KSRP/FUBP2 is a binding protein of GO-Y086, a cytotoxic curcumin analogue

Article abstract of DOI:10.1021/ml1000454

Bis(arylmethylidene)acetone derivatives are an important class of curcumin analogues that exhibit various biological and pharmacological activities. We herein report that GO-Y086, a biotinylated bis(arylmethylidene)acetone, inhibits cancer cell growth. We also show that GO-Y086 specifically interacts with the nuclear protein KSRP/FUBP2 by covalent modification. GO-Y086 markedly suppresses the expression of the c-Myc protein, which plays an important role in cellular proliferation and whose expression is regulated by KSRP/FUBP2.

Full text of DOI:10.1021/ml1000454

KSRP/FUBP2 is the binding protein of GO-Y086, a cytotoxic curcumin
analogue
Hiroyuki Yamakoshi,† Naoki Kanoh,† Chieko Kudo,‡ Atsuko Sato,‡ Kazunori Ueda,†
Makoto Muroi,§ Shunsuke Kon,‡ Masanobu Satake,‡ Hisatsugu Ohori,‡
Chikashi Ishioka,‡ Yoshiteru Ohshima,† Hiroyuki Osada,§ Natsuko Chiba,‡
Hiroyuki ShibataII and Yoshiharu Iwabuchi*†
Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan, Institute of
Development, Aging and Cancer, Tohoku University, Sendai 980-8578, Japan, Chemical Biology Department,
Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan, and Department of Clinical Oncology,
Akita University Hospital, Akita 010-8543, Japan
Supporting Information
Table of Contents:
Experimental Procedures for Biological Analysis
PMF Analysis Results for the GO-Y086 Binding Protein
Alignment of FUBP2/KSRP Protein Sequences
Effects of KSRP Knockdown
S1
S4
S7
S8
S9
S9
S13
General Experimental Procedures for Chemical Synthesis
Experimental Procedures and Compounds Data
1H and 13C Spectra

Experimental Procedures for Biological Analysis
Cell culture
Cells of the colorectal carcinoma line HCT116 were cultured in RPMI1640 supplemented with 10% fetal
bovine serum (FBS). The HEK-293T cell line was cultured in DMEM supplemented with 10% FBS.
Cell growth suppression analysis
HCT116 was obtained from the Cell Resource Center for Biomedical Research (Institute of
Development, Aging and Cancer, Tohoku University, Sendai, Japan). The growth-suppressive effects of
the compounds were measured for 48 h. Cell viability was assayed by quantitation of the uptake and
digestion
of
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
monosodium salt (MTT) according to the manufacturer’s instructions (Dojindo Laboratories, Kumamoto,
Japan) by using a 96-well plate reader, MPR-4Ai (Tosoh Corp., Tokyo, Japan). The percentage cell
growth of the control, which was treated with 1% DMSO alone, was calculated and plotted, and then the
mean growth inhibitory concentration (GI50) value was determined.
Plasmid construction
The GFP-KSRP-FL (full-length), NT (amino-terminus), KH (KH domains), and CT (carboxy-terminus) have
been described by Hall M. P. et al. (Mol. Biol. Cell, 2004, 15, 774). KSRP point mutations were generated
by site-directed mutagenesis using primers containing
a
corresponding mutation (C436A:
5’-CATCCCCACTCACAAGGCTGGGCTGGTCATC-3’; K473R: 5’-GGGACCCCAACTTCAGGTTGTT
CACATC-3’; C500A: 5’-GATCGAGGGTCCTCTCGCCCCAGTTGGACCAGGC-3’; K564R: 5’-GCCG
CTGCAGCTGCTCTGCTTGGGTCATG-3’; K620R: 5’-TACTCTTCCCAGGCCCTAGTGTAGTCCGA
C-3’; K627R: 5’-TGCTGGCCGATCTTTCTGTAATACTCTTC-3’). GFP-KSRP-FL was digested with Pst I
or Not I and Kpn I. The PCR products were cloned into the digested GFP-KSRP-FL (C436A, K473R,
C500A and K564R: Pst I; K620R and K627R: Not I and Kpn I), respectively. For RNAi-Ready
pSIREN-RetroQ-KSRP, complementary oligonucleotides 5’-GATCCGGATCAACCGGAGAGCAAGATT
CAAGAGATCTTGCTCTCCGGTTGATCCTTTTTTT-3’ and 5’-AATTCTCTAGAAAAAAAGGATCAA
CCGGAGAGCAAGATCTCTTGAATCTTGCTCTCCGGTT-3’
were
annealed.
The
annealed
oligonucleotide was ligated into the BamHI and EcoRI sites of RNAi-Ready pSIREN-RetroQ in BD
Knockout RNAi Systems (Clontech, Palo Alto, CA). For the RNAi-Ready pSIREN-RetroQ-control,
Luciferase shRNA Annealed Oligonucleotide (Clontech) was ligated. All constructs were verified by DNA
sequencing.
Reagents and antibodies
The antibodies used for Western blotting were anti-actin monoclonal antibody (A2066; Sigma-Aldrich, St.
Louis, MO), anti-c-Myc monoclonal antibody (9E10; Santa Cruz Biotechnology, Santa Cruz, CA),
anti-biotin monoclonal antibody (BN-34; Sigma-Aldrich), anti-GFP monoclonal antibody (B34; Covance,
Princeton, NJ), anti-FBP2 (KSRP) polyclonal antibody (D-12; Santa Cruz Biotechnology), ECL-anti-mouse
IgG (NA931V; GE Healthcare, Buckinghamshire, UK), and anti-goat IgG-HRP (sc-2020; Santa Cruz
Biotechnology). The resin used for precipitation was SoftLinkTM Soft Release Avidin Resin (Promega,
Madison, WI).
1

Transfection
Transfection with Effectene Transfection Reagent (Qiagen, Valencia, CA) was performed according to the
manufacturer’s instructions.
Precipitation
HCT116 cells were treated with 2 µM GO-Y086 or GO-Y088 for 9 h. After washing three times with PBS,
cells were lysed with low salt lysis buffer (10 mM Hepes, 100 mM NaCl, 0.2% NP40, 10 mM EDTA, pH
7.6) and protease inhibitor mixture (Sigma, St. Louis, MO). The lysed cells were centrifuged at 12,000 rpm
for 10 min and the supernatant was collected as a cell lysate. The cell lysate was incubated with Avidin
Resin at 4 °C for 30 min. The reacted resins were washed with low salt lysis buffer, and the bounded proteins
were eluted with SDS-polyacrylamide gel electrophoresis sample buffer (125 mM tris-HCl, 10%
°
2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue, pH 6.8) at 95 C for 5 min. Samples
were subjected to electrophoresis in 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, probed with
primary and secondary antibodies, and detected by enhanced chemiluminescence.
Cell fractionation
Cell fractionation with ProteoextractTM was performed according to the manufacturer’s instructions.
PMF analysis
One half of the large scale sample (HCT116, 90Φdish x12) was subjected to electrophoresis in 7.5%
SDS-polyacrylamide gel, and stained with Coomassie Brilliant Blue (CBB). The protein-containing region
°
detected in this manner was excised and was washed successively with MilliQ (37 C, 5 x 400 µL),
acetonitrile (2 x 200 µL), and decolorant (50% acetonitrile, 100 mM NH4HCO3) (2 x 100 µL); the sample
was then dried in vacuo. The dried gel was reduced by incubation with 50 µL of reducing agent (100 mM
NH4HCO3, 100 mM dithiothreitol) at 56 °C for 30 min. After removing the excess reducing agent, 100 µL of
an alkylating aqueous solution (100 mM NH4HCO3, 100 mM iodoacetoamide) was added, and the mixture
°
was incubated at 37 C for 30 min. The gel was washed with 100 mM aqueous NH4HCO3 (200 µL) for 10
min, and dried in vacuo. The dried gel was digested by incubation with 10 µL of digest solution (10 mM
Tris-HCl, 25 µg/mL trypsin, pH 8.8) at 4 °C for 45 min. After excess solution was removed, 20 µL of 10 mM
Tris-HCl buffer (pH 8.8) was added. The mixture was incubated at 37 °C overnight. The extract and gel were
then separated. 20 µL of extract solution (5% formic acid, 50% aqueous acetonitrile) was added to the gel,
and the mixture was incubated for 10 min; the extract was then separated from the gel. After this extraction
cycle was repeated, the gel was further extracted with acetonitrile (2 x 20 µL). The combined extracts were
concentrated until the total volume became ca. 5 µL. To the concentrate was added 20 µL of 0.1% aqueous
trifluoroacetic acid. Extracted peptides in the concentrate ware absorbed on a ZipTip® pipette tip (Millipore
Corporation, Bedford, MA), and the tip was washed three times with 0.1% aqueous trifluoroacetic acid. The
washed peptides were eluted with 5 µL of 75% aqueous acetonitrile containing 0.1% trifluoroacetic acid.
The extracted peptides were loaded onto the MALDI target plate by mixing 0.5 µL of solution with the same
volume of a matrix solution, which was prepared fresh by dissolving 10 mg/mL α-cyano-4-hydroxycinnamic
acid in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid. The samples were then allowed to dry.
MALDI-TOF mass and tandem mass measurements were carried out with Ultraflex TOF/TOF mass
2

spectrometer (Bruker Daltonics GmbsH, Bremen, Germany) in reflection mode. The Mascot Search®
program (Matrix Science Inc., Boston, MA) was used to search the SWISS-PLOT database for the peptide
masses.
Quantitative RT-PCR
The total cellular RNA was isolated using an RNeasy Mini Kit (Qiagen). cDNA was synthesized using a
High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real time-PCR was
performed on a CFX96 system (Bio-Rad, Hercules, CA) using primers of c-myc (Assay IDs:
Hs00153408_m1 and Hs99999903_m1) of the TaqMan (r) Gene Expression Assays (Applied Biosystems).
To analyze the expression level of c-myc mRNA, normalization across samples was performed using the
expression of β-actin.
3

PMF analysis results for the GO-Y086-binding protein
Figure SI-1. MALDI-TOF mass spectrum of digested peptides of the GO-Y086-binding protein
4

Table SI-1. PMF analysis results
Match to : FUBP2_HUMAN
Mass : 73063
Score : 177
Sequence Coverage : 36%
Matched peptides are shown in Bold Red
1 MSDYSTGGPP PGPPPPAGGG GGAGGAGGGP PPGPPGAGDR GGGGPCGGGP
51 GGGSAGGPSQ PPGGGGPGIR KDAFADAVQR ARQIAAKIGG DAATTGNNST
101 PDFGFGGQKR QLEDGDQPES KKLASQGDSI SSQLGPIHPP PRTSMTEEYR
151 VPDGMVGLII GRGGEQINKI QQDSGCKVQI SPDSGGLPER SVSLTGAPES
201 VQKAKMMLDD IVSRGRGGPP GQFHDNANGG QNGTVQEIMI PAGKAGLVIG
251 KGGETIKQLQ ERAGVKMILI QDGSQNTNVD KPLRIIGDPY KVQQACEMVM
301 DILRNVTKAG FGDRNEYGSR IGGGIDVPVP RHSVGVVIGR SGEMIKKIQN
351 DAGVRIQFKQ DDGTGPEKIA HIMGPPDRCE HAARIINDLL QSLRSGPPGP
401 PGGPGIPGGR GRGRGQGNWG PGGEMTFSIP THKCGLVIGR GGENVKAINQ
451 QTGAFVEISR QLPPTGTPTS KLFIIRGSPQ QIDHCRQLIE EKIEGPLCPV
501 GPGPGGPGPA GPMGPFNPGP FNQGPPGAPP HAGGPPPHQY PPQGWGNTYP
551 QWQPPAPHDP SKAAAAAADP NAAWAAYYSH YYQQPPGPVP GPAPAPAAPP
601 AQGEPPQPPP TGQSDYTKAW EEYYKKIGQQ PQQPGAPPQQ DYTKAWEEYY
651 KKQAQVATGG GPGAPPGSQP DYSAAWAEYY RQQAAYYGQT PVPGPQPPPT
701 QQGQQQQ
Matched peptide list (27 peptides in total)
(Start-End) Obsd.
Mr (expt.) Mr (calcd) ppm
Miss*
Sequence
(71-80)
(72–80)
1120.5583 1119.5510 1119.5673 -15
992.4568 991.4495 991.4723 -23
1
0
1
0
0
0
0
0
0
1
0
0
0
0
R.KDAFADAVQR.A
K.DAFADAVQR.A
(122–142) 2185.1253 2184.1181 2184.1651 -22
(123–142) 2057.0389 2056.0316 2056.0701 -19
(143–150) 1016.4279 1015.4206 1015.4280 -7
(151–162) 1226.6572 1225.6500 1225.6853 -29
(178–190) 1354.6821 1353.6748 1353.6888 -10
(191–203) 1302.6283 1301.6210 1301.6827 -47
(206–214) 1079.5664 1078.5591 1078.5151 41
(206–216) 1308.6401 1307.6328 1307.6326 0
(217–244) 2791.2276 2790.2203 2790.3144 -34
(217–244) 2807.2376 2806.2303 2806.3093 -28
(267–284) 2042.0251 2041.0178 2041.0626 -22
(285–291) 805.3559 804.3486 804.4381 -111
K.KLASQGDSISSQLGPIHPPPR.T
K.LASQGDSISSQLGPIHPPPR.T
R.TSMTEEYR.V
R.VPDGMVGLIIGR.G
K.VQISPDSGGLPER.S
R.SVSLTGAPESVQK.A
K.MMLDDIVSR.G
K.MMLDDIVSRGR.G (Ox.)**
R.GGPPGQFHDNANGGQNGTVQEIMIPAGK.A
R.GGPPGQFHDNANGGQNGTVQEIMIPAGK.A (Ox.)**
K.MILIQDGSQNTNVDKPLR.I
R.IIGDPYK.V
5

(292–304) 1592.7383 1591.7311 1591.7520 -13
(292–304) 1608.6999 1607.6927 1607.7470 -34
(332–340) 923.5075 922.5002 922.5349 -38
(347–355) 1000.5039 999.4967 999.5461 -49
(348–355) 872.4142 871.4069 871.4512 -51
(369–378) 1106.5573 1105.5500 1105.5702 -18
(369–378) 1122.5148 1121.5075 1121.5652 -51
(385–394) 1184.6847 1183.6774 1183.6924 -13
(447–460) 1533.7790 1532.7717 1532.7947 -15
(627–644) 1980.8871 1979.8798 1979.9701 -46
(645–651) 988.4015 987.3942 987.4338 -40
(645–652) 1116.5340 1115.5267 1115.5287 -2
(653–681) 2952.3230 2951.3157 2951.3474 -11
0
0
0
1
0
0
0
0
0
0
0
1
0
K.VQQACEMVMDILR.N
K.VQQACEMVMDILR.N (Ox.)**
R.HSVGVVIGR.S
K.KIQNDAGVR.I
K.IQNDAGVR.I
K.IAHIMGPPDR.C
K.IAHIMGPPDR.C (Ox.)**
R.IINDLLQSLR.S
K.AINQQTGAFVEISR.Q
K.IGQQPQQPGAPPQQDYTK.A
K.AWEEYYK.K
K.AWEEYYKK.Q
K.QAQVATGGGPGAPPGSQPDYSAAWAEYYR.Q
*Miss : number of missed cleavage; **(Ox.) : oxidation at M residue
6

Figure SI-2. Alignment of FUBP2/KSRP protein sequences found in the SWISS-PLOT database used in the PMF
analysis (PMF) and that used this study (Vector). Highlights in gray are the sites of amino acid mutation. The
highlight in yellow indicates the C-terminal region. The underlined sequence corresponds to peptide fragments
observed in the PMF analysis. The lysine residues designated as K were found intact in the peptide fragments. The
lysine residue designated as K must be intact in order for the peptide fragments to be observed in the PMF
analysis. Thus, the possible GO-Y086-binding sites are indicated by the blue highlights.
PMF
MSDYSTGGPP PGPPPPAGGG GGAGGAGGGP PPGPPGAGDR GGGGPCGGGP GGGSAGGPSQ 60
Vector MSDYSTGGPP PGPPPPAGGG GGAGGAGGGP PPGPPGAGDR GGGGPCGGGP GGGSAGGPSQ 60
PMF
PPGGGGPGIR KDAFADAVQR ARQIAAKIGG DAATTGNNST PDFGFGGQKR QLEDGDQPES 120
Vector PPGGGGPGIR KDAFADAVQR ARQIAAKIGG DAATTVNNST PDFGFGGQKR QLEDGDQPES 120
PMF
KKLASQGDSI SSQLGPIHPP PRTSMTEEYR VPDGMVGLII GRGGEQINKI QQDSGCKVQI 180
Vector KKLASQGDSI SSQLGPIHPP PRTSMTEEYR VPDGMVGLII GRGGEQINKI QQDSGCKVQI 180
PMF
SPDSGGLPER SVSLTGAPES VQKAKMMLDD IVSRGRGGPP GQFHDNANGG QNGTVQEIMI 240
Vector SPDSGGLPER SVSLTGAPES VQKAKMMLDD IVSRGRGGPP GQFHDNANGG QNGTVQEIMI 240
PMF
PAGKAGLVIG KGGETIKQLQ ERAGVKMILI QDGSQNTNVD KPLRIIGDPY KVQQACEMVM 300
Vector PAGKAGLVIG KGGETIKQLQ ERAGVKMILI QDGSQNTNVD KPLRIIGDPY KVQQACEMVM 300
PMF
DILRNVTKAG FGDRNEYGSR IGGGIDVPVP RHSVGVVIGR SGEMIKKIQN DAGVRIQFKQ 360
Vector DILRERDQGG FGDRNEYGSR IGGGIDVPVP RHSVGVVIGR SGEMIKKIQN DAGVRIQFKQ 360
PMF
DDGTGPEKIA HIMGPPDRCE HAARIINDLL QSLRSGPPGP PGGPGIP-GG RGRGRGQGNW 419
Vector DDGTGPEKIA HIMGPPDRCE HAARIINDLL QSLRSGPPGP PGGPGMPPGG RGRGRGQGNW 420
PMF
GP-GGEMTFS IPTHKCGLVI GRGGENVKAI NQQTGAFVEI SRQLPPTGTP TSKLFIIRGS 478
Vector GPPGGEMTFS IPTHKCGLVI GRGGENVKAI NQQTGAFVEI SRQLPPNGDP NFKLFIIRGS 480
PMF
PQQIDHCRQL IEEKIEGPLC PVGPGPGGPG PAGPMGPFNP GPFNQGPPGA PPHAGGPPPH 538
Vector PQQIDHAKQL IEEKIEGPLC PVGPGPGGPG PAGPMGPFNP GPFNQGPPGA PPHAGGPPPH 540
↑497
PMF
QYPPQGWGNT YPQWQPPAPH DPSKAAAAAA DPNAAWAAYY SHYYQQPPGP VPGPAPAPAA 598
Vector QYPPQGWGNT YPQWQPPAPH DPSKAAAAAA DPNAAWAAYY SHYYQQPPGP VPGPAPAPAA 600
PMF
PPAQGEPPQP PPTGQSDYTK AWEEYYKKIG QQPQQPGAPP QQDYTKAWEE YYKKQAQVAT 658
Vector PPAQGEPPQP PPTGQSDYTK AWEEYYKKIG QQPQQPGAPP QQDYTKAWEE YYKKQAQVAT 660
PMF
GGGPGAPPGS QPDYSAAWAE YYRQQAAYYG QTPVPG-PQP PPTQQGQQQQ - 707
Vector GGGPGAPPGS QPDYSAAWAE YYRQQAAYYG QTPGPGGPQP PPTQQGQQQA Q 711
7

Effects of KSRP knockdown
Figure SI-3. siRNA knockdown of KSRP reduced the expression of c-Myc. HEK293T cells were
transfected with RNAi-Ready pSIREN-RetroQ-control (lane 1) or RNAi-Ready pSIREN-RetroQ-KSRP
(lane 2) to produce control siRNA or KSRP siRNA in cells, respectively. Cell lysates were prepared 48 h
after transfection, and then analyzed by Western blotting using anti-KSRP, anti-c-Myc, and β-actin
antibodies.
Figure SI-4. Relative cell growth of the HEK-293T cells that were transfected with RNAi-Ready
pSIREN-RetroQ-control or RNAi-Ready pSIREN-RetroQ-KSRP. Cell growth was evaluated by MTT assay.
8

General Experimental Procedures for Chemical Synthesis
All reactions were carried out under an atmosphere of argon unless otherwise specified. Anhydrous solvents
were transferred via syringe to flame-dried glassware, which had been cooled under a stream of dry nitrogen.
Ethereal solvents and dichloromethane (anhydrous; Kanto Chemical Co. Inc., Osaka, Japan) were used as
received. All other solvents were dried and distilled by standard procedures. Yields refer to
chromatographically and spectroscopically (1H NMR) homogeneous materials unless otherwise stated.
Reagents were purchased at the highest commercial quality and used without further purification unless
otherwise stated.
The reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm Merck silica gel
plates (60F-254) using UV light as the visualizing agent and p-anisaldehyde in ethanol/aqueous
H2SO4/CH3CO2H for staining. Column chromatography was performed using silica gel 60 particle size
0.063-0.210 mm. The eluents employed are reported as volume : volume percentages.
Proton nuclear magnetic resonance (1H NMR) spectra were recorded using a JEOL JMN-AL400 (400 MHz),
and a JEOL JNM-ECA500 (500 MHz) spectrometer. Chemical shift (δ) is reported in parts per million (ppm)
downfield relative to tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Multiplicities are
reported using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad;
app, Apparent. Carbon-13 nuclear magnetic resonance (13C NMR) spectra were recorded using a JEOL
JMN-AL400 (100 MHz) and a JEOL JNM-ECA500 (125 MHz) spectrometer. Chemical shifts are reported
in ppm relative to the center of CDCl3.
Melting points were determined using a Yazawa BY-2 melting point apparatus and are reported uncorrected.
Infrared spectra were obtained on a JASCO FT-IR-410 at 4.0 cm-1 resolution and are reported in
wavenumbers. High resolution mass spectra (HRMS) were recorded on a JMS-AX500 or JMS-700. Low
resolution mass spectra (LRMS) were recorded on a JEOL JMS-DX303.
Experimental Procedures and Compounds Data
10-Bromodecanol (2)
A mixture of 1,10-decandiol (1.5 g, 8.6 mmol) and 48% HBr (aq.) (1.2 ml) in toluene (26 ml) was refluxed
for 2 days. The mixture was allowed to cool to room temperature and then H2O and AcOEt were added. The
organic layer was separated, and the aqueous layer was extracted with AcOEt. The combined organic layers
were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (AcOEt / hexane = 1 / 1) to afford 2 (1.9 g, 92% yield) as a
colorless oil.
2: FT-IR (CHCl3) νmax 3336 cm-1; 1H-NMR (400 MHz, CDCl3) δ 3.64 (2H, t, J = 6.5 Hz), 3.41 (2H, t, J = 6.8
Hz), 1.85 (2H, quintet, J = 7.7 Hz), 1.56 (2H, quintet, J = 7.2 Hz), 1.42 (2H, m), 1.20-1.40 (12H, m);
13C-NMR (100 MHz, CDCl3) δ 63.0, 34.0, 32.8, 32.8, 29.4, 29.3, 29.3, 28.7, 28.1, 25.7; LRMS (EI) m/z 235
([M-H]+); HRMS (EI) calcd. for C10H20BrO 235.0698, found 235.0686.
10-Azidodecanol (3)
A mixture of bromide 2 (1.8 g, 7.6 mmol) and sodium azide (740 mg, 11.4 mmol) in DMF (25 ml) was
°
heated for 6 h at 50 C. The mixture was allowed to cool to room temperature and then H2O and Et2O were
added. The organic layer was separated, and the aqueous layer was extracted with Et2O. The combined
9

organic layers were washed with brine, dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (AcOEt / hexane = 2 / 3) to afford 3 (1.5 g, 97%
yield) as a colorless oil.
3: FT-IR (CHCl3) νmax 2095 cm-1; 1H-NMR (400 MHz, CDCl3) δ 3.64 (2H, t, J = 6.7 Hz), 3.26 (2H, t, J = 7.0
Hz), 1.58 (4H, m), 1.20-1.40 (13H, m); 13C-NMR (100 MHz, CDCl3) δ 63.0, 51.5, 32.8, 29.4, 29.4, 29.3,
29.1, 28.8, 26.7, 25.7; LRMS (FAB) m/z 200 ([M+H]+); HRMS (FAB) Calcd. for C10H22N3O 200.1763,
Found 200.1764.
3-(10-Azidodecyloxy)propyne (4)
To a solution of alcohol 3 (500 mg, 2.5 mmol) in DMF (13 ml) was added sodium hydride (60% in oil, 130
°
mg, 2.8 mmol) at 0 C. After stirring for 10 min, propargyl bromide (0.2 ml, 2.8 mmol) was added to the
reaction mixture and the reaction mixture was stirred at room temperature for 6 h. The mixture was quenched
with H2O and diluted with Et2O. The organic layer was separated, and the aqueous layer was extracted with
Et2O. The combined organic layer was washed with brine, dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography (AcOEt / hexane = 1 / 2) to
afford 4 (505 mg, 85% yield) as a yellow oil.
4: IR (CHCl3) νmax 3305, 2096 cm-1; 1H-NMR (400 MHz, CDCl3) δ 4.13 (2H, d, J = 2.3 Hz), 3.50 (2H, t, J =
6.6 Hz), 3.25 (2H, t, J = 7.0 Hz), 2.41 (1H, t, J = 2.3 Hz), 1.59 (4H, m), 1.35-1.29 (12H, m); 13C-NMR (100
MHz, CDCl3) δ 80.0, 74.0, 70.3, 58.0, 51.5, 29.5, 29.4, 29.4, 29.3, 29.1, 28.8, 26.7, 26.0; LRMS (FAB) m/z
238 ([M+H]+); HRMS (FAB) Calcd. for C13H24N3O 238.1919, Found 238.1927.
5-(2-Oxohexahydrothieno[3,4-d]imidazol-6-yl)pentanoic
acid (10-prop-2-ynyloxydecyl)amide (5)
To a solution of azide 4 (0.2 g, 0.84 mmol) in THF (5.6 ml) and H2O was added PPh3 (440 mg, 1.7 mmol).
After stirring for 1 h at room temperature, MgSO4 was added. The mixture was filtered through a filter paper
and concentrated under reduced pressure. To a solution of the residue and (+)-biotin (247 mg, 1.0 mmol) in
DMF (3.7 ml) were added DMAP (220 mg, 1.7 mmol) and N-(3-dimethylaminopropyl)-
N’-ethylcarbodiimide hydrochloride (340 mg, 1.8 mmol). After stirring for 12 h at room temperature, H2O
was added and the solution was diluted with AcOEt. The organic layer was separated, and the aqueous layer
was extracted with AcOEt. The combined organic layer was washed with brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography (MeOH
/ AcOEt = 15 / 100) to afford 5 (277 mg, 75% yield) as a colorless amorphous solid.
5: [α]D +105.52 (c 0.95, MeOH); IR (CHCl3) νmax 3296, 1701 cm-1; H-NMR (400 MHz, CDCl3) δ 6.26
(0.5H, s), 6.03 (0.5H, s), 5.91 (0.5H, s), 5.80 (1H, s), 5.40 (0.5H, s), 5.21 (0.5H, s), 4.52 (1H, m), 4.32 (1H,
m), 4.13 (2H, d, J = 2.4 Hz), 3.51 (2H, t, J = 6.8 Hz), 3.20 (3H, m), 2.91 (1H, dd, J = 13.0, 5.0 Hz), 2.74 (1H,
d, J = 12.8 Hz), 2.42 (1H, t, J = 2.4 Hz), 2.20 (2H, t, J = 7.3 Hz), 1.22-1.74 (22H, m); 13C-NMR (100 MHz,
CDCl3) δ 173.0, 173.0, 163.5, 163.4, 80.1, 74.1, 70.3, 61.8, 60.2, 58.0, 55.6, 55.5, 40.6, 39.6, 36.1, 29.7, 29.5,
29.5, 29.4, 29.3, 28.2, 28.1, 27.0, 26.1, 25.7, 25.7; LRMS (EI) m/z 437 (M+); HRMS (EI) Calcd. for
C23H39N3O3S 437.2712, Found 437.2711.
31
1
10

GO-Y086
To a mixture of GO-Y085 (0.35 g, 2.1 mmol) and alkyne 5 (156 mg, 0.36 mmol) in MeCN (10 ml) was
added CuI (3 mg, 0.018 mmol). After stirring for 12 h at room temperature, H2O was added and diluted with
AcOEt. The organic layer was separated, and the aqueous layer was extracted with AcOEt. The combined
organic layer was washed with brine, dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (MeOH / CHCl3 = 15 / 100) to afford GO-Y086
(194 mg, 60% yield) as a yellow amorphous solid.
GO-Y086: [α]D23 + 16.4 (c 0.25, CHCl3); IR (CHCl3) νmax 3296, 2929, 1701, 1648, 1618, 1583, 1503, 1462,
1
1419, 1278, 1244, 1629 cm-1; H-NMR (500 MHz, CDCl3) δ 8.04 (1H, s), 7.67 (1H, d, J = 15.9 Hz), 7.64
(1H, d, J = 15.9 Hz), 6.98 (2H, d, J = 15.9 Hz), 6.85 (2H, s), 6.82 (2H, s), 5.80 (1H, brs). 5.77 (1H, brs), 5.07
(1H, brs), 4.71 (2H, t, J = 5.0 Hz), 4.65 (2H, s), 4.50 (1H, m), 4.39 (2H, t, J = 5.0 Hz), 4.31 (1H, m), 3.92
(6H, s), 3.90 (3H, s), 3.85 (6H, s), 3.53 (2H, t, J = 6.2 Hz), 3.21 (2H, m), 3.14 (1H, m), 2.90 (1H, dd, J =
12.9, 5.0 Hz), 2.72 (1H, d, J = 12.9 Hz), 2.18 (2H, t, J = 7.8 Hz), 1.68-1.58 (6H, m), 1.49-1.40 (4H, m), 1.25
(12H, m); 13C-NMR (125 MHz, CDCl3) δ 188.4, 172.9, 163.6, 153.5, 153.3, 145.2, 143.5, 143.0, 140.5,
138.2, 131.0, 130.2, 125.1, 124.7, 124.1, 105.7, 105.3, 71.3, 70.8, 64.4, 61.7, 61.0, 60.1, 56.2, 56.0, 55.5,
50.5, 40.5, 39.5, 36.0, 29.6, 29.6, 29.4, 29.4, 29.4, 29.2, 28.1, 28.1, 26.9, 26.1, 25.6; LRMS (FAB) m/z 907
([M+H]+); HRMS (FAB) Calcd. for C47H67N6O10S 907.4639, Found : 907.4626.
2-Phenoxyethanol (7)
A mixture of phenol (0.3 g, 3.2 mmol), K2CO3 (1.3 g, 9.6 mmol) and ethylene bromohydrin (0.34 ml, 4.8
°
mmol) in DMF (25 ml) was heated for 6 h at 90 C. The mixture was cooled to room temperature and then
H2O was added and the mixture was diluted with Et2O. The organic layer was separated, and the aqueous
layer was extracted with Et2O. The combined organic layers were washed with brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography (AcOEt
/ hexane = 1 / 1) to afford 7 (0.21 g, 47% yield) as a colorless oil.
1
7: IR (CHCl3) νmax 3359, 1245 cm-1; H-NMR (400 MHz, CDCl3) δ7.31-7.16 (2H, m), 6.99-6.91 (3H, m),
4.08 (2H, t, J = 4.5 Hz), 3.96 (2H, m), 2.17 (1H, s); 13C-NMR (100 MHz, CDCl3) δ 158.6, 129.5, 121.1,
114.5, 69.1, 61.5; LRMS (EI) m/z 138 ([M-H]+); HRMS (EI) Calcd. for C8H10O2 138.0681, Found 138.0685.
(2-Azidoethoxy)benzene (8)
To a mixture of alcohol 8 (200 mg, 1.5 mmol) and triethylamine (0.4 ml, 2.9 mmol) in CH2Cl2 (7 ml) was
slowly added mesyl chloride (0.13 ml, 1.7 mmol). After stirring for 3 h at room temperature, H2O was added
and the solution was diluted with CHCl3. The organic layer was separated, and the aqueous layer was
extracted with CHCl3. The combined organic layers were washed with brine, dried over MgSO4 and
concentrated under reduced pressure. A mixture of the residue and sodium azide (280 mg, 4.3 mmol) in
°
DMF (3.6 ml) was heated for 3 h at 50 C. The mixture was allowed to cool to room temperature and then
H2O and Et2O were added. The organic layer was separated, and the aqueous layer was extracted with Et2O.
11

The combined organic layers were washed with brine, dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (AcOEt / hexane = 1 / 4) to afford 8
(0.23 g, 97% yield) as a colorless oil.
1
8: IR (CHCl3) νmax 2109, 1242 cm-1; H-NMR (400 MHz, CDCl3) δ 7.29 (2H, m), 7.00 (1H, t, J = 7.5 Hz),
6.93 (2H, m), 4.15 (2H, t, J = 5.0 Hz), 3.59 (2H, t, J = 5.0 Hz); 13C-NMR (100 MHz, CDCl3) δ 158.2, 129.5,
121.3, 114.6, 66.8, 50.2; LRMS (EI) m/z 163 ([M-H]+); HRMS (EI) Calcd. for C8H9N3O 163.0746, Found
163.0740.
GO-Y088
To a mixture of azide 8 (20 mg, 0.012 mmol) and 5 (18 mg, 0.041 mmol) in MeCN (0.4 ml) was added CuI
(0.4 mg, 0.002 mmol). After stirring for 5 days at room temperature, the mixture was concentrated under
reduced pressure. The residue was purified by silica gel column chromatography (MeOH / CHCl3 = 1 / 10) to
afford GO-Y088 (12 mg, 50% yield) as a colorless amorphous oil.
GO-Y088: [α]D + 34.8 (c 0.20, CHCl3); IR (CHCl3) νmax 3303, 1702, 1639, 1462, 1215 cm-1; H-NMR
(400 MHz, CDCl3) δ 7.75 (1H, s), 7.27 (2H, dd, J = 8.0, 7.5 Hz), 6.98 (1H, t, J = 7.5 Hz), 6.87 (2H, d, J =
8.0 Hz), 6.30 (1H, brs), 5.97 (1H, brs), 5.52 (1H, brs). 4.77 (2H, t, J = 4.9 Hz), 4.61 (2H, s), 4.50 (1H, m),
4.36 (2H, t, J = 4.9 Hz), 4.31 (1H, m), 3.50 (2H, t, J = 6.8 Hz), 3.16 (2H, m), 3.14 (1H, m), 2.90 (1H, dd, J =
12.8, 4.9 Hz), 2.73 (1H, d, J = 12.8 Hz), 2.20 (2H, t, J = 7.5 Hz), 1.77-1.42 (10H, m), 1.26 (12H, m);
13C-NMR (100 MHz, CDCl3) δ 173.0, 163.8, 157.8, 145.6, 129.7, 123.6, 121.7, 114.5, 70.8, 66.3, 64.2, 61.8,
60.2, 55.5, 49.8, 40.6, 39.6, 36.1, 29.6, 29.4, 29.4, 29.4, 29.3, 28.2, 28.1, 26.9, 26.1, 25.7; LRMS (EI) m/z
600 (M+); HRMS (FAB) Calcd. for C31H48N6O4S 600.3458, Found 600.3466.
23
1
12

ꢂ ꢁ ꢂ ꢂ ꢂ
ꢈ ꢁ ꢆ ꢂ ꢂ
ꢈ ꢁ ꢆ ꢉ ꢀ
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ꢈ ꢁ ꢅ ꢇ ꢆ
ꢈ ꢁ ꢅ ꢅ ꢈ
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ꢈ ꢁ ꢄ ꢃ ꢃ
ꢈ ꢁ ꢄ ꢉ ꢅ
ꢈ ꢁ ꢉ ꢈ ꢀ
ꢈ ꢁ ꢉ ꢆ ꢄ
ꢈ ꢁ ꢉ ꢄ ꢅ
ꢈ ꢁ ꢉ ꢀ ꢇ
ꢈ ꢁ ꢉ ꢉ ꢊ
ꢈ ꢇ ꢁ ꢂ ꢇ
ꢇ ꢁ ꢂ ꢀ
ꢇ ꢁ ꢂ ꢆ
ꢇ ꢁ ꢂ ꢂ
ꢆ ꢁ ꢆ ꢊ ꢂ
ꢆ ꢁ ꢅ ꢂ ꢀ
ꢆ ꢁ ꢅ ꢇ ꢄ
ꢆ ꢁ ꢃ ꢇ ꢅ
ꢆ ꢁ ꢃ ꢅ ꢂ
ꢆ ꢁ ꢃ ꢄ ꢃ
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13

ꢇ ꢄ ꢁ ꢃ ꢊ ꢅ
ꢇ ꢉ ꢁ ꢈ ꢆ ꢂ
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ꢇ ꢊ ꢁ ꢆ ꢇ ꢆ
ꢇ ꢊ ꢁ ꢆ ꢆ ꢊ
ꢇ ꢊ ꢁ ꢅ ꢆ ꢉ
ꢆ ꢇ ꢁ ꢀ ꢃ ꢆ
ꢆ ꢇ ꢁ ꢀ ꢊ ꢃ
ꢆ ꢆ ꢁ ꢊ ꢊ ꢉ
ꢃ ꢆ ꢁ ꢂ ꢆ ꢅ
ꢀ ꢃ ꢁ ꢃ ꢀ ꢊ
ꢀ ꢀ ꢁ ꢂ ꢂ ꢂ
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14

ꢂ ꢁ ꢂ ꢂ ꢈ
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ꢈ ꢁ ꢄ ꢄ ꢂ
ꢈ ꢁ ꢄ ꢃ ꢀ
ꢈ ꢁ ꢄ ꢉ ꢈ
ꢈ ꢁ ꢄ ꢊ ꢊ
ꢈ ꢁ ꢃ ꢈ ꢀ
ꢈ ꢆ ꢁ ꢇ ꢆ
ꢅ ꢁ ꢂ ꢈ
ꢆ ꢁ ꢇ ꢅ ꢂ
ꢆ ꢁ ꢇ ꢄ ꢀ
ꢆ ꢁ ꢇ ꢀ ꢅ
ꢇ ꢁ ꢂ ꢂ
ꢇ ꢁ ꢂ ꢈ
ꢆ ꢁ ꢃ ꢇ ꢆ
ꢆ ꢁ ꢃ ꢅ ꢂ
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15

ꢇ ꢄ ꢁ ꢃ ꢉ ꢄ
ꢇ ꢃ ꢁ ꢃ ꢀ ꢆ
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ꢇ ꢊ ꢁ ꢆ ꢆ ꢊ
ꢇ ꢊ ꢁ ꢆ ꢄ ꢃ
ꢇ ꢊ ꢁ ꢅ ꢆ ꢉ
ꢆ ꢇ ꢁ ꢀ ꢄ ꢄ
ꢄ ꢈ ꢁ ꢅ ꢃ ꢇ
ꢃ ꢆ ꢁ ꢂ ꢈ ꢀ
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ꢈ ꢁ ꢃ ꢈ ꢈ
ꢅ ꢁ ꢆ ꢆ
ꢇ ꢁ ꢅ ꢂ ꢀ
ꢇ ꢁ ꢅ ꢈ ꢆ
ꢇ ꢁ ꢅ ꢈ ꢊ
ꢂ ꢁ ꢉ ꢊ
ꢆ ꢁ ꢇ ꢆ ꢄ
ꢆ ꢁ ꢇ ꢄ ꢆ
ꢆ ꢁ ꢇ ꢀ ꢈ
ꢆ ꢁ ꢅ ꢊ ꢂ
ꢆ ꢁ ꢄ ꢂ ꢀ
ꢆ ꢁ ꢄ ꢇ ꢅ
ꢇ ꢁ ꢈ ꢇ
ꢇ ꢁ ꢂ ꢆ
ꢅ ꢁ ꢈ ꢇ ꢊ
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ꢇ ꢃ ꢁ ꢂ ꢆ ꢊ
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ꢇ ꢊ ꢁ ꢆ ꢆ ꢊ
ꢇ ꢊ ꢁ ꢆ ꢄ ꢃ
ꢇ ꢊ ꢁ ꢆ ꢊ ꢀ
ꢇ ꢊ ꢁ ꢅ ꢀ ꢈ
ꢄ ꢈ ꢁ ꢅ ꢃ ꢇ
ꢄ ꢀ ꢁ ꢊ ꢉ ꢂ
ꢀ ꢂ ꢁ ꢇ ꢄ ꢈ
ꢀ ꢆ ꢁ ꢊ ꢉ ꢂ
ꢀ ꢃ ꢁ ꢃ ꢀ ꢊ
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ꢉ ꢂ ꢁ ꢂ ꢅ ꢄ
18

ꢂ ꢁ ꢂ ꢂ ꢂ
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ꢈ ꢁ ꢄ ꢃ ꢊ
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ꢈ ꢁ ꢃ ꢈ ꢉ
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ꢈ ꢁ ꢀ ꢅ ꢃ
ꢈ ꢁ ꢀ ꢃ ꢂ
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ꢇ ꢁ ꢇ ꢂ ꢂ
ꢇ ꢁ ꢇ ꢈ ꢊ
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ꢅ ꢁ ꢆ ꢈ ꢊ
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ꢅ ꢁ ꢄ ꢈ ꢄ
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ꢄ ꢁ ꢅ ꢂ ꢅ
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ꢄ ꢁ ꢊ ꢈ ꢅ
ꢃ ꢁ ꢂ ꢆ ꢈ
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ꢈ ꢇ ꢁ ꢃ ꢇ
ꢆ ꢁ ꢅ ꢀ
ꢃ ꢁ ꢇ ꢆ
ꢈ ꢁ ꢊ ꢄ
ꢂ ꢁ ꢊ ꢅ
ꢈ ꢁ ꢂ ꢅ
ꢈ ꢁ ꢂ ꢊ
ꢆ ꢁ ꢈ ꢊ
ꢇ ꢁ ꢇ ꢊ
ꢇ ꢁ ꢂ ꢉ
ꢈ ꢁ ꢂ ꢆ
ꢈ ꢁ ꢂ ꢂ
ꢂ ꢁ ꢆ ꢀ
ꢂ ꢁ ꢅ ꢀ
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ꢇ ꢄ ꢁ ꢃ ꢉ ꢃ
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ꢇ ꢃ ꢁ ꢊ ꢄ ꢅ
ꢇ ꢉ ꢁ ꢈ ꢂ ꢃ
ꢇ ꢉ ꢁ ꢈ ꢄ ꢄ
ꢇ ꢊ ꢁ ꢇ ꢊ ꢊ
ꢇ ꢊ ꢁ ꢅ ꢂ ꢃ
ꢇ ꢊ ꢁ ꢅ ꢉ ꢉ
ꢇ ꢊ ꢁ ꢄ ꢈ ꢆ
ꢇ ꢊ ꢁ ꢃ ꢄ ꢆ
ꢆ ꢃ ꢁ ꢂ ꢉ ꢊ
ꢆ ꢊ ꢁ ꢄ ꢃ ꢇ
ꢅ ꢂ ꢁ ꢄ ꢄ ꢉ
ꢄ ꢄ ꢁ ꢅ ꢉ ꢀ
ꢄ ꢄ ꢁ ꢄ ꢃ ꢈ
ꢄ ꢉ ꢁ ꢂ ꢇ ꢇ
ꢃ ꢂ ꢁ ꢈ ꢄ ꢅ
ꢃ ꢈ ꢁ ꢀ ꢉ ꢆ
ꢀ ꢂ ꢁ ꢆ ꢈ ꢉ
ꢀ ꢅ ꢁ ꢂ ꢀ ꢊ
ꢀ ꢃ ꢁ ꢀ ꢈ ꢆ
ꢀ ꢀ ꢁ ꢂ ꢆ ꢅ
ꢀ ꢀ ꢁ ꢆ ꢄ ꢄ
ꢉ ꢂ ꢁ ꢂ ꢀ ꢈ
ꢈ ꢃ ꢆ ꢁ ꢄ ꢄ ꢀ
ꢈ ꢃ ꢆ ꢁ ꢀ ꢄ ꢅ
ꢈ ꢀ ꢇ ꢁ ꢊ ꢃ ꢅ
ꢈ ꢀ ꢆ ꢁ ꢂ ꢆ ꢉ
20

ꢂ ꢁ ꢂ ꢂ ꢂ
ꢈ ꢁ ꢇ ꢄ ꢂ
ꢈ ꢁ ꢅ ꢆ ꢀ
ꢈ ꢁ ꢅ ꢄ ꢇ
ꢈ ꢁ ꢅ ꢃ ꢀ
ꢈ ꢁ ꢄ ꢀ ꢃ
ꢈ ꢁ ꢄ ꢊ ꢈ
ꢈ ꢁ ꢃ ꢂ ꢄ
ꢈ ꢁ ꢃ ꢃ ꢃ
ꢈ ꢁ ꢃ ꢉ ꢂ
ꢈ ꢆ ꢁ ꢈ ꢀ
ꢅ ꢁ ꢄ ꢈ
ꢇ ꢁ ꢅ ꢇ
ꢇ ꢁ ꢂ ꢉ
ꢃ ꢁ ꢀ ꢊ
ꢇ ꢁ ꢈ ꢀ ꢇ
ꢇ ꢁ ꢈ ꢉ ꢉ
ꢇ ꢁ ꢀ ꢈ ꢆ
ꢇ ꢁ ꢀ ꢆ ꢉ
ꢇ ꢁ ꢉ ꢉ ꢉ
ꢇ ꢁ ꢉ ꢊ ꢉ
ꢇ ꢁ ꢊ ꢈ ꢆ
ꢇ ꢁ ꢊ ꢇ ꢆ
ꢆ ꢁ ꢈ ꢇ ꢇ
ꢆ ꢁ ꢈ ꢆ ꢀ
ꢆ ꢁ ꢈ ꢅ ꢀ
ꢆ ꢁ ꢈ ꢃ ꢇ
ꢆ ꢁ ꢈ ꢉ ꢀ
ꢆ ꢁ ꢇ ꢂ ꢇ
ꢆ ꢁ ꢇ ꢈ ꢄ
ꢆ ꢁ ꢇ ꢇ ꢊ
ꢆ ꢁ ꢄ ꢇ ꢂ
ꢆ ꢁ ꢄ ꢆ ꢆ
ꢆ ꢁ ꢄ ꢅ ꢃ
ꢆ ꢁ ꢉ ꢅ ꢃ
ꢆ ꢁ ꢊ ꢂ ꢆ
ꢆ ꢁ ꢊ ꢇ ꢂ
ꢅ ꢁ ꢇ ꢊ ꢄ
ꢅ ꢁ ꢆ ꢂ ꢇ
ꢅ ꢁ ꢆ ꢂ ꢉ
ꢅ ꢁ ꢆ ꢈ ꢊ
ꢅ ꢁ ꢆ ꢀ ꢊ
ꢅ ꢁ ꢆ ꢉ ꢉ
ꢅ ꢁ ꢆ ꢊ ꢉ
ꢅ ꢁ ꢅ ꢉ ꢉ
ꢅ ꢁ ꢅ ꢊ ꢉ
ꢅ ꢁ ꢄ ꢂ ꢆ
ꢅ ꢁ ꢄ ꢈ ꢅ
ꢅ ꢁ ꢃ ꢄ ꢂ
ꢅ ꢁ ꢀ ꢂ ꢅ
ꢅ ꢁ ꢀ ꢈ ꢄ
ꢅ ꢁ ꢀ ꢇ ꢄ
ꢄ ꢁ ꢂ ꢃ ꢉ
ꢄ ꢁ ꢀ ꢀ ꢆ
ꢄ ꢁ ꢉ ꢂ ꢄ
ꢃ ꢁ ꢉ ꢈ ꢊ
ꢃ ꢁ ꢉ ꢄ ꢅ
ꢃ ꢁ ꢊ ꢃ ꢆ
ꢃ ꢁ ꢊ ꢊ ꢄ
ꢂ ꢁ ꢊ ꢊ
ꢂ ꢁ ꢊ ꢀ
ꢈ ꢁ ꢈ ꢂ
ꢇ ꢁ ꢈ ꢄ
ꢇ ꢁ ꢇ ꢇ
ꢃ ꢁ ꢂ ꢇ
ꢃ ꢁ ꢂ ꢄ
ꢆ ꢁ ꢈ ꢉ
ꢈ ꢁ ꢂ ꢇ
ꢈ ꢁ ꢉ ꢊ
ꢈ ꢁ ꢂ ꢃ
ꢈ ꢁ ꢊ ꢂ
ꢇ ꢁ ꢈ ꢉ
ꢂ ꢁ ꢊ ꢂ
ꢈ ꢁ ꢊ ꢀ
ꢈ ꢁ ꢉ ꢃ
ꢇ ꢁ ꢂ ꢃ
ꢇ ꢁ ꢂ ꢂ
ꢀ ꢁ ꢇ ꢃ ꢄ
ꢀ ꢁ ꢃ ꢇ ꢊ
ꢀ ꢁ ꢃ ꢄ ꢈ
ꢀ ꢁ ꢃ ꢃ ꢈ
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21

ꢇ ꢄ ꢁ ꢃ ꢅ ꢃ
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ꢇ ꢉ ꢁ ꢂ ꢃ ꢄ
ꢇ ꢉ ꢁ ꢈ ꢇ ꢃ
ꢇ ꢊ ꢁ ꢇ ꢆ ꢇ
ꢇ ꢊ ꢁ ꢆ ꢄ ꢅ
ꢇ ꢊ ꢁ ꢆ ꢉ ꢄ
ꢇ ꢊ ꢁ ꢅ ꢇ ꢆ
ꢇ ꢊ ꢁ ꢄ ꢊ ꢉ
ꢇ ꢊ ꢁ ꢃ ꢆ ꢃ
ꢆ ꢃ ꢁ ꢂ ꢇ ꢆ
ꢆ ꢊ ꢁ ꢅ ꢊ ꢄ
ꢅ ꢂ ꢁ ꢅ ꢊ ꢄ
ꢄ ꢂ ꢁ ꢅ ꢊ ꢈ
ꢄ ꢄ ꢁ ꢅ ꢀ ꢅ
ꢄ ꢄ ꢁ ꢊ ꢊ ꢆ
ꢄ ꢃ ꢁ ꢇ ꢈ ꢅ
ꢃ ꢂ ꢁ ꢈ ꢇ ꢊ
ꢃ ꢂ ꢁ ꢊ ꢃ ꢉ
ꢃ ꢈ ꢁ ꢀ ꢆ ꢊ
ꢃ ꢅ ꢁ ꢆ ꢄ ꢃ
ꢀ ꢂ ꢁ ꢀ ꢄ ꢂ
ꢀ ꢈ ꢁ ꢆ ꢈ ꢄ
ꢀ ꢃ ꢁ ꢀ ꢅ ꢈ
ꢀ ꢀ ꢁ ꢂ ꢂ ꢂ
ꢀ ꢀ ꢁ ꢇ ꢄ ꢇ
ꢈ ꢂ ꢄ ꢁ ꢇ ꢊ ꢅ
ꢈ ꢂ ꢄ ꢁ ꢃ ꢊ ꢈ
ꢈ ꢇ ꢅ ꢁ ꢂ ꢄ ꢈ
ꢈ ꢇ ꢅ ꢁ ꢃ ꢊ ꢇ
ꢈ ꢇ ꢄ ꢁ ꢈ ꢆ ꢅ
ꢈ ꢆ ꢂ ꢁ ꢈ ꢀ ꢉ
ꢈ ꢆ ꢈ ꢁ ꢂ ꢂ ꢇ
ꢈ ꢆ ꢉ ꢁ ꢇ ꢈ ꢆ
ꢈ ꢅ ꢂ ꢁ ꢄ ꢈ ꢉ
ꢈ ꢅ ꢆ ꢁ ꢂ ꢇ ꢉ
ꢈ ꢅ ꢆ ꢁ ꢅ ꢉ ꢃ
ꢈ ꢅ ꢄ ꢁ ꢈ ꢃ ꢄ
ꢈ ꢄ ꢆ ꢁ ꢆ ꢇ ꢊ
ꢈ ꢄ ꢆ ꢁ ꢅ ꢀ ꢅ
ꢈ ꢃ ꢆ ꢁ ꢄ ꢀ ꢀ
ꢈ ꢀ ꢇ ꢁ ꢊ ꢆ ꢆ
ꢈ ꢉ ꢉ ꢁ ꢆ ꢊ ꢇ
22

ꢂ ꢁ ꢂ ꢂ ꢂ
ꢂ ꢁ ꢊ ꢅ
ꢇ ꢁ ꢈ ꢀ ꢄ
ꢆ ꢁ ꢊ ꢄ ꢇ
ꢆ ꢁ ꢊ ꢃ ꢂ
ꢅ ꢁ ꢂ ꢃ ꢉ
ꢅ ꢁ ꢂ ꢉ ꢂ
ꢅ ꢁ ꢂ ꢊ ꢈ
ꢇ ꢁ ꢂ ꢊ
ꢇ ꢁ ꢂ ꢀ
ꢃ ꢁ ꢊ ꢂ ꢇ
ꢃ ꢁ ꢊ ꢂ ꢉ
ꢃ ꢁ ꢊ ꢈ ꢈ
ꢃ ꢁ ꢊ ꢈ ꢅ
ꢃ ꢁ ꢊ ꢇ ꢄ
ꢃ ꢁ ꢊ ꢆ ꢂ
ꢃ ꢁ ꢊ ꢆ ꢆ
ꢃ ꢁ ꢊ ꢆ ꢀ
ꢃ ꢁ ꢊ ꢅ ꢀ
ꢃ ꢁ ꢊ ꢅ ꢊ
ꢃ ꢁ ꢊ ꢄ ꢇ
ꢃ ꢁ ꢊ ꢃ ꢉ
ꢃ ꢁ ꢊ ꢉ ꢅ
ꢃ ꢁ ꢊ ꢉ ꢃ
ꢃ ꢁ ꢊ ꢉ ꢊ
ꢀ ꢁ ꢇ ꢄ ꢈ
ꢀ ꢁ ꢇ ꢃ ꢅ
ꢀ ꢁ ꢇ ꢀ ꢂ
ꢀ ꢁ ꢇ ꢀ ꢄ
ꢀ ꢁ ꢇ ꢉ ꢆ
ꢀ ꢁ ꢇ ꢉ ꢊ
ꢀ ꢁ ꢇ ꢊ ꢇ
ꢀ ꢁ ꢇ ꢊ ꢊ
ꢀ ꢁ ꢆ ꢂ ꢄ
ꢀ ꢁ ꢆ ꢈ ꢈ
ꢀ ꢁ ꢆ ꢈ ꢀ
ꢆ ꢁ ꢂ ꢇ
ꢇ ꢁ ꢂ ꢂ
23

ꢃ ꢈ ꢁ ꢅ ꢀ ꢂ
ꢃ ꢊ ꢁ ꢂ ꢀ ꢅ
ꢀ ꢃ ꢁ ꢃ ꢀ ꢊ
ꢀ ꢀ ꢁ ꢂ ꢂ ꢂ
ꢀ ꢀ ꢁ ꢆ ꢇ ꢈ
ꢈ ꢈ ꢅ ꢁ ꢄ ꢅ ꢃ
ꢈ ꢇ ꢈ ꢁ ꢈ ꢈ ꢆ
ꢈ ꢇ ꢊ ꢁ ꢄ ꢂ ꢉ
ꢈ ꢄ ꢉ ꢁ ꢄ ꢉ ꢄ
24

ꢂ ꢁ ꢂ ꢂ ꢂ
ꢈ ꢁ ꢄ ꢅ ꢇ
ꢆ ꢁ ꢄ ꢀ ꢆ
ꢆ ꢁ ꢄ ꢉ ꢄ
ꢆ ꢁ ꢄ ꢊ ꢉ
ꢇ ꢁ ꢈ ꢈ
ꢇ ꢁ ꢈ ꢆ
ꢅ ꢁ ꢈ ꢆ ꢆ
ꢅ ꢁ ꢈ ꢅ ꢄ
ꢅ ꢁ ꢈ ꢄ ꢉ
ꢃ ꢁ ꢊ ꢂ ꢅ
ꢃ ꢁ ꢊ ꢈ ꢈ
ꢃ ꢁ ꢊ ꢈ ꢇ
ꢃ ꢁ ꢊ ꢈ ꢄ
ꢃ ꢁ ꢊ ꢇ ꢀ
ꢃ ꢁ ꢊ ꢆ ꢆ
ꢃ ꢁ ꢊ ꢆ ꢄ
ꢃ ꢁ ꢊ ꢆ ꢊ
ꢃ ꢁ ꢊ ꢃ ꢈ
ꢃ ꢁ ꢊ ꢃ ꢅ
ꢃ ꢁ ꢊ ꢃ ꢃ
ꢃ ꢁ ꢊ ꢉ ꢇ
ꢃ ꢁ ꢊ ꢊ ꢉ
ꢀ ꢁ ꢂ ꢂ ꢈ
ꢀ ꢁ ꢇ ꢅ ꢄ
ꢀ ꢁ ꢇ ꢀ ꢇ
ꢀ ꢁ ꢇ ꢀ ꢉ
ꢀ ꢁ ꢇ ꢉ ꢆ
ꢀ ꢁ ꢇ ꢊ ꢈ
ꢀ ꢁ ꢇ ꢊ ꢃ
ꢀ ꢁ ꢆ ꢂ ꢂ
ꢀ ꢁ ꢆ ꢂ ꢃ
ꢀ ꢁ ꢆ ꢈ ꢆ
ꢀ ꢁ ꢆ ꢈ ꢉ
ꢇ ꢁ ꢂ ꢂ
ꢈ ꢁ ꢂ ꢇ
ꢇ ꢁ ꢂ ꢂ
25

ꢄ ꢂ ꢁ ꢈ ꢃ ꢊ ꢊ
ꢃ ꢃ ꢁ ꢉ ꢅ ꢅ ꢂ
ꢀ ꢃ ꢁ ꢃ ꢀ ꢊ ꢂ
ꢀ ꢀ ꢁ ꢂ ꢂ ꢂ ꢂ
ꢀ ꢀ ꢁ ꢆ ꢇ ꢈ ꢂ
ꢈ ꢈ ꢅ ꢁ ꢄ ꢉ ꢃ ꢊ
ꢈ ꢇ ꢈ ꢁ ꢆ ꢅ ꢆ ꢉ
ꢈ ꢇ ꢊ ꢁ ꢄ ꢅ ꢈ ꢂ
ꢈ ꢄ ꢉ ꢁ ꢇ ꢂ ꢃ ꢄ
26

ꢂ ꢁ ꢂ ꢂ ꢂ ꢂ
ꢈ ꢁ ꢇ ꢄ ꢃ ꢃ
ꢈ ꢁ ꢅ ꢈ ꢀ ꢃ
ꢈ ꢁ ꢅ ꢆ ꢀ ꢈ
ꢈ ꢁ ꢅ ꢄ ꢄ ꢅ
ꢈ ꢁ ꢅ ꢀ ꢆ ꢈ
ꢈ ꢁ ꢄ ꢅ ꢇ ꢈ
ꢈ ꢁ ꢄ ꢄ ꢊ ꢈ
ꢈ ꢁ ꢄ ꢀ ꢉ ꢂ
ꢈ ꢁ ꢄ ꢊ ꢄ ꢈ
ꢈ ꢁ ꢃ ꢇ ꢅ ꢅ
ꢈ ꢁ ꢃ ꢅ ꢇ ꢈ
ꢈ ꢁ ꢃ ꢄ ꢊ ꢉ
ꢈ ꢁ ꢃ ꢀ ꢊ ꢆ
ꢈ ꢁ ꢃ ꢉ ꢃ ꢂ
ꢈ ꢁ ꢀ ꢂ ꢅ ꢆ
ꢈ ꢁ ꢀ ꢈ ꢅ ꢈ
ꢈ ꢁ ꢀ ꢆ ꢅ ꢇ
ꢈ ꢁ ꢀ ꢄ ꢈ ꢊ
ꢈ ꢁ ꢀ ꢃ ꢊ ꢂ
ꢇ ꢁ ꢈ ꢀ ꢄ ꢉ
ꢇ ꢁ ꢈ ꢊ ꢅ ꢀ
ꢇ ꢁ ꢇ ꢈ ꢆ ꢀ
ꢇ ꢁ ꢀ ꢈ ꢄ ꢀ
ꢇ ꢁ ꢀ ꢅ ꢉ ꢂ
ꢇ ꢁ ꢉ ꢀ ꢊ ꢉ
ꢇ ꢁ ꢉ ꢊ ꢇ ꢂ
ꢇ ꢁ ꢊ ꢈ ꢈ ꢄ
ꢇ ꢁ ꢊ ꢇ ꢅ ꢆ
ꢆ ꢁ ꢈ ꢈ ꢉ ꢊ
ꢆ ꢁ ꢈ ꢆ ꢀ ꢇ
ꢆ ꢁ ꢈ ꢅ ꢉ ꢉ
ꢆ ꢁ ꢈ ꢃ ꢀ ꢈ
ꢆ ꢁ ꢈ ꢉ ꢇ ꢆ
ꢆ ꢁ ꢇ ꢂ ꢂ ꢂ
ꢆ ꢁ ꢇ ꢈ ꢃ ꢄ
ꢆ ꢁ ꢇ ꢆ ꢇ ꢆ
ꢆ ꢁ ꢅ ꢉ ꢅ ꢊ
ꢆ ꢁ ꢄ ꢂ ꢇ ꢂ
ꢆ ꢁ ꢄ ꢈ ꢊ ꢂ
ꢅ ꢁ ꢇ ꢊ ꢂ ꢈ
ꢅ ꢁ ꢆ ꢂ ꢉ ꢅ
ꢅ ꢁ ꢆ ꢈ ꢊ ꢊ
ꢅ ꢁ ꢆ ꢅ ꢄ ꢃ
ꢅ ꢁ ꢆ ꢄ ꢊ ꢂ
ꢅ ꢁ ꢆ ꢀ ꢈ ꢇ
ꢅ ꢁ ꢅ ꢉ ꢃ ꢄ
ꢅ ꢁ ꢅ ꢊ ꢊ ꢊ
ꢅ ꢁ ꢄ ꢈ ꢀ ꢂ
ꢅ ꢁ ꢃ ꢈ ꢉ ꢇ
ꢅ ꢁ ꢀ ꢅ ꢉ ꢉ
ꢅ ꢁ ꢀ ꢃ ꢈ ꢃ
ꢅ ꢁ ꢀ ꢀ ꢆ ꢉ
ꢄ ꢁ ꢄ ꢈ ꢃ ꢀ
ꢄ ꢁ ꢊ ꢀ ꢈ ꢉ
ꢃ ꢁ ꢆ ꢂ ꢈ ꢇ
ꢈ ꢇ ꢁ ꢄ ꢂ
ꢅ ꢁ ꢂ ꢃ
ꢃ ꢁ ꢅ ꢊ
ꢇ ꢁ ꢈ ꢉ
ꢈ ꢁ ꢂ ꢃ
ꢈ ꢁ ꢂ ꢉ
ꢆ ꢁ ꢆ ꢆ
ꢇ ꢁ ꢈ ꢃ
ꢈ ꢁ ꢂ ꢄ
ꢇ ꢁ ꢈ ꢊ
ꢈ ꢁ ꢈ ꢇ
ꢇ ꢁ ꢂ ꢄ
ꢇ ꢁ ꢈ ꢃ
ꢈ ꢁ ꢂ ꢂ
ꢈ ꢁ ꢂ ꢃ
ꢂ ꢁ ꢊ ꢊ
ꢃ ꢁ ꢉ ꢃ ꢂ ꢃ
ꢃ ꢁ ꢉ ꢉ ꢂ ꢀ
ꢃ ꢁ ꢉ ꢉ ꢇ ꢄ
ꢃ ꢁ ꢊ ꢃ ꢅ ꢊ
ꢃ ꢁ ꢊ ꢃ ꢃ ꢀ
ꢃ ꢁ ꢊ ꢉ ꢆ ꢉ
ꢀ ꢁ ꢂ ꢂ ꢇ ꢈ
ꢀ ꢁ ꢇ ꢃ ꢅ ꢅ
ꢀ ꢁ ꢇ ꢃ ꢉ ꢃ
ꢀ ꢁ ꢇ ꢉ ꢄ ꢀ
ꢀ ꢁ ꢇ ꢊ ꢊ ꢀ
ꢀ ꢁ ꢆ ꢂ ꢅ ꢃ
ꢀ ꢁ ꢀ ꢄ ꢆ ꢃ
ꢇ ꢁ ꢈ ꢈ
ꢈ ꢁ ꢂ ꢀ
ꢇ ꢁ ꢅ ꢃ
ꢈ ꢁ ꢂ ꢂ
27

ꢇ ꢄ ꢁ ꢃ ꢉ ꢃ ꢇ
ꢇ ꢃ ꢁ ꢂ ꢀ ꢆ ꢂ
ꢇ ꢃ ꢁ ꢊ ꢆ ꢀ ꢈ
ꢇ ꢉ ꢁ ꢈ ꢂ ꢄ ꢉ
ꢇ ꢉ ꢁ ꢈ ꢀ ꢊ ꢊ
ꢇ ꢊ ꢁ ꢇ ꢀ ꢅ ꢄ
ꢇ ꢊ ꢁ ꢆ ꢉ ꢊ ꢀ
ꢇ ꢊ ꢁ ꢅ ꢅ ꢀ ꢆ
ꢇ ꢊ ꢁ ꢄ ꢃ ꢇ ꢄ
ꢇ ꢊ ꢁ ꢃ ꢇ ꢉ ꢅ
ꢆ ꢃ ꢁ ꢂ ꢉ ꢂ ꢉ
ꢆ ꢊ ꢁ ꢄ ꢄ ꢆ ꢊ
ꢅ ꢂ ꢁ ꢄ ꢀ ꢅ ꢅ
ꢅ ꢊ ꢁ ꢀ ꢀ ꢄ ꢀ
ꢄ ꢄ ꢁ ꢄ ꢅ ꢄ ꢂ
ꢃ ꢂ ꢁ ꢈ ꢊ ꢄ ꢂ
ꢃ ꢈ ꢁ ꢉ ꢇ ꢅ ꢃ
ꢃ ꢅ ꢁ ꢇ ꢆ ꢃ ꢂ
ꢃ ꢃ ꢁ ꢆ ꢈ ꢂ ꢂ
ꢀ ꢂ ꢁ ꢉ ꢆ ꢃ ꢄ
ꢀ ꢃ ꢁ ꢀ ꢇ ꢈ ꢈ
ꢀ ꢀ ꢁ ꢂ ꢅ ꢇ ꢂ
ꢀ ꢀ ꢁ ꢆ ꢃ ꢆ ꢂ
ꢈ ꢈ ꢅ ꢁ ꢄ ꢅ ꢃ ꢃ
ꢈ ꢇ ꢈ ꢁ ꢀ ꢈ ꢄ ꢈ
ꢈ ꢇ ꢆ ꢁ ꢄ ꢀ ꢄ ꢈ
ꢈ ꢇ ꢊ ꢁ ꢃ ꢀ ꢆ ꢃ
ꢈ ꢅ ꢄ ꢁ ꢃ ꢈ ꢄ ꢆ
ꢈ ꢄ ꢀ ꢁ ꢉ ꢇ ꢂ ꢄ
ꢈ ꢃ ꢆ ꢁ ꢉ ꢈ ꢇ ꢂ
ꢈ ꢀ ꢆ ꢁ ꢂ ꢈ ꢆ ꢆ
28