Synthesis of hydrophobic phase-tagged prolyl peptides featuring rapid reaction/separation

Article abstract of DOI:10.1016/j.tet.2009.07.045

Hydrophobically tagged prolyl peptides were synthesized using an electrochemically modified prolyl moiety. The hydrophobic tag served a useful handle for the repetitive reaction/separation steps during solution-phase peptide synthesis. Furthermore, the ta

Full text of DOI:10.1016/j.tet.2009.07.045

Tetrahedron 65 (2009) 8014–8020
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of hydrophobic phase-tagged prolyl peptides featuring
rapid reaction/separation
*
Kazuhiro Chiba , Mari Sugihara, Kazumi Yoshida, Yuzuru Mikami, Shokaku Kim
Laboratory of Bio-organic Chemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2009
Received in revised form 20 July 2009
Accepted 20 July 2009
Available online 24 July 2009
Hydrophobically tagged prolyl peptides were synthesized using an electrochemically modified prolyl
moiety. The hydrophobic tag served a useful handle for the repetitive reaction/separation steps during
solution-phase peptide synthesis. Furthermore, the tag was also effective as an anchor on solid phases for
the evaluation of activity of immobilized peptides.
Ó 2009 Published by Elsevier Ltd.
1. Introduction
Introduction of the hydrophobic tags at the inert methylene
group of prolyl residues, which are typically located at relatively
Over the past few decades, peptide synthesis based on solid-
phase techniques has grown in popularity to become essential for
automated synthesis and combinatorial chemistry.1–5 Alternatively,
tag-assisted solution-phase organic synthesis has also developed
into an important technique for solid-supported and combinatorial
methodologies.6–8 Efforts to design effective phase-tags that can
improve the purification of the desired products using the solid-
phase extraction (SPE)9–11 have achieved higher reactivity with ease
of separation from solutions. Furthermore, effective phase-tags can
provide the possibility of using routine analytical methods to
monitor reaction processes, and also help directly determine the
structures of products on the liquid-phase-tags. In particular, hy-
drophobic phase-tagging strategy based on SPE featuring ODS-silica
(octadecylsilyl silica) has been recognized as an effective method to
effectively generate target products.
Recently, we have developed a cycloalkane-based thermomor-
phic biphasic reaction system that can readily accomplish the
multi-step synthesis of hydrophobic tag-assisted peptides.12–17
Specifically, benzyl moieties with long alkoxy groups can serve as
effective protective groups of C-terminal peptides and as hydro-
phobic tags that assist selective separation during the on-cooling
phase separation of the cycloalkane solution from excessive re-
agents in polar organic solvents. Additionally, the hydrophobic tags
can also function as anchors for retention on the long alkyl chain-
modified beads in aqueous phases through hydrophobic in-
teractions. Retention on such hydrophobic beads can also provide
for the evaluation of biological and biochemical activities of pep-
tides on the immobilized peptides.
acute positions within a peptide or protein, can be favorable in
minimizing conformational changes of the entire molecule. The
specific site for connecting the tags with ‘hydrophilic’ linkers would
also effectively repel the peptides from the surface of the beads for
the evaluations of the functions of peptides immobilized on the
solid phase. Furthermore, the prolyl residues can function as either
the N- or C-terminal residues of peptides during fragment syn-
theses because of its resistance to isomerization during activation
of the carboxylate moiety.
Previously, we have developed an efficient method for in-
troducing various functional groups at the N-a
0-position of prolyl
moieties via electrochemical oxidation in a LiClO4/CH3NO2 elec-
trolyte solution (Scheme 1).18–21 In this case, our electrochemical
methodology was applied toward the novel synthesis of hydro-
phobically tagged prolyl peptides. Herein, we report the syn-
thesis of targeted hydrophobic phase-tagged prolyl residues
featuring an anodically introduced proline derivative as the key
intermediate. Furthermore, our methodology was employed in
the solution-phase synthesis and activity-evaluations of prolyl
peptides (Fig. 1).
O
-2e-
Nu
O
O
Nu
N
N
N
X
OR'
1.0 M LiClO4/ MeNO2
50 mM AcOH
OR'
OR'
R
O
R
O
R
O
1) -2e-
TMS
2)
CO2Me
N
CO2Me
N
CO2Me
1.0 M LiClO4 / MeNO2
97%
CO2Me
* Corresponding author. Tel.: þ81 42 367 5667; fax: þ81 42 360 7167.
E-mail address: chiba@cc.tuat.ac.jp (K. Chiba).
Scheme 1.
0040-4020/$ – see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.tet.2009.07.045

K. Chiba et al. / Tetrahedron 65 (2009) 8014–8020
8015
O
O
H
H
a
b
C18H37
O
OC18H37
HO
OH
C18H37
O
OC18H37
OC18H37
OC18H37
8
10
OH
9
Scheme 4. (a) C18H37Br, K2CO3, DMI, 70 ꢀC, 20 h (b) Ph3PCH3Br, PhLi, toluene, reflux,
6 h, 58% (two steps).
(5), whereas 10 was synthesized from 2,3,4-trihydroxybenzaldehyde
(8) via octadecylation of the phenol groups followed by Wittig re-
action. A solutionofcis-3 andhydrophobic tags 6, 7, or10 wasrefluxed
in anhydrous CH2Cl2 in the presence of Grubbs catalyst (second
generation),thenpurifiedusingcolumnchromatographytoaffordthe
corresponding hydrophobic phase-tagged proline derivatives (11,
52%; 12, 86%; 13, 9%, respectively) (Scheme 5). In the case of 12, the
acrylic moiety was reactive with the allyl group of the proline de-
rivative, even in the presence of highly hydrophobic groups.
Figure 1. Concept of multi-step solution-phase peptides on a hydrophobic tag with
ease of separation and retention of those immobilized products on the hydrophobic
beads for the activity evaluation.
2. Results and discussion
COOMe
C18H37
O
O
N
O
2.1. Electrochemical synthesis of cis-allyl-proline
Boc
C18H37
11
OC18H37
First, Boc-L-Pro-OMe (1) was electrolyzed at 1.9 V (vs Ag/AgCl) by
O
direct anodic oxidation using a glassy carbon anode and a platinum
cathode in a 1.0 M LiClO4/CH3NO2 electrolyte solution in the presence
of acetic acid (50 mM) at 0 ꢀC (Scheme 2). Upon completion of the
oxidation with the passage of an electrical current of 2.2 F/mol, an
excess amount of allyltrimethylsilane 2 was added. The reaction
COOMe
C18H37
O
COOMe
N
O
N
Boc
Boc
C18H37
O
6,7,10
Glubbs cat. CH2Cl2
OC18H37
OC18H37
12
C18H37
O
O
mixture was allowed to stand at rt for 16 h to give Boc-L-(5-allyl)Pro-
BocN
OMe (3) as an inseparable 1:1 mixture of cis- and trans-isomers in
77% yield.22–27 Subsequently, the isomers were separated using col-
umn chromatography after removal of the Boc protection group. The
purified cis-isomer of 4 (cis-4) was re-protected using Boc2O in
CH3CN to give the cis-isomer of 3 (cis-3) in 68% yield.
C18H37
COOMe
13
Scheme 5. Synthesis of hydrophobically tagged proline derivatives.
Subsequently, based on these preliminary studies, we undertook
the synthesis of a hydrophobic tag that possesses a hydrophilic spacer
with a terminal acrylamide group. When connected to an allylprolyl
group, which would allow peptide-elongation, the hydrophilic spacer
should serve to extending the key peptide units into the aqueous
phases, while repelling from the intramolecular tags that are an-
chored on the solid phases. Accordingly, as shown in Scheme 6, 2-20-
(ethylenedioxy)diethylamine was introduced as a hydrophilic spacer
for 3,4,5-tris(octadecyloxy)benzoic acid (14), followed by the in-
troduction of an acrylamide moiety to give 16 (59% in TWO steps)
(Scheme 6). As shown in Scheme 7, the Grubbs coupling reaction was
carried out by refluxing a solution of hydrophobic tag 16 and cis-3 in
anhydrous CH2Cl2 in the presence of Grubbs catalyst (second gener-
ation). Addition of ethyl vinyl ether afforded the hydrophobic phase-
tagged Boc-proline methyl ester (17), which was precipitated by the
addition of CH3CN. The methyl ester group of 17 was deprotected via
hydrolysis using aqueous 1 M LiOH. The residue was purified using
flash chromatography to give 18 as white powder in 71% yield (from
16) (Scheme 7).
a,b
c
COOMe
N
H
COOMe
COOMe
3
N
N
trans-4
Boc
Boc
1
d
COOMe
COOMe
cis-4
N
N
Boc
H
cis-3
Scheme 2. (a) ꢁ2eꢁ, 1 M LiClO4/CH3NO2, 50 mM AcOH; (b) allylTMS (2); (c) 4 M HCl/
EtOAc, rt; (d) Bc2O, DMAP, CH3CN, rt.
2.2. Synthesis of hydrophobic phase-tagged proline
derivatives
To assess the reactivity of the alkene moiety toward olefin cross-
metathesis, pure cis-3 was reacted with three hydrophobic tags: 1,2,
3-tris-octadecyloxy-5-pent-4-enyloxymethyl-benzene(6), 3,4,5-tris-
octadecyloxybenzyl acrylate (7) (Scheme 3), and 1,2,3-tris-octadecy-
loxy-4-vinylbenzene (10) (Scheme 4), which possess an aliphatic, an
acrylate, and a conjugated (with aromatic ring) alkene as the terminal
moieties, respectively. As shown in Schemes 3 and 4, compounds 6
and 7 were synthesized from 1,2,3-tris-octadecyloxybenzyl alcohol
O
O
C18H37
O
O
C18H37
O
O
NH2
OH
N
H
O
a
C18H37
O
O
C18H37
O
O
a
OH
O
C18H37
C18H37
O
C18H37
C18H37
OC18H37 14
OC18H37
O
15
6
OC18H37
OC18H37
5
O
H
N
b
C18H37
O
O
O
C
18H37
O
O
b
N
H
O
O
O
C18H37
C18H37
16
OC18H37
OC18H37
59% (2 steps)
7
Scheme 6. (a) 2,20-(Ethane-1,2-diylbis(oxy))diethanamine, 14, DIPCI, HOBt, THF, rt;
Scheme 3. (a) 5-Bromo-pent-1-ene, NaH, DMF, rt to 80 C, 5 h, 63%; (b) acrylic acid,
DIPCI, DMAP(cat.), CH2Cl2, reflux, 3 h, 83%.
(b) acryloyl chloride, TEA, THF, rt.

8016
K. Chiba et al. / Tetrahedron 65 (2009) 8014–8020
O
COOMe
N
Tag
Boc
16
cis-3
O
COOR
a
N
17 R = Me
18 R = H
71% (2steps)
b
Tag
Boc
O
H
N
C18H37
C18H37
O
O
O
O
N
H
O
=
O
16
Tag
Figure 2. Microscopic observation of a pyrene-modified tagged peptide retained on
OC18H37
ODS; (a) in the presence of the reaction mixture of 4-(pyren-1-yl)butanoic acid and 21;
(b) in the presence of the reaction mixture of 4-(pyren-1-yl)butanoic acid without 21
as a control.
Scheme 7. (a) Grubbs catalyst second generation, CH2Cl2, reflux; (b) LiOH, THF/water,
35 ꢀC.
Using the hydrophobic phase-tagged Boc-proline 18, the syn-
thesis of a tagged lactotripeptide (VPP) was carried out as a model
reaction to illustrate both N- and C-terminal peptide-bond elon-
gations based on the tagged proline moiety. The C-terminal of 18
was activated at rt using diisopropylcarbodiimide (DIPCI) and
1-hydroxy-1H-benzotriazole (HOBt) (Scheme 8). The subsequent
coupling between activated 18 and NH-Pro-OMe HCl was carried
out in anhydrous THF in the presence of N,N-diisopropylethylamine
(DIPEA). Upon stirring the reaction for 1 h at rt, an excess amount of
CH3CN was poured into the reaction mixture to precipitate the
protected dipeptide 19 in 96% yield. After deprotection of the Boc
group in anhydrous THF using 4 M HCl/EtOAc at rt, and removal of
excess HCl in vacuo, deprotected 19 was redissolved in THF, treated
with DIPEA, then coupled with Fmoc-Val-OH using DIPCI and HOBt.
Upon precipitation by the addition of excess CH3CN, protected tri-
peptide 20 was obtained in 91% yield as a white powder. The Fmoc
group was removed by treatment with LiOH in THF to afford lac-
totripeptide 21 in 93% yield after recrystallization from THF/CH3CN.
Tag
peptide elongation
19
Tag
H-Pro-Pro-OMe
Fmoc-Lys(Boc)-Gly-Tyr(t-But)-Gly-Gly-Phe-Leu
Pro-Pro-OMe
Tag
23
Fmoc-Lys-Gly-Tyr-Gly-Gly-Phe-Leu Pro-Pro-OMe
24
Scheme 9. An example of peptide elongation on the hydrophobic phase-tagged prolyl
peptides.
removing the Boc group of 19, Fmoc-Leu-enkephalin moiety
(Fmoc-Tyr-Gly-Gly-Phe-Leu-OH) was constructed stepwise on the
prolyl NH group. Furthermore, Fmoc-Gly-OH and following Fmoc-
Lys(Boc)-OH were attached in turn to introduce as a terminal hy-
drophilic moiety. Finally, Boc and t-Bu groups on the side chains of
compound 23 were removed to confirm the completion of typical
deprotection reactions of side chains. Each reaction and isolation of
the tagged products were also confirmed to be accomplished by
TLC analysis (CHCl3/MeOH 9:1) in each step, and the final product
24 detected as a single spot on the TLC was analyzed by TOF MS
(ESIMS). These sequential schemes showed that the tagged prolyl
peptides could also be used as platforms for the solution-phase
multi-step peptide syntheses.
Tag
Tag
a
O
BocN
O
BocN
N
O
COOH
19
18
MeOOC
FmocHN
H2N
O
O
d
b,c
Tag
Tag
O
O
N
N
O
N
O
N
20
MeOOC
HOOC
21
3. Conclusion
Scheme 8. (a) HCl-NH-Pro-OMe, DIPCI, HOBt, DIPEA, rt, 96%; (b) 4 M HCl/EtOAc, THF,
rt; (c) Fmoc-Val-OH, DIPCI, HOBt, DIPEA, rt, 91% (two steps); (d) LiOH, THF/water,
35 ꢀC, 93%.
Hydrophobic phase-tagged lactotripeptide (VPP) was synthesized
as a model compound following the methodology of sequential
precipitation. Introduction of a 3,4,5-tris(octadecyloxy)phenyl group
as a hydrophobic tag was successfully accomplished via olefin me-
tathesis between the acrylamide moiety of the hydrophobic tag and
the electrochemically synthesized 5-allylprolyl residue. In the case of
multi-step peptide syntheses, the hydrophobic tag was effective as
a phase-tag by assisting the separation/purification of the product via
precipitation. Furthermore, the tagged peptide was shown to be ef-
fectively anchored on ODS beads, which would allow for evaluating
biochemical interactions of the peptide in aqueous dispersions.
To evaluate the hydrophobic tag as an anchor on hydrophobic
beads dispersed in aqueous solutions, 4-(pyren-1-yl)butanoic acid
was introduced as a fluorescence marker to the N-terminal of 21 to
give 22.28–30 Upon completion of the condensation reaction, ODS
beads were dispersed within the reaction mixture, then washed
with water. Subsequently, the fluorescence of an aqueous disper-
sion of the ODS beads was measured using UV-irradiation (Fig. 2a).
As a control experiment, an aqueous dispersion of ODS beads that
was subjected to a reaction mixture of THF without 21 was also
measured (Fig. 2b). In the absence of 21, the fluorescence was
scarcely observed (Fig. 2b). The fluorescence was also not observed
on the ODS after the reaction of 4-(pyren-1-yl)butanoic acid and
corresponding tripeptide (H-Val-Pro-Pro-OH)31 in the presence of
ODS followed by washing. The presence of fluorescence on the
surface of ODS (Fig. 2a) suggests that the hydrophobic phase-tag-
ged peptide are retained on the surfaces of hydrophobic ODS beads.
In addition, fragment peptide-elongation reactions were per-
formed to confirm the functions of the hydrophobic tag for sepa-
rations in each of muti-step reactions. As shown in Scheme 9, after
4. Experimental section
4.1. Spectroscopic measurements
NMR spectra were measured in CDCl3 using JEOL AL-400 and AL-
600 spectrometers at 400, 600 (1H) MHz and at 100, 150 (13C) MHz,
with tetramethylsilane as the internal standard. MS-spectra (MALDI,
ESI) and infrared spectra were measured on Voyager-DE STR MALDI-
TOF MS, JMS-T100LC AccuTOF, and JEOL WINSPEC-50, respectively.

K. Chiba et al. / Tetrahedron 65 (2009) 8014–8020
8017
4.2. Microscopic observations
of MeOH), then by column chromatography (n-hexane/EtOAc 30:1)
to afford 6 as white powder (705.0 mg, 63% yield). Mp 44–45 ꢀC; 1H
ODS-silica was observed using LEICA MZ 16F equipped with
LEICA 10447160 (10x/21B), LEICA 10447243 (PLANAPO 5.0x/0.50
LWD), under UV-irradiation (LEICA Filter 10447287).
NMR (600 MHz, CDCl3)
d
6.52 (2H, s), 5.82 (1H, ddt, J¼16.9, 10.3,
6.6 Hz), 5.02 (1H, ddt, J¼16.9, 1.8, 1.8 Hz), 4.96 (1H, ddt, J¼10.3, 1.8,
1.0 Hz), 4.39 (2H, s), 3.96 (4H, t, J¼6.6 Hz), 3.92 (2H, t, J¼6.6 Hz),
3.47 (2H, t, J¼6.6 Hz), 2.17–2.12 (2H, m),1.81–1.76 (4H, m),1.76–1.69
(4H, m), 1.49–1.43 (6H, m), 1.37–1.21 (84H, m), 0.88 (9H, t,
4.3. Synthetic procedures
J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
d 153.1, 138.3, 137.6, 133.6,
4.3.1. Boc-
L
-(5-allyl)Pro-OMe (3). A solution of Boc-L-Pro-OMe (1,
114.7, 106.2, 73.4, 73.1, 69.7, 69.1, 31.9, 30.4, 30.3, 29.8, 29.7, 29.7,
1.0 mmol) in 1.0 M LiClO4/anhydrous CH3NO2 (20 mL) was electro-
lyzed under constant current at 0 ꢀC, in the presence of 50 mM acetic
acid, using an undivided cell equipped with a glassy carbon plate
anode (60 mmꢂ20 mm) and a Pt cathode (10 mmꢂ10 mm) under an
Ar atmosphere. Upon passage of 2.2 F/mol, the reaction mixture was
treated with allyltrimethylsilane (2, 3.0 mmol), then allowed to stand
for 16 h at rt. After concentration, the residue was purified using
silica-gel column chromatography (n-hexane/EtOAc 6:1) to afford 3
as a mixture of cis- and trans-isomers (68% yield, colorless oil). 1H
29.6, 29.4, 29.4, 29.0, 26.1, 26.1, 22.7, 14.1; IR (thin film) nmax 3076,
2954, 2848, 1716, 1587, 1502, 1468, 1437, 1335, 1227, 1120 cmꢁ1
;
MALDI-TOF MS (m/z): calcd for C66H125O4 981.9663, found
981.9653.
4.3.5. 3,4,5-Tris-octadecyloxybenzyl acrylate (7). To a solution of 5
(3.33 g, 3.65 mmol) in CH2Cl2 (60 mL) at rt was added a solution of
acrylic acid (0.50 mL, 7.3 mmol), DIPCI (2.3 mL, 114.6 mmol), and
DMAP (134.4 mg, 1.10 mmol). After refluxing for 3 h, the reaction
solution was concentrated in vacuo. The residue was purified by
recrystallization (MeOH), then by silica-gel column chromatogra-
phy (n-hexane/EtOAc 30:1) to afford 7 as a white powder (2.93 mg,
NMR (600 MHz, CDCl3) d 5.85–5.73 (1H, m), 5.10–5.03 (2H, m), 4.34–
4.08 (1H, m), 3.98–3.83 (1H, m), 3.74–3.71 (3H, m), 2.74–2.43 (1H, m),
2.25–2.09 (2H, m), 2.00–1.87 (2H, m), 1.82–1.71 (1H, m), 1.48–1.40
(9H, m); 13C NMR (150 MHz, CDCl3)
d
173.9, 173.7, 173.6, 173.3, 154.3,
83% yield). Mp 54–56 ꢀC; 1H NMR (600 MHz, CDCl3)
d 6.56 (2H, s),
154.2, 153.6, 153.5, 135.3, 135.0, 135.0, 117.3, 117.2, 116.9, 79.9, 79.9,
79.8, 60.3, 60.1, 59.8, 59.7, 59.4, 58.0, 57.9, 57.5, 57.4, 52.0, 52.0, 51.8,
39.1, 38.9, 38.1, 38.1, 29.5, 28.7, 28.4, 28.2, 28.0.
6.45 (1H, dd, J¼17.2, 1.5 Hz), 6.17 (1H, dd, J¼17.2, 10.6 Hz), 5.85 (1H,
dd, J¼10.6, 1.5 Hz), 5.09 (2H, s), 3.96 (4H, t, J¼6.6 Hz), 3.94 (2H, t,
J¼6.6 Hz), 1.82–1.76 (4H, m), 1.76–1.70 (2H, m), 1.49–1.43 (6H, m),
1.37–1.22 (84H, m), 0.88 (9H, t, J¼7.0 Hz); 13C NMR (150 MHz,
4.3.2. cis-NH-
L
-(5-Allyl)Pro-OMe (cis-4). The mixture of the cis-
CDCl3) d 153.2, 138.3, 131.0, 130.7, 128.4, 117.4, 107.0, 73.4, 69.2, 66.7,
and trans-isomers of 3 (0.5 mmol) was treated with 4 M HCl in
EtOAc (60 mL). After stirring for 30 min, the reaction mixture was
concentrated, diluted with 100 ml of EtOAc, then washed with
100 mL of NaHCO3 (satd) and 100 mL of brine. The resulting organic
layer was dried over anhydrous Na2SO4, then concentrated to give 4
as a mixture of diastereomers (169 mg, 83%, cis/trans 1:1), which
were separated using silica-gel column chromatography (Et2O/
MeOH 97:3) to afford pure cis-4 as a colorless oil (45% yield). 1H
31.9, 30.3, 29.8, 29.7, 29.7, 29.7, 29.6, 29.4, 29.4, 29.4, 26.1, 26.1, 22.7,
14.1; IR (thin film) nmax 3064, 2956, 2918, 2848, 1726, 1593, 1512,
1468, 1439, 1336, 1124 cmꢁ1; MALDI-TOF MS (m/z): calcd for
C64H119O5 967.9057, found 967.9042.
4.3.6. 1,2,3-Tris-octadecyloxy-4-vinylbenzene (10). To a solution of
stearyl bromide (6.04 g, 18.1 mmol) in DMI (30 mL) at 70 ꢀC was
added 2,3,4-trihydroxybenzaldehyde (807.6 mg, 5.24 mmol), fol-
lowed by the addition of K2CO3 (3.0 g). After stirring at 70 ꢀC for
20 h, the reaction mixture was treated with water (50 mL), then
extracted, while warm, with n-hexane (100 mL). The n-hexane
layer was dried over anhydrous Na2SO4, then concentrated in
vacuo. The residue was purified by recrystallization (EtOAc/MeOH)
to afford the precursor of 10.
NMR (600 MHz, CDCl3)
d
5.82 (1H, ddd, J¼17.2, 10.3, 7.0 Hz), 5.14
(1H, dd, J¼17.2, 1.5 Hz), 5.08 (1H, dd, J¼10.3, 1.5 Hz), 3.85–3.83 (1H,
m), 3.74 (3H, s), 3.22–3.17 (1H, m), 2.36–2.25 (2H, m), 2.17–2.10 (1H,
m), 1.97–1.88 (2H, m), 1.41–1.33 (1H, m); 13C NMR (150 MHz, CDCl3)
d
175.0, 134.8, 117.3, 59.1, 58.6, 52.2, 39.5, 30.8, 30.0.
4.3.3. cis-Boc- -(5-allyl)Pro-OMe (cis-3). To solution of cis-4
L
a
Separately, to a solution of Ph3PCH3Br (1.57 g, 4.41 mmol) in
anhydrous toluene (80 mL) was added PhLi (2.1 mL, 4.41 mmol) at
0 ꢀC, then allowed to warm to rt while stirring for 1 h. To this so-
lution was added a solution of the precursor of 10 (2.0 g, 2.2 mmol)
in anhydrous toluene (80 mL). After refluxing for 6 h, the reaction
mixture was concentrated in vacuo. The residue was purified by
recystallization (EtOAc/MeOH), then by silica-gel column chroma-
tography (n-hexane/EtOAc 30:1) to afford 10 as a white powder
(43 mg, 2.6 mmol) in anhydrous CH3CN (50 mL) was added Boc2O
(84 mg, 3.8 mmol) and DMAP (22 mg, 0.18 mmol). After stirring at
rt for 1 h, the reaction mixture was diluted with aqueous 5% citric
acid (100 mL), then extracted twice with 50 mL of EtOAc. The
combined organic layer was washed with 100 mL of NaHCO3 (satd)
and brine (100 mL), dried over anhydrous Na2SO4, then concen-
trated in vacuo. The residue was purified using silica-gel column
chromatography (n-hexane/EtOAc 6:1) to afford cis-3 as colorless
(2.76 mg, 58% yield). Mp 53–54 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.16
oil (68% yield). 1H NMR (600 MHz, CDCl3)
d
5.85–5.77 (1H, m), 5.08
(1H, d, J¼8.8 Hz), 6.94 (1H, dd, J¼18.0, 11.0 Hz), 6.63 (1H, d,
J¼8.8 Hz), 5.61 (1H, dd, J¼18.0, 1.5 Hz), 5.15 (1H, dd, J¼11.0, 1.5 Hz),
3.99–3.94 (6H, m), 1.84–1.78 (2H, m), 1.78–1.72 (4H, m), 1.50–1.42
(6H, m), 1.37–1.22 (84H, m), 0.88 (9H, t, J¼7.0 Hz); 13C NMR
(1H, dd, J¼17.2, 1.5 Hz), 5.04 (1H, d, J¼10.3 Hz), 4.35–4.19 (1H, m),
3.98–3.83 (1H, m), 3.74 (3H, s), 2.75–2.59 (1H, m), 2.24–2.15 (2H,
m), 2.00–1.86 (2H, m), 1.82–1.76 (1H, m), 1.48–1.39 (9H, m); 13C
NMR (150 MHz, CDCl3)
d
173.8, 173.6, 154.2, 153.5, 135.3, 116.8, 79.9,
(150 MHz, CDCl3) d 153.2,151.1,141.8,131.4,124.8,120.1,112.4,108.5,
60.1, 59.7, 58.0, 57.9, 52.0, 51.8, 39.1, 38.1, 29.5, 28.7, 28.3, 28.2, 28.0.
74.0, 73.6, 68.7, 31.9, 30.4, 30.3, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4,
26.2, 26.1, 22.7, 14.1; IR (thin film) nmax 3080, 2956, 2916, 2848,
1626, 1595, 1496, 1466, 1375, 1294, 1093 cmꢁ1; MALDI-TOF MS
(m/z): calcd for C62H117O3 909.9003, found 909.9009.
4.3.4. 1,2,3-Tris-octadecyloxy-5-pent-4-enyloxymethyl-benzene
(6). To a heated solution of 5 (1043.4 mg, 1.14 mmol) in DMF
(30 mL) (heated to 80 ꢀC for complete dissolution) were added NaH
(137.2 mg, 3.43 mmol) and 5-bromo-pent-1-ene (0.27 mL,
2.28 mmol). After stirring at 80 ꢀC for 5 h, the reaction mixture was
quenched with 10 mL of methanol followed by addition of 30 mL
of water, then extracted, while warm, with n-hexane (100 mL). The
n-hexane layer was dried over anhydrous Na2SO4, then concen-
trated in vacuo. The residue was purified by recrystallization (20 mL
4.4. General procedure for hydrophobic phase-tagged
proline derivatives by cross-metathesis
To a solution of hydrophobic tag 6, 7, or 10 (0.1 mmol) and cis-3
(0.1 mmol) in anhydrous CH2Cl2 (30 mL) was added Grubbs catalyst
(second generation) 4 (0.005 mmol), then refluxed for 12 h under

8018
K. Chiba et al. / Tetrahedron 65 (2009) 8014–8020
an Ar atmosphere. To this solution was added ethyl vinyl ether
(0.1 mL, 1.0 mmol), then stirred for 1 h to quench the catalyst. The
hydrophobic proline derivative was crystallized with CH3CN. The
resulting crystals were isolated by filtration, rinsed with CH3CN to
remove unreacted cis-300, then purified using silica-gel column
chromatography (n-hexane/EtOAc 20:1) to afford the correspond-
ing hydrophobic phase-tagged proline derivatives (11, 52%; 12, 86%;
13, 9%, respectively).
followed by silica-gel flash chromatography (CHCl3/MeOH 98:2) to
afford 16 as a white powder (1.28 mg, 59% yield). Mp 88–90 ꢀC; 1H
NMR (600 MHz, CDCl3)
d
7.00 (2H, s), 6.59 (1H, t, J¼5.5 Hz), 6.28
(1H, dd, J¼16.9, 1.5 Hz), 6.14 (1H, br), 6.10 (1H, dd, J¼16.9, 10.3 Hz),
5.62 (1H, dd, J¼10.3, 1.5 Hz), 4.02–3.96 (6H, m), 3.68 (2H, t,
J¼5.1 Hz), 3.67–3.62 (6H, m), 3.58 (2H, t, J¼5.1 Hz), 3.52–3.49 (2H,
m), 1.83–1.77 (4H, m), 1.76–1.71 (2H, m), 1.50–1.42 (6H, m), 1.36–
1.23 (84H, m), 0.88 (9H, t, J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
d
167.5, 165.5, 153.0, 141.3, 130.8, 129.4, 126.4, 106.0, 73.5, 70.3, 70.1,
4.4.1. cis-Boc-
enyl]}-OMe (11). White powder. Mp 42–44 ꢀC; 1H NMR (600 MHz,
CDCl3) 6.52 (2H, s), 5.55–5.37 (2H, m), 4.41–4.37 (2H, m), 4.33–
4.18 (1H, m), 4.12–3.90 (7H, m), 3.72 (3H, s), 3.48–3.43 (2H, m),
2.69–2.53 (1H, m), 2.19–2.06 (4H, m), 1.98–1.89 (2H, m), 1.84–1.70
(9H, m), 1.48–1.40 (15H, m), 1.36–1.23 (84H, m), 0.88 (9H, t,
L
-Pro{5-[6-(3,4,5-tris-octadecyloxy-benzyloxy)-hex-2-
69.9, 69.7, 69.4, 39.9, 39.2, 31.9, 30.3, 29.7, 29.6, 29.4, 29.4, 29.3, 26.1,
22.7, 14.1; IR (thin film) nmax 3307, 3253, 3080, 3034, 2954, 2916,
2848, 1659, 1626, 1581, 1551, 1504, 1470, 1425, 1382, 1351, 1236,
1124 cmꢁ1; ESIMS (m/z): 1112 (MþHþ), 1114 (MþNaþ). HR-ESIMS
(m/z) calcd for C70H131N2O7 1111.9956, found 1111.9956.
d
J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
d
173.8, 173.5, 154.1, 153.4,
4.4.5. cis-Boc-L-Pro(5-{5-[3-(2-{2-[2-(3,4,5-tris-octadecyloxy-
153.0, 137.4, 133.5, 132.2, 127.0, 106.0, 79.7, 73.2, 73.1, 69.7, 69.0,
68.9, 60.0, 59.6, 58.3, 51.9, 51.7, 37.8, 36.7, 31.9, 30.3, 29.7, 29.7, 29.6,
29.4, 29.3, 28.7, 28.3, 28.2, 28.0, 26.1, 26.1, 22.6, 14.0; IR (thin film)
nmax 2956, 2916, 2850, 1753, 1701, 1591, 1504, 1468, 1437, 1389,
1115 cmꢁ1; ESIMS (m/z): 1223 (MþHþ), 1245 (MþNaþ). HR-ESIMS
(m/z) calcd for C78H143NNaO8 1245.0711, found 1245.0731.
benzoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-allyl]})-OH
(18). To a solution of hydrophobic tag 16 (0.1 mmol) and cis-3
(0.1 mmol) in anhydrous CH2Cl2 (30 mL) was added Grubbs (cata-
lyst second generation) (0.005 mmol), then refluxed for 12 h under
an Ar atmosphere. To this solution was added ethyl vinyl ether
(0.1 mL, 1.0 mmol), then stirred for 1 h to quench this catalyst. The
tagged proline derivative was crystallized from CH3CN. The crystals
were isolated by filtration, rinsed with CH3CN to remove unreacted
cis-3, dissolved in THF (8 mL), then treated with aqueous 1 M LiOH
(2 mL). After stirring at 35 ꢀC for 24 h, the organic layer was sepa-
rated, then dried over anhydrous Na2SO4. The crude product was
purified by recrystallization (CH3CN), followed by silica-gel flash
chromatography (CHCl3/MeOH 98:2) to afford 18 as a white powder
(95.1 mg, 71% yield). Mp 74–78 ꢀC; 1H NMR (600 MHz, CDCl3)
4.4.2. cis-Boc-
carbonyl)-allyl]}-OMe (12). White powder. Mp 49–51 ꢀC; 1H NMR
(600 MHz, CDCl3) 7.03–6.96 (1H, m), 6.57–6.54 (2H, m), 5.94 (1H,
L-Pro{5-[3-(3,4,5-tris-octadecyloxy-benzyloxy-
d
d, J¼15.8 Hz), 5.05 (2H, s), 4.36–4.20 (1H, m), 4.06–3.97 (1H, m),
3.96 (4H, t, J¼6.6 Hz), 3.93 (2H, t, J¼6.6 Hz), 3.75––3.71 (3H, m),
2.91–2.72 (1H, m), 2.44–2.34 (1H, m), 2.25–2.18 (1H, m), 2.04–1.91
(2H, m), 1.82–1.70 (7H, m), 1.49–1.39 (15H, m), 1.36–1.22 (84H, m),
0.88 (9H, t, J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
d
173.7, 173.6,
d 7.11–7.01 (3H, m), 6.78–6.70 (1H, m), 6.46–6.39 (1H, m), 5.87 (1H,
166.2, 153.1, 146.2, 146.1, 138.2, 138.0, 130.9, 130.8, 123.1, 122.9, 107.1,
106.8, 80.4, 80.2, 73.4, 69.1, 66.5, 66.4, 60.0, 59.6, 57.3, 57.1, 52.2,
52.0, 37.9, 36.6, 31.9, 30.3, 30.2, 29.7, 29.7, 29.6, 29.6, 29.4, 29.4, 29.3,
28.9, 28.7, 28.3, 28.2, 28.0, 26.1, 26.1, 22.7, 14.1; IR (thin film) nmax
2954, 2916, 2850, 1751, 1699, 1589, 1468, 1439, 1390, 1333, 1174,
1119 cmꢁ1; ESIMS (m/z): 1231 (MþNaþ). HR-ESIMS (m/z) calcd for
C68H137NNaO9 1231.0191, found 1231.0196.
d, J¼15.4 Hz), 4.32–4.18 (1H, m), 4.14–4.05 (1H, m), 4.00 (4H, t,
J¼6.6 Hz), 3.98 (2H, t, J¼6.6 Hz), 3.79–3.73 (2H, m), 3.73–3.62 (4H,
m), 3.62–3.57 (2H, m), 3.57–3.50 (2H, m), 3.50–3.41 (2H, m), 3.39–
3.31 (1H, br), 2.89–2.56 (1H, m), 2.38–2.24 (1H, m), 2.20–2.09 (1H,
m), 2.07–1.94 (2H, m), 1.84–1.69 (8H, m), 1.49–1.42 (15H, m), 1.36–
1.22 (84H, m), 0.88 (9H, t, J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
d
173.9, 167.8, 166.1, 155.5, 153.0, 141.2, 138.6, 129.1, 127.2, 106.0,
73.5, 70.2, 70.0, 69.9, 69.3, 68.1, 60.3, 57.4, 39.8, 39.2, 37.2, 31.9, 30.3,
29.7, 29.7, 29.7, 29.6, 29.4, 29.4, 29.3, 28.3, 26.1, 26.1, 22.7, 14.1; IR
(thin film) nmax 3261, 3088, 2954, 2916, 2848,1736,1697,1670,1635,
1581, 1545, 1502, 1470, 1425, 1389, 1350, 1238, 1122 cmꢁ1; ESIMS
(m/z): 1339 (MþHþ), 1345 (MþLiþ), 1361 (MþNaþ). HR-ESIMS (m/z)
calcd for C81H148N3O11 1339.1114, found 1339.1128.
4.4.3. cis-Boc-
OMe (13). White powder. Mp 47.0–47.5 ꢀC; 1H NMR (600 MHz,
CDCl3) 7.13–7.09 (1H, m), 6.66–6.59 (2H, m), 6.13–6.06 (1H, m),
L-Pro{5-[3-(2,3,4-tris-octadecyloxy-phenyl)-allyl]}-
d
4.36–4.20 (1H, m), 4.06–3.97 (1H, m), 3.97–3.92 (6H, t, J¼6.2 Hz),
3.74 (3H, s), 2.85–2.71 (1H, m), 2.41–2.35 (1H, m), 2.21–2.15 (1H,
m), 2.02–1.95 (1H, m), 1.92–1.71 (8H, m), 1.49–1.40 (15H, m), 1.36–
1.24 (84H, m), 0.88 (9H, t, J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
4.4.6. cis-Boc-L-Pro(5-{5-[3-(2-{2-[2-(3,4,5-tris-octadecyloxy-
d
173.9, 173.6, 154.3, 153.6, 152.6, 152.5, 150.6, 141.9, 126.5, 126.4,
benzoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-allyl]})-Pro-OMe
(19). To a solution of 18 (446.8 mg, 0.33 mmol) in THF (30 mL) was
added a mixture of DIPCI (0.10 mL, 0.67 mmol) and HOBt (90.5 mg,
0.67 mmol), then stirred at rt for 90 min. To this reaction mixture
was added a solution of H-Pro-OMe/HCl (82.0 mg, 0.5 mmol) and
DIPEA (0.17 mL, 0.99 mmol) in anhydrous THF (6 mL), then stirred
at rt for 1 h. The product was precipitated by the addition of CH3CN
(30 mL) to afford 19 as a white powder (459.8 mg, 96% yield). Mp
125.9, 125.7, 124.9, 124.7, 120.2, 120.1, 108.6, 79.9, 74.0, 73.6, 68.7,
60.3, 59.8, 58.6, 58.2, 52.1, 51.9, 38.5, 37.7, 31.9, 30.4, 29.7, 29.7, 29.6,
29.6, 29.6, 29.4, 29.4, 29.4, 28.8, 28.5, 28.4, 28.3, 28.1, 26.2, 26.1, 22.7,
14.1; IR (thin film) nmax 2956, 2916, 2850, 1753, 1701, 1470, 1394,
1296, 1173, 1090 cmꢁ1; ESIMS (m/z): 1151 (MþHþ). HR-ESIMS (m/z)
calcd for C74H136NO7 1151.0317, found 1151.0311.
4.4.4. N-{2-[2-(2-Acryloylamino-ethoxy)-ethoxy]-ethyl}-3,4,5-tris-
octadecyloxybenzamide (16). To a solution of 14 (1.82 g, 1.96 mmol)
in anhydrous THF (100 mL) was added a mixture of DIPCI (0.61 mL,
3.93 mmol) and HOBt (531.0 mg, 3.93 mmol). After stirring at rt for
90 min, the reaction mixture was slowly added dropwise to a so-
lution of 2-20-(ethylenedioxy)diethylamine (0.86 mL, 5.89 mmol) in
anhydrous THF (50 mL), then stirred at rt for 1 h. The resulting
intermediate was purified by recrystallization (CH3CN), then
redissolved in anhydrous THF (200 mL). To this solution was added
a mixture of TEA (0.27 mL, 1.96 mmol) and acryloyl chloride
(0.48 mL, 5.89 mmol), then stirred at rt for 30 min. After concen-
tration, the residue was purified by recrystallization (THF/CH3CN),
67–69 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.06–7.00 (2H, m), 6.95–6.91
(1H, br), 6.83–6.73 (1H, m), 6.53–6.26 (1H, br), 5.92–5.85 (1H, m),
4.57–4.39 (2H, m), 4.03–3.95 (7H, m), 3.76–3.42 (16H, m), 2.85–
2.63 (1H, m), 2.52–2.43 (1H, m), 2.21–2.10 (2H, m), 2.03–1.90 (5H,
m), 1.84–1.70 (7H, m), 1.49–1.37 (15H, m), 1.36–1.22 (84H, m), 0.88
(9H, t, J¼7.0 Hz); 13C NMR (150 MHz, CDCl3)
d 172.7, 171.3, 167.4,
166.4, 154.3, 153.3, 153.0, 141.0, 140.8, 129.4, 125.8, 125.7, 105.8, 80.1,
79.8, 73.4, 70.3, 70.2, 70.2, 69.9, 69.9, 69.8, 69.3, 58.8, 58.8, 58.7,
57.7, 57.7, 52.2, 52.1, 46.5, 39.8, 39.1, 37.3, 36.0, 31.9, 30.3, 29.7, 29.6,
29.6, 29.4, 29.4, 29.3, 28.9, 28.8, 28.7, 28.4, 28.3, 28.0, 27.5, 26.1,
24.9, 24.8, 22.7, 14.1; IR (thin film) nmax 3500, 3263, 3078, 2953,
2914, 2848, 1747, 1695, 1668, 1633, 1581, 1543, 1504, 1470, 1394,

K. Chiba et al. / Tetrahedron 65 (2009) 8014–8020
8019
1394, 1338, 1238, 1122 cmꢁ1; ESIMS (m/z): 1450 (MþHþ), 1456
(MþLiþ), 1472 (MþNaþ). HR-ESIMS (m/z) calcd for C87H156N4LiO12
1456.1880, found 1456.1870.
was recovered from the cyclohexane layer (90%). Compound 22:
ESIMS (m/z) calcd for C106H169N5O12Na 1727.27, found 1727.2.
4.4.10. Elongation of peptide chains on the hydrophobic tag. To
a solution of compound 19 (26.2 mg, 0.018 mmol) in 2 ml of CH2Cl2,
4 M HCl/EtOAc (2 ml) was added and stirred for 90 min at rt. After
the completion of the deprotection of Boc group, the solution was
concentrated in vacuo. The residue was then dissolved in dry THF
4.4.7. Fmoc-Val-cis-L-Pro(5-{5-[3-(2-{2-[2-(3,4,5-tris-octadecyloxy-
benzoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-allyl]})-Pro-OMe
(20). To a solution of 19 (272.6 mg, 0.19 mmol) in anhydrous THF
(10 mL) was added 4 M HCl/EtOAc (10 mL). After stirring at rt for
1.5 h, the reaction mixture was concentrated in vacuo to remove
excess HCl. The residue was dissolved in anhydrous THF (20 mL),
treated with DIPEA (0.10 mL, 0.56 mmol), then stirred at rt 3 h. To
this reaction mixture was added a solution of Fmoc-Val-OH
(129.0 mg, 0.38 mmol), DIPCI (0.06 mL, 0.38 mmol), and HOBt
(51.3 mg, 0.38 mmol) in anhydrous THF (5 mL). After stirring at rt
for 1 h, the crude product was precipitated by the addition of
CH3CN (30 mL) to afford 20 as a white powder (288.9 mg, 91%
(2 ml) with DIPEA (13.6 ml, 0.072 mmol), stirred for 10 min at rt. The
reaction mixture was poured into the THF solution of Fmoc-Leu-OH
(0.036 mmol), HBTU (0.036 mmol), HOBt (0.036 mmol) and stirred
for 2 h at rt. Acetonitrile was then poured into the reaction mixture
to give the product as a precipitation. After separation of the pre-
cipitation by filtration, the product was then dissolved in 3 ml of
THF. DBU (0.045 ml) was added to the solution, stirred for 5 min at
rt to complete the deprotection of Fmoc group. The deprotected
product was then isolated as the precipitation by the addition of
acetonitrile and following filtration. According to the condensation
and deprotection, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,
Fmoc-Tyr(t-Bu)-OH, Fmoc-Gly-OH, and Fmoc-Lys (Boc)-OH were
introduced stepwise. Completion of the reactions and purification
of every reaction steps were confirmed by TLC (silica-gel, CHCl3/
MeOH 9:1, yield 67% from compound 19). Boc and t-Bu groups on
Lys and Tyr residues of compound 23 were removed by the acid
treatment with 4 M HCl/EtOAc (4 ml) in CH2Cl2 (2 ml) for 3 h at rt to
give 24. The product was isolated by the precipitation with CH3CN
followed by filtration. The structure of the isolated model com-
yield). Mp 75–78 ꢀC; 1H NMR (600 MHz, CDCl3)
d 7.76 (2H, d,
J¼7.3 Hz), 7.58 (2H, d, J¼7.3 Hz), 7.40 (2H, t, J¼7.3 Hz), 7.31 (2H, t,
J¼7.3 Hz), 6.99 (2H, s), 6.83 (1H, dt, J¼15.0, 7.3 Hz), 6.66 (1H, d,
J¼5.1 Hz), 6.25–6.15 (1H, m), 5.91 (1H, d, J¼15.0 Hz), 5.21 (1H, dd,
J¼9.2, 4.4 Hz), 4.65–4.60 (2H, m), 4.58–4.53 (1H, m), 4.44 (1H, dd,
J¼10.6, 7.0 Hz), 4.29 (1H, dd, J¼10.6, 7.0 Hz), 4.20 (1H, t, J¼7.0 Hz),
4.16 (1H, t, J¼9.2 Hz), 3.99 (4H, t, J¼6.2 Hz), 3.97 (2H, t, J¼6.2 Hz),
3.89–3.83 (1H, m), 3.70 (3H, s), 3.67–3.60 (10H, m), 3.59–3.54 (2H,
m), 3.53–3.45 (2H, m), 2.74–2.60 (1H, m), 2.30–2.19 (1H, m), 2.07–
1.91 (6H, m), 1.82–1.70 (8H, m), 1.49–1.42 (6H, m), 1.36–1.23 (84H,
m), 0.98 (6H, t, J¼6.6 Hz), 0.88 (9H, t, J¼7.0 Hz); 13C NMR (150 MHz,
CDCl3)
d
172.5, 171.1, 170.3, 167.4, 165.5, 156.1, 153.0, 143.7, 143.6,
pound 24 was confirmed by ESIMS. Fmoc-L-Lys-Gly-L-Tyr-Gly-Gly-
141.2, 141.1, 139.4, 129.3, 127.6, 127.0, 126.9, 126.5, 125.1, 124.9, 119.9,
119.8, 105.8, 73.4, 70.2, 70.2, 69.9, 69.8, 69.3, 66.9, 58.9, 58.6, 57.7,
57.6, 52.1, 47.1, 46.6, 39.8, 39.1, 37.2, 31.8, 31.5, 30.3, 29.7, 29.7, 29.6,
29.5, 29.4, 29.3, 29.3, 28.7, 26.3, 26.0, 26.0, 24.8, 22.6,19.9,18.2,14.0;
IR (thin film) nmax 3525, 3444, 3277, 3070, 2954, 2916, 2848, 1747,
1714, 1659, 1633, 1581, 1539, 1470, 1338, 1238, 1122 cmꢁ1; ESIMS
(m/z): 1671 (MþHþ), 1693 (MþNaþ). HR-ESIMS (m/z) calcd for
C102H167N5NaO13 1671.2639, found 1671.2647.
L
-Phe- -Leu- -Pro(5-{5-[3-(2-{2-[2-(3,4,5-Tris-octadecyloxy-benzoyl-
L
L
amino)-ethoxy]-ethoxy}-ethylcarbamoyl)-allyl]})-Pro-OMe (24):
ESIMS (m/z) calcd for C133H209N12O20 2294.57, found 2294.6
(MþHþ).
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology.
4.4.8. NH2-Val-cis-L-Pro(5-{5-[3-(2-{2-[2-(3,4,5-tris-octadecyloxy-
benzoylamino)-ethoxy]-ethoxy}-ethylcarbamoyl)-allyl]})-Pro-OH
(21). To a solution of 20 (152.3 mg, 0.11 mmol) in THF (6 mL) was
added aqueous 1 M LiOH (6 mL), then stirred at 35 ꢀC for 24 h. The
organic layer was separated, then dried over Na2SO4. The product
was precipitated by the addition of CH3CN (20 mL) to give 21 as
a white powder (140.2 mg, 93% yield). Mp 186–188 ꢀC; 1H NMR
Supplementary data
1H and 13C NMR spectra of compound 4, cis-4, trans-4, 6, 7, 10, 11,
12, 13, 16, 18, 19, 20, and 21 and ESIMS spectra of compound 22 and
24 are provided. Supplementary data associated with this article
can be found in the online version, at doi:10.1016/j.tet.2009.07.045
(600 MHz, CDCl3)
d
7.01 (2H, s), 6.82 (1H, t, J¼5.1 Hz), 6.72–6.65
(1H, m), 6.18 (1H, t, J¼5.1 Hz), 5.90 (1H, s), 5.83 (1H, d, J¼15.4 Hz),
4.21–4.15 (1H, m), 4.05 (1H, dd, J¼11.0, 6.6 Hz), 4.03–3.95 (6H, m),
3.83 (1H, s), 3.71–3.60 (9H, m), 3.59–3.55 (2H, m), 3.51–3.46 (2H,
m), 2.77–2.71 (1H, m), 2.64–2.58 (1H, m), 2.28–2.20 (2H, m), 2.12–
1.85 (7H, m),1.84–1.76 (4H, m),1.76–1.70 (2H, m),1.52–1.42 (6H, m),
1.39–1.20 (84H, m), 1.06 (3H, d, J¼6.6 Hz), 0.91 (3H, d, J¼6.6 Hz),
References and notes
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