'O-Acyl isopeptide method' for the efficient synthesis of difficult sequence-containing peptides: use of 'O-acyl isodipeptide unit'

Article abstract of DOI:10.1016/j.tetlet.2006.03.017

A novel 'O-acyl isodipeptide unit', Boc-Thr(Fmoc-Val)-OH 5 has been successfully used for the efficient synthesis of a difficult sequence-containing pentapeptide based on the 'O-acyl isopeptide method', in which racemization-inducible esterification could

Full text of DOI:10.1016/j.tetlet.2006.03.017

Tetrahedron Letters 47 (2006) 3013–3017
‘O-Acyl isopeptide method’ for the efficient synthesis
of difficult sequence-containing peptides: use
of ‘O-acyl isodipeptide unit’
Youhei Sohma, Atsuhiko Taniguchi, Mariusz Skwarczynski, Taku Yoshiya, Fukue Fukao,
Tooru Kimura, Yoshio Hayashi and Yoshiaki Kiso*
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program,
Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
Received 27 January 2006; revised 1 March 2006; accepted 2 March 2006
Abstract—A novel ‘O-acyl isodipeptide unit’, Boc-Thr(Fmoc-Val)-OH 5 has been successfully used for the efficient synthesis of a
difficult sequence-containing pentapeptide based on the ‘O-acyl isopeptide method’, in which racemization-inducible esterification
could be omitted, suggesting that the use of O-acyl isodipeptide units allows the application of this method to fully automated pro-
tocols for the synthesis of long peptides or proteins.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The synthesis of ‘difficult sequence’-containing peptides
is one of the most problematic areas in peptide chemis-
try. These peptides are often obtained with low yield and
purity in solid-phase peptide synthesis (SPPS).1 These
difficult sequences are generally hydrophobic and pro-
mote aggregation in solvents during synthesis and puri-
fication. This aggregation is attributed to intermolecular
hydrophobic interaction and hydrogen bond network
among resin-bound peptide chains, resulting in the
formation of extended secondary structures such as
b-sheets.1
Our studies indicated that the isomerization of the pep-
tide backbone at only one position of the whole peptide
sequence, that is, formation of the ester, significantly
changed the unfavorable secondary structure of the dif-
ficult sequence-containing peptides, leading to improved
coupling and deprotection efficacy during SPPS. Mutter
et al.3a,c and Carpino et al.3b have also confirmed the
efficacy of the ‘O-acyl isopeptide method’. Herein, a
novel ‘O-acyl isodipeptide unit’, Boc-Thr(Fmoc-Val)-
OH 5 was successfully used to efficiently synthesize a
difficult sequence-containing pentapeptide (Ac-Val-Val-
Thr-Val-Val-NH2 3).
In regard to the synthesis of difficult sequence-contain-
ing peptides, we have recently disclosed an ‘O-acyl iso-
peptide method’,2 in which a native amide bond at a
hydroxyamino acid residue, for example, Ser was iso-
merized to the ester bond, followed by an O–N intra-
molecular acyl migration reaction (Fig. 1A). The method
has been successfully applied to the efficient synthesis
of difficult sequence-containing peptides such as Ac-
Val-Val-Ser-Val-Val-NH2 1 (Fig. 1B)2b,d,e and Alzhei-
mer’s disease-related amyloid b peptide (Ab) 1–42.2c–g
In the synthesis of peptide 3 by standard SPPS using
Fmoc-amino acids,4 an undesired peptide, Fmoc-Val-
Val-Thr-Val-Val-NH2 was obtained at a similar rate to
peptide 3 after the final deprotection (Fig. 2A). This
indicated that the Fmoc group of the pentapeptide-resin
was not deprotected during SPPS, similar to what we
have previously reported for the synthesis of 1.2b,d,e This
suggests that the highly hydrophobic nature of Fmoc-
peptide-resin prevented the base from accessing the
Fmoc group, thus forming insoluble micro-aggregates
on the resin. Further purification of 3 in preparative
scale HPLC was laborious due to the extremely low
solubility of the product (the solubility of 3 in H2O,
MeOH, and DMSO being 0.008 0.003, 0.059 0.004
Keywords: O-Acyl isodipeptide unit; O-Acyl isopeptide method;
Difficult sequence; O–N Intramolecular acyl migration.
*
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
and 1.89 0.14 mg mLÀ1
, respectively). When the
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.017

3014
Y. Sohma et al. / Tetrahedron Letters 47 (2006) 3013–3017
A
O
R2
O
H2N
O
H
Xaa OH
n
H
Xaa
N
pH 7.4
N
R1
H
Xaa
OH
m
H
n
H
O
R1
O
–N
intramolecular
acyl migration
N
HO
Xaa: amino acid
H
O
H
Xaa
m
R2
difficult sequence-containing peptides
O-acyl isopeptides
B
O
O
H
H2N
Val Val NH2
N
R1
Val Val NH2
Ac Val Val
pH 7.4
O
H
H
R1
N
H
HO
O
Ac Val
difficult sequence-containing pentapeptides
2 (R1 = H)
4 (R1 = CH3)
1 (R1 = H)
3 (R1 = CH3)
Figure 1. (A) ‘O-Acyl isopeptide method’: the synthetic strategy for difficult sequence-containing peptides via the O–N intramolecular acyl migration
reaction of O-acyl isopeptides, (B) application of the O-acyl isopeptide method for the synthesis of pentapeptides 1 and 3.
and 4ÆTFA was purified using the 0.1% aqueous TFA–
CH3CN system as the eluant to obtain pure 4 with an
isolated yield of 28.0%.
However, a large amount of racemization (21%) of the
esterified Val residue occurred in the DIPCDI–DMAP
method (Fig. 2B), which was confirmed by an indepen-
dent synthesis of H-Thr(Ac-Val-D-Val)-Val-Val-NH2.
This extent of racemization is remarkably higher than
that of the esterification between Val and Ser in 2
Figure 2. HPLC profiles of crude (A) peptide 3 (synthesized by the
standard SPPS) and (B) its O-acyl isopeptide 4. Analytical HPLC was
performed using a C18 reverse phase column (4.6 · 150 mm; YMC
Pack ODS AM302) with a binary solvent system: a linear gradient of
(0.8%),2b,d,e which is probably due to steric hindrance
at the secondary hydroxyl group in Thr as compared to
Ser. We also observed a slightly higher amount of race-
mization (33%) when 1-(mesitylene-2-sulfonyl)-3-nitro-
1,2,4-triazole (MSNT) (3.0 equiv)–N-methylimidazole
(NMI) (12 equiv) in CH2Cl26 was used for the esterifica-
tion, which agrees with a literature report.6c We consid-
ered that the large extent of racemization should be a
serious disadvantage in the synthesis of Thr-containing
peptides using the O-acyl isopeptide method.
CH3CN (0–100% CH3CN, 40 min) in 0.1% aqueous TFA at a flow rate
of 0.9 mL minÀ1 (40 ꢁC), detected at 230 nm.
DMSO solution of 3 was used for HPLC purification,
the overall yield of 3 was only 1.4%.4
For the case of O-acyl isopeptide method,5 Boc-Thr-OH
was coupled to the H-Val-Val-NH-resin, and subse-
quent acylation with Fmoc-Val-OH to the b-hydroxyl
group of Thr was performed using the DIPCDI–DMAP
method in CH2Cl2 to obtain ester. After coupling with
another Val residue, N-acetylation and TFA treatment,
O-acyl isopeptide 4ÆTFA was obtained without forming
Fmoc-containing by-product (Fig. 2B). Hence, the pro-
tected peptide resin was efficiently synthesized with no
interference from the difficult sequences. The results
support our hypothesis that the modification of 3 to
the ester structure 4 changed the secondary structure
of peptide to that more favorable for Fmoc-depro-
tection. Moreover, the solubility of 4ÆTFA in H2O or
MeOH was 46.2 22.7 (5775-fold) or 266.5 65.4
(4517-fold) mg mLÀ1, respectively, higher than that of
N-acyl peptide 3, because of the ionized amino group
in the isopeptide. Accordingly, a solution of 4ÆTFA in
MeOH could easily be applied to preparative HPLC,
To avoid this problem, we decided to adapt an O-acyl
isodipeptide unit, Boc-Thr(Fmoc-Val)-OH 5 (Fig. 3A),
Figure 3. (A) O-Acyl isodipeptide unit 5 and (B) crude isopeptide 4
(synthesized using 5). Analytical HPLC was performed using a C18
reverse phase column (4.6 · 150 mm; YMC Pack ODS AM302) with
binary solvent system: a linear gradient of CH3CN (0–100% CH3CN,
40 min) in 0.1% aqueous TFA at a flow rate of 0.9 mL minÀ1 (40 ꢁC),
detected at 230 nm.

Y. Sohma et al. / Tetrahedron Letters 47 (2006) 3013–3017
3015
i , ii
i , ii
H
H
i , iii
Fmoc N
Fmoc Val
Val
N
6
Rink-Amide-AM resin
O
H
O
H
N
i , ii
H
H
Boc
N
Boc
Ac Val Val
Val Val
CH3
N
Val Val
N
CH3
H
i , iv
H
O
Fmoc Val
O
7
8
O
.
H N
TFA
2
v
vi
Val Val NH2
CH3
3
H
Ac Val Val
O
.
4 TFA
Scheme 1. Reagents and conditions: (i) 20% piperidine/DMF, 20 min; (ii) Fmoc-Val-OH (2.5 equiv), DIPCDI (1,3-diisopropylcarbodiimide,
2.5 equiv), HOBt (2.5 equiv), DMF, 2 h; (iii) 5 (2.5 equiv), DIPCDI (2.5 equiv), HOBt (2.5 equiv), DMF, 2 h; (iv) Ac2O (1.5 equiv), TEA (1.0 equiv),
DMF, 2 h; (v) TFA–m-cresol–thioanisole–H2O (92.5:2.5:2.5:2.5), 90 min; (vi) phosphate buffered saline (PBS), pH 7.4, 25 ꢁC.
which was synthesized by solution phase,7 for the syn-
thesis of 3 based on the O-acyl isopeptide method
(Scheme 1).8 The use of isodipeptide 5 could omit the
racemization-inducing esterification reaction. The O-
acyl isodipeptide 5, which readily solubilized in DMF,
was coupled to the H-Val-Val-NH-resin using the stan-
dard DIPCDI–HOBt method (2 h) to obtain 7. The
completeness of the coupling was verified by the Keiser
test. After coupling with another Val residue followed
by N-acetylation and TFA–m-cresol–thioanisole–H2O
(92.5:2.5:2.5:2.5) treatment, O-acyl isopeptide 4ÆTFA
was obtained. As shown in Figure 3B, HPLC analysis
of crude 4 (synthesized using O-acyl isodipeptide unit
5) exhibited a high purity of the desired product 4 with
no by-product derived from the difficult sequence or
racemization. The use of isodipeptide 5 did not lead to
any additional side reaction. Moreover, since H-Thr-
Val-Val-NH2 was not formed as a by-product, we con-
cluded that (1) the ester bond between Val and Thr
was stable in both piperidine and TFA treatments and
(2) diketopiperazine was not formed when the last Fmoc
group was removed. Consequently, we could obtain
pure 4 without further purification, with an isolated
yield of 44.5%.
Figure 4. (A) A graph and (B) photographs in conversion of O-acyl
isopeptide 4 to 3 via the O–N intramolecular acyl migration in
phosphate buffered saline (pH 7.4, 25 ꢁC).
Compound 4ÆTFA was stable at 4 ꢁC for at least 2 years.
On the other hand, when 4ÆTFA was dissolved and stir-
red in phosphate buffered saline (PBS, pH 7.4) at rt,
quantitative O–N intramolecular acyl migration to the
corresponding parent peptide 3 was observed with no
side reaction (Fig. 4A).9 Isopeptide 4 exhibited more
than 5-fold faster migration with a half-life of 23 min
than that observed in 2 containing Ser (half-life =
2 h).2b,d,e The faster migration in 4 may be attributed
to a unique interlocking effect of the b-methyl group
in Thr, which has conformational restrictions, such as
a gem-effect by geminal methyl substitution.10 Finally,
as depicted in Figure 4B, 3 was formed as a white precip-
itate from 4. The resulting precipitate was centrifuged
and washed with water and methanol to afford highly
pure 3. The overall yield of 3 in O-acyl isopeptide method
was 42.7%.8
In conclusion, ‘O-acyl isopeptide method’ with a novel
O-acyl isodipeptide unit, Boc-Thr(Fmoc-Val)-OH 5, in
which the racemization-inducing esterification reaction

3016
Y. Sohma et al. / Tetrahedron Letters 47 (2006) 3013–3017
DMF (1.5 mL) for 2 h according to the sequence. The
Fmoc-group was removed by 20% piperidine/DMF
(20 min). N-Acetylation was carried out with Ac2O
(14.3 lL, 0.151 mmol) in the presence of TEA (17.5 lL,
0.126 mmol) for 2 h. The peptide was cleaved from the
resin using TFA (4.7 mL) in the presence of thioanisole
(126 lL), m-cresol (126 lL), and distilled water (126 lL)
for 90 min at rt, concentrated in vacuo, washed with Et2O,
centrifuged, suspended with water, and lyophilized to give
the crude peptide (45.9 mg). This crude peptide (20 mg)
was saturated in DMSO, filtered using a 0.46 lm filter
unit, and immediately injected into preparative HPLC
with a 0.1% aqueous TFA–CH3CN system. The desired
fractions were collected and immediately lyophilized to
afford the desired peptide 3 as a white amorphous pow-
der. Yield: 0.4 mg (1.4%); HRMS (FAB): calcd for
C26H49N6O7 (M+H)+: 557.3663, found: 557.3657; HPLC
analysis at 230 nm: purity was higher than 95%.
could be omitted, has been successfully applied to the
efficient synthesis of a difficult sequence-containing pen-
tapeptide by improving the nature of difficult sequence
during SPPS. This suggests that the use of O-acyl iso-
dipeptide units allows the application of ‘O-acyl isopep-
tide method’ to fully automated protocols for the
synthesis of long peptides or proteins.
Acknowledgments
This research was supported in part by the ‘Academic
Frontier’ Project for Private Universities: matching fund
subsidy from MEXT (Ministry of Education, Culture,
Sports, Science and Technology) of the Japanese Gov-
ernment, and the 21st Century COE Program from
MEXT. Y.S. is grateful for Research Fellowships of
JSPS for Young Scientists. M.S. is grateful for Postdoc-
toral Fellowship of JSPS. We thank Ms. Y. Fukusako
and Mr. M. Sasaki for technical assistance. We are
grateful to Ms. K. Oda and Mr. T. Hamada for mass
spectra measurements. We thank Dr. J.-T. Nguyen for
his help in preparing the manuscript.
5. After the preparation of the H-Val-Val-NH-resin (Rink
amide AM resin, 200 mg, 0.126 mmol) in the same manner
as described in the synthesis of 3 using the conventional
method, Boc-Thr-OH (82.9 mg, 0.378 mmol) was coupled
in the presence of DIPCDI (59.2 lL, 0.378 mmol) and
HOBt (57.8 mg, 0.378 mmol) in DMF (1.5 mL). Subse-
quent coupling with Fmoc-Val-OH (128 mg, 0.378 mmol)
to the b-hydroxyl group of Thr was performed using the
DIPCDI
(59.2 lL,
0.378 mmol)–DMAP
(3.1 mg,
0.0252 mmol) method in CH2Cl2 (1.5 mL) for 16 h (·2),
followed by coupling with another Val residue, N-acety-
lation using Ac2O (17.8 lL, 0.15 mmol)–TEA (17.6 lL,
0.126 mmol), TFA (4.7 mL)–thioanisole (128 lL)–m-cre-
sol (128 lL)–distilled water (128 lL) treatment for 90 min
at rt, concentration in vacuo, Et2O wash, centrifugation,
suspension in water, and lyophilization to give the crude
O-acyl isopeptide 4ÆTFA (51.5 mg). This crude peptide
(20 mg) was dissolved in MeOH, filtered using a 0.46 lm
filter unit, and immediately injected into preparative
HPLC with a 0.1% aqueous TFA–CH3CN system. The
desired fractions were collected and immediately lyophi-
lized, affording the desired O-acyl isopeptide 4ÆTFA as a
white amorphous powder (9.2 mg, 28.0%). HRMS (FAB):
calcd for C26H49N6O7 (M+H)+: 557.3663, found:
557.3666; HPLC analysis at 230 nm: purity was higher
than 95%.
References and notes
1. (a) Wo¨hr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato,
T.; Sun, X.; Mutter, M. J. Am. Chem. Soc. 1996, 118,
9218–9227; For a review, see: (b) Sheppard, R. J. Pept.
Sci. 2003, 9, 545–552.
2. (a) Sohma, Y.; Sasaki, M.; Ziora, Z.; Takahashi, N.;
Kimura, T.; Hayashi, Y.; Kiso, Y. Peptides, Peptide
Revolution: Genomics, Proteomics & Therapeutics; Kluwer
Academic: Netherlands, 2003; pp 67–68; (b) Sohma, Y.;
Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Chem.
Commun. 2004, 124–125; (c) Sohma, Y.; Sasaki, M.;
Hayashi, Y.; Kimura, T.; Kiso, Y. Tetrahedron Lett. 2004,
45, 5965–5968; (d) Sohma, Y.; Hayashi, Y.; Skwarczynski,
M.; Hamada, Y.; Sasaki, M.; Kimura, T.; Kiso, Y.
Biopolymers 2004, 76, 344–356; (e) Sohma, Y.; Hayashi,
Y.; Kimura, M.; Chiyomori, Y.; Taniguchi, A.; Sasaki,
M.; Kimura, T.; Kiso, Y. J. Pept. Sci. 2005, 11, 441–451;
(f) Sohma, Y.; Chiyomori, Y.; Kimura, M.; Fukao, F.;
Taniguchi, A.; Hayashi, Y.; Kimura, T.; Kiso, Y. Bioorg.
Med. Chem. 2005, 13, 6167–6174; (g) Taniguchi, A.;
Sohma, Y.; Kimura, M.; Okada, T.; Ikeda, K.; Hayashi,
Y.; Kimura, T.; Hirota, S.; Matsuzaki, K.; Kiso, Y. J. Am.
Chem. Soc. 2006, 128, 696–697.
6. (a) Nielsen, J.; Lyngso, L. O. Tetrahedron Lett. 1996, 37,
8439–8442; (b) Meldal, M.; Svendsen, I. B.; Juliano, L.;
Juliano, M. A.; Del, N. E.; Scharfstein, J. J. Pept. Sci.
1998, 4, 83–91; (c) Tamamura, H.; Kato, T.; Otaka, A.;
Fujii, N. Org. Biomol. Chem. 2003, 1, 2468–2473.
7. EDCÆHCl (104 mg, 0.539 mmol) was added to a stirring
solution of N-(t-butoxycarbonyl)-L-threonine benzyl ester
(Boc-Thr-OBzl)11 (139 mg, 0.449 mmol), N-(9H-fluoren-9-
ylmethoxycarbonyl)-L-valine (Fmoc-Val-OH, 183 mg,
0.539 mmol), and DMAP (5.5 mg, 0.045 mmol) in dry
CHCl3 (10 mL) at 0 ꢁC. The mixture was slowly warmed
to rt over 2 h, stirred additionally for 5 h, diluted with
AcOEt, and washed successively with water, 1 M HCl,
water, a saturated NaHCO3, and brine. The organic layer
was dried over MgSO4 and the solvent was removed in
vacuo. The resulting oil was purified by silica gel column
chromatography (AcOEt–hexane 1:4) to yield Boc-
Thr(Fmoc-Val)-OBzl (266 mg, 0.422 mmol, 94%). Epi-
merization during the above reaction was not detected,
which was confirmed by comparison with independently
synthesized D-valine derivative. After that, Pd/C was
added (12 mg) to the stirring solution of the Boc-
Thr(Fmoc-Val)-OBzl (236 mg, 0.374 mmol) in AcOEt
(10 mL), and the reaction mixture was vigorously stirred
for 3 h (it is worthy to notice that the higher amount of
3. (a) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.;
Santos, S. D.; Mandal, B.; Mimna, R.; Murat, K.; Patiny,
`
L.; Saucede, L.; Tuchscherer, G. Angew. Chem., Int. Ed.
2004, 43, 4172–4178; (b) Carpino, L. A.; Krause, E.;
Sferdean, C. D.; Schumann, M.; Fabian, H.; Bienert, M.;
¨
Beyermann, M. Tetrahedron Lett. 2004, 45, 7519–7523; (c)
Santos, S. D.; Chandravarkar, A.; Mandal, B.; Mimna,
`
R.; Murat, K.; Saucede, L.; Tella, P.; Tuchscherer, G.;
Mutter, M. J. Am. Chem. Soc. 2005, 127, 11888–11889.
4. The peptide 3 was synthesized on Rink amide amino-
methyl (AM) resin (200 mg, 0.126 mmol) according to the
general Fmoc-based solid-phase procedure. After the resin
was washed with DMF (1.5 mL, ·5), Fmoc-Val-OH
(107 mg, 0.315 mmol), and Fmoc-Thr(tBu)-OH (111 mg,
0.315 mmol) were coupled in the presence of 1,3-diisoprop-
ylcarbodiimide (DIPCDI, 49.2 lL, 0.315 mmol) and 1-
hydroxybenzotriazole (HOBt, 48.2 mg, 0.315 mmol) in

Y. Sohma et al. / Tetrahedron Letters 47 (2006) 3013–3017
3017
Pd/C caused by-products formation). The catalyst was
filtered off through Celite. The solvent was removed in
vacuo and the crude product was filtered via silica gel, at
first with AcOEt–hexane 1:2 and then the final product
was washed out by methanol to give pure 5 (186 mg,
0.346 mmol, 92%). HRMS (FAB): calcd for C29H36-
N2O8Na (M+Na)+: 563.2369, found: 563.2373; HPLC
analysis at 230 nm: purity was higher than 95%; NMR
(CD3OD, 400 MHz): d 7.79 (d, 3J(H,H) = 7.3 Hz, 2H,
CH), 7.75–7.66 (m, 2H, CH), 7.38 (t, 3J(H,H) = 7.5 Hz,
2H, CH), 7.33–7.29 (m, 2H, CH), 5.44–5.41 (m, 1H, CH),
4.38 (d, 3J(H,H) = 7.0 Hz, 2H, CH2), 4.25–4.22 (m, 2H,
CH), 4.05–4.01 (m, 1H, CH), 2.11–2.02 (m, 1H, CH), 1.44
found: 557.3666; HPLC analysis at 230 nm: purity was
higher than 95%. Purified O-acyl isopeptide 4ÆTFA
(3.0 mg) was then dissolved in phosphate-buffered saline
(PBS, pH 7.4, 3 mL) at rt and stirred overnight at rt. The
resultant precipitate was centrifuged and washed with
water and MeOH followed by drying in vacuo to give 3 as
a white powder. Yield: 2.4 mg (96%); HRMS (FAB): calcd
for C26H49N6O7 (M+H)+: 557.3663, found: 557.3667;
HPLC analysis at 230 nm: purity was higher than 95%; the
retention time on HPLC (0–100% CH3CN for 40 min,
230 nm) of synthesized product was identical to that of 3,
which was synthesized independently by the conventional
method.4
3
(s, 9H, CH3), 1.25 (d, J(H,H) = 6.4 Hz, 3H, CH3), 0.91,
9. To PBS (495 lL, pH 7.4) were added DMSO (4 lL) and
solution of 4 (1 lL, 10 mM in DMSO), and the mixture
was stirred at rt. At the desired time points, DMSO
(500 lL) was added to the samples and the mixture was
directly analyzed by RP-HPLC. HPLC was performed
using a C18 (4.6 · 150 mm; YMC Pack ODS AM302)
reverse-phase column with a binary solvent system: linear
gradient of CH3CN (0–100%, 40 min) in 0.1% aqueous
TFA at a flow rate of 0.9 mL minÀ1, detected at UV
230 nm.
10. (a) Beesley, R. M.; Ingold, C. K.; Thorpe, J. F. J. Chem.
Soc. 1915, 107, 1080–1106; (b) Matsumoto, H.; Sohma,
Y.; Kimura, T.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem.
Lett. 2001, 11, 605–609.
11. Skwarczynski, M.; Sohma, Y.; Noguchi, M.; Hayashi, Y.;
Kimura, T.; Kiso, Y. J. Org. Chem. 2006, 71, 2542–
2545.
3
0.89 (2d, J(H,H) = 7.7, 7.0 Hz, 6H, CH3).
8. After the preparation of the H-Val-Val-NH-resin (Rink
amide AM resin, 100 mg, 0.071 mmol) in the same manner
described in the synthesis of 3 using the conventional
method, Boc-Thr(Fmoc-Val)-OH 5 (100 mg, 0.18 mmol)
was coupled in the presence of DIPCDI (29.0 lL,
0.18 mmol) and HOBt (28.4 mg, 0.18 mmol) in DMF
(1.5 mL). Subsequent coupling with Fmoc-Val-OH
(62.8 mg, 0.18 mmol), N-acetylation using Ac2O
(10.5 lL, 0.11 mmol)–TEA (10.4 lL, 0.071 mmol), TFA
(2.47 mL)–thioanisole (66.7 lL)–m-cresol (66.7 lL)–dis-
tilled water (66.7 lL) treatment for 90 min at rt, concen-
tration in vacuo, Et2O wash, centrifugation, suspension in
water, and lyophilization gave the pure O-acyl isopeptide
4ÆTFA as a white amorphous powder (21.2 mg, 44.5%).
HRMS (FAB): calcd for C26H49N6O7 (M+H)+: 557.3663,