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Vol. 49, No. 9
removed under reduced pressure [CON1 (50c, 300 min), RF-LF-DN], and mixture was rapidly stirred and transferred to SF where the standard extrac-
the residue was dissolved in ethyl acetate (450 ml), and 5% NaHCO3 aq. tion procedure was performed (washing with ethyl acetate, 40 ml, followed
(800 ml) [RS4-RF, RS2-RF]. The mixture was stirred at high speed before
by acid (0.2 N HCl, 40 ml), and water (40 ml)) [RF1-MIX, BKTETRANK,
being transferred to SF. The standard extraction procedure (washing with 0.2 RF1-ST-OF, ALARM]. The collected product was then removed manually
N HCl acid (500 ml), and water (500 ml)) [RF-MIX to RF-SF, ALARM] was from RF3, and the RFs and lines were washed and dried [SR1-DR to END].
then performed and the organic extract was transferred to the collection flask The excess solvent was evaporated and the crystals of Boc-L-Lys(Z)–D-
(CF) before being removed manually from the automated apparatus. Finally,
the RF and lines were washed and dried [SR1-DR to END]. Excess solvent
Ala–L-Tyr(Bzl)–D-Ala-OBzl were collected on an 11G3 glass filter, washed
twice with diisopropylether and dried to give 9.53 g (91.7%). 1H-NMR (200
was evaporated from the extract to give white crystals, which were filtered MHz, CDCl3): 1.11—1.14 (d, 3H), 1.17—1.20 (d, 3H), 1.23—1.61 (m,
(11G3 glass filter), washed twice with diisopropylether (500 ml) and dried to 15H), 2.84—3.16 (d-d, 2H), 3.13—3.15 (d, 2H), 4.00—4.08 (m, 1H), 4.21—
give 206 g (95%) of Boc-L-Lys(Z)–D-Ala-OBzl.
4.41 (t, 1H), 4.49—4.68 (m, 2H), 5.01 (s, 2H), 5.07 (s, 2H), 5.13 (d, 2H),
b) General Synthesis of Dipeptide Carboxylic Acid by Saponification 5.35 (d, 1H), 6.84—7.40 (m, 19H).
The procedure for saponification of Boc-L-Lys(Z)–D-Ala-OBzl is described
as a typical example. Table 2 lists the chosen subroutines, and the conditions
for the reaction (reaction time, temperature, and other information) were
input to the subroutine [START]. Boc-L-Lys(Z)–D-Ala-OBzl (108 g; 200
mmol), and methanol (600 ml) were put in the RF. Then from RR1, 1 M
sodium hydroxide aqueous solution 240 ml (240 mmol; 1.2 eq) was added at
e) General Procedure for the Peptide Bond Formation to Form Pen-
tapeptides The parallel synthesis of a sub-library of 56 pentapeptides, de-
scribed by the formula Boc-Z–L-Pro–D-Ala–Y–D-Ala-OBzl, where Z is ran-
domly selected from D-Leu, D-Ser(Bzl), D-Glu(O-cHex), D-Lys(Z), D-
Tyr(Bzl), D-Trp, D-Arg(Tos), D-His(Bom), and Y is randomly selected from
L-Leu, L-Ser(Bzl), L-Glu(O-cHex), L-Lys(Z), L-Trp, L-Arg(Tos), and L-
0 °C [RF-ST-ON, RF1-LF-UP, RF1-T-ON (0c), RR1-RF]. The solution was His(Bom), is shown in Fig. 4, as a typical example. First, one of the 7 start-
stirred at room temperature for 300 min. [RF1-T-OF, REA1 (25c, 300 m)],
then cooled to 0 °C, before 1 M hydrochloric acid 240 ml (240 mmol) was
ing tetrapeptides (Boc-L-Pro–D-Ala–Y–D-Ala-OBzl was weighed into each
of the reaction vessels (0.5 mmol; 300—350 mg), and the stock solution of 1
added from RR3. After stirring for 5 min [RF1-T-ON (0c), RR3-RF, RF1-T- M MSA in acetonitrile (2 ml for each reaction (2 mmol; 4 eq) was set in a dis-
OF] the excess solvent was removed [CON1 (40c, 60 min)], and the residue pense reservoir. A stock solution of DIEA in acetonitrile (1.5 ml per reaction
was maintained at 10 °C [RF1-T-ON (10c), MATU]. From SF, 5% NaHCO3 (1.5 mmol)), WSCD in DMF (0.63 ml per reaction (0.63 mmol), and Boc-Z
aq. (400 ml) was added to the residue under rapid stirring to separate the (0.55 mmol per reaction) plus HOBt (84 mg per reaction (0.55 mmol)) in
pure product as a salt from the by-products in the organic layer [SF-RF to DMF (2 ml per reaction) were set in the reagent reservoirs. Following the
SF-DR]. Then, after neutralization with 1 N hydrochloric acid (300 ml), the
procedure in Fig. 4, the robot delivered reagents to the reaction vessels auto-
expected dipeptide carboxylic acid was extracted from the aqueous layer matically, and after the reaction, each sample was analyzed by HPLC. The
with ethyl acetate (450 ml) followed by standard work-up [SR1-RF to SF- reaction mixture was extracted manually from ethyl acetate (10 ml) and 5%
CF, ALARM]. The CF was manually removed from the apparatus, and the NaHCO3 aq. (15 ml), followed by additional extraction (ethyl acetate (10
solvent removed to give Boc-L-Lys(Z)–D-Ala-OH, 110 g (90%) as the dicy-
clohexylamine salt.
c) Catalytic Hydrogenation of Boc-L-Glu(O-cHex)–D-Ala-OBzl A so-
lution of Boc-L-Glu(O-cHex)–D-Ala-OBzl (78.3 g, 160 mmol; 20 mmol for
ml)). The combined organic extract was analyzed by HPLC, and if the purity
of the product was over 80%, it was simply evaporated, whereas if the purity
was under 80%, it was chromatographed on disposable silica-gel tubes.
After evaporation and crystallization, the expected pentapeptides were ob-
each cycleϫ8 cycles) in methanol (320 ml) was set in the RR1. A solution of tained in about 20—80% yield (100—500 mg).
ammonium formate (31.9 g, 480 mmol; 60 mmol for each cycleϫ8 cycles) in
Acknowledgements We wish to thank Dr. Masahiko Fujino for his
helpful advice.
methanol (320 ml) was set in the RR2 and 10% Pd–C (1.5 g) was suspended
in methanol (20 ml) in the RF. For the first cycle, the dipeptide solution (40
ml, 20 mmol) was transferred to the RF from RR1, followed by the ammo-
nium formate solution (40 ml, 60 mmol) from RR2. The reaction mixture
was well mixed by shaking at room temperature for 2 h, and then the solu-
tion containing the deprotected benzyl ester was transferred from the RF
through a teflon filter to the CF. The RF was washed twice with methanol
(40 ml) and the washings were combined in the CF. The methanol solution in
CF was evaporated and then the catalyst in the RF was washed with 1 M
formic acid solution in methanol (80 ml), followed by methanol (80 ml). For
the next cycle, the reagents were transferred to the RF, and the same se-
quence of subroutines was run for an additional 7 cycles. During the runs,
1.5 g of 10% Pd–C was renewed after every 3 cycles. The extracted organic
solvent was dried and removed to give 46.3 g (71%) of Boc-L-Glu(O-cHex)–
D-Ala-OH as a oil.
References and Notes
1) Merrifield R. B., J. Am. Chem. Soc., 85, 2149—2154 (1963).
2) Mutter M., Bayer E., “The Peptides,” Vol. 2, ed. by Gross E., Meien-
hofer J., Academic Press, 1979, pp. 285—332.
3) Bayer E., Mutter M., Nature (London), 237, 512—513 (1972).
4) Erb E., Janda K. D., Brenner S., Proc. Natl. Acad. Sci. U.S.A., 91,
11422—11426 (1994).
5) a) Cork D. G., Sugawara T., Lab. Rob. Autom., 8, 221—230 (1996); b)
Sugawara T., Pharmacia, 31, 1159—1162 (1995).
6) Fukumoto S., Fukushi S., Terao S., Shiraishi M., J. Chem. Soc., Perkin
Trans. 1, 1996, 1021—1026.
7) Berta P. O., Marc C. P., Gemma J. F., Javier C. R., Jordi B. C., PCT Int.
Appl. (2000), WO 0071570, p. 38.
d) General Procedure of the Peptide Bond Formation The procedure
for the peptide bond formation between Boc-L-Lys(Z)–D-Ala-OH and Boc-L-
Tyr(Bzl)–D-Ala-OBzl is described as a typical example. Table 4 lists the
chosen subroutine program, and the conditions for the reaction (reaction
time, temperature, and other information) were input to the subroutine
[START]. Boc-L-Tyr(Bzl)–D-Ala-OBzl (6.39 g; 12.0 mmol) was put as a
powder in the RF1, and Boc-L-Lys(Z)–D-Ala-OH (5.42 g; 12.0 mmol) and
HOBT (2.02 g; 13.2 mmol) were dissolved in DMF (10 ml) in the RF2 [RF1-
ST-ON, RF2-ST-ON]. Then, from RR1, 2 M MSA in acetonitrile solution, 15
ml (30 mmol; 2.5 eq), was added at room temperature [RR1-RF1]. The solu-
tion was then stirred at 40 °C for 60 min [RF1-LF-UP, REA1 (40c, 60 m)].
After cooling the solution to 0 °C, 0.9 M DIEA in acetonitrile, 20 ml (18
mmol), was added from RR2, and stirred for 5 min [RF1-T-ON (0c), RR2-
RF1, MATU]. Then at 0 °C, the solution in RF2 was added to RF1 while
stirring was continued [RF2-RF1, RF6-RF2, RF2-MIX, RF2-RF1, RF2-ST-
OF]. After adding WSCD (2.05 g; 13.2 mmol) in acetonitrile (5 ml) from
RR3, the condensation reaction was carried out at 25 °C in RF1 for 10 h
[RR3-RF1, RF1-T-OF, REA1 (25c, 600 m)]. The excess solvent was re-
moved under reduced pressure [CON1 (40c, 60 min), RF1-LF-DN], and the
residue was first dissolved in THF (20 ml), followed by ethyl acetate (30 ml),
and finally 5% NaHCO3 aq. (40 ml) [RS5-RF1, RS4-RF1, RS2-RF1]. The
8) The name of our large scale automated synthesizer, which is equipped
with a 2 l reaction flask, and a 2 l separatory funnel (Japanese Pat.
Appl. No. 09-134771).
9) Cork D. G., Sugawara T., Lab. Rob. Autom., 7, 301—308 (1995).
10) 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; c) Kiso Y,
Kimura T., Fujiwara Y., Shimokura M., Nishitani A., Chem. Pharm.
Bull., 36, 5024—5027 (1988); d) Fujii N., Funakoshi S., Otaka A.,
Morimoto H., Tamamura H., Carpino L. A., Yajima H., “Peptide
Chemistry 1988,” ed. by Ueki M., Protein Reserch Foundation, Osaka,
1989, pp. 147—152.
11) Sugawara T., Kato S., Okamoto S., J. Autom. Chem., 16, 33—42
(1994).
12) Cork D. G., Kato S., Sugawara T. Linsey J. S., Corkan L. A., Du H.,
Lab. Rob. Autom., 11, 217—223 (1999).
13) Sugawara T., Cork D. G., “Combinatorial Chemistry,” ed. by Fenniri
H., Oxford University Press, New York, 2000, Chapter 13, pp. 377—
400.