Solution-phase automated synthesis of tripeptide derivatives

Article abstract of DOI:10.1248/cpb.49.1138

An improved general method for automated synthesis of tripeptides was developed, in which methanesulfonic acid (MSA) was used in place of trifluoroacetic acid (TFA), thus making it possible to avoid, 1) corrosion of the apparatus by strong acid vapor, 2) formation of emulsions, and 3) use of the restricted solvent, dichloromethane. As an application of the automated synthesis apparatus, 216 fragment tripeptide derivatives were synthesized systematically using the MSA method, in excellent yield and with increased efficiency.

Full text of DOI:10.1248/cpb.49.1138

1138
Chem. Pharm. Bull. 49(9) 1138—1146 (2001)
Vol. 49, No. 9
Solution-Phase Automated Synthesis of Tripeptide Derivatives
a
Noritaka KURODA, ,a Taeko HATTORI,a Chieko KITADA,b and Tohru SUGAWARA
*
Discovery Research Laboratories V,a Discovery Research Division, Takeda Chemical Industries, Ltd., 17–85 Jusohonmachi
2-chome, Yodogawaku, Osaka 532–8686, Japan and Discovery Research Division,b Takeda Chemical Industries, Ltd.,
Wadai-10, Tsukuba, Ibaraki 300–4293, Japan. Received April 4, 2001; accepted June 6, 2001
An improved general method for automated synthesis of tripeptides was developed, in which methanesul-
fonic acid (MSA) was used in place of trifluoroacetic acid (TFA), thus making it possible to avoid, 1) corrosion
of the apparatus by strong acid vapor, 2) formation of emulsions, and 3) use of the restricted solvent,
dichloromethane. As an application of the automated synthesis apparatus, 216 fragment tripeptide derivatives
were synthesized systematically using the MSA method, in excellent yield and with increased efficiency.
Key words automated synthesis; solution-phase peptide synthesis; fragment tripeptide; methanesulfonic acid method; trifluo-
roacetic acid method
As combinatorial chemistry has been rapidly expanding the Boc group, and its subsequent vacuum evaporation,
for solution-phase as well as solid-phase synthesis, several which caused extensive damage to the solenoid valves and
kinds of automated apparatus for both synthesis strategies tubings, resulting in leakages and expensive maintenance.
have been developed.1) When chemists want to synthesize Another problem was the use of dichloromethane for liquid–
large numbers of compounds efficiently using automated ap- liquid extraction. The use of dichloromethane is restricted as
paratus, it is necessary to carefully plan how to apply the ap- it is harmful to the environment, and in addition, its use can
paratus. Most reaction procedures were originally developed lead to further problems due to emulsion formation, which
assuming manual operations, and it might not be suitable to may stop the automated process due to blockages in the dry-
use them directly for an automated synthesis. For successful ing tubes or the inability to separate phases. Also the parallel
automation, it is important to develop new reaction processes processing of the deprotection of the Boc group and the acti-
that are specially suited to automated synthesis apparatus.2) vation of the carboxyl group caused some problems. If the
Recently, new methodologies, such as those using scavenger activated ester was left for long periods (e.g. 3 h), it decom-
resins,3) cation pools or phase tags,4) have been developed for posed and low yields were obtained. In addition, manual op-
automation, and solution-phase laboratory automation is erations were needed during the process, such as when it was
likely to become more and more important as a research tool. necessary to manually restart the process after the activated
In our laboratories, we have developed automated synthe- ester had been made in one vessel, to acylate the amine,
sis systems that can utilize a variety of reaction procedures, which was deprotected in an another vessel. Considering
including those that have been developed for manual opera- these problems, we planned to change the reagent for the
tion. For example, solid reagents can be automatically added deprotection of the Boc group, from TFA to MSA, which
to a vessel using ASRA,5) or a centrifugal separatory funnel would also allow the subsequent liquid–liquid extraction step
can be used to separate emulsions in NEW-TACOS.6) How- to be replaced by a simple stoichiometrical neutralization.
ever, another approach to automation involves developing Dilute MSA has been reported deprotection of the Boc group
new reaction procedures specially for automated synthesis in liquid-phase fragment condensation8) and in solid-phase
apparatus, and recent emphasis has been made on increasing peptide elongation,9) so we focused on how to effectively use
throughput by parallelisation.
it in the automated apparatus.
First we investigated the reaction conditions, and found
In a previous report,7) we described the solution-phase au-
tomated synthesis of fragment peptide derivatives using our that four mol eq of MSA, relative to the starting Boc-amino
unique automated synthesis system. It was used to synthesize acid derivative, was required for deprotection. Dichloro-
systematically a library of all possible tripeptides (125 deriv- methane, diisopropylether, methyl ethyl ketone, or acetonitrile
atives) from 5 different kinds of protected amino acids [tert- could be used as the reaction solvent, but acetonitrile was
Butoxycarbonyl-Leu (Boc-Leu), Boc-Ser(Bzl), Boc-Glu(O- chosen as it could also be used for the following acylation
cHex), Boc-Trp, Boc-Arg(Tos)]. In this report, we describe process. Reaction at 40 °C for 1 h gave complete deprotec-
the automated synthesis of fragment tripeptides, using a new tion, and then excess MSA could be stoichiometrically neu-
methanesulfonic acid (MSA) method, that has improved tralized with diisopropylethylamine. Our new MSA method
compatibility with our automated synthesis apparatus.
was used for the automated synthesis of several tripeptides
and the results were compared to those obtained using the
TFA method (see Table 1). It was clear that the MSA method
Results and Discussion
a) Improved Method for General Synthesis of Tripep- gave higher yields because of the simplicity and reliablity of
tides Automated synthesis of fragment tripeptides was first the process. The complete MSA method, as shown in Chart
carried out following the trifluoroacetic acid (TFA) method 1, was then used to synthesize 216 fragment tripeptides.
as shown in Fig. 1.7) It is a good general method for manual
b) Synthesis of 216 Fragment Tripeptides Figure 2
synthesis, but from the view point of application to the auto- shows the flowchart of a peptide synthesis performed follow-
mated apparatus, it was found to have several demerits. One ing the MSA method, and Table 2 shows the subroutine se-
of these was the necessity to use TFA for the deprotection of quence of the program.7) We planned to synthesize fragment
To whom correspondence should be addressed. e-mail: Kuroda_Noritaka@takeda.co.jp
© 2001 Pharmaceutical Society of Japan

September 2001
1139
Fig. 1. Flowchart of a Peptide Synthesis by the TFA Method
Table 1. Comparison of Yields for the Synthesis of Tripeptide Derivatives
(Boc-A3–A2–A1-OBzl)
-A3–A2–A1-a)
TFA method yield (%)
MSA method yield (%)
EEL
66
64
77
80
71
75
72
81
85
92
LRW
KWA
EWA
LES
Chart 1. Schematic Diagram of the MSA Method
a) AϭAla, EϭGlu(O-cHex), LϭLeu, RϭArg(Tos), WϭTrp, KϭLys(Z).
low isolated yields were observed because of partial salt for-
mation during work-up with 0.2 N HCl, as shown in Fig. 2.
Therefore the work-up procedure was changed for the syn-
thesis of later derivatives (cf. Table 3, APH-RPH, HPW,
HPR, HKA-HKR) to increase the yields. The series of
tripeptides which possess a tyrosyl group in the center were
found to have poor solubility, especially for the lysyl–tyrosyl
derivatives, which were poorly soluble even in dimethylfor-
mamide (DMF). Precipitates were often formed during syn-
thesis, and these caused considerable trouble in the auto-
mated synthesis apparatus, but the problem was overcome by
dissolving the DIEA reagent in a greater volume of DMF. In
tripeptide derivatives having a tryptophyl group, other than at
the N-terminal position, a substituted indole by-product (5—
10%) was formed during removal of the Boc group. Anisole,
thioanisole, or m-cresol was added to the reaction mixture to
tripeptides which possessed a prolyl, tyrosyl or tryptophyl
group in the center of the amino acid, and the other two
amino acids were randomly selected from Boc-Ala, Boc-Leu,
Boc-Ser(Bzl), Boc-Glu(O-cHex), Boc-Pro, Boc-Lys(Z), Boc-
Tyr(Bzl), Boc-Trp, Boc-Arg(Tos), Boc-His(Bom). All yields
are shown in Table 3, and physical data is shown in Table 4.
In the series of tripeptide derivatives with a prolyl group in
the center, cis–trans isomers, relative to the proline ring,
were obtained, especially when the prolyl group was between
two bulky amino acid residues, such as Tyr(Bzl) and Glu(O-
1
cHex). H- and 13C-NMR spectra were measured, and the
cross coupling peaks between the major and minor compo-
nents were observed in nuclear Overhauser effect spectro-
scopy (NOESY) spectra (Tables 5, 6). In the case of the
tripeptide derivatives containing a histidyl group, extremely

1140
Vol. 49, No. 9
Table 2. Subroutine Sequence for the MSA Method
Program name MSA1
1. START
2. RF1-ST-ON
7. RR3-RF1
12. RF1-RF2
17. RF1-LF-DN
22. RF2-LF-DN
27. RF2-ST-OF
32. RF1-LF-UP
37. RF2-LF-DN
3. RR2-RF1
8. RF1-T-OF
13. RS6-RF1
18. RR4-RF2
23. RS4-RF2
28. ALARM
33. RF2-LF-UP
38. END
4. RF1-LF-UP
9. RF2-LF-UP
14. RF1-MIX
19. RF2-T-OF
24. RS2-RF2
29. SR1-DR
5. REA1 (40c, 60 m)
10. RF2-T-ON (0c)
15. RF1-RF2
20. REA2 (25c, 600 m)
25. RF2-MIX
30. BKWASH
6. RF1-T-ON (0c)
11. RF2-ST-ON
16. RF1-ST-OF
21. CON2 (40c, 60 m)
26. BKEXT
31. BKWASH
36. RF1-LF-DN
BKEXT
34. RF1-DRY
35. RF2-DRY
1. BKSTART
6. RF2-SF
11. RF2-SF
16. RF2-MIX
21. BKEND
BKWASH
2. RF2-SF
7. SF-BUBB
12. SF-BUBB
17. RF2-SF
3. SEP-SR1
8. SEP-SR0
13. SEP-SR1
18. SF-BUBB
4. RS3-RF2
9. RS1-RF2
14. SF-RF3
19. SF-RF3
5. RF2-MIX
10. RF2-MIX
15. RS4-RF2
20. SR0-DR
1. BKSTART
6. WS-RF2
11. SF-SR1
2. WS-RR2
7. RF1-BUBB
12. SR1-BUBB
17. BKEND
3. WS-RR3
8. RF2-BUBB
13. SR1-DR
4. WS-RR4
9. RF2-SF
14. RF1-RF2
5. WS-RF1
10. SF-BUBB
15. RF2-SF
16. SF-RF3
Table 3. PercentageYields of Tripeptides
Fig. 2. Flowchart of a Peptide Synthesis by the MSA Method
prevent by-product formation for the synthesis of SYW-HYW
tripeptides. Typically, yields were increased by the addition
of m-cresol from an average 64.5% to 74.7%. (cf. Table 3,
tripeptides in italics.) Acid-labile side chain deprotection
reactions, such as those of benzyl or benzyloxycarbonyl pro- deprotected byproducts were formed for the tripeptides con-
tecting groups by MSA,8,9) were checked by measuring repre- taining Lys(Z) or Ser(Bzl) or Tyr(Bzl) residues. The advan-
sentative NMR spectra of the separated tripeptides. Elemen- tages of the MSA method, found during the synthesis of the
1
tal analysis (Table 4) and H-NMR (Table 7) showed that no tripeptide series, are summarized in Table 8.

September 2001
1141
Table 4. Physical Data of Tripeptide Derivatives (Boc-A3–A2–A1-OBzl)
A3–A2–A1
mp (°C)
[a]D
Anal. Calcd (Found)
Glu–Pro–Ala
His–Pro–Ala
Lys–Pro–Ala
Leu–Pro–Ala
Arg–Pro–Ala
Ser–Pro–Ala
Trp–Pro–Ala
Tyr–Pro–Ala
Ala–Pro–Glu
His–Pro–Glu
Lys–Pro–Glu
Leu–Pro–Glu
Arg–Pro–Glu
Ser–Pro–Glu
Trp–Pro–Glu
Tyr–Pro–Glu
Ala–Pro–His
Glu–Pro–His
Lys–Pro–His
Leu–Pro–His
Arg–Pro–His
Ser–Pro–His
Trp–Pro–His
Tyr–Pro–His
Ala–Pro–Lys
Glu–Pro–Lys
His–Pro–Lys
Leu–Pro–Lys
Arg–Pro–Lys
Ser–Pro–Lys
Trp–Pro–Lys
Tyr–Pro–Lys
Ala–Pro–Leu
Glu–Pro–Leu
His–Pro–Leu
Lys–Pro–Leu
Arg–Pro–Leu
Ser–Pro–Leu
Trp–Pro–Leu
Tyr–Pro–Leu
Ala–Pro–Arg
Glu–Pro–Arg
His–Pro–Arg
Lys–Pro–Arg
Leu–Pro–Arg
Ser–Pro–Arg
Trp–Pro–Arg
Tyr–Pro–Arg
Ala–Pro–Ser
Glu–Pro–Ser
His–Pro–Ser
Lys–Pro–Ser
Leu–Pro–Ser
Arg–Pro–Ser
Trp–Pro–Ser
Tyr–Pro–Ser
Ala–Pro–Trp
Glu–Pro–Trp
His–Pro–Trp
Lys–Pro–Trp
Leu–Pro–Trp
Arg–Pro–Trp
Ser–Pro–Trp
Tyr–Pro–Trp
Ala–Pro–Tyr
Glu–Pro–Tyr
His–Pro–Tyr
Lys–Pro–Tyr
Leu–Pro–Tyr
Arg–Pro–Tyr
Ser–Pro–Tyr
Trp–Pro–Tyr
Glu–Lys–Ala
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Oil
217.0—218.0
73.0—75.0
Amorphous
Amorphous
Oil
Ϫ85.1
Ϫ71.2
Ϫ78.6
Ϫ118.3
Ϫ63.1
Ϫ71.7
Ϫ75.3
Ϫ56
Ϫ86.9
Ϫ49.3
Ϫ52.7
Ϫ80.8
Ϫ43.1
Ϫ47.2
Ϫ62
Ϫ35.6
Ϫ73.4
Ϫ47.4
Ϫ42.3
Ϫ66.4
Ϫ36.5
Ϫ41.8
Ϫ59.8
Ϫ31.8
Ϫ81.2
Ϫ52.5
Ϫ44
Ϫ70.6
Ϫ39.1
Ϫ44.5
Ϫ53.8
Ϫ30.5
Ϫ115.5
Ϫ77.7
Ϫ66.4
Ϫ71.4
Ϫ58.2
Ϫ66.4
Ϫ82.3
Ϫ52.2
Ϫ70.5
Ϫ44.3
Ϫ36.1
Ϫ42.4
Ϫ60.6
Ϫ37
C; 62.87 (62.84) H; 7.74 (7.89) N; 7.10 (6.99)
C; 63.54 (63.40) H; 6.90 (6.97) N; 10.90 (11.03)
C; 63.49 (63.32) H; 7.29 (7.50) N; 8.71 (8.52)
C; 63.78 (63.69) H; 8.03 (8.09) N; 8.58 (8.63)
C; 56.60 (56.42) H; 6.84 (6.44) N; 11.92 (11.74)
C; 65.08 (64.88) H; 7.10 (7.29) N; 7.59 (7.54)
C; 65.65 (65.85) H; 6.84 (6.82) N; 9.88 (9.95)
C; 68.66 (68.69) H; 6.88 (6.73) N; 6.67 (6.76)
C; 62.87 (62.81) H; 7.74 (7.75) N; 7.10 (6.99)
C; 64.43 (64.67) H; 7.21 (7.21) N; 8.95 (8.98)
C; 64.76 (64.62) H; 7.51 (7.58) N; 7.19 (7.22)
C; 63.93 (64.05) H; 8.21 (8.27) N; 6.58 (6.42)
C; 59.55 (59.65) H; 7.07 (7.18) N; 10.16 (10.13)
C; 64.94 (64.96) H; 7.46 (7.55) N; 5.98 (6.01)
C; 66.31 (66.27) H; 7.19 (7.13) N; 7.93 (8.03)
C; 68.64 (68.52) H; 7.20 (7.11) N; 5.46 (5.68)
C; 63.69 (63.54) H; 6.86 (6.90) N; 10.86 (10.90)
C; 63.67 (63.85) H; 7.10 (7.18) N; 9.05 (8.93)
C; 63.78 (63.79) H; 6.96 (6.98) N; 9.92 (10.19)
C; 65.14 (65.09) H; 7.44 (7.35) N; 10.55 (10.43)
C; 60.53 (60.23) H; 6.47 (6.64) N; 12.83 (12.80)
C; 65.76 (65.85) H; 6.73 (6.61) N; 9.35 (9.29)
C; 65.02 (64.73) H; 6.63 (6.25) N; 10.83 (10.73)
C; 68.73 (68.79) H; 6.58 (6.45) N; 8.53 (8.60)
C; 63.93 (63.76) H; 7.26 (7.26) N; 8.77 (8.78)
C; 64.76 (64.40) H; 7.51 (7.51) N; 7.19 (7.13)
C; 64.81 (64.53) H; 6.89 (6.94) N; 10.08 (10.12)
C; 65.27 (64.97) H; 7.70 (7.74) N; 8.23 (8.45)
C; 59.88 (59.84) H; 6.80 (7.05) N; 11.11 (10.90)
C; 66.11 (66.25) H; 7.04 (6.86) N; 7.52 (7.72)
C; 66.91 (66.85) H; 6.82 (6.84) N; 9.29 (9.34)
C; 68.76 (68.65) H; 6.88 (6.95) N; 6.82 (6.86)
C; 63.32 (63.37) H; 8.05 (8.12) N; 8.52 (8.54)
C; 64.29 (64.23) H; 8.19 (8.21) N; 6.62 (6.53)
C; 64.89 (64.71) H; 7.36 (7.30) N; 10.23 (10.25)
C; 64.84 (64.72) H; 7.72 (7.79) N; 8.18 (8.19)
C; 59.32 (59.08) H; 7.19 (7.44) N; 11.53 (11.34)
C; 66.03 (65.99) H; 7.64 (7.66) N; 7.00 (6.89)
C; 67.53 (67.55) H; 7.33 (7.19) N; 9.26 (9.40)
C; 69.26 (69.16) H; 7.38 (7.11) N; 6.21 (6.27)
C; 56.96 (57.04) H; 6.81 (6.92) N; 12.08 (11.92)
C; 58.90 (59.14) H; 7.11 (7.17) N; 10.05 (10.27)
C; 58.72 (58.98) H; 6.61 (6.56) N; 12.45 (12.19)
C; 59.58 (59.49) H; 6.82 (6.73) N; 11.05 (10.96)
C; 58.60 (58.86) H; 7.24 (7.47) N; 11.39 (11.54)
C; 59.91 (59.95) H; 6.66 (6.75) N; 10.48 (10.54)
C; 60.06 (60.00) H; 6.51 (6.44) N; 11.96 (11.57)
C; 62.92 (62.71) H; 6.54 (6.31) N; 9.57 (9.54)
C; 64.04 (63.82) H; 7.17 (7.10) N; 7.47 (7.39)
C; 64.94 (65.01) H; 7.46 (7.43) N; 5.98 (5.88)
C; 64.98 (65.06) H; 6.78 (6.62) N; 9.24 (9.26)
C; 66.11 (66.05) H; 7.04 (7.09) N; 7.52 (7.55)
C; 65.54 (65.56) H; 7.67 (7.58) N; 6.95 (6.97)
C; 60.59 (60.37) H; 6.61 (6.65) N; 10.60 (10.52)
C; 67.79 (67.86) H; 6.66 (6.70) N; 8.32 (8.26)
C; 69.34 (69.54) H; 6.77 (6.78) N; 5.64 (5.83)
C; 65.65 (65.87) H; 6.84 (7.07) N; 9.88 (9.60)
C; 66.65 (66.64) H; 7.17 (7.39) N; 7.97 (7.95)
C; 67.36 (67.24) H; 6.46 (6.63) N; 11.22 (10.94)
C; 66.52 (66.57) H; 6.84 (6.81) N; 9.23 (9.27)
C; 67.03 (67.01) H; 7.36 (7.57) N; 9.20 (9.07)
C; 60.06 (60.28) H; 6.52 (6.22) N; 11.96 (11.66)
C; 67.79 (67.86) H; 6.66 (6.72) N; 8.32 (8.36)
C; 70.10 (69.84) H; 6.55 (6.71) N; 7.43 (7.39)
C; 68.66 (68.48) H; 6.88 (6.71) N; 6.67 (6.75)
C; 68.64 (68.75) H; 7.20 (7.30) N; 5.46 (5.39)
C; 68.80 (68.74) H; 6.57 (6.53) N; 8.54 (8.55)
C; 68.76 (68.52) H; 6.88 (6.96) N; 6.82 (6.93)
C; 69.72 (69.64) H; 7.35 (7.21) N; 6.25 (6.37)
C; 63.58 (63.51) H; 6.49 (6.69) N; 9.67 (9.61)
C; 70.18 (70.35) H; 6.71 (6.42) N; 5.71 (5.62)
C; 70.52 (70.58) H; 6.52 (6.39) N; 7.48 (7.48)
C; 63.81 (63.68) H; 7.50 (7.53) N; 7.44 (7.59)
Oil
Amorphous
Oil
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
149.0—151.0
Amorphous
Amorphous
Amorphous
92.0—93.0
Amorphous
Amorphous
Amorphous
Amorphous
92.0—93.0
117.0—118.0
138.0—139.0
Oil
Oil
Amorphous
Amorphous
Amorphous
Oil
173.0—175.0
Oil
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Oil
Ϫ46.5
Ϫ27.1
Ϫ86.4
Ϫ57.2
Ϫ46
Oil
Amorphous
94.0—95.0
Oil
Oil
Amorphous
Oil
Ϫ54.2
Ϫ76
Ϫ42.2
Ϫ50.7
Ϫ31.8
Ϫ75.1
Ϫ49.4
Ϫ36.2
Ϫ47.3
Ϫ65.6
Ϫ34.6
Ϫ41.9
Ϫ25.9
Ϫ70.3
Ϫ46.6
Ϫ33.1
Ϫ41.9
Ϫ62.4
Ϫ32.4
Ϫ39.8
Ϫ47.9
Ϫ33.1
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
84.0—86.0
66.0—68.0
Amorphous
131.0—132.0
57.0—58.0
Amorphous
75.0—77.0
Amorphous
84.0—86.0

1142
Vol. 49, No. 9
Table 4. (Continued.)
A3–A2–A1
mp (°C)
[a]D
Anal. Calcd (Found)
His–Lys–Ala
Leu–Lys–Ala
Pro–Lys–Ala
Arg–Lys–Ala
Ser–Lys–Ala
Trp–Lys–Ala
Tyr–Lys–Ala
Ala–Lys–Glu
His–Lys–Glu
Leu–Lys–Glu
Pro–Lys–Glu
Arg–Lys–Glu
Ser–Lys–Glu
Trp–Lys–Glu
Tyr–Lys–Glu
Ala–Lys–His
Glu–Lys–His
Leu–Lys–His
Pro–Lys–His
Arg–Lys–His
Ser–Lys–His
Trp–Lys–His
Tyr–Lys–His
Ala–Lys–Leu
Glu–Lys–Leu
His–Lys–Leu
Pro–Lys–Leu
Arg–Lys–Leu
Ser–Lys–Leu
Trp–Lys–Leu
Tyr–Lys–Leu
Ala–Lys–Pro
Glu–Lys–Pro
His–Lys–Pro
Leu–Lys–Pro
Arg–Lys–Pro
Ser–Lys–Pro
Trp–Lys–Pro
Tyr–Lys–Pro
Ala–Lys–Arg
Glu–Lys–Arg
His–Lys–Arg
Leu–Lys–Arg
Pro–Lys–Arg
Ser–Lys–Arg
Trp–Lys–Arg
Tyr–Lys–Arg
Ala–Lys–Ser
Glu–Lys–Ser
His–Lys–Ser
Leu–Lys–Ser
Pro–Lys–Ser
Arg–Lys–Ser
Trp–Lys–Ser
Tyr–Lys–Ser
Ala–Lys–Trp
Glu–Lys–Trp
His–Lys–Trp
Leu–Lys–Trp
Pro–Lys–Trp
Arg–Lys–Trp
Ser–Lys–Trp
Tyr–Lys–Trp
Ala–Lys–Tyr
Glu–Lys–Tyr
His–Lys–Tyr
Leu–Lys–Tyr
Pro–Lys–Tyr
Arg–Lys–Tyr
Ser–Lys–Tyr
Trp–Lys–Tyr
Glu–Tyr–Ala
His–Tyr–Ala
Amorphous
161.0—162.0
73.0—75.0
Amorphous
126.0—128.0
107.5—108.5
125.0—127.0
85.0—87.0
Ϫ28.7
Ϫ44
Ϫ63
C; 64.65 (64.45) H; 6.81 (6.93) N; 10.52 (10.71)
C; 64.20 (64.12) H; 7.70 (7.46) N; 8.56 (8.80)
C; 63.93 (63.72) H; 7.26 (7.38) N; 8.77 (8.73)
C; 58.59 (58.78) H; 6.79 (6.97) N; 11.39 (11.56)
C; 65.16 (65.13) H; 7.01 (6.86) N; 7.79 (7.88)
C; 66.01 (65.82) H; 6.79 (6.81) N; 9.62 (9.74)
C; 67.99 (67.80) H; 6.85 (6.78) N; 7.05 (7.04)
C; 63.81 (63.63) H; 7.50 (7.40) N; 7.44 (7.52)
C; 65.03 (65.23) H; 7.34 (7.08) N; 8.75 (8.95)
C; 64.97 (64.96) H; 7.86 (7.89) N; 7.05 (7.22)
C; 64.76 (64.73) H; 7.51 (7.50) N; 7.19 (7.22)
C; 59.98 (59.81) H; 7.05 (7.25) N; 9.79 (10.12)
C; 65.54 (65.72) H; 7.16 (7.27) N; 6.71 (6.52)
C; 66.42 (66.23) H; 7.08 (6.88) N; 8.07 (8.14)
C; 68.07 (67.84) H; 7.11 (6.91) N; 5.99 (6.07)
C; 63.75 (63.88) H; 6.89 (7.16) N; 10.34 (10.53)
C; 64.00 (63.99) H; 7.16 (7.20) N; 8.78 (8.75)
C; 64.32 (64.48) H; 7.27 (7.29) N; 9.78 (9.75)
C; 64.12 (64.21) H; 6.94 (7.21) N; 9.97 (9.95)
C; 61.31 (60.79) H; 6.50 (6.55) N; 12.14 (12.04)
C; 64.43 (64.57) H; 6.81 (6.69) N; 9.02 (9.32)
C; 66.69 (66.65) H; 6.53 (6.56) N; 10.67 (10.69)
C; 66.72 (66.89) H; 6.70 (6.58) N; 8.34 (8.57)
C; 64.20 (64.06) H; 7.70 (7.66) N; 8.56 (8.47)
C; 64.97 (64.79) H; 7.86 (7.74) N; 7.05 (7.09)
C; 65.35 (65.34) H; 7.21 (7.22) N; 9.94 (10.02)
C; 64.42 (64.45) H; 7.74 (7.78) N; 8.12 (8.07)
C; 59.78 (59.85) H; 7.12 (7.14) N; 11.05 (10.86)
C; 66.30 (66.24) H; 7.42 (7.37) N; 7.36 (7.38)
C; 67.08 (67.00) H; 7.20 (7.19) N; 9.10 (9.11)
C; 68.88 (68.63) H; 7.23 (7.10) N; 6.69 (6.77)
C; 63.45 (63.49) H; 7.10 (7.46) N; 8.97 (8.81)
C; 64.02 (64.16) H; 7.55 (7.52) N; 7.11 (6.97)
C; 64.12 (63.99) H; 6.94 (6.93) N; 9.97 (9.81)
C; 64.84 (64.71) H; 7.72 (7.74) N; 8.18 (8.04)
C; 59.28 (59.18) H; 6.84 (6.81) N; 11.00 (10.87)
C; 66.11 (65.82) H; 7.04 (7.18) N; 7.52 (7.58)
C; 66.52 (66.34) H; 6.84 (6.85) N; 9.23 (9.33)
C; 68.39 (68.17) H; 6.90 (6.82) N; 6.79 (6.90)
C; 58.59 (58.88) H; 6.79 (6.57) N; 11.39 (11.44)
C; 59.98 (60.11) H; 7.05 (7.06) N; 9.79 (9.90)
C; 61.31 (61.05) H; 6.50 (6.31) N; 12.14 (12.43)
C; 59.85 (60.10) H; 7.14 (6.99) N; 10.86 (10.96)
C; 59.58 (59.51) H; 6.82 (6.90) N; 11.05 (10.92)
C; 60.51 (60.85) H; 6.76 (6.67) N; 10.40 (10.14)
C; 61.83 (62.09) H; 6.74 (6.46) N; 11.48 (11.59)
C; 63.60 (63.42) H; 6.55 (6.42) N; 9.44 (9.44)
C; 65.16 (65.05) H; 7.01 (7.03) N; 7.79 (7.90)
C; 65.72 (65.62) H; 7.27 (7.29) N; 6.52 (6.57)
C; 66.22 (66.35) H; 6.46 (6.68) N; 9.42 (9.29)
C; 66.30 (66.20) H; 7.42 (7.36) N; 7.36 (7.46)
C; 66.11 (66.01) H; 7.04 (7.02) N; 7.52 (7.59)
C; 60.85 (61.07) H; 6.67 (6.54) N; 10.14 (10.04)
C; 67.69 (67.81) H; 6.65 (6.54) N; 8.40 (8.38)
C; 69.31 (69.51) H; 6.71 (6.65) N; 6.22 (6.43)
C; 66.01 (66.05) H; 6.79 (6.83) N; 9.62 (9.53)
C; 66.42 (66.39) H; 7.08 (6.86) N; 8.07 (8.09)
C; 65.09 (65.17) H; 6.64 (6.42) N; 10.42 (10.43)
C; 66.30 (66.46) H; 7.25 (7.27) N; 8.99 (9.07)
C; 66.52 (66.48) H; 6.84 (6.71) N; 9.23 (9.19)
C; 60.96 (60.77) H; 6.55 (6.53) N; 11.37 (11.20)
C; 67.69 (67.49) H; 6.65 (6.48) N; 8.40 (8.39)
C; 69.95 (69.81) H; 6.53 (6.49) N; 7.70 (7.73)
C; 67.99 (68.06) H; 6.85 (6.78) N; 7.05 (7.06)
C; 68.07 (68.06) H; 7.11 (6.88) N; 5.99 (6.06)
C; 68.81 (68.55) H; 6.45 (6.57) N; 8.39 (8.57)
C; 68.88 (68.87) H; 7.23 (7.21) N; 6.69 (6.80)
C; 68.76 (68.52) H; 6.88 (6.61) N; 6.82 (6.98)
C; 63.32 (63.13) H; 6.57 (6.42) N; 9.40 (9.44)
C; 69.31 (69.06) H; 6.71 (6.59) N; 6.22 (6.38)
C; 69.26 (69.47) H; 6.58 (6.43) N; 7.62 (7.84)
C; 67.81 (67.90) H; 7.18 (7.08) N; 5.65 (5.80)
C; 68.42 (68.35) H; 6.51 (6.25) N; 8.87 (8.88)
Ϫ24.4
Ϫ25.4
Ϫ32.7
Ϫ20.2
Ϫ33.8
Ϫ20.8
Ϫ32.3
Ϫ49.5
Ϫ16.4
Ϫ17.5
Ϫ22.8
Ϫ12.6
Ϫ28.7
Ϫ18.2
Ϫ24.2
Ϫ39.1
Ϫ18.5
Ϫ16.7
Ϫ18
129.0—130.0
129.0—130.0
97.0—98.0
Amorphous
Amorphous
136.0—138.0
139.0—140.0
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
87.0—88.0
163.0—165.0
139.0—140.0
147.0—149.0
113.0—114.0
98.0—100.0
Amorphous
Amorphous
110.0—113.0
143.0—144.0
123.0—125.0
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
134.0—136.0
Amorphous
Amorphous
Amorphous
Amorphous
101.0—103.0
140.0—141.0
118.0—119.0
147.0—148.0
124.0—126.0
92.0—94.0
Ϫ8.9
Ϫ46.4
Ϫ35
Ϫ25.9
Ϫ61.6
Ϫ24.5
Ϫ25
Ϫ35.2
Ϫ21.5
Ϫ70.8
Ϫ55.2
Ϫ45.7
Ϫ68.8
Ϫ42.6
Ϫ52.6
Ϫ53.5
Ϫ37.7
Ϫ28.8
Ϫ19.4
Ϫ15.6
Ϫ25.4
Ϫ39.1
Ϫ15.3
Ϫ18.5
Ϫ8.7
Ϫ29.5
Ϫ20.3
Ϫ17.7
Ϫ28
Ϫ45.1
Ϫ13.5
Ϫ21.7
Ϫ9.5
87.0—89.0
128.0—129.0
138.0—139.0
93.0—94.0
Ϫ23.3
Ϫ16.6
Ϫ15.3
Ϫ21.1
Ϫ41.6
Ϫ9.8
126.0—128.0
155.0—158.0
Amorphous
Amorphous
Amorphous
Amorphous
154.0—156.0
144.0—145.0
115.0—117.0
141.0—143.0
135.5—136.5
111.0—112.0
Amorphous
104.0—105.0
124.0—125.0
111.0—113.0
149.5—150.5
Ϫ10
Ϫ4.7
Ϫ26.7
Ϫ19.7
Ϫ16.7
Ϫ23.6
Ϫ40.7
Ϫ12.3
Ϫ13.9
Ϫ18.8
Ϫ24.9
Ϫ24.3

September 2001
Table 4. (Continued.)
A3–A2–A1
1143
mp (°C)
[a]D
Anal. Calcd (Found.)
Lys–Tyr–Ala
Leu–Tyr–Ala
Pro–Tyr–Ala
Arg–Tyr–Ala
Ser–Tyr–Ala
Trp–Tyr–Ala
Ala–Tyr–Glu
His–Tyr–Glu
Lys–Tyr–Glu
Leu–Tyr–Glu
Pro–Tyr–Glu
Arg–Tyr–Glu
Ser–Tyr–Glu
Trp–Tyr–Glu
Ala–Tyr–His
Glu–Tyr–His
Lys–Tyr–His
Leu–Tyr–His
Pro–Tyr–His
Arg–Tyr–His
Ser–Tyr–His
Trp–Tyr–His
Ala–Tyr–Lys
Glu–Tyr–Lys
His–Tyr–Lys
Leu–Tyr–Lys
Pro–Tyr–Lys
Arg–Tyr–Lys
Ser–Tyr–Lys
Trp–Tyr–Lys
Ala–Tyr–Leu
Glu–Tyr–Leu
His–Tyr–Leu
Lys–Tyr–Leu
Pro–Tyr–Leu
Arg–Tyr–Leu
Ser–Tyr–Leu
Trp–Tyr–Leu
Ala–Tyr–Pro
Glu–Tyr–Pro
His–Tyr–Pro
Lys–Tyr–Pro
Leu–Tyr–Pro
Arg–Tyr–Pro
Ser–Tyr–Pro
Trp–Tyr–Pro
Ala–Tyr–Arg
Glu–Tyr–Arg
His–Tyr–Arg
Lys–Tyr–Arg
Leu–Tyr–Arg
Pro–Tyr–Arg
Ser–Tyr–Arg
Trp–Tyr–Arg
Ala–Tyr–Ser
Glu–Tyr–Ser
His–Tyr–Ser
Lys–Tyr–Ser
Leu–Tyr–Ser
Pro–Tyr–Ser
Arg–Tyr–Ser
Trp–Tyr–Ser
Ala–Tyr–Trp
Glu–Tyr–Trp
His–Tyr–Trp
Lys–Tyr–Trp
Leu–Tyr–Trp
Pro–Tyr–Trp
Arg–Tyr–Trp
Ser–Tyr–Trp
152.0—155.0
135.0—137.0
Oil
Amorphous
97.0—99.0
162.0—164.0
99.0—101.0
88.0—90.0
145.0—147.0
155.0—156.0
106.0—107.0
105.0—106.0
128.0—129.0
122.0—123.0
96.0—97.0
123.0—124.0
160.0—163.0
118.0—119.0
Amorphous
Amorphous
94.0—95.0
Amorphous
189.0—190.0
136.0—137.0
98.0—99.0
157.0—158.0
112.0—113.0
164.0—165.0
162.0—163.0
145.0—149.0
121.0—122.0
120.0—121.0
145.0—146.0
160.0—161.0
Amorphous
Amorphous
105.0—107.0
150.0—151.0
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
82.0—84.0
Amorphous
99.0—101.0
Amorphous
140.0—142.0
120.0—121.0
129.0—131.0
158.0—159.0
149.0—150.0
Amorphous
Amorphous
150.0—151.0
Amorphous
125.0—126.0
192.0—193.0
148.0—150.0
Amorphous
Amorphous
Amorphous
Amorphous
Ϫ22.1
Ϫ34.4
Ϫ44.6
Ϫ16.2
Ϫ16
C; 67.99 (67.79) H; 6.85 (6.61) N; 7.05 (7.14)
C; 68.82 (68.74) H; 7.34 (7.15) N; 6.51 (6.67)
C; 68.17 (68.25) H; 6.91 (7.01) N; 6.63 (6.74)
C; 62.69 (62.41) H; 6.46 (6.37) N; 9.97 (10.11)
C; 69.37 (69.16) H; 6.67 (6.58) N; 5.92 (6.07)
C; 70.18 (70.08) H; 6.45 (6.31) N; 7.79 (7.90)
C; 67.81 (67.94) H; 7.18 (7.02) N; 5.65 (5.71)
C; 67.14 (67.32) H; 6.91 (6.93) N; 7.39 (7.43)
C; 68.07 (67.97) H; 7.11 (6.92) N; 5.99 (6.08)
C; 68.77 (68.53) H; 7.57 (7.49) N; 5.35 (5.40)
C; 68.64 (68.59) H; 7.20 (7.01) N; 5.46 (5.58)
C; 63.52 (63.43) H; 6.77 (6.70) N; 8.55 (8.62)
C; 69.24 (69.16) H; 7.00 (6.82) N; 4.94 (5.07)
C; 69.91 (69.83) H; 6.81 (6.79) N; 6.52 (6.63)
C; 68.42 (68.10) H; 6.51 (6.43) N; 8.87 (8.89)
C; 66.79 (66.94) H; 7.06 (6.76) N; 7.21 (7.53)
C; 67.93 (67.97) H; 6.62 (6.33) N; 8.49 (8.54)
C; 69.29 (69.02) H; 6.91 (6.67) N; 8.42 (8.55)
C; 69.18 (68.89) H; 6.55 (6.50) N; 8.58 (8.68)
C; 62.54 (62.56) H; 6.39 (6.04) N; 10.61 (10.80)
C; 69.35 (69.30) H; 6.44 (6.28) N; 7.78 (7.97)
C; 68.30 (68.34) H; 6.38 (6.29) N; 9.02 (9.16)
C; 67.99 (67.98) H; 6.85 (6.62) N; 7.05 (7.08)
C; 68.07 (67.95) H; 7.11 (6.81) N; 5.99 (6.06)
C; 67.32 (67.54) H; 6.66 (6.50) N; 8.41 (8.50)
C; 68.88 (68.83) H; 7.23 (7.15) N; 6.69 (6.75)
C; 68.76 (68.55) H; 6.88 (6.70) N; 6.82 (6.90)
C; 63.87 (63.65) H; 6.53 (6.40) N; 9.48 (9.55)
C; 69.31 (69.17) H; 6.71 (6.47) N; 6.22 (6.24)
C; 69.95 (69.70) H; 6.53 (6.61) N; 7.70 (7.77)
C; 68.82 (68.86) H; 7.34 (7.11) N; 6.51 (6.59)
C; 68.77 (68.66) H; 7.57 (7.44) N; 5.35 (5.59)
C; 68.55 (68.73) H; 6.95 (6.90) N; 8.33 (8.51)
C; 68.88 (68.87) H; 7.23 (7.05) N; 6.69 (6.85)
C; 69.72 (69.45) H; 7.35 (7.20) N; 6.25 (6.20)
C; 63.14 (63.32) H; 6.88 (6.77) N; 9.40 (9.60)
C; 70.28 (70.22) H; 7.10 (6.98) N; 5.59 (5.62)
C; 71.03 (71.01) H; 6.89 (6.87) N; 7.36 (7.47)
C; 68.17 (68.21) H; 6.91 (6.86) N; 6.63 (6.78)
C; 68.21 (68.31) H; 7.22 (7.13) N; 5.43 (5.63)
C; 68.43 (68.44) H; 6.60 (6.65) N; 8.49 (8.79)
C; 68.39 (68.26) H; 6.90 (6.73) N; 6.79 (6.97)
C; 69.72 (69.45) H; 7.35 (7.17) N; 6,25 (6.37)
C; 63.58 (63.30) H; 6.49 (6.43) N; 9.67 (9.77)
C; 69.34 (69.61) H; 6.77 (6.55) N; 5.64 (5.68)
C; 70.10 (70.24) H; 6.55 (6.55) N; 7.43 (7.54)
C; 62.03 (61.78) H; 6.51 (6.51) N; 9.86 (9.93)
C; 63.52 (63.36) H; 6.77 (6.71) N; 8.55 (8.63)
C; 63.63 (63.62) H; 6.31 (6.25) N; 10.79 (10.81)
C; 63.32 (63.48) H; 6.57 (6.45) N; 9.40 (9.45)
C; 63.78 (63.60) H; 6.83 (6.82) N; 9.50 (9.57)
C; 63.58 (63.27) H; 6.49 (6.60) N; 9.67 (9.84)
C; 64.54 (64.38) H; 6.37 (6.51) N; 8.85 (8.94)
C; 63.98 (64.22) H; 6.30 (6.14) N; 10.04 (9.97)
C; 69.37 (69.30) H; 6.67 (6.59) N; 5.92 (5.94)
C; 69.24 (69.06) H; 7.00 (7.11) N; 4.94 (4.95)
C; 69.70 (69.47) H; 6.41 (6.38) N; 7.82 (7.83)
C; 69.31 (69.17) H; 6.71 (6.72) N; 6.22 (6.22)
C; 70.28 (70.17) H; 7.10 (6.80) N; 5.59 (5.68)
C; 70.18 (70.09) H; 6.71 (6.83) N; 5.71 (5.73)
C; 64.29 (64.14) H; 6.39 (6.28) N; 8.82 (8.89)
C; 71.34 (71.14) H; 6.35 (6.31) N; 6.79 (6.84)
C; 69.31 (69.60) H; 6.51 (6.53) N; 7.70 (7.77)
C; 69.55 (69.55) H; 6.83 (6.74) N; 6.49 (6.55)
C; 69.64 (69.93) H; 6.29 (6.20) N; 9.19 (9.30)
C; 69.95 (69.85) H; 6.53 (6.48) N; 7.70 (7.68)
C; 70.86 (70.68) H; 6.90 (6.84) N; 7.35 (7.33)
C; 70.10 (69.86) H; 6.55 (6.52) N; 7.43 (7.47)
C; 63.40 (63.34) H; 6.34 (6.16) N; 9.95 (9.83)
C; 70.57 (70.89) H; 6.41 (6.30) N; 6.72 (6.80)
Ϫ32.3
Ϫ25.4
Ϫ17.1
Ϫ16.1
Ϫ24.2
Ϫ35.5
Ϫ10.6
Ϫ11.7
Ϫ22.7
Ϫ18.3
Ϫ12.5
Ϫ10.6
Ϫ16.2
Ϫ27.4
Ϫ7.5
Ϫ7.4
Ϫ17.1
Ϫ22.3
Ϫ17.4
Ϫ17.9
Ϫ23.9
Ϫ31.9
Ϫ10.7
Ϫ11.5
Ϫ21.4
Ϫ34.1
Ϫ24.8
Ϫ24.3
Ϫ23.9
Ϫ46
Ϫ17.9
Ϫ17.4
Ϫ34.9
Ϫ55
Ϫ43.5
Ϫ39.5
Ϫ38.6
Ϫ54.9
Ϫ30.4
Ϫ38
Ϫ46.3
Ϫ18.1
Ϫ12.1
Ϫ10.6
Ϫ12.1
Ϫ18
Ϫ25.3
Ϫ7.2
Ϫ16.9
Ϫ16.8
Ϫ14.2
Ϫ12.3
Ϫ10
Ϫ17.7
Ϫ27.4
Ϫ6.9
Ϫ20.1
Ϫ12.3
Ϫ7.2
Ϫ10
Ϫ6
Ϫ10.9
Ϫ27.1
Ϫ4.3
Ϫ4.2

1144
Vol. 49, No. 9
Table 5. 1H Chemical Shifts of Boc-Tyr(Bzl)–Pro–Glu(O-c-Hex)-OBzl
Kind of proton
Major
Minor (d)
Kind of proton
Major
Minor (d)
Tyr
NH
aH
bH
5.50
4.61
2.83, 2.98
7.16
6.89
4.48
1.88, 2.15
1.84, 1.98
3.19, 3.56
7.47
5.22
4.15
2.69, 2.77
6.97
6.85
3.45
1.06, 2.06
1.44, 1.59
3.42
Boc
Bzl
CH3
CH2
1.39
5.00
5.14, 5.18
1.38
5.00
5.00, 5.11
Ring 2,6
Ring 3,5
aH
Ring
1
2,6
3,5
4
7.26—7.41
c–Hex
4.70
4.73
Pro
Glu
1.1—1.8
1.15, 1.45
}
bH
gH
1.30, 1.64
dH
NH
8.28
aH
4.62
2.13, 2.00
2.35
4.55
2.11, 2.34
2.34
bH
gH
Solvent: CDCl3, major : minorϭ2 : 1 at 22 °C. 3Jϭ8.5,1) 4.9,2) 7.6,3) 8.3 Hz.4)
Table 6. 13C Chemical Shifts of Boc–Tyr(Bzl)–Pro–Glu(O-c-Hex)-OBzl
Kind of carbon
Major
Minor
Kind of carbon
Major
Minor
Tyr
Ca
Cb
53.42
38.30
54.25
37.64
Boc
Bzl
CH3
C
CϭO
CH2
Ring 1
28.30
79.50
157.68
28.30
80.16
157.91
Ring 1
Ring 2,6
Ring 3,5
Ring 4
Ca
128.55
130.55
114.76
155.16
59.96
27.66
25.05
47.28
51.83
27.27
30.39
171.00
171.52
171.98
128.58
130.38
115.22
155.75
60.58
30.49
21.89
46.50
52.21
25.63
31.16
171.10
171.64
171.89
69.84, 67.08
135.34, 137.06
128.24
69.80, 66.92
135.56, 136.80
128.18
128.34
Pro
Ring 2—6
128.45
128.53
72.84
31.47
23.63
25.26
{
{
Cb
Cg
c–Hex
1
72.74
31.50
23.63
25.30
Cd
2, 6
3, 5
4
Glu
Ca
Cb
Cg
CϭO
{
{
Solvent: CDCl3, major : minorϭ2 : 1 at 22 °C.
Conclusion
our automated apparatus in order to apply it to a wide variety
An improved MSA method was developed for automated of standard reaction procedures. However, developing new
synthesis of peptides, in which it was possible to avoid 1) chemical processes, or modifying established manual ones, is
corrosion of the apparatus, 2) formation of emulsions, and 3) also very important for us to increase the scope, efficiency
use of dichloromethane. By this method, we synthesized 216 and reliability of automated synthesis.
fragment tripeptide derivatives in excellent yield. We have
hitherto paid much attention to developing the hardware of

September 2001
1145
Table 7. 1H–NMR Spectrum Data of Tripeptide Derivatives (Boc-A3–A2–A1-OBzl)
A3–A2–A1
Solvent
Chemical shift (200 MHz) : d (ppm)
Lys–Pro–Leu
DMSO–d6
0.82 (3H, d, Jϭ6.2 Hz), 0.89 (3H, d, Jϭ6.2 Hz), 1.37 (9H, s), 1.47—1.89 (16H, m),
3.00 (2H, d, Jϭ3.4 Hz), 3.37—3.70 (2H, m), 4.01—4.39 (2H, m), 5.02 (2H, s), 5.01 (2H, d, Jϭ2.6 Hz),
6.90 (1H, d, Jϭ7.8 Hz), 7.25—7.45 (10H, m), 8.24 (1H, d, Jϭ7.8 Hz)
Lys–Pro–Ser
Tyr–Pro–Lys
Trp–Pro–Lys
Ser–Lys–Pro
Arg–Lys–Glu
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
1.26—2.21 (18H, m), 3.12 (2H, d, Jϭ5.4 Hz), 3.54—3.74 (4H, m), 4.41 (2H, d, Jϭ8.6 Hz),
4.50—4.88 (3H, m), 5.05 (2H, s), 5.14 (2H, d, Jϭ10.4 Hz), 5.21—5.48 (2H,m), 6.96 (1H, d, Jϭ8.4 Hz),
7.19—7.42 (15H, m)
1.25—1.49 (15H, m), 1.88—2.15 (2H, m), 2.68—2.98 (2H, m), 3.10—3.75 (3H, m),
4.34—4.68 (3H, m), 5.01 (2H, s), 5.05—5.15 (2H,m), 5.17 (2H, d, Jϭ4.2 Hz), 5.59 (1H, d, Jϭ8.4 Hz),
5.81 (1H, s), 6.75—7.10 (5H, m), 7.30—7.44 (16H, m)
1.23—1.51 (15H, m), 1.78—2.18 (4H, m), 2.95—3.65 (6H, m), 4.39—4.41 (1H, m),
4.49—4.61 (1H, m), 4.71—4.85 (1H, m), 5.05—5.25 (4H,m), 5.58—5.75 (1H, m),
6.89—7.20 (16H, m), 7.68 (1H, d, Jϭ4.2 Hz), 8.56 (1H, s)
1.43 (9H, s), 1.48—1.74 (6H, m), 1.88—2.28 (4H, m), 3.11 (2H, d, Jϭ5.4 Hz),
3.53—3.78 (4H, m), 3.92—3.98 (1H, m), 4.21—4.35 (1H, m), 4.50 (2H, s), 4.57 (1H, d, Jϭ4.6 Hz),
4.75 (1H, d, Jϭ4.6 Hz), 5.07 (2H, s), 5.10—5.45 (3H, m), 7.30—7.44 (15H, m)
1.23—1.56 (24H, m), 1.78—2.02 (6H, m), 2.25—2.40 (3H, m), 3.05—3.25 (4H, m),
4.28—4.41 (1H, m), 4.45—4.61 (1H, m), 4.62—4.78 (1H, m), 5.06 (2H, s), 5.08 (2H, d, Jϭ4.8 Hz),
5.21—5.32 (1H, m), 5.45—5.53 (1H, d, Jϭ4.8 Hz), 6.38 (2H, br s), 7.20 (2H, d, Jϭ6.2 Hz),
7.32 (12H, s), 7.75 (2H, d, Jϭ6.2 Hz)
Trp–Lys–Pro
Ala–Lys–His
Leu–Tyr–Pro
Glu–Tyr–Trp
Ser–Tyr–His
His–Tyr–Lys
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
1.23—1.56 (4H, m), 1.43 (9H, s), 1.84—2.23 (4H, m), 2.96—3.20 (3H, m), 3.36—3.62 (3H, m),
4.35—4.41 (1H, s), 4.43 (1H, d, Jϭ4.8 Hz), 4.50—4.66 (1H, m), 5.04 (2H, m),
5.11—5.18 (2H, d, Jϭ4.8 Hz), 5.12 (2H, s), 6.42 (1H, d, Jϭ4.8 Hz), 6.72—6.86 (1H, d, Jϭ6.2 Hz),
7.04—7.39 (13H, m), 7.57 (1H, d, Jϭ7.2 Hz), 9.12 (1H, s)
1.27 (3H, d, Jϭ4.6 Hz), 1.15—1.78 (6H,m), 1.42 (9H, s), 3.18 (2H, t, Jϭ4.4 Hz),
4.11 (1H, t, Jϭ4.8 Hz), 4.33 (1H, t, Jϭ4.8 Hz), 4.37 (2H, s), 4.90 (1H, q, Jϭ4.8 Hz),
5.08 (2H, s), 5.10 (2H, s), 5.02—5.30 (2H, m), 5.21 (2H, s), 6.78 (1H, s), 6.78 (1H, d, Jϭ4.2 Hz),
7.22—7.42 (16H, m)
0.90 (6H, d, Jϭ5.2 Hz), 1.43 (9H, s), 1.48—2.23 (8H, m), 2.80—3.21 (3H, m),
3.45—3.58 (1H, m), 4.48—4.58 (2H, m), 4.61—4.92 (2H, m), 5.02 (2H, s), 5.01—5.15 (1H, m),
5.20 (2H, s), 6.72 (1H, d, Jϭ4.8 Hz), 6.85 (2H, d, Jϭ7.8 Hz), 7.14 (2H, d, Jϭ7.8 Hz),
7.28—7.48 (10H, m), 6.78 (1H, d, Jϭ4.2 Hz), 7.22—7.42 (16H, m)
1.21—1.85 (4H, m), 1.42 (9H, s), 2.22—2.34 (2H, m), 2.94 (2H, d, Jϭ6.6 Hz),
3.33 (2H, d, Jϭ6.6 Hz), 3.92—4.08 (1H, m), 4.51—4.96 (3H, m), 5.00 (2H, s),
5.06 (2H, s), 6.39—6.43 (1H, m), 6.61 (1H, d, Jϭ8.4 Hz), 6.71 (1H, s), 6.82 (2H, d, Jϭ8.4 Hz),
7.06 (2H, d, Jϭ8.4 Hz), 7.28—7.46 (12H, m), 8.31 (1H, br s)
1.43 (9H, s), 2.98 (2H, d, Jϭ6.6 Hz), 3.08 (2H, d, Jϭ6.6 Hz), 3.61—3.78 (1H, m),
3.82—3.98 (1H, m), 4.21—4.40 (1H, m), 4.39 (2H, s), 4.55 (2H, s), 4.58—4.70 (1H, m),
4.82—4.95 (1H, m) 5.02 (2H, s), 5.15 (2H, s), 5.25 (2H, s), 5.39 (1H, d, Jϭ4.8 Hz),
6.77—7.04 (7H, m), 7.31—7.51 (21H, m)
1.15—1.85 (6H, m), 1.39 (9H, s), 2.80—3.20 (6H, m), 4.32—4.58 (3H, m), 4.47 (2H, s),
4.97 (2H, s), 5.08 (2H, s), 5.12 (2H, s), 5.19 (2H, brs), 5.29—5.41 (1H, m),
5.85—5.99 (1H, s), 6.35 (1H, d, Jϭ8.4 Hz), 6.55 (1H, m), 6.82 (2H, d, Jϭ8.6 Hz),
6.99 (2H, d, Jϭ8.6 Hz), 7.27—7.46 (23H, m)
Table 8. Improvement and Merits of the MSA Method
Industries Ltd., and the starting amino acid derivatives were commercially
available reagents from Peptide Institute Inc. Column chromatography was
carried out on Silica Gel 60 (70—230 mesh, ASTM, Merck). The starting
dipeptides (27 kinds) were synthesized in our large-scale automated synthe-
sis apparatus (FUTOSHI).11) Typical synthesis for one dipeptide, Boc-
Pro–Ala-OBzl, was as follows: In a 2 l three-necked flask, MSA (98 g) in
acetonitrile (600 ml) was added to Boc-Ala-OBzl (112 g, 400 mmol) in ace-
tonitrile (400 ml) at room temperature and the reaction mixture was stirred
for 1 h. After the reaction mixture was cooled to 0 °C, diisopropylethylamine
(78.6 g, 600 mmol) was added, and then Boc-Pro (86.1 g, 400 mmol), HOBt
(67.4 g, 440 mmol), and WSCD (68.4 g, 440 mmol) in acetonitrile (100 ml)–
DMF (200 ml) was added. After the reaction mixture was stirred for 16 h,
solvents were evaporated-off, and the residue was extracted from ethyl ac-
etate–5% sodium hydrogen carbonate, and washed with 0.2 N hydrochloric
acid and water. The organic layer was dried, and evaporated, and recrystal-
lized from diisopropylether affording 124.6 g (83%) of the expected product.
General Procedure of Peptide Bond Formation The procedure for
Improvement
Effect (Merit)
Avoid use of trifluoroacetic acid
(volatile, corrosive acid)
Easy handling and less damage
to the apparatus
Avoid use of dichloromethane
Friendly to the environment
Decrease of the number of extractions Increased reliability
and drying processes
(ca. 90%→ca. 99%)
Generation of the free amine and
activated ester at the same time
Higher yield (70.1→77.4%)
Easy set up and simpler process
Higher throughput
Experimental
Computer-controlled automated synthesis systems (TAFT, EASY, peptide bond formation between Boc-Leu and Boc-Pro–Lys(Z)-OBzl is de-
ASTRO)10) were used to synthesize oligopeptide derivatives in solution. Op- scribed as a typical example. Table 2 lists the subroutine sequence by which
tical rotation values (in MeOH) were measured with a Nihon Bunko DIP-
the apparatus was controlled. In the reaction flask1 (RF1), to a powder of
370 spectrometer. All melting points were taken with a Yanagimoto micro Boc-Pro–Lys(Z)-OBzl (1.70 g, 3.0 mmol) was added MSA in acetonitrile
melting point apparatus and are uncorrected. NMR spectra were measured (12 ml, 12 mmol) from reagent reservoir 2 (RR2) [RF1-ST-ON, RR2-RF1].
on a Varian Gemini-200 or a JOEL JNM-GX400 FT NMR spectrometer (in Then the reaction mixture was stirred at 40 °C for 1 h [RF1-LF-UP, REA1
CDCl3). Solvents were of special or first grade from Wako Pure Chemical (40c, 60 m)]. The solution was cooled to 0 °C, and ethyldiisopropylamine in

1146
Vol. 49, No. 9
acetonitrile (10 ml, 9 mmol) was added from RR3 to neutralize the excess
acid [RF1-T-ON (0c), RR3-RF1]. After the neutralization, the reaction mix-
ture was transferred to RF2 where Boc-Leu (0.75 g, 3 mmol) and HOBt
(505 mg, 3.3 mmol) were stored at 0 °C [RF1-T-OF, RF2-LF-UP, RF2-T-ON
(0c), RF2-ST-ON, RF1-RF2, RS6-RF1, RF1-MIX, RF1-RF2, RF1-ST-OF,
RF1-LF-DN]. Then water-soluble carbodiimide (1.2 g) in DMF (5 ml) was
added from RR4 [RR4-RF2, RF2-ST-OF] before the reaction mixture was
stirred at room temperature for 10 h [REA2 (25c, 600 m)]. After the reaction
was completed, the solvents were removed under vacuum for 1 h [CON2
(40c, 60 m)], and extracted with ethyl acetate–5% sodium hydrogen carbon-
ate, followed by the usual work-up [RF2-LF-DN, RS4-RF2, RS2-RF2, RF2-
MIX, BKEXT, RF2-ST-OF]. The collection flask containing the expected
tripeptide was taken from the apparatus, and chromatographed on silica gel
(eluent; hexane–AcOEt (2 : 1)) to give the expected product as an amor-
phous solid (1.72 g, 84%).
H., Lab. Rob. Autom., 11, 217—223 (1999).
2) a) Yoshida J., “Combinatorial Chemistery,” Kagakudojin, Japan, 1997,
pp. 146—162; b) Orita A., Yasui Y., Otera J., Organic Process Re-
search & Development, 4, 337—341 (2000).
3) Ferritto R., Seneci P., Drugs Future, 23, 643—654 (1998).
4) a) Yoshida J., Suga S., Suzuki S., Kinomura N., Yamashita A., Fuji-
wara K., J. Am. Chem. Soc., 121, 9546—9549 (1999); b) Yoshida J.,
Itami K., Mitsudo K., Suga S., Tetrahedron Lett., 40, 3403—3406
(1999).
5) Cork D. G., Kato S., Sugawara T., Lab. Rob. Autom., 7, 301—308
(1995).
6) The name of our medium-scale automated synthesizer, which is
equipped with a centrifugal separation funnel (Japanese Pat. Appl. No.
09-134771).
7) Sugawara T., Kobayashi K., Okamoto S., Kitada C., Fujino M., Chem.
Pharm. Bull., 43, 1272—1280 (1995).
Acknowledgements We wish to thank Drs. Masahiko Fujino and Tune-
hiko Fukuda for their helpful advice. We also express our thanks to Mrs.
Mika Murabayashi for measuring NMR spectra.
8) a) Yajima H., Ogawa H., Fujii N., Funakoshi S., Chem. Pharm. Bull.,
25, 740—747 (1977); b) Kai Y., Nakashita M., Suwa K., Sano A.,
Ikeda Y., Ueki Y., Ono K., “Peptide Chemistry 1988,” ed. by Ueki M.,
Protein Reserch Foundation, Osaka, 1989, pp. 91—96.
References and Notes
9) a) Kiso Y., Fujiwara Y., Kimura T., Nishitani A., Akaji K., Int. J. Pep-
tide & Protein Res., 40, 308—314 (1992); b) Kiso Y., Kimura T., Fuji-
wara Y., Shimokura M., Nishitani A., Chem. Pharm. Bull., 36, 5024—
5027 (1988); c) Fujii N., Funakoshi S., Otaka A., Morimoto H., Tama-
mura H., Carpino L. A., Yajima H., “Peptide Chemistry 1988,” ed. by
Ueki M., Protein Reserch Foundation, Osaka, 1989, pp. 147—152.
10) Sugawara T., Kato S., Okamoto S., J. Autom. Chem., 16, 33—42
(1994).
1) a) Cork D. G., Sugawara T., Lab. Rob. Autom., 8, 221—230 (1996); b)
Christensen J. W., Brown S. F., Healy M. L., Peterson H. H., “Combi-
natorial Chemistry and High Through-put Screening,” Coronado, CA,
Jan. 29—31 (1996); c) Dewitt S. H., Kiely J. S., Stankovic C. J.,
Schroeder M. C., Cody D. M. R., Pavia M. R., Proc. Natl. Acad. Sci.
U.S.A., 90, 6909—6913 (1993); d) Cargill J. F., Maiefski R. R., Lab.
Rob. Autom., 8, 139—148 (1996); e) Cheng S., Comer D. D., Williams
J. P., Myers P. L., Boger D. L., J. Am. Chem. Soc., 118, 2567—2572
(1996); f ) Cork D. G., Sugawara T., Lindsey J. S., Corkan L. A., Du
11) The name of our large-scale automated synthesizer, which is equipped
with a 2 l reaction flask, and a 2 l separation funnel.