"o-Acyl isopeptide method" for peptide synthesis: Synthesis of forty kinds of "o-acyl isodipeptide unit" Boc-Ser/Thr(Fmoc-Xaa)-OH

Article abstract of DOI:10.1039/b702284k

The O-acyl isopeptide method has recently received attention as an efficient synthetic method for peptides. Herein, forty kinds of "O-acyl isodipeptide unit" Boc-Ser/Thr(Fmoc-Xaa)-OH (1-40) were effectively synthesized in two-steps without epimerization. The O-acyl isodipeptide units are important building blocks to enable the routine use of the O-acyl isopeptide method. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b702284k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
“O-Acyl isopeptide method” for peptide synthesis: synthesis of forty kinds of
“O-acyl isodipeptide unit” Boc-Ser/Thr(Fmoc-Xaa)-OH†
Taku Yoshiya,a Atsuhiko Taniguchi,a Youhei Sohma,a,b Fukue Fukao,a Setsuko Nakamura,a Naoko Abe,a
Nui Ito,a Mariusz Skwarczynski,a Tooru Kimura,a Yoshio Hayashia and Yoshiaki Kiso*a
Received 13th February 2007, Accepted 12th April 2007
First published as an Advance Article on the web 25th April 2007
DOI: 10.1039/b702284k
The O-acyl isopeptide method has recently received attention as an efficient synthetic method for
peptides. Herein, forty kinds of “O-acyl isodipeptide unit” Boc-Ser/Thr(Fmoc-Xaa)-OH (1–40) were
effectively synthesized in two-steps without epimerization. The O-acyl isodipeptide units are important
building blocks to enable the routine use of the O-acyl isopeptide method.
Val-Val-NH2 and Alzheimer’s disease-related amyloid b peptide
Introduction
(Ab) 1–42.2a–f Moreover, a “Click Peptide”, which provides a
Solid-phase peptide synthesis (SPPS) has been routinely used for
chemical synthesis of peptides. Specifically, the development of
new basis to investigate pathological functions of amyloid b
peptides in Alzheimer’s disease by inducible activation of its self-
automated SPPS has enabled rapid and convenient preparation
assembly, represents a valuable use of the isopeptide method in
chemical biology-oriented research.2g,i,j,l Furthermore we devel-
oped a “racemization-free segment condensation” based on the
O-acyl isopeptide method, in which an N-segment possessing a
C-terminal O-acyl isopeptide structure with a urethane-protected
Ser/Thr residue was employed for the segment condensation.2k,l
Our studies indicated that modification of the peptide backbone
to an ester structure at only one position within the whole
peptide sequence significantly changed the unfavorable secondary
structure of the native peptides, leading to improved coupling and
deprotection efficacy during SPPS. Shortly after our disclosure,
the O-acyl isopeptide method began to be utilized by several other
groups, especially Mutter et al.,3 Carpino et al.,4 and Bo¨rner and
co-workers,5 indicating that the O-acyl isopeptide method is widely
advantageous for peptide preparation.
However, epimerization during esterification has been a draw-
back in the solid-phase synthesis of O-acyl isopeptides. During
the synthesis of O-acyl isopeptide H-Thr(Ac-Val-Val)-Val-Val-
NH2, epimerization at the esterified Val residue occurred during
esterification using the 1,3-diisopropylcarbodiimide (DIPCDI)–
N,N-dimethylaminopyridine (DMAP) method and the crude
deprotected mixture contained 21% of D-Val diastereomer.2h Thus,
to solve this problem, we designed an “O-acyl isodipeptide unit”,
Boc-Ser/Thr(Fmoc-Xaa)-OH (Fig. 2) based on the hypothesis
that esterification-derived epimerization should be suppressed in
solution due to the faster coupling rate as compared to that
on a solid support. Along this line, we previously used Boc-
Thr(Fmoc-Val)-OH for synthesizing a small difficult sequence-
containing Ac-Val-Val-Thr-Val-Val-NH2 based on the O-acyl
isopeptide method.2h
of target peptides. However, the synthesis of “difficult sequence”-
containing peptides is still a problematic area, and the peptides of-
ten have low synthetic yield and purity in SPPS.1 Current examples
of such sequences include amyloidogenic peptides and membrane
peptides that are difficult to synthesize and handle in various
conditions because of their high self-assembling characters. The
difficult sequences are generally hydrophobic and are prone
to aggregate in solvent during synthesis and purification. This
aggregation is attributed to inter-/intra-molecular hydrophobic
interactions and the hydrogen bond network among resin-bound
peptide chains, resulting in the formation of extended secondary
structures such as b-sheets.1 To solve this problem, Mutter et al.
developed building blocks, named “pseudo-prolines”, which are
dipeptide derivatives, including Ser/Thr-derived oxazolidines or
Cys-derived thiazolidines.1a Sheppard and Johnson et al. also
reported building blocks, 2-hydroxy-4-methoxybenzyl (Hmb), as a
protecting group for the backbone amide nitrogen.1b These special
building blocks were designed to disrupt the secondary structure
formed by inter-/intra-chain interactions.
In 2003, we discovered that the presence of an O-acyl instead of
N-acyl residue within the peptide backbone significantly changed
the secondary structure of the native peptide. Moreover, the target
peptide was subsequently generated by an O–N intramolecular
acyl migration reaction. These findings led to the development
of a novel method, called the “O-acyl isopeptide method”,2 for
the synthesis of peptides containing difficult sequences (Fig. 1).
The method has been successfully applied to efficiently synthesize
difficult sequence-containing peptides such as Ac-Val-Val-Ser-
In the present study, we report the synthesis of forty kinds of O-
acyl isodipeptide unit, Boc-Ser/Thr(Fmoc-Xaa)-OH 1–40, with
all naturally coded amino acids (Fig. 2). Interestingly, we did not
observe any epimerized D-derivative in each ester bond formation.
Additionally, the synthesized isodipeptide units were successfully
applied to synthesize a bioactive influenza A virus-related peptide
containing difficult sequence. The synthesis of O-acyl isodipep-
tide units required only two simple reactions with good total
aDepartment of Medicinal Chemistry, Center for frontier Research in Medic-
inal Science, 21st Century COE program, Kyoto Pharmaceutical University,
Yamashina-ku, Kyoto, 607-8412, Japan. E-mail: kiso@mb.kyoto-phu.ac.jp;
Fax: +81 75 591 9900; Tel: +81 75 595 4635
bDepartment of Physical Chemistry, 21st Century COE program, Kyoto
Pharmaceutical University, Yamashina-ku, Kyoto, 607-8412, Japan
† Electronic supplementary information (ESI) available: Experimental
procedure and chemical data for O-acyl isodipeptide units and intermedi-
ates. See DOI: 10.1039/b702284k
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Fig. 1 The “O-acyl isopeptide method”: synthetic method for the preparation of peptides through O–N intramolecular acyl migration reaction of O-acyl
isopeptides.
Fig. 2 General structure of O-acyl isodipeptide units 1–40.
yields (except for the case of Cys). Using isodipeptide units, the
epimerization-inducing esterification step on the resin could be
omitted. Hence, the set of O-acyl isodipeptide units developed
herein are be important building blocks to enable the routine use
of the O-acyl isopeptide method.
of Boc-Thr(Fmoc-Val)-OH also indicated that the Val-Thr ester
forming reaction in solution was sufficiently fast to avoid epimer-
ization, while 21% of D-Val derivative was observed in the solid-
phase synthesis.2h Hence, we envisioned synthesizing a set of O-
acyl isodipeptide units with all naturally coded amino acids by
carefully evaluating whether epimerization could be avoided in
the esterification with various amino acid residues.
Results and discussion
As a drawback of the O-acyl isopeptide method, we recently
found that epimerization at the esterified amino acid residue
occurred during the ester bond-forming reaction on the resin.
We postulated the esterification in solution should suppress the
extent of D-derivative formation due to faster coupling rate as
compared to that on a solid support. Our previous synthesis
Synthesis of O-acyl isodipeptide units
Forty kinds of the O-acyl isodipeptide units 1–40 (Fig. 2) were syn-
thesized according to Scheme 1. Fmoc-amino acid was coupled to
the b-hydroxyl group of Boc-Ser/Thr-OBzl6 using N-ethyl-Nꢀ-(3-
dimethylaminopropyl)-carbodiimide (EDC)–DMAP method in
Scheme
1
Synthetic scheme for the “O-acyl isodipeptide unit”. Reagents and conditions: (i) Fmoc-Xaa-OH, EDC·HCl (N-ethyl-
Nꢀ-(3-dimethylaminopropyl)-carbodiimide·HCl), DMAP (N,N-dimethylaminopyridine), dry CHCl3, overnight; (ii) 10% Pd/C (10 w/w%), H2, AcOEt,
overnight (for 1–7, 10–20, 21–27, 30–40); (iii) 10% Pd/C (60 w/w%), ammonium formate (10 eq.), EtOH–H2O (95:5), 40 ◦C, 3 h (for 8 and 28, iii × 2 for
9 and 29).
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dry CHCl3 to obtain the corresponding Boc-Ser/Thr(Fmoc-Xaa)-
OBzl (41–80). Although this esterification was mainly performed
overnight, the reaction could be stopped after an appropriate time
(maybe a few hours) with TLC monitoring. Subsequently, the O-
Bzl esters of the fully protected dipeptides were removed using
Pd/C under hydrogen atmosphere to afford the corresponding
Boc-Ser/Thr(Fmoc-Xaa)-OH (1–7, 10–27 and 30–40). The O-
Bzl ester of Boc-Ser/Thr(Fmoc-Cys(Trt))-OBzl (48 and 68) and
Boc-Ser/Thr(Fmoc-Met)-OBzl (49 and 69), which contain sulfur
atoms, were deprotected using catalytic transfer hydrogenation
(CTH)7,8 at 40 ◦C with ammonium formate as a hydrogen donor
to afford Boc-Ser/Thr(Fmoc-Cys(Trt))-OH (8 and 28) and Boc-
Ser/Thr(Fmoc-Met)-OH (9 and 29). Boc-Ser/Thr(Fmoc-D-Xaa)-
OBzl (81–118) were also synthesized in a similar manner described
in Scheme 1.
In Tables 1 and 2, we summarize the isolated yield, ex-
tent of epimerization and some physicochemical data of Boc-
Ser/Thr(Fmoc-Xaa)-OBzl (41–80), Boc-Ser/Thr(Fmoc-D-Xaa)-
OBzl (81–118) and Boc-Ser/Thr(Fmoc-Xaa)-OH (1–40). All Boc-
Ser/Thr(Fmoc-Xaa)-OBzl (41–80) were obtained in high yields
(70–99%). Treatment of the protected isodipeptide units (41–47,
50–67, 70–80) with Pd/C–H2 also gave the desired dipeptides in
high yields of 75–99%. However, in the treatment of Cys- and
Met-containing protected isodipeptide (68 and 69, respectively)
with Pd/C–H2, the desired compound (either 28 or 29) was not
obtained due to the catalyst poisoning8 of the sulfur atom. We
therefore adopted the catalytic transfer hydrogenation (CTH)
method with ammonium formate as a hydrogen donor, which
is reported as a powerful hydrogenolysis system under mild
conditions,8 for Bzl ester deprotection of 48, 49, 68 and 69. In the
CTH method, although a small amount of decomposition of Fmoc
was observed under mass spectra analysis, each desired product
was readily obtained after HPLC purification. Deprotection by
the CTH method resulted in moderate yields: 58% for 8, 30% for
28, 70% for 9, 73% for 29, respectively.
Fig. 3 Evaluation of epimerization during esterification. (A-a) crude
Boc-Ser(Fmoc-Ser(tBu))-OBzl (46); (A-b) analytical HPLC of mix-
ture of L-Ser derivative 46 and D-Ser derivative 85; (B-a) crude
Boc-Thr(Fmoc-Cys(Trt))-OBzl (68); (B-b) analytical HPLC of mix-
ture of L-Cys derivative 68 and D-Cys derivative 106; (C-a) crude
Boc-Thr(Fmoc-Arg(Pmc))-OBzl (77); (C-b) analytical HPLC of mixture
of L-Arg derivative 77 and D-Arg derivative 115. 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 (60–100% CH3CN, 40 min) in 0.1% aqueous TFA at a flow
rate of 0.9 mL min−1 (40 ◦C), detected at 230 nm for separation 46
and 85; ChiralcelꢀR OD normal phase column (4.6 × 250 mm; Daicel
Chemical Ind., Ltd, Tokyo, Japan) with an isocratic solvent system:
n-hexane–isopropanol or n-hexane–ethanol at a flow rate of 0.9 mL min−1
(room temperature), detected at 230 nm for separation 68 and 106
(n-hexane : ethanol = 7 : 3), 77 and 115 (n-hexane : isopropanol =
7 : 3).
Thus, the synthesis of O-acyl isodipeptide units generally
requires only simple two reactions (Scheme 1) with moderate
to excellent total yields (55%–99%). The synthesis of O-acyl
isodipeptide units would be simpler than that of other building
blocks such as pseudo-prolines1a and Hmb,1b for synthesizing
difficult sequence-containing peptides.
Study of epimerization during esterification
In the synthesis of thirty-eight kinds of protected isodipep-
tide units 42–60 and 62–80 with various Fmoc-protected amino
acids, we examined whether epimerization occurred during the
esterification reaction. To evaluate this matter, we synthesized
all corresponding D-derivatives 81–118. Interestingly, no de-
tectable epimerized product 81–118 was observed in crude 42–
60 and 62–80, even in the esterification with epimerization-
favored Cys9 and His10 residues, indicating that epimerization
did not occur in any ester bond-forming reaction with EDC–
DMAP,11 although whether epimerization had occurred could
not be determined in the case of Boc-Ser(Fmoc-Met)-OBzl
(49) and Boc-Ser(Fmoc-His(Trt))-OBzl (55) because separation
of L-derivative and independently synthesized D-derivative was
achieved by neither RP-HPLC nor chiral HPLC. Fig. 3 shows the
HPLC profiles of crude Boc-Ser(Fmoc-Ser(tBu))-OBzl (46), Boc-
Thr(Fmoc-Cys(Trt))-OBzl (68) and Boc-Thr(Fmoc-Arg(Pmc))-
OBzl (77). These results suggest that the coupling rate of O-
acylation with Fmoc-Xaa-OH is commonly sufficiently fast to
avoid epimerization, while products from solid-phase synthesis
exhibited large amounts of undesired diastereomers.2h
Applying the O-acyl isodipeptide unit to solid-phase
peptide synthesis
The synthesized O-acyl isodipeptide unit, Boc-Thr(Fmoc-Phe)-
OH (38) was applied to SPPS of influenza A virus matrix M1
58–66 (H-GILGFVFTL-OH, 119)12 containing a difficult se-
quence. In standard SPPS (see Experimental section), the difficult
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Table 1 Data for 1–20, 41–60, 81–99 (serine series)
HRMS (FAB)d
Calcd
Structure
Yielda (%)
Epimerizationb
RP-HPLCc tR/min
Found
Boc-Ser(Fmoc-Gly)-OBzl (41)
96
91
>99
97
89
93
85
82
>99
92
93
84
97
>99
98
98
>99
>99
>99
89
>99
77
—
34.9
29.2
35.6
35.6
29.8
36.4
36.9
32.7
38.5
38.4
33.2
38.6
38.5
33.1
38.5
38.2
32.5
39.8
39.3
34.4
42.1
42.1
40.2
38.6
37.6
32.6
38.5
38.4
32.0
38.3
38.2
33.3
40.6
41.0
36.9
38.9
38.7
33.4
40.6
40.9
36.6
35.2
34.7
31.9
38.0
38.0
33.1
38.8
38.7
34.5
37.9
38.3
32.7
40.7
38.9
36.4
41.2
41.3
37.5
597.2213
507.1743
611.2369
611.2369
521.1900
639.2682
639.2682
549.2213
653.2839
653.2839
563.2369
653.2839
653.2839
563.2369
683.2945
683.2945
593.2475
697.3101
697.3101
607.2632
885.3186
885.3186
795.2716
671.2403
671.2403
581.1934
637.2526
637.2526
547.2056
711.2894
711.2894
621.2424
896.3523
896.3523
806.3054
725.3050
725.3050
636.2581
910.3680
910.3680
820.3210
919.3683
919.3683
829.3213
768.3472
768.3472
678.3003
962.3986
962.3986
872.3516
687.2682
687.2682
597.2213
759.3258
759.3258
669.2788
826.3316
826.3316
736.2846
597.2217
507.1735
611.2373
611.2366
521.1907
639.2685
639.2678
549.2217
653.2844
653.2845
563.2365
653.2835
653.2835
563.2373
683.2939
683.2939
593.2471
697.3096
697.3096
607.2639
885.3179
885.3179
795.2723
671.2407
671.2410
581.1927
637.2531
637.2531
547.2061
711.2889
711.2900
621.2420
896.3528
896.3528
806.3058
725.3044
725.3057
636.2585
910.3673
910.3677
820.3204
919.3688
919.3688
829.3208
768.3468
768.3480
678.2997
962.3978
962.3978
872.3525
687.2678
687.2678
597.2220
759.3262
759.3262
669.2783
826.3321
826.3311
736.2840
Boc-Ser(Fmoc-Gly)-OH (1)
—
Boc-Ser(Fmoc-Ala)-OBzl (42)
Boc-Ser(Fmoc-D-Ala)-OBzl (81)
Boc-Ser(Fmoc-Ala)-OH (2)
N.D.e
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
Boc-Ser(Fmoc-Val)-OBzl (43)
Boc-Ser(Fmoc-D-Val)-OBzl (82)
Boc-Ser(Fmoc-Val)-OH (3)
Boc-Ser(Fmoc-Leu)-OBzl (44)
Boc-Ser(Fmoc-D-Leu)-OBzl (83)
Boc-Ser(Fmoc-Leu)-OH (4)
Boc-Ser(Fmoc-Ile)-OBzl (45)
Boc-Ser(Fmoc-D-allo-Ile)-OBzl (84)
Boc-Ser(Fmoc-Ile)-OH (5)
Boc-Ser(Fmoc-Ser(tBu))-OBzl (46)
Boc-Ser(Fmoc-D-Ser(tBu))-OBzl (85)
Boc-Ser(Fmoc-Ser(tBu))-OH (6)
Boc-Ser(Fmoc-Thr(tBu))-OBzl (47)
Boc-Ser(Fmoc-D-allo-Thr(tBu))-OBzl (86)
Boc-Ser(Fmoc-Thr(tBu))-OH (7)
Boc-Ser(Fmoc-Cys(Trt))-OBzl (48)
Boc-Ser(Fmoc-D-Cys(Trt))-OBzl (87)
Boc-Ser(Fmoc-Cys(Trt))-OH (8)
Boc-Ser(Fmoc-Met)-OBzl (49)
Boc-Ser(Fmoc-D-Met)-OBzl (88)
Boc-Ser(Fmoc-Met)-OH (9)
Boc-Ser(Fmoc-Pro)-OBzl (50)
Boc-Ser(Fmoc-D-Pro)-OBzl (89)
Boc-Ser(Fmoc-Pro)-OH (10)
Boc-Ser(Fmoc-Asp(OtBu))-OBzl (51)
Boc-Ser(Fmoc-D-Asp(OtBu))-OBzl (90)
Boc-Ser(Fmoc-Asp(OtBu))-OH (11)
Boc-Ser(Fmoc-Asn(Trt))-OBzl (52)
Boc-Ser(Fmoc-D-Asn(Trt))-OBzl (91)
Boc-Ser(Fmoc-Asn(Trt))-OH (12)
Boc-Ser(Fmoc-Glu(OtBu))-OBzl (53)
Boc-Ser(Fmoc-D-Glu(OtBu))-OBzl (92)
Boc-Ser(Fmoc-Glu(OtBu))-OH (13)
Boc-Ser(Fmoc-Gln(Trt))-OBzl (54)
Boc-Ser(Fmoc-D-Gln(Trt))-OBzl (93)
Boc-Ser(Fmoc-Gln(Trt))-OH (14)
Boc-Ser(Fmoc-His(Trt))-OBzl (55)
Boc-Ser(Fmoc-D-His(Trt))-OBzl (94)
Boc-Ser(Fmoc-His(Trt))-OH (15)
Boc-Ser(Fmoc-Lys(Boc))-OBzl (56)
Boc-Ser(Fmoc-D-Lys(Boc))-OBzl (95)
Boc-Ser(Fmoc-Lys(Boc))-OH (16)
Boc-Ser(Fmoc-Arg(Pmc))-OBzl (57)
Boc-Ser(Fmoc-D-Arg(Pmc))-OBzl (96)
Boc-Ser(Fmoc-Arg(Pmc))-OH (17)
Boc-Ser(Fmoc-Phe)-OBzl (58)
—
N.D.
—
—
58
89
>99
70
98
Not determinedf
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
89
>99
>99
>99
>99
>99
>99
96
98
>99
99
85
>99
89
78
97
81
>99
93
99
94
>99
88
>99
>99
>99
91
>99
93
>99
>99
91
—
Not determinedf
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
Boc-Ser(Fmoc-D-Phe)-OBzl (97)
Boc-Ser(Fmoc-Phe)-OH (18)
Boc-Ser(Fmoc-Tyr(tBu))-OBzl (59)
Boc-Ser(Fmoc-D-Tyr(tBu))-OBzl (98)
Boc-Ser(Fmoc-Tyr(tBu))-OH (19)g
Boc-Ser(Fmoc-Trp(Boc))-OBzl (60)
Boc-Ser(Fmoc-D-Trp(Boc))-OBzl (99)
Boc-Ser(Fmoc-Trp(Boc))-OH (20)
a Isolated yield. b Epimerization was evaluated by comparison with authentic D-derivatives. c 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 min−1 (40 ◦C), detected at 230 nm. d (M + Na)+. e Not detected. f In these cases, we couldn’t separate L-derivative and
D-derivative by HPLC. g Ref. 2k.
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Table 2 Data for 21–40, 61–80, 100–118 (Threonine series)
HRMS (FAB)d
Calcd
Structure
Yielda (%)
Epimerizationb
RP-HPLCc tR/min
Found
Boc-Thr(Fmoc-Gly)-OBzl (61)
99
95
81
86
98
94
89
92
>99
96
96
92
>99
94
>99
89
93
86
65
83
97
>99
30
>99
>99
73
86
>99
96
>99
>99
94
>99
>99
78
74
88
90
>99
>99
89
72
>99
77
—
35.8
30.8
36.1
36.0
30.5
39.2
38.4
33.5
39.3
39.1
34.3
39.1
39.0
33.0
39.4
39.6
34.0
40.3
40.0
35.0
43.2
43.2
40.5
37.2
36.6
33.5
37.8
37.7
31.6
40.2
38.7
35.1
39.7
40.2
36.1
38.7
39.0
33.9
41.9
40.7
36.9
35.3
35.3
31.7
37.8
37.8
35.1
39.0
38.3
37.0
39.3
38.6
33.5
40.8
39.7
35.0
40.8
40.7
37.0
611.2369
521.1900
603.2706 (M + H)+
625.2526
535.2056
653.2839
653.2839
563.2369
667.2995
667.2995
577.2526
667.2995
667.2995
577.2526
697.3101
697.3101
607.2632
711.3258
711.3258
621.2788
899.3342
899.3342
809.2873
685.2560
685.2560
595.2090
651.2682
651.2682
561.2213
725.3050
725.3050
635.2581
910.3680
910.3680
820.3210
739.3207
739.3207
649.2737
924.3836
924.3836
834.3367
933.3839
933.3839
843.3370
782.3629
782.3629
692.3159
976.4142
976.4142
886.3673
679.3019 (M + H)+
701.2839
611.2369
773.3414
773.3414
683.2945
840.3472
840.3472
750.3003
611.2375
521.1904
603.2703
625.2522
535.2051
653.2833
653.2845
563.2373
667.2999
667.2991
577.2520
667.2991
667.2991
577.2520
697.3107
697.3105
607.2624
711.3252
711.3253
621.2792
899.3348
899.3348
809.2867
685.2555
685.2554
595.2097
651.2688
651.2676
561.2220
725.3057
725.3046
635.2585
910.3675
910.3675
820.3205
739.3214
739.3201
649.2728
924.3830
924.3830
834.3362
933.3836
933.3836
843.3374
782.3624
782.3634
692.3154
976.4137
976.4138
886.3669
679.3015
701.2843
611.2375
773.3418
773.3420
683.2939
840.3476
840.3476
750.3007
Boc-Thr(Fmoc-Gly)-OH (21)
—
Boc-Thr(Fmoc-Ala)-OBzl (62)f
N.D.e
—
Boc-Thr(Fmoc-D-Ala)-OBzl (100)
Boc-Thr(Fmoc-Ala)-OH (22)f
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
—
N.D.
—
Boc-Thr(Fmoc-Val)-OBzl (63)
Boc-Thr(Fmoc-D-Val)-OBzl (101)
Boc-Thr(Fmoc-Val)-OH (23)g
Boc-Thr(Fmoc-Leu)-OBzl (64)
Boc-Thr(Fmoc-D-Leu)-OBzl (102)
Boc-Thr(Fmoc-Leu)-OH (24)
Boc-Thr(Fmoc-Ile)-OBzl (65)
Boc-Thr(Fmoc-D-allo-Ile)-OBzl (103)
Boc-Thr(Fmoc-Ile)-OH (25)
Boc-Thr(Fmoc-Ser(tBu))-OBzl (66)
Boc-Thr(Fmoc-D-Ser(tBu))-OBzl (104)
Boc-Thr(Fmoc-Ser(tBu))-OH (26)
Boc-Thr(Fmoc-Thr(tBu))-OBzl (67)
Boc-Thr(Fmoc-D-allo-Thr(tBu))-OBzl (105)
Boc-Thr(Fmoc-Thr(tBu))-OH (27)
Boc-Thr(Fmoc-Cys(Trt))-OBzl (68)
Boc-Thr(Fmoc-D-Cys(Trt))-OBzl (106)
Boc-Thr(Fmoc-Cys(Trt))-OH (28)
Boc-Thr(Fmoc-Met)-OBzl (69)
Boc-Thr(Fmoc-D-Met)-OBzl (107)
Boc-Thr(Fmoc-Met)-OH (29)
Boc-Thr(Fmoc-Pro)-OBzl (70)
Boc-Thr(Fmoc-D-Pro)-OBzl (108)
Boc-Thr(Fmoc-Pro)-OH (30)
Boc-Thr(Fmoc-Asp(OtBu))-OBzl (71)
Boc-Thr(Fmoc-D-Asp(OtBu))-OBzl (109)
Boc-Thr(Fmoc-Asp(OtBu))-OH (31)
Boc-Thr(Fmoc-Asn(Trt))-OBzl (72)
Boc-Thr(Fmoc-D-Asn(Trt))-OBzl (110)
Boc-Thr(Fmoc-Asn(Trt))-OH (32)
Boc-Thr(Fmoc-Glu(OtBu))-OBzl (73)
Boc-Thr(Fmoc-D-Glu(OtBu))-OBzl (111)
Boc-Thr(Fmoc-Glu(OtBu))-OH (33)
Boc-Thr(Fmoc-Gln(Trt))-OBzl (74)
Boc-Thr(Fmoc-D-Gln(Trt))-OBzl (112)
Boc-Thr(Fmoc-Gln(Trt))-OH (34)
Boc-Thr(Fmoc-His(Trt))-OBzl (75)
Boc-Thr(Fmoc-D-His(Trt))-OBzl (113)
Boc-Thr(Fmoc-His(Trt))-OH (35)
Boc-Thr(Fmoc-Lys(Boc))-OBzl (76)
Boc-Thr(Fmoc-D-Lys(Boc))-OBzl (114)
Boc-Thr(Fmoc-Lys(Boc))-OH (36)
Boc-Thr(Fmoc-Arg(Pmc))-OBzl (77)
Boc-Thr(Fmoc-D-Arg(Pmc))-OBzl (115)
Boc-Thr(Fmoc-Arg(Pmc))-OH (37)
Boc-Thr(Fmoc-Phe)-OBzl (78)
98
>99
96
79
55
>99
94
97
Boc-Thr(Fmoc-D-Phe)-OBzl (116)
Boc-Thr(Fmoc-Phe)-OH (38)
96
—
N.D.
—
—
N.D.
—
Boc-Thr(Fmoc-Tyr(tBu))-OBzl (79)
Boc-Thr(Fmoc-D-Tyr(tBu))-OBzl (117)
Boc-Thr(Fmoc-Tyr(tBu))-OH (39)
Boc-Thr(Fmoc-Trp(Boc))-OBzl (80)
Boc-Thr(Fmoc-D-Trp(Boc))-OBzl (118)
Boc-Thr(Fmoc-Trp(Boc))-OH (40)
>99
>99
76
94
96
91
—
a Isolated yield. b Epimerization was evaluated by comparison with authentic D-derivatives. c 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 min−1 (40 ◦C), detected at 230 nm. d (M + Na)+. e Not detected. f Ref. 4b. g Ref. 2h.
1724 | Org. Biomol. Chem., 2007, 5, 1720–1730
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Fig. 4 HPLC profile of crude (A) peptide 119 (synthesized by standard SPPS), (B) O-acyl isopeptide 123 (synthesized without O-acyl isodipeptide unit),
and (C) peptide 119 (synthesized using O-acyl isopeptide method with Boc-Thr(Fmoc-Phe)-OH 38).
sequence-derived byproduct, Fmoc-GILGFVFTL-OH, was ob-
tained after final deprotection (Fig. 4A).
from Boc-Ser/Thr-OBzl. Interestingly, we did not observe any
epimerization in all ester bond-forming reactions between Fmoc-
Xaa-OH and Boc-Ser/Thr-OBzl. Additionally, the synthesized
isodipeptide unit was successfully applied in synthesizing bioactive
influenza A virus-related peptide with difficult sequences. Analysis
of the crude peptide revealed high purity of the product with no
by-product derived from the difficult sequence and epimerization.
Moreover, the use of isodipeptide did not lead to any additional
side reaction. Hence, using isodipeptide units, the epimerization-
inducing esterification on the resin could be omitted. A set of
O-acyl isodipeptide units developed herein would be important
building blocks to enable the routine use of the O-acyl isopeptide
method.
On the other hand, in the O-acyl isopeptide method without the
O-acyl isodipeptide unit (Scheme 2A), Fmoc-Phe-OH was coupled
to Boc-Thr-Leu-resin (2-chlorotrityl resin) using the DIPCDI–
DMAP method in dry CH2Cl2. After continuous couplings
of Fmoc-amino acids and TFA treatment, O-acyl isopeptide
123·TFA was obtained without any undesired residue (Fig. 4B).
Hence, the protected peptide resin was efficiently synthesized
with no interference from the difficult sequences. The result is
comparable with our previous observation that modification of
difficult sequence-containing peptide to ester structure changed
the secondary structure of the peptide to that which is more
favorable for Fmoc-deprotection.2 As expected, a large amount
(7.6%) of isopeptide epimer H-Thr(H-Gly-Ile-Leu-Gly-Phe-Val-
D-Phe)-Leu-OH was observed in the final deprotected mixture
(Fig. 4B), which was confirmed by an independent synthesis of
H-Thr(H-Gly-Ile-Leu-Gly-Phe-Val-D-Phe)-Leu-OH.
Experimental
General procedures
In these contexts, we synthesized 119 based on the O-acyl
isopeptide method using O-acyl isodipeptide unit 38 (Scheme 2B).
The use of isodipeptide unit 38 omitted the epimerization-inducing
esterification reaction on the resin. O-Acyl isodipeptide unit 38,
which readily solubilized in DMF, was coupled to H-Leu-resin (2-
chlorotrityl resin) using the standard DIPCDI–HOBt method to
obtain 121. The completeness of coupling was verified by Kaiser
test. After assembly of the remaining residues followed by TFA
treatment, O-acyl isopeptide 123·TFA was obtained. 123·TFA was
further dissolved and stirred in phosphate buffer (pH 7.4) at rt
to induce quantitative O–N intramolecular acyl migration to the
corresponding target peptide 119 with a half-life of approximately
1 min. As shown in Fig. 4C, HPLC analysis of crude 119
(synthesized using O-acyl isodipeptide unit 38) exhibited a high
purity of the desired product with no by-product derived from the
difficult sequence or epimerization. The use of isodipeptide 38 did
not lead to any additional side reaction. After HPLC purification,
the overall yield of 119·TFA was 73%.
Fmoc-amino acid side-chain protections were selected as follows:
tBu (Asp, Glu, Ser, Thr, Tyr), Boc (Lys), Pmc (Arg), Trt (Cys,
Asn, Gln, His). All protected amino acids and resins were
purchased from Calbiochem-Novabiochem Japan Ltd. (Tokyo).
Other chemicals were purchased from commercial suppliers,
Wako Pure Chemical Ind., Ltd. (Osaka, Japan), Nacalai Tesque
(Kyoto, Japan), and Aldrich Chemical Co., Inc. (Milwaukee, WI)
and were used without further purification. Analytical thin-layer
chromatography (TLC) was performed using Merck 105715 silica
gel 60 F254 precoated plate (0.25 thickness). Visualization of the
chromatogram was by UV illumination (254 nm), phosphomolyb-
dic acid, and ninhydrin, as appropriate. Merck 107734 silica gel 60
(70–230 mesh) was used for column chromatography. 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 in 0.1% aqueous TFA at a flow
rate of 0.9 mL min−1 (temperature: 40 ◦C), detected at 230 nm.
Preparative HPLC was carried out on a C18 reverse phase column
(20 × 250 mm; YMC Pack ODS SH343-5) with a binary solvent
system: a linear gradient of CH3CN in 0.1% aqueous TFA at a flow
rate of 5.0 mL min−1 (temperature: 40 ◦C), detected at 230 nm.
To separate diastereomers, chiral HPLC analysis was performed
with JASCO HPLC systems consisting of following: pump, 880-
Conclusion
Forty kinds of “O-acyl isodipeptide unit” Boc-Ser/Thr(Fmoc-
Xaa)-OH 1–40 with all naturally occurring amino acids were
synthesized in two-steps without epimerization (29–99%), starting
R
PU; detector, 875-UV, measured at 230 nm; column, Chiralcelꢀ
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Scheme 2 Synthesis of (A) O-acyl isopeptide of influenza A virus matrix M1 58–66 (123) by theO-acyl isopeptide method without O-acyl isodipeptide unit,
(B) influenza A virus matrix M1 58–66 (119) by the O-acyl isopeptide method with O-acyl isodipeptide unit (38). Reagents and conditions: (i) 20%
piperidine–DMF, 20 min; (ii) Boc-Thr-OH (2.5 eq.), DIPCDI (1,3-diisopropylcarbodiimide, 2.5 eq.), HOBt (1-hydroxybenzotriazole, 2.5 eq.), DMF, 2 h;
(iii) Fmoc-Phe-OH (3 eq.), DIPCDI (1,3-diisopropylcarbodiimide, 3 eq.), DMAP (N,N-dimethylaminopyridine, 0.2 eq.), dry CH2Cl2, 16 h (×2);
(iv) Fmoc-Xaa-OH (2.5 eq.), DIPCDI (2.5 eq.), HOBt (2.5 eq.), DMF, 2 h; (v) TFA-m-cresol–thioanisole–H2O (92.5 : 2.5 : 2.5 : 2.5), 90 min;
(vi) Boc-Thr(Fmoc-Phe)-OH (38, 2.5 eq.), DIPCDI (2.5 eq.), HOBt (2.5 eq.), DMF, 2 h (vii) phosphate buffer, pH 7.4, 25 ◦C; (viii) HPLC purification.
OD normal phase column (4.6 × 250 mm; Daicel Chemical Ind.,
Ltd, Tokyo, Japan); mobile phase, n-hexane–isopropanol or n-
hexane–ethanol; flow rate, 0.9 mL min−1 (room temperature).
Solvents used for HPLC were of HPLC grade. NMR spectra
were recorded on a JEOL 300 MHz instrument, or a 400 MHz
Varian UNITY INOVA 400NB spectrometer, and calibrated using
tetramethylsilane (TMS) as an internal reference. MALDI-TOF
MASS spectra were recorded on Voyager DE-RP using a-cyano-
4-hydroxycinnamic acid as a matrix. FAB-MS was performed
on a JEOL JMS-SX102A spectrometer equipped with the JMA-
DA7000 data system.
2 mL) in the presence of 1,3-diisopropylcarbodiimide (DIPCDI,
2.5 eq.) and 1-hydroxybenzotriazole (HOBt, 2.5 eq.) with a reac-
tion time of 2 h at room temperature. The resins were then washed
with DMF (1.5 mL, ×5) and the completeness of each coupling
was verified by the Kaiser test. Na-Fmoc deprotection was carried
out by treatment with piperidine (20% v/v in DMF) (2 mL, 1 min ×
1 and 20 min × 1), followed by washing with DMF (1.5 mL,
×10) and chloroform (1.5 mL, ×5). After complete elongation of
the peptide chains, the peptide resins were washed with methanol
(1.5 mL, ×5) and dried for at least 2 h in vacuo. The peptides were
then cleaved from the resin with TFA in the presence of thioanisole,
m-cresol and distilled water (92.5 : 2.5 : 2.5 : 2.5) for 90 min at room
temperature, concentrated in vacuo, and precipitated with diethyl
ether (4–8 mL) at 0 ◦C followed by centrifugation at 4000 rpm for
5 min (×3). The resultant peptides were dissolved or suspended in
water and lyophilized for at least 12 h.
Solid phase peptide synthesis
In general, the peptide chains were assembled by sequential
coupling of activated Na-Fmoc-amino acid (2.5 eq.) in DMF (1.5–
1726 | Org. Biomol. Chem., 2007, 5, 1720–1730
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Boc-Ser(Fmoc-Gly)-OBzl (41). EDC·HCl (0.53 g, 2.76 mmol)
was added to a stirring solution of N-(tert-butoxycarbonyl)-L-
serine benzyl ester (Boc-Ser-OBzl) (0.68 g, 2.30 mmol), N-(9H-
fluoren-9-ylmethoxycarbonyl)-L-glycine (Fmoc-Gly-OH, 0.82 g,
2.76 mmol), and DMAP (56.3 mg, 0.46 mmol) in dry CHCl3
HRMS (FAB): calcd. for C33H36N2O8Na (M + Na)+: 611.2369,
found: 611.2366.
Boc-Ser(Fmoc-Ala)-OH (2). 2 was synthesized in a similar
manner to 1. Yield: 89%; HPLC analysis at 230 nm: purity was
92% (tR = 29.8 min); 1H NMR (CD3OD, 300 MHz) d 7.78 (d, J =
7.3 Hz, 2H), 7.67 (t, J = 6.1 Hz, 2H), 7.40–7.28 (m, 4H), 4.58–
4.47 (m, 1H), 4.40–4.33 (m, 2H), 4.30–4.19 (m, 4H), 1.44–1.37
(m, 12H); HRMS (FAB): calcd. for C26H30N2O8Na (M + Na)+:
521.1900, found: 521.1907.
◦
(60 mL) at 0 C. The mixture was slowly warmed to rt over 2 h,
stirred additionally overnight, diluted with AcOEt, and washed
successively with water, 1 N HCl, water, 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) to yield Boc-
Ser(Fmoc-Gly)-OBzl (1.26 g, 2.20 mmol, 96%). HPLC analysis at
230 nm: purity was higher than 95% (retention time (tR) = 34.9
Boc-Ser(Fmoc-Leu)-OBzl (44). EDC·HCl (0.19 g, 0.97 mmol)
was added to a stirring solution of N-(tert-butoxycarbonyl)-L-
serine benzyl ester (Boc-Ser-OBzl) (0.24 g, 0.81 mmol), N-(9H-
fluoren-9-ylmethoxycarbonyl)-L-leucine (Fmoc-Leu-OH, 0.34 g,
0.97 mmol), and DMAP (9.9 mg, 0.081 mmol) in dry CHCl3
1
min); H NMR (CD3OD, 300 MHz) d 7.78 (d, J = 7.3 Hz, 2H),
7.65 (d, J = 7.3 Hz, 2H), 7.43–7.25 (m, 9H), 5.20 (d, J = 12.4 Hz,
1H), 5.12 (d, J = 12.4 Hz, 1H), 4.54–4.31 (m, 5H), 4.26–4.18 (m,
1H), 3.90–3.73 (m, 2H), 1.40 (s, 9H); HRMS (FAB): calcd. for
C32H34N2O8Na (M + Na)+: 597.2213, found: 597.2217.
◦
(10 mL) at 0 C. The mixture was slowly warmed to rt over 2 h,
stirred additionally overnight, diluted with AcOEt, and washed
successively with water, 1 N HCl, water, 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 : 4) to yield Boc-
Ser(Fmoc-Leu)-OBzl (0.51 g, 0.81 mmol, >99%). Epimerization
during the synthesis was not detected, confirmed by comparison
with independently synthesized D-leucine derivative 83. HPLC
Boc-Ser(Fmoc-Gly)-OH (1). Pd/C (115 mg) was added to a
stirring solution of Boc-Ser(Fmoc-Gly)-OBzl (1.15 g, 2.00 mmol)
in AcOEt (50 mL), and the reaction mixture was vigorously stirred
under a hydrogen atmosphere overnight. 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 Boc-Ser(Fmoc-Gly)-OH (0.89 g, 1.83 mmol, 91%). HPLC
1
analysis at 230 nm: purity was 98% (tR = 38.5 min); H NMR
(CD3OD, 300 MHz) d 7.78 (d, J = 7.5 Hz, 2 H), 7.68–7.63 (m, 2
H), 7.40–7.26 (m, 9 H), 5.18–5.07 (m, 2 H), 4.49–4.35 (m, 5 H),
4.23–4.16 (m, 2 H), 1.69–1.53 (m, 3 H), 1.40 (s, 9 H), 0.93 (d, J =
6.4 Hz, 3 H), 0.90 (d, J = 6.4 Hz, 3 H); HRMS (FAB): calcd. for
C36H42N2O8Na (M + Na)+: 653.2839, found: 653.2845.
1
analysis at 230 nm: purity was 95% (tR = 29.2 min); H NMR
(CD3OD, 300 MHz) d 7.78 (d, J = 7.3 Hz, 2H), 7.66 (d, J =
7.4 Hz, 2H), 7.42–7.26 (m, 4H), 4.55–4.46 (m, 1H), 4.41–4.19 (m,
5H), 3.94–3.81 (m, 2H), 1.42 (s, 9H); HRMS (FAB): calcd. for
C25H28N2O8Na (M + Na)+: 507.1743, found: 507.1735.
Boc-Ser(Fmoc-D-Leu)-OBzl (83). 83 was synthesized in a
similar manner to 44. Yield: 95%; HPLC analysis at 230 nm:
purity was 93% (tR = 38.4 min); 1H NMR (CD3OD, 300 MHz) d
7.78 (d, J = 7.4 Hz, 2 H), 7.67–7.61 (m, 2 H), 7.40–7.27 (m, 9 H),
5.19 (d, J = 12.3 Hz, 1 H), 5.13 (d, J = 12.3 Hz, 1 H), 4.49–4.35
(m, 5 H), 4.23–4.15 (m, 2 H), 1.70–1.51 (m, 3 H), 1.39 (s, 9 H),
0.92 (d, J = 6.6 Hz, 3 H), 0.89 (d, J = 6.6 Hz, 3 H); HRMS (FAB):
calcd. for C36H42N2O8Na (M + Na)+: 653.2839, found: 653.2844.
Boc-Ser(Fmoc-Ala)-OBzl
(42). EDC·HCl
(77.9
mg,
0.41 mmol) was added to a stirring solution of N-(tert-
butoxycarbonyl)-L-serine benzyl ester (Boc-Ser-OBzl) (50 mg,
0.17 mmol), N-(9H-fluoren-9-ylmethoxycarbonyl)-L-alanine
(Fmoc-Ala-OH, 126.5 mg, 0.41 mmol), and DMAP (2.1 mg,
0.017 mmol) in dry CHCl3 (3 mL) at 0 ◦C. The mixture was
slowly warmed to rt over 2 h, stirred additionally overnight,
diluted with AcOEt, and washed successively with water, 1 N
HCl, water, 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
(CHCl3 : methanol 90 : 1) to yield Boc-Ser(Fmoc-Ala)-OBzl
(101.7 mg, 0.17 mmol, >99%). Epimerization during the synthesis
was not detected, confirmed by comparison with independently
synthesized D-alanine derivative 81. HPLC analysis at 230 nm:
purity was 88% (tR = 35.6 min); 1H NMR (CD3OD, 300 MHz) d
7.78 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 7.1 Hz, 2H), 7.40–7.27 (m,
9H), 5.19–5.07 (m, 2H), 4.52–4.45 (m, 2H), 4.42–4.28 (m, 3H),
4.23–4.08 (m, 2H), 1.47–1.32 (m, 12H); HRMS (FAB): calcd. for
C33H36N2O8Na (M + Na)+: 611.2369, found: 611.2373.
Boc-Ser(Fmoc-Leu)-OH (4). 4 was synthesized in a similar
manner to 1. Yield: 93%; HPLC analysis at 230 nm: purity was
higher than 95% (tR = 33.2 min); 1H NMR (CD3OD, 300 MHz)
d 7.78 (d, J = 7.4 Hz, 2 H), 7.70–7.65 (m, 2 H), 7.40–7.28 (m, 4
H), 4.53–4.49 (m, 1 H), 4.37–4.20 (m, 6 H), 1.73–1.57 (m, 3 H),
1.40 (s, 9 H), 0.95 (d, J = 6.2 Hz, 3 H), 0.91 (d, J = 6.2 Hz, 3 H);
HRMS (FAB): calcd. for C29H36N2O8Na (M + Na)+: 563.2369,
found: 563.2365.
Boc-Ser(Fmoc-Ser(tBu))-OBzl (46). 46 was synthesized in a
similar manner to 44. Epimerization during the synthesis was not
detected, confirmed by comparison with independently synthe-
sized D-serine derivative 85. Yield: 98%; HPLC analysis at 230 nm:
purity was higher than 95% (tR = 38.5 min); 1H NMR (CD3OD,
300 MHz) d 7.79 (d, J = 7.7 Hz, 2H), 7.69–7.64 (m, 2H), 7.41–7.27
(m, 9H), 5.20 (d, J = 12.5 Hz, 1H), 5.12 (d, J = 12.3 Hz, 1H),
4.53–4.21 (m, 7H), 3.73–3.68 (m, 1H), 3.61–3.56 (m, 1H), 1.40 (s,
9 H), 1.17 (s, 9H); HRMS (FAB): calcd. for C37H44N2O9Na (M +
Na)+: 683.2945, found: 683.2939.
Boc-Ser(Fmoc-D-Ala)-OBzl (81). 81 was synthesized in a
similar manner to 42. Yield: 97%; HPLC analysis at 230 nm:
purity was 95% (tR = 35.6 min); 1H NMR (CD3OD, 300 MHz) d
7.78 (d, J = 7.5 Hz, 2H), 7.67–7.64 (m, 2H), 7.40–7.27 (m, 9H),
5.20 (d, J = 12.2 Hz, 1H), 5.12 (d, J = 12.2 Hz, 1H), 4.52–4.44 (m,
2H), 4.43–4.26 (m, 3H), 4.24–4.12 (m, 2H), 1.45–1.26 (m, 12H);
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Boc-Ser(Fmoc-D-Ser(tBu))-OBzl (85). 85 was synthesized in
a similar manner to 44. Yield: 98%; HPLC analysis at 230 nm:
purity was higher than 95% (tR = 38.2 min); 1H NMR (CD3OD,
300 MHz) d 7.79 (d, J = 7.4 Hz, 2H), 7.68–7.64 (m, 2H), 7.40–7.27
(m, 9H), 5.22–5.12 (m, 2H), 4.51–4.06 (m, 7H), 3.71–3.67 (m, 1H),
3.61–3.57 (m, 1H), 1.40 (s, 9 H), 1.15 (s, 9H); HRMS (FAB): calcd.
for C37H44N2O9Na (M + Na)+: 683.2945, found: 683.2939.
HRMS (FAB): calcd. for C45H44N2O8SNa (M + Na)+: 795.2716,
found: 795.2723.
Boc-Thr(Fmoc-Cys(Trt))-OBzl (68). 68 was synthesized in
a similar manner to 103. Epimerization during the synthesis
was not detected, confirmed by comparison with independently
synthesized D-cysteine derivative 106. Yield: 97%; HPLC analysis
at 230 nm: purity was higher than 95% (tR = 43.2 min); 1H NMR
(CD3OD, 300 MHz) d 7.78 (d, J = 7.7 Hz, 2 H), 7.67–7.63 (m, 2
H), 7.39–7.18 (m, 24 H), 5.42–5.31 (m, 1 H), 5.04 (d, J = 12.4 Hz,
1 H), 4.97 (d, J = 12.4 Hz, 1 H), 4.37–4.20 (m, 4 H), 3.90–3.85 (m,
1 H), 2.67–2.56 (m, 1 H), 2.51–2.41 (m, 1 H), 1.43 (s, 9 H), 1.17 (d,
J = 6.2 Hz, 3 H); HRMS (FAB): calcd. for C53H52N2O8SNa (M +
Na)+: 899.3342, found: 899.3348.
Boc-Ser(Fmoc-Ser(tBu)-OH (6). 6 was synthesized in a similar
manner to 1. Yield: >99%; HPLC analysis at 230 nm: purity was
higher than 95% (tR = 32.5 min); 1H NMR (CD3OD, 300 MHz)
d 7.79 (d, J = 7.5 Hz, 2H), 7.69–7.66 (m, 2H), 7.40–7.28 (m, 4H),
4.54–4.21 (m, 7H), 3.81–3.76 (m, 1H), 3.65–3.60 (m, 1H), 1.42 (s,
9H), 1.19 (s, 9H); HRMS (FAB): calcd. for C30H38N2O9Na (M +
Na)+: 593.2475, found: 593.2471.
Boc-Thr(Fmoc-D-Cys(Trt))-OBzl (106). 106 was synthesized
in a similar manner to 103. Yield: >99%; HPLC analysis at 230 nm:
purity was higher than 95% (tR = 43.2 min); 1H NMR (CD3OD,
300 MHz) d 7.78 (d, J = 7.4 Hz, 2 H), 7.68–7.61 (m, 2 H), 7.38–
7.19 (m, 24 H), 5.41–5.30 (m, 1 H), 5.02 (d, J = 12.2 Hz, 1 H),
4.92 (d, J = 12.2 Hz, 1 H), 4.35–4.32 (m, 3 H), 4.24–4.20 (m, 1
H), 3.83–3.79 (m, 1 H), 2.65–2.48 (m, 2 H), 1.42 (s, 9 H), 1.19 (d,
J = 6.2 Hz, 3 H); HRMS (FAB): calcd. for C53H52N2O8SNa (M +
Na)+: 899.3342, found: 899.3348.
Boc-Ser(Fmoc-Cys(Trt))-OBzl (48). 48 was synthesized in a
similar manner to 42. Epimerization during the synthesis was not
detected, confirmed by comparison with independently synthe-
sized D-cysteine derivative 87. Yield: >99%; HPLC analysis at
1
230 nm: purity was higher than 95% (tR = 42.1 min); H NMR
(CD3OD, 300 MHz) d 7.81–7.73 (m, 2H), 7.70–7.60 (m, 2H), 7.43–
7.13 (m, 24H), 5.12–5.01 (m, 2H), 4.45–4.17 (m, 5H), 4.14–4.02 (m,
1H), 3.91–3.79 (m, 1H), 2.70–2.44 (m, 2H), 1.38 (s, 9H); HRMS
(FAB): calcd. for C52H50N2O8SNa (M + Na)+: 885.3186, found:
885.3179.
Boc-Thr(Fmoc-Cys(Trt))-OH (28). 28 could not be synthe-
sized in the same manner to 21 because the sulfur atom at the
cysteine resisted the deprotection of the O-benzyl group. Thus we
adopted catalytic transfer hydrogenation (CTH) to deprotect the
benzyl group. Pd/C (6 mg) was added to a stirring solution of the
Boc-Thr(Fmoc-Cys(Trt))-OBzl (10 mg, 0.011 mmol, 68) in EtOH
(1.0 mL) including H2O (100 lL) in the presence of ammonium
formate (7 mg) as hydrogen donor, and the reaction mixture was
vigorously stirred for 3 h under H2 atmosphere at 40 ◦C. Once the
catalyst was filtered off using 0.46 lm filter unit, Pd/C (6 mg) and
ammonium formate (7 mg) were added again, and the reaction
mixture was vigorously stirred for 3 h under H2 atmosphere at
40 ◦C. Then, the catalyst was filtered off through Celite. The
solvent was removed in vacuo and the crude compound dissolved in
DMSO, filtered using 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, affording pure Boc-Thr(Fmoc-Cys(Trt))-OH (2.7 mg,
0.003 mmol, 30%). HPLC analysis at 230 nm: purity was higher
than 95% (tR = 40.5 min); 1H NMR (CD3OD, 300 MHz) d 7.78 (d,
J = 7.4 Hz, 2 H), 7.70–7.64 (m, 2 H), 7.40–7.20 (m, 19 H), 5.37–
5.28 (m, 1 H), 4.37–4.28 (m, 2 H), 4.27–4.20 (m, 2 H), 3.97–3.88
(m, 1 H), 2.73–2.60 (m, 1 H), 2.54–2.42 (m, 1 H), 1.44 (s, 9 H), 1.18
(d, J = 5.5 Hz, 3 H); HRMS (FAB): calcd. for C46H46N2O8SNa
(M + Na)+: 809.2873, found: 809.2867.
Boc-Ser(Fmoc-D-Cys(Trt))-OBzl (87). 87 was synthesized in
a similar manner to 42. Yield: 77%; HPLC analysis at 230 nm:
purity was higher than 95% (tR = 42.1 min); 1H NMR (CD3OD,
300 MHz) d 7.81–7.73 (m, 2H), 7.69–7.60 (m, 2H), 7.42–7.13
(m, 24H), 5.09–4.98 (m, 2H), 4.45–4.16 (m, 6H), 3.82–3.71 (m,
1H), 2.74–2.51 (m, 2H), 1.38 (s, 9H); HRMS (FAB): calcd. for
C52H50N2O8SNa (M + Na)+: 885.3186, found: 885.3179.
Boc-Ser(Fmoc-Cys(Trt))-OH (8). 8 could not be synthesized
in the same manner to 1 because the sulfur atom at the cysteine
resisted the deprotection of the O-benzyl group. Thus we adopted
catalytic transfer hydrogenation (CTH) to deprotect the benzyl
group. Pd/C (6 mg) was added to the stirring solution of the
Boc-Ser(Fmoc-Cys(Trt))-OBzl (10 mg, 0.012 mmol, 48) in EtOH
(1.0 mL) including H2O (100 lL) in the presence of ammonium
formate (7 mg) as hydrogen donor, and the reaction mixture was
vigorously stirred for 3 h under H2 atmosphere at 40 ◦C. Once the
catalyst was filtered off using 0.46 lm filter unit, Pd/C (6 mg) and
ammonium formate (7 mg) were added again, and the reaction
mixture was vigorously stirred for 3 h under H2 atmosphere at
◦
40 C. Then, the Pd/C was filtered off using 0.46 lm filter unit,
then, next portions of Pd/C (6 mg) and ammonium formate
(7 mg) were added, and the reaction mixture was vigorously
stirred for 3 h under H2 atmosphere at 40 ◦C. Again catalyst
was filtered off through Celite. The solvent was removed in vacuo
and the crude compound dissolved in DMSO, filtered using
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, affording
pure Boc-Ser(Fmoc-Cys(Trt))-OH (4.5 mg, 0.0058 mmol, 58%).
HPLC analysis at 230 nm: purity was higher than 95% (tR = 40.2
Boc-Thr(Fmoc-Arg(Pmc))-OBzl (77). 77 was synthesized in
a similar manner to 103. Epimerization during the synthesis
was not detected, confirmed by comparison with independently
synthesized D-arginine derivative 115. Yield: 79%; HPLC analysis
1
at 230 nm: purity was 94% (tR = 39.0 min); H NMR (CD3OD,
300 MHz) d 7.78 (d, J = 7.5 Hz, 2H), 7.65–7.60 (m, 2H), 7.39–7.24
(m, 8H), 5.44–5.41 (m, 1H), 5.10 (d, J = 12.2 Hz, 1H), 5.01 (d, J =
12.2 Hz, 1H), 4.39–4.34 (m, 3H), 4.20 (t, J = 6.6 Hz, 1H), 4.06–
4.03 (m, 1H), 3.16–3.11 (m, 1H), 2.62 (t, J = 6.8 Hz, 2H), 2.57 (s,
3H), 2.55 (s, 3H), 2.08 (s, 3H), 1.78 (t, J = 6.8 Hz, 2H), 1.72–1.66
1
min); H NMR (CD3OD, 300 MHz) d 7.79 (d, J = 7.4 Hz, 2H),
7.73–7.63 (m, 2H), 7.46–7.18 (m, 18H), 4.49–4.16 (m, 6H), 3.97–
3.84 (m, 1H), 2.76–2.65 (m, 1H), 2.64–2.52 (m, 1H), 1.39 (s, 9H);
1728 | Org. Biomol. Chem., 2007, 5, 1720–1730
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(m, 1H), 1.60–1.46 (m, 3H), 1.42 (s, 9H), 1.27 (s, 6H), 1.21 (d,
J = 6.4 Hz, 3H); HRMS (FAB): calcd. for C51H63N5O11SNa (M +
Na)+: 976.4142, found: 976.4137.
Synthesis of H-Thr(H-Gly-Ile-Leu-Gly-Phe-Val-Phe)-Leu-OH
(123) by the O-acyl isopeptide method without O-acyl isodipep-
tide unit (O-acyl isopeptide of influenza A virus matrix M1 58–66).
Fmoc-Leu-resin was prepared in the same manner described above
(2-chrolotrityl resin, 200 mg, 0.19 mmol). After deprotection of the
Fmoc group by 20% piperidine in DMF, Boc-Thr-OH (103.6 mg,
0.47 mmol) was coupled using DIPCDI (74.1 lL, 0.47 mmol) and
HOBt (72.1 mg, 0.47 mmol). Subsequent coupling with Fmoc-
Phe-OH (219.7 mg, 0.57 mmol) to the b-hydroxyl group of Thr
was performed using the DIPCDI (88.8 lL, 0.56 mmol)–DMAP
(4.6 mg, 0.038 mmol) method in dry CH2Cl2 (1.5 mL) for 16 h (×2).
Subsequent amino acid residues were coupled after removing each
Fmoc group using 20% piperidine for 20 min (resin: 269.2 mg).
The resulting protected peptide-resin (142.7 mg) was treated with
TFA (2.8 mL)–thioanisole (71.3 lL)–m-cresol (71.3 lL)–distilled
water (71.3 lL) for 90 min at rt, concentration in vacuo, Et2O wash,
centrifugation, suspension in water, and lyophilization to give the
crude O-acyl isopeptide 123·TFA (108.6 mg). TOF-MS: calcd. for
C49H75N9O11Na (M + Na)+: 989.17, found: 989.20; HPLC analysis
at 230 nm: purity was 93%.
Boc-Thr(Fmoc-D-Arg(Pmc))-OBzl (115). 115 was synthesized
in a similar manner to 62. Yield: 55%; HPLC analysis at 230 nm:
purity was 96% (tR = 38.3 min); 1H NMR (CD3OD, 300 MHz) d
7.78 (d, J = 7.7 Hz, 2H), 7.65 (t, J = 6.4 Hz, 2H), 7.39–7.26 (m,
9H), 5.41–5.38 (m, 1H), 5.12 (d, J = 12.4 Hz, 1H), 5.03 (d, J =
12.4 Hz, 1H), 4.41–4.35 (m, 3H), 4.21 (t, J = 6.8 Hz, 1H), 4.05–
4.01 (m, 1H), 3.13–3.09 (m, 2H), 2.62 (t, J = 6.8 Hz, 2H), 2.57
(s, 3H), 2.55 (s, 3H), 2.07 (s, 3H), 1.77 (t, J = 6.8 Hz, 2H), 1.69–
1.62 (m, 1H), 1.50–1.44 (m, 3H), 1.40 (s, 9H), 1.28–1.21 (m, 9H);
HRMS (FAB): calcd. for C51H63N5O11SNa (M + Na)+: 976.4142,
found: 976.4138.
Boc-Thr(Fmoc-Arg(Pmc))-OH (37). 37 was synthesized in a
similar manner to 21. Yield: >99%; HPLC analysis at 230 nm:
1
purity was 91% (tR = 37.0 min); H NMR (CD3OD, 300 MHz)
d 7.78 (d, J = 7.4 Hz, 2H), 7.66 (t, J = 7.6 Hz, 2H), 7.40–7.27
(m, 4H), 5.41–5.38 (m, 1H), 4.44–4.10 (m, 5H), 3.21–3.12 (m, 2H),
2.63 (t, J = 6.9 Hz, 2H), 2.57 (s, 3H), 2.55 (s, 3H), 2.08 (s, 3H), 1.79
(t, J = 6.9 Hz, 2H), 1.72–1.68 (m, 1H), 1.62–1.45 (m, 3H), 1.41 (s,
9H), 1.38 (s, 6H), 1.21 (d, J = 5.3 Hz, 3H); HRMS (FAB): calcd.
for C44H57N5O11SNa (M + Na)+: 886.3673, found: 886.3669.
Except for 1, 2, 4, 6, 8, 28, 37, 41, 42, 44, 46, 48, 68, 77, 81,
83, 85, 87, 106, 115, experimental procedures and chemical data
of all O-acyl isodipeptide units and their intermediates shown in
Tables 1 and 2 (3, 5, 7, 9–27, 29–36, 38–40, 43, 45, 47, 49–67,
69–76, 78–80, 82, 84, 86, 88–105, 107–114, 116–118) are described
in the electronic supplementary information (ESI).†
Synthesis of influenza A virus matrix M1 58–66 (119) by the
O-acyl isopeptide method with O-acyl isodipeptide unit
Fmoc-Leu-resin was prepared in the same manner described
above (2-chlorotrityl chloride resin, 70 mg, 0.11 mmol). After
deprotection of the Fmoc group by 20% piperidine in DMF, Boc-
Thr(Fmoc-Phe)-OH (72.1 mg, 0.15 mmol, 38) was coupled using
DIPCDI (23.1 lL, 0.15 mmol) and HOBt (22.6 mg, 0.15 mmol).
Subsequent amino acid residues were coupled after removing each
Fmoc group using 20% piperidine for 20 min (resin: 113.8 mg). The
resulting protected isopeptide-resin treated with TFA (2.1 mL)–
thioanisole (56.9 lL)–m-cresol (56.9 lL)–H2O (56.9 lL) (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 a crude isopeptide 123·TFA (51.1 mg). This isopeptide
(5.5 mg) was dissolved in phosphate buffer and stirred overnight
at rt and lyophilized to give the crude 119. This crude peptide
was dissolved in DMSO, filtered using 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, affording the desired peptide 119·TFA as
a white amorphous powder (5.0 mg, 73%). TOF-MS: calcd. for
C49H75N9O11Na (M + Na)+: 989.17, found: 989.26; HPLC analysis
at 230 nm: purity was 94%.
Synthesis of influenza A virus matrix M1 58–66 (119) by SPPS.
Chlorotrityl chloride resin (200 mg, 0.3 mmol) and Fmoc-Leu-OH
(265.1 mg, 0.75 mmol) were mixed in a manual solid-phase reactor
under an argon atmosphere and stirred for 2.5 h in the presence of
DIPEA (130.6 lL, 0.75 mmol) in 1,2-dichloroethane (1.5 mL). Af-
ter washing with DMF (1.5 mL, ×5), capping was performed with
MeOH (200 lL) in the presence of DIPEA (52.5 lL, 0.3 mmol) in
DMF for 20 min. After washing with DMF (×5), DMF–H2O (1 :
1, ×5), CHCl3 (×2) and MeOH (×2) followed by drying in vacuo,
the loading ratio (0.18 mmol) was determined photometrically
from the amount of Fmoc chromophore liberated upon treatment
with 50% piperidine–DMF for 30 min at 37 ◦C. Sequential Fmoc-
protected amino acids (0.45 mmol) were manually coupled in the
presence of DIPCDI (70.2 lL, 0.44 mmol) and HOBt (68.4 mg,
0.45 mmol) for 2 h in DMF (1.5 mL) after removal of each Fmoc
group by 20% piperidine–DMF for 20 min (resin: 346.7 mg).
The resulting protected peptide-resin (346.7 mg) was treated with
TFA (6.7 mL)–m-cresol (173.3 lL)–thioanisole (173.3 lL)–H2O
(173.3 lL) for 90 min, concentrated in vacuo, washed with diethyl
ether, centrifuged, suspended with water, and lyophilized to give
the crude peptide 119 (161.1 mg). This crude peptide (20 mg)
was dissolved in DMSO, filtered using 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, affording the desired peptide 119·TFA as
a white amorphous powder (12.6 mg, 53%). TOF-MS: calcd. for
C49H76N9O11 (M + H)+: 967.17, found: 968.55; HPLC analysis at
230 nm: purity was higher than 95%.
Acknowledgements
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 Government, 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
Postdoctoral Fellowship of JSPS. We thank Ms M. Tsukuda and
Mr Y. Chiyomori 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 English correction.
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128, 7722–7723; (b) J. Hentschel and H. G. Bo¨rner, J. Am. Chem. Soc.,
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6 M. Skwarczynski, Y. Sohma, M. Noguchi, Y. Hayashi, T. Kimura and
Y. K i s o , J. Org. Chem., 2006, 71, 2542–2545.
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