Synthesis and biological activity of optimized belactosin C congeners

Article abstract of DOI:10.1039/c1ob05661a

Successful biochemical studies of the natural products belactosin A and C as well as their more stable acylated derivatives have proved them to be powerful proteasome inhibitors and thereby potential candidates as pharmacologically relevant active compoun

Full text of DOI:10.1039/c1ob05661a

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PAPER
Synthesis and biological activity of optimized belactosin C congeners†
Vadim S. Korotkov,a Antje Ludwig,b Oleg V. Larionov,a Alexander V. Lygin,a Michael Grollc and
Armin de Meijere*a
Received 27th April 2011, Accepted 11th August 2011
DOI: 10.1039/c1ob05661a
Successful biochemical studies of the natural products belactosin A and C as well as their
more stable acylated derivatives have proved them to be powerful proteasome inhibitors and thereby
potential candidates as pharmacologically relevant active compounds. In order to understand their
structure–biological activity relations in detail and to find ways of improving their biological activity, four
new modified belactosin congeners have been synthesized and tested. One of them (compound 6) turned
out to be a more potent inhibitor against HeLa cells than the known proteasome inhibitor MG132.
that 2-isobutylmalic acid monophenylthio ester 4 (Fig. 1) as a key
building block could be easily prepared in enantiomerically pure
Introduction
Because of their potent proteasome inhibitory effects1 and other
important biological activities of the natural products belactosin
A and C and especially several of their acylated as well as
otherwise modified congeners, these b-lactone derivatives have
attracted considerable interest.1–3 Despite this interest, however,
only a small number of detailed investigations concerning their
biological activities have been carried out so far.4,5
form.6b Subsequent peptide coupling of 4 with the corresponding
dipeptide and ensuing in situ b-lactone ring closure provided the
diprotected derivatives of belactosins A and C and homobelac-
tosin C, 1, 2 and 3, respectively, which were eventually completely
deprotected.
In order to better understand their proteasome inhibitory power
and the important structure–activity relationships, the prevail-
ing interactions between such molecules and the proteasome
should be known. Towards this end, a protected homobelactosin
C was initially cocrystallized with the 20S proteasome from
Saccharomyces Cerevisiae, and the structure of this crystal was
elucidated by X-ray diffraction.1 On the basis of this analy-
sis it was decided which groups in the inhibitor should be
modified in order to improve potentially favorable interactions
with the proteasome. In this paper we report the synthesis and
detailed structure–activity investigations of some new belactosin C
congeners.
Fig. 1 The naturally occurring proteasome inhibitors belactosin A and
C, its non-natural analogue homobelactosin C and a key building block 4
in our total syntheses of 1–3.6b
Computer modelling of possible modifications of the belactosin
structure showed that attachment of a 2-naphthylethoxycarbonyl
(CNAP) group onto the amino terminus in the alanine residue in
belactosin C could be favorable for hydrophobic interactions and
thus improve its biological activity. On the other hand, introduc-
tion of a 4-carboxybenzyl residue capable of both hydrophilic and
hydrophobic interactions, onto the ornithine part of belactosin C
could also lead to an improved biological activity.
To test these hypotheses, we prepared the analogues 6, 11 and
15 of belactosin C (2) as well as the known derivative 16 according
to our previously published strategy.6b Compound 6 with a 2-
naphthylmethoxycarbonyl protective group on the amino termi-
nus was obtained starting from the 2-naphthylmethoxycarbonyl-
protected alanine (CNAP-Ala-OH) 5 by coupling it with the
appropriately decorated ornithine and subsequently installing
Chemistry
Six different total syntheses of members of the belactosin family
have been reported up to date.6 Our own approach to the
belactosins A 1 and C 2 as well as homo-C 3 relied on the fact
aInstitut fu¨r Organische und Biomolekulare Chemie der Georg-August-
Universita¨t Go¨ttingen, Tammannstrasse 2, D-37077 Go¨ttingen, Germany.
E-mail: Armin.deMeijere@chemie.uni-goettingen.de
bCharite´ Universita¨tsmedizin Berlin CCM Med. Klinik fu¨r Kardiologie und
Angiologie Kardiologisches Forschungslabor Ziegelstrasse 5-9, D-10117
Berlin, Germany
cCenter for Integrated Protein Science at the Department Chemie, Lehrstuhl
fu¨r Biochemie, Technische Universita¨t Mu¨nchen, Garching, D-85747,
Germany
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05661a
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the b-lactone moiety by a one-pot acylation/b-lactonization6b
reaction employing the building block 4 (Scheme 1).
Scheme 1 Synthesis of the new belactosin C derivative 6 with a
(2-naphthylmethoxy)carbonyl substituent at the amino terminus. Reagents
and conditions: (a) H2N-Orn(Boc)-OBn, EDC, HOAt, TMP, 0 to 20 ◦C,
24 h, 77%. (b) (1) HCl, EtOAc, r.t., 12 h (2) 4, TMP, HOAt, EDC, -20 to
20 ◦C, 48 h, 33%.
Scheme 2 Synthesis of the belactosin C analogues 10 and 11 with
functionally substituted benzyl ester moieties. Reagents and conditions:
(a) KOH, tBuOH, 50 ◦C, 3 h, 63%. (b) Bu4NBH4, r.t., 18 h, 75%. (c)
Fmoc-Orn(Boc)-OH, DCC, CH2Cl2, 0 ◦C, 6 h, 74%. (d) (1) Et2NH, THF,
3 h; (2) Cbz-Ala-OH, EDC, TMP, HOAt, 0 to 20 ◦C, 24 h, 65%. (e) (1)
HCl, EtOAc, r.t., 3 h; (2) 4, TMP, HOAt, EDC, -20 to 20 ◦C, 48 h, 37%.
(f ) CF3CO2H, CH2Cl2, -18 ◦C, 18 h, 99%.
The synthesis of the belactosin C analogues 10 and 11 con-
taining a tert-butoxycarbonyl (10) and a free carboxylic acid
group, respectively, in the benzyl ester moiety, commenced with
partial hydrolysis of di-tert-butyl terephthalate 7 followed by a
chemoselective reduction of the free carboxylic acid group with
tetrabutylammonium borohydride to furnish the benzyl alcohol 8
(Scheme 2).
The DCC-mediated condensation of 8 with the correspondingly
protected ornithine gave rise to the N-terminally Boc-protected
intermediate 9. This was converted into the tripeptide 10 by way
of Fmoc removal, peptide coupling with Cbz-Ala-OH, selective
removal of the N-Boc group, and the one-pot acylation/b-
lactonization reaction as a key step. Cleavage of the tert-butyl ester
under acidic conditions (trifluoroacetic acid in dichloromethane)
yielded the desired belactosin C congener 11 with a free carboxylic
acid functionality in the benzyl ester moiety.
the dipeptide 13, which after hydrogenolytic deprotection was
condensed with the hydroxyacid monothioester 4 to give the
diprotected belactosin C derivative 14. Complete deprotection
was effected upon treatment with trifluoroacetic acid to yield the
hydrotrifluoroacetate salt 15 of belactosin C.
The belactosin C derivative 16 was synthesized according to the
previously published protocol.6b
Thus, the novel derivatives of belactosin C 6, 11 and 15 were
prepared in sizable quantities employing our original synthetic
protocol.6b
Results and discussion
Finally, the stable and storable crystalline hydrotrifluoroacetate
salt of belactosin C 15 was prepared along the route outlined
in Scheme 3. Thus, H-Orn(Cbz)-OtBu 12 was transformed into
To investigate the effect of the obtained belactosin C congeners
on proteasomal activity in mammalian cells, HeLa cells were
treated with varying concentrations of compounds 6, 10, 14, and
Scheme 3 Preparation of the belactosin C hydrotrifluoroacetate salt. Reagents and conditions: (a) Boc-Ala-OH, EDC, HOAt, TMP, 0 to 20 ◦C, 24 h,
87%. (b) (1) H2/Pd/C, r.t., 16 h; (2) 4, EDC, HOAt, TMP, -20 to 20 ◦C, 48 h, 57%. (c) CF3CO2H, CH2Cl2, -18 ◦C, 18 h, 99%.
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Fig. 2 Biological activities of belactosin C congeners 6, 14 and 16 in comparison to MG132. A) HeLa cells were treated with the indicated concentrations
of MG132 and belactosin C congeners 6, 14 and 16 or solvent for 4 h. Cells were lysed, followed by measurement of chymotrypsin-like (ChTL),
trypsin-like (TL) and caspase-like (CaspL) activities in the in vitro activity assay employing fluorogenic substrates. Activities are expressed as percentage
of a solvent-treated control. Values are given as the means of three independent experiments SEM. B) Ear fibroblasts of UbG76V-GFP1 transgenic mice
were treated with increasing concentrations of MG132 and belactosin C congeners 6, 14, and 16 or solvent (C) for 4 h. Accumulation of degradation-prone
proteins was analyzed in cell lysates by western blot, using anti-GFP and anti-ubiquitin antibodies. Amido black staining of western blot membranes
served as a control for equal protein loading (LC).
16 for 4 h as well as with the widely used proteasome inhibitor
MG132. Subsequently, an in vitro activity assay was performed
in cell lysates employing the fluorogenic substrates SSLVY-AMC,
BzVGR-AMC and ZLLE-AMC to measure the chymotrypsin-
like (ChTL), the trypsin-like (TL) and the caspase-like (CaspL)
activities respectively. All compounds dose dependently inhibited
proteasomal degradation. The ChTL activity was predominantly
affected by all substances. As shown in Fig. 2A, the inhibitory
effects of compounds 14 and 16 were similar to those of MG132,
albeit MG132 was more effective in concentrations below 0.25 mM.
Compound 6 turned out to be the most effective inhibitor.
Notably, besides its potent inhibitory effect on the ChTL activity,
compound 6 showed the most pronounced effect of all tested
substances on TL activity, in particular in concentrations above
0.25 mM. Compound 10 displayed a considerably lower inhibitory
potency compared to MG132.
The accumulation of polyubiquitinated proteins or proteins
that contain a constitutively active degradation signal is one
characteristic effect of proteasome inhibition in cells. To detect
the levels of degradation-prone proteins that accumulated upon
incubation with belactosin C congeners, fibroblasts obtained
from ears of UbG76V-GFP1 mice were treated with varying
concentrations of compounds 6, 14, 16, and MG132 for 4 h.
UbG76V-GFP1 mice contain the green fluorescent protein (GFP)
fused to a constitutively active degradation signal (UbG76V),
thereby allowing us to monitor the accumulation of this specific
reporter protein in addition to overall polyubiquitinated protein.
Following treatment, cell lysates were analyzed by western blot,
using anti-GFP and anti-ubiquitin antibodies. As shown in Fig.
2 B, a concentration-dependent accumulation of endogenous
polyubiquitinated proteins, as well as elevated levels of UbG76V
-
GFP were caused by substances 6, 14, and 16, thereby confirming
their inhibitory effect on proteasomal activity in living cells. In
concentrations below 1mM, compounds 14 and 16 lead to a
considerably lower accumulation of polyubiquitinated proteins
and UbG76V-GFP compared to MG132. It is noteworthy that a
similar effect was observed for compound 6, which appeared to be
a more potent inhibitor compared to MG132 as estimated in the
in vitro activity assay.
In order to get insight into the mechanism of biological activities
of these compounds, compound 6 was co-crystallized with the
yeast 20S proteasome (see Fig. 3).
Yeast 20S proteasome crystals were soaked with compound 6
for 24 h at a final concentration of 5 mM. Data collected from the
soaked crystals were evaluated by molecular replacement using the
coordinates of the yeast 20S proteasome.7 Subsequent bulk solvent
correction and positional refinement performed with CNS yielded
an Rfree of 23.7% (Table S1†). The 2Fo-Fc-electron density map
calculated after density averaging visualized compound 6 in the
primed site of the chymotrypsin-like substrate binding channel,
for which it exerts a high selectivity (Fig. 3A). In close analogy
to what has been observed for bis-protected homobelactosin C
(hBelC),1 the carbonyl carbon atom of the ligand derived from
the beta-lactone ring is covalently bound to the free hydroxyl
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the novel chemical, biological and structural insights into the
binding mechanism of belactosin C derivatives might contribute
to the development of new anti-inflammatory or anti-cancer drugs
targeting the proteasome.
Conclusion
Four novel belactosin congeners have been synthesized according
to an established protocol6b in order to check the hypothesis
concerning their structure–activity relations based on the com-
puter modelling of their complexes with the proteasome. The best
substances turned out to have an improved biological activity
against HeLa cells at least comparable or even superior to the
activity of the known proteasome inhibitor MG132. The most
active compound 6 is more potent than MG132, thus becoming
a promising candidate for further investigations and possible
application in cancer therapy. A detailed investigation of the
complex of 6 with the proteasome showed an identical binding
mode (and thus mechanism of its activity) as the known b-lactone
inhibitor homobelactosin C.
Experimental section
Fig. 3 The belactosin C derivative 6 specifically binds to the chymotryp-
tic-like active site by formation of an ester. (a) Stereorepresentation of
the chymotryptic-like active site of the yeast 20S proteasome (colored in
bisque) in its complex with 6 (colored in green). The covalent linkage of the
inhibitor with b5-Thr1Og is drawn in magenta. The electron density map
(colored in grey) is contoured from 1s around Thr-1 (colored in black)
with 2Fo - Fc coefficients after twofold averaging. Temperature factor
refinement indicates full occupancies of the inhibitor-binding site except
for the 2-naphthylmethoxycarbonyl side chain. 6 has been omitted for
phasing. Amino acid residues stabilizing 6 are colored in black. (b) Surface
representation of the chymotryptic-like active site in its complex with 6
covalently bound to Thr-1 (depicted in white). Surface colors indicate
positive and negative electrostatic potentials contoured from 15 kT/e
(intense blue) to -15 kT/e (intense red). (c) Structural superposition of 6
and bisbenzyl-protected homobelactosin C (hBelC), including Thr-1 with
respect to subunit b5. 6 is shown in green, hBelC as well as the active site
Thr1 are drawn in black.
Synthesis of belactosin C congeners
General remarks. 1H and 13C NMR spectra were recorded
at 250, 300, 600 (1H), and 75.5, 151 MHz (13C), additional
APT (Attached Proton Test)] with Bruker AM 250, Var-
ian AMX 300 and Inova 600 instruments on compounds in
CDCl3 solutions if not otherwise specified, chemical shifts are
reported as d values in ppm, coupling constants J in Hz.
IR: Bruker IFS 66 (FT-IR) spectrometer, measured as KBr
pellets or as films between KBr plates. MS (ESI): Finnigan
MAT 95 spectrometer. Optical rotations: Perkin-Elmer 241
digital polarimeter, 1 dm cell. Starting materials: CH2Cl2 was
distilled from P4O10. 4-(tert-Butyloxycarbonyl)phenylmethanol
(8) was synthesized from di-tert-butyl terephthalate (7)
according to a published procedure.10 (2S)-[(2S)-Benzyloxy-
carbonylaminopropionylamino]-5-{[(3S)-((1S)-methylpropyl)-4-
oxo-oxetane-(2R)-carbonyl]amino}pentanoic acid benzyl ester
(16) was synthesized according to a known procedure.6b All other
chemicals were used as commercially available. All reactions were
performed under an atmosphere of dry nitrogen. Organic extracts
were dried over Na2SO4.
group of the catalytic N-terminal Thr1Oy of subunit b5 (Fig. 3B).
Except for the interaction of the isoleucine side chain with the
S1 specificity pocket, the molecule targets only the primed site
of the substrate binding channel. Structural superposition of 6
and hBelC bound to the subunit b5 reveals that shortening of
the linker by a methylene group in 6 compared to hBelC only
affects the conformation of the inhibitor backbone but not that
of the side chains. The perfect match of the benzyl side chains
of both compounds indicates an important proteasomal primed
site for substrate binding at this position (Fig. 3C). The restricted
size of the primed specificity pockets in the yeast b1- and b2-
subunits8 explains the selectivity of 6 and hBelC to only bind
by their extended benzyl side chain to the chymotrypsin-like
active site. Furthermore, comparison of the primary sequences of
mammalian subunits b5 and b5i reveals a conspicuous alteration in
this primed specificity channel: Ser115 and Glu116 of the human
constitutive subunit b5 are replaced by Glu and His in subunit
b5i, respectively. The putative primed substrate binding channels
in the b1- and b2-subunits modelled on the basis of subunit
b5 show similar architectures and exhibit similar significant
differences between constitutive and immune subunits.9 Thus,
((2S)-Naphthylmethoxycarbonylamino))propionic acid (CNAP-
Ala) (5). To a stirred ice-cold solution of L-alanine (1.8 g,
20 mmol) and NaOH (1.6 g, 40 mmol) in H2O (10 mL) was added
2-naphthylmethyl chloroformate11 (CNAP–Cl) (6.3 g, 29 mmol).
Then water (10 mL) and THF (10 mL) were added, upon which
the solution became clear. The reaction mixture was stirred at r. t.
for 2 h, and the reaction was then quenched by adding a saturated
solution of NaHCO3 (20 mL). The mixture was extracted with
Et2O (1 ¥ 30 mL), the ethereal phases were discarded, and the
pH value of the aqueous phase was adjusted to 2–3 with 12 M
aqueous HCl. Then it was extracted EtOAc (3 ¥ 30 mL), and the
combined organic phases were dried. Evaporation of the solvent
gave◦4.4 g (80%) of the acid 5 as a colorless solid, m.p. 132–
1
133 C. – H NMR (500 MHz, CD3OD): 1.48 (3 H, d, J = 7
Hz), 4.31 (1 H, q, J = 7 Hz), 5.32 (2 H, s), 7.50–7.56 (3 H, m),
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7.86–7.92 (4 H, m) ppm. – 13C NMR (125.7 MHz): 17.8 (CH3),
50.8 (CH), 67.6 (CH2), 126.5 (CH), 127.1 (CH), 127.2 (CH), 127.6
(CH), 128.6 (CH), 128.9 (CH), 129.1 (CH), 134.4 (C), 134.6 (C),
135.6 (C), 158.4 (C), 176.5 (C) ppm. – IR (KBr): n = 3329 cm-1,
3048, 1692, 1535, 1461, 1253, 1076, 820, 737, 623. – [a]2D0 -8.1
General procedure (GP2) for Boc-removal and subsequent se-
quential acylation/b-lactonization. To a solution of the respective
dipeptide (1.56 mmol) in EtOAc (3 mL) was added 3 N HCl in
EtOAc (12 mL). The mixture was stirred at r. t. for 12 h, then
concentrated under reduced pressure at 40 ◦C. The resulting crude
solid product was dried in vacuo (0.05 mbar) at r. t. for 3 h, then
dissolved in DMF (6 mL) and the solution cooled to -30 ◦C.
A solution of the thioester 4 (440 mg, 1.57 mmol) in CH2Cl2
(20.6 mL) was added first, then HOAt (255 mg, 1.54 mmol), TMP
(377 mg, 3.11 mmol), as well as EDC (561 mg, 3.11 mmol), and
the resulting mixture was stirred at -30 ◦C for 6 h, then at r. t. for
30 h. It was then washed with 1 N aqueous KHSO4 (80 mL), dried
and concentrated under reduced pressure. The oily residue was
purified by column chromatography [SiO2 (60 g), hexane–EtOAc
(1 : 2)] to give the desired product.
+
(c 1.0, MeOH). – MS (DCI) m/z = 564 ([2 M + NH4 ], 4), 291
+
([M + NH4 ], 100). – Calcd. for C15H15NO4: C 65.92%, H 5.53%,
N 5.13%, found: C 65.65%, H 5.31%, N 4.89%.
General procedure (GP1) for Fmoc-deprotection of the benzyl
esters and subsequent peptide condensation. To a solution of the
respective benzyl ester (1.70 mmol) in THF (3.3 mL) was added at
r.t. Et2NH (3.3 mL). The mixture was stirred at r. t. for 1 h, then
another 2 mL of Et2NH was added, and the mixture was stirred
for an additional 2 h. The volatiles were removed under reduced
pressure at 35 ◦C. The oily residue was azeotropically distilled off
with toluene (2 ¥ 8 mL) under reduced pressure at 45–50 ◦C, and
the crude amine was taken up in CH2Cl2 (5 mL).
(2S)-[(2S)-(2-Naphthyl)methoxycarbonylaminopropionylamino]-
5-{[(3S)-((1S)-methylpropyl)-4-oxo-oxetane-(2R)-carbonyl]ami-
no}pentanoic acid benzyl ester (6). Compound 6 was prepared
according to GP2 from 2-naphthylmethoxycarbonyl-(S)-alanyl-
(S)-(Nd-tert-butyloxycarbonyl)ornithine benzyl ester (900 mg,
1.56 mmol), the thioester 4 (440 mg, 1.57 mmol), TMP (377 mg,
3.11 mmol), HOAt (255 mg, 1.54 mmol) and EDC (561 mg,
3.11 mmol) in dichloromethane (20.6 mL). Yield 320 mg (33%),
Rf 0.46 [hexane–EtOAc (1 : 2)]. – 1H NMR (300 MHz): 0.88 (3 H,
t, J = 7 Hz), 0.97 (3 H, d, J = 7 Hz), 1.20–1.27 (1 H, m), 1.37 (3 H,
d, J = 7 Hz), 1.35–1.48 (2 H, m), 1.54–1.64 (2 H, m), 1.78–1.92 (2
H, m), 3.05–3.12 (1 H, m), 3.20–3.28 (1 H, m), 3.53 (1 H, dd, J =
8, 5 Hz), 4.33–4.40 (1 H, m), 4.48 (1 H, d, J = 5 Hz), 4.57–4.62 (1
H, m), 5.09–5.18 (2 H, m), 5.20–5.27 (2 H, m), 5.66–5.70 (1 H,
d, J = 8 Hz), 6.82 (1 H, m), 7.03 (1 H, d, J = 8 Hz), 7.29–7.37 (4
H, m), 7.40–7.44 (1 H, m), 7.46–7.49 (2 H, m), 7.78–7.84 (4 H,
m) ppm. – 13C NMR (75.5 MHz): 18.2 (CH3), 25.5 (CH2), 27.9
(CH3), 28.2 (CH3), 29.6 (CH2), 40.4 (CH2), 50.1 (CH), 52.3 (CH),
66.5 (C), 80.0 (C), 82.2 (CH2), 128.0 (CH), 128.4 (CH), 136.5
(CH), 155.4 (C), 156.5 (C), 171.0 (C), 172.5 (C) ppm. – IR (film):
n = 3306 cm-1, 3059, 2964, 2877, 1830, 1734, 1717, 1700, 1696,
1684, 1669, 1653, 1539, 1250, 1098, 1072, 908, 734. – [a]2D0 = +5.4
(c 0.96, CHCl3). – MS (ESI) positive ion mode: m/z = 1285 ([2
M + Na+], 100), 654 ([M + Na+], 90). Negative ion mode: m/z =
676 ([M + HCOO-], 100), 630 ([M – H+], 30). – HRMS (ESI)
[M + H+]. – Calcd. for C35H42N3O8 632.2972, found 632.2966.
A separate flask was charged with the corresponding protected
alanine (2.06 mmol), CH2Cl2 (7.5 mL) and HOAt (0.26 g,
1.92 mmol). EDC (0.32 g, 2.04 mmol) was added dropwise at
0
0
◦C within 10 min, and the resulting mixture was stirred at
◦C for 20 min. To the solution of the prepared crude amine
in CH2Cl2 was added TMP (0.66 g, 0.72 mL, 5.43 mmol), and the
resulting suspension was added through a cannula to the stirred
reaction mixture. This was left to attain r.t. within 16 h and then
concentrated under reduced pressure. The residue was taken up in
EtOAc (30 mL). The cloudy solution was washed with 1 N aqueous
KHSO4 solution (2 ¥ 30 mL), and saturated aqueous NaHCO3
solution (2 ¥ 30 mL), then dried and concentrated under reduced
pressure. The oily residue was taken up in EtOAc (5 mL) and
purified by column chromatography [SiO2 (50 g), hexane–EtOAc
1 : 1] to give the desired dipeptide.
2-Naphthylmethoxycarbonyl-(S)-alanyl-(S)-(Nd-tert-butyloxy-
carbonyl)ornithine benzyl ester [CNAP-Ala-Orn(Boc)-OBn]. 2-
Naphthylmethoxycarbonyl-(S)-alanyl-(S)-(Nd-tert-butyloxycar-
bonyl)ornithine benzyl ester (756 mg, 77%) was prepared from
5 (560 mg, 2.06 mmol) and Nd-(tert-butyloxycarbonyl)-Na -
(9H-9-fluorenyloxycarbonyl)-L-ornithine benzyl ester (900 mg,
1.70 mmol) according to GP1 as a colorless solid. Rf 0.40 [hexane–
EtOAc (1 : 1)], m. p. 112–113 ◦C. – 1H NMR (300 MHz): 1.30–1.50
(2 H, m), 1.38 (3 H, d, J = 7 Hz), 1.41 (9 H, s), 1.60–1.92 (2 H,
m), 2.90–3.11 (2 H, m), 4.26–4.40 (1 H, m), 4.53–4.64 (1 H, m),
4.65–4.80 (1 H, m), 5.07–5.20 (2 H, m), 5.21–5.32 (2 H, m), 5.64–
5.77 (1 H, br m), 7.05–7.12 (1 H, m), 7.28–7.40 (4 H, m), 7.40–7.52
(3 H, m), 7.75–7.85 (4 H, m) ppm. – 13C NMR (75.5 MHz): 18.6
(CH3), 26.0 (CH2), 28.3 (CH3), 28.9 (CH2), 39.7 (CH2), 50.4 (CH),
52.1 (CH), 67.0 (CH2), 67.1 (CH2), 79.2 (C), 125.7 (CH), 126.1
(CH), 126.2 (CH), 127.0 (CH), 127.6 (CH), 127.9 (CH), 128.2
(CH), 128.3 (CH), 128.4 (CH), 128.5 (CH), 133.0 (C), 133.1 (C),
133.6 (C), 135.1 (C), 155.9 (C), 156.1 (C), 171.7 (C), 172.3 (C)
ppm. – IR (film): n = 3336 cm-1, 2926, 1718, 1700, 1685, 1669,
1653, 1521, 1507, 1457, 1368, 1251, 1159, 1027, 912, 738. – [a]D20
-6.0 (c 1.0, CHCl3). – MS (ESI) positive ion mode: m/z = 1177 ([2
M + Na+], 100), 600 ([M + Na], 40). Negative ion mode: m/z =
622 ([M + CH3COO-], 100). – HRMS (ESI) [M + H+]. – Calcd. for
C33H40N3O7 578.2866, found 494.2861. – Calcd. for C32H39N3O7: C
66.53%, H 6.80%, N 7.27%, found: C 60.44%, H 6.65%, N 7.08%.
5-tert-Butyloxycarbonylamino-(2S)-(9H-fluoren-9-ylmethoxy-
carbonylamino)pentanoic acid (4-tert-butyloxycarbonylphenyl)-
methyl
ester
(9). Dicyclohexylcarbodiimide(1360
mg,
6.60 mmol) was added at 0 ◦C to a mixture of 5-tert-
butoxycarbonylamino-(2S)-(9H)-fluoren-9-ylmethoxycarbonyla-
mino)pentanoic acid (1530 mg, 3.37 mmol) and 4-(tert-
butyloxycarbonyl)phenylmethanol 8 (700 mg, 3.37 mmol) in
CH2Cl2 (6.5 mL) and DMF (3.5 mL). The reaction mixture
was stirred at r. t. for 3 h, then the solvent was distilled off
under reduced pressure, and the residue was purified by column
chromatography, yielding 800 mg (37%) of the product (Rf 0.28
[hexane–Et2O (1 : 1)]) along with 350 mg (50%) of starting alcohol
(Rf 0.45 [hexane–Et2O (1 : 1)]). – 1H NMR (300 MHz): 1.43 (9 H,
s), 1.58 (9 H, s), 1.88–2.00 (4 H, m), 3.04–3.20 (2 H, m), 3.40–3.54
(1 H, m), 4.08–4.24 (1 H, m), 4.30–4.44 (2 H, m), 5.07 (2 H, s),
5.19–5.22 (2 H, m), 7.26–7.44 (6 H, m), 7.56–7.64 (2 H, m), 7.76
(2 H, d, J = 8 Hz), 7.96 (2 H, d, J = 8 Hz) ppm. – 13C NMR
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(75.5 MHz): 24.9 (CH2), 25.6 (CH2), 28.1 (CH3), 28.4 (CH3), 29.6
(CH2), 33.9 (CH2), 47.0 (CH), 47.1 (CH), 66.5 (CH2), 66.9 (CH2),
81.2 (C), 120.0 (CH), 127.0 (CH), 127.7 (CH), 129.7 (CH), 141.2
(C), 141.3 (C), 141.1 (C), 143.6 (C), 156.7 (CO), 165.2 (CO),
167.6 (CO), 172.1 (CO) ppm. – IR (film): n = 3334 cm-1, 2923,
1710, 1700, 1685, 1669, 1653, 1521, 1507, 1457, 1368, 1251, 1159,
1027, 912, 738. – [a]2D0 -6.0 (c 1.0, CHCl3). – MS (ESI) m/z = 683
([M + K+], 100), 622 ([M + Na+], 80). – Calcd. for C32H39N3O7: C
66.53%, H 6.80%, N 7.27%, found: C 60.44%, H 6.65%, N 7.08%.
(CH), 128.5 (CH), 129.7 (CH), 132.0 (C), 136.0 (C), 139.3 (C),
168.2 (C), 169.2 (C), 171.5 (C), 172.5 (C) ppm. – IR (film): n =
3297 cm-1, 2933, 2877, 1836, 1734, 1717, 1700, 1684, 1669, 1653,
1559, 1540, 1293, 1256, 1167, 1112, 756. – [a]2D0 = +1.6 (c 1.8,
CHCl3). – MS (ESI): m/z = 1385 ([2 M + Na+], 48), 704 ([M +
Na+], 100), 682 ([M + H+], 7). – HRMS (ESI) [M + NH4 ]. –
+
Calcd. for C36H51N4O10 699.3605, found 699.3600.
(2S)-[(2S)-Benzyloxycarbonylaminopropionylamino]-5-{[(3S)-
((1S)-methylpropyl)-4-oxooxetane-(2R)-carbonyl]amino}pentanoic
acid 4-carboxylphenylmethyl ester (11). To a solution of the
product 10 (15 mg, 0.22 mmol) in CH2Cl2 (1 mL) was added
trifluoroacetic acid (1 mL). The mixture was left overnight at
-15 ◦C in a freezer. All the volatiles were distilled off under
reduced pressure, and the residue was dried in vacuo. Yield 13 mg
(99%). – 1H NMR (300 MHz): 0.91 (3 H, t, J = 7 Hz), 1.02 (3 H,
d, J = 7 Hz) 1.20–1.30 (2 H, m), 1.37 (3 H, d, J = 7 Hz), 1.44–1.53
(1 H, m), 1.58–1.68 (2 H, m), 1.82–1.95 (2 H, m), 3.15–3.35 (2 H,
m), 3.54 (1 H, dd, J = 8, 5 Hz), 4.28–4.38 (1 H, m), 4.57–4.63 (1
H, m), 4.59 (1 H, d, J = 5 Hz), 5.02–5.12 (2 H, m), 5.15–5.25 (1
H, m), 5.84–5.90 (1 H, m), 6.71–6.77 (1 H, m), 6.90–7.00 (1 H,
m), 7.27–7.43 (7 H, m), 8.05 (2 H, d, J = 8 Hz), 9.00–9.40 (1 H,
br s) ppm. – 13C NMR (75.5 MHz): 11.0 (CH3), 16.2 (CH3), 25.2
(CH2), 26.5 (CH2), 29.7 (CH2), 33.7 (CH), 38.6 (CH2), 50.6 (CH),
52.1 (CH), 63.0 (CH2), 66.5 (CH2), 67.3 (CH), 70.6 (CH), 127.8
(CH), 127.9 (CH), 128.5 (CH), 129.4 (C), 130.5 (CH), 132.0 (C),
135.9 (C), 140.8 (C), 169.0 (C), 169.2 (C), 170.1 (C), 171.3 (C)
ppm. – IR (film): n = 3022 cm-1, 1844, 1792, 1772, 1734, 1717,
1700, 1696, 1684, 1669, 1653, 1559, 1539, 1457, 1250, 1098, 734. –
[a]2D0 -5.5 (c 0.65, CHCl3). – MS (ESI): m/z = 1273 ([2 M + Na+],
100), 648 ([M + Na+], 90). HRMS (ESI) [M + H+]. – Calcd. for
C32H40N3O10 626.2714, found 626.2708.
(S)-Benzyloxycarbonylalanyl-(S)-(Nd -tert-butyloxycarbonyl)-
ornithine 4-(tert-butoxycarbonyl)phenylmethyl ester. (S)-Ben-
zyloxycarbonylalanyl-(S)-(Nd-tert-butyloxycarbonyl)ornithine 4-
(tert-butoxycarbonyl)phenylmethyl ester (343 mg, 44%) was pre-
pared from 9 (800 mg, 1.24 mmol) and Cbz-Ala (330 mg,
1.42 mmol) according to GP1 as a colorless solid. Rf 0.11 [hexane–
EtOAc (2 : 1)], m. p. 118–119 ◦C. – 1H NMR (300 MHz): 1.38 (3
H, d, J = 7 Hz), 1.41 (7.7 H, s), 1.43 (1.3 H, s), 1.37 (9 H, s), 1.38–
1.76 (2 H, m), 1.87–1.96 (2 H, m), 3.00–3.17 (2 H, m), 3.80–3.96
(1 H, m), 4.10–4.32 (1 H, m), 4.55–4.69 (1 H, m), 5.05–5.11 (2 H,
m), 5.15–5.19 (2 H, m), 5.47–5.57 (1 H, br m), 6.93–7.02 (1 H, br
m), 7.30–7.40 (7 H, m), 7.97 (2 H, d, J = 8 Hz) ppm. – 13C NMR
(75.5 MHz): 18.4 (CH3), 25.8 (CH2), 28.1 (CH3), 28.3 (CH3), 29.3
(CH2), 39.7 (CH2), 50.5 (CH), 52.2 (CH), 66.5 (CH2), 67.0 (CH2),
79.4 (C), 81.2 (C), 127.7 (CH), 128.0 (CH), 128.2 (CH), 128.9
(CH), 129.7 (CH), 132.0 (C), 136.2 (C), 139.6 (C), 156.2 (C), 156.7
(C), 157.3 (C), 165.3 (C), 172.3 (C) ppm. – IR (film): n = 3336
cm-1, 2926, 1718, 1700, 1685, 1669, 1653, 1521, 1507, 1457, 1368,
1251, 1159, 1027, 912, 738. – [a]2D0 -6.0 (c 1.0, CHCl3). – MS (ESI)
positive ion mode: m/z = 1177 ([2 M + Na+], 100), 600 ([M +
Na], 40). Negative ion mode: m/z = 622 ([M + CH3COO-], 100). –
HRMS (ESI) [M + H+]. – Calcd. for C33H46N3O9 628.3234, found
628.3229.
(S)-tert-Butyloxycarbonyl-alanyl-(S)-(Nd-benzyloxycarbonyl)-
ornithine tert-butyl ester [Boc-Ala-Orn(Cbz)-OtBu] (13).
The dipeptide 13 (2.60 g, 84%) was prepared from N-
tert-butyloxycarbonyl-(S)-alanine (1.44 g, 7.62 mmol) and
(Nd-benzyloxycarbonyl)-(S)-ornithine tert-butyl ester (2.00 g,
6.30 mmol) according to GP1 as a yellow viscous oil. Rf 0.25
(2S)-[(2S)-Benzyloxycarbonylaminopropionylamino]-5-{[(3S)-
((1S)-methylpropyl)-4-oxooxetane-(2R)-carbonyl]amino}pentanoic
acid 4-(tert-butyloxycarbonyl)phenylmethyl ester (10). A solu-
tion of (S)-benzyloxycarbonylalanyl-(S)-(Nd-tert-butyloxycar-
bonyl)ornithine
4-(tert-butoxycarbonyl)phenylmethyl
ester
(340 mg, 0.54 mmol) and triethylsilane (316 mg, 2.70 mmol) in
EtOAc (6 mL) was added to a saturated solution of HCl in EtOAc
(5 mL), and the mixture was stirred for 3 h. The volatiles were
distilled off, and the residue was dried under reduced pressure
(0.05 mbar). A solution of the dry solid in DMF (3 mL) was
treated with a solution of the thioester 4 (157 mg, 0.56 mmol)
in CH2Cl2 (6 mL), TMP (146 mg, 1.21 mmol), HOAt (100 mg,
0.60 mmol) and EDC (200 mg, 1.11 mmol) in CH2Cl2 (2 mL)
according to GP2 to yield 130 mg (35%) of the product 10 as a
glassy colorless solid, Rf 0.30 [hexane–EtOAc (1 : 2)]. – 1H NMR
(300 MHz): 0.90 (3 H, t, J = 7 Hz), 1.01 (3 H, d, J = 7 Hz)
1.20–1.30 (2 H, m), 1.35 (3 H, d, J = 7 Hz), 1.44–1.53 (1 H, m),
1.59 (9 H, s), 1.58–1.68 (2 H, m), 1.82–1.95 (2 H, m), 3.14–3.21
(1 H, m), 3.25–3.33 (1 H, m), 3.40–3.46 (1 H, m), 3.54 (1 H, dd,
J = 8, 5), 4.28–4.34 (1 H, m), 4.52 (1 H, d, J = 5 Hz), 4.57–4.63
(1 H, m), 5.00–5.10 (2 H, m), 5.12–5.22 (1 H, m), 5.50–5.54 (1 H,
m), 6.71–6.77 (1 H, m), 6.90–6.95 (1 H, m), 7.27–7.40 (7 H, m),
7.95 (2 H, d, J = 8 Hz) ppm. – 13C NMR (75.5 MHz): 10.9 (CH3),
16.2 (CH3), 18.3 (CH3), 25.3 (CH2), 26.5 (CH2), 28.1 (CH3), 29.6
(CH2), 38.3 (CH2), 50.4 (CH), 51.7 (CH), 62.8 (C), 66.6 (CH),
66.9 (CH), 70.6 (CH), 81.18 (C), 127.7 (CH), 128.0 (CH), 128.1
1
[hexane–EtOAc (1 : 1)] – H NMR (300 MHz): 1.26 (3 H, d, J =
7 Hz), 1.34 (9 H, s), 1.37 (9 H, s), 1.38–1.76 (4 H, m), 3.14–3.22
(2 H, m), 4.10–4.20 (2 H, br m), 4.38–4.47 (1 H, m), 5.07 (2 H,
s), 5.09–5.19 (1 H, br m), 6.80 (1 H, d, J = 6 Hz), 7.30–7.34 (5
H, m) ppm. – 13C NMR (75.5 MHz): 18.2 (CH3), 25.5 (CH2),
27.9 (CH3), 28.2 (CH3), 29.6 (CH2), 40.4 (CH2), 50.1 (CH), 52.3
(CH), 66.5 (C), 80.0 (C), 82.2 (CH2), 128.0 (CH), 128.4 (CH),
136.5 (CH), 155.4 (C), 156.5 (C), 171.0 (C), 172.5 (C) ppm. – IR
(film): n = 3336 cm-1, 2926, 1718, 1700, 1685, 1669, 1653, 1521,
1507, 1457, 1368, 1251, 1159, 1027, 912, 738. – [a]2D0 -3.0 (c 1.0,
CHCl3). – MS (ESI) positive ion mode: m/z = 1009 ([2 M + Na+],
100), 516 ([M + Na+], 34). Negative ion mode: m/z = 552 ([M +
CH3COO-], 70), 538 ([M + HCOO-], 100), 492 ([M - H], 48). –
HRMS (ESI) [M + H+] – calcd. for C25H40N3O7 494.2866, found
494.2861. – Calcd. for C25H40N3O7: C 60.83%, H 7.96%, N 8.51%,
found: C 60.60%, H 7.86%, N 8.29%.
(2S)-[(2S)-tert-Butyloxycarbonylaminopropionylamino]-5-{[(3S)-
((1S)-methylpropyl)-4-oxooxetane-(2R)-carbonyl]amino}pentanoic
acid tert-butyl ester (14). A mixture of the dipeptide 13 (1.0 g,
2.1 mmol) and Pd/C (50 mg, 10%) in methanol (50 mL) was
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stirred at r. t. overnight under an atmosphere of hydrogen, then
it was filtered through a pad of Celite, and the volatiles were
removed in vacuo. The crude amine obtained was treated with the
thioester 4 (510 mg, 1.83 mmol), TMP (438 mg, 3.6 mmol), HOAt
(255 mg, 1.8 mmol) and EDC (561 mg, 3.6 mmol) according to
GP2 to give 400 mg (38%) of 14. Rf 0.28 [hexane–EtOAc (1 : 1)].
– 1H NMR (300 MHz): 0.91 (3 H, t, J = 7 Hz), 1.03 (3 H, d, J = 7
Hz), 1.32 (3 H, d, J = 7 Hz), 1.40 (9 H, s), 1.42 (9 H, s), 1.48–1.64
(4 H, m), 1.78–1.86 (1 H, m), 1.88–2.00 (1 H, m), 3.20–3.40 (2
H, m), 3.58 (1 H, dd, J = 9, 5 Hz), 4.10–4.20 (1 H, m), 4.36–4.44
(1 H, m), 4.57 (1 H, d, J = 5 Hz), 5.16–5.22 (1 H, m), 6.80–6.88
(1 H, m) ppm. – 13C NMR (75.5 MHz): 10.9 (CH3), 16.3 (CH3),
18.2 (CH), 25.0 (CH3), 28.2 (CH3), 29.6 (CH2), 33.7 (CH3), 38.5
(CH2), 50.0 (CH), 52.1 (CH), 62.8 (CH), 70.7 (CH), 80.0 (C),
82.3 (C), 155.5 (C), 168.1 (C), 169.2 (C), 170.8 (C), 172.6 (C)
ppm. – IR (film): n = 3318 cm-1, 2976, 2934, 2878, 1837, 1669,
1540, 1507, 1457, 1368, 1251, 1161, 1098, 738. – [a]2D0 = +9.7 (c
1.5, CHCl3). – MS (ESI) positive ion mode: m/z = 1049 ([2 M +
Na+], 100), 536 ([M + Na+], 64). Negative ion mode: m/z = 558
([M + HCOO-], 100), 512 ([M - H], 26). – HRMS (ESI) [M +
H+]. – Calcd. for C25H44N3O8 514.3128, found 514.3123.
1000 rpm for 5 min. The pellet was resuspended in 1 mL of
DMEM) supplemented with 10% FCS, 100 U mL-1 penicillin,
and 100 mg mL-1 streptomycin and transferred into a well of a 6
well-plate. Fibroblasts had grown after 1–2 days and covered the
well bottom after 3–5 days.
2. Assay of proteasomal activity. Proteasome chymotrypsin-
like, trypsin-like and caspase-like activities from HeLa cell lysates
were determined fluorometrically in a spectramax GEMINI-
EM (MolecularDevices) by using synthetic peptides linked to
the fluorophor methylcoumarine. ChTL activity was measured
by SLLVY-AMC hydrolysis, TL by BzVGR-AMC and CaspL
activity by ZLLE-AMC hydrolysis with 360-nm excitation and
460-nm emission wavelengths.
Cells were treated for indicated times with indicated substances
or solvent (DMSO) as a control. Cells were subsequently washed
with PBS, and then scraped and lysed under hypotonic con-
ditions with repeated cycles of thawing and freezing in liquid
nitrogen. Lysates were centrifuged, and the protein content of
the supernatant was estimated by BCA◦protein assay (Pierce).
Lysates were incubated for 30 min at 37 C in incubation buffer
containing an ATP-regenerating system (225 mM Tris–HCl, pH
8.2, 45 mM KCl, 7.5 mM Mg(CH3COO)2, 7.5 mM MgCl2, 1.1
mM dithiothreitol, 6 mM ATP, 5 mM phosphocreatine, 0.2 unit
of phosphocreatinekinase) and 0.2 mM of the appropriate fluo-
rogenic substrate. Enzymatic activity was normalized to protein
concentration and expressed as a percentage of activity of the
solvent-treated control. The values are given as the means of three
independent experiments S.E.M.
(2S)-[(2S)-Aminopropionylamino]-5-{[(3S)-((1S)-methylprop-
yl)4-oxooxetane-(2R)-carbonyl]amino}pentanoic acid (15). The
tripeptide 14 (75 mg, 0.15 mmol) was mixed with CH2Cl2 (2 mL)
and trifluoroacetic acid (2 mL) at 0 ◦C, and this mixture was kept at
-15 ◦C overnight. The volatiles were distilled off, and the obtained
hydrotrifluoroacetate salt was dried in vacuo. Yield 71 mg (99%).
– 1H NMR (CD3OD, 300 MHz): 0.94 (3 H, t, J = 7 Hz), 1.03 (3 H,
d, J = 7 Hz), 1.25–1.32 (1 H, m), 1.52 (3 H, d, J = 7 Hz), 1.60–1.71
(2 H, m), 1.90–2.00 (2 H, m), 3.25–3.32 (2 H, m), 3.65 (1 H, dd,
J = 8, 4 Hz), 3.93–4.00 (1 H, m), 4.40–4.46 (1 H, m), 4.70 (1 H,
d, J = 4 Hz) ppm. 13C NMR (75.5 MHz): 11.3 (CH3), 16.6 (CH3)
17.5 (CH3), 26.7 (CH2), 27.7 (CH2), 29.6 (CH2), 36.9 (CH), 39.7
(CH2), 50.2 (CH), 53.4 (CH), 63.8 (CH), 72.9 (CH), 170.7 (C),
171.0 (C), 171.1 (C), 174.5 (C) ppm. – IR (film): n = 3420 cm-1,
2967, 2934, 1830, 1684, 1540, 1457, 1362, 1203, 1140. – [a]2D0 -3.6
(c 1.1, MeOH). – MS (ESI) positive ion mode: m/z = 715 ([2 M +
H+], 14), 358 ([M + H+], 100). Negative ion mode: m/z = 713 ([2 M
- H], 96), 356 ([M - H], 100). – HRMS (ESI) [M + H+]. – Calcd.
for C16H28N3O6 358.1978, found 358.1973.
3. Western blot. Cells were lysed in extraction buffer con-
taining 50 mM Tris/HCl 5 (pH 7.4), 150 mM KCl, 5 mM glucose,
50 mM EDTA, 1 mM PMSF, 2 mM DTT 2, and 1% Triton X-100.
Total protein (20 mg per lane) was subjected to SDS-PAGE and
membranes were probed with the respective antibodies: rabbit
anti-GFP (Epitomics); rabbit anti-Ubi (DAKO). Bands were
visualized by employing the ECL detection system (Amersham).
Amido black staining of membranes served as control for equal
protein loading.
4. Co-crystallisation. Crystals of the 20S proteasome from
S. cerevisiae were grown in hanging drops at 24 ◦C as has
previously been described13 and incubated for 48 h with the
chemical compound 6 at 10 mM. The protein concentration used
for the crystallization was 45 mg mL-1 in Tris-HCl (10 mM, pH
7.5) and EDTA (1 mM). The drops contained 1 mL of protein and
1 xmL of the reservoir solution, containing 20 mM of magnesium
acetate, 100 mM of morpholino-ethanesulfonic acid (pH 6.9) and
10% of MPD.
Investigation of biological activities of the new
belactosin C congeners
Methods
1. Cell culture. HeLa cells (epithelial cells derived from
human cervix carcinoma) were obtained from the American Type
Culture Collection. The cells were maintained in RPMI (Gibco,
Karlsruhe, Germany) supplemented with 10% FCS, 100 U mL-1
penicillin, and 100 mg mL-1 streptomycin in a humidified 5% CO2
atmosphere.
Primary mouse ear fibroblasts were isolated from UbG76V GFP1
mice.12 Mouse ears were washed in 70% EtOH for 2 min and cut
into small pieces in DMEM using a scalpel. Pieces were transferred
into a 15 mL tube containing 2 mL of 0.25% trypsin. After
incubation for 1 h at 37 ◦C, trypsin was inactivated by adding
5 mL of DMEM/10%FCS. The suspension was centrifuged at
The space group belongs to P21 with cell dimensions of a =
◦
˚
˚
˚
134.9 A, b = 301.6 A, c = 144.2 A and b = 112.9 (see Table
˚
S1†). Data to 2.7 A for the proteasome : inhibitor-complex were
˚
collected using synchrotron radiation with l = 1.0 A at the X06SA-
beamline in SLS/Villingen/Switzerland. Crystals were soaked
in a cryoprotecting buffer (30% MPD, 20 mM of magnesium
acetate, 100 mM of morpholino-ethanesulfonic acid pH 6.9) and
frozen in a stream of liquid nitrogen gas at 100 K (Oxford Cryo
Systems). X-ray intensities were evaluated employing the XDS
program package.14 The anisotropy of diffraction was corrected
by an overall anisotropic temperature factor by comparing
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observed and calculated structure amplitudes using the pro-
gram CNS.15,16 Electron density was improved by averaging and
back transforming the reflections 10 times over the twofold
noncrystallographic symmetry axis using the program package
MAIN.17 Conventional crystallographic rigid body, positional and
temperature factor refinements were carried out with CNS using
the yeast 20S proteasome structure as a starting model.7 Modelling
experiments were performed with the program MAIN with current
crystallographic values of Rcryst = 0.212, Rfree = 0.237 18 (Table S1†).
5 T. Muthny, M. Koverik, L. Sispera, A. de Meijere, O. V. Lari-
onov, I. Tilser and M. Holecek, J. Physiol. Biochem., 2009, 65,
137.
6 (a) A. Armstrong and J. N. Scutt, Chem. Commun., 2004, (5), 510;
(b) O. V. Larionov and A. de Meijere, Org. Lett., 2004, 6, 337; (c) G.
Kumaraswamy, M. Padmaja, B. Markondaiah, N. Jena, B. Sridhar and
M. U. Kiran, J. Org. Chem., 2006, 71, 337; (d) S. W. Cho and D. Romo,
Org. Lett., 2007, 9, 1537; (e) G. Kumaraswamy and B. Markondaiah,
Tetrahedron Lett., 2007, 48, 1707; (f) K. Yoshida, K. Yamaguchi, T.
Sone, Y. Unno, A. Asai, H. Yokosawa, A. Matsuda, M. Arisawa and S.
Shuto, Org. Lett., 2008, 10, 3571.
7 M. Groll, L. Ditzel, J. Lo¨we, D. Stock, M. Bochtler, H. D. Bartunik
and R. Huber, Nature, 1997, 386, 463.
8 L. Borissenko and M. Groll, Chem. Rev., 2007, 107,
687.
Accession number. The atomic coordinates and structure fac-
tors have been deposited in the Protein Data Bank (entry code
3TDD).
9 L. Borissenko and M. Groll, Biol. Chem., 2007, 388, 947.
˚
10 (a) M. Graffner-Nordberg, J. Marelius, S. Ohlsson, A. Persson, G.
Acknowledgements
˚
Swedberg, P. Andersson, S. E. Andersson, J. Aqvist and A. Hallberg,
J. Med. Chem., 2000, 43, 3852; (b) S. Narasimhan, S. Swarnalakshmi
and R. Balakumar, Synth. Commun., 2000, 30, 941.
11 2-Naphthylmethyl chloroformate (CNAP-Cl) was prepared according
to: E. A. Papageorgiou, M. J. Gaunt, J.-q. Yu and J. B. Spencer, Org.
Lett., 2000, 2, 1049.
12 K. Lindsten, V. Mene´ndez-Benito, M. G. Masucci and N. P. Dantuma,
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13 M. Groll and R. Huber, Methods Enzymol., 2005, 398, 329.
14 W. Kabsch, J. Appl. Cryst., 1993, 26, 795.
15 A. T. Bru¨nger, P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, R. W.
Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, R.
J. Read, L. M. Rice, T. Simonson and G. L. Warren, Acta Crystallogr
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16 A. Bru¨nger, X-PLOR version 3.1. A system for X-ray crystallography
and NMR, Yale University Press, New Haven, (1992).
17 D. Turk, Improvement of a program for molecular graphics and
manipulation of electron densities and its application for protein struc-
ture determination, Dissertation, Technische Universitaet Muenchen,
(1992).
The authors thank the staff of the X06DA-beamline at the
Paul Scherrer Institute, SLS, Villingen, Switzerland for assistance
during data collection. V. S. K., O. V. L. and A. V. L. are grateful
to the Degussa Foundation (Evonik Industries AG) for doctoral
student fellowships.
Notes and references
1 M. Groll, O. V. Larionov, M. Huber and A. de Meijere, Proc. Natl.
Acad. Sci. U. S. A., 2006, 103, 4576.
2 A. Asai, A. Hasegawa, K. Ochiai, Y. Yamashita and T. Mizukami, J.
Antibiot., 2000, 53, 81.
3 A. Asai, T. Tsujita, S. V. Sharma, Y. Yamashita, S. Akinaga, M.
Funakoshi, H. Kobayashi and T. Mizukami, Biochem. Pharmacol.,
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4 K. Yoshida, K. Yamaguchi, A. Mizuno, Y. Unno, A. Asai, T. Sone, H.
Yokosawa, A. Matsuda, M. Arisawa and S. Shuto, Org. Biomol. Chem.,
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18 A. T. Bru¨nger, Nature, 1992, 355, 472.
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