Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant
Identifieur interne : 000E47 ( Pmc/Corpus ); précédent : 000E46; suivant : 000E48Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant
Auteurs : Kenichi Akaji ; Hiroyuki Konno ; Mari Onozuka ; Ayumi Makino ; Hiroyuki Saito ; Kazuto NosakaSource :
- Bioorganic & Medicinal Chemistry [ 0968-0896 ] ; 2008.
Abstract
The mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. The tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde inhibitor of the protease.
Url:
DOI: 10.1016/j.bmc.2008.09.057
PubMed: 18845442
PubMed Central: 7126698
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant</title><author><name sortKey="Akaji, Kenichi" sort="Akaji, Kenichi" uniqKey="Akaji K" first="Kenichi" last="Akaji">Kenichi Akaji</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author><author><name sortKey="Konno, Hiroyuki" sort="Konno, Hiroyuki" uniqKey="Konno H" first="Hiroyuki" last="Konno">Hiroyuki Konno</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author><author><name sortKey="Onozuka, Mari" sort="Onozuka, Mari" uniqKey="Onozuka M" first="Mari" last="Onozuka">Mari Onozuka</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author><author><name sortKey="Makino, Ayumi" sort="Makino, Ayumi" uniqKey="Makino A" first="Ayumi" last="Makino">Ayumi Makino</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan</nlm:aff></affiliation></author><author><name sortKey="Saito, Hiroyuki" sort="Saito, Hiroyuki" uniqKey="Saito H" first="Hiroyuki" last="Saito">Hiroyuki Saito</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan</nlm:aff></affiliation></author><author><name sortKey="Nosaka, Kazuto" sort="Nosaka, Kazuto" uniqKey="Nosaka K" first="Kazuto" last="Nosaka">Kazuto Nosaka</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18845442</idno><idno type="pmc">7126698</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7126698</idno><idno type="RBID">PMC:7126698</idno><idno type="doi">10.1016/j.bmc.2008.09.057</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000E47</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000E47</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant</title><author><name sortKey="Akaji, Kenichi" sort="Akaji, Kenichi" uniqKey="Akaji K" first="Kenichi" last="Akaji">Kenichi Akaji</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author><author><name sortKey="Konno, Hiroyuki" sort="Konno, Hiroyuki" uniqKey="Konno H" first="Hiroyuki" last="Konno">Hiroyuki Konno</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author><author><name sortKey="Onozuka, Mari" sort="Onozuka, Mari" uniqKey="Onozuka M" first="Mari" last="Onozuka">Mari Onozuka</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author><author><name sortKey="Makino, Ayumi" sort="Makino, Ayumi" uniqKey="Makino A" first="Ayumi" last="Makino">Ayumi Makino</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan</nlm:aff></affiliation></author><author><name sortKey="Saito, Hiroyuki" sort="Saito, Hiroyuki" uniqKey="Saito H" first="Hiroyuki" last="Saito">Hiroyuki Saito</name><affiliation><nlm:aff id="aff2">Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan</nlm:aff></affiliation></author><author><name sortKey="Nosaka, Kazuto" sort="Nosaka, Kazuto" uniqKey="Nosaka K" first="Kazuto" last="Nosaka">Kazuto Nosaka</name><affiliation><nlm:aff id="aff1">Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</nlm:aff></affiliation></author></analytic><series><title level="j">Bioorganic & Medicinal Chemistry</title><idno type="ISSN">0968-0896</idno><idno type="eISSN">1464-3391</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Graphical abstract</title><p>The mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Bioorg Med Chem</journal-id><journal-id journal-id-type="iso-abbrev">Bioorg. Med. Chem</journal-id><journal-title-group><journal-title>Bioorganic & Medicinal Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0968-0896</issn><issn pub-type="epub">1464-3391</issn><publisher><publisher-name>Elsevier Ltd.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18845442</article-id><article-id pub-id-type="pmc">7126698</article-id><article-id pub-id-type="publisher-id">S0968-0896(08)00909-7</article-id><article-id pub-id-type="doi">10.1016/j.bmc.2008.09.057</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Akaji</surname><given-names>Kenichi</given-names></name><email>akaji@koto.kpu-m.ac.jp</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib><contrib contrib-type="author"><name><surname>Konno</surname><given-names>Hiroyuki</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Onozuka</surname><given-names>Mari</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Makino</surname><given-names>Ayumi</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Saito</surname><given-names>Hiroyuki</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Nosaka</surname><given-names>Kazuto</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Department of Chemistry, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kita-ku, Kyoto 603-8334, Japan</aff><aff id="aff2"><label>b</label>Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Tel./fax: +075 465 7659. <email>akaji@koto.kpu-m.ac.jp</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>26</day><month>9</month><year>2008</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>1</day><month>11</month><year>2008</year></pub-date><pub-date pub-type="epub"><day>26</day><month>9</month><year>2008</year></pub-date><volume>16</volume><issue>21</issue><fpage>9400</fpage><lpage>9408</lpage><history><date date-type="received"><day>19</day><month>8</month><year>2008</year></date><date date-type="rev-recd"><day>22</day><month>9</month><year>2008</year></date><date date-type="accepted"><day>23</day><month>9</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008 Elsevier Ltd. All rights reserved.</copyright-statement><copyright-year>2008</copyright-year><copyright-holder>Elsevier Ltd</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract abstract-type="graphical"><title>Graphical abstract</title><p>The mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. The tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde inhibitor of the protease.</p><fig id="d32e1117" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract><p>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 <italic>K</italic><sub>m</sub> of 33.8 μM and <italic>k</italic><sub>cat</sub> of 4753 s<sup>−1</sup>. Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10<sup>6</sup>. 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.</p></abstract><kwd-group><title>Keywords</title><kwd>SARS</kwd><kwd>3CL protease</kwd><kwd>Protease inhibitor</kwd><kwd>Peptide aldehyde</kwd></kwd-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Severe acute respiratory syndrome (SARS), a life-threatening form of pneumonia, is caused by a new coronavirus (SARS CoV).<xref rid="bib1" ref-type="bibr">1</xref>, <xref rid="bib2" ref-type="bibr">2</xref>, <xref rid="bib3" ref-type="bibr">3</xref> 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 positive-sense, single-stranded RNA virus featuring the largest viral RNA genome known to date.<xref rid="bib4" ref-type="bibr">4</xref>, <xref rid="bib5" ref-type="bibr">5</xref> 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 polypeptides.<xref rid="bib6" ref-type="bibr"><sup>6</sup></xref> The key enzyme in this processing is a 33-kDa protease called 3C-like protease (3CL protease), because its substrate specificity is similar to that of the picornavirus 3C protease.<xref rid="bib7" ref-type="bibr">7</xref>, <xref rid="bib8" ref-type="bibr">8</xref> The SARS 3CL protease is a cysteine protease with a chymotrypsin fold, and cleaves precursor proteins at as many as 11 conserved sites involving a conserved Gln at the P1 position and a small amino acid (Ser, Ala, or Gly) at the P1’ position with varying efficiency.<xref rid="bib9" ref-type="bibr">9</xref>, <xref rid="bib10" ref-type="bibr">10</xref> The 3CL protease exists as a homodimer and each 33-kDa protomer has its own active site containing a Cys-His catalytic dyad (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A). Interactions 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 domain-II of another protomer to fold the dimeric structure. Although some studies reported that the addition of several residues to the C-terminus of mature 3CL protease did not significantly affect its activity, the kinetic properties and specificity of the mature protease have not been well described.<fig id="fig1"><label>Figure 1</label><caption><p>(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-β-barrel fold similar to that of a chymotrypsin-type protease. The C-terminal part (201–303, Domain-III) has five α-helices. (B) Fusion protein composed of MBP, His<sub>6</sub> and a Flag-tag, and 3CL protease.</p></caption><graphic xlink:href="gr1"></graphic></fig></p><p>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.<xref rid="bib11" ref-type="bibr">11</xref>, <xref rid="bib12" ref-type="bibr">12</xref>, <xref rid="bib13" ref-type="bibr">13</xref>, <xref rid="bib14" ref-type="bibr">14</xref>, <xref rid="bib15" ref-type="bibr">15</xref>, <xref rid="bib16" ref-type="bibr">16</xref>, <xref rid="bib17" ref-type="bibr">17</xref> 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.<xref rid="bib8" ref-type="bibr">8</xref>, <xref rid="bib18" ref-type="bibr">18</xref>, <xref rid="bib19" ref-type="bibr">19</xref>, <xref rid="bib20" ref-type="bibr">20</xref>, <xref rid="bib21" ref-type="bibr">21</xref> 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 reversible inhibitors containing an aldehyde group as a functional group have been reported.<xref rid="bib22" ref-type="bibr"><sup>22</sup></xref> 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 <italic>Escherichia coli</italic> 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 protease containing a C-terminal His-tag. The potency makes it possible to evaluate newly synthesized peptide-aldehyde inhibitors quantitatively by conventional HPLC.</p></sec><sec><label>2</label><title>Results and discussion</title><sec><label>2.1</label><title>Expression of the mature 3CL protease</title><p>We first intended to obtain the mature 3CL protease from a fusion protein containing maltose-binding protein (MBP), His<sub>6</sub> and a Flag-tag, and mature 3CL protease (MBP-His tag-3CL protease, <xref rid="fig1" ref-type="fig">Fig. 1</xref>B). The corresponding plasmid was introduced into <italic>E. coli</italic>, and the desired fusion protein of 75-kDa was obtained as a major product of the total cell lysate (<xref rid="fig2" ref-type="fig">Fig. 2</xref>A, lane 1). The fusion protein was easily purified with a cobalt affinity column to give a single major band on SDS–PAGE (<xref rid="fig2" ref-type="fig">Fig. 2</xref>A, 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-NH<sub>2</sub>) containing the P<sub>1</sub>/P<sub>2</sub> cleavage site of 3CL protease, probably because of an N-terminal fused large protein. After digestion of the fusion protein with enterokinase, the N-terminal 42-kDa fragment containing the MBP was detected, but the desired 33-kDa protein corresponding to the mature 3CL protease was hardly detected (<xref rid="fig2" ref-type="fig">Fig. 2</xref>A, 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 (<xref rid="fig2" ref-type="fig">Fig. 2</xref>B; peak 1, [M+H]<sup>+</sup> 593.365 (Calcd. 593.353) for H-Ser-Gly-Phe-Arg-Lys-NH<sub>2</sub>; peak 2, [M+H]<sup>+</sup> 618.288 (Calcd. 618.347) for H-Thr-Ser-Ala-Val-Leu-Gln-OH). This result suggests that the mature 3CL protease was temporarily produced by the digestion of the 75-kDa fusion protein by the enterokinase.<fig id="fig2"><label>Figure 2</label><caption><p>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 μg of protein) of IPTG-induced bacterial cells; lane 2, metal affinity resin-treated fraction (3 μg); lane 3, enterokinase-treated (0.1 U for 2 h) fraction (3 μg). (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 μg of protein) of IPTG-induced bacterial cells; lane 2, metal affinity resin-treated fraction (4 μg). (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 μg); 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 μg).</p></caption><graphic xlink:href="gr2"></graphic></fig></p><p>We then tried to directly express the 3CL protease containing a C-terminal His-tag as described previously.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> The necessary plasmid was constructed from the previous plasmid, and introduced into <italic>E. coli</italic>. The desired 34-kDa product containing the mature 3CL protease was not detected as a major product (<xref rid="fig2" ref-type="fig">Fig. 2</xref>C, 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 (<xref rid="fig2" ref-type="fig">Fig. 2</xref>C, lane 2, <xref rid="fig2" ref-type="fig">Fig. 2</xref>D, lane 1). The immunostaining, however, decreased in intensity gradually and a 14-kDa protein emerged after placement at room temperature (<xref rid="fig2" ref-type="fig">Fig. 2</xref>D, lane 2), or overnight incubation with enterokinase (<xref rid="fig2" ref-type="fig">Fig. 2</xref>D, lane 3). These results strongly suggest that mature 3CL protease is susceptible to degradation by proteolysis.</p><p>To identify the probable cleavage site, enterokinase-based digestion of the 75-kDa fusion protein, MBP-His tag-3CL protease (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, 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 (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lane 2) as probable products of the proteolysis. The faint band of 33-kDa disappeared with prolonged treatment over 60 min (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lanes 3–4). The newly emerged 13-kDa protein seemed to be the same product obtained from the C-terminal His-tagged 3CL protease (<xref rid="fig2" ref-type="fig">Fig. 2</xref>D, 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.<fig id="fig3"><label>Figure 3</label><caption><p>Time course of enterokinase (0.05 U)-based digestion of the metal affinity resin-treated fusion protein containing the mature 3CL protease (10 μg): lane 1, untreated; lane 2, 15 min; lane 3, 30 min; lane 4, 60 min.</p></caption><graphic xlink:href="gr3"></graphic></fig></p><p>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 α-helical domain-III (201–303) of mature 3CL protease (<xref rid="fig1" ref-type="fig">Fig. 1</xref>). In a homodimeric structure of mature 3CL protease, the α-helical domain is expected to be responsible for orienting the amino terminus of one monomer to a position in which it can interact with the active site of the other monomer.<xref rid="bib23" ref-type="bibr"><sup>23</sup></xref> It was also reported that the expression of the N-terminal catalytic region alone yields a protein with reduced catalytic efficiency.<xref rid="bib18" ref-type="bibr"><sup>18</sup></xref> Thus, the expected biological role of the α-helical domain is consistent with our finding that proteolysis at the 188Arg/189Gln site rapidly reduces the catalytic efficiency of mature 3CL protease.</p></sec><sec><label>2.2</label><title>Expression and characterization of R188I mutant 3CL protease</title><p>To obtain an active and stable 3CL protease, we then constructed 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 <italic>E. coli</italic> 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 (<xref rid="fig4" ref-type="fig">Fig. 4</xref>A, 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 (<xref rid="fig4" ref-type="fig">Fig. 4</xref>A, lane 3). The 3CL-R188I protease was further purified by anion exchange chromatography at pH 5.5: the R188I protease with a calculated p<italic>I</italic> of 6.24 flowed through and the MBP-His<sub>6</sub>-Flag of p<italic>I</italic> 5.08 was absorbed on the resin as expected (<xref rid="fig4" ref-type="fig">Fig 4</xref>A, 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 (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B, lane 1). A single step of purification with metal affinity resin was enough to obtain a highly purified product (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B, lane 2). In this case, about 0.13 mg of the purified C-terminal His-tagged protein was obtained from 100 ml of culture. These purified mutant proteins with or without a C-terminal 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.<fig id="fig4"><label>Figure 4</label><caption><p>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 μg of protein) of bacterial cells expressing the N-terminal MBP-His-Flag-tagged protease; lane 2, metal affinity resin-treated fraction (6 μg); lane 3, enterokinase-treated fraction (4 μg); lane 4, DEAE–Sepharose column flow-through fraction (3 μg); lane 5, DEAE–Sepharose column-retained fraction (3 μg). (B) C-terminal His-tagged protease: lane 1, crude extract (20 μg of protein) of IPTG-induced bacterial cells; lane 2, metal affinity resin-treated fraction (2 μg).</p></caption><graphic xlink:href="gr4"></graphic></fig></p><p>The catalytic activities of purified 3CL-R188I proteases were examined using three different substrates (SO1, SO3, and SR1). SO1 containing the P<sub>1</sub>/P<sub>2</sub> cleavage site, the N-terminal self-cleavage site of the protease, is reported to be the most suitable substrate, a canonical substrate, for 3CL protease.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> SO3 is an undecapeptide containing the non-canonical P<sub>3</sub>/P<sub>4</sub> 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 <xref rid="fig2" ref-type="fig">Fig. 2</xref>B. The initial digestion rate was calculated from the decrease in the initial amount of substrate, and each kinetic parameter was calculated by plotting [S]/<italic>v</italic> against [S].</p><p>As summarized in <xref rid="tbl1" ref-type="table">Table 1</xref>, the catalytic ability of 3CL-R188I protease was found to be extreme as compared to that of a previously reported mature 3CL protease containing a C-terminal His tag<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref>, especially the <italic>k</italic><sub>cat</sub> values; 12.2 min<sup>−1</sup> for mature 3CL protease and 4753 s<sup>−1</sup> for the 3CL-R188I. The activity of the mutant relative to the mature protease was nearly 10<sup>6</sup>. Addition of a C-terminal His-tag lowered the relative activity to 0.03 owing to a significant decrease in the <italic>k</italic><sub>cat</sub> 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 additions as expected from the maturation analysis.<xref rid="bib24" ref-type="bibr"><sup>24</sup></xref><table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Catalytic parameters and relative efficiency of the SARS 3CL R188I mutant protease for three different peptides</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Tag of protease</th><th>Substrate</th><th><italic>K</italic><sub>m</sub> (μM)</th><th><italic>k</italic><sub>cat</sub> (s<sup>−1</sup>)</th><th><italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>m</sub> (μM s<sup>−1</sup>)</th><th>Relative rate</th></tr></thead><tbody><tr><td>None</td><td>SO1</td><td align="char">33.8</td><td align="char">4753</td><td align="char">156.9</td><td align="char">1.00</td></tr><tr><td>C-terminal His tag</td><td>SO1</td><td align="char">31.8</td><td align="char">153</td><td align="char">4.8</td><td align="char">0.03</td></tr><tr><td>None</td><td>SO3</td><td align="char">50.5</td><td align="char">455</td><td align="char">9.1</td><td align="char">0.06</td></tr><tr><td>None</td><td>SR1</td><td align="char">46.0</td><td align="char">76</td><td align="char">2.0</td><td align="char">0.01</td></tr></tbody></table></table-wrap></p><p>The relative efficiency for SO3 and SR1 was 0.06 and 0.01, respectively, when compared with the <italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>m</sub> for SO1. The relative efficiency for SO3 vs. SO1 was exactly the same as reported for the mature protease.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> The relative efficiency for SR1 was nearly the same as that reported for cleavage at the P<sub>5</sub>/P<sub>6</sub> site, another non-canonical cleavage site of mature 3CL protease.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> 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 derived from the 3CL protease (<xref rid="fig2" ref-type="fig">Fig. 2</xref>D, lane 4). From these results, we suppose that 3CL protease can digest itself. Application of the mutant 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.</p></sec><sec><label>2.3</label><title>Peptide-aldehyde inhibitors</title><p>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 amide<xref rid="bib25" ref-type="bibr"><sup>25</sup></xref> prepared 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(OBu<sup>t</sup>)-Ala-Val-Leu-Gln(Trt)-N(OMe)Me, was then reduced with LiAlH<sub>4</sub> and the remaining side-chain protecting groups were removed with TFA (<xref rid="sch1" ref-type="fig">Scheme 1</xref>). The deprotected product was purified by HPLC to yield a product showing a single peak. A <sup>1</sup>H 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 reduction with lithium aluminum hydride did not proceed as expected. The LiAlH<sub>4</sub> attacked the methoxy group instead of the carbonyl carbon probably because of the large steric hindrance of the Trt group at the C-terminal Gln side chain, and gave a corresponding methylamide derivative (<xref rid="sch1" ref-type="fig">Scheme 1</xref>).<fig id="sch1"><label>Scheme 1</label><graphic xlink:href="gr6"></graphic></fig></p><p>To avoid this undesired reaction, we then synthesized the peptide-aldehyde using the Gln(Me<sub>2</sub>) 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.<xref rid="bib26" ref-type="bibr"><sup>26</sup></xref> Starting from Fmoc-Glu-OBu<sup>t</sup>, dimethylamine was condensed to the side-chain carboxyl group. Then, the α-<italic>tert</italic>-butyl ester was cleaved and converted to the Weinreb amide. Subsequent peptide chain elongation was conducted as before using a combination of Fmoc-deprotection with diethylamine and coupling with BOP (<xref rid="sch2" ref-type="fig">Scheme 2</xref>). The resulting hexapeptide amide was then reduced with LiAlH<sub>4</sub>. After deprotection with TFA, the crude product was purified by HPLC to give a single peak on analytical HPLC. MALDI-TOF-MS and NMR analyses gave results confirming the aldehyde structure. Peptide aldehydes containing truncated chains were similarly synthesized from the corresponding intermediates and purified.<fig id="sch2"><label>Scheme 2</label><graphic xlink:href="gr7"></graphic></fig></p><p>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 mutant protease in the presence of various concentrations of peptide-aldehyde, and the reaction mixture was analyzed by HPLC as in <xref rid="fig2" ref-type="fig">Fig. 2</xref>B. From the decrease in SO1 at the respective concentration of inhibitor, a sigmoidal dose-response curve was obtained (<xref rid="fig5" ref-type="fig">Fig. 5</xref>), and each IC<sub>50</sub> value was estimated from each sigmoid curve. As summarized in <xref rid="tbl2" ref-type="table">Table 2</xref>, the tetrapeptide sequence was long enough for inhibitory activity. The activity increased in parallel with chain length; the hexapeptide aldehyde had an IC<sub>50</sub> of 26 μM. E-64<xref rid="bib27" ref-type="bibr"><sup>27</sup></xref>, a typical cysteine protease inhibitor, showed nearly the same inhibitory activity (36% inhibition at 5.6 mM) as Ac-Val-Leu-Gln(Me<sub>2</sub>)-CHO, which suggests that the peptide-aldehyde derivatives would be a promising reversible SARS 3CL protease inhibitor. Detailed structure–activity studies are now underway.<fig id="fig5"><label>Figure 5</label><caption><p>A typical sigmoidal dose–response curve used for estimation of the IC<sub>50</sub> value of Ac-Thr-Ser-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO.</p></caption><graphic xlink:href="gr5"></graphic></fig><table-wrap position="float" id="tbl2"><label>Table 2</label><caption><p>Inhibitory activities of peptide-aldehydes for SARS 3CL R188I mutant protease</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Peptide aldehyde</th><th>IC<sub>50</sub></th></tr></thead><tbody><tr><td>Ac-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</td><td>∼ 6 mM</td></tr><tr><td>Ac-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</td><td>155 μM</td></tr><tr><td>Ac-Ser-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</td><td>37 μM</td></tr><tr><td>Ac-Thr-Ser-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</td><td>26 μM</td></tr></tbody></table></table-wrap></p></sec></sec><sec><label>3</label><title>Experimental</title><p>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 fusion protein on enterokinase treatment.</p><p>E-64 was purchased from Peptide Institute, Inc. (Osaka, Minoh, Japan). Fmoc amino acid derivatives were purchased form Watanabe 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 × 150 mm or 10 × 250 mm) reversed phase column. The product was eluted with a gradient of CH<sub>3</sub>CN containing 0.1% TFA in 0.1% aqueous TFA solution. <sup>1</sup>H (300 MHz) and <sup>13</sup>C (75 MHz) NMR spectra were recorded on a Bruker AM-300. Chemical shifts are expressed in parts per million relative to TMS (0 ppm) or CHCl<sub>3</sub> (7.28 ppm for <sup>1</sup>H and 77.0 ppm for <sup>13</sup>C). High resolution 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.</p><sec><label>3.1</label><title>Expression of mature 3CL proteases</title><p>The full-length 3CL protease-coding sequence was amplified from pMAL-3CL with 5′-ccggctagcGGTTTTAGGAAAATGGCATTCC containing a NheI site and 5′-ccgctcgagTTGGAAGGTAACACCAGAG containing a XhoI site. The PCR product was cloned directly into pGEM T-easy (Promega) to produce pGEM-3CL. Another expression 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.</p><p>The pMAL-c2X-based plasmid and pET-21a(+)-based plasmid were transformed into <italic>E. coli</italic> strains DH5α and BL21(DE3)pLys, respectively. Bacterial cells were grown overnight at 37 °C in 10 ml of LB medium containing 50 μg/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-β-<sc>d</sc>-thiogalactopyranoside (IPTG) to produce the recombinant protein. The cells were then pelleted and resuspended in 20 ml of solution L (50 mM Na<sub>2</sub>HPO<sub>4</sub> pH 7.0, and 300 mM NaCl) containing 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride and 10 μl/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 14,000<italic>g</italic> 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 imidazole. 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).</p></sec><sec><label>3.2</label><title>Identification of degradation products from mature 3CL-protease</title><p>The affinity resin-purified fraction (12 μg protein) of the N-terminal 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 Coomassie blue staining, the 20- and 13-kDa bands were dissected and subjected to Edman sequencing for determination of the N-terminal.<xref rid="bib28" ref-type="bibr"><sup>28</sup></xref> This analysis was performed using an Applied Biosystems 491 Protein Sequencer at APRO Life Science Institute (Naruto, Japan).</p></sec><sec><label>3.3</label><title>Expression of 3CL-R188I mutant proteases</title><p>Site-directed mutagenesis was performed by the inverse PCR method described by Stemmer and Morris.<xref rid="bib29" ref-type="bibr"><sup>29</sup></xref> The primers used to generate point mutations were 5′-atctctCGTCTCCA<italic>T</italic>ACAAACTGCACAGGCTG (nucleotide in italics is mutated) and 5′-atctctCGTCTCTGT<italic>A</italic>TGTCAACAAATGGACC, and PCR was performed with the plasmid pGEM-3CL as the template. After PCR, the ends of the full-length linearized plasmids were digested with BsmBI (CGTCTCN’NNNN), leaving 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 unintentional changes.</p><p>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 CaCl<sub>2</sub>. After the enterokinase was removed from the solution 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 Sepharose (GE Healthcare) equilibrated with the same buffer. The target protease flowed though the Sepharose resin and the MBP-His-Flag fragment was retained in the resin. Optionally, the bound protein could be eluted with the same buffer containing 0.5 M KCl.</p><p>The metal affinity resin-treated fraction of the C-terminal His-tagged 3CL protease was used as the substrate to examine degradation. After incubating the fraction (about 4 μg protein) for 14 h at room temperature with 0.05 U of enterokinase or with 0.5 μg of 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 Immun-Blot assay kit (Bio-Rad).</p></sec><sec><label>3.4</label><title>Substrate peptides</title><p>The substrate peptides (SO1; H-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH<sub>2</sub>: SO3; H-Lys-Val-Ala-Thr-Val-Gln-Ser-Lys-Met-Ser-Asp-NH<sub>2</sub>: and SR1; H-Gly-Pro-Phe-Val-Asp-Arg-Gln-Thr-Ala-Gln-Ala-Ala-Gly-Thr-Asp-Thr-NH<sub>2</sub>) were synthesized using Fmoc-based solid-phase peptide synthesis (SPPS)<xref rid="bib30" ref-type="bibr"><sup>30</sup></xref> starting with Rink amide resin (4-(2′,4′-dimethoxyphenyl-Fmoc aminomethyl)phenoxy resin).<xref rid="bib31" ref-type="bibr"><sup>31</sup></xref> SPPS was achieved by a combination of Fmoc deprotection using 20% piperidine/DMF and a coupling reaction using a standard diisopropylcarbodiimide/HOBt protocol. Each peptide was purified by preparative HPLC after cleavage from the resin by treatment with TFA thioanisole (10:1) at 25 °C for 3 h. Homogeneity was further confirmed by MALDI-TOF-MS. SO1; HPLC [Cosmosil 5C18 AR column (4.6 × 150 mm), 1.0 ml/min, CH<sub>3</sub>CN (10–20%)/30 min] rt 19.62 min, <italic>m</italic>/<italic>z</italic> 1192.714 for [M+H]<sup>+</sup> (calcd 1192.681 for C<sub>52</sub>H<sub>90</sub>N<sub>17</sub>O<sub>15</sub>). SO3; HPLC [Cosmosil 5C18 AR column (4.6 × 150 mm), 1.0 ml/min, CH<sub>3</sub>CN (10–20%)/30 min] rt 8.58 min, <italic>m</italic>/<italic>z</italic> 1192.288 for [M+H]<sup>+</sup> (calcd 1192.637 for C<sub>49</sub>H<sub>90</sub>N<sub>15</sub>O<sub>17</sub>S). SR1; HPLC [Cosmosil 5C18 AR column (4.6 × 150 mm), 1.0 ml/min, CH<sub>3</sub>CN (10–50%)/30 min] rt 11.07 min, <italic>m</italic>/<italic>z</italic> 1633.758 for [M+H]<sup>+</sup> (calcd 1633.794 for C<sub>68</sub>H<sub>109</sub>N<sub>22</sub>O<sub>25</sub>).</p></sec><sec><label>3.5</label><title>Enzymatic activity</title><p>The proteolytic activities of 3CL proteases were detected by a cleavage assay of SO1 using analytical HPLC. Each peptide in 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 monitored by analytical HPLC [Cosmosil 5C18 (4.6 × 150 mm), 1.0 ml/min, CH<sub>3</sub>CN (10–20%)/30 min]. Each product of hydrolysis was identified by MALDI-TOF MS.</p><p>Kinetic parameters, <italic>K</italic><sub>m</sub> and <italic>k</italic><sub>cat</sub>, 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 168 μM). The final concentration of the enzyme (10 to 170 nM for the mutant protease) and the digestion time (15 to 90 min) varied 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. <italic>K</italic><sub>m</sub> was calculated from the plot of [S]/<italic>v</italic> vs. [S], where [S] is the concentration of the substrate and <italic>υ</italic> is the initial reaction rate. All reactions were repeated three times and the results were averaged.</p></sec><sec><label>3.6</label><title>Inhibitors</title><sec><label>3.6.1</label><title>Fmoc-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>HN(Me)<sub>2.</sub> 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-OBu<sup>t</sup> (1.28 g, 3 mmol) in CH<sub>2</sub>Cl<sub>2</sub> (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% NaHCO<sub>3</sub>, and brine, and then dried over MgSO<sub>4</sub>. The solvent was evaporated and the residue was purified by silica-gel column chromatography using CHCl<sub>3</sub>: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 stirring 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 dissolved 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 solvent was evaporated. The residue was dissolved in AcOEt, and the organic phase was washed with 5% citric acid, 5% NaHCO<sub>3</sub>, and brine, and then dried over MgSO<sub>4</sub>. The solvent was evaporated and the residue was purified by silica-gel column chromatography using CHCl<sub>3</sub>:MeOH = 10:0.5 to yield the desired product as an oil. Yield 1.13 g (85%), [α]<sub>D</sub><sup>27</sup> +46.7 (c 0.24, CHCl<sub>3</sub>), <sup>1</sup>H NMR (CDCl<sub>3</sub>) <italic>δ</italic>; 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, <italic>J</italic> = 7.1 Hz, 1H), 4.35 (m, 2H), 4.76 (br s, 1H), 5.91 (d, <italic>J</italic> = 8.1 Hz, 1H), 7.31 (d, <italic>J</italic> = 7.5 Hz, 2H), 7.39 (t, <italic>J</italic> = 7.5 Hz, 2H), 7.61 (br t, <italic>J</italic> = 8.3 Hz, 2H), 7.75 (d, <italic>J</italic> = 8.3 Hz, 2H); <sup>13</sup>C NMR <italic>δ</italic>; 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 C<sub>24</sub>H<sub>29</sub>N<sub>3</sub>O<sub>5</sub>Na, Found. 462.126 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.2</label><title>Fmoc-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>Et<sub>2</sub>NH (8.6 ml) was added to Fmoc-Gln-N(OMe)Me (0.73 g, 1.66 mmol) in CH<sub>3</sub>CN (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 CH<sub>2</sub>Cl<sub>2</sub> (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% NaHCO<sub>3</sub>, and brine, and then dried over MgSO<sub>4</sub>. The solvent was evaporated and the residue was purified by silica-gel column chromatography using CHCl<sub>3</sub>:MeOH = 10:0.5 to yield the desired product as an oil. Yield 0.60 g (91%), [α]<sub>D</sub><sup>27</sup> −27.6 (c 1.1, CHCl<sub>3</sub>), <sup>1</sup>H NMR (CDCl<sub>3</sub>) <italic>δ</italic>; 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, <italic>J</italic> = 6.9 Hz, 1H), 4.29–4.42 (m, 2H), 4.92 (br s, 1H), 5.40 (d, <italic>J</italic> = 8.4 Hz, 1H), 7.32 (t, <italic>J</italic> = 7.5 Hz, 2H), 7.39 (t, <italic>J</italic> = 7.5 Hz, 2H), 7.59 (d, <italic>J</italic> = 7.5 Hz, 2H), 7.75 (d, <italic>J</italic> = 7.5 Hz, 2H); <sup>13</sup>C NMR <italic>δ</italic>; 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 C<sub>30</sub>H<sub>40</sub>N<sub>4</sub>O<sub>6</sub>Na, Found. 575.174 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.3</label><title>Fmoc-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>Fmoc-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me (0.60 g, 1.09 mmol) in CH<sub>3</sub>CN (6 ml) was treated with Et<sub>2</sub>NH (5.6 ml) as above. To the N<sup>α</sup>-deprotected product in CH<sub>2</sub>Cl<sub>2</sub>–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 CHCl<sub>3</sub>:MeOH–10:0.5 to yield the desired product as an oil. Yield 0.61 g (86%), [α]<sub>D</sub><sup>27</sup> −11.6 (c 1.0, CHCl<sub>3</sub>), <sup>1</sup>H NMR (CDCl<sub>3</sub>) <italic>δ</italic>; 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, <italic>J</italic> = 7.2 Hz, 1H), 4.21 (t, <italic>J</italic> = 6.9 Hz, 1H), 4.31–4.53 (m, 3H), 4.92 (br s, 1H), 5.58 (br d, <italic>J</italic> = 8.7 Hz, 1H), 6.56 (br d, <italic>J</italic> = 7.5 Hz, 1 H), 7.30 (t, <italic>J</italic> = 7.5 Hz, 2H), 7.39 (t, <italic>J</italic> = 7.5 Hz, 2H), 7.59 (d, <italic>J</italic> = 7.5 Hz, 2H), 7.75 (d, <italic>J</italic> = 7.5 Hz, 2H); <sup>13</sup>C NMR <italic>δ</italic>; 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 C<sub>35</sub>H<sub>49</sub>N<sub>5</sub>O<sub>7</sub>Na, Found. 674.211 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.4</label><title>Fmoc-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>Fmoc-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me (0.60 g, 0.92 mmol) in CH<sub>2</sub>Cl<sub>2</sub>–CH<sub>3</sub>CN (3 ml–3 ml) was treated with Et<sub>2</sub>NH (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 chromatography using CHCl<sub>3</sub>:MeOH = 10:0.5 to yield the desired product as an oil. Yield 0.51 g (77%), [α]<sub>D</sub><sup>27</sup> −30.3 (c 0.90, CHCl<sub>3</sub>), <sup>1</sup>H NMR (CDCl<sub>3</sub>) <italic>δ</italic>; 0.87 (d, <italic>J</italic> = 6.6 Hz, 6H), 0.93 (d, <italic>J</italic> = 6.6 Hz, 6H), 1.36 (d, <italic>J</italic> = 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, <italic>J</italic> = 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, <italic>J</italic> = 7.2 Hz, 2H), 7.37 (t, <italic>J</italic> = 7.4 Hz, 2H), 7.60 (d, <italic>J</italic> = 7.5 Hz, 2H), 7.74 (d, <italic>J</italic> = 7.5 Hz, 2H), 7.95 (br s, 1H); <sup>13</sup>C NMR <italic>δ</italic>; 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 C<sub>38</sub>H<sub>54</sub>N<sub>6</sub>O<sub>8</sub>Na, Found. 745.199 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.5</label><title>Fmoc-Ser(Bu<sup>t</sup>)-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>Fmoc-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me (0.56 g, 0.77 mmol) in CH<sub>2</sub>Cl<sub>2</sub>–CH<sub>3</sub>CN (3 ml–3 ml) was treated with Et<sub>2</sub>NH (4.0 ml) as above. To the product in DMF (5 ml), were added Fmoc-Ser(Bu<sup>t</sup>)-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-gel column chromatography using CHCl<sub>3</sub>:MeOH = 10:0.5 to yield the desired product as an oil. Yield 0.58 g (86%), [α]<sub>D</sub><sup>27</sup> −22.8 (c 0.15, CHCl<sub>3</sub>), <sup>1</sup>H NMR (CDCl<sub>3</sub>) <italic>δ</italic>; 0.84–0.92 (m, 12H), 1.15 (s, 9H), 1.34 (d, <italic>J</italic> = 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 (s, 3H), 3.47 (t, <italic>J</italic> = 7.8 Hz, 1H), 3.71 (t, <italic>J</italic> = 7.8 Hz, 1H), 3.73 (s, 3H), 4.21 (t, <italic>J</italic> = 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, <italic>J</italic> = 7.4 Hz, 2H), 7.37 (br t, <italic>J</italic> = 7.4 Hz, 2H), 7.62 (br d, <italic>J</italic> = 6.9 Hz, 2H), 7.74 (br d, <italic>J</italic> = 7.2 Hz, 2H), 7.83 (br s, 1H), 8.03 (br s, 1H); <sup>13</sup>C NMR δ; 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 C<sub>45</sub>H<sub>66</sub>N<sub>7</sub>O<sub>10</sub>Na, Found. 888.253 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.6</label><title>Fmoc-Thr(Bu<sup>t</sup>)-Ser(Bu<sup>t</sup>)-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>Fmoc-Ser(Bu<sup>t</sup>)-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me (0.57 g, 0.66 mmol) in CH<sub>2</sub>Cl<sub>2</sub>–CH<sub>3</sub>CN (3 ml–3 ml) was treated with Et<sub>2</sub>NH (3.4 ml) as above. To the product in DMF (5 ml), were added Fmoc-Thr(Bu<sup>t</sup>)-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 H<sub>2</sub>O was added. The resulting precipitate was washed with 5% citric acid, 5% NaHCO<sub>3</sub>, and H<sub>2</sub>O. The crude product was reprecipitated from DMF/Et<sub>2</sub>O to yield the desired product as an amorphous solid. Yield 0.40 g (59%), [α]<sub>D</sub><sup>27</sup> −13.2 (c 0.68, CHCl<sub>3</sub>), <sup>1</sup>H NMR (CDCl<sub>3</sub>) <italic>δ</italic>; 0.93-0.95 (br s, 12H), 1.17 (s, 12H), 1.31 (s, 9H), 1.41 (br d, <italic>J</italic> = 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, <italic>J</italic> = 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, <italic>J</italic> = 5.7 Hz, 2H), 7.77 (br d, <italic>J</italic> = 6.3 Hz, 2H); <sup>13</sup>C NMR <italic>δ</italic>; 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. 1045.252 for C<sub>53</sub>H<sub>81</sub>N<sub>8</sub>O<sub>12</sub>Na, Found. 1045.426 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.7</label><title>Ac-Thr(Bu<sup>t</sup>)-Ser(Bu<sup>t</sup>)-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me</title><p>Fmoc-Thr(Bu<sup>t</sup>)-Ser(Bu<sup>t</sup>)-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me (78 mg, 75 μmol) in CH<sub>2</sub>Cl<sub>2</sub>–CH<sub>3</sub>CN (1.5 ml–1.5 ml) was treated with Et<sub>2</sub>NH (0.59 ml) as above. To the product in DMF (3 ml), were added Ac<sub>2</sub>O (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 H<sub>2</sub>O was added. The crude product was reprecipitated from DMF/Et<sub>2</sub>O to yield the desired product as an amorphous solid. Yield 40 mg (63%), [α]<sub>D</sub><sup>27</sup> −17.5 (c 0.20, DMF), <sup>1</sup>H NMR (DMSO-<italic>d</italic><sub>6</sub>) <italic>δ</italic>; 0.80–0.88 (m, 12H), 1.04 (d, <italic>J</italic> = 6.3 Hz, 3H), 1.10 (s, 9H), 1.11 (d, <italic>J</italic> = 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, <italic>J</italic> = 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); <sup>13</sup>C NMR <italic>δ</italic>; 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 C<sub>40</sub>H<sub>74</sub>N<sub>8</sub>O<sub>11</sub>Na, Found. 865.476 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.8</label><title>Ac-Thr-Ser-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</title><p>LiAlH<sub>4.</sub> (30 mg) was added to Ac-Thr(Bu<sup>t</sup>)-Ser(Bu<sup>t</sup>)-Ala-Val-Leu-Gln(Me)<sub>2</sub>-N(OMe)Me (30 mg, 36 μmol) in DMF–THF (1.5 ml–1.5 ml), and the mixture was stirred at 25 °C for 90 min. The mixture was filtered and the solvent was evaporated. The crude product was purified by HPLC [Cosmosil 5C18 (10 × 250 mm), 3.0 ml/min, CH<sub>3</sub>CN (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%) <sup>1</sup>H NMR (CD<sub>3</sub>CN containing D<sub>2</sub>O) <italic>δ</italic>; 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-TOF-MS; Calcd. 806.501 for C<sub>38</sub>H<sub>69</sub>N<sub>7</sub>O<sub>10</sub>Na, Found. 806.121 for [M+Na]<sup>+</sup>.</p><p>Anisole (15 μl, 6.0 mmol) and TFA (0.5 ml) were added to the above powder (7.1 mg, 9 μmol), and the mixture was stirred at 25 °C for 2 h. The TFA was evaporated and ether (5 ml) and 0.1% aq.TFA (5 ml) were added to the residue. The aqueous phase was washed with ether (5 ml) twice and the solvent was removed by lyophilization. The crude product was purified by HPLC [Cosmosil 5C18 (10 × 250 mm), 3.0 ml/min, CH<sub>3</sub>CN (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 × 150 mm), 1 ml/min, CH<sub>3</sub>CN (10–40%)/30 min], <sup>1</sup>H NMR (CD<sub>3</sub>CN containing D<sub>2</sub>O) <italic>δ</italic>; 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 C<sub>30</sub>H<sub>53</sub>N<sub>7</sub>O<sub>10</sub>Na, Found. 694.337 for [M+Na]<sup>+</sup>.</p><p>The following truncated peptide-aldehyde derivatives were similarly prepared from the above intermediates.</p></sec><sec><label>3.6.9</label><title>Ac-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</title><p>HPLC rt, 18.64 min [Cosmosil 5C18 (4.6 × 150 mm), 1 ml/min, CH<sub>3</sub>CN (10–60%)/30 min], <sup>1</sup>H NMR (CD<sub>3</sub>CN) <italic>δ</italic>; 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, <italic>J</italic> = 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, <italic>J</italic> = 7.8 Hz, 2H), 6.96 (br d, <italic>J</italic> = 8.7 Hz, 1H), 9.42 (br s); MALDI-TOF-MS; Calcd. 435.259 for C<sub>20</sub>H<sub>46</sub>N<sub>4</sub>O<sub>5</sub>Na, Found. 435.955 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.10</label><title>Ac-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</title><p>HPLC rt, 12.64 min [Cosmosil 5C18 (4.6 × 150 mm), 1 ml/min, CH<sub>3</sub>CN (10–60%)/30 min], <sup>1</sup>H NMR (D<sub>2</sub>O) <italic>δ</italic>; 0.86–0.94 (m, 12H), 1.33 (d, <italic>J</italic> = 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, <italic>J</italic> = 8.1 Hz, 1H), 4.28 (q, <italic>J</italic> = 7.2 Hz, 1H), 4.38–4.42 (m, 2H), 9.41 (br s); MALDI-TOF-MS; Calcd. 506.296 for C<sub>23</sub>H<sub>41</sub>N<sub>5</sub>O<sub>6</sub>Na, Found. 506.216 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.11</label><title>Ac-Ser-Ala-Val-Leu-NHCH(CH<sub>2</sub>CH<sub>2</sub>CON(CH<sub>3</sub>)<sub>2</sub>)-CHO</title><p>HPLC rt, 14.38 min [Cosmosil 5C18 (4.6 × 150 mm), 1 ml/min, CH<sub>3</sub>CN (10–40%)/30 min], <sup>1</sup>H NMR (CD<sub>3</sub>CN containing D<sub>2</sub>O) <italic>δ</italic>; 0.85–0.92 (m, 12H), 1.36 (d, <italic>J</italic> = 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 C<sub>26</sub>H<sub>46</sub>N<sub>6</sub>O<sub>8</sub>Na, Found. 593.258 for [M+Na]<sup>+</sup>.</p></sec><sec><label>3.6.12</label><title>Ac-Ser-Ala-Val-Leu-Gln-NHCH<sub>3</sub></title><p>Ac-Ser(Bu<sup>t</sup>)-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 LiAlH<sub>4</sub> as stated above. The products were absorbed on Diaion HP20 and eluted with CH<sub>3</sub>CN:H<sub>2</sub>O = 3:1. The solvent was removed by lyophilization, and the product was partially purified by HPLC [Cosmosil 5C18 (10 × 250 mm), 3 ml/min, CH<sub>3</sub>CN (40–80%)/60 min]. An aliquot of the product was treated with TFA as stated above and purified by HPLC [Cosmosil 5C18 (10 × 250 mm), 3 ml/min, CH<sub>3</sub>CN (10–40%)/60 min]. HPLC rt, 10.95 min [Cosmosil 5C18 (4.6 × 150 mm), 1 ml/min, CH<sub>3</sub>CN (10–70%)/30 min], <sup>1</sup>H NMR (DMSO-<italic>d</italic><sub>6</sub>) <italic>δ</italic>; 0.81–0.91 (m, 12H), 1.21 (d, <italic>J</italic> = 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, <italic>J</italic> = 4.5 Hz, 3H), 3.52–3.55 (m, 2H), 4.07–4.33 (m, 4H), 4.98 (br t, <italic>J</italic> = 5.1 Hz, 1H), 7.69–7.73 (m, 2H), 7.94–8.14 (m, 4H); MALDI-TOF-MS; Calcd. 594.323 for C<sub>25</sub>H<sub>45</sub>N<sub>7</sub>O<sub>8</sub>Na, Found. 594.268 for [M+Na]<sup>+</sup>.</p></sec></sec><sec><label>3.7</label><title>Inhibitory activity</title><p>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 monitored by analytical HPLC, as described above. Inhibitory activity was evaluated using the corresponding IC<sub>50</sub> value obtained from the sigmoid dose-response curve. 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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.</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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