Preparation and biological properties of biotinylated PhTX derivatives

Article abstract of DOI:10.1016/S0968-0896(99)00054-1

We report the synthesis of several highly functionalized biotinylated philanthotoxin (PhTX) analogues (7, 8, 10, 13-16) designed on the basis of earlier structure-activity relationship studies. Despite the extensive modifications, the binding to nicotinic acetylcholine receptor (nAChR) is in the low micromolar range according to an inhibition assay using ³H-thienylcyclohexyl-piperidine (TCP). A patch clamp functional assay gave comparable results. Compounds exemplified by 16, which consists of a biotinylated ligand linked to a bifunctional photoaffinity probe (BPP), represent a new type of probe which should find use in photo-cross-linking studies of ligand-receptor interactions. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00054-1

Bioorganic & Medicinal Chemistry 7 (1999) 1181±1194
Preparation and Biological Properties of Biotinylated
PhTX Derivatives
Masaru Hashimoto,y Ying Liu, Kan Fang, Hong-yu Li, Giuseppe Campiani{
and Koji Nakanishi*
Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
Received 16 October 1998; accepted 2 January 1999
AbstractÐWe report the synthesis of several highly functionalized biotinylated philanthotoxin (PhTX) analogues (7, 8, 10, 13±16)
designed on the basis of earlier structure±activity relationship studies. Despite the extensive modi®cations, the binding to nicotinic
3
acetylcholine receptor (nAChR) is in the low micromolar range according to an inhibition assay using H-thienylcyclohexyl-piper-
idine (TCP). A patch clamp functional assay gave comparable results. Compounds exempli®ed by 16, which consists of a biotiny-
lated ligand linked to a bifunctional photoanity probe (BPP), represent a new type of probe which should ®nd use in photo-cross-
linking studies of ligand±receptor interactions. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
bifunctional photoanity probe BPP (Fig. 2), with two
di€erent photolabile groups at sites A and B, was
designed to serve as a new general probe for cross-linking
studies of ligand/receptor complexes in the following
manner:17 (1) The ligand, which is attached to a biotin
group via a spacer, and the receptor are incubated to
form the complex. (2) Irradiation at 350 nm under neu-
tral conditions leads to carbene generation at site A
which cross-links to the protein receptor. (3) The recep-
tor is cleaved chemically or enzymatically. (4) The cross-
linked and non-cross-linked peptide fragments are
separated using the biotin tag by avidin anity chro-
matography. (5) The separated cross-linked peptide
fragment(s) is irradiated again at 350 nm, but under
mildly basic conditions, upon which the BPP group is
cleaved at site B, thus leaving only the nitrophenolic
marker cross-linked to the peptide(s), which is then
sequenced by MS.18,19 We have also shown that BPP
can be linked to a primary amino group by reductive
amination of the Schi€ base formed between BPP alde-
hyde 2-[5-nitro-3-(3-tri¯uoromethyl-1,2-diazirin-3-yl)-
phenoxy]ethanal 19 (Scheme 1) and the primary amine.
Earlier studies showed that introduction of terminal
alkyl groups in the polyamine chain did not decrease
the binding activity so far as the amino groups are
not transformed into amides.9,15,17 Therefore, it can
be expected that a BPP-linked molecule such as that
depicted in Figure 2 or Scheme 1, 16, despite multiple
modi®cations, should retain reasonable activity. Assay
results indeed con®rmed this speculation (see below).
This unique molecule could play an important role in
determining the cross-linking sites in ligand/receptor
interaction studies.
Recognition of the gating mechanism of ion channels
and interests in the three-dimensional structural features
of these proteins have stimulated active studies in recent
years.1±3 Neurotoxins are powerful probes in these stu-
dies,4 and in many cases chemically modi®ed neurotoxins
are providing considerable structural information of the
corresponding receptors.5,6 Philanthotoxin-433 (1)
(PhTX-433,7,8 the numerals denote number of methy-
lene groups between the amino groups, Fig. 1), a
polyamine amide toxin isolated from the venom of
Philanthus triangulum, and its analogues are non-
competitive antagonists of the nicotinic acetylcholine
and glutamate receptors (nAChR, GluR).9±14 Analo-
gues carrying ¯uorine and porphyrin labels have also
been synthesized and submitted to biophysical and
spectroscopic studies in order to investigate their inter-
actions with nAChR,15 while PhTX derivatives with 125
labels have been employed for preliminary photoanity
labeling studies of nAChR.16
I
We recently developed a new bifunctional photoanity
probe (BPP),17 which should ®nd use in photo-cross-
linking studies of ligand/receptor interactions. The
Key words: Philanthotoxin; biotin-labeling; nAChR; photoanity.
y*Corresponding author.
Present address: Department of Biochemistry and Bioscience,
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki,
036-8561, Japan.
{
Present address: Dipartimento Farmaco Chimico Tecnologico,
Siena University, Banchi di Sotto 55, 53100, Siena, Italy.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00054-1

1182
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
photo-cross-linked peptide fragments by agarose-avidin
anity chromatography. In order to incorporate a bio-
tin moiety for anity chromatography purposes and yet
cope with the solubility problem, a polar triethylene
glycol spacer was inserted between the biotin moiety
and the long hydrophobic acyclic chain (region III). By
this means, compound 13, which was expected to be the
least soluble, was soluble in water at concentrations
around 1 mM.
The length of the spacer should be suciently long so
that both the biotin and PhTX moieties have sucient
space to reach their respective binding pockets.26 The
15
biotin±avidin complexes are strongly bound (Kd=10
)
and the dissociation process is slow. In cases where the
BPP group is used for cross-linking studies, it is not
necessary to dissociate the biotin-linked ligand±peptide
fragment from the avidin column since the peptide is
cleaved at site B of BPP, so that the cross-linked peptide
fragment(s) linked to the nitrophenol marker is eluted.
However, in cases where BPP is not employed, the bio-
tin-ligand-peptide fragment has to be dissociated from
the avidin column. In order to cope with such cases,
analogues with a weaker biotin/avidin association were
prepared by converting the biotin into their sulfoxides
based on the work of Leonard.27 The following analo-
gues have been synthesized: bio-C10-PhTX343 (7), its
lysine adduct bio-C10-PhTX343-Lys (8), the diastereo-
meric pair of sulfoxides (O)-bio-C10-PhTX343 (10a and
10b), the iodine derivative bio-C10-PhTX(I2)343 (13),
the lysine adduct bio-C10-PhTX(I2)343-Lys (14), its b-
sulfoxide b(O)-bio-C10-PhTX(I2)343 (15), and ®nally
bio-C10-PhTX343-BPP (16).
Figure 1. Structures of the natural philan-thotoxin-433 (PhTX-433)
and PhTX-343.
Extensive studies on structure±activity relationship (SAR)
of PhTX with respect to nAChR and GluR have been
carried out systematically by dividing PhTX into four
regions I±IV.9,13,15,20,21 Since the activity of PhTX-343
and PhTX-433 are comparable,7 the symmetric sper-
mine was incorporated as the polyamine chain for ease
of synthesis (region I), and then elongated with a lysine
unit (region II) to enhance binding.9 Replacement of the
butyryl amide moiety (region III) with longer aliphatic
amides enhances binding activity while substitution of this
moiety with polar functions results in loss of activity.22
On the other hand, an excessively long hydrophobic
chain led to poor aqueous solubility, thus making such
analogues unsuited for biological applications.
Here we report the preparation and binding properties
of bioactive biotinylated philanthotoxins, including
analogue 16 which incorporates the BPP group. Biotin,
with an anity to avidin of Kd=10 15, has been used
extensively in bioorganic studies23±25 and subsequent cross-
linking studies to elucidate ligand±receptor 3-D interac-
tions. This type of biotinylated PhTXs and other ligands
can be utilized in a variety of experiments, including
puri®cation of receptors or selective sequestering of the
Results and Discussion
The syntheses are summarized in Schemes 1 and 2.
Alcohol 2 was prepared from triethylene glycol by: (i)
mono-acetylation of triethylene glycol (Ac2O, Py,
CH2Cl2, 74%); (ii) mesylation of the resulting alcohol
(MsCl, Et3N, CH2Cl2, 73%); (iii) substitution by azide
Figure 2. Design of Philanthotoxin derivatives.

M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
1183
Scheme 1.
Scheme 2.

1184
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
(NaN3, H2O, EtOH); and (iv) deacetylation (NH4OH,
MeOH, 97%, 2 steps). A straightforward manipulation
of 1,10-decanediol provided 3 via: (i) mono-MPM ether
formation (NaH, MPMCl, DMF, 55%); (ii) mesylation
of the resulting alcohol (MsCl, Et3N, CH2Cl2, 99%);
and (iii) substitution with iodine (NaI, acetone, 99%).
After coupling of units 2 and 3 with aqueous NaOH in
the presence of Bu4NBr,28 the MPM group was
removed with DDQ.29 The regenerated hydroxyl group
was then transformed into a carboxylic group stepwise
by Dess±Martin reagent30 followed by NaClO2. The
carboxylic acid thus obtained was coupled with tyrosine
methyl ester 4 employing combined usage of WSCI31
and HOBt,32 to give adduct 5. Reduction of the azide
group in 5 was achieved using palladium catalyzed
hydrogenation, to provide the corresponding amine,33
which was treated with biotin p-nitrophenyl ester34
without chromatographic puri®cation to yield bio-C10-
Tyr-OMe 6. Spermine was then coupled by DCC after
saponi®cation of the methyl ester in 6, to furnish bio-
C10-PhTx343 (7). Treatment of 7 with one equivalent
of Na,-Ne-di-t-Boc-L-lysine hydroxysuccinimide ester
gave rise to the coupling product in good yield.
Removal of the Boc groups by TFA in the presence of
thiophenol yielded bio-C10-PhTX343-Lys (8).
with I2 in the presence of NaHCO3 resulted in forma-
tion of 2,6-diiodophenol, giving 11 in high yield.
Deprotection of the Boc group followed by coupling
with biotin p-nitrophenol ester gave rise to 12. After
saponi®cation of the methyl ester moiety of 12 with
aqueous NaOH, coupling with spermine and then
with lysine proceeded smoothly, producing Bio-C10-
PhTX(I2)343 (13) and Bio-C10-PhTX(I2)-343-Lys (14).
It is worth noting that when hydrolysis of the methyl
ester in 12 was performed under ambient atmosphere, b-
stereoselective oxidation of the biotin part took place to
give the b-sulfoxide, which was then reacted with sper-
mine to give b(O)-bio-C10-PhTX(I2)343 (15). Finally,
treatment of 7 with BPP aldehyde and sodium cyano-
borohydride in MeOH in the presence of TFA gave the
desired product 16 in 82% yield.
The stereochemistry of biotin sulfoxide moieties was
assigned as follows (Table 1). Leonard demonstrated
the a-selective sulfoxide formation of the biotin function
employing NBS in anhydrous MeOH.27 In order to
ascertain the stereochemistry in 10a and 10b biotin
methyl ester (17) and biotin b-phenethyl amide (18)
were oxidized with NBS and with NaIO4. Both oxida-
tions proceeded stereoselectively to yield products with
1
similar but not identical H NMR spectra; the spectra
Compound 6 was also transformed into the diastereo-
meric sulfoxides 9a and 9b. Stereoselective oxidation
into a-sulfoxide 9a was performed by the procedure of
Leonard,27 who reported that N-bromosuccinimide
(NBS) oxidation of certain biotins in anhydrous methanol
resulted in stereoselective formation of a-sulfoxybiotins.
As it was found that simultaneous bromination of the
ortho position of phenol moiety of tyrosine took place
when 6 was treated with NBS, it was necessary to reduce
the electron density of the aromatic ring. Thus the phe-
nol function was ®rst acylated as a methyl carbonate by
ClCO2Me and pyridine in CH2Cl2, and the product
submitted to NBS oxidation to yield the a-sulfoxide
selectively in 85% yield; subsequent cleavage of the
methyl carbonate and methyl ester under basic aqueous
of 10a and 10b were also close but not identical. The
con®gurations were determined by comparison of che-
mical shifts as well as coupling constants in the 1H
NMR spectra.35,36 The Hd signal of the NBS oxidation
product 17a appeared at a higher ®eld compared to 17,
while all other signals of the biotin moiety underwent a
low ®eld shift in both 17a and 17b. The same was true
for 18 and 10 series. In addition, clear di€erences in
coupling constants were also observed for JHa±Hc
.
Namely, those of 17, 18, and 7 were in the range from
0.4 to 0.8 Hz, while those of the sulfoxides prepared by
NBS (a-sulfoxide) and NaIO4 (b-sulfoxide) were 0.0 and
2.2±2.5 Hz, respectively. As there was no exception in
the relationships between oxidation methods and 1H NMR
data, sulfoxides obtained by NBS and NaIO4 oxidation
were assigned a- and b-con®gurations, respectively.
1
conditions provided the desired 9a. The H NMR spec-
tra of the product showed that only a small amount of
b-sulfoxide was formed (see below for assignment of
sulfoxide stereochemistry). b-Sulfoxide 9b was synthe-
sized by saponi®cation of 6 under aqueous basic condi-
tions, followed by NaIO4 treatment which occurred
stereoselectively from the convex side of the biotin
bicyclic ring system, giving rise to b-selective oxidation.
Linking of spermine to 9a and 9b was performed in the
same manner as in the synthesis of 7, giving rise to the
diastereomeric pair of a(O)-bio-C10-PhTX343 (10a and
its isomer 10b, respectively).
Biotin functions were introduced into the molecules in
order to take advantage of the biotin±avidin anity in
chromatographic separation. Although it was expected
that the terminal biotin group in these compounds is
suciently separated from the bulk of the molecule so
that it can bind with avidin, it was necessary to check
this point because it was possible that the strongly basic
polyamine moiety could interact with avidin to interfere
with the binding. Thus, a displacement assay using the
dye 2-(40-hydroxyphenylazo)-benzoic acid (HABA)37
was employed to measure their anity to avidin. It is
known that HABA also associates with avidin
(Ka=6Â10 6 M at pH 4.7). Upon complexation with
avidin, HABA exhibits intense UV absorption with
e=35,500 at 500 nm, while the released free HABA
shows e=480 at 500 nm. Thus when compounds with
stronger anity for avidin than HABA are added to the
avidin±HABA complex, HABA is released from avidin,
giving rise to a change in the UV pro®le. Titration of
the avidin±HABA complex with aqueous solutions of
Synthesis of derivatives with iodine on the tyrosine rings
was next carried out. Oxidation of the phenol group was
performed before introducing the biotin-containing
portion, since the sul®de function in biotin is more sus-
ceptible to most oxidative conditions than phenols. The
azide group of 5 was therefore ®rst transformed into the
corresponding N-Boc protected amine by sequential
palladium catalyzed reduction and protection by
(Boc)2O. It was found that treatment of the product

M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
Table 1. 1H NMR Data of some biotin derivatives and their sulfoxides
1185
Methods (yield %, a:b)
Haa
Hb
Hc
Hd
He
JHa±Hc (Hz)
17
17a
17b
Sul®de
Ab (90%,>10:1)d
Bc (85%,1:4)
4.51
4.91
4.70
2.94
3.01
3.09
2.75
3.40
3.55
3.22
2.87
3.17
4.32
4.76
4.61
0.8
0.0
2.2
18
18a
18b
Sul®de
Ab (82%,>10:1)d
Bc (92%,1:10)
4.51
4.87
4.80
2.95
3.00
3.21
2.63
3.41
3.63
3.20
2.83
3.23
4.30
4.72
4.64
0.8
0.0
2.4
7
10a
10b
Sul®de
A
b (92%,16:1)
Bc (66%,<1:10)d
4.51
4.86
4.70
2.94
3.02
3.09
2.72
3.42
3.54
3.24
2.83
3.21
4.32
4.73
4.61
0.4
0.0
2.5
Chemical shifts and coupling constants are reported in ppm (versus TMS) and Hz, respectively (400 MHz in CD3OD). Small di€erences in chemical
shifts (max‹0.10 ppm) from reported data in authentic methyl ester series were observed. Our own observations are reported here.
aFor convenience, the numbering for biotin is arbitrary and does not follow the IUPAC nomenclature.
bCondition (A) NBS in anhydrous MeOH, room temperature.
cCondition (B) NaIO4 in aqueous MeOH, room temperature.
dNo isomer was observed by 1H NMR. The selectivities were determined right after oxidations by NMR spectra.
biotinylated PhTXs resulted in a linear decrease in A500
against the volume of PhTX solution added. This
showed that all compounds (7, 8, 10, 13±16) formed
stable avidin complexes with dissociation constants
below the sensitivity limit of this method (Kd<10 9),38
and could be used in avidin anity chromatography to
facilitate photoanity labeling studies. However, the
anity was not high enough for purposes of receptor
puri®cation.39
low mM range. (2) Compound 6 without the PhTX
moiety can be regarded as a control for the biotin-
spacer chain. It is inactive. (3) However, linking of the
biotin-spacer chain and the PhTX moiety as in 7 bio-
C10-PhTX343 gave rise to a 37-fold potentiation over
PhTX 343. In contrast, the activity of the moiety lack-
ing the biotin-spacer chain (i.e., C10-PhTX343) was only
10-fold that of PhTX 343.21 The reason for this syner-
gistic enhancement is unclear. (4) Other biotinylated
analogues 8, 13, 14, 15, and 16 also exhibit a 30- to 50-
fold enhancement in activity over PhTX. (5) Results
with sulfoxides are varied. In the case of 10a and 10b
sulfoxidation signi®cantly diminishes activity, whereas
with 15 the activity remains more or less the same.
A radioligand competition assay was next performed to
determine the binding activity of analogues with
nAChR based on the protocol developed by Eldefrawi
and co-workers with minor modi®cations.15,40 In this
3
study, the commercially available H-thienylcyclohexyl-
piperidine (3H-TCP), a recently characterized potent
noncompetitive antagonist (channel blocker) of
nAChR,41 was employed as the radioligand instead of
3H-histrionicotoxin, which is dicult to obtain. The
sensitivity and reproducibility of the system were con-
®rmed using known antagonists of nAChR including
amantadine, chlorpromazine and tri¯uoperazine.15,40
IC50 values of all compounds tested were found from
the inhibition curves obtained (Fig. 3) and are shown in
Table 2. All experiments were carried out in triplicate
with <10% relative errors.
The results of these assays can be summarized as follows.
Figure 3. Selected inhibitory curves of philanthotoxin derivatives.
.
(Conc.: concentration (M); A: 6; B: PhTX-343; C: 7 avidin complex;
D: 7.)
(1) All biotinylated analogues of PhTX exhibit stronger
binding than native PhTX-433, with IC50 values in the

1186
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
Table 2. Summary of binding activity of biotinylated PhTX derivatives with nAChR
Free compounds
Avidin complexes
Relative activity
Compound
IC50 (mM)
Relativea activity
IC50 (mM)
PhTX-433
PhTX-343
75
75
1
1
±
±
1
1
6
7
8
10a
10b
13
14
15
16
bio-C10-Tyr-OMe
bio-C10-PhTX343
>300
2.0
1.5
8.0
14
2.6
1.5
1.4
2.5
<0.25
37
50
9.4
5.3
29
50
53
30
>300
6.0
35
18
33
15
29
20
22
<0.25
12.5
2.2
4.2
2.3
5.0
2.6
3.7
3.5
bio-C10-PhTX343-Lys
a(O)-bio-C10-PhTX343
b(O)-bio-C10-PhTX343
bio-C10-PhTX(I2)343
bio-C10-PhTX(I2)343-Lys
b(O)-bio-C10-PhTX(I2)343
bio-C10-PhTX343-BPP
aRelative activities were calculated as the ratio: (IC50 of PhTX433):(IC50 of new compounds). The lower the IC50, the higher the relative activity.
(6) SAR of PhTX showed that attachment of Lys in
region 2 led to a sevenfold enhancement in activity,21
whereas in the case of biotinylated derivatives (7 versus
8, 13 versus 14), the enhancement is less than twofold.
Bis-iodination of the tyrosine moiety increased the
activity by eightfold in PhTX;42 however, in the bio-
tinylated derivatives, the activity remains unchanged (8
versus 14) or is slightly decreased (7 versus 13) after
iodination. (7) Note that the activity of biotinylated
analogue 16 carrying the BPP moiety is 30-fold that of
PhTX. (8) Relative activities of the avidin complex of
biotinylated PhTX analogues are weaker than the non-
complexed free forms but are still noteworthy. Pre-
liminary patch clamp assays performed by Dr. Matthew
Brierley, Nottingham University, on TE671 cells with
overexpressed nAChR receptor gave the following
results: at a concentration of 10 6 M acetylcholine and
100 mV, the inhibition on the transmembrane currents
caused by 10 mM solutions of PhTX analogues was: 1
47.4%, 6 17.0%, 7 85.4%, 10a 62.0%, 10b 52.1%, and
14 58.6%. The results from this functional assay are in
good agreement with binding assay results.
Paragon 1000FT IR spectrophotometer with NaCl cell
or KBr pellets. Optical rotations were measured by
Jasco DIP-1000 digital polarimeter. Measurements of
high and low resolution mass (MS) spectra were per-
formed on Nermag R10-10 or Jeol JMS-DX 303HF
mass spectrometers. FAB mass spectra were collected
using 3-nitrobenzoylalcohol (NBA) as matrix. Analy-
tical thin layer chromatography was carried out by pre-
coated silica gel plates (Merck Art 7754). Prep TLC
plates used were Analtech Uniplate silica gel GF Pre-
parative Layers 20Â20 cm (1000 microns). Silica gel
used for column chromatography was Select Scienti®c
silica gel Art 162824.
All reactions were carried out under rt and argon
atmosphere unless speci®ed. Throughout the experimental
section, the following abbreviations are used: (Ac2O)
acetic anhydride; (MsCl) methanesulfonylchloride; (HOBT)
1-hydroxybenzotriazole; (WSCI) 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride; (DCC)
dicyclo-hexylcarbodiimide; (Boc2O) di-t-butyl dicarbo-
nate; (PhSH) thiophenol; (THF) tetrahydrofuran; (i-
PrOH) 2-propanol; (i-PrNH2) isopropylamine; aqueous
(aq). THF was distilled from sodium benzophenone
ketyl before use. Commercially available anhydrous
grade CH2Cl2 and DMF were purchased from Aldrich.
Other solvents were used without further puri®cation.
a,e-bis(t-butoxycarbonyl)lysine hydroxysuccinimide ester
were purchased from Sigma.
In summary, the synthesis of a series of novel biotinyl-
ated PhTX analogues has been carried out. Binding
assays showed that these compounds and their avidin
complexes exhibit reasonable binding activities against
nAChR. The 30-fold enhanced activity of analogue 16
incorporating the bifunctional photoanity probe BPP
should be noted; this demonstrates that such analogues
should be useful for streamlining the frequently tedious
photo-cross-linking studies of ligand±receptor 3-D
structural studies.
2-(2-Azidoethoxy)ethoxyethanol (2). To a solution of
triethylene glycol (6.00 mL, 6.78 g, 45.0 mmol) and
pyridine (6.0 mL. 5.88 g, 74 mmol) in CH2Cl2 (30 mL)
was added Ac2O (3.00 mL, 3.24 g, 31.8 mmol) at 0 ꢀC.
The mixture was allowed to warm to rt and stirred for
another 12 h. After concentration, the residue was
puri®ed by column chromatography (silica gel, acetone:
CH2Cl2, 25:75) to a€ord 2-[2-(2-hydroxyethoxy)ethoxy]-
Experimental
General
1
Melting points are uncorrected. H NMR spectra were
measured on Bruker DMX 300, DMX 400, or DMX
500 spectrometers. Chemical shifts are reported in ppm
down ®elds from the peak of tetramethylsilane as an
internal standard. Splitting patterns are designated as
``s, d, t, q, quint, m, and br'', indicating ``singlet, doub-
let, triplet, quartet, quintet, multiplet and broad'',
respectively. IR spectra were recorded on Perkin±Elmer
ethyl acetate (4.40 g, 22.9 mmol, 74% based on Ac2O) as
1
an oil. IR (®lm) 3030, 2930, 1600, 1455, 1030, 905 cm
.
1H NMR (300 MHz, CDCl3) d 2.10 (3H, s), 2.22 (1H,
br), 3.63 (2H, dd, J=4.0, 5.9 Hz), 3.70 (8H, m), 4.25
(2H, dd, J=4.6, 5.9 Hz). MS (EI) m/z (%) 193 (9.3,
MH+), 87 (100, AcOCH2CH2+). HRMS (EI) found
193.1074, calcd for C8H17O5 (MH+) 193.1076.

M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
1187
To a mixture of the above obtained acetate (1.92 g,
9.99 mmol) and Et3N (1.8 mL, 13.9 mmol) in CH2Cl2
(10 mL) was added MsCl (950 mL, 1.38 g, 12.0 mmol) at
78 ꢀC. After stirring at 78 ꢀC for 2 h, the mixture was
poured into water and extracted with AcOEt. The
organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was
puri®ed with column chromatography (silica gel, acet-
one:CH2Cl2, 5:95) to give 2-[2-(2-methanesulfoxy-
ethoxy)ethoxy]ethyl acetate (2.61 g, 9.67 mmol, 97%) as
an oil. IR (®lm) 2880, 1740, 1250, 1175, 1055, 1030, 970
20.7 mmol) at 78 ꢀC. After 10 min, the cooling bath
was removed and the mixture was further stirred for 1 h
at rt. The mixture was poured into water and was
extracted with ether. The ethereal solution was washed
with brine, dried over Na2SO4, and concentrated. The
residue was puri®ed with column chromatography
(silica gel, AcOEt:hexane, 20:80) to give 10-methane-
sulfoxydecanyl MPM ether (5.00 g, 13.4 mmol, 99%) as
needles. Mp <30 ꢀC (from cold ether/hexane). IR (®lm,
compound melted under measurement condition)
2930, 2850, 1515, 1360, 1250, 1170, 1100, 1030, 995
1
1
cm 1. H NMR (300 MHz, CDCl3) d 2.10 (3H, s), 3.09
cm 1. H NMR (300 MHz, CDCl3) d 1.30 (12H, m),
(3H, s), 3.69 (6H, m), 3.79 (2H, dd, J=3.9, 4.6 Hz), 4.23
(2H, dd, J=3.9, 4.9 Hz), 4.40 (2H, dd, J=3.6, 4.9 Hz).
MS (EI) m/z (%) 271 (10, MH+), 210 (3.7, [M+-
AcOH]), 166 (20, [M+-AcOCH2CH2OH]), 123 (100,
MsOCH2CH2+). HRMS (EI) found 271.0850, calcd for
C9H19SO7 (MH+) 271.0852.
1.60 (2H, quint, J=6.7 Hz), 1.76 (2H, quint, J=6.7 Hz),
3.02 (3H, s), 3.45 (2H, t, J=6.7 Hz), 3.83 (3H, s), 4.24
(2H, t, J=6.7 Hz), 4.45 (2H, s), 6.90 (2H, brd,
J=8.0 Hz), 7.28 (2H, brd, J=8.0 Hz). MS (EI) m/z (%)
372 (5.0, M+), 137 (100, MPMO+), 121 (100, MPM+).
HRMS (EI) found 372.1973, calcd for C18H32O5S (M+)
372.1972.
The mesylate obtained above (4.00 g, 14.8 mmol) and
sodium azide (2.30 g, 35.0 mmol) in a mixture of EtOH
(35 mL) and H2O (17 mL) was stirred at 80 ꢀC for 3 h.
After ethanol was removed in vacuo, the mixture was
poured into AcOEt and washed with water. The organic
layer was washed with brine, dried with Na2SO4, and
concentrated. The residue was diluted with MeOH
(20 mL) and 30% aq NH4OH (20 mL), and the mixture
was stirred for another 12 h. After concentration, the
residue obtained was puri®ed by column chromato-
graphy (silica gel, acetone:CH2Cl2, 25:75) to give 2
A suspension of the above mesylate (5.00 g, 13.4 mmol)
and NaI (12.0 g, 56 mmol, 4.2 equiv) in acetone (60 mL)
was re¯uxed with stirring for 3 h. After removing the
solvent, the residue was diluted with water containing
small amount of Na2S2O3 and extracted with ether. The
ethereal solution was washed with brine, dried over
Na2SO4, and concentrated. The residue was puri®ed
with column chromatography (silica gel, AcOEt:hexane,
4:96) to give 3 (5.35 g, 13.2 mmol, 99%) as an oil. IR
(®lm) 2930, 2850, 1515, 1360, 1250, 1170, 1100, 1030,
995 cm 1. 1H NMR (300 MHz, CDCl3) d 1.35 (12H, m),
1.65 (2H, quint, J=6.7 Hz), 1.84 (2H, quint, J=6.7 Hz),
3.21 (2H, t, J=6.7 Hz), 3.45 (2H, t, J=6.7 Hz), 3.83
(3H, s), 4.45 (2H, s), 6.90 (2H, brd, J=8.0 Hz), 7.28
(2H, brd, J=8.0 Hz). MS (EI) m/z (%) 404 (27, M+),
121 (100, MeOC6H4CH2+). HRMS (EI) found
404.1209, calcd for C18H29O2I (M+) 404.1209.
(2.52 g, 14.4 mmol, 97%, 2 steps) as an oil. IR (®lm)
1
3450, 2870, 2110, 1120 cm
.
1H NMR (300 MHz,
CDCl3), d 3.42 (2H, t, J=5.2 Hz), 3.64 (2H, dd, J=3.9,
5.9 Hz), 3.69 (6H, m), 3.76 (2H, dd, J=3.9, 5.9 Hz), MS
(EI) m/z (%) 176 (17, MH+), 119 (100, [M+-N3CH2]).
HRMS (EI) found 176.1031, calcd for C6H14N3 (MH+)
176.1036.
10-Iododecanyl MPM ether (3). To a suspension of
NaH (washed with hexane, 730 mg, 30.4 mmol) in DMF
(20 mL) was added 1,10-decanediol (5.00 g, 28.7 mmol).
After the mixture was stirred at 30ꢀC for 1 h, p-methoxy-
benzylchloride (MPMCl) (4.3 mL, 4.95 g, 31.6 mmol)
was added and the whole mixture was stirred for
another 6 h. The mixture was poured into water and
extracted with ether. The ethereal solution was washed
with brine, dried over Na2SO4, and concentrated. The
residue obtained was puri®ed with column chromato-
graphy (silica gel, AcOEt:hexane, 20:80) to give the 10-
hydroxydecanyl MPM ether (4.61 g, 15.6 mmol, 55%)
as a pellet. Mp 46.5±47.5 ꢀC (from ether/hexane). IR
(KBr), 3420, 2930, 2850, 1515, 1460, 1245, 1105, 1025
N-10-(2-[2-(2-Aminoethoxy)ethoxy]ethoxy)decanoyl-(L)-
tyrosine methyl ester (5). A suspension of 2 (1.20 g,
6.25 mmol), 3 (3.50 g, 8.66 mmol), and tetrabutyl-
ammonium bromide (160 mg, 1.55 mmol) in 50% aq
NaOH (10 mL) was stirred at 60 ꢀC for 5 h. The mixture
was poured into water and extracted with ether. The
ethereal extract was washed with brine, dried over
Na2SO4 and concentrated. The residue was puri®ed
with column chromatography (silica gel, AcOEt:hexane,
20:80) to give 10-(2-[2-(2-azidoethoxy)ethoxy]ethoxy)-
decanyl MPM ether (2.47 g, 5.47 mmol, 88% based on
1
2) as an oil. IR (®lm) 2930, 2860, 1510, 1250, 1100 cm
.
1H NMR (300 MHz, CDCl3) d 1.29 (12H, m), 1.59 (4H,
m), 3.41 (2H, t, J=4.8 Hz), 3.45 (2H, t, J=6.6 Hz), 3.46
(2H, t, J=6.6 Hz), 3.60 (2H, m), 3.68 (8H, m), 3.82 (3H,
s), 4,45 (2H, m), 6.90 (2H, brt, J=8.7 Hz), 7.27 (2H, brt,
J=8.7 Hz). MS (CI, NH3) m/z 469 (M+NH4+).
HRMS (FAB) found 474.2944, calcd for C24H41
N3O5Na (M+Na+) 474.2946.
1
cm 1. H NMR (300 MHz, CDCl3) d 1.30 (12H, m),
1.45 (1H, br), 1.62 (4H, m), 3.45 (2H, t, J=6.7 Hz), 3.65
(2H, t, J=6.7 Hz), 3.82 (3H, s), 4.45 (2H, s), 6.90 (2H,
brd, J=8.0 Hz), 7.29 (2H. brd, J=8.0 Hz). MS (EI) m/z
(%) 294 (12, M+), 137 (30, MeOC6H4CH2O+), 121
(100, MeOC6H4CH2+). HRMS (EI) found 294.2199,
calcd for C18H30O3 (M+) 294.2196.
A mixture of the above adduct (2.30 g, 5.10 mmol)
and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
(1.50 g, 6.60 mmol) in a mixture of CH2Cl2 (30 mL) and
H2O (3.0 mL) was stirred for 1 h. The mixture was then
poured into water and extracted with ether. The ethereal
A mixture of the mono MPM ether thus obtained (4.0 g,
13.6 mmol) and Et3N (4.0 mL, 3.12 g, 30.9 mmol) in
CH2Cl2 (20 mL) was stirred with MsCl (1.6 mL, 2.36 g,

1188
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
solution was washed with brine, dried over MgSO4, and
concentrated. The residue was puri®ed with column
chromatography (silica gel, acetone:CH2Cl2, 10:90) to give
10-(2-[2-(2-azidoethoxy)ethoxy]ethoxy)decanol (1.66 g,
5.02 mmol, 98%) as an oil. IR (®lm) 3450, 2930, 2105,
1H NMR (300 MHz, CDCl3) d 1.30 (10H, m), 1.59 (4H,
m), 2.19 (1H, dt, J=7.2, 14.4 Hz), 2.22 (1H, dt, J=7.2,
14.4 Hz), 2.99 (1H, dd, J=6.3, 14.1 Hz), 3.12 (1H, dd,
J=5.4, 14.1 Hz), 3.38 (2H, t, J=5.2 Hz), 3.50 (2H, t,
J=6.3 Hz), 3.65 (10H, m), 3.77 (3H, s), 4.86 (1H, ddd,
J=5.4, 6.3, 10.8 Hz), 5.91 (1H, brd, J=10.8 Hz), 6.77,
and 6.94 (each 2H, brd, J=8.7 Hz). MS (EI) m/z (%)
523 (1.0, MH+), 491 (0.5, [M+-MeO]), 463 (1.1, [M+-
MeO-N2]), 345 (40, [M+-HOC6H4CH2CHCO2Me]),
196 (22, [Tyr-OMe++H]), 178 (73, [TyrOMe+-NH3]),
107 (100, HOC6H4CH2+). HRMS (FAB) found
523.3123, calcd for C26H43N4O7(MH+) 523.3134.
1
1300, 1120 cm 1. H NMR (300MHz, CDCl3) d 1.29
(12H, m), 1.56 (4H, m), 3.40 (2H, brt, J=4.8Hz), 3.46
(2H, t, J=6.9Hz), 3.60 (2H, m), 3.67 (10H, m). MS (EI)
m/z (%) 332 (16, MH+), 160 (base peak). HRMS (EI)
found 332.2546, calcd for C16H34N3O4 (MH+) 332.2551.
A solution of the obtained alcohol (1.66 g, 5.02 mmol) in
CH2Cl2 (30 mL) was stirred with Dess±Martin reagent30
(2.70 g, 6.53 mmol) for 1 h. The mixture was poured into
a 1:1 mixture of saturated NaHCO3 and 5% Na2S2O3
solution, and was extracted with ether. The ethereal
solution was washed with brine, dried over Na2SO4 and
concentrated. The residue was puri®ed with column
chromatography (silica gel, AcOEt:hexane, 23:77) to
provide 10-(2-[2-(2-azidoethoxy)ethoxy]ethoxy)decanal
Bio-C10-Tyr-OMe (6). A suspension of 5 (98.0 mg,
187 mmol) and 20% Pd(OH)2/C (10 mg) in MeOH
(5.0 mL) was stirred vigorously under H2 atmosphere
for 1 h. After removal of the catalyst by ®ltration
through a pad of Celite, the ®ltrate was concentrated to
give the amine in almost pure form. The obtained amine
was diluted with CH2Cl2 (3.0 mL) and THF (3.0 mL),
and was stirred with (+)-biotin p-nitrophenyl ester
(71.3 mg, 195 mmol) and Et3N (40 mL) for 10 h. The
mixture was concentrated and the residue was puri®ed
with column chromatography (silica gel, MeOH:AcOEt,
10:90) to give 6 (125 mg, 173 mmol, 93%) as an oil. [a]d20
+29.9ꢀ (c 1.26, MeOH). IR (®lm) 3280, 2930, 2860,
(1.31 g, 3.98 mmol) as an oil. IR (®lm) 2930, 2105, 1720,
1
1460, 1350, 1300, 1125 cm
.
1H NMR (300 MHz,
CDCl3) d 1.31 (10H, m), 1.64 (4H, m), 2.43 (2H, dt, J=
1.8, 7.4 Hz), 3.41 (2H, brt, J=5.1 Hz), 3.46 (2H, t, J=
6.8 Hz), 3.60 (2H, m), 3.68 (8H, m), 9.77 (1H, t, J=
1.8 Hz). MS (CI, NH3) m/z 347 (M+NH4+). HRMS
(FAB) found 330.2398, calcd for C16H32N3O4 (MH+)
330.2395.
1
1690, 1655, 1545, 1520, 1460, 1245, 1105, 830 cm 1. H
NMR (400 MHz, CD3OD) d 1.30 (10, m), 1.60 (10H,
m), 2.18 (2H, t, J=7.6 Hz), 2.24 (2H, t, J= 7.6 Hz), 2.73
(1H, brd, J=13.6 Hz), 2.88 (1H, dd, J=8.8, 13.6), 2.94
(1H, dd, J=5.2, 13.6), 3.07 (1H, dd, J=5.6, 13.6 Hz),
3.23 (1H, ddd, J=4.4, 6.0, 9.2 Hz), 3.38 (2H, brq,
J=5.2 Hz), 3.49 (2H, t, J=6.8 Hz), 3.56 (2H, t,
J=5.6 Hz), 3.60 (2H, m), 3.65 (6H, m), 3.71 (3H, s),
4.32 (1H, dd, J=4.4, 8.0 Hz), 4.51 (1H, dd, J=5.2,
8.0 Hz), 4.62 (1H, m), 6.72, 7.03 (each 2H, brd, J=
8.4 Hz), 8.00 (1H, brt, J=5.2 Hz), 8.19 (1H, brd, J=
8.0 Hz). MS (CI, NH3) m/z 723 (MH+), 740 (M+
NH4+). HRMS (FAB) found 723.4005, calcd for
C36H59N4O9S (MH+) 723.4006.
To a mixture of the aldehyde thus obtained (1.31 g,
3.98 mmol) and 2-methyl-2-butene (2 M in THF,
5.0 mL) in t-BuOH (12 mL) was added a mixture of
NaClO2 (983 mg, 5.17 mmol) and NaH2PO4 (1.0 g) in
H2O (5.0 mL). The mixture was stirred for 30 min and
then THF and t-BuOH were removed in vacuo at
<20 ꢀC. The residue was diluted with 1% NaOH and
washed with ether. The aq layer was acidi®ed to pH 3
with 2 M HCl and extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4 and
concentrated to give pure 10-(2-[2-(2-azidoethoxy)-
ethoxy]ethoxy)decanoic acid (1.25 g, 3.62 mmol, 91%)
as an oil. IR (®lm) 3200, 2930, 2860, 2105, 1735, 1710,
Bio-C10-PhTX343 (7).
A solution of 6 (125 mg,
1
1460, 1290, 1120 cm 1. H NMR (300 MHz, CDCl3) d
173 mmol) in a mixture of MeOH (1.0 mL) and aq
NaOH (15 mg, 375 mmol, in 1.0 mL) was stirred for 4 h.
After MeOH was removed in vacuo at <20 ꢀC, the
mixture was acidi®ed to pH 2 by aqueous HCl and was
kept at 0 ꢀC for 10 h. The ®ne crystalline product was
collected as the corresponding carboxylic acid in almost
pure form (92.0 mg, 129 mmol, 75%). Mp 103±105 ꢀC
(®ne needles, from H2O). [a]2d0 +4.95ꢀ (c 0.75, MeOH).
IR (KBr) 3300, 2920, 2850, 1720, 1640, 1535, 1240, 1100
1.31 (10H, m), 1.60 (4H, m), 2.36 (2H, t, J=7.5 Hz),
3.41 (2H, brt, J=4.8 Hz), 3.47 (2H, t, J=6.6 Hz), 3.60
(2H, m), 3.69 (8H, m). MS (EI) m/z (%) 346 (7.0,
MH+), 328 (5.0, [M+-H2O]), 259 (25, [M+-N3CH2
CH2O]), 215 (26, [M+-N3CH2CH2OCH2CH2O]), 160
(base peak). HRMS (EI) found 346.2347, calcd for
C16H31N3O5 (MH+) 346.2344.
1
A mixture of the carboxylic acid thus prepared (1.20 g,
3.47 mmol), HOBT (700 mg, 4.58 mmol), (l)-tyrosine
methyl ester 4 (900 mg, 4.61 mmol), Et3N (1.0 mL,
720 mg, 7.13 mmol), and WSCI (850 mg, 4.45 mmol) in
CH2Cl2 (10.0 mL) was stirred for 18 h. The mixture was
poured into 2 M HCl and extracted with AcOEt. The
organic layer was washed with brine, dried over Na2SO4
and concentrated. The crude material was puri®ed with
column chromatography (silica gel, acetone:CH2Cl2,
6:94) to give 5 (1.55 g, 2.96 mmol, 94%) as an oil. [a]d20
cm 1. H NMR (400 MHz, CD3OD) d 1.30 (10H, m),
1.60 (10H, m), 2.17 (2H, t, J=7.6 Hz), 2.24 (2H, t,
J=7.2 Hz), 2.73 (1H, d, J=12.8 Hz), 2.87 (1H, dd,
J=9.2, 14.0 Hz), 2.94 (1H, dd, J=4.8, 12.8 Hz), 3.13
(1H, dd, J=4.8, 14.0 Hz), 3,22 (1H, m), 3.37 (2H, q,
J=5.2 Hz), 3.50 (2H, t, J=6.8 Hz), 3.56 (2H, t, J=
5.6 Hz), 3.61 (2H, m), 3.66 (6H, m), 4.32 (1H, dd,
J=4.4, 7.6 Hz), 4.51 (1H, dd, J=4.8, 7.6 Hz), 4.62 (1H,
b), 6.71, 7.06 (each 2H, brd, J=8.4 Hz), 8.01 (1H, br).
MS (CI, NH3) m/z 726 (M+NH4+), 709 (MH+), 691
(MH+-H2O). HRMS (FAB) found 709.3842, calcd for
C35H56N4O9S (MH+) 709.3849.
+47.9ꢀ (c 1.04, CHCl3). IR (®lm) 3315, 2930, 2860,
1
2105, 1740, 1655, 1510, 1440, 1225, 1125, 830 cm
.

M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
1189
A mixture of the above carboxylic acid (16.0 mg,
22.6 mmol) and p-nitrophenol (4.0 mg, 28.7 mmol) in
THF (1.0 mL) was stirred with DCC (6.2 mg, 30.1 mmol)
for 12 h, then a solution of spermine (12 mg, 59 mmol) in
THF (1.0 mL) was added and the whole mixture was
stirred for another 2 h. The mixture was adsorbed on
silica gel column intact. Successive elution with MeOH
and i-PrOH:MeOH (5:95) gave p-nitrophenol and 7
(12.0 mg, 13.7 mmol) as an oil, respectively. [a]2d0 +19.3ꢀ
(c 1.05, MeOH). IR (®lm) 3280, 2930, 1690, 1655, 1560,
1545, 1250, 1106 cm 1. 1H NMR (500 MHz, CD3OD) d
1.30 (10H, m), 1.60 (28H, m), 2.20 (2H, t, J=7.2 Hz),
2.24 (2H, t, J=7.2 Hz), 2.52 (2H. t, J=7.2 Hz), 2.60
(2H, t, J=6.8 Hz), 2.67 (2H, t, J=6.8 Hz), 2.72 (1H, d,
J=12.4 Hz), 2.76 (2H, t, J=7.4 Hz), 2.81 (1H, dd,
J=8.0, 13.6 Hz), 2.94 (1H, dd, J=4.4, 12.4 Hz), 2.98
(1H, dd, J=7.2, 13.6 Hz), 3.16 (1H, dd, J=6.8, 13.6 Hz),
3.23 (1H, m), 3.24 (1H, dd, J=6.8, 13.6 Hz), 3.81 (2H, t,
J=5.6 Hz), 3.50 (2H, t, J=6.4 Hz), 3.56 (2H, t, J=
5.6 Hz), 3.61 (2H, m), 3.67 (6H, m), 4.32 (1H, dd,
J=4.4, 8.0 Hz), 4.49 (1H, dd, J=7.2, 8.0 Hz), 4.51 (1H,
ddd, J=0.4, 4.4, 8.0 Hz), 6.71, 7.06 (each 2H, brd,
J=8.4 Hz). MS (CI, NH3) m/z 893 (MH+), 875 (MH+-
H2O), 836 (MH+-CH2=CHCH2NH2). HRMS (FAB)
found 893.5878, calcd for C45H80N8O8S (MH+),
893.5903.
found 1021.6849, calcd for C51H92N10O9S (MH+)
1021.6854.
N-10-[2-(2-[2-((ꢀ-Sulfoxybiotinylamino)ethoxy]ethoxy)-
ethoxy]decanoyl-(L)-tyrosine (9ꢀ). With similar treat-
ment as mentioned above in the preparation of 7,
hydrolysis of 6 a€orded the corresponding carboxylic
acid. A suspension of the obtained carboxylic acid
(33.0 mg, 46.6 mmol) and NaIO4 (11 mg, 51.4 mmol) in a
mixture of MeOH (2.0 mL) and H2O (400 mL) was stir-
red for 10 h. After MeOH was removed at <20 ꢀC, the
mixture was poured into brine (10 mL) containing HCl
(2 M, 50 mL) and Na2S2O3 (saturated, 20 mL), and was
extracted with n-BuOH. The organic solution was dried
over MgSO4 and concentrated to give 9b. 1H NMR
(400 MHz, CDCl3) d 1.29 (10 H, m), 1.55 (6H, m), 1.72
(2H, quint, J=7.5 Hz), 1.82 (2H, m), 2.16, 2.21 (each
1H, dt, J=7.4, 14.1 Hz), 2.28 (2H, t, J=7.2 Hz), 2.87
(1H, dd, J=8.9, 13.9 Hz), 3.12 (1H, dd, J=6.4, 13.9 Hz),
3.27 (2H, m), 3.39 (2H, t, J=5.4 Hz)m 3.49 (2H, t,
J=6.6 Hz), 3.60 (m, 12H), 4.51 (1H, brd, J=4.6,
8.5 Hz), 4.61 (1H, dd, J=5.3, 8.9 Hz), 4.72 (1H, ddd,
J=2.2, 6.4, 8.9 Hz), 6.70, 7.03 (each 2H, brd, J=
8.5 Hz).
ꢀ(O)-Bio-C10-PhTX343 (10ꢀ). The coupling between 9b
and spermine was carried out using p-nitrophenol and
DCC analogously to the preparation of 7, a€ording 10b
Bio-C10-PhTX343-Lys (8). Compound
7
(9.6 mg,
10.8 mmol) was stirred with a,e-bis-Boc-(l)-lysine N-
hydroxysuccinimide ester (4.9 mg, 11.3 mmol) in DMF
(1.0 mL) for 2 h. The mixture was puri®ed by column
chromatography (silica gel, i-PrNH2:MeOH, 7:93) to
give the coupling product (12.5 mg, 10.2 mmol, 95%) as
(66%) as an oil. [a]2d0 +6.9ꢀ (c 0.670, MeOH). IR (KBr)
1
3280, 2930, 1685, 1655, 1460, 1200 cm
.
1H NMR
(400 MHz, CD3OD), d 1.30 (10H, m), 1.60 (28H, m),
2.16, 2.21 (each 1H, dt, J=7.4, 14.1 Hz), 2.28 (2H, t,
J=7.2 Hz), 2.56 (2H, t, J=7.2 Hz), 2.60±2.85 (13H, m),
2.98 (1H, dd, J=7.0, 13.7 Hz), 3.09 (1H, dd, J=6.5,
13.8 Hz), 3.20 (4H, m), 3.39 (2H, t, J=5.6 Hz), 3.50
(2H, t, J=6.6 Hz), 3.54 (1H, dd, J=2.2, 13.8 Hz), 3.6
(12H, m), 4.47 (1H, t, J=7.2 Hz), 4.61 (1H, dd, J=5.3,
8.8 Hz), 4.70 (1H, ddd, J=2.5, 6.5. 8.8 Hz), 6.71, 7.06
(each 2H, brd, J=8.4 Hz). MS (FAB) m/z 909 (MH+),
852 (MH+-CH2=CHCH2NH2). HRMS (FAB) found
909.5844, calcd for C45H80N8O9S (MH+) 909.5852.
1
an oil. H NMR (400 MHz, CD3OD) d 1.31 (12H, m),
1.46, 1.47 (each 9H, s), 1.6 (22H, m), 2.20 (2H, t,
J=7.2 Hz), 2.24 (2H, t, J=7.2 Hz), 2.53 (2H, t, J=
7.6 Hz), 2.64 (6H. m), 2.72 (1H, d, J=12.4 Hz), 2.82
(1H, dd, J=8.4, 13.6 Hz), 2.94 (1H, dd, J=5.2, 12.4 Hz),
2.98 (1H, dd, J=7.2, 13.6 Hz), 3.05 (2H, t, J=6.4 Hz),
3.23 (5H, m), 3.38 (2H, t, J=5.6 Hz), 3.50 (2H, t,
J=7.2 Hz), 3.56 (2H, t, J=5.2 Hz), 3.61 (2H, m), 3.66
(6H, m), 3.96 (1H, br), 4.32 (1H, dd, J=4.4, 7.6 Hz),
4.49 (7.2, 8.4 Hz), 4.51 (1H, dd, J=5.2, 7.6 Hz), 6.72,
7.07 (each 2H, d, J=8.4 Hz)
ꢁ(O)-Bio-C10-PhTX343 (10ꢀ). A solution of 6 (38.0 mg,
52.6 mol) in CH2Cl2 (1.0 mL) was stirred with methyl
chloroformate (5 mL, 6.1 mg, 65.0 mmol) in the presence
of pyridine (10 mL, 9.8 mg, 123 mmol) for 3 h. The mix-
ture was poured into brine and extracted with AcOEt.
The organic solution was dried over MgSO4 and con-
centrated. The residue was puri®ed by column chroma-
tography (silica gel, MeOH:CH2Cl2, 10:90) to give the
corresponding (methoxycarbonyl)tyrosine methyl ester
(25 mg, 32.1 mmol, 59%) as ®ne needles. Mp 95±96 ꢀC
(from AcOEt-hexane). [a]2d2 +49.9ꢀ (c 1.00, CHCl3). IR
(KBr) 3300, 2930, 2860, 1710, 1650, 1545, 1280, 1125
A solution of the above adduct (12.5 mg, 10.2 mmol) in a
mixture of PhSH (20 mL) and TFA (1.0 mL) was stirred
for 1 h. After concentration, the residue was puri®ed
with Amberlite IRA410 (OH form, eluted with H2O)
to give the product in free amine state with small
amount of phenyl disul®de. Further puri®cation with
column chromatography (silica gel, iPrNH2:MeOH,
15:85) a€orded compound 8 (6.5 mg, 6.3 mmol, 62%) as
1
an oil. H NMR (400 MHz, CD3OD) d 1.31 (12H, m),
1
1.6 (22H, m) 2.20 (2H, t, J=7.2 Hz), 2.24 (2H, t, J=
7.2 Hz), 2.53 (2H, t, J=7.2 Hz), 2.65 (6H, m), 2.73 (2H,
t, J=7.2 Hz), 2.73 (1H, d, 12.8 Hz), 2.81 (1H, dd,
J=8.0, 14.0 Hz), 2.94 (1H, dd, J=4.8, 12.8 Hz), 2.99
(1H, dd, J=7.2, 14.0 Hz), 3.2 (4H, m), 3.29 (2H, t, J=
6.8 Hz), 3.38 (2H, t, J=5.6 Hz), 3.50 (2H, t, J=6.8 Hz),
3.57 (2H, t, J=5.6 Hz), 3.61 (2H, m), 3.67 (6H, m), 6.72,
7.06 (each 2H, d, J=8.1 Hz), MS (CI, NH3) m/z 1021
(MH+), 1002 (M+-H2O), 893 (MH+-Lys). HRMS (FAB)
cm 1. H NMR (400 MHz, CDCl3) d 1.30 (10H, m),
1.45 (2H, m), 1.58 (4H, m), 1.70 (4H, m), 2.22 (4H, m),
2.75 (1H, d, J=12.8 Hz), 2.92 (1H, dd, J=4.4, 12.8 Hz),
3.10 (1H, dd, J=6.4, 14.0 Hz), 3.15 (1H, dd, J=6.4,
14.0 Hz), 3.16 (1H, m), 3.46 (4H, m), 3.60 (4H, m), 3.65
(6H, m), 3.74 (3H, s), 3.92 (3H, s), 4.33 (1H, dd, J=4.4,
7.6 Hz), 4.52 (1H, dd, J=5.2, 7.6 Hz), 4.88 (1H, ddd,
J=6.4, 6.6, 8.0 Hz), 5.34 (1H, br), 6.40 (1H, brd,
J=7.6 Hz), 6.76 (1H, brt, J=5.2 Hz), 7.12 (4H, m). MS

1190
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
(FAB) m/z 781 (MH+), 803 (M+Na+). HRMS (FAB)
found 803.3883, calcd for C38H60N4O11SNa (M+Na+),
803.3876.
(silica gel, acetone:CH2Cl2) to give the Boc protected
amine (108 mg, 181 mmol, 95%) as an oil. 1H NMR
(400 MHz, CDCl3) d 1.28 (10 H, m), 1.47 (9H, s), 1.59
(4H, m), 2.14 (1H, dd, J=7.2, 14.4 Hz), 2.22 (1H, dd,
J=6.8, 14.4 Hz), 3.01 (1H, dd, J=6.4, 14.4 Hz), 3.14
(1H, dd, J=5.6, 14.4 Hz), 3.54 (4H, m), 3.66 (8H, m),
3.78 (3H, s), 4.88 (1H, dt, J=6.4, 5.6 Hz), 5.1 (1H, br),
5.86 (1H, brd, J=8.0 Hz). 6.78, 6.95 (each 2H,
J=8.1 Hz). MS (CI, NH3) m/z 614 (M+NH4+), 597
(MH+), 540 (MH+-C(CH3)3), 496 (M+-Boc). HRMS
(FAB) found 619.3587, calcd for C31H53N2O9 (MH+)
597.3753.
A solution of the ester prepared above (45.0 mg,
57.7 mmol) in MeOH (2.0 mL) was stirred with N-bro-
mosuccinimide (NBS) (12.0 mg, 67.4 mmol) for 12 h.
After concentration, the residue was puri®ed with col-
umn chromatography (silica gel, MeOH:CH2Cl2, 10:90)
to give the corresponding a-sulfoxybiotinyl ester
(39.0 mg, 48.9 mmol, 85%) as an oil. [a]2d2 11.6ꢀ (c 1.15,
MeOH). IR (®lm) 3315, 2930, 2855, 2472, 1750, 1700,
1
1685, 1655, 1640, 1460 cm
.
1H NMR (400 MHz,
CD3OD) d 1.30 (10H, m), 1.54 (2H, quint, J=7.2 Hz),
1.59 (2H, quint, J=6.8 Hz), 1.65 (2H, m), 1.77 (2H, m),
1.96 (1H, m), 2.15 (1H, m), 2.18 (2H, t, J=7.2 Hz), 2.29,
2.31 (each 1H, dt, J=14.0, 7.2 Hz), 2.86 (1H, dd,
J=9.2, 6.0 Hz), 2.99 (1H, dd, J=9.2, 14.0 Hz), 3.00
(1H, ddd, J=0.6, 6.8, 16.0 Hz), 3.20 (1H, dd, J=5.4,
14.0 Hz), 3.39 (2H, t, J=5.6 Hz), 3.40 (1H, d, J=
16.0 Hz), 3.49 (2H, t, J=6.0 Hz), 3.57 (2H, t, J=
5.6 Hz), 3.60 (2H, m), 3.65 (6H, m), 3.73 (3H, s), 3.88
(3H, m), 4.71 (1H, dd, J=5.4, 9.2 Hz), 4.73 (1H, dd,
J=6.0, 8.4 Hz), 4.86 (1H, dd, J=6.8, 8.4 Hz), 7.12, 7.26
(each 2H, brs, J=8.8 Hz). MS (FAB) m/z 819
(M+Na+), 797 (MH+). HRMS (FAB) found 819.3857,
calcd for C38H60N4O12SNa (M+Na+) 819.3825.
To a suspension of the Boc protected amine (40.0 mg,
67.1 mmol) in MeOH (3.0 mL), NaHCO3 (36 mg,
428 mmol in 1.0 mL H2O) and iodine (70.0 mg, 275 mmol
in 1.0 mL MeOH) was added successively. After stirring
vigorously for 30 min, 5% aq Na2S2O3 (2.0 mL) was
added to the mixture to decompose excess iodine. The
mixture was poured into water and extracted with
AcOEt. The organic solution was washed with brine,
dried over MgSO4 and concentrated. Puri®cation by
column chromatography (silica gel, acetone:CH2Cl2)
gave 11 (55.4 mg, 65.3 mmol, 97%) as an oil. IR (®lm)
3320, 2930, 2860, 1745, 1710, 1680, 1650, 1540, 1460,
1365, 1275, 1250, 1170, 1125 cm 1. 1H NMR (400 MHz,
CDCl3) d 1.32 (10H, m), 1.47 (9H, s), 1.62 (4H, m), 2.20
(1H, dd, J=7.2, 14.8 Hz), 2.25 (1H, dd, J=7.6, 14.8 Hz),
2.97 (1H, d, J=5.6, 14.0 Hz), 3.07 (1H, dd, J=5.6,
14.0 Hz), 3.33 (2H, bq, J=5.2 Hz), 3.48 (2H, t, J=
6.8 Hz), 3.56 (2H, t, J=5.2 Hz), 3.62 (2H, m), 3.66 (6H,
m), 3.78 (3H, s), 4.82 (1H, dt, J=7.6, 5.6 Hz), 5.06 (1H,
br), 5.82 (1H, brs), 5.97 (1H, brd, J=7.6 Hz), 7.44 (2H,
s). MS (CI, NH3) m/z 866 (M+NH4+), 849 (MH+),
848 (M+).
The a-sulfoxybiotinyl ester prepared above (39 mg,
48.9 mmol) and NaOH (7.0 mg, 175 mmol) was stirred in
a mixture of MeOH (2.0 mL) and H2O (300 mL) for 6 h.
After MeOH was removed at <20 ꢀC, the mixture was
poured into brine (10 mL) containing 2 M HCl (30 mL)
and extracted with n-BuOH. The organic solution was
dried with MgSO4, concentrated and the residue was
diluted with DMF (2.0 mL). This DMF solution was
treated with p-nitrophenol, DCC, and then spermine
analogously to preparation of 7 to give 10a (25.0 mg,
38.0 mmol, 62%) as an oil. [a]2d2 2.2ꢀ (c 0.600, MeOH).
N-10-[2-(2-[2-(Biotinylamino)ethoxy]ethoxy)ethoxy]decan-
oyl-(L)-(3,5-diiodo)tyrosine methyl ester (12). A solution
of 11 (58.0 mg, 68.8 mmol) in a mixture of PhSH (20 mL)
and TFA (1.0 mL) was stirred for 1 h. The mixture was
concentrated and then diluted with CH2Cl2 (1.0 mL).
Biotin p-nitrophenyl ester (28 mg, 76.7 mmol) was then
added and the mixture was stirred for 6 h, washed with
brine, dried over Na2SO4, and concentrated. Puri®ca-
tion with column chromatography (silica gel, MeOH:
CH2Cl2, 7:93) a€orded 12 (58.0 mg, 59.4 mmol, 86%) as
an oil. IR (®lm) 3290, 2930, 2860, 1730, 1695, 1645,
1555, 1540, 1240, 1205, 1130 cm 1. 1H NMR (400 MHz,
CD3OD) d 1.31 (10H, m), 1.6 (10H, m), 2.18 (1H, dd,
J=6.8, 14.0 Hz), 2.22 (1H, dd, J=6.4, 14.0 Hz), 2.24
(2H, t, J=7.6 Hz), 2.73 (1H, J=12.8 Hz), 2.80 (1H, dd,
J=10.0, 14.0 Hz), 2.94 (1H, dd, J=4.0, 12.8 Hz), 3.08
(1H, dd, J=5.0, 14.0 Hz), 3.22 (1H, m), 3.38 (2H, t,
J=5.2 Hz), 3.50 (2H, t, J=6.8 Hz), 3.57 (2H, t, J=
5.6 Hz), 3.61 (2H, m), 3.66 (6H, m), 3.74 (3H, s), 4.32
(1H, dd, J=4.4, 8.0 Hz), 4.51 (1H, dd, J=4.8, 8.0 Hz),
4.63 (1H, dd, J=5.0, 10.0 Hz), 7.61 (2H, s). MS (FAB)
m/z 975(MH+). HRMS (FAB) found 975.1920, calcd
for C36H57N4O9I2S (MH+) 975.1930.
1
IR (KBr) 3280, 2930, 1655, 1565, 1462 cm 1. H NMR
(400 MHz, CD3OD) d 1.30 (10H, m), 1.60 (26H, m),
1.97 (1H, m), 2.18 (1H, m), 2.20 (2H, t, J=7.2 Hz), 2.27,
2.33 (each 1H, dt, J=7.4, 14.1 Hz), 2.51 (2H, t,
J=7.3 Hz), 2.61 (2H, t, J=6.4 Hz), 2.62-2.73 (6H, m),
2.74 (2H, t, J=7.2 Hz), 2.81 (1H, dd, J=8.4, 13.6 Hz),
2.88 (2H, m), 2.98 (1H, t, J=6.6, 13.2 Hz), 3.02 (1H, t,
J=6.7, 13.2 Hz), 3.19, 3.22 (each 1H, m), 3.39 (2H, t,
J=5.8 Hz), 3.42 (1H, d, J=13.2 Hz), 3.49 (2H, t,
J=6.6 Hz), 3.58 (2H, t, J=5.5 Hz), 3.63 (6H, m), 4.49
(1H, t, J=6.6 Hz), 4.73 (1H, dd, J=6.0, 8.4 Hz), 4.86
(1H, dd, J=6.7, 8.4 Hz), 6.71, 7.06 (each 2H, brd, J=
8.4 Hz). MS (FAB) m/z 909 (MH+). HRMS (FAB)
found 909.5858, calcd for C45H80N8O9S (MH+)
909.5852.
N-10-(2-[2-(2-N-Boc-Aminoethoxy)ethoxy]ethoxy)decan-
oyl-3,5-diiodo-(L)-tyrosine methyl ester (11). Compound
5 (100 mg, 191 mmol) was hydrogenated by 20%
Pd(OH)2/C (10 mg) analogously to the preparation of 6.
The crude product was diluted with CH2Cl2 (2.0 mL),
Boc2O (44.0 mg, 200 mmol) was added, and the whole
mixture was further stirred for 12 h. After concentration,
the residue was puri®ed by column chromatography
Bio-C10-PhTX(I2)343 (13). A solution of 12 (50 mg,
50.1 mmol) in MeOH (1.0 mL) was stirred with aq
NaOH (6.0 mg, in 50 mL) for 3 h. Aq TFA solution was

M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
1191
added into the mixture until acidic, and the mixture was
concentrated in vacuo under Ar atmosphere. The
residue was puri®ed by Amberlite IRA410. After elution
of the impurity by H2O, subsequent elution with 10%
aq AcOH a€orded the carboxylic acid as an oil. IR
8. 11.0 mg of the above prepared adduct (6.8 mmol)
a€orded 14 (5.0 mg, 3.9 mmol, 58%) as an oil. IR (®lm)
3270, 2930, 1680, 1645, 1555, 1455, 1125 cm
1
.
1H
NMR (400 MHz, CD3OD) d 1.35 (10H, m), 1.48 (2H,
quint, J=7.0 Hz), 1.65 (18H, m), 1.95 (2H, quint,
J=7.2 Hz), 2.23 (1H, dd, J=7.6, 14.0 Hz), 2.25 (2H, t,
J=7.2 Hz), 2.27 (1H, dd, J=7.6, 14.0 Hz), 2.49 (2H, m),
2.71 (1H), 2.73 (1H, d, J=12.5 Hz), 2.74 (1H, dd,
J=9.5, 13.0 Hz), 2.78 (1H, dd, J=6.0, 13.3 Hz), 2.86
(2H, t, J=7.5 Hz), 2.92 (4H, m), 2.94 (1H, dd, J=4.5,
12.5 Hz), 3.23 (1H, m), 3.36 (1H, t, J=7.0 Hz), 3.38
(2H, t, J=5.5 Hz), 3.41 (1H), 3.49 (2H, t, J=7.0 Hz),
3.58 (2H, t, J=6.0 Hz), 3.60 (2H, m), 3.65 (6H, m), 4.33
(1H, dd, J=3.0, 7.5 Hz), 4.36 (1H, dd, J=6.0, 9.5 Hz),
4.51 (1H, dd, J=4.5, 7.5 Hz), 7.50 (2H, s). HRMS
(FAB) found 1245.4591, calcd for C51H91N10O9I2S
(MH+) 1245.4600.
(®lm) 3280, 2920, 1695, 1645, 1555, 1540, 1455, 1240,
1
1090 cm
.
1H NMR (500 MHz, CD3OD) d 1.22
(2H, m), 1.31 (8H, m), 1.6 (10H, m), 2.16 (1H, dd,
J=6.8, 14.0 Hz), 2.24 (1H, dd, J=6.4, 14.0 Hz), 2.24
(2H, t, J=7.5 Hz), 2.73 (1H, J=13.0 Hz), 2.82 (1H, dd,
J=9.6, 14.0 Hz), 2.95 (1H, dd, J=5.1, 13.0 Hz), 3.13
(1H, dd, J=4.7, 14.0 Hz), 3.23 (1H, m), 3.38 (2H, t,
J=5.0 Hz), 3.49 (2H, t, J=6.5 Hz), 3.57 (2H, t,
J=5.5 Hz), 3.61 (2H, m), 3.66 (6H, m), 3.74 (3H, s),
4.34 (1H, dd, J=4.4, 7.6 Hz), 4.52 (1H, dd, J=5.1,
7.6 Hz), 4.62 (1H, dd, J=4.7, 9.6 Hz), 7.62 (2H, s). MS
(FAB) m/z 983 (M+Na+), 961 (MH+). HRMS (FAB)
found 961.1818, calcd for C35H55N4O9I2S (MH+),
961.1773.
ꢀ(O)-Bio-C10-PhTX(I2) 343 (15). A solution of 12
(46.0 mg, 47.2 mmol) in MeOH (1.0 mL) was stirred with
aq NaOH (4.0 mg, in 20 mL) under ambient atmosphere
for 4 h. Aq TFA solution was added into the mixture
until acidic, and the mixture was concentrated. The
residue obtained was puri®ed by silica gel column chro-
matography. Successive elution with AcOH:CH2Cl2:
MeOH (0.3:50:50) and AcOH:CH2Cl2:MeOH (1:50:50)
gave the corresponding biotinyl carboxylic acid (12 mg,
12.4 mmol, 26%) and b-sulfoxybiotinyl carboxylic acid
The carboxylic acid obtained above (40 mg, 41.7 mmol)
was coupled with spermine using p-nitrophenol and
DCC analogously to the preparation of 7, a€ording 13
(20 mg, 17.5 mmol, 42%) as a caramel. IR (®lm) 3280,
2920, 2860, 1695, 1680, 1660, 1645, 1555, 1455, 1305,
1
1120 cm 1. H NMR (500 MHz, CD3OD) d 1.35 (10H,
m), 1.60 (8H, m), 1.92 (2H, quint, J=7.5 Hz), 2.24 (4H,
m), 2.47 (2H, m), 2.68 (1H, t, J=6.1 Hz), 2.73 (1H, d,
J=12.5 Hz), 2.75 (1H, dd, J=9.3, 13.5 Hz), 2.78 (1H,
dd, J=6.0, 13.5 Hz), 2.84 (2H, t, J=6.5 Hz), 2.93 (6H,
m), 3.23 (1H, m), 3.38 (2H, t, J=5.5 Hz), 3.40 (1H), 3.49
(2H, t, J=6.5 Hz), 3.57 (2H, t, J=5.5 Hz), 3.59 (2H, m),
3.64 (6H, m), 4.33 (1H, dd, J=4.5, 7.9), 4.35 (1H, dd,
J=6.1, 9.3 Hz), 5.51 (1H, dd, J=4.5, 7.6 Hz), 7.49 (2H,
s). MS (FAB) 1167 (M+Na+), 1145 (MH+). HRMS
(FAB) found 1145.3875, calcd for C45H79N8O8I2S
(MH+) 1145.3827.
1
(30 mg, 30.7 mmol, 65%) as oil, respectively. H NMR
(400 MHz, CD3OD) d 1.25 (2H, m), 1.30 (8H, m), 1.58
(6H, m), 1.73 (2H, quint, J=7.2 Hz), 1.90 (2H, m), 2.18
(1H, dd, J=7.2, 13.6 Hz), 2.22 (1H, dd, J=8.0, 13.6 Hz),
2.28 (2H, t, J=7.2 Hz), 2.82 (1H, dd, J=9.2, 14.0 Hz),
3.10 (1H, d, J=14.0 Hz), 3.15 (2H, m), 3.39 (2H, t,
J=5.2 Hz), 3.50 (2H, t, J=6.8 Hz), 3.56 (1H, dd,
J=2.0, 14.0 Hz), 3.68 (2H, t, J=5.6 Hz), 3.60 (2H,
m), 3.65 (67H, m), 4.49 (1H, br), 4.63 (1H, dd, J=
5.2, 8.8 Hz), 4.73 (1H, ddd, J=2.0, 6.4, 8.8 Hz), 7.62
(2H, s).
Bio-C10-PhTX(I2)-343-Lys (14).
A solution of 12
(12.0 mg, 10.4 mmol) in DMF (1.0 mL) was stirred with
a,e-bis-N-Boc-(l)-lysine N-hydroxysuccinimide ester
(4.8 mg, 10.8 mmol) for 2 h. Column chromatography
(silica gel, i-PrNH2:MeOH, 3:97) gave the adduct
(15.0 mg, 10.2 mmol, 92%) as an oil. IR (®lm) 3280,
2920, 2850, 1695, 1650, 1540, 1455, 1250, 1170, 1120
Coupling between the b-sulfoxybiotinyl carboxylic acid
(30 mg, 30.7 mmol) and spermine was carried out using
p-nitrophenol and DCC analogously to the preparation
of 7, giving 10.0 mg of 15 (8.6 mmol, 28%) as a caramel.
1H NMR (400 MHz, CD3OD) d 1.35 (10H, m), 1.55±1.75
(14H, m), 1.9 (2H, m), 1.98 (2H, quint, J=6.8 Hz), 2.26
(4H, m), 2.54 (2H, m), 2.70±3.20 (13H, m), 3.38 (2H, t,
5.2 Hz), 3.43 (1H), 3.49 (2H, t, J=6.8 Hz), 3.5 (1H),
3.68 (2H, t, J=5.6 Hz), 3.60 (2H, m), 3.65 (67H, m),
4.37 (1H, 1H, dd, J=6.2, 9.5 Hz), 4.61 (1H, dd, J=
5.3, 8.9 Hz), 4.70 (1H, ddd, J=2.2, 6.5, 8.9 Hz), 7.52
(2H, s).
1
cm 1. H NMR (400 MHz, CD3OD) d 1.35 (10H, m),
1.56, 1.47 (each 9H, s), 1.50 (4H, m), 1.65 (16H, m), 1.95
(2H, quint, J=7.2 Hz), 2.23 (1H, dd, J=7.6, 14.0 Hz),
2.25 (2H, t, J=7.2 Hz), 2.27 (1H, dd, J=7.6, 14.0 Hz),
2.46 (2H, m), 2.68 (2H, t, J=6.0 Hz), 2.74 (1H, d,
J=12.8 Hz), 2.74 ( 1H, dd, J=9.6, 13.2 Hz), 2.77 (1H,
dd, J=5.2, 13.2 Hz), 2.91 (5H, m), 2.96 (1H, dd,
J=4.8, 12.8 Hz), 3.05 (2H, t, J=6.8 Hz), 3.22 (1H,
m), 3.38 (2H, t, J=5.6 Hz), 3.41 (1H), 3.49 (2H, t,
J=6.8 Hz), 3.57 (2H, t, J=5.6 Hz), 3.59 (2H, m), 3.65
(6H, m), 3.97 (1H, brd, J=4.4, 6.0 Hz), 4.33 (1H, dd,
J=4.4, 7.6 Hz), 4.37 (1H, dd, J=6.0, 9.6 Hz), 4.51
(1H, dd, J=4.4, 7.6 Hz), 7.50 (2H, s). HRMS (FAB)
found 1473.5859, calcd for C61H107N10O13I2S (MH+)
1473.5826.
Bio-C10-PhTX343-BPP (16). In darkness, 0.25 M
methanolic TFA solution was added into a mixture of 7
(4.40 mg, 4.95 mmol) and BBP aldehyde 19 (1.30 mg,
4.50 mmol) in MeOH (2.0 mL) until neutral. Then
NaBH3CN (310 mg, 4.95 mmol) was added and the mix-
ture was stirred for 12 h and concentrated. The residue
was puri®ed by preparative silica gel TLC developed
with i-PrNH2:MeOH (5:95) to give 16 (4.30 mg,
4.54 mmol, 82%). Rf=0.5. 1H NMR (300 MHz, CD3OD)
a 1.20±1.82 (28H, m), 2.17 (2H, t, J=8.0 Hz), 2.21 (2H,
Deprotection of Boc groups was carried out using TFA
and PhSH analogously to the preparation of compound

1192
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
t, J=7.3 Hz), 2.53 (2H, t, J=7.2 Hz), 2.65 (2H, brt,
J=6.8 Hz), 2.69±2.83 (8H, m), 2.91 (3H, m), 3.03 (2H, t,
J=5.1 Hz), 3.20 (8H, m), 3.35 (2H, t, J=5.4 Hz), 3.46
(2H, t, J=6.6 Hz), 3.53 (2H, t, J=5.4 Hz), 3.58 (2H, m),
3.62 (6H, m), 4.22 (2H, t, J=5.2 Hz), 4.29 (1H, dd,
J=4.4, 7.9 Hz), 4.47 (2H, m), 6.69, 7.04 (each 2H, d,
J=8.5 Hz), 7.18 (1H, brs), 7.69 (1H, brs), 7.91 (1H, t,
J=2.0 Hz). 19F-NMR (282 MHz, CD3OD, ppm versus
CFCl3) d 65.24. HRMS (FAB) found 1166.6255, calcd
for C55H87N11F3O11S (MH+) 1166.6274.
dd, J=6.8, 8.4 Hz), 7.24 (5H, m). MS (CI, NH3) m/z
381 (M+NH4+), 364 (MH+).
ꢀ-Sulfoxybiotin methyl ester (17ꢀ). Biotin methyl ester
17 (10.0 mg, 38.7 mmol) was treated with NaIO4
analogously to the preparation of 10b. Column chro-
matography (silica gel, MeOH:CH2Cl2, 20:80) gave 17b
(9.0 mg, 32.8 mmol, 85%) as a caramel (a:b=1:4, by
1
NMR). H NMR (400 MHz, CD3OD) d 1.60 (2H, m),
1.75 (2H, m), 1.92 (2H, m), 2.41 (2H, t, J=7.4 Hz), 3.09
(1H, dd, J=6.5, 13.1 Hz), 3.17 (1H, m), 3.55 (1H, dd,
J=2.2, 13.1 Hz), 3.69 (3H, s), 4.61 (1H, dd, J=5.3,
8.8 Hz), 4.70 (1H, ddd, J=2.2, 6.5, 8.8 Hz).
Biotin methyl ester (17). A solution of biotin (400 mg,
1.64 mmol) in MeOH (5.0 mL) was stirred with tri-
methyl-silyldiazomethane (2 M in hexane, 2.0 mL) at
0 ꢀC for 10 h. After concentration, the residue was
recrystallized with AcOEt:MeOH to give biotin methyl
ester 17 (370 mg, 1.43 mmol, 87%). 1H NMR (400 MHz,
CD3OD) d 1.45 (2H, quint, J=7.5 Hz), 1.67 (4H, m),
2.37 (2H, t, J=7.4 Hz), 2.75 (1H, dd, J=0.8, 12.7 Hz),
2.94 (1H, dd, J=5.0, 12.7 Hz), 3.22 (1H, m), 3.69 (3H,
s), 4.32 (1H, dd, J=4.5, 7.8 Hz), 4.51 (1H, ddd, J=0.8,
5.0, 7.8 Hz).
ꢀ-Sulfoxybiotin ꢀ-phenethylamide (18ꢀ). Biotinamide
18 (21.0 mg, 60.4 mmol) was treated with NaIO4 analo-
gously to the preparation of 10b. Column chromato-
graphy (silica gel, MeOH:CH2Cl2, 50:50) gave 18b
(20.0 mg, 55.1 mmol, 91%) as a caramel (a/b=1/4, by
1
NMR). H NMR (400 MHz, CD3OD) d 1.43 (2H, m),
1.61 (2H, m), 1.82 (2H, m), 2.21 (2H, t, J=7.3 Hz), 2.84
(2H, t, J=6.9 Hz), 3.21 (1H, dd, J=6.5, 13.5 Hz), 3.23
(1H, dt, J=5.4, 7.6 Hz), 3.49 (2H, dt, J=1.3, 6.9 Hz),
3.63 (1H, dd, J=2.4, 13.5 Hz), 4.64 (1H, dd, J=5.4,
9.0 Hz), 4.80 (1H, ddd, J=2.4, 6.5, 9.0 Hz), 7.35 (5H,
m). MS (CI, NH3) m/z 381 (M+NH4+), 364 (MH+). In
the following procedures, all preparations containing
proteins were kept on ice and all operations were carried
out at 4 ꢀC whenever possible. Total protein concentra-
tions were carried out using bicinchoninic acid (BCA)
Biotin ꢀ-phenethylamide (18). To a solution of biotin p-
nitrophenyl ester (200 mg, 547 mmol) in CH2Cl2 (2.0 mL)
was added b-phenethyl-amine (100 mL, 795 mmol) at
0 ꢀC. After stirring for 30 min, the mixture was poured
into saturated NaHCO3 and extracted with AcOEt. The
organic solution was washed with 2 M NaHCO3 and
brine successively, dried over MgSO4 and concentrated.
The residue was puri®ed with column chromatography
(silica gel, MeOH:AcOEt, 30:70) to give 18 as amor-
phous powder (150 mg, 431 mmol, 79%). 1H NMR
(400 MHz, CD3OD) d 1.40 (2H, m), 1.60 (2H, m), 1.70
(2H, m), 2.18 (2H, t, J=7.4 Hz), 2.63 (1H, d, J=12.8 Hz),
2.81 (1H, d, J=7.3 Hz), 2.95 (1H, dd, J=4.9, 12.8 Hz),
3.20 (1H, ddd, J=4.5, 5.9, 8.9 Hz), 3.42 (1H, dt, J =1.5,
7.3 Hz), 4.30 (1H, dd, J=4.5, 7.9 Hz), 4.51 (1H, ddd,
J=0.8, 4.9, 7.9 Hz), 7.25 (5H, m). MS (CI, NH3), m/z
365 (M+NH4+), 348 (MH+).
3
assay following Sigma procedure TPRO-562. H-TCP
was purchased from DuPont NEN.
Tissue preparation and 3H-TCP binding assay. The
nAChR enriched membrane was obtained from electro-
plax of Torpedo californica following published proto-
cols.15,40 Tissue frozen at 78 ꢀC was thawed and
minced in bu€er (5 mM Tris, 1 mM EDTA, 0.1 mM
phenylmethylsulfonyl ¯uoride (PMSF), 154 mM NaCl)
to give a tissue homogenate. The homogenate was cen-
trifuged at 1000Âg for 10 min and the supernatant
obtained was then centrifuged at 35,000Âg for 60 min.
The pellet thus obtained was resuspended in bu€er and
kept on ice. Each batch of tissue preparation was used
in binding assay within a week.
ꢁ-Sulfoxybiotin methyl ester (17ꢁ). Biotin methyl ester
17 (100 mg, 387 mmol) was treated with NBS analo-
gously to the preparation of 10a. Column chromato-
graphy (silica gel, MeOH:CH2Cl2, 20:80) a€orded 17a
(95 mg, 346 mmol, 90%) as a caramel. 1H NMR
(400 MHz, CD3OD) d 1.70 (4H, m), 1.95 (1H, m), 2.13
(1H, m), 2.44 (2H, t, J=7.2 Hz), 2.87 (1H, m), 3.01 (1H,
dd, J=6.4, 15.2 Hz), 3.40 (1H, d, J=15.2 Hz), 3.62 (3H,
s), 4.76 (1H, dd, J=6.0, 8.4 Hz), 4.91 (1H, dd, J=6.4,
About 75mg of total protein was added into a mixture of
bu€er, carbamylcholine (®nal concentration at 100 mM),
3H-TCP (6nmol) and ligands (amantadine, chlorproma-
zine and tri¯uoperazine and biotin-PhTXs, ®nal con-
centration ranging from 0.03 mM to 300 mM), to a ®nal
volume of 500 mL. Total bound radioactivity was con-
trolled to be less than 5% of the total radioactivity by
adjusting the amount of tissue used. After incubation for
30s at rt, the mixture was ®ltered through GF/B glass
®ber ®lters (diameter 2.5 cm) presoaked in 0.05%
polyethyleneimide. The ®lter was washed extensively
with 0.9% NaCl (3 mLÂ4 times), immersed in Fisher
ScintiVerse scintillation cocktail and counted on a Beck-
man LS-3801 liquid scintillation counter. Each ligand
concentration was tested in triplicate with standard var-
iation at less than 10%. The count per minute (cpm)
values were analyzed and plotted to ®nd the IC50 value.
1
8.4 Hz). b-Isomer were not detected in this H NMR
spectrum.
ꢁ-Sulfoxybiotin ꢀ-phenethylamide (18ꢁ). Biotinamide
18 (21.3 mg, 61.4 mmol) was treated with NBS analo-
gously to the preparation of 10a. Column chromato-
graphy (silica gel, acetone:MeOH, 50:50) gave 18a
(18.3 mg, 50.4 mmol, 82%) as a caramel (a/b, 16/1, by
1
NMR). H NMR (400 MHz, CD3OD) d 1.60 (2H, m),
1.73 (2H, m), 1.94 (1H, m), 2.13 (1H, m), 2.25 (1H, dt,
J=2.3, 7.3 Hz), 2.83 (1H, t, J=7.3 Hz), 2.83 (1H, m),
3.00 (1H, dd, J=6.8, 15.4 Hz), 3.41 (1H, d, J=15.4 Hz),
3.45 (2H, m), 4.72 (1H, dd, J=5.9, 8.4 Hz), 4.87 (1H,

M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
1193
Partial puri®cation of enriched membrane by immobilized
avidin-biotin-PhTX complex. One mM solution of com-
pound 7 (bio-C10-PhTX343) was incubated with avidin
(monomeric) immobilized on 4% cross-linked agarose
(Sigma) at 4 ꢀC under end-to-end stirring for 8 h. The
suspension was ®ltered through a ®lter column, and the
®ltrate was measured in the HABA assay to determine
its concentration of 7. The concentration di€erence
before and after complexation was used to obtain con-
centration of 7 on agarose, which is ca. 85 mM. The
agarose was washed twice with bu€er (0.15 M NaCl,
0.01 M NaH2PO4, pH 6.8) and then reequilibrated with
0.01 M NaH2PO4, pH 6.8. In the batch operation, a
solution of nAChR enriched membrane solubilized in
2% sodium cholate (total protein concentration at
1.5 mg/mL) was incubated with this resin at 4 ꢀC for 8 h.
After ®ltration, the resin was washed twice with 0.01%
Triton X-100 in neutral bu€er, treated with electro-
phoresis sample solution and analyzed by 10% poly-
acrylamide gel electrophoresis. Results showed that
bands corresponding to nAChR were enriched by the
anity resin but other proteins were also present. In the
column method, 5 mL of resin (wet volume) was packed
into a plastic column, the nAChR enriched membrane
solubilized in 2% sodium cholate was adsorbed onto the
resin and eluted with a phosphate bu€er (0.01 M
NaH2PO4, pH 6.8). The fractions were concentrated
under vacuum centrifugation, dialyzed against 0.5 mM
Tris bu€er (pH 7.2) for 6 h, and were analyzed by 10%
polyacrylamide gel electrophoresis. Results showed
that nAChR was eluted at 5±6 times of the void volume.
The dissociation constant of nAChR on this resin was
calculated to be ca. 20 mM under the experimental
condition.43
Bukownik, R.; Kallimopoulos, T. A.; Usherwood, P.; Eldefrawi,
A. T.; Eldefrawi, M. E. Pure & Appl. Chem. 1990, 62, 1223.
10. Benson, J. A.; Kaufmann, L.; Hue, B.; Pelhate, M.;
Schurmann, F.; Gsell, L.; Piek, T. Comp. Biochem. Physiol.
1993, 105C, 303.
11. McCormick, K. D.; Meinwald, J. J. Chem. Ecol. 1993, 19,
2411.
12. Blagbrough, I. S.; Moya, E.; Taylor, S. Biochem. Soc.
Trans. 1994, 22, 888.
13. Nakanishi, K.; Huang, D.; Monde, K.; Tokiwa, Y.; Fang,
K.; Liu, Y.; Jiang, H.; Huang, X.; Matile, S.; Usherwood, P.
N. R.; Berova, N. ACS Symp. Ser. 1997, 658, 339.
14. Bahring, R.; Mayer, M. L. J. Physiol. (London) 1998, 509,
635.
15. Nakanishi, K.; Huang, X.; Jiang, H.; Liu, Y.; Fang, K.;
Huang, D.; Choi, S.-K.; Katz, E.; Eldefrawi, M. Bioorg. Med.
Chem. 1997, 5, 1969.
16. Choi, S.-K.; Kalivretenos, A. G.; Usherwood, P. N. R.;
Nakanishi, K. Chem. & Biol. 1995, 2, 23.
17. Fang, K.; Hashimoto, M.; Jockusch, S.; Turro, N. J.;
Nakanishi, K. J. Am. Chem. Soc. 1998, 120, 8543.
18. Orlando, R.; Kenny, P. T. M.; Moquin-Pattey, C.; Lerro,
K. A.; Nakanishi, K. Org. Mass Spectrom. 1993, 28, 1395.
19. Kaufmann, R.; Kirsch, D.; Tourmann, J. L.; Machold, J.;
Hucho, F.; Utkin, Y. U.; Tsetlin, V. Eur. Mass Spectrom.
1995, 1, 313.
20. Bruce, M.; Bukownik, R.; Eldefrawi, A. T.; Eldefrawi, M.
E.; Goodnow, R.; Kallimopoulos, T.; Konno, K.; Nakanishi,
K.; Niwa, M.; Usherwood, P. Toxicon 1990, 28, 1333.
21. Nakanishi, K.; Choi, S.-K.; Hwang, D.; Lerro, K.;
Orlando, M.; Kalivretenos, A. G.; Eldefrawi, A.; Eldefrawi,
M.; Usherwood, P. N. R. Pure & Appl. Chem. 1994, 66, 671.
22. Nakanishi, K.; Goodnow, R.; Kalivretenos, A.; Choi,
S.-K.; Fushiya, S.; Chiles, G.; Eldefrawi, A.; Eldefrawi, M.;
Usherwood, P. ``Synthetic and Bioorganic Studies with Phi-
lanthotoxin Analogues''; Alfred Benzon Symposium, 1992,
Munksgaard, Copenhagen.
23. Wilchek, M.; Bayer, E. A. Methods in Enzymology 1990,
184, 123.
24. Gilbert, B. A.; Rando, R. R. J. Am. Chem. Soc. 1995, 117,
8061.
Acknowledgements
25. Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. J. Am.
Chem. Soc. 1998, 120, 453.
26. Redeuilh, G.; Secco, C.; Baulieu, E. E. J. Biol. Chem.
1985, 260, 3996.
27. Liu, F.-T., Leonard, N. J. J. Am. Chem. Soc. 1979, 101, 996.
28. Freedman, H. H.; Dubois, R. A. Tetrahedron Lett. 1975,
38, 3251.
The authors are grateful to Professor Peter Usherwood
and Dr. Matthew Brierley, University of Nottingham,
for the path clamp assay data, and to Professors
Mohyee and Amira Eldefrawi, University of Maryland
at Baltimore, for discussions on the TCP binding assay.
29. Oikawa, Y.; Yoshioka, T., Yonemitsu, O. Tetrahedron
Lett. 1982, 23, 885.
References and Notes
30. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
31. Sheehan, J. C.; Preston, J.; Cruickshank, P. A. J. Am.
Chem. Soc. 1965, 87, 2494.
1. Unwin, N. Nature 1995, 373, 37.
2. Unwin, N. J. Mol. Biol. 1996, 257, 586.
3. Nakanishi, S.; Masu, M. Annu. Rev. Biophys. Biomol. 1994,
23, 319.
32. Konig, W.; Geiger, R. Chem. Ber. 1973, 106, 3626.
33. Scriven, E. F. Chem. Rev. 1988, 88, 297.
4. Hucho, F. Angew. Chem. Int. Ed. Engl. 1995, 34, 39.
5. Yoshida, E.; Nakayama, H.; Hatanaka, Y.; Kanaoka, Y.
Chem. Pharm. Bull. 1990, 38, 982.
6. Lin, S.; Chi, S.; Chang, L.; Kuo, K.; Chang, C. J. Biochem.
(Tokyo) 1995, 118, 297.
7. Eldefrawi, A. T.; Eldefrawi, M. E.; Kono, K.; Mansour, N.
A.; Nakanishi, K.; Oltz, E.; Usherwood, P. N. R. Proc. Natl.
Acad. Sci. USA 1988, 85, 4910.
8. Piek, T.; Fokkens, R. H.; Karst, H.; Kruck, C.; Lind, A.;
Van Marle, J.; Nakajima, K.; Nibbering, N. M. M.;
Shinozaki, H.; Spanier, M. W.; Tong, Y. C. Neurotox '88
Molecular Basis of Drug & Pesticide Action.; Elsevier: Amster-
dam, 1988.
34. Bodanszky, M.; Meienhofer, J.; Vigneaud, V. J. Am.
Chem. Soc. 1960, 82, 3195.
35. Lett, R.; Marquet, A. Tetrahedron Lett. 1971, 30, 2851.
36. Lett, R.; Marquet, A. Tetrahedron Lett. 1971, 30, 2855.
37. Green, N. M. Methods in Enzymology 1970, 48A, 418.
38. Green, N. M. Adv. Protein Chem. 1975, 29, 85.
39. Notes: An anity resin for nAChR was prepared by
incubating an aqueous solution of bio-C10-PhTX343 7 with
monomeric avidin on agarose and removing unbound
protein by centrifugation. The dissociation constant of the
resin±nAChR complex was estimated to ca. 20 mM (see
Experimental.).
40. Rozental, R.; Scoble, G. T.; Albuquerque, E. X.; Idriss,
M.; Sherby, S.; Sattelle, D. B.; Nakanishi, K.; Konno, K.;
9. Nakanishi, K.; Goodnow, R.; Konno, K.; Niwa, M.;

1194
M. Hashimoto et al. / Bioorg. Med. Chem. 7 (1999) 1181±1194
Eldefrawi, A.; Eldefrwai, M. J. Pharmacol. Exp. Therap. 1989,
249, 123.
41. Katz, E.; Cortes, V.; Eldefrawi, M. E.; Eldefrawi, A. T.
Toxicol. Appl. Pharmacol. 1997, 146, 146.
42. Anis, N.; Sherby, S.; Goodnow, R.; Niwa, M.; Konno, K.;
Kallimopoulos, T.; Bukownik, R.; Nakanishi, K.; Usherwood,
P.; Eldefrawi, A.; Eldefrawi, M. J. Pharmacol. Exp. Therap.
1990, 254, 764.
43. Mohr, P.; Pommerening, K. Anity Chromatography
Practical and Theoretical Aspects. Marcel Dekker: 1985.