'O-Acyl isopeptide method': racemization-free segment condensation in solid phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2006.09.001

We disclosed a novel 'racemization-free segment condensation' based on the 'O-acyl isopeptide method' in which an N-segment including C-terminal O-acyl isopeptide structure with urethane-protected Ser/Thr residue was employed for the segment condensation,

Full text of DOI:10.1016/j.tetlet.2006.09.001

Tetrahedron Letters 47 (2006) 7905–7909
‘O-Acyl isopeptide method’: racemization-free
segment condensation in solid phase peptide synthesis
Taku Yoshiya,a Youhei Sohma,a,b Tooru Kimura,a Yoshio Hayashia and Yoshiaki Kisoa,*
aDepartment of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program,
Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
bDepartment of Physical Chemistry, 21st Century COE Program, Kyoto Pharmaceutical University, Yamashina-ku,
Kyoto 607-8412, Japan
Received 8 August 2006; revised 31 August 2006; accepted 1 September 2006
Available online 25 September 2006
Abstract—We disclosed a novel ‘racemization-free segment condensation’ based on the ‘O-acyl isopeptide method’ in which an N-
segment including C-terminal O-acyl isopeptide structure with urethane-protected Ser/Thr residue was employed for the segment
condensation, suggesting that the use of this method contributes to the effective convergent synthesis of long peptides/proteins.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Total chemical synthesis of peptides/proteins is of great
significance to understand biological functions. Toward
this purpose, many kinds of convergent synthetic meth-
ods have been reported.1 However, a fundamental draw-
back of convergent synthesis is that racemization at the
C-terminal residue of an N-segment occurs during the
condensation reaction with the C-segment. In ‘segment
condensation’,1g–r which is one of the important meth-
ods in convergent synthesis, a large amount of racemiza-
tion is generally involved. Particularly, in solid phase
segment condensation,1k–r the lower reactivity causes
a higher extent of racemization as compared with
solution phase synthesis. That is because, in contrast
to urethane-protected amino acids, peptides easily form
chirally labile oxazolones upon C-terminal carboxyl
activation, limiting the N-segment to contain either a
C-terminal Gly or Pro residue.1j,o,r
successfully applied to efficiently synthesize difficult
sequence-containing peptides such as Alzheimer’s dis-
ease-related amyloid b peptide (Ab) 1–42.2c–g,i Our
studies indicated that isomerization of the peptide back-
bone at only one position in the whole peptide sequence,
that is, formation of a single ester bond, significantly
changed the unfavorable secondary structure of the dif-
ficult sequence-containing peptide, leading to improved
coupling and deprotection efficacy during SPPS. Mutter
et al.,3 Carpino et al.,4 and Bo¨rner and co-workers5 have
also confirmed the efficacy of the ‘O-acyl isopeptide
method’. Moreover, very recently, we designed a novel
‘O-acyl isodipeptide unit’, that is, Boc-Ser/Thr(Fmoc-
Xaa)-OH (Fig. 1B).2h,i The use of O-acyl isodipeptide
units, in which the racemization-inducing esterification
reaction on resin could be omitted, allows the ‘O-acyl
isopeptide method’ to fully automated protocols for
the synthesis of peptides/proteins.
We have recently disclosed a novel ‘O-acyl isopeptide
method’2 in which a native amide bond at a hydroxy-
amino acid residue, for example, Ser, was isomerized
to an ester bond, followed by an O–N intramolecular
acyl migration reaction (Fig. 1A). The method has been
Herein, we disclosed a novel ‘racemization-free segment
condensation’ based on the ‘O-acyl isopeptide method’
(Fig. 2B). We conceived the idea that the N-segment,
which possesses a C-terminal O-acyl isopeptide struc-
ture, could be coupled to the N-terminal amino group
of a C-segment without any undesired racemization
because the isopeptide structure includes a urethane-
protected Ser/Thr residue. Thus, during the activation
of the carboxyl group of the isopeptide, the formation of
racemization-inducing oxazolones should be remarkably
suppressed.
Keywords: O-Acyl isodipeptide unit; O-Acyl isopeptide method; O–N
intramolecular acyl migration; Racemization-free; Segment condensa-
tion.
*
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.09.001

7906
T. Yoshiya et al. / Tetrahedron Letters 47 (2006) 7905–7909
A
B
O
H2N
R2
O
Xaa OH
n
H
N
pH 7.4
H
Xaa
R1
H
N
Xaa OH
n
O
O–N
intramolecular
acyl migration
m
H
H
O
R1
N
O
HO
Xaa: amino acid
R1 = H; Ser
1 = CH3; Thr
H
H
Xaa
m
O
R2
R
difficult sequence-containing peptides
(hydrophobic)
-acyl isopeptides
(hydrophilic)
O
H
O
N
OH
R1
O
O
H
H
N
O
O
R
1 = H, R2 = CH2p-C6H4OC(CH3)3; Boc–Ser(Fmoc–Tyr(
tBu))–OH 2
O
R2
R1 = CH3, R2 = CH(CH3)2; Boc–Thr(Fmoc–Val)–OH 3
Boc–Ser/Thr(Fmoc–Xaa)–OH
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) ‘O-acyl isodipeptide units’.
hydroxybenzotriazole, 2.5 equiv) method to obtain
R2
O
A
H
Fmoc-Tyr(tBu)-Ser(tBu)-Phe-O-resin. After the pro-
tected peptide resin was deprotected with TFA, the
resulting crude 1 was analyzed by HPLC. As a result,
3.0% of Fmoc-Tyr-D-Ser-Phe-OH was detected in crude
1 (Fig. 3A), which was confirmed by an independent
synthesis of the D-Ser derivative. This result indicated
that racemization at the activated Ser residue occurred
during segment condensation.
N
peptide A
N
OH
+
H2N
peptide B
H
O
R1
H
HO
C-segment
N-segment
segment
condensation
R2
O
H
N
∗
peptide B
peptide A
N
N
H
H
R1
O
HO
deprotection/
cleavage
H
racemization mixture
On the other hand, in segment condensation based on
the ‘O-acyl isopeptide method’,7 O-acyl isodipeptide
unit, Boc-Ser(Fmoc-Tyr(tBu))-OH8 (2, Fig. 1B) was
coupled to H-Phe-O-resin using the DIPCDI(2.5
equiv)–HOBt(2.5 equiv) method to obtain Boc-Ser-
(Fmoc-Tyr(tBu))-Phe-O-resin. After deprotection with
TFA, the obtained isopeptide H-Ser(Fmoc-Tyr)-Phe-
OHÆTFA was treated with phosphate buffer (pH 7.4)
to induce an O–N intramolecular acyl migration to
afford 1. In HPLC analysis of crude 1, no detectable
racemized compound Fmoc-Tyr-D-Ser-Phe-OH was
observed (Fig. 3B), indicating that the O-acyl isodipep-
O
B
H
N
Boc
H
OH
R1
H
+
H2N
peptide B
O
peptide A
N
O
R2
N-segment
C-segment
O
H
N
Boc
peptide B
N
R1
segment
condensation
H
O
H
H
N
peptide A
O
R2
1
A
B
1
deprotection/
cleavage
R2
O
H
N
peptide A
N
peptide B
N
H
H
O
R1
O–N intramolecular
acyl migration
HO
H
No racemized compound
at Ser residue
peptides/proteins
Figure 2. (A) A standard segment condensation; (B) a novel ‘racemi-
zation-free segment condensation’ based on the ‘O-acyl isopeptide
method’.
Fmoc–Tyr–
D-Ser–Phe–OH
(3.0%)
15
20
25
15
20
25
retention time (min)
retention time (min)
Figure 3. HPLC profiles of crude peptide Fmoc-Tyr-Ser-Phe-OH (1)
synthesized using (A) the standard segment condensation and (B) ‘O-
acyl isopeptide method’-based segment condensation. Analytical
To evaluate this hypothesis, we first selected Fmoc-Tyr-
Ser-Phe-OH (1) as a model. As a comparative study, 1
was synthesized by the standard segment condensation
method.6 Fmoc-Tyr(tBu)-Ser(tBu)-OH was coupled to
H-Phe-O-resin (2-chlorotrityl resin) using the DIP-
CDI(1,3-diisopropylcarbodiimide, 2.5 equiv)–HOBt(1-
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 CH3CN (35–55% CH3CN, 40 min) in 0.1%
aqueous TFA at a flow rate of 0.9 mL minÀ1 (40 ꢁC), detected at
230 nm.

T. Yoshiya et al. / Tetrahedron Letters 47 (2006) 7905–7909
7907
7
Ac–Val–Val–
D
-allo-Thr–Val–Val–NH2
(37.5%)
4
No detectable
H– allo-Thr(Ac–Val–Val)–Val–Val–NH2
D
-
back ground
15
16
17
18
19
retention time (min)
Figure 4. HPLC profile of crude 4 synthesized using a standard
segment condensation. 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 CH3CN (0–100% CH3CN,
40 mm) in 0.1% aqueous TFA at a flow rate of 0.9 mL minÀ1 (40 ꢁC),
detected at 230 nm.
5
10 15 20 25 30 35 40
retention time (min)
Figure 5. HPLC profile of crude isopeptide 7 (Rt = 17.0 min) synthe-
sized using the ‘O-acyl isopeptide method’-based segment condensa-
tion. The retention time of H-D-allo-Thr(Ac-Val-Val)-Val-Val-NH2,
which was synthesized independently, was 17.8 min. 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
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.
tide unit could be introduced to the amino group on the
resin without any racemization at the activated Ser res-
idue in the isopeptide structure.
To further elucidate the efficacy of this ‘O-acyl isopep-
tide method’-based segment condensation, pentapeptide
an isolated yield of 69%. As shown in Figure 5, HPLC
analysis of crude 7 exhibited a high purity of the desired
product without any byproduct derived from racemiza-
tion at Thr, which was confirmed by an independent
synthesis of H-D-allo-Thr(Ac-Val-Val)-Val-Val-NH2.
Moreover, the use of an N-segment with a C-terminal
isopeptide did not lead to any additional side reaction.
Isopeptide 7 was converted to 4 in phosphate buffered
saline at pH 7.4.2h These results reveal that a protected
O-acyl isopeptide with a C-terminal Boc-Thr residue
could be introduced to the peptide resin without any
racemization at the activated Thr residue, in contrast
to the standard method using Ac-Val-Val-Thr(tBu)-
OH that involved a significant amount of racemization
during condensation to the solid support.
2h,i
Ac-Val-Val-Thr-Val-Val-NH2
(4) was adopted. In
the condensation of Ac-Val-Val-Thr(tBu)-OH with H-
Val-Val-NH-resin (as a standard segment condensa-
tion),9 a large amount of racemization (37.5%) at the
activated Thr residue occurred during the DIPCDI–
HOBt segment condensation (Fig. 4), which was
confirmed by an independent synthesis of Ac-Val-Val-
D-allo-Thr-Val-Val-NH2. In contrast, in the ‘O-acyl
isopeptide method’-based segment condensation
(Scheme 1),10 N-segment Boc-Thr(Ac-Val-Val)-OH,11
which was synthesized using O-acyl isodipeptide unit
Boc-Thr(Fmoc-Val)-OH 3 (Fig. 1B),2h was coupled to
C-segment H-Val-Val-NH-resin (5) to obtain isopeptide
resin 6. The DIPCDI(2.5 equiv)–HOBt(2.5 equiv)
method in DMF (2 h) was employed for segment con-
densation, in which N-segment Boc-Thr(Ac-Val-Val)-
OH was readily solubilized. The completeness of the
coupling was verified by the Keiser test. After TFA
treatment, O-acyl isopeptide 7ÆTFA was obtained with
In summary, we herein developed a novel ‘racemization-
free segment condensation’ based on the ‘O-acyl isopep-
tide method’ with the successful synthesis of small pep-
tides. This method allows the use of an N-segment
( i , ii ) x 2
H
H
N
iii
Fmoc
N
Val Val
H
i
Rink-Amide AM resin
5
O
O
H
H
N
iv
TFA•H2N
N
Boc
Val Val NH2
Val Val
CH3
CH3
H
Ac Val Val
O
O
Ac Val Val
H
6
7•TFA
O
H
N
v
Ac Val Val
Val Val NH2
CH3
H
HO
4
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) Boc-Thr(Ac-Val-Val)-OH (2.5 equiv), DIPCDI (2.5 equiv), HOBt (2.5 equiv), DMF, 2 h; (iv) TFA–m-
cresol–thioanisole–H2O (92.5:2.5:2.5:2.5), 90 min; (v) phosphate buffered saline, pH 7.4, 25 ꢁC.

7908
T. Yoshiya et al. / Tetrahedron Letters 47 (2006) 7905–7909
Shimohigashi, Y., Ed.; 2005, pp 51–54; (r) Goulas, S.;
Gatos, D.; Barlos, K. J. Pept. Sci. 2006, 12, 116–123.
2. (a) Sohma, Y.; Sasaki, M.; Ziora, Z.; Takahashi, N.;
Kimura, T.; Hayashi, Y.; Kiso, Y. Peptides, Peptide
Revolution: Genomics, Proteomics and Therapeutics; Klu-
wer 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; (h) Sohma, Y.; Taniguchi,
A.; Skwarczynski, M.; Yoshiya, T.; Fukao, F.; Kimura,
T.; Hayashi, Y.; Kiso, Y. Tetrahedron Lett. 2006, 47,
3013–3017; (i) Sohma, Y.; Kiso, Y. ChemBiochem, pub-
lished on web on 17-Aug-2006.
possessing a C-terminal Ser/Thr residue for segment
condensation, without any racemization, as a result of
the C-terminal O-acyl isopeptide structure with a ure-
thane-protected Ser/Thr residue. Thus, in the synthesis
of long peptides/proteins, racemization-free segment
condensation becomes possible at not only the C-termi-
nal Gly/Pro but also Ser/Thr residues of the N-segment.
Additionally, final deprotected peptides/proteins synthe-
sized using the ‘O-acyl isopeptide method’-based seg-
ment condensation are effectively purified by HPLC,
because a simple isomerization to an O-acyl isopeptide
remarkably and temporarily changes the physicochemi-
cal properties of the native peptide, and an O–N intra-
molecular acyl migration triggers the native amide
bond formation under physiological conditions.2 Exam-
ples of such studies include membrane peptides/proteins
that are difficult to handle in various conditions because
of their high self-assembling characters.
Acknowledgments
3. (a) Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.;
Santos, S. D.; Mandal, B.; Mimna, R.; Murat, K.; Patiny,
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) from the Japanese
Government, and The 21st Century COE Program from
MEXT. Y.S. is grateful for the Research Fellowships
of JSPS for Young Scientists. We are grateful to Ms.
K. Oda for mass spectra measurements. We thank Dr.
J.-T. Nguyen and Dr. Y. Hamada for his help in prepar-
ing the manuscript.
`
L.; Saucede, L.; Tuchscherer, G. Angew. Chem., Int. Ed.
2004, 43, 4172–4178; (b) 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. (a) Carpino, L. A.; Krause, E.; Sferdean, C. D.; Schu-
¨
mann, M.; Fabian, H.; Bienert, M.; Beyermann, M.
Tetrahedron Lett. 2004, 45, 7519–7523; (b) Coin, I.;
Do¨lling, R.; Krause, E.; Bienert, M.; Beyermann, M.;
Sferdean, C. D.; Carpino, L. A. J. Org. Chem. 2006, 71,
6171–6177.
5. Hentschel, J.; Krause, E.; Bo¨rner, H. G. J. Am. Chem.
Soc. 2006, 128, 7722–7723.
References and notes
6. Protected peptide Fmoc-Tyr(tBu)-Ser(tBu)-OH (2.5 equiv)
was coupled to H-Phe-O-resin (2-chlorotrityl resin,
0.055 mmol) in the presence of DIPCDI (2.5 equiv) and
HOBt (2.5 equiv) in DMF for 2 h. The peptide was
cleaved from the resin using TFA–thioanisole–m-cresol–
H2O (92.5:2.5:2.5:2.5) for 90 min at rt, concentrated in
vacuo, washed with Et2O, centrifuged, suspended with
water, and lyophilized to give the crude Fmoc-Tyr-Ser-
Phe-OH 1. ESI-MS: calcd for (M+Na)+: 660.2, found:
660.0. The retention time on HPLC (0–100% CH3CN for
40 min, 230 nm) of synthesized product was identical to
that of 1 which was synthesized independently by the
standard Fmoc-based SPPS.
1. (a) Kiso, Y.; Yajima, H. In Peptides: Synthesis, Structures,
and Applications; Gutte, B., Ed.; Academic Press: San
Diego, 1995; pp 39–91; (b) Bang, D.; Pentelute, B. L.;
Kent, S. B. H. Angew. Chem., Int. Ed. 2006, 45, 3985–
3988; (c) Lu, Y.-A.; Tam, J. P. Org. Lett. 2005, 7, 5003–
5006; (d) Muralidharan, V.; Muir, T. W. Nature Methods
2006, 3, 429–438; (e) Ohta, Y.; Itoh, S.; Shigenaga, A.;
Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett. 2006, 8, 467–
470; (f) Kawakami, T.; Sumida, M.; Nakamura, K.;
Vorherr, T.; Aimoto, S. Tetrahedron Lett. 2005, 46, 8805–
8807; (g) Akaji, K.; Tamai, Y.; Kiso, Y. Tetrahedron 1997,
53, 567–584; (h) Kawakami, T.; Hasegawa, K.; Teruya,
K.; Akaji, K.; Horiuchi, M.; Inagaki, F.; Kurihara, Y.;
Uesugi, S.; Aimoto, S. J. Pept. Sci. 2001, 7, 474–487; (i)
7. O-Acyl isodipeptide unit, Boc-Ser(Fmoc-Tyr(tBu))-OH8 2
(2.5 equiv) was coupled to H-Phe-O-resin (2-chlorotrityl
resin, 0.058 mmol) in the presence of DIPCDI (2.5 equiv)
and HOBt (2.5 equiv) in DMF for 2 h. The crude H-
Ser(Fmoc-Tyr)-Phe-OH was obtained in a same depro-
tection manner described in Ref. 6. The isopeptide was
dissolved in phosphate buffer and stirred for 6 h at rt to
give the crude 1. ESI-MS: calcd for (M+H)+: 638.2,
found: 638.0. The retention time on HPLC (0–100%
CH3CN for 40 min, 230 nm) of synthesized product was
identical to that of 1 which was synthesized independently
by the standard Fmoc-based SPPS.
8. EDCÆHCl (623 mg, 3.25 mmol) was added to a stirring
solution of Boc-Ser-OBzl12 (400 mg, 1.35 mmol), Fmoc-
Tyr(tBu)-OH (1.5 g, 3.25 mmol), and DMAP (16.6 mg,
0.136 mmol) in dry CHCl3 (40 mL) at 0 ꢁC. The mixture
was slowly warmed to rt over 2 h, stirred additionally for
´
Inui, T.; Bodi, J.; Nishio, H.; Nishiuchi, Y.; Kimura, T.
Lett. Pept. Sci. 2002, 8, 319–330; (j) Makino, T.;
Matsumoto, M.; Suzuki, Y.; Kitajima, Y.; Yamamoto,
K.; Kuramoto, M.; Minamitake, Y.; Kangawa, K.;
Yabuta, M. Biopolymers 2005, 79, 238–247; (k) Benz, H.
Synthesis 1994, 337–358; (l) Habermann, J.; Kunz, H.
Tetrahedron Lett. 1998, 39, 4797–4800; (m) Hamuro, Y.;
Scialdone, M. A.; DeGrado, W. F. J. Am. Chem. Soc.
1999, 121, 1636–1644; (n) Barlos, K.; Gatos, D. Biopoly-
mers 1999, 51, 266–278; (o) Kitagawa, K.; Aida, C.;
Fujiwara, H.; Yagami, T.; Futaki, S. Chem. Pharm. Bull.
2001, 49, 958–963; (p) Nishiuchi, Y.; Nishio, H.; Ishimaru,
M.; Kimura, T. In Peptide Science 2004, Shimohigashi, Y.,
Ed.; 2005, pp 131–134.; (q) Do¨rner, B.; Steinauer, R.;
´ ´
Barthelemy, S.; White, P. D. In Peptide Science 2004,

T. Yoshiya et al. / Tetrahedron Letters 47 (2006) 7905–7909
7909
16 h, diluted with AcOEt, and washed successively with
water, 1 M HCl, water, a saturated NaHCO3, and brine.
The organic layer was dried over Na2SO4 and the solvent
was removed in vacuo. The resulting oil was purified by
silica gel column chromatography (AcOEt–hexane 1:3.5)
was obtained in a similar deprotection manner described
in Ref. 6. ESI-MS: calcd for (M+Na)+: 579.4, found:
579.3. The retention time on HPLC (0–100% CH3CN for
40 min, 230 nm) of the synthesized product was identical
to that of 4 which was synthesized previously.2h
to
yield
Boc-Ser(Fmoc-Tyr(tBu))-OBzl
(889 mg,
10. After the preparation of the H-Val-Val-NH-resin (Rink-
amide AM resin, 0.009 mmol), Boc-Thr(Ac-Val-Val)-
OH11 (2.5 equiv) was coupled in the presence of DIPCDI
(2.5 equiv) and HOBt (2.5 equiv) in DMF for 2 h at rt.
The crude O-acyl isopeptide 7ÆTFA was obtained in a
similar deprotection manner described in Ref. 6. Yield:
69% (calculated from the original loading of Rink-amide
AM resin). HRMS (FAB): calcd for C26H45N6O7
(M+H)+: 557.3663, found: 557.3656; HPLC analysis at
230 nm: purity was higher than 95%. The retention time
on HPLC (0–100% CH3CN for 40 min, 230 nm) of the
synthesized product was identical to that of 7 which was
synthesized previously.2h
1.23 mmol, 91%). After that, Pd/C was added (87 mg) to
the stirring solution of the Boc-Ser(Fmoc-Tyr(tBu))-OBzl
(856 mg, 1.18 mmol) in AcOEt (36 mL), and the reaction
mixture was vigorously stirred under a hydrogen atmo-
sphere for 16 h. 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 1:2.5 and then the final product was washed out by
methanol to give pure 2 (708 mg, 1.09 mmol, 93%).
HRMS (FAB): calcd for C36H42N2O9Na (M+Na)+:
669.2788, found: 669.2783; HPLC analysis at 230 nm:
purity was higher than 95%; NMR (CD3OD, 300 MHz): d
7.77 (d, J = 7.4 Hz, 2H), 7.63–7.60 (m, 2H), 7.38 (t,
J = 7.1 Hz, 2H), 7.32–7.27 (m, 2H), 7.13 (d, J = 8.3 Hz,
2H), 6.85 (d, J = 8.4 Hz, 2H), 4.84–4.09 (m, 7H), 3.25–
3.15 (m, 1H), 2.91–2.83 (m, 1H), 1.41 (s, 9H), 1.22 (s, 9H).
11. After O-acyl isodipeptide unit, Boc-Thr(Fmoc-Val)-OH2h
3 was loaded to 2-chlorotrityl resin, subsequent coupling
with Fmoc-Val-OH, N-acetylation using Ac2O, 0.1% TFA
treatment, and HPLC purification gave pure Boc-Thr(Ac-
Val-Val)-OH. ESI-MS: calcd for (M+Na)+: 482.3, found:
482.1.
9. The
protected
peptide
Ac-Val-Val-Thr(tBu)-OH
(2.5 equiv) was coupled to H-Val-Val-NH-resin (Rink-
amide AM resin, 0.065 mmol) in the presence of DIPCDI
(2.5 equiv) and HOBt (2.5 equiv) in DMF for 2 h. Crude 4
12. Skwarczynski, M.; Sohma, Y.; Noguchi, M.; Hayashi, Y.;
Kimura, T.; Kiso, Y. J. Org. Chem. 2006, 71, 2542–2545.