Development of supramolecular organo-gel based on tripeptide skeletons

Article abstract of DOI:10.1248/cpb.58.680

Boc-Ser-Val-Gly-OCH₂Ph (31) showed high gelation abilities in the aromatic solvents, particularly in toluene. The minimum gelation concentration of 31 in toluene was 10 mg/ml, suggesting that 2500 molecules of toluene were immobilized by each m

Full text of DOI:10.1248/cpb.58.680

680
Regular Article
Chem. Pharm. Bull. 58(5) 680—684 (2010)
Vol. 58, No. 5
Development of Supramolecular Organo-Gel Based on Tripeptide
Skeletons
Eriko AZUMA, Kouji KURAMOCHI, and Kazunori TSUBAKI*
Graduate School of Life and Environmental Science, Kyoto Prefectural University; Sakyo, Kyoto 606–8522, Japan.
Received December 25, 2009; accepted February 6, 2010; published online February 10, 2010
Boc-Ser-Val-Gly-OCH2Ph (31) showed high gelation abilities in the aromatic solvents, particularly in
toluene. The minimum gelation concentration of 31 in toluene was 10 mg/ml, suggesting that 2500 molecules of
toluene were immobilized by each molecule of the tripeptide 31. The FT-IR data indicated that formation of an-
tiparallel b-sheets through intermolecular hydrogen bonding was central to the generation of nanofibers during
gelation.
Key words supramolecular gel; Boc-Ser-Val-Gly-OCH2Ph; nanofiber
Low molecular weight (supramolecular) gels have at- mogeneous solution. The solutions were stored in an incuba-
tracted attention in the fields of supramolecular chemistry tor at 20 °C for 24 h. When the tube was inverted and tapped,
and material chemistry over the past two decades.1—6) The the absence of a fluid phase was defined as a gel (transparent
gel phase is a wettable viscoelastic physical state that shares or opaque gel), and a crumpled jelly object was defined as a
some properties of both liquid and solid phases. Gelation oc- partial gel. Table 1 shows the results of the first screening
curs when self-organizing gelators produce an entangled and the minimum gelation concentration for those tripep-
three-dimensional network of fibers that constrains the mo- tide/solvent combinations that yielded a gel.
bility of the embedded solvent molecules. Low molecular
The results shown in Table 1 indicate that toluene and
weight gelators based on amino acids,7—10) ureas,11—13) chlorobenzene, which have aromatic rings, showed the high-
amides,14—16) and saccharides17—19) have been developed, and est propensity for gelation among the solvents assayed, sug-
the responses of these gels to photo irradiation,20,21) ultra- gesting that a p–p interaction between the aromatic rings of
sonic frequencies,22,23) and biologically active agents10,24,25) the solvent and the benzyl group of the tripeptide played a
have been described. However, organic reactions (covalent crucial role in gelation. The tripeptide sequences X-Val-Gly
bond forming reactions) in the gel phase have received rela- 7—9 and X-Ala-Ala 13—15 displayed inherently high gela-
tively little attention.26,27) We are trying to develop novel or- tion abilities for these solvents.
ganic reactions in a supramolecular gel phase using a dual-
A new series of tripeptides with the sequences X-Val-Gly
functional molecule that is capable of catalyzing reaction as and X-Ala-Ala were synthesized and examined in detail. For
well as forming gel. In this paper, we describe the synthesis the N-terminal amino acid (X), serine and glutamine were se-
and gelation properties of several tripeptide derivatives, lected because of their hydrogen bonding abilities, and
which form the backbone of the dual-functional molecule.
phenylalanine was selected for its ability to engage in p–p
N-t-Butoxycarbonyl (Boc) and O-benzyl protected tripep- stacking interactions. Tripeptides 28—33 were synthesized
tide gelators, composed of Gly, Ala, and Val, were designed via the route described in Chart 1. The results from a second
because this combination was previously and accidentally gelation screening assay, using the additional solvents o-, m-,
shown to yield interesting gelation properties.28) Twenty- and p-xylenes as aromatic solvents in addition to the solvents
seven tripeptides, 1—27, based on various combinations of assayed in the first screening, are summarized in Table 2.
Gly, Ala, and Val, were synthesized by repeated condensa-
Among these tripeptides, Boc-Ser-Val-Gly-OCH2Ph (31)
tion and deprotection in the liquid phase (Chart 1).29—31) showed excellent gelation properties in the aromatic solvents,
Each amino acid benzyl ester (third fragment) was reacted particularly in toluene (Fig. 1a), most likely because of the
with a Boc-protected amino acid (second fragment) under
1-hydroxybenzotriazole hydrate (HOBt·H2O)/1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDAC·
HCl)/triethylamine conditions in 27% to quantitative yields.
The dipeptide was subsequently deprotected by removal of
the Boc group in HCl/dioxane to give an O-benzyl dipeptide.
A Boc-protected amino acid (first fragment) was then intro-
duced to the dipeptide under similar conditions to afford the
desired tripeptides 1—27 in yields of 15—99%.
The gelation abilities of the 27 tripeptide derivatives in
each of ten organic solvents (hexane, EtOAc, CHCl3,
CH2Cl2, acetone, acetonitrile, EtOH, MeOH, toluene, and
chlorobenzene) were surveyed as follows. Each tripeptide/
solvent combination was mixed in a microtube and (as ap-
propriate) irradiated with ultrasound for 30 min at tempera-
tures ranging from room temperature to 60 °C to afford a ho- Chart 1. Synthetic Route for Tripeptides 1—27
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: tsubaki@kpu.ac.jp

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681
Table 1. Gelation Studies of Tripeptides 1—27 in Various Organic Solvents
Compd.
No.
Boc-XXX-OCH2Ph
Hexane
EtOAc
CHCl3
CH2Cl2
Acetone
CH3CN
EtOH
MeOH
Toluene
Cl–C6H5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
G-G-G
A-G-G
V-G-G
G-A-G
A-A-G
V-A-G
G-V-G
A-V-G
V-V-G
G-G-A
A-G-A
V-G-A
G-A-A
A-A-A
V-A-A
G-V-A
A-V-A
V-V-A
G-G-V
A-G-V
V-G-V
G-A-V
A-A-V
V-A-V
G-V-V
A-V-V
V-V-V
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
p
h
h
h
p
h
h
h
h
h
h
h
p
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
p
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
i
h
h
h
h
h
i
i
h
h
h
h
h
h
h
h
h
h
p
h
PG
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
i
i
O (100)
h
h
i
O (50)
O (33)
PG
PG
h
PG
p
O (33)
O (50)
O (100)
i
i
PG
i
h
h
i
PG
h
h
h
h
T (100)
h
h
i
T (33)
O (66)
O (33)
T (33)
h
PG
h
T (66)
O (50)
O (33)
PG
PG
O (100)
O (50)
PG
O (100)
PG
h
h
h
h
h
h
h
h
PG
h
h
p
p
h
h
h
h
h
h
h
h
PG
h
h
h
h
h
h
h
h
h
O (100)
O (100)
h
h
h
h
h
h
h
h
h
h
i
h
O (100)
h
h
h
PG
h
h
h
h
h
h
h
h
i
i
i
i
PG
T (50)
i
O (100)
h
h
h
h
h
h
h
h
h
h
h
p
h
h
h
h
i
i
h
h
h
h
h
h
h
PG
i
O (33)
PG
i
i
O (100)
The concentration of the tripeptide was up to 100 mg/ml. The minimum gelation concentration (mg/ml) is shown in a parenthesis. h: homogeneous solution; T: transparent gel;
O: opaque gel; PG: partial gel; p: precipitation; i: insoluble.
Table 2. Gelation Studies of Tripeptides 28—33 in Various Organic Solvents
Compd.
Boc-XXX-OCH2Ph Hexane EtOAc
CHCl3 CH2Cl2 Acetone CH3CN
EtOH
MeOH Toluene Cl–C6H5 o-Xylene m-Xylene p-Xylene
No.
28
29
30
31
32
33
S-A-A
F-A-A
Q-A-A
S-V-G
F-V-G
Q-V-G
i
i
i
i
i
i
i
h
i
h
h
h
h
h
i
h
h
i
i
h
h
i
h
h
h
h
h
i
h
h
h
h
h
i
i
i
i
i
i
i
O (50)
O (33)
i
i
i
i
O (100)
i
i
i
i
h
h
i
h
h
i
h
h
i
h
h
i
O (10)
T (33)
O (33)
p
O (33)
O (33)
O (33)
i
i
i
i
i
i
i
i
The concentration of the tripeptide was up to 100 mg/ml. The minimum gelation concentration (mg/ml) is shown in a parenthesis. h: homogeneous solution; T: transparent gel;
O: opaque gel; PG: partial gel; p: precipitation; i: insoluble.
additional hydrogen bonding capacity introduced by the hy- quired solvent removal under reduced pressure, which in-
droxy group of serine. The minimum gelation concentration duced the nanofibers to form a microcrystalline phase. 3D
of 31 in toluene was 10 mg/ml, suggesting that 2500 mole- optical imaging was used to follow the nanofiber-to-micro-
cules of toluene were immobilized by each molecule of the crystal transition (Fig. 1d). A digital optical microscope, in
tripeptide 31.
which the gel remained wet, also revealed long fibers (Fig.
Boc-Ser-Val-Gly-OCH2Ph (31) yielded the best gelation 1e).
properties, and the morphological characteristics of its
The intermolecular interactions involved in stabilizing the
1
toluene gel were observed by scanning electron microscopy toluene gel of 31 were characterized by H-NMR. The short
(SEM), three-dimensional (3D) laser scanning optical mi- transversal relaxation time in the gel state prevented collec-
croscopy and digital optical microscopy (Fig. 1). SEM re- tion of the gel state NMR spectrum of gelator 31. We there-
vealed a plate-like microcrystalline structure (1 mm in width, fore inferred the intermolecular interactions and driving
Fig. 1b). 3D laser microscopy revealed a network of entan- force for gelation of 31 by measuring the solution-phase
gled nanofibers, the minimum height of which was estimated NMR spectrum as a function of temperature and concentra-
to be 60—70 nm (Fig. 1c). The imaging conditions were re- tion of 31 (Fig. 2). At 40 °C, 5 mg/ml 31 in toluene d-8, three
sponsible for the observation of two morphologies, micro- signals corresponding to the NH protons (Ha, Hb, Hc indi-
crystals or nanofibers, for the toluene gel of 31. 3D optical cated in the structure of Fig. 2) were observed at d 7.18 (Hb),
imaging permitted observation of the fibers under wet condi- 6.69 (Hc), and 5.67 (Ha), respectively (all shifts are given in
tions (in the presence of solvent). The SEM measurement re- ppm). As the concentration of 31 increased and/or the tem-

682
Vol. 58, No. 5
Fig. 3. Time Course IR Spectra of 31 during the Phase Transition from a
Solution to a Gel
10 mg/ml 31 in toluene d-8, NaCl liquid cell (path length: 0.1 mm), * denotes an ab-
sorption peak from the toluene d-8.
hydrogen bonding network and gelation, therefore, appeared
to be closely related.
The solution-to-gel phase transition was tracked by FT-IR
using a liquid cell with toluene d-8 (Fig. 3).
Fig. 1. The Morphologies of the Toluene Gel of 31
(a) A photograph of a toluene gel of 31 (10 mg/ml). (b) SEM image of microcrystals
of 31, formed during sample drying. (c, d) 3D scanning laser images of the toluene gel
(wet state). (e) Digital microscopy of the toluene gel (wet state).
The time course IR spectra of 31 at 3200—3450 cmꢀ1 (in
the region of amide A) is shown in Fig. 3a. As the solution
phase transitioned to the gel phase, the strength of the free
NH stretch at 3410 cmꢀ1 decreased and the strength of the
hydrogen bonding NH stretch (3319 cmꢀ1) increased with
an isosbestic point. At 1500—1800 cmꢀ1 (amide I and II
regions, Fig. 3b), the strength of the free carbonyl stretch at
1678 cmꢀ1 decreased with a concomitant increase in the
strength of the hydrogen bonding carbonyl stretch at 1645
cmꢀ1, with several isosbestic points. Yamada and coworkers
reported that parallel or antiparallel b-sheet secondary struc-
tures of (tri-)peptides could be distinguished by the presence
or absence, respectively, of the characteristic IR absorption at
1690 cmꢀ1 in addition to the absorptions at 1630 cmꢀ1 and
1527 cmꢀ1, independent of the sequence.32,33) Taking into
consideration of their data, in the solution phase, tripeptide
31 absorbed at 3410, 1678, and 1495 cmꢀ1, indicating the
absence of specific secondary structure. In contrast, the gel
phase showed absorptions at 3318, 1681, 1645, and 1527
cmꢀ1, suggesting the formation of antiparallel b sheets.
In conclusion, we synthesized 27 tripeptides (combina-
tions of Gly, Ala, and Val) for a first screening assay, and 6
tripeptides for a second screening assay, to test for gelation in
organic solvents. We identified Boc-Ser-Val-Gly-OCH2Ph
Fig. 2. 1H-NMR (400 MHz) Spectra of Tripeptide 31 in Toluene d-8
perature was reduced, the chemical shifts of the amidic NH (31) as an excellent gelator for aromatic solvents. Boc-Ser-
protons shifted dramatically downfield. This behavior was as- Val-Gly-OCH2Ph (31) yielded particularly high gelation abil-
cribed to the formation of hydrogen bonds between these ities in toluene, with a minimum gelation concentration of
amidic protons and the carbonyl oxygen. An increase in the 10 mg/ml. The FT-IR data suggested that formation of an-

May 2010
683
(ABq, 12.7 Hz, JABꢁ12.2 Hz, 2H), 5.03 (br, 1H), 4.61 (qd, Jꢁ7.3 Hz,
Jꢁ7.3 Hz, 1H), 4.26 (dd, Jꢁ8.4 Hz, Jꢁ6.8 Hz, 1H), 3.91 (dd, Jꢁ6.5 Hz,
Jꢁ6.5 Hz, 1H), 2.19—2.15 (m, 2H), 1.45 (s, 9H), 1.41 (d, Jꢁ7.3 Hz, 3H),
0.97—0.90 (m, 12H); 13C-NMR (68 MHz, CDCl3) d: 172.2, 172.0, 170.7,
155.8, 135.2, 128.4, 128.3, 128.1, 80.8, 67.1, 60.2, 58.4, 48.1, 31.3, 30.8,
28.3, 19.3, 19.2, 18.2, 18.0, 17.5; MS (EI) m/z (rel. intensity) 477 [Mꢂ, (8)],
404 (5), 299 (11), 271 (36), 200 (36), 116 (67), 72 (100), 57 (22); HR-MS
(EI) Calcd for C25H39O6N3 (Mꢂ) 435.2839, Found 477.2847.
tiparallel b-sheets through intermolecular hydrogen bonding
was central to the generation of nanofibers during gelation.
Identification and characterization of the gelation properties
of 31 lay the foundation for developing a dual-functional
molecule that displays both catalytic activity and gelation
ability.
Experimental
Boc-Val-Ala-Val-OCH2Ph (24): Viscous oil; IR (KBr) 3292, 2968, 1734,
1714, 1645, 1528, 1391, 1367, 1166, 1018, 752, 698 cmꢀ1 1H-NMR
;
Tripeptides 1—33 were synthesized by the procedure described in the
text.29—31) FT-IR spectra of 31 were collected on a JASCO FT/IR-4200,
using a NaCl liquid cell (0.1 mm path length) with toluene d-8. SEM, 3D
scanning laser microscopy, and digital optical microscopy were performed
using a VE-9800 SEM, a VK-9700 Generation II Color Laser Scanning Mi-
croscope, and a VHX-1000 (KEYENCE) digital optical microscope, respec-
tively.
(270 MHz, CDCl3) d: 7.36—7.30 (m, 5H), 6.87—6.75 (br, 2H), 5.21 (br,
1H), 5.17 (ABq, 23.1 Hz, JABꢁ12.4 Hz, 2H), 4.60—4.53 (m, 2H), 3.95 (br,
1H), 2.24—2.05 (m, 2H), 1.44 (s, 9H), 1.36 (d, Jꢁ6.8 Hz, 3H), 0.98—0.84
(m, 12H); 13C-NMR (68 MHz, CDCl3) d: 172.0, 171.6, 171.3, 155.7, 135.2,
128.4, 128.3, 128.2, 80.0, 67.0, 57.3, 48.8, 31.2, 31.0, 28.3, 19.3, 19.0, 18.2,
17.7, 17.5; MS (EI) m/z (rel. intensity) 477 [Mꢂ, (9)], 342 (13), 305 (13),
271 (20), 215 (25), 172 (37), 116 (97), 91 (76), 72 (100), 57 (29); HR-MS
(EI) Calcd for C25H39O6N3 (Mꢂ) 447.2839, Found 477.2837.
Boc-Ala-Val-Val-OCH2Ph (26): Viscous oil; IR (KBr) 3299, 3069, 2969,
1742, 1714, 1648, 1549, 1391, 1367, 1248, 1170, 1048, 1026, 698 cmꢀ1; 1H-
NMR (270 MHz, CDCl3) d: 7.36—7.31 (m, 5H), 6.80—6.71 (br, 1H),
6.50—6.36 (br, 1H), 5.16 (ABq, 20.1 Hz, JABꢁ12.2 Hz, 2H), 5.07—4.86 (br,
1H), 4.57 (dd, Jꢁ8.8 Hz, Jꢁ4.6 Hz, 1H), 4.24 (br, 1H), 4.22—4.10 (br, 1H),
2.18 (m, 2H), 1.44 (s, 9H), 1.34 (d, Jꢁ7.3 Hz, 3H), 0.94—0.84 (m, 12H);
13C-NMR (68 MHz, CDCl3) d: 172.8, 171.3, 171.2, 155.3, 135.2, 128.4,
128.2, 79.7, 66.9, 58.6, 57.0, 50.0, 31.1, 30.9, 28.3, 19.2, 19.0, 18.9, 18.3,
17.7; MS (EI) m/z (rel. intensity) 477 [Mꢂ, (6)], 333 (9), 291 (8), 271 (11),
243 (21), 187 (22), 172 (15), 91 (50), 72 (100), 57 (14); HR-MS (EI) Calcd
for C25H39O6N3 (Mꢂ) 447.2838, Found 477.2831.
Characterization Data of New Compounds Boc-Val-Gly-Gly-
OCH2Ph (3): Pale yellow viscous oil; IR (neat) 3307, 3067, 2968, 2933,
1751, 1656, 1526, 1366, 1174, 1032, 752 cmꢀ1; 1H-NMR (270 MHz, CDCl3)
d: 7.32—7.33 (m, 5H), 7.06 (br, 1H), 6.82 (br, 1H), 5.17 (s, 2H), 5.07 (br,
1H), 4.16—3.95 (m, 4H), 3.87 (dd, Jꢁ6.8 Hz, Jꢁ6.8 Hz, 1H), 2.19—2.11
(m, 1H), 1.41 (s, 9H), 0.98 (d, Jꢁ6.8 Hz, 3H), 0.94 (d, Jꢁ6.8 Hz, 3H); 13C-
NMR (68 MHz, CDCl3) d: 172.5, 169.4, 156.1, 135.0, 128.5, 128.3, 128.2,
80.2, 67.1, 60.6, 42.9, 41.3, 30.6, 28.3, 19.3, 18.1; MS (EI) m/z (rel. inten-
sity) 421 [Mꢂ, (2)], 365 (5), 201 (14), 172 (75), 116 (100), 72 (86), 57 (34);
HR-MS (EI) Calcd for C21H31O6N3 (Mꢂ) 421.2213, Found 421.2207.
Boc-Val-Ala-Gly-OCH2Ph (6): Clear viscous oil; IR (KBr) 3299, 3068,
2975, 1756, 1642, 1528, 1391, 1173, 1019, 698, 419 cmꢀ1 1H-NMR
;
(270 MHz, CDCl3) d: 7.38—7.30 (m, 5H), 6.78 (br, 1H), 6.49 (br, 1H), 5.17
(s, 2H), 5.00 (br, 1H), 4.55 (qd, Jꢁ7.3 Hz, Jꢁ7.3 Hz, 1H), 4.05 (d,
Jꢁ5.4 Hz), 3.91 (dd, Jꢁ6.2 Hz, Jꢁ6.2 Hz, 1H), 2.21—2.07 (m, 1H), 1.43 (s,
9H), 1.40 (d, Jꢁ7.3 Hz, 3H), 0.96 (d, Jꢁ7.0 Hz, 3H), 0.91 (d, Jꢁ6.8 Hz,
3H); 13C-NMR (68 MHz, CDCl3) d: 180.7, 172.2, 171.5, 169.3, 155.9,
135.0, 128.5, 128.4, 128.2, 80.0, 67.1, 60.0, 48.7, 41.3, 31.0, 28.3, 19.3,
18.3, 17.8; MS (EI) m/z (rel. intensity) 435 [Mꢂ, (2)], 365 (5), 243 (14), 172
(53), 116 (100), 91 (76), 72 (78), 57 (32); HR-MS (EI) Calcd for
C22H33O6N3 (Mꢂ) 435.2369, Found 435.2371.
Boc-Ser-Ala-Ala-OCH2Ph (28): White solid; mp 91 °C; IR (KBr) 3311,
3068, 2979, 2933, 1743, 1649, 1531, 1455, 1367, 1251, 1166, 1061, 752,
698 cmꢀ1 1H-NMR (270 MHz, CDCl3) d: 7.36—7.33 (m, 5H), 6.83 (br,
;
1H), 6.76 (br, 1H), 5.45 (br, 1H), 5.16 (ABq, 11.1 Hz, JABꢁ12.2 Hz, 2H),
4.60 (ABC, 12.6 Hz, Jꢁ7.3 Hz, Jꢁ7.3 Hz, 1H), 4.47 (ABC, 12.6 Hz,
Jꢁ7.3 Hz, Jꢁ7.3 Hz, 1H), 4.20 (br, 1H), 4.01 (m, 1H), 3.63 (m, 1H), 3.32
(br, 1H), 1.45 (s, 9H), 1.45—1.39 (m, 6H); 13C-NMR (68 MHz, CDCl3) d:
172.4, 171.9, 170.9, 155.6, 135.1, 128.4, 128.3, 128.0, 80.3, 67.1, 63.1,
55.4, 49.2, 48.3, 28.3, 18.0, 17.9; MS (EI) m/z (rel. intensity) 437 [Mꢂ,
(0.7)], 407 (30), 351 (19), 259 (15), 203 (22), 175 (32), 131 (31), 91 (100),
57 (41); HR-MS (EI) Calcd for C21H31O7N3 (Mꢂ) 437.2162, Found
437.2167.
Boc-Gly-Gly-Ala-OCH2Ph (10): Viscous oil; IR (neat) 3307, 3068, 2979,
2937, 1661, 1528, 1367, 1251, 1162, 751, 698 cmꢀ1 1H-NMR (270 MHz,
;
CDCl3) d: 7.25—7.34 (m, 5H), 6.86—6.76 (br, 1H), 6.75—6.65 (br, 1H),
5.18—5.10 (br, 1H), 5.17 (ABq, 8.5 Hz, JABꢁ12.2 Hz, 2H), 4.61 (qd,
Jꢁ7.0 Hz, Jꢁ7.0 Hz, 1H), 3.99 (ABC, 32.4 Hz, Jꢁ16.7 Hz, Jꢁ5.4 Hz, 2H),
3.82 (d, Jꢁ5.7 Hz, 2H), 1.45 (s, 9H), 1.43 (d, Jꢁ7.0 Hz, 3H); 13C-NMR
(68 MHz, CDCl3) d: 172.3, 170.3 168.7, 156.1, 135.1, 128.3, 128.1, 127.7,
79.9, 66.9, 48.2, 44.0, 42.6, 20.2, 17.5; MS (FABꢂ, Glycerol) m/z (rel. in-
tensity) 435 [(MꢂGlycerolꢂH)ꢂ, (1)], 394 [(MꢂH)ꢂ, 8], 338 (12), 229
(40), 173 (62), 91 (67), 72 (100), 57 (21); HR-MS (EI) Calcd for
C19H28O6N3 (MꢂH)ꢂ 394.1978, Found 394.1979.
Boc-Gln-Ala-Ala-OCH2Ph (30): White solid; mp 167—170 °C; IR (KBr)
3320, 3067, 2979, 1738, 1657, 1528, 1454, 1166, 1051, 698 cmꢀ1; 1H-NMR
(270 MHz, CDCl3+CD3OD) d: 7.38—7.33 (m, 5H), 5.16 (ABq, 11.5 Hz,
J
ABꢁ12.2 Hz, 2H), 4.51 (q, Jꢁ7.3 Hz, 1H), 4.37 (q, Jꢁ7.3 Hz, 1H), 4.10—
4.03 (m, 1H), 2.28 (t, Jꢁ7.7 Hz, 2H), 2.06—1.90 (m, 2H), 1.44 (s, 9H), 1.42
(d, Jꢁ7.3 Hz, 3H), 1.36 (d, Jꢁ7.0 Hz, 3H); 13C-NMR (68 MHz,
CDCl3+CD3OD) d: 175.8, 172.4, 172.1, 171.8, 155.7, 134.9, 128.1, 127.9,
127.6, 79.7, 66.7, 53.5, 48.5, 47.9, 31.1, 27.8, 17.3, 16.8; MS (EI) m/z (rel.
intensity) 478 [Mꢂ, (5)], 216 (30), 201 (55), 173 (30), 145 (40), 91 (100);
HR-MS (EI) Calcd for C23H34O7N4 (Mꢂ) 478.2428, Found 478.2437.
Boc-Ser-Val-Gly-OCH2Ph (31): White solid; mp 113—114 °C; IR (KBr)
3310, 3072, 2970, 1746, 1649, 1527, 1367, 1173, 1062, 698 cmꢀ1; 1H-NMR
(270 MHz, CDCl3) d: 7.38—7.31 (m, 5H), 6.97 (br d, Jꢁ8.5 Hz, 1H), 6.88
(br, 1H), 5,54 (br d, Jꢁ6.5 Hz, 1H), 5.16 (s, 2H), 4.33 (dd, Jꢁ5.9 Hz,
Jꢁ8.5 Hz, 1H), 4.21 (br, 1H), 4.09—4.03 (m, 2H), 4.00 (br, 1H), 3.70—3.60
(m, 1H), 3.36 (br, 1H), 2.32—2.25 (m, 1H), 1.44 (s, 9H), 1.02—0.88 (m,
6H); 13C-NMR (68 MHz, CDCl3) d: 171.5, 169.7, 155.8, 134.9, 128.5,
128.4, 128.2, 80.4, 67.2, 63.0, 58.7, 55.3, 41.2, 30.2, 28.3, 19.3, 17.7; MS
(EI) m/z (rel. intensity) 451 [Mꢂ, (0.4)], 421 (28), 365 (12), 259 (19), 203
(43), 159 (39), 91 (91), 72 (100), 57 (30); HR-MS (EI) Calcd for
C22H33O7N3 (Mꢂ) 451.2318, Found 451.2325.
Boc-Val-Gly-Ala-OCH2Ph (12): White solid; mp 107—109 °C; IR (KBr)
3341, 2966, 1742, 1690, 1644, 1523, 1390, 1247, 1166, 1047, 753,
652 cmꢀ1 1H-NMR (270 MHz, CDCl3) d: 7.36—7.38 (m, 5H), 7.00 (br,
;
1H), 6.92 (br, 1H), 5.16 (ABq, 9.0 Hz, JABꢁ12.4 Hz, 2H), 5.13 (br, 1H),
4.61 (qd, Jꢁ7.3 Hz, Jꢁ7.3 Hz, 1H), 4.15—4.07 (m, 1H), 3.98—3.87 (m,
1H), 1.43 (s, 9H), 1.42 (d, Jꢁ4.9 Hz, 3H), 0.98—0.91 (m, 6H); 13C-NMR
(68 MHz, CDCl3) d: 172.4, 172.3, 168.5, 155.8, 135.2, 128.4, 128.2, 127.9,
79.9, 67.1, 60.2, 48.3, 43.0, 30.8, 28.3, 19.3, 17.9; MS (EI) m/z (rel. inten-
sity) 435 [Mꢂ, (2)], 379 (8), 257 (11), 172 (54), 116 (100), 91 (78), 72 (79),
57 (38); HR-MS (EI) Calcd for C22H33O6N3 (Mꢂ) 435.2370, Found
435.2372.
Boc-Gly-Val-Ala-OCH2Ph (16): Viscous oil; IR (KBr) 3296, 2973, 1748,
1705, 1640, 1543, 1367, 1163, 1050, 750, 695 cmꢀ1 1H-NMR (270 MHz,
;
CDCl3) d: 7.30—7.36 (m, 5H), 6.88 (d, Jꢁ8.9 Hz, 1H), 6.75 (d, Jꢁ7.3Hz,
1H), 5.31—5.23 (m, 1H), 5.16 (ABq, 13.0 Hz, JABꢁ12.2 Hz, 2H), 4.60 (qd,
Jꢁ7.3 Hz, Jꢁ7.3 Hz, 1H), 4.30 (dd, Jꢁ8.9 Hz, Jꢁ8.9 Hz, 1H), 3.81 (ABC,
23.0 Hz, JABꢁ17.0 Hz, Jꢁ5.4 Hz, 2H), 2.19—2.02 (m, 1H), 1.44 (s, 9H),
1.42 (d, Jꢁ7.3 Hz, 3H), 0.93 (d, Jꢁ6.8 Hz, 3H), 0.91 (d, Jꢁ7.0 Hz, 3H); 13C-
NMR (68 MHz, CDCl3) d: 172.1, 171.0, 169.8, 155.9, 135.1, 128.3, 128.1,
128.0, 79.8, 66.9, 58.2, 48.1, 44.0, 31.3, 28.2, 19.0, 18.2, 17.6; MS (EI) m/z
(rel. intensity) 435 [Mꢂ, (1)], 379 (4), 257 (12), 229 (40), 173 (62), 91 (67),
72 (100), 57 (21); HR-MS (EI) Calcd for C22H33O6N3 (Mꢂ) 435.2369,
Found 435.2366.
Acknowledgments The authors are sincerely grateful to Prof. Takeo
Kawabata and Dr. Yoshiharu Uruno (Institute for Chemical Research, Kyoto
University) for the AMBER calculations, Ms. Kyoko Ohmine for collecting
the temperature-dependent 1H-NMR data, and Mr. Kenichi Ochi
(KEYENCE, Japan) for assistance with microscopic imaging.
References and Notes
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Boc-Val-Val-Ala-OCH2Ph (18): White solid; mp 171—173 °C; IR (KBr)
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1
NMR (270 MHz, CDCl3) d: 7.32—7.35 (m, 5H), 6.47—6.62 (br, 2H), 5.17

684
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