA solvation model.[63] The line search parameter
was set from 1 to 0 to choose the direction of minimization steps, default
values were used for the remaining parameters. Convergence (RMSD
gradient=0.05 kJmolꢀ1 ꢄꢀ1) was reached after a maximum of 10,000 iter-
ations. For the restrained modeling up to 30 distances calculated from
NOE signals were set as distance constraints with a ꢁ10% flat potential
interval. Calculations starting from varying initial geometries gave consis-
tent results and each unique conformation was found on average 20–30
times. The final conformations were submitted to a clustering by using
Schrçdingerꢅs XCluster program. Clustering criterion was the atomic
root-mean-square (RMS) with all heavy ring atoms and the first heavy
atom of each substituent as comparison set. Subsequently, the hierarchy
level corresponding to an RMS of 0.1 ꢄ was chosen and its representa-
tive structures, the cluster centers, exported. Images were created by
using PyMol 0.99 from Delano Scientific. Overlays were generated based
on all heavy atoms in the macrocycle by using Schrçdinger Maestro 7.5.
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Inhibition studies: The human VHR (ꢃ12.5 mgmLꢀ1) was prepared ac-
cording to published procedures.[51] 10 mm stock solutions of inhibitors
were prepared in DMSO. The assay buffer consisted of 25 mm MOPS,
5 mm EDTA, 1 mm DTE, and 0.025% NP-40. The pH was adjusted to
6.5 and the buffer was filtered and degassed. 10 mm pNPP was used as
substrate. In a 96-well plate a dilution series of 50 mL inhibitor was pipet-
ted. After addition of 30 mL VHR (final dilution 1:1600), the mixture was
incubated at 378C for 30 min. As soon as 20 mL pNPP was added, the ex-
tinction was monitored for 40 min in 2 min steps at 405 nm. Subsequently,
an endpoint measurement was performed. To this end, 10 mL 2n NaOH
were added to each well and the extinction was measured again. All ex-
periments were carried out in triplicate, including the known PTP inhibi-
tor IV[64] as control. To determine the IC50 values, experimental data
were analyzed by using the ORIGIN (Microcal Software) program.
Malachite green test:[59] The malachite green solution was prepared by
addition of malachite green oxalate (350 mg, 0.38 mmol) and ammonium
molybdate (3.2 g, 2.59 mmol) to 33% HCl (45 mL). The mixture was di-
luted with water to 150 mL, stirred for 12 h, and filtrated twice. The same
assay conditions as for the IC50 determination without addition of the
substrate were applied. After 45 min, 50 mL malachite green solution was
added and the mixture was incubated for an additional 15 min at RT. For
calibration, a series of defined Na2HPO4 concentrations were measured.
The phosphate concentration was monitored by UV-absorbance at
620 nm.
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This research was supported by the Zentrum fꢀr Angewandte Chemische
Genomic, the Max-Planck Gesellschaft (to H. Waldmann) and the DFG
(Emmy-Noether Young Investigator grant to H.-D. Arndt). S. Wetzel was
supported by a Novartis graduate fellowship. We thank Dr. K. Alexan-
drov (Max-Planck-Institute Dortmund) for enzyme preparation and pu-
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Received: April 11, 2008
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