Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant

Article abstract of DOI:10.1016/j.bmc.2008.09.057

The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K_m of 33.8 μM and k_cat of 4753 s^-1. Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10⁶. Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.

Full text of DOI:10.1016/j.bmc.2008.09.057

Bioorganic & Medicinal Chemistry 16 (2008) 9400–9408
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL
protease as a proteolysis-resistant mutant
a
a
b
b
a
Kenichi Akaji a, , Hiroyuki Konno , Mari Onozuka , Ayumi Makino , Hiroyuki Saito , Kazuto Nosaka
*
a Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan
b Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme
for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to deg-
radation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to pro-
Received 19 August 2008
Revised 22 September 2008
Accepted 23 September 2008
Available online 26 September 2008
teolysis. The R188I mutant digested a conserved undecapeptide substrate with a Km of 33.8 lM and kcat of
4753 sꢀ1. Compared with the value reported for the mature protease containing a C-terminal His-tag, the
relative activity of the mutant was nearly 106. Novel peptide-aldehyde derivatives containing a side-
chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results
indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-
aldehyde derivatives.
Keywords:
SARS
3CL protease
Protease inhibitor
Peptide aldehyde
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
dues to the C-terminus of mature 3CL protease did not significantly
affect its activity, the kinetic properties and specificity of the ma-
Severe acute respiratory syndrome (SARS), a life-threatening
form of pneumonia, is caused by a new coronavirus (SARS
CoV).1–3 Although the primary SARS epidemic affecting about
8500 patients and leaving 800 dead was eventually controlled,
no effective therapy exists for this viral infection. SARS CoV is a po-
sitive-sense, single-stranded RNA virus featuring the largest viral
RNA genome known to date.4,5 The genomic RNA produces two
large proteins with overlapping sequences, polyproteins 1a
(ꢁ450 kDa) and 1ab (ꢁ750 kDa), which are auto-catalytically
cleaved by two or three viral proteases to yield functional polypep-
tides.6 The key enzyme in this processing is a 33-kDa protease
called 3C-like protease (3CL protease), because its substrate speci-
ficity is similar to that of the picornavirus 3C protease.7,8 The SARS
3CL protease is a cysteine protease with a chymotrypsin fold, and
cleaves precursor proteins at as many as 11 conserved sites involv-
ing a conserved Gln at the P1 position and a small amino acid (Ser,
Ala, or Gly) at the P1’ position with varying efficiency.9,10 The 3CL
protease exists as a homodimer and each 33-kDa protomer has its
own active site containing a Cys-His catalytic dyad (Fig. 1A). Inter-
actions between the C-terminal domain-III of each protomer play
an important role in the formation of this dimer. The N-terminal
end of one protomer is also expected to interact with the do-
main-II of another protomer to fold the dimeric structure.
Although some studies reported that the addition of several resi-
ture protease have not been well described.
Due to its functional importance in the viral life cycle, the 3CL
protease is considered an attractive target for the structure-based
design of drugs against SARS.11–17 Several crystal structures of the
SARS 3CL protease with or without inhibitors have also been used
to evaluate various types of inhibitors for this protease.8,18–21 Most
inhibitors contain a functional group such as a chloromethyl
ketone, Michael acceptor, or epoxide that can react irreversibly
with the active-site cysteine residue. Few studies regarding revers-
ible inhibitors containing an aldehyde group as a functional group
have been reported.22 In the course of our own studies on the SARS
3CL protease and its inhibitors, we found that the mature protease
could be further processed into two fragments devoid of catalytic
activity. Here, we report on high level of expression in Escherichia
coli and the purification of a proteolysis-resistant mutant of the
SARS 3CL protease. The kinetic characterization of this mutant
revealed remarkable potency compared with the mature 3CL prote-
ase containing a C-terminal His-tag. The potency makes it possible
to evaluate newly synthesized peptide-aldehyde inhibitors quanti-
tatively by conventional HPLC.
2. Results and discussion
2.1. Expression of the mature 3CL protease
We first intended to obtain the mature 3CL protease from a fu-
sion protein containing maltose-binding protein (MBP), His6 and a
* Corresponding author. Tel./fax: +075 465 7659.
E-mail address: akaji@koto.kpu-m.ac.jp (K. Akaji).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.057

K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
9401
tected, but the desired 33-kDa protein corresponding to the mature
3CL protease was hardly detected (Fig. 2A, lane 3). To confirm
whether the 33-kDa protein was produced or not, we then added
enterokinase in the presence of a protease substrate; the substrate
SO1 was mixed with the fusion protein prior to the addition of the
enterokinase. In this reaction mixture, the substrate completely
disappeared within 1 h, and expected fragment peptides were
clearly identified by MALDI-TOF-MS (Fig. 2B; peak 1, [M+H]+
593.365 (Calcd. 593.353) for H-Ser-Gly-Phe-Arg-Lys-NH2; peak 2,
[M+H]+ 618.288 (Calcd. 618.347) for H-Thr-Ser-Ala-Val-Leu-Gln-
OH). This result suggests that the mature 3CL protease was tempo-
rarily produced by the digestion of the 75-kDa fusion protein by
the enterokinase.
We then tried to directly express the 3CL protease containing a
C-terminal His-tag as described previously.9 The necessary plasmid
was constructed from the previous plasmid, and introduced into
E. coli. The desired 34-kDa product containing the mature 3CL pro-
tease was not detected as a major product (Fig. 2C, lane 1) in our
experiment. After purification with a cobalt column, a small
amount of 34-kDa product was detected by Coomassie staining
and Western blotting using anti-His-tag antibody (Fig. 2C, lane 2,
Fig. 2D, lane 1). The immunostaining, however, decreased in inten-
sity gradually and a 14-kDa protein emerged after placement at
room temperature (Fig. 2D, lane 2), or overnight incubation with
enterokinase (Fig. 2D, lane 3). These results strongly suggest that
mature 3CL protease is susceptible to degradation by proteolysis.
To identify the probable cleavage site, enterokinase-based
digestion of the 75-kDa fusion protein, MBP-His tag-3CL protease
(Fig. 3, lane 1), was carefully examined. After 15 min of treatment
with a small amount of enterokinase, the desired 33-kDa product
was detected as a faint band by Coomassie staining. Two additional
bands at 20- and 13-kDa were also detected (Fig. 3, lane 2) as prob-
able products of the proteolysis. The faint band of 33-kDa disap-
peared with prolonged treatment over 60 min (Fig. 3, lanes 3–4).
The newly emerged 13-kDa protein seemed to be the same product
obtained from the C-terminal His-tagged 3CL protease (Fig. 2D,
lanes 2–3). By blotting the 13-kDa protein to a polyvinylidene
difluoride(PVDF), the N-terminal sequence of the product was
determined to be Gln-Thr-Ala-Gln-Ala, which shows that mature
3CL protease is cleaved at the 188Arg/189Gln site.
Figure 1. (A) Amino acid sequence of SARS CoV 3CL protease: the catalytic dyad is
defined by His 41 and Cys 145, which is similar to the arrangement found in other
coronavirus proteases. The N-terminal part (1–184, Domain I and II) is composed of
a two-b-barrel fold similar to that of a chymotrypsin-type protease. The C-terminal
part (201–303, Domain-III) has five
a-helices. (B) Fusion protein composed of MBP,
His6 and a Flag-tag, and 3CL protease.
Flag-tag, and mature 3CL protease (MBP-His tag-3CL protease,
Fig. 1B). The corresponding plasmid was introduced into E. coli,
and the desired fusion protein of 75-kDa was obtained as a major
product of the total cell lysate (Fig. 2A, lane 1). The fusion protein
was easily purified with a cobalt affinity column to give a single
major band on SDS–PAGE (Fig. 2A, lane 2). The 75-kDa product
did not cleave SO1, an undecapeptide substrate (H-Thr-Ser-Ala-
Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH2) containing the P1/P2 cleav-
age site of 3CL protease, probably because of an N-terminal fused
large protein. After digestion of the fusion protein with enteroki-
nase, the N-terminal 42-kDa fragment containing the MBP was de-
Figure 2. Expression of mature 3CL protease: (A) SDS–PAGE (10%) of MBP-His-Flag-tagged 3CL protease stained with Coomassie brilliant blue; lane M, molecular standard
proteins (Bio-Rad); lane 1, crude extract (20 g of protein) of IPTG-induced bacterial cells; lane 2, metal affinity resin-treated fraction (3 g); lane 3, enterokinase-treated
(0.1 U for 2 h) fraction (3 g). (B) (a) HPLC profile of the substrate SO1 in the reaction buffer, (b) HPLC profile of the mixture of 75-kDa fusion protein, enterokinase, and SO1
after 4 h of treatment. Peak 1 derives from the C-terminal cleavage fragment of SO1 and peak 2 derives from the N-terminal cleavage fragment. (C) SDS–PAGE (15%) of C-
terminal His-tagged 3CL protease stained with Coomassie brilliant blue; lane M, molecular standard proteins (Bio-Rad); lane 1, crude extract (20 g of protein) of IPTG-
induced bacterial cells; lane 2, metal affinity resin-treated fraction (4 g). (D) Western blotting of C-terminal His-tagged 3CL protease using anti-His-tag antibody; lane M,
molecular standard proteins (Bio-Rad); lane 1, metal affinity resin-treated fraction (4 g); lane 2, after overnight incubation at room temperature; lane 3, after incubation
with enterokinase (0.05 U); lane 4, after incubation with purified 3CL-R188I protease (0.5 g).
l
l
l
l
l
l
l

9402
K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
Figure 4. SDS–PAGE of R188I mutant 3CL protease: samples at each purification
step were separated by 10% (A) or 15% (B) SDS–PAGE and stained with Coomassie
brilliant blue. Lanes M indicate molecular standard proteins (Bio-Rad). (A) Untagged
protease: lane 1, crude extract (20
terminal MBP-His-Flag-tagged protease; lane 2, metal affinity resin-treated fraction
(6 g); lane 3, enterokinase-treated fraction (4 g); lane 4, DEAE–Sepharose column
flow-through fraction (3 g); lane 5, DEAE–Sepharose column-retained fraction
(3 g). (B) C-terminal His-tagged protease: lane 1, crude extract (20 g of protein)
of IPTG-induced bacterial cells; lane 2, metal affinity resin-treated fraction (2 g).
lg of protein) of bacterial cells expressing the N-
Figure 3. Time course of enterokinase (0.05 U)-based digestion of the metal affinity
resin-treated fusion protein containing the mature 3CL protease (10
untreated; lane 2, 15 min; lane 3, 30 min; lane 4, 60 min.
lg): lane 1,
l
l
l
l
l
l
The 188Arg/189Gln site is in a long loop region (residue 185–
200) which connects a chymotrypsin-like catalytic domain I–II
(1–184) and C-terminal
3CL protease (Fig. 1). In a homodimeric structure of mature 3CL
protease, the -helical domain is expected to be responsible for
a-helical domain-III (201–303) of mature
His tag were concentrated by ultrafiltration, and an equal volume
of glycerol was added. Each solution was stored at ꢀ20 °C without
any loss of catalytic activity for at least a year.
a
orienting the amino terminus of one monomer to a position in
which it can interact with the active site of the other monomer.23
It was also reported that the expression of the N-terminal catalytic
region alone yields a protein with reduced catalytic efficiency.18
Thus, the expected biological role of the a-helical domain is consis-
tent with our finding that proteolysis at the 188Arg/189Gln site
The catalytic activities of purified 3CL-R188I proteases were
examined using three different substrates (SO1, SO3, and SR1).
SO1 containing the P1/P2 cleavage site, the N-terminal self-cleav-
age site of the protease, is reported to be the most suitable sub-
strate,
a
canonical substrate, for 3CL protease.9 SO3 is an
undecapeptide containing the non-canonical P3/P4 cleavage site
of 3CL protease, and SR1 is a hexadecapeptide containing a newly
found proteolytic site (188Arg/189Gln). Each substrate, at different
concentrations, was incubated with the mutant protease at 37 °C,
and the cleavage reaction was monitored with analytical HPLC as
in Fig. 2B. The initial digestion rate was calculated from the de-
crease in the initial amount of substrate, and each kinetic parame-
ter was calculated by plotting [S]/v against [S].
As summarized in Table 1, the catalytic ability of 3CL-R188I
protease was found to be extreme as compared to that of a previ-
ously reported mature 3CL protease containing a C-terminal His
tag9, especially the kcat values; 12.2 minꢀ1 for mature 3CL protease
and 4753 sꢀ1 for the 3CL-R188I. The activity of the mutant relative
to the mature protease was nearly 106. Addition of a C-terminal
His-tag lowered the relative activity to 0.03 owing to a significant
decrease in the kcat value. The results suggest that the additional
sequences at the N- and C-terminus of mature 3CL protease
remarkably affect the catalytic activities even with several addi-
tions as expected from the maturation analysis.24
rapidly reduces the catalytic efficiency of mature 3CL protease.
2.2. Expression and characterization of R188I mutant 3CL
protease
To obtain an active and stable 3CL protease, we then con-
structed a mutant in which Arg-188 was substituted with Ile
(3CL-R188I) by site-directed mutagenesis. This substitution was
selected to remove the ionic guanidine group without remarkably
changing the steric effect of the Arg side-chain. Similar to wild-
type 3CL protease, 3CL-R188I protease was expressed in E. coli as
a fusion protein, MBP-His-tag-R188I mutant. The 75-kDa fusion
protein was obtained as a major product, and easily purified with
metal affinity resin (Fig. 4A, lanes 1 and 2). After enterokinase
treatment, the desired 33-kDa product corresponding to
3CL-R188I protease was intensely stained with Coomassie, and
no proteolytic product was detected (Fig. 4A, lane 3). The
3CL-R188I protease was further purified by anion exchange chro-
matography at pH 5.5: the R188I protease with a calculated pI of
6.24 flowed through and the MBP-His6-Flag of pI 5.08 was
absorbed on the resin as expected (Fig 4A, lanes 4 and 5). About
0.10 mg of the purified untagged protein was obtained from
100 ml of bacterial culture. A 34-kDa protein corresponding to
3CL-R188I with the C-terminal His tag was also directly produced
as a major product (Fig. 4B, lane 1). A single step of purification
with metal affinity resin was enough to obtain a highly purified
product (Fig. 4B, lane 2). In this case, about 0.13 mg of the purified
C-terminal His-tagged protein was obtained from 100 ml of cul-
ture. These purified mutant proteins with or without a C-terminal
Table 1
Catalytic parameters and relative efficiency of the SARS 3CL R188I mutant protease
for three different peptides
Tag of protease
Substrate
Km
M)
kcat
kcat/Km
M sꢀ1
)
Relative
rate
(
l
(sꢀ1
)
(
l
None
C-terminal His tag
None
SO1
SO1
SO3
SR1
33.8
31.8
50.5
46.0
4753
153
455
76
156.9
4.8
9.1
1.00
0.03
0.06
0.01
None
2.0

K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
9403
The relative efficiency for SO3 and SR1 was 0.06 and 0.01,
respectively, when compared with the kcat/Km for SO1. The relative
efficiency for SO3 vs. SO1 was exactly the same as reported for the
mature protease.9 The relative efficiency for SR1 was nearly the
same as that reported for cleavage at the P5/P6 site, another non-
canonical cleavage site of mature 3CL protease.9 This suggests that
mature 3CL protease can digest the precursor protein of SARS CoV
at the Arg188/Gln189 site as at non-canonical cleavage sites. Thus,
we examined the cleavage of mature 3CL protease by the mutant.
Incubation of the C-terminal His-tagged mature 3CL protease with
the purified 3CL-R188I protease gave the degradation product de-
rived from the 3CL protease (Fig. 2D, lane 4). From these results, we
suppose that 3CL protease can digest itself. Application of the mu-
tant protease of the same size as the mature 3CL protease would be
effective to accelerate the development of efficient inhibitors for
the SARS CoV 3CL protease.
To avoid this undesired reaction, we then synthesized the pep-
tide-aldehyde using the Gln(Me2) derivative. Use of the side-chain-
protected Gln is also effective at preventing equilibrium between
the desired C-terminal aldehyde and the corresponding hemiaminal
formed with an unprotected amide at the Gln side-chain.26 Starting
from Fmoc-Glu-OBut, dimethylamine was condensed to the side-
chain carboxyl group. Then, the a-tert-butyl ester was cleaved and
converted to the Weinreb amide. Subsequent peptide chain elonga-
tion was conducted as before using a combination of Fmoc-depro-
tection with diethylamine and coupling with BOP (Scheme 2). The
resulting hexapeptide amide was then reduced with LiAlH4. After
deprotection with TFA, the crude product was purified by HPLC to
give a single peak on analytical HPLC. MALDI-TOF-MS andNMR anal-
yses gave results confirming the aldehyde structure. Peptide alde-
hydes containing truncated chains were similarly synthesized
from the corresponding intermediates and purified.
The inhibitory effects of newly synthesized peptide-aldehyde
inhibitors were assayed using the purified untagged 3CL-R188I
protease. The substrate peptide SO1 was incubated with the mu-
tant protease in the presence of various concentrations of pep-
tide-aldehyde, and the reaction mixture was analyzed by HPLC as
in Fig. 2B. From the decrease in SO1 at the respective concentration
of inhibitor, a sigmoidal dose-response curve was obtained (Fig. 5),
and each IC50 value was estimated from each sigmoid curve. As
summarized in Table 2, the tetrapeptide sequence was long en-
ough for inhibitory activity. The activity increased in parallel with
2.3. Peptide-aldehyde inhibitors
As a suitable candidate for a reversible-inhibitor, we selected a
peptide-aldehyde containing the SO1 sequence. To obtain highly
homogeneous derivatives efficiently, we constructed the aldehyde
group by reduction of the corresponding Weinreb amide25 pre-
pared by solution phase synthesis. Thus, starting from Fmoc-
Gln(Trt)-N(OMe)Me, peptide chain elongation was conducted
using a combination of Fmoc-deprotection with diethylamine
and a coupling reaction using BOP. The resulting peptapeptide
Weinreb amide, Ac-Ser(OBut)-Ala-Val-Leu-Gln(Trt)-N(OMe)Me,
was then reduced with LiAlH4 and the remaining side-chain pro-
tecting groups were removed with TFA (Scheme 1). The deprotec-
ted product was purified by HPLC to yield a product showing a
single peak. A 1H NMR spectrum of the product, however, gave
no aldehyde signal, but showed an additional methyl signal at
2.57 ppm. MALDI-TOF-MS also showed a higher molecular weight
than expected. These spectral data strongly suggest that the reduc-
tion with lithium aluminum hydride did not proceed as expected.
The LiAlH4 attacked the methoxy group instead of the carbonyl car-
bon probably because of the large steric hindrance of the Trt group
at the C-terminal Gln side chain, and gave a corresponding methyl-
amide derivative (Scheme 1).
chain length; the hexapeptide aldehyde had an IC50 of 26 lM. E-
6427, a typical cysteine protease inhibitor, showed nearly the same
inhibitory activity (36% inhibition at 5.6 mM) as Ac-Val-Leu-
Gln(Me2)-CHO, which suggests that the peptide-aldehyde deriva-
tives would be a promising reversible SARS 3CL protease inhibitor.
Detailed structure–activity studies are now underway.
3. Experimental
The pMAL-c2X-based (NEB BioLabs) bacterial expression vector,
pMAL-3CL, was kindly provided by Dr. Norio Yamamoto and Prof.
Naoki Yamamoto (Tokyo Medical and Dental University). pMAL-
3CL encodes a 75-kDa protein containing a full-length SARS CoV
3CL protease with a maltose-binding protein, a six-histidine tag,
and a Flag tag (MBP-His-Flag) at the N-terminus, and is designed
to release mature 3CL protease with a free N-terminus from the fu-
sion protein on enterokinase treatment.
E-64 was purchased from Peptide Institute, Inc. (Osaka, Minoh,
Japan). Fmoc amino acid derivatives were purchased form Watan-
abe Chemical, Inc. (Hiroshima, Japan). Analytical and preparative
HPLCs were performed using a HITACHI ELITE LaChrom system
(OD, 220 nm) equipped with the Nacalai tesque COSMOSIL 5C18-
AR-II (4.6 ꢂ 150 mm or 10 ꢂ 250 mm) reversed phase column.
The product was eluted with a gradient of CH3CN containing
0.1% TFA in 0.1% aqueous TFA solution. 1H (300 MHz) and 13C
(75 MHz) NMR spectra were recorded on a Bruker AM-300. Chem-
ical shifts are expressed in parts per million relative to TMS
(0 ppm) or CHCl3 (7.28 ppm for 1H and 77.0 ppm for 13C). High res-
olution MALDI TOF-MS mass spectra were obtained on a Bruker
Autoflex-II. Optical rotations were recorded on a HORIBA SEPA-
300 polarimeter at the sodium D line.
3.1. Expression of mature 3CL proteases
The full-length 3CL protease-coding sequence was amplified
from pMAL-3CL with 50-ccggctagcGGTTTTAGGAAAATGGCATTCC
containing a NheI site and 50-ccgctcgagTTGGAAGGTAACACCAGAG
containing a XhoI site. The PCR product was cloned directly into
pGEM T-easy (Promega) to produce pGEM-3CL. Another expression
Scheme 1.

9404
K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
Scheme 2.
The pMAL-c2X-based plasmid and pET-21a(+)-based plasmid
were transformed into E. coli strains DH5 and BL21(DE3)pLys,
respectively. Bacterial cells were grown overnight at 37 °C in
10 ml of LB medium containing 50 g/ml ampicillin, pelleted, and
grown for 2 h in 100 ml of fresh medium. The cells were further
shaken at 28 °C for 2 h with 0.5 mM isopropyl-b- -thiogalactopy-
a
l
D
ranoside (IPTG) to produce the recombinant protein. The cells were
then pelleted and resuspended in 20 ml of solution L (50 mM
Na2HPO4 pH 7.0, and 300 mM NaCl) containing 10 mM imidazole,
1 mM phenylmethylsulfonyl fluoride and 10 ll/ml of Protease
Inhibitors Cocktail for purification of Histidine-Tagged Proteins
(Sigma), and sonicated 4 times in ice-cold water using a Bioruptor
(Cosmo Bio, Tokyo, Japan) at 200 W for 30 s each time with a 120-s
interval. Cell debris was removed by centrifugation at 14,000g for
20 min, and the supernatant served as a crude extract. The crude
extract was applied to a 1 ml bed volume of TALON Metal Affinity
Resin (Clontech) equilibrated with solution L containing 10 mM
imidazole. After being washed with 20 ml of the same solution,
the protease was eluted with solution L containing 125 mM imid-
azole. Combined eluted fractions (1.3 ml) were concentrated to
0.2 ml during exchange of the buffer with 20 mM Tris–HCl pH
7.5 by ultrafiltration using Centricon YM-10 (10 kDa cutoff,
Millipore).
Figure 5. A typical sigmoidal dose–response curve used for estimation of the IC50
value of Ac-Thr-Ser-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO.
Table 2
Inhibitory activities of peptide-aldehydes for SARS 3CL R188I mutant protease
Peptide aldehyde
IC50
3.2. Identification of degradation products from mature 3CL-
protease
Ac-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
ꢁ 6 mM
Ac-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
Ac-Ser-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
Ac-Thr-Ser-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
155 lM
37
l
M
M
The affinity resin-purified fraction (12 lg protein) of the N-ter-
26
l
minal MBP-His-Flag-tagged 3CL protease was incubated with 1 U
of enterokinase for 30 min at 21 °C, and then separated on 15%
SDS–PAGE gels and transferred to a PVDF membrane. After Coo-
massie blue staining, the 20- and 13-kDa bands were dissected
and subjected to Edman sequencing for determination of the N-ter-
minal.28 This analysis was performed using an Applied Biosystems
491 Protein Sequencer at APRO Life Science Institute (Naruto,
Japan).
vector, pET-3CL, was made by inserting the NheI–XhoI fragment
corresponding to the 3CL protease-coding fragment of pGEM-3CL
into the NheI/XhoI site of pET-21a(+) (Novagen), and encodes a
34-kDa protein containing a full-length 3CL protease with three
N-terminal amino acids (Met-Ala-Ser) and two C-terminal amino
acids (Leu-Glu) plus a His tag.

K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
9405
3.3. Expression of 3CL-R188I mutant proteases
20 mM Tris–HCl buffer pH 7.5 containing 7 mM DTT was incubated
with the protease at 37 °C and the cleavage reaction was moni-
tored by analytical HPLC [Cosmosil 5C18 (4.6 ꢂ 150 mm), 1.0 ml/
min, CH3CN (10–20%)/30 min]. Each product of hydrolysis was
identified by MALDI-TOF MS.
Kinetic parameters, Km and kcat, were determined by initial rate
measurements of the hydrolysis of each substrate at 37 °C. Each
reaction was initiated by adding the protease to various solutions
containing different final concentrations of the substrate (0 to
Site-directed mutagenesis was performed by the inverse PCR
method described by Stemmer and Morris.29 The primers used to
generate point mutations were 50-atctctCGTCTCCATACAAACTGCAC
AGGCTG (nucleotide in italics is mutated) and 50-atctctCGTCTCTGTA
TGTCAACAAATGGACC, and PCR was performed with the plasmid
pGEM-3CL as the template. After PCR, the ends of the full-length lin-
earized plasmids were digested with BsmBI (CGTCTCN’NNNN), leav-
ing plasmids with compatible overhangs at both ends. Expression
vectors pMAL-3CL-R188I and pET-3CL-R188I were constructed by
replacing the short SphI–StyI fragment of pMAL-3CL and the short
NheI–XhoI fragment of pET-3CL with the corresponding fragments
of pGEM-3CL-R188I, respectively. Sequencing was carried out to
confirm the presence of only the desired mutation and no uninten-
tional changes.
168 lM). The final concentration of the enzyme (10 to 170 nM
for the mutant protease) and the digestion time (15 to 90 min) var-
ied because the cleavage activity differed for each substrate. After
the reaction, aliquots were analyzed by HPLC. The initial digestion
rates were calculated from the decrease in the peak area of each
substrate. Km was calculated from the plot of [S]/v vs. [S], where
[S] is the concentration of the substrate and
t is the initial reaction
For purification of the untagged 3CL-R188 L protease, the metal
affinity resin-treated fraction of the N-terminal MBP-His-Flag-
tagged protease (about 1.5 mg protein) was further incubated with
17 U of recombinant enterokinase (Novagen) for 2 h at 21 °C in
1 ml of a buffer containing 20 mM Tris–HCl pH 7.4, 50 mM NaCl,
and 2 mM CaCl2. After the enterokinase was removed from the solu-
tion using EKapture Agarose (Novagen), the buffer was changed to
20 mM Bis–Tris pH 5.5 using a Centricon YM-10. The enterokinase
treated fraction was then applied to a 1 ml bed volume of DEAE Se-
pharose (GE Healthcare) equilibrated with the same buffer. The tar-
get protease flowed though the Sepharose resin and the MBP-His-
Flag fragment was retained in the resin. Optionally, the bound pro-
tein could be eluted with the same buffer containing 0.5 M KCl.
The metal affinity resin-treated fraction of the C-terminal His-
tagged 3CL protease was used as the substrate to examine degrada-
rate. All reactions were repeated three times and the results were
averaged.
3.6. Inhibitors
3.6.1. Fmoc-Gln(Me)2-N(OMe)Me
HN(Me)2. HCl salt (0.29 g, 3.56 mmol), BOP (1.59 g, 3.56 mmol),
and DIEA (1.91 ml, 12 mmol) were added to Fmoc-Glu-OBut
(1.28 g, 3 mmol) in CH2Cl2 (10 ml), and the mixture was stirred
at 25 °C for 2 h. The solvent was evaporated and the residue was
dissolved in AcOEt. The organic phase was washed with 5% citric
acid, 5% NaHCO3, and brine, and then dried over MgSO4. The sol-
vent was evaporated and the residue was purified by silica-gel col-
umn chromatography using CHCl3:MeOH = 10:0.5. The desired
product, without further purification, was treated with TFA
(7.0 ml) in the presence of anisole (0.65 ml, 6.0 mmol). After stir-
ring at 25 °C for 1 h, the TFA was evaporated at 25 °C. The residue
was washed with hexane, and dried in vacuo. The residue was dis-
solved in DMF (10 ml), and HN(OMe)Me HCl salt (0.38 g,
3.90 mmol), BOP (1.72 g, 3.90 mmol), and DIEA (1.70 ml, 11 mmol)
were added. The mixture was stirred at 25 °C for 8 h, and the sol-
vent was evaporated. The residue was dissolved in AcOEt, and
the organic phase was washed with 5% citric acid, 5% NaHCO3,
and brine, and then dried over MgSO4. The solvent was evaporated
and the residue was purified by silica-gel column chromatography
tion. After incubating the fraction (about 4
room temperature with 0.05 U of enterokinase or with 0.5
l
g protein) for 14 h at
g of
l
purified 3CL-R188L protease, the solution was separated on 15%
SDS–PAGE gels and electroblotted onto a PVDF membrane. The
His-tagged protease and its cleaved product were visualized using
an anti-His-Tag monoclonal antibody (Novagen), a biotinylated
anti-mouse IgG (Vector), and an amplified alkaline phosphatase Im-
mun-Blot assay kit (Bio-Rad).
3.4. Substrate peptides
using CHCl3:MeOH = 10:0.5 to yield the desired product as an oil.
27
The substrate peptides (SO1; H-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-
Phe-Arg-Lys-NH2: SO3; H-Lys-Val-Ala-Thr-Val-Gln-Ser-Lys-Met-Ser-
Asp-NH2: and SR1; H-Gly-Pro-Phe-Val-Asp-Arg-Gln-Thr-Ala-Gln-
Ala-Ala-Gly-Thr-Asp-Thr-NH2) were synthesized using Fmoc-based
solid-phase peptide synthesis (SPPS)30 starting with Rink amide resin
(4-(20,40-dimethoxyphenyl-Fmoc aminomethyl)phenoxy resin).31
SPPS was achieved by a combination of Fmoc deprotection using
20% piperidine/DMF and a coupling reaction using a standard diiso-
propylcarbodiimide/HOBt protocol. Each peptide was purified by pre-
parative HPLC after cleavage from the resin by treatment with TFA
thioanisole (10:1) at 25 °C for 3 h. Homogeneity was further con-
firmed by MALDI-TOF-MS. SO1; HPLC [Cosmosil 5C18 AR column
(4.6 ꢂ 150 mm), 1.0 ml/min, CH3CN (10–20%)/30 min] rt 19.62 min,
m/z 1192.714 for [M+H]+ (calcd 1192.681 for C52H90N17O15). SO3;
HPLC [Cosmosil 5C18 AR column (4.6 ꢂ 150 mm), 1.0 ml/min, CH3CN
(10–20%)/30 min] rt 8.58 min, m/z 1192.288 for [M+H]+ (calcd
1192.637 for C49H90N15O17S). SR1; HPLC [Cosmosil 5C18 AR column
(4.6 ꢂ 150 mm), 1.0 ml/min, CH3CN (10–50%)/30 min] rt 11.07 min,
m/z 1633.758 for [M+H]+ (calcd 1633.794 for C68H109N22O25).
Yield 1.13 g (85%), [
a]
+46.7 (c 0.24, CHCl3), 1H NMR (CDCl3)
D
d; 2.00 (m, 1H), 2.16 (m, 1H), 2.42 (m, 2H), 2.94 (s, 3H), 2.95 (s,
3H), 3.22 (s, 3H), 3.79 (s, 3H), 4.22 (t, J = 7.1 Hz, 1H), 4.35 (m,
2H), 4.76 (br s, 1H), 5.91 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 7.5 Hz,
2H), 7.39 (t, J = 7.5 Hz, 2H), 7.61 (br t, J = 8.3 Hz, 2H), 7.75 (d,
J = 8.3 Hz, 2H); 13C NMR d; 27.72, 29.13, 32.29, 35.63, 37.24,
47.27, 51.04, 61.72, 67.09, 120.05, 125.23, 125.34, 127.15, 127.78,
141.37, 141.40, 143.89, 144.12, 156.39, 162.44, 171.99; MALDI-
TOF-MS; Calcd. 462.201 for C24H29N3O5Na, Found. 462.126 for
[M+Na]+.
3.6.2. Fmoc-Leu-Gln(Me)2-N(OMe)Me
Et2NH (8.6 ml) was added to Fmoc-Gln-N(OMe)Me (0.73 g,
1.66 mmol) in CH3CN (8 ml), and the mixture was stirred at 25 °C
for 40 min. The solvent was evaporated and the residue was dried
in vacuo. The residue was dissolved in CH2Cl2 (5 ml). Fmoc-Leu-OH
(0.49 g, 1.39 mmol), BOP (0.61 g, 1.39 mmol), and DIEA (0.44 ml,
2.77 mmol) were added to the solution and the mixture was stirred
at 25 °C for 8 h. The solvent was evaporated, and the residue was
dissolved in AcOEt. The organic phase was washed with 5% citric
acid, 5% NaHCO3, and brine, and then dried over MgSO4. The sol-
vent was evaporated and the residue was purified by silica-gel col-
3.5. Enzymatic activity
The proteolytic activities of 3CL proteases were detected by a
cleavage assay of SO1 using analytical HPLC. Each peptide in
umn chromatography using CHCl3:MeOH = 10:0.5 to yield the
27
desired product as an oil. Yield 0.60 g (91%), [
a]
D
ꢀ27.6 (c 1.1,

9406
K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
CHCl3), 1H NMR (CDCl3) d; 0.94 (s, 3H), 0.96 (s, 3H), 1.53–1.63 (m,
2H), 1.66–1.72 (m, 1H), 2.00 (br s, 1H), 2.14–2.21 (m, 1H), 2.88 (s,
3H), 2.90 (s, 3H), 3.21 (s, 3H), 3.79 (s, 3H), 4.19–4.24 (m, 1H), 4.22
(d, J = 6.9 Hz, 1H), 4.29–4.42 (m, 2H), 4.92 (br s, 1H), 5.40 (d,
J = 8.4 Hz, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.59
(d, J = 7.5 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H); 13C NMR d; 22.06,
23.12, 24.77, 26.77, 29.18, 32.33, 35.70, 37.20, 42.19, 47.32,
49.64, 53.66, 61.71, 67.09, 120.06, 120.08, 125.22, 127.20, 127.82,
141.40, 143.97, 144.02, 156.19, 171.80, 172.36, 172.43; MALDI-
TOF-MS; Calcd. 575.285 for C30H40N4O6Na, Found. 575.174 for
[M+Na]+.
(s, 3H), 3.47 (t, J = 7.8 Hz, 1H), 3.71 (t, J = 7.8 Hz, 1H), 3.73 (s, 3H),
4.21 (t, J = 7.1 Hz, 1H), 4.29–4.50 (m, 4H), 4.83 (br s, 1H), 4.94 (br
s, 1H), 5.09 (br s, 1H), 6.39 (br s, 1H), 7.26 (br t, J = 7.4 Hz, 2H),
7.37 (br t, J = 7.4 Hz, 2H), 7.62 (br d, J = 6.9 Hz, 2H), 7.74 (br d,
J = 7.2 Hz, 2H), 7.83 (br s, 1H), 8.03 (br s, 1H); 13C NMR d; 18.51,
19.26, 22.36, 22.79, 24.95, 27.35, 27.90, 29.20, 31.57, 35.42,
37.09, 41.80, 47.15, 48.99, 51.67, 54.98, 58.70, 61.70, 62.20,
67.05, 119.89, 125.22, 127.02, 127.65, 141.26, 143.92, 162.34,
170.02, 171.00, 172.00; MALDI-TOF-MS; Calcd. 888.041 for
C45H66N7O10Na, Found. 888.253 for [M+Na]+.
3.6.6. Fmoc-Thr(But)-Ser(But)-Ala-Val-Leu-Gln(Me)2-N(OMe)Me
Fmoc-Ser(But)-Ala-Val-Leu-Gln(Me)2-N(OMe)Me (0.57 g, 0.66
mmol) in CH2Cl2–CH3CN (3 ml–3 ml) was treated with Et2NH
(3.4 ml) as above. To the product in DMF (5 ml), were added Fmoc-
Thr(But)-OH (0.33 g, 0.80 mmol), BOP (0.35 g, 0.80 mmol), and DIEA
(0.25 ml, 1.57 mmol). The mixture was stirred at 25 °C for 1 h and
H2O was added. The resulting precipitate was washed with 5% citric
acid, 5% NaHCO3, and H2O. The crude product was reprecipitated
3.6.3. Fmoc-Val-Leu-Gln(Me)2-N(OMe)Me
Fmoc-Leu-Gln(Me)2-N(OMe)Me (0.60 g, 1.09 mmol) in CH3CN
a
(6 ml) was treated with Et2NH (5.6 ml) as above. To the N -depro-
tected product in CH2Cl2–DMF (2.5 ml–2.5 ml), were added Fmoc-
Val-OH (0.44 g, 1.30 mmol), BOP (0.58 g, 1.30 mmol), and DIEA
(0.41 ml, 2.58 mmol). The mixture was stirred at 25 °C for 8 h
and the crude product isolated as above was purified by silica-
gel column chromatography using CHCl3:MeOH–10:0.5 to yield
from DMF/Et2O to yield the desired product as an amorphous solid.
27
27
the desired product as an oil. Yield 0.61 g (86%), [
a
]
D
ꢀ11.6 (c
Yield 0.40 g (59%), [
a
]
D
ꢀ13.2 (c 0.68, CHCl3), 1H NMR (CDCl3) d;
1.0, CHCl3), 1H NMR (CDCl3) d; 0.86 (m, 12H), 1.49–1.65 (m, 2H),
1.97 (br s, 1H), 2.12–2.17 (m, 2H), 2.37–2.39 (m, 2H), 2.92 (s,
3H), 2.94 (s, 3H), 3.19 (s, 3H), 3.76 (s, 3H), 4.03 (br t, J = 7.2 Hz,
1H), 4.21 (t, J = 6.9 Hz, 1H), 4.31–4.53 (m, 3H), 4.92 (br s, 1H),
5.58 (br d, J = 8.7 Hz, 1H), 6.56 (br d, J = 7.5 Hz, 1 H), 7.30 (t,
J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.75
(d, J = 7.5 Hz, 2H); 13C NMR d; 17.97, 19.38, 22.10, 23.03, 24.83,
29.20, 31.25, 35.73, 37.28, 41.79, 47.32, 49.56, 52.01, 60.61,
61.71, 67.24, 120.08, 125.22, 127.21, 127.84, 141.43, 144.05,
156.58, 171.32, 171.85, 172.36; MALDI-TOF-MS; Calcd. 674.353
for C35H49N5O7Na, Found. 674.211 for [M+Na]+.
0.93-0.95 (br s, 12H), 1.17 (s, 12H), 1.31 (s, 9H), 1.41 (br d,
J = 6.0 Hz, 3H), 1.67 (br s, 3H), 1.93 (br s, 1H), 2.13 (br s, 1H), 2.38
(br s, 3H), 2.92 (s, 3H), 2.97 (s, 3H), 3.20 (s, 3H), 3.51 (br s, 1H), 3.77
(s, 3H), 3.87 (br d, J = 7.8 Hz, 1H), 4.22 (br s, 4H), 4.42 (br s, 4H),
4.96 (br s, 1H), 6.78–6.81 (m, 2H), 7.14–7.41 (m, 7H), 7.58 (br d,
J = 5.7 Hz, 2H), 7.77 (br d, J = 6.3 Hz, 2H); 13C NMR d; 17.28, 17.35,
17.89, 19.33, 21.48, 23.24, 24.73, 27.45, 28.30, 29.08, 29.65, 35.51,
37.23, 40.50, 47.22, 49.11, 50.49, 51.89, 54.52, 59.07, 60.83, 61.58,
66.72, 67.19, 120.08, 124.98, 125.08, 127.12, 127.84, 141.39,
143.61, 143.75, 156.07, 162.36, 169.63, 171.05, 171.35, 171.94,
172.31;MALDI-TOF-MS; Calcd. 1045.252for C53H81N8O12Na, Found.
1045.426 for [M+Na]+.
3.6.4. Fmoc-Ala-Val-Leu-Gln(Me)2-N(OMe)Me
Fmoc-Val-Leu-Gln(Me)2-N(OMe)Me (0.60 g, 0.92 mmol) in
CH2Cl2–CH3CN (3 ml–3 ml) was treated with Et2NH (4.8 ml) as
above. To the product in DMF (5 ml), were added Fmoc-Ala-OH
(0.34 g, 1.09 mmol), BOP (0.49 g, 1.09 mmol), and DIEA (0.35 ml,
2.20 mmol). The mixture was stirred at 25 °C for 8 h and the crude
product isolated as above was purified by silica-gel column chroma-
3.6.7. Ac-Thr(But)-Ser(But)-Ala-Val-Leu-Gln(Me)2-N(OMe)Me
Fmoc-Thr(But)-Ser(But)-Ala-Val-Leu-Gln(Me)2-N(OMe)Me (78 mg,
75 lmol) in CH2Cl2–CH3CN (1.5 ml–1.5 ml) was treated with Et2NH
(0.59 ml) as above. To the product in DMF (3 ml), were added Ac2O
(0.14 ml, 1.48 mmol) and DIEA (0.24 ml, 1.48 mmol). The mixture
was stirred at 25 °C for 8 h and H2O was added. The crude product
tography using CHCl3:MeOH = 10:0.5 to yield the desired product as
was reprecipitated from DMF/Et2O to yield the desired product as
27
27
an oil. Yield 0.51 g (77%), [
a]
D
ꢀ30.3 (c 0.90, CHCl3), 1H NMR
an amorphous solid. Yield 40 mg (63%), [
a]
ꢀ17.5 (c 0.20, DMF),
D
(CDCl3) d; 0.87 (d, J = 6.6 Hz, 6H), 0.93 (d, J = 6.6 Hz, 6H), 1.36 (d,
J = 6.9 Hz, 3H), 1.51–1.67 (m, 3H), 1.95–2.14 (m, 3H), 2.33–2.38 (m,
2H), 2.88 (s, 6H), 3.17(s, 3H), 3.72 (s, 3H), 4.20 (t, J = 7.2 Hz, 1H),
4.28–4.41 (m, 2H), 4.46 (br s, 1H), 4.57 (br s, 1H), 4.83 (br s, 1H),
5.08 (br s, 1H), 6.21 (br s, 1H), 7.24 (t, J = 7.2 Hz, 2H), 7.37 (t,
J = 7.4 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H), 7.95
(br s, 1H); 13C NMR d; 18.66, 19.42, 22.65, 22.88, 25.04, 27.92,
29.34, 31.51, 32.33, 35.58, 37.20, 42.06, 47.27, 49.01, 50.59, 51.76,
58.92, 61.79, 67.19, 120.06, 125.32, 127.17, 127.81, 141.39, 144.05,
162.49, 171.03, 172.07, 172.94; MALDI-TOF-MS; Calcd. 745.390 for
C38H54N6O8Na, Found. 745.199 for [M+Na]+.
1H NMR (DMSO-d6) d; 0.80–0.88 (m, 12H), 1.04 (d, J = 6.3 Hz, 3H),
1.10 (s, 9H), 1.11 (d, J = 6.6 Hz, 3H), 1.17 (s, 9H), 1.36–1.46 (m, 2H),
1.53–1.60 (m, 1H), 1.63–1.83 (m, 2H), 1.91 (s, 3H), 1.97 (m, 1H),
2.30 (br t, J = 7.4 Hz, 2H), 2.80 (s, 3H), 2.90 (s, 3H), 3.10 (s, 3H),
3.41–3.46 (m, 1H), 3.51–3.59 (m, 1H), 3.70 (s, 3H), 3.87–3.90 (m,
1H), 4.10–4.15 (m, 1H), 4.30–4.39 (m, 4H), 4.71 (br s, 1H), 7.61–
7.97 (m, 6H); 13C NMR d; 18.49, 18.92, 19.64, 21.94, 22.97, 23.54,
24.40, 27.57, 28.45, 30.75, 35.29, 37.01, 48.61, 53.60, 57.75, 58.10,
62.25, 67.50, 169.89, 170.98, 171.64, 172.23;MALDI-TOF-MS;Calcd.
865.538 for C40H74N8O11Na, Found. 865.476 for [M+Na]+.
3.6.8. Ac-Thr-Ser-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
3.6.5. Fmoc-Ser(But)-Ala-Val-Leu-Gln(Me)2-N(OMe)Me
LiAlH4. (30 mg) was added to Ac-Thr(But)-Ser(But)-Ala-Val-Leu-
Fmoc-Ala-Val-Leu-Gln(Me)2-N(OMe)Me (0.56 g, 0.77 mmol) in
CH2Cl2–CH3CN (3 ml–3 ml) was treated with Et2NH (4.0 ml) as
above. To the product in DMF (5 ml), were added Fmoc-Ser(But)-
OH (0.36 g, 0.94 mmol), BOP (0.41 g, 0.94 mmol), and DIEA
(0.30 ml, 1.89 mmol). The mixture was stirred at 25 °C for 2 h
and the crude product isolated as above was purified by silica-
Gln(Me)2-N(OMe)Me (30 mg, 36 lmol) in DMF–THF (1.5 ml–
1.5 ml), and the mixture was stirred at 25 °C for 90 min. The mix-
ture was filtered and the solvent was evaporated. The crude prod-
uct was purified by HPLC [Cosmosil 5C18 (10 ꢂ 250 mm), 3.0 ml/
min, CH3CN (15–60%)/30 min]. The solvent of the desired peak
(rt, 42.0 min) was removed by lyophilization to yield a white fluffy
powder. Yield 5.2 mg (19%) 1H NMR (CD3CN containing D2O) d;
0.82–0.93 (m, 12H), 1.10–1.14 (m, 3H), 1.14 (s, 9H), 1.17 (s, 9H),
1.31–1.36 (m, 3H), 1.56–1.64 (m, 3H), 2.00 (s, 3H), 2.10–2.17 (m,
1H), 2.28–2.33 (m, 2H), 2.85 (br s, 3H), 2.97 (br s, 3H), 3.61–3.70
(m, 2H), 3.97–4.02 (m, 1H), 4.10–4.24 (m, 6H), 9.41 (br s); MALDI-
gel column chromatography using CHCl3:MeOH = 10:0.5 to yield
27
the desired product as an oil. Yield 0.58 g (86%), [
a
]
D
ꢀ22.8 (c
0.15, CHCl3), 1H NMR (CDCl3) d; 0.84–0.92 (m, 12H), 1.15 (s, 9H),
1.34 (d, J = 6.6 Hz, 3H), 1.55–1.63 (m, 3H), 1.89–1.99 (m, 1H),
2.07–2.17 (m, 2H), 2.36 (br s, 2H), 2.88 (s, 3H), 2.89 (s, 3H), 3.17

K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
9407
TOF-MS; Calcd. 806.501 for C38H69N7O10Na, Found. 806.121 for
[M+Na]+.
Anisole (15
above powder (7.1 mg, 9
3.7. Inhibitory activity
l
l, 6.0 mmol) and TFA (0.5 ml) were added to the
Inhibitory activity was examined using the substrate SO1.
Cleavage of the substrate by the purified mutant 3CL protease in
the presence of various concentrations of the inhibitor was moni-
tored by analytical HPLC, as described above. Inhibitory activity
was evaluated using the corresponding IC50 value obtained from
the sigmoid dose-response curve. Reactions were repeated three
times and the results were averaged.
l
mol), and the mixture was stirred at
25 °C for 2 h. The TFA was evaporated and ether (5 ml) and 0.1% aq.T-
FA (5 ml) were added to the residue. The aqueous phase was washed
with ether (5 ml) twice and the solvent was removed by lyophiliza-
tion. The crude product was purified by HPLC [Cosmosil 5C18
(10 ꢂ 250 mm), 3.0 ml/min, CH3CN (10–40%)/30 min]. The solvent
of the desired peak (rt, 25.6 min) was removed by lyophilization to
yield a white fluffy powder. Yield 1.8 mg (30%), HPLC rt, 14.36 min
[Cosmosil 5C18 (4.6 ꢂ 150 mm), 1 ml/min, CH3CN (10–40%)/
30 min], 1H NMR (CD3CN containing D2O) d; 0.84–0.94 (m, 12H),
1.14–1.17 (m, 3H), 1.33–1.37 (m, 3H), 1.56–1.63 (m, 3H), 2.02 (s,
3H), 2.02–2.09 (m, 1H), 2.32–2.35 (m, 2H), 2.84–2.87 (m, 3H),
2.94–2.99 (m, 3H), 3.74–3.76 (m, 1H), 3.81–3.85 (m, 1H), 4.17–
4.33 (m, 6H), 4.77–4.82 (m, 1H), 9.44 (br s); MALDI-TOF-MS; Calcd.
694.375 for C30H53N7O10Na, Found. 694.337 for [M+Na]+.
Acknowledgments
This work was supported in part by Grant-in-aid for Scientific
Research 18590010 from the Japan Society for the Promotion of
Science. We are grateful to Prof. Naoki Yamamoto and Dr. Norio
Yamamoto of Tokyo Medical and Dental University, Department
of Molecular Virology for kindly providing the plasmid pMAL-3CL.
The following truncated peptide-aldehyde derivatives were
similarly prepared from the above intermediates.
References and notes
1. Lee, N.; Hui, D.; Wu, A.; Chan, P.; Cameron, P.; Joynt, F. M.; Ahuja, A.; Yung, M.
Y.; Leung, C. B.; To, K. F.; Szeto, M. D.; Leu, C. C.; Chung, S.; Sung, J. J. Y. N. Engl. J.
Med. 2003, 348, 1986–1994.
2. Drosten, C.; Günther, S.; Preiser, W.; Ven der Werf, S.; Brodt, H. R.; Becker, S.;
Rabenau, H.; Panning, M.; Kolensnikova, L.; Fouchier, R. A. M.; Berger, A.;
Burguière, A. M.; Cinatl, J.; Eickmann, M.; Escriou, N.; Grywna, K.; Kramme, S.;
Manuguerra, J.; Müller, S.; Rickerts, V.; Stürmer, M.; Vieth, S.; Klenk, H. D.;
Osterhaus, A. D. M. E.; Schmitz, H.; Doerr, H. W. N. Engl. J. Med. 2003, 348, 1967–
1976.
3. Ksiazek, T. G.; Erdman, D.; Goldsmith, C. S.; Zaki, S. R.; Peret, T.; Emery, S.; Tong,
S.; Urbani, C.; Comer, J. A.; Lim, W.; Rollin, P. E.; Dowell, S. F.; Ling, A. E.;
Humphrey, C. D.; Shieh, W. J.; Guarner, J.; Paddock, C. D.; Rota, P.; Fields, B.;
DeRisi, J.; Yang, J. Y.; Cox, N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.; Anderson,
L. J. N. Engl. J. Med. 2003, 348, 1953–1966.
4. Rota, P. A.; Oberste, M. S.; Monroe, S. S.; Nix, W. A.; Campagnoli, R.; Icenogle, J. P.;
Penaranda, S.; Bankamp, B.; Maher, K.; Chem, M. H.; Tong, W.; Tamin, A.; Lowe, L.;
Frace, M.; DeRisi, J. L.; Chen, Q.; Wang, D.; Erdman, D. D.; Peret, T. C.; Burns, C.;
Ksiazek, T. G.; Rollin, P. E.; Sanchez, A.; Liffick, S.; Holloway, B.; Limor, J.;
McCaustland, K.; Olsen-Rasmussen, M.; Fouchier, R.; Gunther, S.; Osterhaus, A.
D.; Drosten, C.; Pallansch, M. A.; Anderson, L. J.; Bellini, W. J. Science 2003, 300,
1394–1399.
5. Marra, M. A.; Jones, S. J.; Astell, C. R.; Holt, R. A.; Brooks-Wilson, A.; Butterfield,
Y. S.; Khattra, J.; Asano, J. K.; Barber, S. A.; Chan, S. Y.; Cloutier, A.; Coughlin, S.
M.; Freeman, D.; Girn, N.; Griffith, O. L.; Leach, S. R.; Mayo, M.; McDonald, H.;
Montgomery, S. B.; Pandoh, P. K.; Petrescu, A. S.; Robertson, A. G.; Schein, J. E.;
Siddiqui, A.; Smailus, D. E.; Sott, J. M.; Yang, G. S.; Plummer, F.; Andonov, A.;
Artsob, H.; Bastien, N.; Bermard, K.; Booth, T. F.; Bowness, D.; Czub, M.; Drebot,
M.; Fernando, L.; Flick, R.; Garbutt, M.; Gray, M.; Grolla, A.; Jones, S.; Feldmann,
H.; Meyers, A.; Kabani, A.; Li, Y.; Normand, S.; Stroher, U.; Tipples, G. A.; Tyler,
S.; Vogrig, R.; Ward, D.; Watson, B.; Brunham, R. C.; Krajden, M.; Petric, M.;
Skowronski, D. M.; Upton, C.; Roper, R. L. Science 2003, 300, 1399–1404.
6. Thiel, V.; Ivanov, K. A.; Putics, Á.; Hertzig, T.; Schelle, B.; Bayer, S.; Weibbrich, B.;
Snijder, E. J.; Rabenau, H.; Doerr, H. W.; Gorbalenya, A. E.; Ziebuhr, J. J. Gen. Viol.
2003, 84, 2305–2315.
3.6.9. Ac-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
HPLC rt, 18.64 min [Cosmosil 5C18 (4.6 ꢂ 150 mm), 1 ml/min,
CH3CN (10–60%)/30 min], 1H NMR (CD3CN) d; 0.86–0.99 (m,
12 H), 1.51–1.64 (m, 3H), 2.03 (s, 3H), 2.31–2.37 (m, 2H), 3.98 (br
t, J = 5.9 Hz, 1H), 4.27–4.33 (m, 1H), 4.55–4.63 (m, 1H), 4.78–4.86
(m, 1H), 6.82 (br d, J = 7.8 Hz, 2H), 6.96 (br d, J = 8.7 Hz, 1H), 9.42
(br s); MALDI-TOF-MS; Calcd. 435.259 for C20H46N4O5Na, Found.
435.955 for [M+Na]+.
3.6.10. Ac-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
HPLC rt, 12.64 min [Cosmosil 5C18 (4.6 ꢂ 150 mm), 1 ml/min,
CH3CN (10–60%)/30 min], 1H NMR (D2O) d; 0.86–0.94 (m, 12H),
1.33 (d, J = 7.2 Hz, 3H), 1.57–1.67 (m, 3H), 2.00 (s, 3H), 2.00–2.22
(m, 3H), 2.45–2.49 (m, 2H), 2.92 (s, 3H), 3.04 (s, 3H), 4.08 (d,
J = 8.1 Hz, 1H), 4.28 (q, J = 7.2 Hz, 1H), 4.38–4.42 (m, 2H), 9.41 (br
s); MALDI-TOF-MS; Calcd. 506.296 for C23H41N5O6Na, Found.
506.216 for [M+Na]+.
3.6.11. Ac-Ser-Ala-Val-Leu-NHCH(CH2CH2CON(CH3)2)-CHO
HPLC rt, 14.38 min [Cosmosil 5C18 (4.6 ꢂ 150 mm), 1 ml/min,
CH3CN (10–40%)/30 min], 1H NMR (CD3CN containing D2O) d;
0.85–0.92 (m, 12H), 1.36 (d, J = 7.5 Hz, 3H), 1.56–1.66 (m, 3H),
1.98 (s, 3H), 2.07–2.10 (m, 1H), 2.30–2.35 (m, 2H), 2.86 (s, 3H),
2.99 (s, 3H), 3.64–3.69 (m, 1H), 3.77–3.83 (m, 1H), 4.00–4.02 (m,
1H), 4.19–4.30 (m, 23H), 9.44 (br s); MALDI-TOF-MS; Calcd.
593.328 for C26H46N6O8Na, Found. 593.258 for [M+Na]+.
7. Anand, K.; Palm, G. J.; Mesters, J. R.; Siddell, S. G.; Ziebuhr, J.; Hilgenfeld, R.
EMBO J. 2002, 21, 3213–3224.
8. Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Science 2003,
300, 1763–1767.
9. Fan, K.; Wei, P.; Feng, Q.; Chen, S.; Huang, C.; Ma, L.; Lai, B.; Pei, J.; Liu, Y.; Chen,
J.; Lai, L. J. Biol. Chem. 2004, 279, 1637–1642.
10. Huang, C.; Wei, P.; Fan, K.; Liu, Y.; Lai, L. Biochemistry 2004, 43, 4568–4574.
11. Kaeppler, U.; Stiefl, N.; Schiller, M.; Vicik, R.; Breuning, A.; Schmitz, W.;
Rupprecht, D.; Schmuck, C.; Baumann, K.; Ziebuhr, J.; Schirmeister, T. J. Med.
Chem. 2005, 48, 6832–6842.
12. Ghosh, A. K.; Xi, K.; Ratia, K.; Santarsiero, B. D.; Fu, W.; Harcourt, B. H.; Rota, P. A.;
Baker, S. C.; Johnson, M. E.; Mesecar, A. D. J. Med. Chem. 2005, 48, 6767–6771.
13. Shie, J. J.; Fang, J. M.; Kuo, C. J.; Kuo, T. H.; Liang, P. H.; Huang, H. J.; Yang, W. W.;
Lin, C. H.; Chen, J. L.; Wu, Y. T.; Wong, C. H. J. Med. Chem. 2005, 48, 4469–4473.
14. Chen, L. R.; Wang, Y. C.;Lin, Y. W.; Chou, S. Y.; Chen, S. F.; Liu, L. T.; Wu, Y. T.; Kuo, C.
J.; Chen, T. S. S.; Juang, S. H. Bioorg. Med. Chem. Lett. 2005, 15, 3058–3062.
15. Shie, J. J.; Fang, J. M.; Kuo, T. H.; Kuo, C. J.; Liang, P. H.; Huang, H. J.; Wu, Y. T.;
Jan, J. T.; Cheng, Y. S.; Wong, C. H. Bioorg. Med. Chem. 2005, 13, 5240–5252.
16. Jain, R. P.; Petterson, H. I.; Zhang, J.; Aull, K. D.; Fortin, P. D.; Huitema, C.; Eltis, L.
D.; Parrish, J. C.; James, M. N. G.; Wishart, D. S.; Vederas, J. C. J. Med. Chem. 2004,
47, 6113–6116.
17. Ghosh, A. K.; Xi, K.; Grum-Tokars, V.; Xu, X.; Ratia, K.; Fu, W.; Houser, K. V.;
Baker, S. C.; Johnson, M. E.; Mesecar, A. D. Bioorg. Med. Chem. Lett. 2007, 17,
5876–5880.
18. Bacha, U.; Barrila, J.; Velazquez-Campoy, A.; Leavitt, S. A.; Freire, E. Biochemistry
2004, 43, 4906–4912.
3.6.12. Ac-Ser-Ala-Val-Leu-Gln-NHCH3
Ac-Ser(But)-Ala-Val-Leu-Gln(Trt)-N(OMe)Me was prepared
similarly as above starting from Fmoc-Gln(Trt)-N(OMe)Me. The
protected pentapeptide Weinreb amide (0.20 g, 0.22 mmol) in
DMF (10 ml) was treated with LiAlH4 as stated above. The products
were absorbed on Diaion HP20 and eluted with CH3CN:H2O = 3:1.
The solvent was removed by lyophilization, and the product was
partially purified by HPLC [Cosmosil 5C18 (10 ꢂ 250 mm), 3 ml/
min, CH3CN (40–80%)/60 min]. An aliquot of the product was trea-
ted with TFA as stated above and purified by HPLC [Cosmosil 5C18
(10 ꢂ 250 mm), 3 ml/min, CH3CN (10–40%)/60 min]. HPLC rt,
10.95 min [Cosmosil 5C18 (4.6 ꢂ 150 mm), 1 ml/min, CH3CN (10–
70%)/30 min], 1H NMR (DMSO-d6) d; 0.81–0.91 (m, 12H), 1.21 (d,
J = 7.2 Hz, 3H), 1.44–1.49 (m, 2H), 1.52–1.73 (m, 2H), 1.86 (s, 3H),
1.89–2.06 (m, 4H), 2.57 (d, J = 4.5 Hz, 3H), 3.52–3.55 (m, 2H),
4.07–4.33 (m, 4H), 4.98 (br t, J = 5.1 Hz, 1H), 7.69–7.73 (m, 2H),
7.94–8.14 (m, 4H); MALDI-TOF-MS; Calcd. 594.323 for C25H45N7O8-
Na, Found. 594.268 for [M+Na]+.

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K. Akaji et al. / Bioorg. Med. Chem. 16 (2008) 9400–9408
19. Chou, C. Y.; Chang, H. C.; Hsu, W. C.; Lin, T. Z.; Lin, C. H.; Chang, G. G.
Biochemistry 2004, 43, 14958–14970.
20. Lee, T. W.; Cherney, M. M.; Huitema, C.; Liu, J.; James, K. E.; Powers, J. C.; Eltis, L.
D.; James, M. N. G. J. Mol. Biol. 2005, 353, 1137–1151.
21. Yang, S.; Chen, S. J.; Hsu, M. F.; Wu, J. D.; Tseng, C. T. K.; Liu, Y. F.; Chen, H. C.;
Kuo, C. W.; Wu, C. S.; Chang, L. W.; Chen, W. C.; Liao, S. Y.; Chang, T. Y.; Hung, H.
H.; Shr, H. L.; Liu, C. Y.; Huang, Y. A.; Chang, L. Y.; Hsu, J. C.; Peters, C. J.; Wang,
A. H. J.; Hsu, M. C. J. Med. Chem. 2006, 49, 4971–4980.
24. Hsu, M. F.; Kuo, C. J.; Chang, K. T.; Chang, H. C.; Chou, C. C.; Ko, T. P.; Shr,
H. L.; Chang, G. G.; Wang, A. H. J.; Liang, P. H. J. Biol. Chem. 2005, 280,
31257–31266.
25. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815–3818.
26. Sydnes, M. O.; Hayashi, Y.; Sharma, V. K.; Hamada, T.; Bacha, U.; Barrila, J.;
Freire, E.; Kiso, Y. Tetrahedron 2006, 62, 8601–8609.
27. Hanada, K.; Tamai, M.; Yamagishi, M.; Ohmura, S.; Sawada, J.; Tanaka, I. Agric.
Biol. Chem. 1978, 42, 523–528.
22. Al-Gharabli, S.; AliShah, S. T.; Weik, S.; Schmidt, M. F.; Mesters, J. R.; Kuhn, D.;
Klebe, G.; Hilgenfeld, R.; Rademann, J. ChemBioChem 2006, 7, 1048–1055.
23. Yang, H.; Yang, M.; Ding, Y.; Liu, Y.; Lou, Z.; Zhou, Z.; Sun, L.; Mo, L.; Ye, S.; Pang,
H.; Gao, G. F.; Anand, K.; Bartlam, M.; Hilgenfeld, R.; Rao, Z. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 13190–13195.
28. Matsudaira, P. J. Biol. Chem. 1987, 262, 10035–10038.
29. Stemmer, W. P. C.; Morris, S. K. BioTechniques 1992, 13, 214–220.
30. Atherton, E.; Wellings, D. In Synthesis of Peptides and Peptidemimetics E22a;
Goodman, M., Ed.; Georg Thieme Verlag: Stuttgart, 2004; pp 740–754.
31. Rink, H. Tetrahedron Lett. 1987, 28, 3787–3790.