Orthogonality and compatibility between Tsc and Fmoc amino-protecting groups

Article abstract of DOI:10.1016/j.tet.2004.12.052

New deprotection conditions that provide a complete orthogonality between Tsc and Fmoc amino-protecting groups are described. The potential of these orthogonal deprotection conditions was then demonstrated by the efficient solid-phase synthesis of branched peptides 20 and 21 using doubly protected amino acids such as Tsc-Lys(Fmoc)-OH 4c and Fmoc-Lys(Tsc)-OH 4d.

Full text of DOI:10.1016/j.tet.2004.12.052

Tetrahedron 61 (2005) 2493–2503
Orthogonality and compatibility between Tsc and Fmoc
amino-protecting groups
*
Jin Seok Choi, Hunhui Kang, Nakcheol Jeong and Hogyu Han
Department of Chemistry, Korea University, Seoul 136-701, South Korea
Received 9 December 2004; revised 21 December 2004; accepted 22 December 2004
Available online 1 February 2005
Abstract—New deprotection conditions that provide a complete orthogonality between Tsc and Fmoc amino-protecting groups are
described. The potential of these orthogonal deprotection conditions was then demonstrated by the efficient solid-phase synthesis of branched
peptides 20 and 21 using doubly protected amino acids such as Tsc-Lys(Fmoc)-OH 4c and Fmoc-Lys(Tsc)-OH 4d.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
2.1. Synthesis of Tsc- and Fmoc-protected amino acids
and esters
Recently, we reported the 2-(4-trifluoromethylphenylsulfo-
nyl)ethoxycarbonyl (Tsc) function as a novel base-labile
amino-protecting group (Fig. 1).1 The Tsc group differs
from the 9-fluorenylmethoxycarbonyl (Fmoc) group regard-
ing its less sensitivity to premature deprotection when
installed on the exocyclic amino group of the hetero-
aromatic pyrrole (Py) and imidazole (Im) amino acids. The
higher efficiency of Tsc compared to Fmoc in the solid-
phase synthesis of pyrrole-imidazole polyamides envisions
its promising use for protecting numerous amines.
In order to develop orthogonal deprotection conditions
required for the Tsc/Fmoc strategy, Tsc- and Fmoc-
protected amino acids and esters were prepared (Fig. 1
and Scheme 1). Tsc- and Fmoc-protected pyrrole (1a, 1b),
imidazole (2a, 2b), and phenylalanine (3a, 3b) amino esters
were synthesized by direct introduction of Tsc and Fmoc
into the corresponding amino esters 13, 14, and 15,
respectively. Tsc-protected pyrrole 1c and imidazole 2c
amino acids were prepared from esters 13 and 14,
respectively via base-resistant 2-(4-trifluoromethylphenyl-
thio)ethoxycarbonyl (Ttc) protection.1 Basic ester hydroly-
sis of Ttc-protected esters followed by subsequent oxidative
conversion of Ttc into Tsc afforded the desired amino acids
1c and 2c. An alternative preparation of Tsc-Phe-OEt 3a
from Ttc-Phe-OEt was performed in a similar manner.
Fmoc-protected pyrrole 1d and imidazole 2d amino acids
were synthesized as described.4 Lysine amino esters (4a,
4b) and acids (4c, 4d) with Na-Tsc/N3-Fmoc or Na-Fmoc/
N3-Tsc protecting groups were prepared by direct introduc-
tion of Tsc into H-Lys(Fmoc)-OMe 16 and Fmoc-Lys-OMe
17b amino esters and H-Lys(Fmoc)-OH and Fmoc-Lys-OH
amino acids. It is noteworthy that Tsc-protected amino acids
could be prepared using Tsc-Cl or convertible Ttc-Cl.
The development of orthogonal amino-protecting groups or
deprotection conditions allows new strategies for the solid-
and solution-phase syntheses of more complex peptides and
scaffolds.2 Although a large number of orthogonal strategies
are available, a combination of amino-protecting groups
both orthogonal and compatible under basic deprotection
conditions is rare.3 Here, we report a dual Tsc/Fmoc strategy
that provides a convenient procedure for both the orthogonal
and compatible use of such amino-protecting groups under
basic deprotection conditions. Our Tsc/Fmoc strategy was
successfully applied to the synthesis of branched peptides
by use of amino acids bearing the Tsc/Fmoc dyad.
2.2. Chemical and thermal stability of Tsc and Fmoc in
the presence of N,N-diisopropylethylamine
Keywords: Tsc; Fmoc; Amino-protecting group; Orthogonal deprotection;
Branched peptide.
* Corresponding author. Tel.: C82 2 3290 3134; fax: C82 2 3290 3121;
e-mail: hogyuhan@korea.ac.kr
In order to implement the Tsc/Fmoc strategy, orthogonal
deprotection conditions were required. We reasoned that
this should be possible given the different deprotection rates
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.12.052

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J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
Scheme 1. Reagents and conditions: (a) 10% Pd/C, 40 psi H2, EtOAc (13)
or DMF (14), rt; (b) Tsc-Cl (1a, 69%; 2a, 82%; 3a, 88%; 4a, 92%; 4b, 69%)
or Fmoc-Cl (1b, 88%; 2b, 83%; 3b, 77%), DIEA, CH2Cl2, rt; (c) (i) 0.1 N
LiOH, THF/H2O (1:1), 0 8C, (ii) Tsc-Cl, NaHCO3, 1,4-dioxane, rt, (4c,
85%); (d) CH2N2, CH2Cl2, rt, 74%; (e) 20% TFA, CH2Cl2, rt, (17b from
17a, 88%); (f) (i) 9% TFA, CH2Cl2, rt, (ii) Tsc-Cl, NaHCO3, 1,4-dioxane,
rt, (4d, 38%).
Figure 1. Structure of Tsc-Cl and Fmoc-Cl (A), Tsc- and Fmoc-protected
amino acids and esters (B), and various bases tested for deprotecting Tsc
and Fmoc groups (C).
prone to premature deprotection than their Fmoc analogs
at higher temperature (O95% versus !50% intact after
2 h at 50 8C). Therefore, Tsc is superior to Fmoc in
chemical and thermal stability when used to protect the
exocyclic amino group of heteroaromatic amino acids.
The higher stability of Tsc-Py-OH compared to Tsc-Im-
OH is reversed in the case of Fmoc-Py-OH and Fmoc-
Im-OH. Taken together, all these results indicate that the
deprotection rates of Tsc and Fmoc are dependent on
bases, temperature, and amino acids. Therefore, proper
choice of deprotection conditions would permit the
orthogonality between Tsc and Fmoc in addition to
their compatibility affordable by piperidine treatment.
of Tsc and Fmoc under basic conditions. To test such a
possibility, we examined the premature deprotection of Tsc-
and Fmoc-protected pyrrole and imidazole amino acids 1c,
1d, 2c, and 2d in the presence of N,N-diisopropylethylamine
(DIEA, 5) as a mild base (Fig. 2).1,5 While both Tsc and
Fmoc were completely cleaved within 5 min at room
temperature by treatment with 20% (v/v) piperidine in
DMF, their cleavage by 0.5 M DIEA in DMF proceeded
much slower. Intriguingly, moreover, the difference in their
deprotection rates is clearly significant. Careful HPLC
monitoring of the decomposition of Tsc-Py-OH 1c and Tsc-
Im-OH 2c in the presence of DIEA at 25 8C revealed that
O98% of the Tsc group remained intact even after 4 h,
whereas about 60 and 10% of the Fmoc groups were
eliminated from Fmoc-Py-OH 1d and Fmoc-Im-OH 2d,
respectively. Tsc-Py-OH and Tsc-Im-OH were also less
2.3. Orthogonal deprotection conditions for Tsc and
Fmoc groups
To develop orthogonal deprotection conditions for Tsc and
Fmoc, a set of deprotection conditions were evaluated using

J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
2495
Figure 2. Effect of DIEA (5) on the chemical stability of amino acids at 25
or 50 8C. Time courses for the premature deprotection of amino-protecting
groups in Tsc-Py-OH 1c (:), Tsc-Im-OH 2c (&), Fmoc-Py-OH 1d (6),
and Fmoc-Im-OH 2d (,) amino acids (0.1 M) were performed by their
treatment with DIEA (0.5 M) in DMF at 25 8C (solid lines) or 50 8C (dashed
lines). The amount of intact amino acids was determined by analytical
HPLC on a Nova-Pakw C18 reverse-phase column (3.9!150 mm, 4 mm,
Waters, Milford, MA) with UV monitoring at 280 nm under gradient
conditions: 0–20 min, 5% MeCN/min, 1 mL/min flow rate.
the Tsc- (1a–3a) and Fmoc-protected (1b–3b) amino esters
and basic reagents 6–12 (Table 1 and Scheme 2).6 Among
secondary and tertiary amines 6–11, 1-methylpyrrolidine
(9) showed a selective preference for the deprotection of
Fmoc-protected amino esters 1b–3b (Table 1). Complete
and selective removal of Fmoc while maintaining Tsc in
both heteroaromatic and aliphatic amino esters 1b–3b was
accomplished by using 50% 1-methylpyrrolidine in DMF
for 1 h at 25 8C. We then attempted to develop conditions
for the deprotection of Tsc without removing Fmoc. We
found that LiOH is the reagent of choice for such purposes
(Scheme 2). Fast cleavage of Tsc-protected amino ester 3a
was achieved in high yield (O99%) using 0.1 N aqueous
Scheme 2. Reagents and conditions: (a) 0.1 N LiOH, THF/H2O (1:1), 0 8C,
5 min, (15, 85%, 15a, 15%, 18a, 77% from 3a; 16, 92%, 16a, 8% from 4a;
17b, 75%, 17c, 20% from 4b); (b) 50% 1-methylpyrrolidine, DMF, 25 8C,
1 h, (16b, O95%; 17d, O98%); (c) 0.5 M DIEA, DMF, 50 8C, 4 h, (16c,
82%; 17d, 87%).
Table 1. Base sensitivities of the amino-protecting groups in Tsc-protected (1a, 2a, and 3a) and Fmoc-protected (1b, 2b, and 3b) amino estersa
1a
2a
3a
1b
2b
3b
6
5%
H
H
H
L
L
M
L
L
L
L
L
L
L
L
L
L
L
L
H
H
H
M
M
M
L
L
L
L
L
L
L
L
L
L
L
L
H
H
H
L
L
L
L
L
M
L
L
L
L
L
L
L
L
L
H
H
H
M
M
M
H
H
H
H
H
H
M
M
M
L
H
H
H
M
M
M
H
H
H
H
H
H
M
M
M
L
H
H
H
L
L
L
M
H
H
L
M
H
M
M
M
L
20%
50%
5%
20%
50%
5%
20%
50%
5%
20%
50%
5%
20%
50%
5%
20%
50%
7
8
9
10
11
L
L
L
L
L
L
a Deprotection of amino esters 1a–3a and 1b–3b by various bases 6–11 was detected by HPLC monitoring of intact amino esters at 254 nm. Base sensitivity
was classified based on the % amount of decomposed amino esters after their treatment (0.1 M) with bases (5, 20, and 50%) in DMF at room temperature for
the indicated time: H (O90%, 5 min), H (O90%, 30 min), M (O90%, 2 h), M (O50%/!90%, 2 h), L (O10%/!50%, 2 h), L (!10%, 2 h).

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J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
LiOH/THF at 0 8C for 5 min wherein the occurrence of
epimerization is negligible.6a–d Consequently, the best
orthogonality between Tsc and Fmoc in terms of reactivity
and selectivity in solution could therefore be achieved if
Fmoc is deprotected with 50% 1-methylpyrrolidine in DMF
at 25 8C for 1 h whereas Tsc is deprotected with 0.1 N LiOH
in THF/H2O (1:1) at 0 8C for 5 min.
ester hydrolysis. Thus, when 3a was treated with 0.1 N
LiOH, we obtained a product mixture of ethyl ester 15
(85%) and carboxylic acid 15a (15%). The same thing
happened with 4a and 4b that were treated with 0.1 N LiOH:
92% of ester 16 and 8% of acid 16a from 4a; 75% of ester
17b and 20% of acid 17c from 4b (Scheme 2, and Fig. 3a
and d). However, ester hydrolysis by LiOH is not expected
to cause a problem in its usage for Tsc deprotection in the
solid-phase peptide synthesis unless the Tsc/Fmoc/ester
triad is needed due to ester groups in the side chain and/or
resin linkage. In addition, it is unlikely that ester groups in
the side chain limit the use of Tsc/Fmoc in tandem for
preparing the Tsc/Fmoc/ester triad since base-labile Tsc
could be introduced directly or via base-resistant Ttc.9
The orthogonality of such conditions for Tsc/Fmoc
deprotection was further demonstrated using doubly pro-
tected amino esters such as Tsc-Lys(Fmoc)-OMe 4a and
Fmoc-Lys(Tsc)-OMe 4b (Scheme 2). Complete and selec-
tive cleavage of Tsc in the presence of Fmoc occurred in
their treatment with LiOH/THF (Fig. 3). In addition, the
selectivity of 1-methylpyrrolidine for complete Fmoc
cleavage in the presence of Tsc proved to be excellent in
both 4a and 4b. In the case of 4a, however, 16b rather than
the desired Tsc-Lys-OMe was obtained as a major product.
Presumably, the initially formed Tsc-Lys-OMe underwent
the rapid conversion to 16b by intermolecular Michael-like
addition,3j,7 which would occur more slowly in the solid-
phase synthesis. Formation of free amines was accompanied
by concomitant release of 18a upon treatment of 3a with
LiOH. However, the negligible amount (!1%) of 18b and
18c was detected in the treatment of 4a and 4b with
1-methylpyrrolidine, respectively.8
2.4. Synthesis of branched peptides using the Tsc/Fmoc
strategy
The potential of these orthogonal deprotection conditions
was then demonstrated by their use for the solid-phase
synthesis of branched peptides (Scheme 3). First, the
branched peptide 20 was synthesized on the Fmoc-Rink
Amide MBHA resin through two methods A and B via 19a
and 19b. Resin-bound peptides 19a and 19b can be
branched off due to the presence of Tsc-Lys(Fmoc)-OH 4c
and Fmoc-Lys(Tsc)-OH 4d at their N-terminus, respec-
tively. Methods A and B differ in the order of applying Tsc
and Fmoc deprotection conditions. Tsc deprotection
The deprotection of Tsc with LiOH proceeded faster than
Figure 3. LC-MS analysis of a reaction mixture containing acid 16a (23.17 min, b) and ester 16 (29.93 min, c) from 4a (a) or acid 17c (25.66 min, e) and ester
17b (29.75 min, f) from 4b (d). The reaction mixture was prepared by treating 4a (a) or 4b (d) with 0.1 N LiOH in THF/H2O (1:1) at 0 8C for 5 min according to
Scheme 2 and experimental 3.1.16 and 3.1.19.

J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
2497
such choices afforded peptide 21 in high isolated yield (up
to 63%) (Scheme 3, Method C and Fig. 5).
In conclusion, we have shown that 1-methylpyrrolidine and
LiOH are the reagents of choice for mild and orthogonal
deprotection of Fmoc and Tsc, respectively. The suitability
of such conditions was demonstrated by their use for the
efficient synthesis of branched peptides. Our new Tsc/Fmoc
strategy should therefore greatly extend the scope of the Tsc
group to dual protections under mild basic conditions. We
anticipate that Tsc/Fmoc will be a potential alternative to
standard Dde/Fmoc, particularly where use of the Dde
amino-protecting group is not possible.3d–f
3. Experimental
3.1. General
1H and 13C NMR spectra were recorded on a Varian
Mercury 300 or a Bruker Avance 500 NMR spectrometer.
Chemical shifts (d) are reported in parts per million (ppm)
with reference to tetramethylsilane or solvent and coupling
constants (J) are reported in hertz (Hz). High-resolution
mass spectra (HRMS) were recorded on a JEOL JMS-
AM505WA mass spectrometer using fast atom bombard-
ment (FAB) or chemical ionization (CI) techniques. Matrix-
assisted laser desorption ionization time-of-flight (MALDI-
TOF) mass spectra were recorded on an Applied Biosystems
Voyager-DEe STR mass spectrometer. HPLC analysis was
performed on a Waters 600 HPLC system equipped with a
2487 dual l absorbance detector. LC-MS analysis was
performed on a Hewlett-Packard HP-1100 HPLC system
and a Micromass QUATTRO LC triple quadrupole tandem
mass spectrometer. Thin-layer chromatography (TLC) was
performed on silica gel 60 F254 precoated plates (0.25 mm
thickness, Merck). Flash chromatography was carried out on
silica gel 60 (230–400 mesh, Merck). Reagent-grade
chemicals were purchased from Aldrich, Fluka, Junsei,
and TCI and used as received unless otherwise specified.
Tetrahydrofuran (THF) was distilled from sodium benzo-
phenone ketyl under N2. N,N-Dimethylformamide (DMF)
was distilled from calcium hydride in vacuo. Dichloro-
methane was distilled from calcium hydride under N2.
Scheme 3. Reagents and conditions of method A: (a) 0.1 N LiOH,
THF/H2O (1:1), 0 8C, 10 min (LiOH); (b) Ac-Phe-OH for 19a or Ac-Gly-
OH for 19b, PyBOP, DIEA, DMF, rt (coupling); (c) Ac2O, DIEA, CH2Cl2,
rt (capping); (d) 50% 1-methylpyrrolidine, DMF, rt, 1 h (MP); (e) Ac-Gly-
OH for 19a or Ac-Phe-OH for 19b, coupling; (f) capping; (g) TFA, rt, (20
via 19a, 18%; 20 via 19b, 15%). Method B: (a) MP; (b) Ac-Gly-OH for 19a
or Ac-Phe-OH for 19b, coupling; (c) capping; (d) LiOH; (e) Ac-Phe-OH for
19a or Ac-Gly-OH for 19b, coupling; (f) capping; (g) TFA, rt, (20 via 19a,
29%; 20 via 19b, 17%). Method C: (a) 19b, MP; (b) Fmoc-Ser(But)-OH,
coupling; (c) capping and then two repeats of (a)–(c); (d) MP; (e) Fmoc-
Phe-OH, coupling; (f) capping; (g) MP; (h) capping; (i) LiOH!2 or 50%
piperidine, rt, 10 min; (j) Tsc-Ala-OH, coupling; (k) capping and then two
repeats of (i)–(k); (l) LiOH!2 or 50% piperidine, rt, 10 min; (m) Fmoc-
Gly-OH, coupling; (n) capping; (o) 50% piperidine, rt, 10 min; (p) capping;
(q) TFA, rt, (21 via 19b, 40% (LiOH) and 63% (piperidine)).
followed by Fmoc deprotection was carried out in method A
whereas their deprotection order was reversed in method B.
Peptides thus prepared were purified by reverse-phase
HPLC. All the conditions afforded peptide 20 in O98%
yield of each coupling step and overall recoveries between
15 and 29%. All observed molecular masses characterized
by MALDI-TOF mass spectrometry agreed to within 0.1%
of the calculated peptide mass. These results indicate that
our Tsc/Fmoc orthogonal strategy is suitable for the
preparation of branched peptides regardless of the position
of Tsc and Fmoc on two amino groups of lysine and their
deprotecting order.
3.1.1. Tsc-Py-OMe (1a). To a solution of 13 (3 g,
16.3 mmol) in EtOAc (40 mL) was added 10% Pd/C
(50 mg). After stirring at room temperature for 10 h under
40 psi H2, 10% Pd/C was removed by filtration through
Celite 545 followed by washing with EtOAc and MeOH and
then solvent evaporation. To a stirred solution of the
resulting amine in dry CH2Cl2 (20 mL) was added N,N-
diisopropylethylamine (DIEA, 4.11 mL, 23.6 mmol) and
then Tsc-Cl1 (5.67 g, 17.9 mmol) at 0 8C. After stirring at
room temperature for 7 h, the reaction mixture was
quenched with H2O and extracted with CH2Cl2. The
combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc/n-hexaneZ1:1) to give 1a (4.90 g,
69%) as a white solid. TLC (EtOAc/n-hexaneZ1:1) RfZ
0.33; 1H NMR (300 MHz, DMSO-d6) d 9.30 (brs, 1H), 8.15
(d, JZ8.1 Hz, 2H), 8.01 (d, JZ8.4 Hz, 2H), 7.02 (d, JZ
2.1 Hz, 1H), 6.61 (d, JZ1.8 Hz, 1H), 4.36 (t, JZ5.6 Hz,
HPLC and MALDI-TOF analyses showed that the cleaner
preparation of the crude peptides was achieved using 4d (via
19b) compared to 4c (via 19a) at the branched position of 20
(Fig. 4, I, II versus III, IV). However, the crude peptide
mixture prepared with 4d (via 19b) contained greater
amounts of the Tsc-containing byproduct 20c than the
desired 20 (Fig. 4, III and IV). This result indicates that
treatment of 4d in 19b with 0.1 N LiOH for 10 min at 0 8C is
not efficient enough to give the complete deprotection of
Tsc in the solid-phase peptide synthesis. Based on these
results, Tsc deprotection twice with LiOH or once with
piperidine was used in conjunction with 4d for the efficient
synthesis of the longer branched peptide 21. As expected,

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J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
Figure 4. HPLC (left) and MALDI-TOF (right) analyses of a crude mixture containing peptide 20 (24.86–25.61 min in left) and byproducts 20a–c. The crude
mixture was prepared with 4c (via 19a) using method A (I) or B (II) or 4d (via 19b) using method A (III) or B (IV). HPLC analysis was performed on a C18
reverse-phase column (4.6!250 mm, 5 mm particle size, TP silica, Vydac, Hesperia, CA) with a linear H2O/MeCN gradient containing 0.1% (v/v) TFA:
0–5 min 100% H2O, 5–35 min 2.67% MeCN/min, 1 mL/min flow rate, 210 nm.

J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
2499
and then solvent evaporation. To a stirred solution of the
resulting amine in dry CH2Cl2 (30 mL) was added N,N-
diisopropylethylamine (1.93 mL, 11.1 mmol) and then Tsc-
Cl (3.52 g, 11.1 mmol) at 0 8C. After stirring at room
temperature for 7 h, the reaction mixture was quenched with
H2O and extracted with CH2Cl2. The combined organic
layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/
n-hexaneZ3:1) to give 2a (3.7 g, 82%) as a white solid.
TLC (EtOAc/n-hexaneZ3:1) RfZ0.40; 1H NMR
(300 MHz, DMSO-d6) d 9.99 (brs, 1H), 8.12 (d, JZ
8.1 Hz, 2H), 7.97 (d, JZ8.1 Hz, 2H), 7.20 (s, 1H), 4.34 (brt,
2H), 4.24 (q, JZ7.2 Hz, 2H), 3.87 (brs, 5H), 1.27 (t, JZ
7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) d 158.37,
152.46, 143.20, 137.42, 133.40 (q, JZ31.8 Hz), 131.01,
128.85, 126.54, 123.30 (q, JZ272.3 Hz), 113.59, 60.46,
57.79, 54.14, 35.42, 14.08; HRMS (FABC) for
C17H19F3N3O6S (MHC), calcd 450.0947, found 450.0957.
Figure 5. HPLC (I) and MALDI-TOF (II) analyses of a crude mixture
containing peptide 21. The crude mixture was prepared with 4d (via 19b)
using method C (piperidine). The HPLC condition is identical to that of
Fig. 4 except for using a linear gradient of 3.33% rather than 2.67%
MeCN/min during 5–35 min.
3.1.4. Fmoc-Im-OEt (2b). To a solution of 14 (2 g,
10.1 mmol) in DMF (20 mL) was added 10% Pd/C
(45 mg). After stirring at room temperature for 10 h under
40 psi H2, 10% Pd/C was removed by filtration through
Celite 545 followed by washing with EtOAc and MeOH and
then solvent evaporation. To a stirred solution of the
resulting amine in dry CH2Cl2 (20 mL) was added N,N-
diisopropylethylamine (1.93 mL, 11.1 mmol) and then
Fmoc-Cl (2.87 g, 11.1 mmol) at 0 8C. After stirring at
room temperature for 4 h, the reaction mixture was
quenched with H2O and extracted with CH2Cl2. The
combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc/n-hexaneZ1:2) to give 2b (3.27 g,
83%) as a white solid. TLC (EtOAc/n-hexaneZ1:1) RfZ
2H), 3.86 (t, JZ5.7 Hz, 2H), 3.80 (s, 3H), 3.72 (s, 3H); 13
C
NMR (75 MHz, DMSO-d6) d 160.68, 152.45, 143.23,
133.48 (q, JZ32.3 Hz), 128.80, 126.61 (q, JZ3.6 Hz),
123.35 (q, JZ273.5 Hz), 122.32, 119.28, 118.82, 107.53,
57.52, 54.21, 50.92, 36.16; HRMS (FABC) for
C17H17F3N2O6S (MC), calcd 434.0759, found 434.0765.
3.1.2. Fmoc-Py-OMe (1b). To a solution of 13 (3 g,
16.3 mmol) in EtOAc (40 mL) was added 10% Pd/C
(50 mg). After stirring at room temperature for 10 h under
40 psi H2, 10% Pd/C was removed by filtration through
Celite 545 followed by washing with EtOAc and MeOH
and then solvent evaporation. To a stirred solution of
the resulting amine in dry CH2Cl2 (20 mL) was added
N,N-diisopropylethylamine (4.11 mL, 23.6 mmol) and then
Fmoc-Cl (4.64 g, 17.9 mmol) at 0 8C. After stirring at room
temperature for 4 h, the reaction mixture was quenched with
H2O and extracted with CH2Cl2. The combined organic
layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/
n-hexaneZ1:3) to give 1b (5.41 g, 88%) as a white solid.
TLC (EtOAc/n-hexaneZ1:1) RfZ0.62; 1H NMR
(300 MHz, DMSO-d6) d 9.51 (brs, 1H), 7.90 (d, JZ
7.5 Hz, 2H), 7.73 (d, JZ7.5 Hz, 2H), 7.43 (td, JZ7.2,
0.6 Hz, 2H), 7.35 (td, JZ7.4, 1.1 Hz, 2H), 7.12 (d, JZ
1.5 Hz, 1H), 6.69 (d, JZ1.8 Hz, 1H), 4.48 (d, JZ6.6 Hz,
2H), 4.28 (t, JZ6.3 Hz, 1H), 3.81 (s, 3H), 3.72 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 160.67, 153.28, 143.81,
140.81, 127.63, 127.10, 125.03, 122.71, 120.16, 119.28,
118.82, 107.45, 65.37, 50.91, 46.70, 36.17; HRMS (FABC)
for C22H20N2O4 (MC), calcd 376.1423, found 376.1414.
1
0.39; H NMR (300 MHz, DMSO-d6) d 10.41 (brs, 1H),
7.89 (d, JZ7.5 Hz, 2H), 7.76 (d, JZ6.6 Hz, 2H), 7.41 (t,
JZ7.4 Hz, 2H), 7.32 (t, JZ7.4 Hz, 3H), 4.26 (m, 5H), 3.89
(s, 3H), 1.28 (t, JZ6.8 Hz, 3H); 13C NMR (75 MHz,
DMSO-d6) d 158.38, 153.27, 143.70, 140.72, 137.78,
131.10, 127.70, 127.11, 125.44, 120.12, 113.62, 66.13,
60.51, 46.45, 35.43, 14.07; HRMS (FABC) for
C22H22N3O4 (MHC), calcd 392.1610, found 392.1605.
3.1.5. Ttc-Phe-OEt. To a stirred solution of 15-HCl (0.5 g,
2.18 mmol) in dry CH2Cl2 (15 mL) was added N,N-
diisopropylethylamine (0.55 mL, 3.16 mmol) and then
Ttc-Cl1 (0.93 g, 3.27 mmol) at 0 8C. After stirring at room
temperature for 4 h, the reaction mixture was quenched with
H2O and extracted with CH2Cl2. The combined organic
layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/
n-hexaneZ1:1) to give the title compound (778 mg, 81%)
as a white solid. TLC (EtOAc/n-hexaneZ1:2) RfZ0.52; 1H
NMR (300 MHz, CDCl3) d 7.52 (d, JZ8.1 Hz, 2H), 7.41 (d,
JZ8.4 Hz, 2H), 7.27 (m, 3H), 7.13 (dd, JZ7.8, 1.8 Hz, 2H),
5.20 (d, JZ8.1 Hz, 1H), 4.61 (dt, JZ8.0, 6.1 Hz, 1H), 4.23
(t, JZ6.8 Hz, 2H), 4.17 (q, JZ7.1 Hz, 2H), 3.17 (t, JZ
7.1 Hz, 2H), 3.13 (dd, JZ13.5, 5.4 Hz, 1H), 3.07 (dd, JZ
13.8, 5.7 Hz, 1H), 1.24 (t, JZ7.1 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 171.37, 155.24, 140.86 (q, JZ1.4 Hz),
135.65, 129.28, 128.54, 127.87, 127.79 (q, JZ32.6 Hz),
127.11, 125.77 (q, JZ3.8 Hz), 124.04 (q, JZ271.8 Hz),
63.00, 61.54, 54.73, 38.26, 31.24, 14.07; HRMS (FABC)
3.1.3. Tsc-Im-OEt (2a). To a solution of 14 (2 g,
10.1 mmol) in DMF (20 mL) was added 10% Pd/C
(45 mg). After stirring at room temperature for 10 h under
40 psi H2, 10% Pd/C was removed by filtration through
Celite 545 followed by washing with EtOAc and MeOH

2500
J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
for C21H23F3NO4S (MHC), calcd 442.1300, found
442.1294.
0 8C. After stirring at room temperature for 3 h, the reaction
mixture was quenched with H2O and extracted with
CH2Cl2. The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The residue was purified
by flash chromatography (EtOAc/n-hexaneZ1:1) to give 4a
(290 mg, 92%) as a white solid. TLC (EtOAc/n-hexaneZ
3.1.6. Tsc-Phe-OEt (3a). To a stirred solution of 15-HCl
(1.5 g, 6.53 mmol) in dry CH2Cl2 (20 mL) was added N,N-
diisopropylethylamine (1.69 mL, 9.70 mmol) and then Tsc-
Cl (2.27 g, 7.18 mmol) at 0 8C. After stirring at room
temperature for 7 h, the reaction mixture was quenched with
H2O and extracted with CH2Cl2. The combined organic
layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/
n-hexaneZ1:1) to give 3a (2.72 g, 88%) as a white solid.
Alternatively, 3a was synthesized by oxidation of Ttc-Phe-
OEt with H2O2/NaMoO4 in acetone.1 To a solution of Ttc-
Phe-OEt (1.2 g, 2.71 mmol) in acetone (30 mL) was added
0.3 M aqueous Na2MoO4 (0.8 mL, 0.24 mmol) and 30%
aqueous H2O2 (1.5 mL). After stirring at room temperature
for 18 h, the reaction mixture was extracted with CH2Cl2.
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc/n-hexaneZ1:1) to give 3a (1.18 g,
91%) as a white solid. TLC (EtOAc/n-hexaneZ1:1) RfZ
1
2:1) RfZ0.29; H NMR (500 MHz, DMSO-d6) d 8.12 (d,
JZ8.0 Hz, 2H), 8.01 (d, JZ8.0 Hz, 2H), 7.87 (d, JZ
7.5 Hz, 2H), 7.66 (d, JZ7.5 Hz, 2H), 7.49 (d, JZ7.5 Hz,
1H), 7.40 (t, JZ7.3 Hz, 2H), 7.31 (t, JZ7.3 Hz, 2H), 7.24
(t, JZ5.3 Hz, 1H), 4.27 (m, 3H), 4.22 (dd, JZ12.5, 6.0 Hz,
1H), 4.19 (t, JZ6.8 Hz, 1H), 3.88 (td, JZ8.0, 5.2 Hz, 1H),
3.77 (t, JZ5.3 Hz, 2H), 3.59 (s, 3H), 2.93 (q, JZ6.3 Hz,
2H), 1.64–1.47 (m, 2H), 1.38–1.30 (m, 2H), 1.25–1.18 (m,
2H); 13C NMR (125 MHz, DMSO-d6) d 172.58, 156.02,
155.22, 143.88, 143.26, 140.68, 133.41 (q, JZ32.4 Hz),
128.79, 127.52, 126.97, 126.55 (q, JZ3.4 Hz), 125.05,
123.34 (q, JZ272.8 Hz), 120.05, 65.10, 57.53, 54.19, 53.74,
51.74, 46.73, 39.66, 30.18, 28.79, 22.54; HRMS (FABC)
for C32H34F3N2O8S (MHC), calcd 663.1988, found
663.1979.
1
0.51; H NMR (300 MHz, CDCl3) d 8.03 (d, JZ8.4 Hz,
3.1.9. Fmoc-Lys(Boc)-OMe (17a). To a stirred solution of
17 (500 mg, 1.07 mmol) in dry CH2Cl2 (15 mL) was added
diazomethane (0.8 M in dry diethyl ether) at 0 8C. After
stirring at room temperature for 2 h, the reaction mixture
was quenched with MeOH and extracted with CH2Cl2/H2O.
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc/n-hexaneZ2:1) to give 17a
(380 mg, 74%) as a white solid. TLC (EtOAc/n-hexaneZ
2H), 7.79 (d, JZ8.4 Hz, 2H), 7.26 (m, 3H), 7.09 (dd, JZ
7.8, 1.5 Hz, 2H), 5.09 (d, JZ8.1 Hz, 1H), 4.48 (dt, JZ8.2,
6.2 Hz, 1H), 4.38 (t, JZ5.9 Hz, 2H), 4.15 (q, JZ7.1 Hz,
2H), 3.47 (td, JZ5.9, 1.8 Hz, 2H), 3.08 (dd, JZ14.1,
6.0 Hz, 1H), 3.00 (dd, JZ13.7, 6.5 Hz, 1H), 1.22 (t, JZ
7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 171.01, 154.38,
142.86 (q, JZ1.2 Hz), 135.50, 135.34 (q, JZ33.2 Hz),
129.10, 128.72, 128.45, 127.04, 126.32 (q, JZ3.7 Hz),
122.95 (q, JZ273.2 Hz), 61.48, 58.08, 55.23, 54.59, 37.91,
13.92; HRMS (FABC) for C21H23F3NO6S (MHC), calcd
474.1198, found 474.1213.
1
2:1) RfZ0.58; H NMR (500 MHz, DMSO-d6) d 7.88 (d,
JZ7.5 Hz, 2H), 7.74 (d, JZ7.5 Hz, 1H), 7.71 (dd, JZ7.5,
3.0 Hz, 2H), 7.41 (t, JZ7.5 Hz, 2H), 7.32 (t, JZ7.5 Hz,
2H), 6.76 (t, JZ5.5 Hz, 1H), 4.29 (m, 2H), 4.22 (t, JZ
7.0 Hz, 1H), 3.99 (td, JZ8.5, 5.0 Hz, 1H), 3.61 (s, 3H), 2.89
(q, JZ4.5 Hz, 2H), 1.70–1.56 (m, 2H), 1.36 (s, 9H), 1.32–
1.23 (m, 4H); 13C NMR (125 MHz, DMSO-d6) d 172.89,
156.07, 155.53, 143.76, 143.72, 140.69, 127.58, 127.00,
125.18, 120.05, 77.30, 65.57, 53.82, 51.75, 46.63, 39.67,
30.32, 28.97, 28.22, 22.76; HRMS (FABC) for
C27H35N2O6 (MHC), calcd 483.2495, found 483.2498.
3.1.7. Fmoc-Phe-OEt (3b). To a stirred solution of 15-HCl
(1.5 g, 6.53 mmol) in dry CH2Cl2 (20 mL) was added N,N-
diisopropylethylamine (1.69 mL, 9.70 mmol) and then
Fmoc-Cl (1.86 g, 7.18 mmol) at 0 8C. After stirring at
room temperature for 4 h, the reaction mixture was
quenched with H2O and extracted with CH2Cl2. The
combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (EtOAc/n-hexaneZ1:3) to give 3b (2.1 g,
77%) as a white solid. TLC (EtOAc/n-hexaneZ1:2) RfZ
3.1.10. Fmoc-Lys-OMe (17b). To a solution of 17a
(300 mg, 0.622 mmol) in CH2Cl2 (16 mL) was added
trifluoroacetic acid (TFA, 4.0 mL) in one portion. After
stirring at room temperature for 3 h, the reaction mixture
was concentrated at 30 8C in vacuo to give 17b (210 mg,
1
0.45; H NMR (300 MHz, CDCl3) d 7.75 (d, JZ7.2 Hz,
2H), 7.55 (dd, JZ6.8, 5.0 Hz, 2H), 7.38 (t, JZ7.2 Hz, 2H),
7.29 (td, JZ7.4, 1.2 Hz, 2H), 7.25 (m, 3H), 7.10 (dd, JZ
6.6, 1.2 Hz, 2H), 5.35 (d, JZ7.8 Hz, 1H), 4.65 (dt, JZ8.2,
6.0 Hz, 1H), 4.43 (dd, JZ10.4, 7.1 Hz, 1H), 4.32 (dd, JZ
10.4, 6.8 Hz, 1H), 4.19 (t, JZ6.6 Hz, 1H), 4.16 (q, JZ
7.2 Hz, 2H), 3.14 (dd, JZ13.4, 5.6 Hz, 1H), 3.08 (dd, JZ
13.5, 5.7 Hz, 1H), 1.22 (t, JZ7.2 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 171.40, 155.46, 143.76, 143.65, 141.20,
135.71, 129.28, 128.45, 127.61, 127.00, 126.96, 125.04,
124.97, 119.90, 119.88, 66.81, 61.44, 54.72, 47.05, 38.18,
14.04; HRMS (FABC) for C22H26NO4 (MHC), calcd
416.1862, found 416.1856.
1
88%) as a white solid. H NMR (500 MHz, DMSO-d6) d
7.89 (d, JZ7.5 Hz, 2H), 7.74 (m, 3H), 7.70 (t, JZ6.5 Hz,
2H), 7.41 (t, JZ7.3 Hz, 2H), 7.33 (t, JZ7.5 Hz, 2H), 4.34
(dd, JZ10.5, 7.0 Hz, 1H), 4.29 (dd, JZ10.5, 7.0 Hz, 1H),
4.22 (t, JZ7.0 Hz, 1H), 4.00 (td, JZ8.5, 5.0 Hz, 1H), 3.62
(s, 3H), 2.75 (q, JZ5.5 Hz, 2H), 1.72–1.57 (m, 2H), 1.56–
1.47 (m, 2H), 1.39–1.29 (m, 2H); 13C NMR (125 MHz,
DMSO-d6) d 172.77, 156.10, 143.75, 143.72, 140.72,
127.61, 127.02, 125.16, 125.13, 120.10, 65.55, 53.60,
51.85, 46.63, 38.52, 30.04, 26.42, 22.36; HRMS (FABC)
for C22H27N2O4 (MHC), calcd 383.1971, found 383.1976.
3.1.8. Tsc-Lys(Fmoc)-OMe (4a). To a stirred solution of
16-HCl (200 mg, 0.477 mmol, Novabiochem, La Jolla, CA)
in dry CH2Cl2 (20 mL) was added triethylamine (73 mL,
0.525 mmol) and then Tsc-Cl (181 mg, 0.573 mmol) at
3.1.11. Fmoc-Lys(Tsc)-OMe (4b). To a stirred solution of
17b (600 mg, 1.57 mmol) in dry CH2Cl2 (20 mL) was added
N,N-diisopropylethylamine (272 mL, 1.57 mmol) and then

J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
2501
Tsc-Cl (596 mg, 1.88 mmol) at 0 8C. After stirring at room
temperature for 4 h, the reaction mixture was quenched with
H2O and extracted with CH2Cl2. The combined organic
layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/
n-hexaneZ1:1) to give 4b (715 mg, 69%) as a white solid.
TLC (EtOAc/n-hexaneZ2:1) RfZ0.29; 1H NMR
(500 MHz, DMSO-d6) d 8.11 (d, JZ8.0 Hz, 2H), 8.01 (d,
JZ8.0 Hz, 2H), 7.88 (d, JZ7.5 Hz, 2H), 7.73 (d, JZ
7.5 Hz, 1H), 7.70 (dd, JZ6.8, 1.8 Hz, 2H), 7.41 (t, JZ
7.5 Hz, 2H), 7.32 (t, JZ7.5 Hz, 2H), 6.93 (t, JZ5.3 Hz,
1H), 4.32–4.20 (m, 5H), 3.97 (td, JZ8.1, 5.0 Hz, 1H), 3.76
(t, JZ5.5 Hz, 2H), 3.61 (s, 3H), 2.82 (q, JZ6.0 Hz, 2H),
1.67–1.54 (m, 2H), 1.29–1.22 (m, 4H); 13C NMR
(125 MHz, DMSO-d6) d 172.86, 156.05, 155.09, 143.75,
143.72, 143.38, 140.69, 133.32 (q, JZ32.0 Hz), 128.75,
127.58, 126.99, 126.51 (q, JZ3.7 Hz), 125.16, 123.36 (q,
JZ272.8 Hz), 120.06, 65.56, 57.16, 54.35, 53.76, 51.76,
46.61, 39.67, 30.21, 28.71, 22.62; HRMS (FABC) for
C32H34F3N2O8S (MHC), calcd 663.1988, found 663.1998.
combined organic layers were dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (MeOH/CH2Cl2Z1:9) to give 4d (720 mg,
38%) as a white solid. TLC (MeOH/CH2Cl2Z1:9) RfZ
1
0.28; H NMR (500 MHz, DMSO-d6) d 12.54 (brs, 1H),
8.11 (d, JZ8.0 Hz, 2H), 8.01 (d, JZ8.0 Hz, 2H), 7.88 (d,
JZ7.0 Hz, 2H), 7.71 (d, JZ7.5 Hz, 2H), 7.57 (d, JZ
8.0 Hz, 1H), 7.40 (t, JZ7.5 Hz, 2H), 7.31 (t, JZ7.0 Hz,
2H), 6.93 (t, JZ5.8 Hz, 1H), 4.27–4.19 (m, 5H), 3.88 (td,
JZ8.5, 4.0 Hz, 1H), 3.76 (t, JZ5.5 Hz, 2H), 2.82 (q, JZ
6.0 Hz, 2H), 1.68–1.53 (m, 2H), 1.29–1.22 (m, 4H); 13C
NMR (125 MHz, DMSO-d6) d 173.90, 156.11, 155.10,
143.80, 143.77, 143.41, 140.69, 140.67, 133.33 (q, JZ
32.1 Hz), 128.77, 127.60, 127.03, 126.53 (q, JZ3.4 Hz),
125.24, 125.22, 123.37 (q, JZ272.8 Hz), 120.06, 65.55,
57.17, 54.36, 53.76, 46.63, 39.67, 30.35, 28.79, 22.79;
HRMS (FABC) for C31H32F3N2O8S (MHC), calcd
649.1831, found 649.1822.
3.1.14. Tsc-Ala-OH. To a solution of H-Ala-OH (250 mg,
2.81 mmol) in 1 M aqueous NaHCO3 (20 mL) was added
1,4-dioxane (20 mL) and then Tsc-Cl (1.07 g, 3.37 mmol) at
08C. After stirring at room temperature for 2 h, the reaction
mixture was quenched with 1 N aqueous HCl and extracted
with EtOAc. The combined organic layers were dried over
MgSO4 and concentrated in vacuo. The residue was
recrystallized from diethyl ether and n-hexane to afford
the title compound (515 mg, 50%) as a white solid. TLC
(MeOH/CH2Cl2Z1:9) RfZ0.34; 1H NMR (500 MHz,
DMSO-d6) d 12.52 (brs, 1H), 8.12 (d, JZ8.5 Hz, 2H),
8.02 (d, JZ8.0 Hz, 2H), 7.29 (d, JZ7.5 Hz, 1H), 4.29 (m,
1H), 4.20 (m, 1H), 3.86 (m, 1H), 3.77 (m, 2H), 1.17 (d, JZ
7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) d 174.03,
154.86, 143.36, 133.42 (q, JZ32.4 Hz), 128.83, 126.58 (q,
JZ3.7 Hz), 123.39 (q, JZ273.3 Hz), 57.48, 54.32, 49.07,
16.88; HRMS (FABC) for C13H15F3NO6S (MHC), calcd
370.0572, found 370.0571.
3.1.12. Tsc-Lys(Fmoc)-OH (4c).10 To a cooled (0 8C)
solution of 16-HCl (302 mg, 0.72 mmol) in THF (7 mL)
was added dropwise over 3 min a solution of LiOH (56 mg,
2.34 mmol) in H2O (7 mL). After stirring at 0 8C for 50 min
(the time for complete reaction as judged by TLC (MeOH/
CH2Cl2Z1:9)), the reaction mixture was adjusted to pH 7
by adding saturated aqueous NH4Cl. To the resulting
suspension was added 1,4-dioxane (20 mL), 1 M aqueous
NaHCO3 (0.86 mL, 0.86 mmol), and then Tsc-Cl
(273.6 mg, 0.864 mmol) at 0 8C. After stirring at room
temperature overnight, the reaction mixture was quenched
with 1 N aqueous HCl and extracted with EtOAc. The
combined organic layers were dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (MeOH/CH2Cl2Z1:9) to give 4c (397 mg,
85%) as a white solid. TLC (MeOH/CH2Cl2Z1:8) RfZ
0.31; 1H NMR (500 MHz, DMSO-d6) d 8.12 (d, JZ8.5 Hz,
2H), 8.01 (d, JZ9.0 Hz, 2H), 7.87 (d, JZ7.5 Hz, 2H), 7.66
(d, JZ7.5 Hz, 2H), 7.40 (t, JZ7.5 Hz, 2H), 7.31 (t, JZ
7.3 Hz, 2H), 7.27 (d, JZ8.0 Hz, 1H), 7.25 (t, JZ5.8 Hz,
1H), 4.27 (m, 3H), 4.21 (dd, JZ12.5, 5.5 Hz, 1H), 4.18 (t,
JZ5.8 Hz, 1H), 3.78 (td, JZ9.0, 5.3 Hz, 1H), 3.75 (t, JZ
6.0 Hz, 2H), 2.94 (q, JZ6.3 Hz, 2H), 1.65–1.46 (m, 2H),
1.40–1.30 (m, 2H), 1.26–1.18 (m, 2H); 13C NMR
(125 MHz, DMSO-d6) d 173.63, 156.05, 155.25, 143.91,
143.30, 140.71, 133.40 (q, JZ32.4 Hz), 128.83, 127.56,
127.01, 126.58 (q, JZ3.7 Hz), 125.10, 123.40 (q, JZ
273.0 Hz), 120.08, 65.15, 57.46, 54.27, 53.79, 46.76, 39.67,
30.35, 28.88, 22.70; HRMS (FABC) for C31H32F3N2O8S
(MHC), calcd 649.1831, found 649.1844.
3.1.15. Deprotection of Tsc-Phe-OEt with LiOH. To a
cooled (0 8C) solution of 3a (17.0 mg, 0.036 mmol) in THF
(0.35 mL) was added dropwise over 1 min 0.2 N aqueous
LiOH (0.35 mL, 0.070 mmol). After stirring at 0 8C for
10 min (the time for complete reaction as judged by TLC
(EtOAc/n-hexaneZ1:5)), the reaction mixture was adjusted
to pH 7 by adding saturated aqueous NH4Cl and then
extracted with EtOAc. The combined organic layers were
dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (EtOAc/
n-hexaneZ1:5) to give 18a (7.0 mg, 77%) as a white solid.
TLC (EtOAc/n-hexaneZ1:4) RfZ0.27; 1H NMR
(300 MHz, CDCl3) d 8.10 (d, JZ8.4 Hz, 2H), 7.89 (d, JZ
8.1 Hz, 2H), 4.17 (dd, JZ3.8, 2.3 Hz, 1H), 3.49 (dd, JZ5.4,
2.1 Hz, 1H), 3.19 (dd, JZ5.4, 3.6 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 140.30 (q, JZ1.2 Hz), 136.05 (q, JZ
33.2 Hz), 129.42, 126.53 (q, JZ3.6 Hz), 122.97 (q, JZ
273.2 Hz), 63.05, 45.43; HRMS (CIC) for C9H8F3O3S
(MHC), calcd 253.0146, found 253.0142.
3.1.13. Fmoc-Lys(Tsc)-OH (4d).10 To a solution of 17
(1.39 g, 2.96 mmol) in CH2Cl2 (40 mL) was added
trifluoroacetic acid (4.0 mL) in one portion. After stirring
at room temperature for 3 h, the reaction mixture was
concentrated at 30 8C in vacuo to give Fmoc-Lys-OH
(1.09 g, O99%) as a white solid. To a stirred solution of
crude Fmoc-Lys-OH in 1,4-dioxane (40 mL) was added 1 M
aqueous NaHCO3 (3.55 mL, 3.55 mmol) and then Tsc-Cl
(1.12 g, 3.55 mmol) at 0 8C. After stirring at room
temperature for 8 h, the reaction mixture was quenched
with 1 N aqueous HCl and extracted with EtOAc. The
3.1.16. Deprotection of Tsc-Lys(Fmoc)-OMe with LiOH.
To a cooled (0 8C) solution of 4a (5.9 mg, 9 mmol) in THF
(87 mL) was added dropwise over 1 min 0.2 N aqueous
LiOH (87 mL, 17.4 mmol). After stirring at 0 8C for 5 min,
the reaction mixture (50 mL) was adjusted to pH 1 by adding

2502
J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
1 N aqueous HCl (100 mL) and then analyzed on a LC-MS
using a C18 reverse-phase column (4.6!250 mm, 5 mm
particle size, TP silica, Vydac, Hesperia, CA) with a linear
H2O/MeCN gradient containing 0.1% (v/v) AcOH: 0–5 min
100% H2O, 5–35 min 2.67% MeCN/min, 1 mL/min flow
rate, 254 nm, RvZ23.17 mL (16a, 8%) and 29.93 mL (16,
92%) (Fig. 3).
3.1.20. Deprotection of Fmoc-Lys(Tsc)-OMe with
1-methylpyrrolidine. To a stirred solution of 4b
(50.0 mg, 75.4 mmol) in dry DMF (189 mL) was added
1-methylpyrrolidine (189 mL, 1.82 mmol) at room tempera-
ture. After stirring at 25 8C for 1 h, the reaction mixture was
directly purified by flash chromatography (EtOAc/
n-hexaneZ2:1 and then MeOH/CH2Cl2Z1:9) to give 17d
(32.5 mg, 98%) and 18c. 17d: TLC (MeOH/CH2Cl2Z1:9)
RfZ0.39; 1H NMR (300 MHz, CDCl3) d 8.08 (d, JZ8.1 Hz,
2H), 7.86 (d, JZ8.4 Hz, 2H), 4.54 (t, JZ5.7 Hz, 1H), 4.41
(t, JZ5.7 Hz, 2H), 3.72 (s, 3H), 3.50 (t, JZ5.9 Hz, 2H),
3.43 (dd, JZ7.5, 5.3 Hz, 1H), 3.06 (q, JZ6.5 Hz, 2H),
1.78–1.30 (m, 6H); HRMS (FABC) for C17H24F3N2O6S
(MHC), calcd 441.1307, found 441.1321. 18c: TLC
(EtOAc/n-hexaneZ2:1) RfZ0.30; 1H NMR (300 MHz,
CDCl3) d 8.10 (d, JZ8.4 Hz, 2H), 7.88 (d, JZ8.4 Hz,
2H), 4.06 (t, JZ5.4 Hz, 2H), 3.40 (t, JZ5.3 Hz, 2H);
HRMS (FABC) for C9H10F3O3S (MHC), calcd 255.0303,
found 255.0303.
3.1.17. Deprotection of Tsc-Lys(Fmoc)-OMe with
1-methylpyrrolidine. To a stirred solution of 4a
(50.0 mg, 75.4 mmol) in dry DMF (189 mL) was added
1-methylpyrrolidine (189 mL, 1.82 mmol) at room tempera-
ture. After stirring at 25 8C for 1 h, the reaction mixture was
directly purified by flash chromatography (MeOH/
CH2Cl2Z1:30 and then MeOH/CH2Cl2Z1:9) to give 16b
(48.4 mg, 95%) and 18b. 16b: TLC (MeOH/CH2Cl2Z1:9)
RfZ0.43; 1H NMR (300 MHz, CDCl3) d 8.08 (d, JZ8.1 Hz,
2H), 8.07 (d, JZ8.1 Hz, 2H), 7.87 (d, JZ8.1 Hz, 2H), 7.86
(d, JZ8.4 Hz, 2H), 5.11 (d, JZ8.4 Hz, 1H), 4.43 (t, JZ
5.8 Hz, 2H), 4.21 (td, JZ7.8, 5.3 Hz, 1H), 3.74 (s, 3H), 3.50
(t, JZ5.9 Hz, 2H), 3.32 (t, JZ6.3 Hz, 2H), 3.03 (t, JZ
6.3 Hz, 2H), 2.56 (t, JZ6.9 Hz, 2H), 1.83–1.72 (m, 2H),
1.65–1.53 (m, 2H), 1.50–1.41 (m, 2H); HRMS (FABC) for
C26H31F6N2O8S2 (MHC), calcd 677.1426, found 677.1425.
18b: TLC (MeOH/CH2Cl2Z1:9) RfZ0.33; 1H NMR
(300 MHz, CDCl3) d 8.07 (d, JZ8.7 Hz, 2H), 7.83 (d, JZ
8.4 Hz, 2H), 3.35 (t, JZ7.5 Hz, 2H), 2.87 (t, JZ7.4 Hz,
2H), 2.39 (m, 4H), 1.65 (m, 4H); HRMS (FABC) for
C13H16F3NO2S (MC), calcd 307.0854, found 307.0866.
3.1.21. Deprotection of Fmoc-Lys(Tsc)-OMe with DIEA.
To a solution of 4b (66.3 mg, 0.10 mmol) in dry DMF
(1 mL) was added N,N-diisopropylethylamine (87.1 mL,
0.50 mmol) at room temperature. After heating at 50 8C for
4 h, the reaction mixture was quenched with H2O and
extracted with EtOAc. The combined organic layers were
dried over Na2SO4 and concentrated in vacuo. The residue
was purified by flash chromatography (MeOH/CH2Cl2Z
1:9) to give 17d as a white solid (38.3 mg, 87%). TLC
(MeOH/CH2Cl2Z1:9) RfZ0.39; 1H NMR (300 MHz,
CDCl3) d 8.08 (d, JZ8.1 Hz, 2H), 7.86 (d, JZ8.4 Hz,
2H), 4.47 (t, JZ5.4 Hz, 1H), 4.41 (t, JZ5.9 Hz, 2H), 3.73
(s, 3H), 3.50 (t, JZ5.9 Hz, 2H), 3.43 (dd, JZ7.5, 5.4 Hz,
1H), 3.06 (q, JZ6.5 Hz, 2H), 1.78–1.38 (m, 6H); HRMS
(FABC) for C17H24F3N2O6S (MHC), calcd 441.1307,
found 441.1305.
3.1.18. Deprotection of Tsc-Lys(Fmoc)-OMe with DIEA.
To a solution of 4a (66.3 mg, 0.10 mmol) in dry DMF
(1 mL) was added N,N-diisopropylethylamine (87.1 mL,
0.50 mmol) at room temperature. After heating at 50 8C for
4 h, the reaction mixture was quenched with H2O and
extracted with EtOAc. The combined organic layers were
dried over Na2SO4 and concentrated in vacuo. The residue
was purified by flash chromatography (MeOH/CH2Cl2Z
1:9) to give 16c (32.5 mg, 82%) as a white solid. TLC
(MeOH/CH2Cl2Z1:9) RfZ0.27; 1H NMR (300 MHz,
CDCl3) d 8.07 (d, JZ8.1 Hz, 2H), 7.86 (d, JZ8.7 Hz,
2H), 3.72 (s, 3H), 3.44 (dd, JZ7.5, 5.4 Hz, 1H), 3.32 (t, JZ
6.5 Hz, 2H), 3.04 (t, JZ6.5 Hz, 2H), 2.57 (t, JZ6.9 Hz,
2H), 1.61–1.38 (m, 6H); 13C NMR (75 MHz, CDCl3) d
176.47, 142.90 (q, JZ1.3 Hz), 135.53 (q, JZ33.3 Hz),
128.65, 126.48 (q, JZ3.7 Hz), 123.05 (q, JZ273.5 Hz),
56.01, 54.26, 51.97, 49.21, 42.90, 34.59, 29.54, 23.24;
HRMS (FABC) for C16H24F3N2O4S (MHC), calcd
397.1409, found 397.1395.
3.2. Peptide synthesis
Peptides 20 and 21 were synthesized on the Fmoc-Rink
Amide MBHA resin in a stepwise fashion by a manual
solid-phase method as described.1,11 Fmoc-Rink Amide
MBHA resin (loading 0.64 mmol/g resin, copolystyrene-1%
DVB, 100–200 mesh, 31.3 mg, 20 mmol, Novabiochem, La
Jolla, CA) was placed in a glass reaction vessel (5 mL, fitted
with a glass frit (G3, 20–30 mm, Iwaki)) and swollen in
CH2Cl2 for 5 min followed by drainage for use with
standard Fmoc chemistry. Amino acids (50 mmol for 4c,
4d, and Tsc-Ala-OH or 60 mmol for other amino acids) were
activated with PyBOP (31.2 mg, 60 mmol) in DMF (2 mL)
and coupled in the presence of N,N-diisopropylethylamine
(DIEA, 13.9 mL, 80 mmol) with shaking (wrist action
shaker, Burrell) at room temperature for 3 h. Deprotection
of the Fmoc group was performed using 50% (v/v)
piperidine for 10 min or 50% (v/v) 1-methylpyrrolidine
for 1 h in DMF with shaking at room temperature.
Deprotection of the Tsc group was performed using 50%
(v/v) piperidine for 10 min or 0.1 N LiOH in cooled (4 8C)
THF/H2O (1:1) for 10 min (or !2) with shaking at room
temperature. Unreacted free amines were capped by
acetylation with acetic anhydride (9.4 mL, 100 mmol) and
DIEA (17.4 mL, 100 mmol) in CH2Cl2 (2 mL) with shaking
at room temperature for 15 min. All peptides were
3.1.19. Deprotection of Fmoc-Lys(Tsc)-OMe with LiOH.
To a cooled (0 8C) solution of 4b (5.9 mg, 9 mmol) in THF
(87 mL) was added dropwise over 1 min 0.2 N aqueous
LiOH (87 mL, 17.4 mmol). After stirring at 0 8C for 5 min,
the reaction mixture (50 mL) was adjusted to pH 1 by adding
1 N aqueous HCl (100 mL) and then analyzed on a LC-MS
using a C18 reverse-phase column (4.6!250 mm, 5 mm
particle size, TP silica, Vydac, Hesperia, CA) with a linear
H2O/MeCN gradient containing 0.1% (v/v) AcOH: 0–5 min
100% H2O, 5–35 min 2.67% MeCN/min, 1 mL/min flow
rate, 254 nm, RvZ25.66 mL (17c, 20%) and 29.75 mL
(17b, 75%) (Fig. 3).

J. Seok Choi et al. / Tetrahedron 61 (2005) 2493–2503
2503
2004, 126, 8598–8599. (d) Opatz, T.; Liskamp, R. M. J. Org.
Lett. 2001, 3, 3499–3502. (e) Yang, R.; Prorok, M.; Castellino,
F. J.; Weliky, D. P. J. Am. Chem. Soc. 2004, 126,
´
14722–14723. (f) Hamze, A.; Martinez, J.; Hernandez, J.-F.
J. Org. Chem. 2004, 69, 8349–8402.
acetylated at the N terminus and amidated at the C terminus.
All couplings were monitored using a ninhydrin assay.
A solvent wash (5 mL!3) with DMF, MeOH, CH2Cl2, and
then DMF was performed right after deprotection, coupling,
and capping. An additional CH2Cl2 wash was employed
right before and after capping. Peptide resin cleavage and
side-chain deprotection were performed in a single step
using trifluoroacetic acid (TFA, 1 mL) at room temperature
for 2 h. After evaporation of the solvent or precipitation
with ether followed by centrifugation, crude peptides were
dissolved in DMF (1 mL). HPLC purification was achieved
using a C18 reverse-phase column (10!250 mm, 5 mm
particle size, TP silica, Vydac, Hesperia, CA) with a linear
H2O/MeCN gradient containing 0.1% (v/v) TFA: 0–5 min
100% H2O, 5–35 min 3.33% MeCN/min, 3 mL/min flow
rate, 210/254 nm. Peptides were recovered upon lyophiliza-
tion of the appropriate fractions as a solid (Scheme 3 and
Figs. 4 and 5): 20 via 19a, method A, 2.6 mg, 18% recovery;
20 via 19a, method B, 4.2 mg, 29%; 20 via 19b, method A,
2.3 mg, 15%; 20 via 19b, method B, 2.5 mg, 17%; 21 via
19b, method C, 9.6 mg, 40% (LiOH for Tsc deprotection)
and 15.1 mg, 63% (piperidine for Tsc deprotection, Fig. 5).
Purity of the peptide was determined to be O98% by
analytical HPLC on a C18 reverse-phase column (4.6!
250 mm, 5 mm particle size, TP silica, Vydac, Hesperia,
CA) with a linear H2O/MeCN gradient containing 0.1%
(v/v) TFA: 0–5 min 100% H2O, 5–35 min 3.33% MeCN/
min, 1 mL/min flow rate, 210/254 nm, RvZ22.50 mL (20)
and 23.09 mL (21). The molecular mass of each peptide was
measured using MALDI-TOF mass spectrometry: 20,
C39H49N7O7Na/C39H49N7O7K (MNaC)/(MKC), calcd
750.3586/766.3325, found 750.8125/766.7810; 21,
C57H79N13O16Na/C57H79N13O16K (MNaC/MKC), calcd
1224.5660/1240.5399, found 1224.4465/1240.4049.
3. (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in
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Acknowledgements
This work was financially supported by the Korea Research
Foundation Grant (KRF-2003-015-C00377). Fellowship
support from the BK21 and CRM programs (J.S.C. and
H.K.) is gratefully acknowledged.
7. (a) Farrera-Sinfreu, J.; Royo, M.; Albericio, F. Tetrahedron
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addition is indicated by the absence of byproducts containing
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3-amino function of lysine when 4a in 19a was treated with
1-methylpyrrolidine in the solid-phase synthesis of peptide 20
(Fig. 4, II).
References and notes
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