Preparation and conformational study of CF3-containing enkephalin-derived oligopeptide

Article abstract of DOI:10.1016/j.tet.2007.11.085

Incorporation of (2S,3S)-4,4,4-trifluorothreonine (F₃-Thr) instead of Thr in the enkephalin-derived hexapeptide led to the apparent conformational alteration due to the strong electron-withdrawing effect of the trifluoromethyl group by comparison with the original compound on the basis of their various NMR measurements.

Full text of DOI:10.1016/j.tet.2007.11.085

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 1888e1894
www.elsevier.com/locate/tet
Preparation and conformational study of CF3-containing
enkephalin-derived oligopeptide
*
Takamasa Kitamoto, Shunsuke Marubayashi, Takashi Yamazaki
Department of Applied Chemistry, Graduate School of Engineering, Tokyo University of Agriculture and Technology,
2-24-16 Nakacho, Koganei 184-8588, Japan
Received 23 October 2007; received in revised form 22 November 2007; accepted 26 November 2007
Available online 21 December 2007
Abstract
Incorporation of (2S,3S)-4,4,4-trifluorothreonine (F3-Thr) instead of Thr in the enkephalin-derived hexapeptide led to the apparent confor-
mational alteration due to the strong electron-withdrawing effect of the trifluoromethyl group by comparison with the original compound on
the basis of their various NMR measurements.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Fluorinated amino acid; Peptides; Conformations; NOESY spectra
1. Introduction
the most important factors for determining whether a specific
donor is accepted as a substrate or not, this issue seems not to
have been paid significant attention thus far.4 Based on such an
idea, we have started our investigation to clarify how much
a fluorinated substituent in amino acids affects the original
peptidic conformation. For this purpose, we have selected
the hexapeptide, Tyr-D-Ser-Gly-Phe-Leu-Thr 1 (DSLET5)
known as the enkephalin-related peptide selectively bound to
the d opioid receptor. The fluorinated enkephalin-type peptides
have been synthesized by utilization of, for instance, fluorine-
containing phenylalanines6 or leucines,7 and their interesting
biological activities were reported to be appeared when fluo-
rine atoms were introduced at a judicious position. However,
the relationship between substitution by fluorine and confor-
mational change as well as the biological activity has not nec-
essarily been clarified yet, and we therefore decided to start
our research.
Completion of decoding of the human genomic base se-
quence in 2003 opened a new route to obtain the three-
dimensional shapes and functions of the target active sites
responsible for some specific diseases with the aid of compu-
tational technique.1,2 Detailed analyses of such information
will in the near future enable us to rationally design more
effective drugs by installing appropriate elements so as to con-
struct firm interaction with these reaction sites. When peptidic
compounds are regarded as donors, introduction of fluorine-
containing amino acids3 would become one of the most useful
methods for control of their conformations and physical prop-
erties. For example, a fluorine atom with three sets of lone
pairs or a fluorinated substituent will endow ability to form
hydrogen bonding (HB) and cause unfavorable electronic re-
pulsion with other proximate electronegative atoms and/or
groups, which would more or less bring about the conforma-
tional change of the original compounds. Although three-
dimensional shapes of molecules are considered as one of
2. Results and discussion
2.1. Synthesis and conformational analysis of
hexapeptide 2
In this study, most of peptide syntheses were performed by
the solution-phase method using WSC$HCl8 (water soluble
* Corresponding author. Tel./fax: þ81 42 388 7038.
E-mail address: tyamazak@cc.tuat.ac.jp (T. Yamazaki).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.11.085

T. Kitamoto et al. / Tetrahedron 64 (2008) 1888e1894
1889
carbodiimide; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride)) in the presence of HOBt$H2O9 (1-hydroxyben-
zotriazole hydrate) as the representative condensation reagent
partner, which usually attained good to excellent yields. How-
ever, preparation of Boc-Tyr-D-SereOBn 8 was the exception
and only ca. 50% yield of 8 was furnished. Then, we have
turned our attention to DMT-MM10 (4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-ethylmorpholinium chloride) because other
reagents like well-known DPPA (diphenylphosphoryl azide) re-
sulted in almost no improvement. DMT-MM did not generally
require any additional base for activation, but this was not the
case here. Addition of 1.1 equiv of base was required to neu-
tralize the trifluoroacetic acid salt of the N-terminal amine
component in substrates, and a 1.1 equiv more amount was
found to be necessary for effective transformation. After sev-
eral experiments, conveniently prepared CDMT11 (2-chloro-
4,6-dimethoxy-1,3,5-triazine), the precursor of DMT-MM,
was selected for this condensation in the presence of 2.2 equiv
of NMM (N-methylmorpholine).12 As a result, this step pro-
ceeded under the usual atmosphere in a conventional AcOEt
solvent to attain 85% isolated yield of the product 8. Combina-
tion of 7 and 8 in a usual manner furnished the desired hexa-
peptide 2 in 79% yield (Scheme 1).
long-range correlations between Tyr and Thr as well as Boc
in Tyr and LeueCH3 groups apart from five and six residues,
respectively, which assumed its partial helix- or spherical-type
structure. The cross peak between Tyr phenol and Thr hydroxy
protons led to speculation that TyreOH proton and ThreOH
oxygen worked as the HB donor and acceptor, respectively,
when their acidity was considered.13 If this was the case, it
was strongly anticipated that substitution of ThreCH3 hydro-
gen atoms for fluorine should effectively lower the electron
density of F3-ThreOH oxygen, which would apparently result
in weakening the HB between Tyr and Thr. This idea was
qualitatively supported by the acidity difference between
CH3CH2OH and CF3CH2OH of DpKa¼3.1,13,14 as well as
HB stabilization energy difference of PhOH with Thr or F3-Thr
of 2.40 kcal/mol in favor of the former by ab initio calcu-
lation15 using the B3LYP/6-311þþG** level of theory with
considering the DMSO solvent effect by the SCI-PCM model.
2.2. Synthesis and conformational analysis of fluorine-
containing hexapeptide 3
The synthesis of the compound 3 with a CF3 group was ini-
tiated by construction of unnatural (2S,3S)-4,4,4-trifluorothreo-
nine 4 (F3-Thr) following the method reported by Soloshonok
et al. (Scheme 2).16 N-Benzylated L-proline was condensed
with 2-aminobenzophenone to afford (S)-2-[(N-benzylprolyl)-
amino]benzophenone (BPB) 10. This chiral auxiliary 10
was then treated with glycine and Ni(NO3)$6H2O and the re-
sultant GlyeNieBPB 11 in turn was allowed to react with
CF3CHO to give F3eNi complex 12. Hydrolysis of 12 led to
recovery of 10 and isolation of F3-Thr 4 whose stereoselectiv-
ity was determined to be at least 96.0% de17 and 91.7% ee.18
The obtained 4 was readily converted to the BoceF3-ThreOBn
14 by protection of both at the N- and C-terminals in a usual
manner.19 After deprotection of N-terminal of 14 and C-termi-
nal of 15, their condensation reaction using WSC$HCl in
the presence of HOBt$H2O and Et3N in DMF produced the
requisite fluorine-containing hexapeptide 3 (Scheme 3). This
condensation has been studied in detail under various
conditions: more polar solvents and bulkier bases were found
inappropriate, and moreover, CDMT and DMT-MM, which re-
corded an excellent yield for the preparation of 8, could not
improve the situation. The condensation reaction between
non-fluorinated Thr and the pentapeptide 15 eventually led to
the formation of the desired 3 in 20% isolated yield. This unsat-
isfactory result was in part as a result of epimerized byproduct
with very close Rf value preventing ready purification.
The NOESY spectrum of 2 observed in DMSO-d6 (Fig. 1)
indicated that its conformation possessed two characteristic
WSC HCl,
Boc Phe
HOBt H2O
Et3N, CH2Cl2
Boc Phe Leu OBn
5
Leu OBn
TsOH
(97%)
CF3CO2H
CH2Cl2, 0 °C
WSC HCl, HOBt H2O
Et3N, CH2Cl2 (96%)
Boc Gly Phe Leu OBn
6
WSC HCl, HOBt H2O
Et3N, CH2Cl2 (76%)
H2, Pd/C
MeOH
Boc Gly Phe Leu Thr OBn
7
Boc
Tyr
CDMT, NMM
AcOEt (85%)
Boc Tyr D-Ser OBn
8
D-Ser OBn TsOH
CF3CO2H
7
CH2Cl2, 0 °C
WSC HCl, HOBt H2O
Et3N, DMF (79%)
2
H2, Pd/C
MeOH
8
Scheme 1. Preparation of BoceTyr-D-Ser-Gly-Phe-Leu-ThreOBn 2.
It would be reasonable to prepare the pentapeptide 15 for
realization of the efficient construction of 2 and 3, while the
condensation of 15 after benzyl deprotection and ThreOBn
was unfortunately failed, which obliged us to select different
routes to get access to the both targets.
In spite of an unacceptable isolated yield, its NMR study
was carried out. As shown in Figure 2, as our expectation,
entry of three fluorine atoms indeed altered the original
three-dimensional shape and the unique long-range HB be-
tween TyreOH and ThreOH found for the prototype 2 that
HO
CH3
OBn
OH
Ph
O
O
O
O
H
H
N
N
N
H
N
H
N
H
HN
O
O
HO
Boc
Medium
Figure 1. NOESY correlations of 2.
Strong
Weak

1890
T. Kitamoto et al. / Tetrahedron 64 (2008) 1888e1894
O
2-aminobenzophenone, SOCl2
CH2Cl2, rt, overnight.
glycine, Ni(NO3) 6H2O, KOH
MeOH, 60 °C, 1 h
BnCl, KOH
i-PrOH, 40 °C, 7 h
Bn
N
CO2H
CO2H
N
H
N
Bn
N
H
Ph
O
9
10 (85% from Pro)
OH
O
O
OH
O
O
H
H
CF3CHO, MeONa
aq. HCl, MeOH
reflux, 15 min.
N
N
CO2H
Ni
N
CF3
Ni
N
N
F3C
H
MeOH, 60 °C, 15 min
N
NH2
(68% from 10)
O
O
4
11
12
Scheme 2. Preparation of (2S,3S)-4,4,4-trifluorothreonine 4.
Table 1
Concentration dependance of chemical shifts of 3
(Boc)2O,
Na2CO3
BnBr,
NaHCO3
Boc F3-Thr
13
Boc F3-Thr OBn
14
4
Chemical shifta (ppm)
Db (ppm)
1,4-Dioxane,
H2O
DMF
(74% from 4)
d20
d0.1
CF3CO2H
TyreNH
D-SereNH
GlyeNH
PheeNH
LeueNH
ThreNH
TyreOH
a
6.894
7.940
8.119
8.066
8.212
8.445
9.152
6.898
7.941
8.121
8.066
8.266
8.450
9.147
0.004
0.001
0.002
0.000
0.054
0.005
ꢁ0.005
6
CH2Cl2,
0 °C
WSC HCl,
HOBt H2O
Et3N, DMF
(94%)
Boc Tyr D-Ser Gly Phe Leu OBn
15
H2, Pd/C
MeOH
8
CF3CO2H
CH2Cl2, 0 °C
Chemical shifts obtained by 20 mM and 0.1 mM solutions.
14
b
Differences between d20 and d0.1
.
WSC HCl, HOBt H2O
Et3N, DMF (20%)
3
structure 3 did not basically contain intermolecular interaction
and their observed HB would be constructed at least mainly in
an intramolecular manner.
H2, Pd/C
MeOH
15
Scheme 3. Preparation of BoceTyr-D-Ser-Gly-Phe-Leu-F3-ThreOBn 3.
To collect further conformational information, we tried to
identify, which protons were responsible for HB by investigat-
ing chemical shift alteration under different solvent polarity
(Fig. 3).20 In our experiment, CDCl3 and DMSO-d6 were
applied as the low and high polarity solvents, respectively,
with the contents of the less polar former solvent of 0, 20, 40,
60, and 80%. A proton forming a stronger HB usually shows
lower sensitivity toward the solvent polarity to be observed
with smaller chemical shift difference even under larger polar-
ity change. As a result, it was determined that HB forming pro-
tons were the amide protons of D-Ser, Gly, Phe, and the hydroxy
proton of D-Ser in both 2 and 3. Moreover, the more acidic
hydroxy proton of F3-Thr in 3 was also found to participate
whose phenomenon was anticipated by incorporation of
a strongly electron-withdrawing trifluoromethyl group, which
should effectively decrease the electron density of F3-ThreOH,
and resulted in increase of acidity of this OH group.
Examination of temperature dependance of these amide and
hydroxy protons was also carried out on the basis of the proton
NMR spectra obtained at 25, 35, and 45 ꢀC (Table 2). Ishida
and co-workers have reported on this issue and a proton
involved in HB showed a high probability of the chemical shift
difference D (ppb/K) less than 4.21 With referring to this
empirical threshold, both of GlyeNH and Fn-ThreOH (n¼0
and 3) were expected to form intramolecular HB and in
OH
Ph
O
HO
CF3
OBn
O
O
O
H
H
N
N
N
H
N
H
N
H
HN
O
O
HO
Boc
Medium
Weak
Strong
Figure 2. NOESY correlations of 3.
was completely disappeared in this material 3. Instead, the
strong correlation between protons CHC(O)NH was noticed
in all the amide linkage and very few cross-peaks between
amino acids were observed, which would support the straight-
type shape with the trans conformation at each amide bond.
Since there is a possibility that the conformation of 3 would
possess a b-sheet type structure by construction of the inter-
1
molecular interaction with the other molecule. H NMR spec-
trum was measured in a 0.1 mM solution by 200-fold dilution
of the previous sample, whose chemical shifts of the amide
and hydroxy protons with the ones in a 20 mM solution
were shown in Table 1. In these instances, chemical shift
differences D (ppm) were almost constant except for the
LeueNH proton. These results led us to conviction that the

T. Kitamoto et al. / Tetrahedron 64 (2008) 1888e1894
1891
10.0
9.0
8.0
7.0
6.0
5.0
4.0
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3. Conclusion
In summary, we have succeeded in preparation of trifluoro-
methyl-containing oligopeptide 3, and clarified that CF3-incor-
poration at a judicious position of 2 does lead to a significant
conformational change by its strong electron-withdrawing ef-
fect at least in the present case. Accumulation of such types of
information will eventually lead to control of the three-dimen-
sional (3D) shapes. Therefore, we are attempting to analyze
three-dimensional structures of these materials, and similar
investigation about other peptidic targets has been in progress
in this laboratory.
4. Experimental section
4.1. General
0
20
40
60
80
0
20
40
60
80
CDCl3 / %
CDCl3 / %
Most of reactions where an organic solvent was employed
were performed under argon with magnetic stirring using
flame-dried glassware. Anhydrous CH2Cl2 was purchased and
used without further purification. DMF was freshly distilled
from CaH2. Unless otherwise noted, materials were obtained
from commercial suppliers and were used without further puri-
fication. All protected amino acids (AAs) for peptide syntheses
were prepared by tert-butyloxycarbonylation of an amino group
or benzyl esterification of a carboxyl group of non-protected
AAs. Analytical thin layer chromatography (TLC) was rou-
tinely usedformonitoringreactions bygenerallyusingamixture
of n-hexane and ethyl acetate (v/v). Spherically-shaped neutral
silica gel (63e210 mm or 40e50 mm) was employed for column
chromatography or flush column chromatography, respectively.
1H, 13C, and 19F NMR spectra were recorded (1H: 500, 400, or
300 MHz; 13C: 125, 100, or 75 MHz; 19F: 376 or 283 MHz) at
room temperature whose data were reported as follows: chemi-
cal shift (d scale) in parts per million (ppm) downfield from
Me4Si (d¼0.00) used as an internal standard, number of protons
(integration), multiplicity (singlet, s; doublet, d; triplet, t; quar-
tet, q; multiplet, m; broad peak, br; etc.), and coupling constant
(J, Hz). Infrared (IR) spectra were reported in wave number
(cmꢁ1). Optical rotation was measured as a MeOH solution
with a cell of 50 mm length. FAB mass spectra were measured
in a positive ion mode.
(a) F0-hexapeptide 2
(b) F3-hexapeptide 3
Tyr-NH
Phe-NH
Tyr-OH
Gly-NH
Fn-Thr-NH
Fn-Thr-OH
D-Ser-NH
Leu-NH
D-Ser-OH
Figure 3. Chemical shift of the specific protons under different solvent ratios;
(a) non-fluorinated hexapeptide 2; (b) trifluorinated hexapeptide 3.
Table 2
Temperature dependance of 2 and 3
F0-hexapeptide 2
F3-hexapeptide 3
D1
D2
D1
D2
LeueNH
GlyeNH
4.9
3.5
3.9
6.2
5.0
5.4
4.6
4.0
4.0
4.9
3.5
3.5
5.8
4.8
5.6
4.5
4.1
3.9
4.9
3.7
4.2
7.6
4.6
5.0
4.3
3.8
4.0
4.7
4.1
4.4
7.7
4.6
5.5
4.7
3.8
4.7
PheeNH
Fn-ThreNH
D-SereNH
TyreNH
TyreOH
Fn-ThreOH
D-SereOH
Chemical shift difference D1 (ppb/K)¼ꢁ{d (35 ꢀC)ꢁd (25 ꢀC)}/10; chemical
shift difference D2 (ppb/K)¼ꢁ{d (45 ꢀC)ꢁd (35 ꢀC)}/10.
addition, PheeNH and D-SereOH in 2 were also suggested to
be in a similar situation but with weaker interaction. The re-
sults from polarity and temperature dependance allowed us
to convince the intramolecular HB formations of GlyeNH,
PheeNH, and D-SereOH in 2, and GlyeNH and F3-ThreOH
in 3. Now, we are trying to analyze each of these conforma-
tions using a method such as X-ray crystallographic analysis
in the solid state because the extensive computation by
MM2 as well as MOPAC with considering solvent effects
failed to obtain appropriate conformers, which could consis-
tently explain a variety of NMR data described above. Since
deprotection of 2 and 3 has not been succeeded yet, we are still
concentrating our attention for attainment of the smooth cleav-
age of the protective groups, which will furnish an important
information on the relationship between fluorine substitution
and conformational change as well as their biological activity.
4.2. Experimental procedure
4.2.1. General procedure for the coupling of two amino
acids
Preparation of Boc-Phe-LeueOBn (5) is described as the
representative example. Boc-Phe (13.3 g, 50.0 mmol) and
LeueOBn$TsOH (21.6 g, 55.0 mmol) in anhydrous CH2Cl2
(70 mL) were added to a 200 mL two-necked round-bottomed
flask under argon. The mixture was cooled to 0 ꢀC with an
ice/water bath, and WSC$HCl (10.6 g, 55.1 mmol) and
HOBt$H2O (8.44 g, 55.1 mmol) were added in one portion,
followed by the slow addition of Et3N (7.7 mL, 55 mmol). Af-
ter 0.5 h, the cold bath was removed, and the reaction mixture
was stirred overnight at room temperature. The reaction

1892
T. Kitamoto et al. / Tetrahedron 64 (2008) 1888e1894
mixture was then concentrated on a rotary evaporator and the
residue was diluted with water. The aqueous layer was ex-
tracted with AcOEt (three times) and the combined organic
layer was successively washed with 5% NaHCO3 aq (three
times), 5% citric acid aq (three times), and brine (once). After
this typical workup procedure, the extracts were dried over an-
hydrous MgSO4, filtered, and evaporated. After purification by
column chromatography, the dipeptide compound 5 (22.8 g,
48.7 mmol) was obtained as white foam. Data for 5:22 yield
BoceF3-Thr (13) (3.34 g, 12.2 mmol, 88.1% yield) as oily
residue, which was used for the next step.
To a mixture of 13 (1.63 g, 5.98 mmol) and NaHCO3
(1.53 g, 18.2 mmol) in DMF (24 mL) was added BnBr
(0.81 mL, 6.8 mmol), and the reaction mixture was stirred
overnight at room temperature. The usual workup procedure
and purification by column chromatography afforded 14
(1.84 g, 5.05 mmol) as an oil. Data for 14: yield 84.5% from
1
4; Rf 0.65 (n-hexaneeAcOEt¼2:1); H NMR d (DMSO-d6)
1
97.4%; Rf 0.51 (n-hexaneeAcOEt¼2:1); H NMR d (CDCl3)
1.44 (9H, s), 4.05 (1H, br s), 4.56 (1H, br s), 4.67 (1H, dd,
J¼2.5, 9.0 Hz), 5.23 (2H, s), 5.343 (1H, d, J¼9.0 Hz),
7.26e7.39 (5H, m); 13C NMR d 28.0, 53.6, 67.8, 69.3 (q,
J¼31.7 Hz), 80.7, 123.9 (q, J¼283.1 Hz), 128.0, 128.4,
128.5, 134.7, 156.0, 169.4; 19F NMR d (CDCl3) ꢁ78.0 (d,
J¼6.8 Hz); IR (neat) n 678, 694, 735, 775, 850, 908, 941,
1028, 1055, 1108, 1140, 1151, 1248, 1335, 1365, 1452,
0.86 (3H, d, J¼6.4 Hz), 0.88 (3H, d, J¼6.1 Hz), 1.40 (9H, s),
1.45e1.62 (3H, m), 3.05 (2H, d, J¼6.7 Hz), 4.35 (1H, q, J¼
5.8 Hz), 4.60 (1H, dt, J¼5.2, 8.4 Hz), 5.01 (1H, br s), 5.11 (1H,
d, J¼12.2 Hz), 5.15 (1H, d, J¼12.2 Hz), 6.30 (1H, d, J¼
8.2 Hz), 7.18e7.28 (5H, m), 7.32e7.38 (5H, m); 13C NMR
d (CDCl3) 21.8, 22.6, 24.5, 28.1, 38.0, 41.3, 50.8, 55.4, 66.8,
80.0, 126.7, 128.1, 128.3, 128.4, 128.5, 129.3, 135.3, 136.5,
155.4, 171.1, 172.2.
1500, 1513, 1675, 1702, 1720, 1738, 2950, 3300 cmꢁ1
;
[a]2D9 þ17.2 (c 1.00, MeOH). Anal. Calcd for C16H20F3NO5:
C, 52.89; H, 5.55; N, 3.86. Found: C, 53.30; H, 5.91; N, 3.99.
4.2.2. General procedure for the deprotection of the
N-terminal Boc group
4.3. Experimental data
The N-terminal Boc deprotection of 5 is described as the
representative example. Compound 5 (4.69 g, 10.0 mmol) in
CH2Cl2 (4.0 mL) was added to a 50 mL round-bottomed flask
at room temperature. The solution was cooled to 0 ꢀC with an
ice/water bath, and CF3CO2H (7.7 mL, 100 mmol) was slowly
added, and the mixture was stirred at 0 ꢀC for 2.5 h. Excess
CF3CO2H was then removed as an azeotropic mixture with
CH2Cl2. Boc-deprotected HePhe-LeueOBn$CF3CO2H thus
obtained was used for the next condensation step without
further purification.
4.3.1. BoceGly-Phe-LeueOBn (6)23
1
Yield 95.6%; Rf 0.54 (n-hexaneeAcOEt¼1:2); H NMR
d (CDCl3) 0.85 (3H, d, J¼6.4 Hz), 0.86 (3H, d, J¼6.4 Hz),
1.39e1.62 (3H, m), 1.42 (9H, s), 3.02 (1H, dd, J¼6.7, 13.7 Hz),
3.09 (1H, dd, J¼6.4, 14.0 Hz), 3.72 (1H, dd, J¼5.3, 16.9
Hz), 3.77 (1H, dd, J¼5.6, 16.9 Hz), 4.56 (1H, dt, J¼5.4,
8.5 Hz), 4.72 (1H, q, J¼7.1 Hz), 5.11 (1H, d, J¼12.2 Hz),
5.14 (1H, d, J¼12.5 Hz), 5.19 (1H, t, J¼5.6 Hz), 6.53 (1H, br
s), 6.82 (1H, d, J¼7.9 Hz), 7.16e7.25 (5H, m), 7.32e7.38
(5H, m); 13C NMR d (CDCl3) 21.8, 22.5, 24.6, 28.2, 38.2,
40.9, 43.9, 50.9, 54.0, 66.8, 79.8, 126.7, 128.1, 128.2, 128.3,
128.4, 129.2, 135.3, 136.3, 155.9, 169.5, 170.7, 172.1.
4.2.3. General procedure for the deprotection of the
C-terminal BnO group
4.3.2. BoceGly-Phe-Leu-ThreOBn (7)24
The C-terminal BnO deprotection of BoceGly-Phe-Leue
OBn (6) is described as the representative example. At 0 ꢀC,
6 (0.549 g, 1.04 mmol) in MeOH (6.0 mL) was added to
10% Pd/C (0.020 g, 1.8 mol %) under argon in a 10 mL two-
necked round-bottomed flask. After argon was replaced with
hydrogen at the same temperature, the reaction mixture was
stirred overnight at room temperature. The catalyst was then
removed by filtration, and the filtrate was concentrated on a ro-
tary evaporator. BoceGly-Phe-LeueOH was used for the next
condensation reaction without further purification.
1
Yield 75.7%; Rf 0.81 (AcOEt); H NMR d (CDCl3) 0.84
(3H, d, J¼6.4 Hz), 0.87 (3H, d, J¼5.8 Hz), 1.17 (3H, d,
J¼6.4 Hz), 1.35 (9H, s), 1.45e1.52 (3H, m), 3.03 (1H,
dd, J¼5.8, 14.0 Hz), 3.15 (1H, dd, J¼6.1, 14.0 Hz), 3.68 (1H,
dd, J¼5.3, 16.9 Hz), 3.73 (1H, dd, J¼4.1, 16.9 Hz), 4.30e4.34
(1H, m), 4.62 (1H, dd, J¼3.4, 8.8 Hz), 4.66 (1H, q,
J¼4.9 Hz), 4.73 (1H, q, J¼6.4 Hz), 5.17 (1H, d, J¼12.5 Hz),
5.20 (1H, d, J¼12.2 Hz), 5.42 (1H, br s), 7.14 (1H, d,
J¼6.7 Hz), 7.12e7.36 (10H, m), 7.25 (1H, d, J¼7.3 Hz), 7.45
(1H, d, J¼8.8 Hz); 13C NMR d (CDCl3) 19.6, 22.1, 22.4, 24.4,
28.2, 38.0, 40.8, 44.0, 51.9, 54.2, 58.1, 67.2, 68.0, 80.2, 126.9,
128.1, 128.2, 128.5, 128.5, 129.2, 135.2, 135.9, 156.3, 170.1,
170.5, 171.2, 172.7.
4.2.4. Preparation of BoceF3-ThreOBn (14)19
F3-threonine 4 (2.40 g, 13.9 mmol), 1,4-dioxane (28 mL),
H2O (28 mL), and Na2CO3 (1.63 g, 15.3 mmol) were added
to a 300 mL round-bottomed flask. The mixture was cooled
to 0 ꢀC with an ice/water bath, and (Boc)2O (3.39 g,
15.5 mmol) was added in one portion. After 1 h, the cold
bath was removed, and the reaction mixture was stirred over-
night at room temperature. The reaction mixture was then con-
centrated on a rotary evaporator and the residue was diluted
with water. The usual workup procedure and evaporation of
the solvent after drying with anhydrous Na2SO4 furnished
4.3.3. BoceTyr-D-SereOBn (8)
Yield 84.6%; Rf 0.71 (AcOEt); 1H NMR d (CDCl3) 1.40 (9H,
s), 2.94 (2H, d, J¼6.7 Hz), 3.74 (1H, dd, J¼3.2, 11.4 Hz), 3.85
(1H, d, J¼11.3 Hz), 4.33 (1H, q, J¼7.3 Hz), 4.59 (1H, br), 5.14
(1H, d, J¼12.2 Hz), 5.17 (1H, d, J¼12.2 Hz), 5.26 (1H, d,
J¼7.6 Hz), 6.73 (2H, d, J¼8.5 Hz), 6.88 (1H, d, J¼7.3 Hz),
7.02 (2H, d, J¼8.2 Hz), 7.30e7.37 (5H, m); 13C NMR

T. Kitamoto et al. / Tetrahedron 64 (2008) 1888e1894
1893
d (CDCl3) 28.2, 37.8, 54.7, 56.2, 62.3, 67.5, 80.7, 115.6, 127.5,
128.0, 128.4, 128.4, 128.5, 130.3, 135.0, 155.8, 170.1, 171.9;
IR (KBr) n 694, 732, 851, 1020, 1163, 1235, 1364, 1453,
1508, 1634, 2300, 2910, 3250 cmꢁ1; [a]D21 ꢁ7.46 (c 1.05,
MeOH); mp 118e123 ꢀC. Anal. Calcd for C24H30N2O7: C,
62.87; H, 6.59; N, 6.11. Found: C, 62.68; H, 6.80; N, 6.34.
1.29 (9H, s), 1.51e1.66 (3H, m), 2.63 (1H, dd, J¼9.9, 13.6 Hz),
2.70 (1H, dd, J¼10.1, 13.7 Hz), 2.86 (1H, dd, J¼4.4, 13.7
Hz), 2.96 (1H, dd, J¼3.7, 14.0 Hz), 3.45e3.49 (1H, m), 3.56e3.62
(1H, m), 3.71 (1H, dd, J¼6.0, 16.6 Hz), 4.13 (1H, dt, J¼4.6,
8.8 Hz), 4.25 (1H, q, J¼6.0 Hz), 4.33 (1H, dt, J¼5.2,
8.2 Hz), 4.55 (1H, dt, J¼3.4, 9.1 Hz), 5.00 (1H, t, J¼5.3 Hz),
5.10 (1H, d, J¼12.0 Hz), 5.13 (1H, d, J¼12.5 Hz), 6.63 (2H,
d, J¼7.9 Hz), 6.86 (1H, d, J¼8.2 Hz), 7.02 (2H, d,
J¼8.2 Hz), 7.16e7.38 (10H, m), 7.93 (1H, d, J¼7.3 Hz),
8.02 (1H, d, J¼8.5 Hz), 8.07 (1H, t, J¼5.5 Hz), 8.43 (1H, d,
J¼7.6 Hz), 9.13 (1H, s); 13C NMR d (DMSO-d6) 21.4, 22.8,
24.3, 28.2, 36.7, 37.7, 39.7, 42.1, 50.6, 53.8, 55.1, 56.2, 61.7,
66.0, 78.3, 114.9, 126.4, 127.9, 127.9, 128.1, 128.1, 128.5,
128.5, 129.2, 130.2, 136.0, 137.8, 155.4, 155.8, 168.5, 170.5,
171.4, 172.2; IR (KBr) n 409, 592, 698, 748, 820, 1053, 1169,
1250, 1367, 1454, 1516, 1658, 2349, 2958, 3064, 3307, 3662,
3789, 3811 cmꢁ1; [a]D21 ꢁ8.61 (c 1.02, MeOH); mp 149e
152 ꢀC; HRMS-FAB (m/z): [M]þ calcd for C41H53N5O10,
775.3792; found, 775.3788.
4.3.4. BoceTyr-D-Ser-Gly-Phe-Leu-ThreOBn (2)
Yield 78.7%; Rf 0.83 (AcOEteMeOH¼2:1); 1H NMR
d (DMSO-d6) 0.82 (3H, d, J¼6.5 Hz), 0.86 (3H, d, J¼6.0 Hz),
1.09 (3H, d, J¼6.5 Hz), 1.29 (9H, s), 1.44e1.52 (2H, m),
1.56e1.63 (1H, m), 2.63 (1H, dd, J¼11.0, 12.5 Hz), 2.74
(1H, dd, J¼10.5, 12.5 Hz), 2.86 (1H, dd, J¼3.0, 13.0 Hz), 3.02
(1H, dd, J¼2.8, 13.8 Hz), 3.58 (1H, dd, J¼5.5, 11.5 Hz),
3.60 (1H, dd, J¼5.5, 16.8 Hz), 3.72 (1H, dd, J¼5.3, 16.8 Hz),
4.13 (1H, dt, J¼4.0, 9.0 Hz), 4.19 (1H, dd, J¼6.2, 8.8 Hz), 4.26
(1H, td, J¼5.0, 6.0 Hz), 4.25 (1H, dd, J¼3.0, 8.5 Hz), 4.44
(1H, dt, J¼6.0, 8.5 Hz), 4.55 (1H, br t, J¼8.5 Hz), 4.99 (1H, t,
J¼5.8 Hz), 5.02 (1H, d, J¼5.50 Hz), 5.10 (1H, d, J¼12.5 Hz),
5.14 (1H, d, J¼12.5 Hz), 6.63 (2H, d, J¼7.0 Hz), 6.88 (1H, d,
J¼8.2 Hz), 7.03 (2H, d, J¼7.5 Hz), 7.16e7.23 (5H, m), 7.31e
7.37 (5H, m), 7.94 (1H, d, J¼9.5 Hz), 7.96 (1H, d, J¼7.5 Hz),
8.00 (1H, d, J¼8.5 Hz), 8.07 (1H, t, J¼5.5 Hz), 8.20 (1H, d,
J¼8.0 Hz), 9.14 (1H, s); 13C NMR d (DMSO-d6) 20.1, 21.6,
23.0, 24.1, 28.1, 36.6, 37.6, 40.7, 42.0, 51.0, 53.8, 54.9, 56.1,
57.8, 61.7, 65.9, 66.3, 78.2, 114.8, 126.2, 127.7, 127.9, 128.0,
128.3, 129.2, 130.1, 135.9, 137.7, 155.3, 155.7, 168.4, 170.2,
170.4, 170.8, 171.9, 172.4; IR (KBr) n 411, 453, 488, 580,
698, 746, 831, 1022, 1080, 1105, 1163, 1252, 1367, 1388,
1454, 1516, 1649, 2335, 2362, 2871, 2931, 2958, 3032, 3066,
3292, 3811, 3847, 3857 cmꢁ1; [a]D21 ꢁ8.32 (c 0.23, MeOH);
mp 160e163 ꢀC; HRMS-FAB (m/z): [M]þ calcd for
C45H60N6O12, 876.4269; found, 876.4233.
4.3.8. BoceTyr-D-Ser-Gly-Phe-Leu-F3-ThreOBn (3)
Yield 20.0%; Rf 0.62 (AcOEteMeOH¼9:1); 1H NMR
d (DMSO-d6) 0.74 (3H, d, J¼6.0 Hz), 0.77 (3H, d,
J¼6.0 Hz), 1.29 (9H, s), 1.18e1.37 (3H, m), 2.62 (1H, dd,
J¼10.0, 14.0 Hz), 2.77 (1H, dd, J¼9.0, 13.5 Hz), 2.87 (1H,
dd, J¼4.0, 13.5 Hz), 2.94 (1H, dd, J¼5.8, 13.2 Hz), 3.47
(1H, ddd, J¼6.3, 12.2, 23.0 Hz), 3.60 (1H, ddd, J¼5.2,
10.0, 20.0 Hz), 3.64 (1H, dd, J¼5.5, 17.2 Hz), 3.70 (1H,
dd, J¼5.8, 17.2 Hz), 4.13 (1H, dt, J¼4.5, 9.0 Hz), 4.27
(1H, dt, J¼5.5, 7.0 Hz), 4.48 (1H, dt, J¼8.7, 9.5 Hz), 4.19
(1H, dd, J¼2.1, 7.1 Hz), 4.62 (1H, dt, J¼6.5, 8.0 Hz),
4.91 (1H, dd, J¼2.2, 9.2 Hz), 5.06 (1H, t, J¼5.2 Hz),
5.15 (1H, d, J¼12.5 Hz), 5.20 (1H, d, J¼12.5 Hz), 6.63
(2H, d, J¼8.5 Hz), 6.90 (1H, d, J¼8.5 Hz), 7.01 (1H, d,
J¼7.5 Hz), 7.03 (2H, d, J¼8.0 Hz), 7.16e7.26 (5H, m),
7.32e7.38 (5H, m), 7.94 (1H, d, J¼8.0 Hz), 8.06 (1H, d,
J¼8.5 Hz), 8.12 (1H, t, J¼5.8 Hz), 8.20 (1H, d, J¼9.0 Hz),
8.44 (1H, d, J¼9.0 Hz), 9.17 (1H, s); 13C NMR d (DMSO-
d6) 21.2, 23.3, 23.9, 28.1, 36.6, 38.2, 40.7, 42.0, 50.6,
51.7, 54.0, 54.8, 56.1, 61.7, 66.8, 68.3 (q, J¼30.2 Hz),
78.2, 114.8, 124.4 (q, J¼281.8 Hz), 126.3, 127.8, 127.9,
128.0, 128.1, 128.4, 128.4, 129.2, 130.2, 135.5, 137.4,
155.3, 155.7, 168.4, 168.7, 170.4, 171.9, 172.6; 19F NMR
d (DMSO-d6) ꢁ76.0 (d, J¼7.9 Hz); IR (KBr) n 833, 908,
949, 1032, 1049, 1101, 1165, 1252, 1338, 1367, 1390,
1454, 1512, 1624, 1670, 1745, 1890, 2343, 2388, 2872,
2935, 2964, 3034, 3068, 3284 cmꢁ1; [a]D21 þ15.7 (c 0.09,
MeOH); mp 101e105 ꢀC. Anal. Calcd for C45H57F3N6O12:
C, 58.06; H, 6.17; N, 9.03. Found: C, 57.64; H, 6.31;
N, 8.79.
4.3.5. (S)-2-[(N-Benzylprolyl)amino]benzophenone
(BPB) (10)16c
1
Yield 85.0%; Rf 0.70 (n-hexaneeAcOEt¼2:1); H NMR
d (CDCl3) 1.73e1.86 (2H, m), 1.96 (1H, ddd, J¼4.4, 8.4,
16.8 Hz), 2.25 (1H, ddd, J¼9.8, 13.0, 18.1 Hz), 2.41 (1H, dt, J¼
6.8, 9.6 Hz), 3.21 (1H, ddd, J¼2.4, 6.8, 9.2 Hz), 3.32 (1H, dd,
J¼4.7, 10.2 Hz), 3.59 (1H, d, J¼12.8 Hz), 3.92 (1H, d, J¼
12.8 Hz), 7.07e7.62 (12H, m), 7.78 (1H, d, J¼7.9 Hz), 8.56
(1H, d, J¼8.5 Hz), 11.52 (1H, s); 13C NMR d (CDCl3) 24.1,
30.9, 53.8, 59.8, 68.2, 121.4, 122.1, 125.2, 127.0, 128.1, 128.2,
129.0, 130.1, 132.5, 132.5, 133.3, 138.1, 138.5, 139.1, 174.6,
198.0.
4.3.6. F3-Threonine (4)16e
1
Yield 65.2%; H NMR (DMSO-d6) d 2.86 (1H, dd, J¼
10.5, 12.5 Hz), 3.10 (1H, d, J¼13.0 Hz), 4.37 (1H, br s); 13C
NMR (DMSO-d6) d 52.9, 67.7 (q, J¼30.17 Hz), 125.4
(q, J¼281.04 Hz), 169.4; 19F NMR d (D2O) ꢁ77.4 (d,
J¼6.8 Hz).
Acknowledgements
Partial financial support by the Asahi Glass Foundation was
acknowledged. T.K. is also grateful to the 21st Century Center
of Excellence (COE) program of the Future Nano-Materials
Research and Education Project for fellowship.
4.3.7. BoceTyr-D-Ser-Gly-Phe-LeueOBn (15)
1
Yield 94.2%; Rf 0.68 (n-hexaneeAcOEt¼1:5); H NMR
d (DMSO-d6) 0.83 (3H, d, J¼6.4 Hz), 0.89 (3H, d, J¼6.4 Hz),

1894
T. Kitamoto et al. / Tetrahedron 64 (2008) 1888e1894
M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
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´
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¨
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