Total enzymatic synthesis of the cholecystokinin octapeptide (CCK-8)

Article abstract of DOI:10.1002/hlca.200890128

The enzymatic synthesis of the cholecystokinin octapeptide (CCK-8) is reported. The target octapeptide CCK-8 is the minimum active sequence with the same biological activity as naturally occurring cholecystokinin and is a potential therapeutic agent in the control of gastrointestinal function as well as a drug candidate for the treatment of epilepsy and obesity. The protected CCK-8 was obtained by incubation of Bz-Arg-Asp(OEt)-Tyr-Met-OAl and Gly-Trp-Met-Asp(OMe)-Phe-NH₂ with immobilized α-chymotrypsin. The Bz-Arg group was used as an N-terminal protecting group in the synthesis of the tripeptide fragment. The protected CCK-8 was treated with trypsin to remove the Bz-Arg group successfully. Free or immobilized enzymes were used as catalysts. The effect of the acyl donor ester structure, the C(α) protecting group of the nucleophile, reaction media, enzyme, and the carrier of the enzymes on the outcome of the coupling reaction was studied.

Full text of DOI:10.1002/hlca.200890128

Helvetica Chimica Acta – Vol. 91 (2008)
983
Total Enzymatic Synthesis of the Cholecystokinin Octapeptide
(CCK-8)
by Rajendra Joshia), Liping Mengb), and Heiner Eckstein*a)
a) Institute of Organic Chemistry, Auf der Morgenstelle 18, D-72076 Tübingen
(phone: þ 49-7071-83261; e-mail: heiner.eckstein@uni-tuebingen.de)
b) Pharmacy School of Tongji Medical College, HUST, Wuhan, P. R. China
The enzymatic synthesis of the cholecystokinin octapeptide (CCK-8) is reported. The target
octapeptide CCK-8 is the minimum active sequence with the same biological activity as naturally
occurring cholecystokinin and is a potential therapeutic agent in the control of gastrointestinal function
as well as a drug candidate for the treatment of epilepsy and obesity. The protected CCK-8 was obtained
by incubation of Bz-Arg-Asp(OEt)-Tyr-Met-OAl and Gly-Trp-Met-Asp(OMe)-Phe-NH2 with immobi-
lized a-chymotrypsin. The Bz-Arg group was used as an N-terminal protecting group in the synthesis of
the tripeptide fragment. The protected CCK-8 was treated with trypsin to remove the Bz-Arg group
successfully. Free or immobilized enzymes were used as catalysts. The effect of the acyl donor ester
structure, the C(a) protecting group of the nucleophile, reaction media, enzyme, and the carrier of the
enzymes on the outcome of the coupling reaction was studied.
Introduction. – Cholecystokinin (CCK) is a polypeptide hormone of 33 amino acid
residues and is involved in the food absorption and digestion. It is also widely
distributed in the vertebrate central nervous system. The cholecystokinin C-terminal
octapeptide (CCK26–33, CCK-8) is the minimum active sequence with the same
biological activity as the naturally occurring cholecystokinin. It is a potential
therapeutic agent in the control of gastrointestinal function [1] and is a drug candidate
for the treatment of epilepsy [2], obesity [3], and inflammatory diseases [4]. In contrast
to the chemical synthesis, in enzymatic peptide synthesis, there occurs no racemization,
and all the coupling steps can be conducted under mild conditions with a minimum of
side-chain protection. The enzyme-catalyzed peptide synthesis is widely reported in the
literature [5 – 7]. Many efforts were devoted in the past to the enzymatic synthesis of
CCK-8 [8 – 10]. Unfortunately, there is no general common protocol for the enzymatic
peptide synthesis. To suppress the competing hydrolytic reaction, the undesired
oligomerization and product cleavage, each coupling step needs to be optimized. A
series of reaction parameters were investigated to find the best condition for each
peptide-bond formation such as acyl donor, nucleophile, reaction media, and enzyme.
In our previous work [11 – 13], we reported the enzymatic synthesis of fragments of
CCK-8. We hereby report the enzymatic synthesis of the protected (Bz-Arg-Asp(OEt)-
Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2) and N-terminal free octapeptide (Asp-
(OEt)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2). As it is difficult to estimate the
optimal fragments leading to a successful condensation, different fragments of CCK-8
were synthesized. In this communication, we report only the successful coupling steps.
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 91 (2008)
Bz-Arg-Asp(OEt)-Tyr-Met-OAl was synthesized by stepwise chain elongation
from the N- to the C-terminus. The protected pentapeptide obtained by fragment
condensation between Phac-Gly-Trp-Met-OAl and Asp(OMe)-Phe-NH2 was subjected
to the enzymatic cleavage of the Phac group by penicillin G amidase (PGA) to obtain
the N-terminal free pentapeptide Gly-Trp-Met-Asp(OMe)-Phe-NH2. The final frag-
ment coupling in the [3 þ (3 þ 2)] approach was successful by coupling Bz-Arg-
Asp(OEt)-Tyr-Met-OAl with Gly-Trp-Met-Asp(OMe)-Phe-NH2 (Scheme).
Scheme. Selected Strategy for the Synthesis of the N-Terminal Free Octapeptide CCK-8 (CHY:
a-chymotrypsin)
One of the objectives was to use the N-terminal protecting groups, which are
cleaved by enzymes under mild conditions. In the present work, phenylacetyl (Phac)
and benzoyl-arginine (Bz-Arg) were used as enzymatically labile N-terminal protecting
groups. We have found earlier that the cleavability of the Phac group depends on the
peptide sequence, and, in special cases, it cannot be cleaved at all. Therefore, Bz-Arg
was used as an alternative N-terminal protecting group as suggested by Glass [14]. The
Bz-Arg group was introduced and removed easily with trypsin. Trypsin is highly specific
towards basic amino acids in the P1 position, and, because there is no basic amino acid
in the target sequence, the use of Bz-Arg as a protecting group is possible. The
enzymatic removal of the N-terminal protecting group from protected CCK-8 is not yet
reported. Fite et al. [15] described the synthesis of the Phac-protected CCK-8.
However, the cleavage of the Phac group is not reported. Other authors [8 – 10] used
chemically cleavable N-terminal protecting groups.

Helvetica Chimica Acta – Vol. 91 (2008)
985
In this work, the reactions were catalyzed by immobilized enzymes as far as
possible. For each reaction, the enzymatic preparation leading to the maximum product
yield was selected. Substrate concentrations and acyl donor/nucleophile ratios were
chosen to obtain a good product yield and to minimize the oligomerization.
The selection of the nucleophile ester was made taking into account its reactivity as
well as the possibility that the product of the reaction would be used as the acyl donor
for the next step. For instance, the methionine allyl ester was chosen as a nucleophile to
prepare the allyl ester at the C-terminal end of the peptide. The peptide allyl ester was
then used directly as an acyl donor without any chemical modification in the following
fragment condensation step. The use of allyl esters in fragment condensation is
reported in [15].
Results and Discussion. – Synthesis of Bz-Arg-Asp(OEt)-Tyr-Met-OAl. The
protecting group Bz-Arg was introduced easily to Asp(OEt)-OEt according to the
method described elsewhere [16]. The reaction was catalyzed by trypsin/Eupergit C.
Enzymatic introduction and cleavage of protecting groups have advantages over
chemical methods due to their selectivity, specificity, and mild operation conditions. To
shift the equilibrium of the enzymatically catalyzed synthesis to the product side, an
excess of one reactant and the highest possible concentration is needed. In the stepwise
elongation from Bz-Arg-OEt · HCl to Bz-Arg-Asp(OEt)-Tyr-Met-OAl, easily avail-
able nucleophiles (amino acid esters) were used in excess. The reaction between Bz-
Arg-OEt · HCl and Asp(OEt)-OEt · HCl under solvent-free condition similar to the
method described by Cerovsky [17] to yield Bz-Arg-Asp(OEt)-OEt was kinetically
controlled. After a reaction time of 4 h, the yield of isolated Bz-Arg-Asp(OEt)-OEt
was 71.5%. If the reaction was not terminated at its optimal point, side reactions were
observed. Two by-products were due to the esterase activity of trypsin, which
hydrolyzed the esters of both starting material (Bz-Arg-OEt · HCl) and product (Bz-
Arg-Asp(OEt)-OEt) giving rise to Bz-Arg-OH and Bz-Arg-Asp(OEt)-OH, respec-
tively. Because of the proteolytic action of trypsin/Eupergit C, Bz-Arg-OH has also
resulted from cleavage of the product Bz-Arg-Asp(OEt)-OEt. Both by-products could
be kept below 10% by monitoring the kinetic of the reaction with HPLC. The
immobilized trypsin could be used at least three times. When the reaction was carried
out in Tris · HCl buffer (0.1m, pH 8.0), the only products were Bz-Arg-OH and Bz-Arg-
Asp(OEt)-OH. For the extension of this sequence to Bz-Arg-Asp(OEt)-Tyr-OH, two
different acyl donors were chosen in solvent-free conditions in the presence of papain.
The first acyl donor, Bz-Arg-Asp(OEt)-OH did not react with Tyr-OMe · HCl, but the
diester Bz-Arg-Asp(OEt)-OEt reacted with Tyr-OMe · HCl to give the product in
30.5% yield. This is generally the case in peptide synthesis where peptide esters react
better than peptides with a free carboxylic acid function. From the kinetics of papain-
catalyzed peptide synthesis, esterified acyl donors are preferable than acyl donors with
a free carboxy group [18]. When the molar ratio of Bz-Arg-Asp(OEt)-OEt and Tyr-
OMe · HCl was 1:4 and 1:1.5, the yield of Bz-Arg-Asp(OEt)-(Tyr)n-OH (n ¼ 2, 3)
determined by HPLC was 10and 5%, respectively. The sequence was further extended
to Bz-Arg-Asp(OEt)-Tyr-Met-OAl by reacting Bz-Arg-Asp(OEt)-Tyr-OH and Met-
OAl · Tos in MeCN containing 3.75% Tris · HCl buffer (0.1m, pH 8.1). Kinetically
controlled peptide synthesis with a free carboxylic acid is not reported often but similar

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Helvetica Chimica Acta – Vol. 91 (2008)
cases were observed many times in our previous work. A hydrolyzed product (Bz-Arg-
Asp(OEt)-Tyr-Met-OH) was also formed, but its yield was kept below 5% by
optimizing the conditions. In this reaction, the amount of buffer played a crucial role to
direct the reaction either towards the target product or towards the hydrolyzed product.
When the reaction was conducted in Tris · HCl buffer (0.1m, pH 8.1) containing 10%
MeCN, only Bz-Arg-Asp(OEt)-Tyr-Met-OH was formed. However, when the concen-
tration of MeCN was increased to 96.2%, Bz-Arg-Asp(OEt)-Tyr-Met-OAl was isolated
in a good yield (63.9%). For the final fragment coupling, Bz-Arg-Asp(OEt)-Tyr-Met-
OAl was used.
Synthesis of CCK-5 (Gly-Trp-Met-Asp(OMe)-Phe-NH2). Capellas et al. [19]
reported the reactivity difference among carboxamidomethyl (OCam), benzyl (OBzl)
and methyl (OMe) esters of Z-Gly-Trp during the coupling reaction with Met-OEt in
organic media. They reported that the methyl ester was least reactive and carbox-
amidomethyl ester was the most reactive. We also observed that Phac-Gly-OMe could
not be coupled with Trp-OMe catalyzed by immobilized papain. As expected, the
OCam ester could be used and the dipeptide was isolated in 90% yield. For the
extension of this sequence from Phac-Gly-Trp-OMe to Phac-Gly-Trp-Met-OAl, two
conditions were studied. When the reaction between Phac-Gly-Trp-OMe and Met-
OAl · Tos was carried out in AcOEt containing 0.2% Tris · HCl buffer (0.1m, pH 8.2) in
presence of a-chymotrypsin, only 3% of the product (Phac-Gly-Trp-Met-OAl) but
20% of the by-product (Phac-Gly-Trp-OH) was obtained. However under solvent-free
condition, the condensation reaction was successful. Basic, neutral, and acidic solvent-
free systems were created by using the salts KHCO3/Na2CO3 · 10 H2O, Na2SO4 · 10 H2O,
and KHSO4/Na2SO4 · 10 H2O, respectively. The highest yield was 60.1% after 4 h by
using the basic salt system.
The synthesis of another fragment, Asp(OMe)-Phe-NH2, was carried out as
reported elsewhere [11]. In an earlier investigation of our group [11], a tripeptide
OCam ester (Phac-Gly-Trp-Met-OCam) was condensed with the dipeptide amide
(Asp(OMe)-Phe-NH2). For the synthesis of the tripeptide fragment, Phac-Gly-Trp-
OMe was coupled with Met-OEt · HCl. The conversion of the ethyl ester to the OCam
ester needed three steps. Here, we report the successful application of the allyl ester.
The tripeptide allyl ester could be obtained by using Met-OAl instead of Met-OEt. The
pentapeptide Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2 was obtained by direct fragment
condensation of the tripeptide allyl ester Phac-Gly-Trp-Met-OAl and the dipeptide
amide Asp(OMe)-Phe-NH2. The condensation of Phac-Gly-Trp-Met-OAl with Asp-
(OMe)-Phe-NH2 · HCl was investigated in different reaction systems (Table). No
reaction took place under the solvent-free conditions and in MeCN. However, the
coupling was observed in AcOEt containing a small amount of buffer and Et3N. The
yield of Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2 was 24.3% (HPLC) when the reaction
was carried out in AcOEt containing 0.35% Tris · HCl buffer. When the Tris · HCl
buffer was replaced by borax buffer containing Et3N, the yield increased to 34.2%. The
best result (66.7%) was obtained when a buffer of Tris, borax, and Et3N was added.
The selective deprotection of Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2 was con-
ducted with immobilized PGA. Unfortunately, the yield of the reaction was low.
Synthesis of Bz-Arg-Protected CCK-8 [3 þ (3 þ 2)]. The crucial fragment con-
densation between Bz-Arg-Asp(OEt)-Tyr-Met-OAl and Gly-Trp-Met-Asp(OMe)-

Helvetica Chimica Acta – Vol. 91 (2008)
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Table. Isolated Amount (HPLC) of Product (Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2), By-product
(Phac-Gly-Trp-Met-OH), and Acyl Donor (Phac-Gly-Trp-Met-OAl). Acyl donor and nucleophile were
mixed in different reaction systems. a-Chymotrypsin/Celite-545 was used to catalyze the reaction.
Reaction System (Conditions)
Reactants
Time [h] Conversion [%]a)
Product By-
Acyl donor Nucleophile
Acyl
[mmol]
[mmol]
product donor
Solvent free
0.1
0.2
72
–
15.3
42.9
(0.2 mmol KHCO3,
0.04 mmol Na2CO3 · 10 H2O)
MeCN Systemb)
(2 ml MeCN, 10 ml 0 .1m Tris · HCl, pH 8.1)
AcOEt System
0.1
0.05
0.1
0.2
0.1
0.2
0.5
72
96
72
95
–
–
82.9
36.8
3.9
–
24.3
34.2
66.7
12.0
9.5
2.0
(2 ml AcOEt, 7 ml 0 .1m Tris · HCl, pH 8.1)
AcOEt Systemb)
(2 ml AcOEt, 8 ml 0 .1m Borax, pH 8.2)
AcOEt Systemc)
0.2
(6 ml AcOEt, 240 ml 0 .1m Borax,
pH 8.2, 400 ml 0 .1m Tris · HCl, pH 8.1)
a) Determined by HPLC. b) 10 ml of Et3N were added. c) 20 ml of Et3N were added.
Phe-NH2 with a-chymotrypsin/Eupergit C gave a protected CCK-8. Unexpectedly, free
a-chymotrypsin did not catalyze the reaction. However, when the enzyme was
immobilized on Eupergit C or Celite-545, 17 and 5% yield was obtained, respectively.
The main cause of the low yield was due to the hydrolysis of the allyl ester of Bz-Arg-
Asp(OEt)-Tyr-Met-OAl to Bz-Arg-Asp(OEt)-Tyr-Met-OH. It was tried to control the
hydrolytic action of the enzyme by lowering the H2O content in the system; however,
the yield could not be improved. Unlike in other coupling steps reported here, the acyl
donor in this reaction was added in double molar ratio to the nucleophile because of
much easier preparation of Bz-Arg-Asp(OEt)-Tyr-Met-OAl than Gly-Trp-Met-
Asp(OMe)-Phe-NH2.
Cleavage of Bz-Arg from Protected CCK-8. The action of trypsin is restricted to the
linkage of the carboxy group of arginine or other basic amino acids. As expected,
trypsin cleaved the Arg-Asp bond with the formation of Bz-Arg-OH and the
deprotected peptide. But several other peptide bonds were also attacked, especially
after longer reaction times. However, applying ꢁtosyl-amido-2-phenylethyl chloro-
methyl ketoneꢂ (TPCK)-treated trypsin resulted in highly selective cleavage and
relatively pure Asp(OEt)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2 was obtained as
evidenced by ESI-MS (Fig. 1). Fig. 2 shows the ESI-MS of the target octapeptide after
purification. At pH 7, there was no cleavage at all but at a pH of 8.5, the cleavage
underwent smoothly. In contrast to the Phac group, the Bz-Arg protecting group
proved to be useful in longer peptide sequences provided they are free of basic amino
acids.
Conclusions. – A complete enzymatic synthetic route for the N-terminal protected
and deprotected CCK-8 has been developed. The cleavability of the enzymatically

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Fig. 1. ESI-MS of the reaction mixture of the aqueous suspension of protected CCK-8 and TPCK-treated
trypsin at pH 8.5
Fig. 2. ESI-MS of Asp(OEt)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2 (CCK-8)
labile Phac group depends on the peptide sequence, and sometimes it cannot be cleaved
at all. It could be demonstrated that the Bz-Arg group is superior to other reported N-
terminal protecting groups in CCK-8 syntheses, as it is cleaved easily with trypsin.
Additionally, it could be shown that the fully enzymatic peptide synthesis even of
longer peptides is possible if all available condensation systems are applied. Especially
in some cases, the solvent-free conditions proved to be very useful. The enzymatic
technology, ꢁGreen Chemistryꢂ, is a versatile alternative to the chemical peptide
synthesis.
Experimental Part
Materials. Papain (EC 3.4.22.2) from Carica papaya (H2O-soluble, 30000 USP-U/mg using casein as
substrate) and a-chymotrypsin (EC 3.4.21.1) from bovine pancreas (Type II, 3 ꢀ crystallized, lyophilized
powder, 350U/mg using N-acetyl-l-tyrosine ethyl ester (ATEE) as substrate) were obtained from
Merck. Thermolysin (EC 3.4.24.2) from Bacillus thermoproteolyticus rokko (Protease X, lyophilized
powder containing calcium and sodium buffer salts, 50U/mg protein, casein assay) was from Sigma.
Trypsin (EC 3.4.21.4) from porcine pancreas (crystallized, lyophilized, 40U/mg using N-benzoyl-l-
arginine ethyl ester (BAEE) as substrate) was obtained from Merck. ꢁTosyl-amido-2-phenylethyl
chloromethyl ketoneꢂ (TPCK)-treated trypsin (EC 3.4.21.4) from bovine pancreas (10138 U/mg), Celite-

Helvetica Chimica Acta – Vol. 91 (2008)
989
545 (particle size 20– 45 mm) and penicillin G amidase (PGA) immobilized on Eupergit C (109 U/g)
were obtained from Fluka. Nucleosil C18 (5 mm) and Polygosil C18 (50– 60 mm) were from Macherey-
Nagel. a-Chymotrypsin was adsorbed on Celite-545 according to Basso et al. [20]. a-Chymotrypsin was
immobilized on Eupergit C as mentioned below. Eupergit C (10g) washed with KH 2PO4 buffer (1m,
pH 8.0) was added to the soln. of a-chymotrypsin (600 mg) in KH2PO4 buffer (1m, 100 ml, pH 8.0). After
rotating for 3 d, NaCl (1m, 100 ml) was added to the suspension. The solvent was sucked off and the a-
chymotrypsin/Eupergit C was washed with KH2PO4 buffer (0.05m, pH 8.0) and stored at ꢁ 208. Trypsin
was immobilized on Eupergit C as reported elsewhere [21]. The vinyl acetate resin was epoxidized
according to Burg et al. [22]. Papain was immobilized on VA-Epoxy as reported elsewhere [23].
Methionine allyl ester tosylate (Met-OAl · Tos) was purchased from Fluka. All l-amino acids were gifts
from Degussa. Phac-Gly-OCam was synthesized according to Martinez et al. [24][25]. Bz-Arg-OH was
synthesized according to the standard procedure [26][27]. The amino acid derivatives were prepared by
standard procedures in our laboratory. All other chemicals and solvents used were of anal. grade.
Anal. HPLC. The HPLC system consisted of Gilson pumps 305 and 307, the Gilson control software
712 and a Merck LaChrom-7400 detector set to 260nm. The anal. columns (100 ꢀ 2 mm) were filled with
Nucleosil C18 (5 mm) and the flow rate was adjusted to 0.3 ml/min.
The MeOH solvent system consisted of 0.05m AcONH4 (pH 6.5) (A) and 80% MeOH/H2O
containing 0.05m AcONH4 (B). The gradient elution systems are listed below.
HPLC System i: Gradient elution from 40to 55% B over 24 min: 0– 3.60min, 40% B; 3.60 –
7.50min, 40– 55% B; 7.50– 16.50min, 55% B; 16.50– 17.20min, 55 – 40% B; 17.20 – 24.00 min, 40% B.
HPLC System ii: Gradient elution from 40to 85% B over 19.50min: 0– 5.00min, 40%
B; 5.00 –
8.00 min, 40 – 85% B; 8.00 – 12.00 min, 85% B; 12.00 – 13.00 min, 85 – 35% B; 13.00 – 16.00 min, 35%
B; 16.00 – 17.00 min, 35 – 40% B; 17.00 – 19.50 min, 40% B.
HPLC System iii: Gradient elution from 55 to 85% B over 22 min: 0– 4.00min, 55% B; 4.0 0 –
7.00 min, 55 – 85% B; 7.00 – 11.50 min, 85% B; 11.50– 12.50min, 85 – 50% B; 12.50– 13.51 min, 50% B;
13.51 – 14.50min, 50– 55% B; 14.50– 22.00min, 55% B.
The MeCN solvent system consisted of 0.1% aq. TFA (A) and 80% MeCN/H2O containing 0.1%
TFA (B). The used gradient systems are listed below.
HPLC System iv: Gradient elution from 35 to 60% B over 19.50min: 0– 4.30min, 35% B; 4.30 –
5.50min, 35 – 60% B; 5.50 – 13.00 min, 60% B; 13.00 – 14.20 min, 60 – 35% B; 14.20– 19.50min, 35% B.
HPLC System v: Gradient elution from 40to 85% B over 19.50 min: 0 – 5.00 min, 40% B; 5.0 0 –
8.00 min, 40 – 85% B; 8.00 – 12.00 min, 85% B; 12.00 – 13.00 min, 85 – 35% B; 13.00 – 16.00 min, 35%
B; 16.00 – 17.00 min, 35 – 40% B; 17.00 – 19.50 min, 40% B.
HPLC System vi: Gradient elution from 50to 85% B over 19.50min: 0– 5.00min, 50%
B; 5.0 0 –
8.00 min, 50 – 85% B; 8.00 – 12.00 min, 85% B; 12.00 – 13.00 min, 85 – 45% B; 13.00 – 16.00 min, 45%
B; 16.00 – 17.00 min, 45 – 50% B; 17.00 – 19.50 min, 50% B.
Prep. HPLC. The HPLC system was the same as in the anal. HPLC. The columns (250 ꢀ 8 mm) were
filled with Nucleosil C18 (7 mm), and the flow rate was set to 4 ml/min. The solvent system consisted of
0.1% aq. TFA (A) and 80% MeCN/H2O containing 0.1% TFA (B).
Prep. Chromatography (MPLC). The samples were loaded to the column with a peristaltic pump P-1
(Pharmacia Fine Chemicals), and the elution was achieved with a ProMinent electronic E-0803 pump.
The UV absorption of the eluent was monitored with a ISCO Model UA-5 detector set to 254 nm, and
fractions of ca. 20ml were collected with a ISCO-328 fraction collector. The column (30 ꢀ 4 cm) was
filled with Polygosil C18 (50– 60 mm), and the flow rate was set to 5 ml/min.
The solvent system consisted of aq. 0.05m AcONH4 (pH 6.5) (A) and MeOH containing 25 ml 2m
AcONH4 per 1000 ml (B). The peptides were eluted with a step gradient, beginning with a MeOH
concentration of the sample up to 80% B according to the elution of the products.
Peptide Syntheses. Bz-Arg-Asp(OEt)-OEt. The synthesis of Bz-Arg-Asp(OEt)-OEt was carried out
under solvent-free conditions. Bz-Arg-OEt · HCl (2.1 g, 6 mmol), Asp(OEt)-OEt · HCl (2.7 g, 12 mmol),
Na2CO3 · 10 H 2O (0.8 g, 2.8 mmol), Na2CO3 (0.8 g, 7.6 mmol), and grinded NaOH (0.34 g, 8.4 mmol)
were mixed thoroughly with a glass rod. Trypsin/Eupergit C (4 g) was added, and the mixture was stirred
manually every 20min. The reaction was monitored with HPLC system ii. When the reaction was
complete, the mixture was diluted with 15 ml of 80% EtOH containing 2 ml of AcOH, and the pH was

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Helvetica Chimica Acta – Vol. 91 (2008)
adjusted to 7. This suspension was then transferred to a sintered glass frit and washed with 80% EtOH
until the product was extracted completely from the solid residue. The filtrate was evaporated to dryness
in vacuo. The remaining residue was dissolved in 40% aq. MeOH and separated by MPLC. The pooled
fractions of Bz-Arg-Asp(OEt)-OEt were lyophilized twice yielding a white powder (1.925 g, 71.5%, tR
11.83 min in HPLC system ii). The product was verified by comparing with the reference sample of our
laboratory.
Bz-Arg-Asp(OEt)-Tyr-OH. Bz-Arg-Asp(OEt)-OEt (450mg, 1 mmol), Tyr-OMe · HCl (350mg,
1.5 mmol), KHCO3 (1.0g, 10mmol), Na 2SO4 · 10 H2O (75 mg, 0.25 mmol), EDTA (0.1m, 50 ml), and 2-
sulfanylethanol (60 ml) were mixed thoroughly. Free papain (80mg) was added, and the mixture was
stirred manually every 20min. The reaction was monitored with HPLC system i. When the reaction was
complete, the mixture was diluted with 20ml of H 2O, transferred into a sintered glass frit, and washed
with H2O until the filtrate was neutral. The remaining filter cake was washed with pure MeOH, and the
filtrate was collected. The filtrate was evaporated to dryness in vacuo. The obtained residue was dissolved
in 35% aq. MeOH and separated by MPLC. The pooled fractions of Bz-Arg-Asp(OEt)-Tyr-OH were
lyophilized twice yielding a white powder (179 mg, 30.5%). tR 7.0min (HPLC system i). FAB-MS
(C28H36N6O8; calc. 584.3): 585.2 ([M þ H]þ), 607.2 ([M þ Na]þ).
The by-products were isolated and characterized by FAB-MS as Bz-Arg-Asp(OEt)-Tyr-Tyr-OH
(13 mg, 1.7%, tR 9.0min in HPLC system i; FAB-MS (C37H45N7O10; calc. 747.3): 748.2 ([M þ H]þ), 770.3
([M þ Na]þ)) and Bz-Arg-Asp(OEt)-Tyr-Tyr-Tyr-OH (7 mg, 0.7%). tR 11.0min (HPLC system i). FAB-
MS (C46H54N8O12; calc. 910.4): 911.5 ([M þ H]þ), 933.7 ([M þ Na]þ).
Bz-Arg-Asp(OEt)-Tyr-Met-OAl. Bz-Arg-Asp(OEt)-Tyr-OH (180mg, 0.3 mmol) and Met-OAl · Tos
(272 mg, 0.75 mmol) were dissolved in MeCN (8 ml) containing Tris · HCl buffer (0.1m, 30 0ml, pH 8.1).
To this soln., a-chymotrypsin/Celite-545 (500 mg) was added, and the mixture was stirred. The reaction
was monitored with HPLC system iv. After 47 h, the reaction was stopped by adding a few drops of
AcOH, and the mixture was filtered to remove a-chymotrypsin/Celite-545. The filter cake was washed
with pure MeOH, and the filtrate was collected and evaporated to dryness in vacuo. The residue was
dissolved in 45% aq. MeOH and separated by MPLC. The pooled fractions were lyophilized yielding a
white powder of Bz-Arg-Asp(OEt)-Tyr-Met-OAl (290mg, 63.9%). tR 10.09 min (HPLC system iv).
FAB-MS (C36H49N7O9S; calc. 755.3): 756.2 ([M þ H]þ), 778.2 ([M þ Na]þ).
Phac-Gly-Trp-OMe. This dipeptide was synthesized in a yield of 90% according to the method
described elsewhere [11].
Phac-Gly-Trp-Met-OAl. Phac-Gly-Trp-OMe (158 mg, 0.4 mmol), Met-OAl · Tos (200 mg,
0.55 mmol), KHCO3 (40mg, 0.4 mmol), and Na 2CO3 · 10 H2O (35 mg, 0.12 mmol) were mixed
thoroughly. a-Chymotrypsin/Celite-545 (150mg) was added and stirred manually every 20min. The
reaction was monitored with HPLC system v. The mixture was then washed with H2O until neutrality to
remove the salts. The filter cake was suspended in 100 ml of warm 80% EtOH, sonicated to extract the
tripeptide, and filtered. The solvent was evaporated to dryness in vacuo. The tripeptide ester Phac-Gly-
Trp-Met-OAl was obtained by recrystallization with EtOH as a white solid (134 mg, 60.1%). M.p. 164 –
1678. tR 11.63 min (HPLC system v). FAB-MS (C29H34N4O5S; calc. 550.2): 551.2 ([M þ H]þ), 573.1
([M þ Na]þ).
Z-Asp(OMe)-Phe-NH2. The synthesis of this dipeptide was performed according to the procedure
reported elsewhere [11]. However, the workup was different. The precipitated white product Z-
Asp(OMe)-Phe-NH2 was isolated by filtration, and washed with 5% cold citric acid (3 ꢀ 100 ml) and cold
H2O (3 ꢀ 100 ml) successively. Finally, the product was air-dried: 5.87 g (96.8%). M.p. 180 – 1838. tR
11.57 min (HPLC system iii).
Asp(OMe)-Phe-NH2. Z-Asp(OMe)-Phe-NH2 (4 g, 9.3 mmol) was completely dissolved in warm
MeOH (700 ml) followed by addition of HCl (6m, 1.7 ml) and then of 10% Pd/C (200 mg) catalyst. The
mixture was hydrogenated in two portions in a 500-ml shaked-bottle with H2. The reaction was
monitored with HPLC system iii. The reaction was complete within 20min. The hydrogenated portions
were combined, filtered, and the filtrate was concentrated in vacuo to yield Asp(OMe)-Phe-NH2 · HCl as
a white solid (2.97 g, 96.4%). M.p. 155 – 1588. tR 2.10min (HPLC system iii).
Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2. Phac-Gly-Trp-Met-OAl (110mg, 0.2 mmol) and Asp-
(OMe)Phe-NH2 · HCl (165 mg, 0.5 mmol) were dissolved in AcOEt (6 ml) containing borax buffer

Helvetica Chimica Acta – Vol. 91 (2008)
991
(0.1m, 240 ml, pH 8.2), Tris · HCl buffer (0.1m, 40 0ml, pH 8.1), and Et3N (20 ml). To this soln., a-
chymotrypsin/Celite-545 (600 mg) was added, and the mixture was stirred. The reaction was monitored
with HPLC system vi. After 95 h, the reactant Phac-Gly-Trp-Met-OAl had completely vanished, and the
HPLC conversion of the target product was 66.7%, and, thereafter, the concentration of the product
started to decrease. The reaction was stopped by adding a few drops of AcOH. The mixture was filtered,
and the filter cake was washed with 80% MeOH followed by addition of AcOEt/H2O 4 :1 until the
product was extracted completely. The combined filtrates were dried in vacuo, and the crude product was
dissolved in MeOH and separated on a Sephadex LH-20 (25 – 100 mm, 90 ꢀ 4 cm) column, equilibrated
with MeOH, followed by elution with MeOH. Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2 eluted first, and
the pooled fractions were collected and dried in vacuo yielding a white solid (85 mg, 54.0%). tR 6.11 min
(HPLC system vi). FAB-MS (C40H47N7O8S; calc. 785.3): 786.2 ([M þ H]þ).
Cleavage of Phac from Phac-Gly-Trp-Met-Asp(OMe)-Phe-NH2. Phac-Gly-Trp-Met-Asp(OMe)-
Phe-NH2 (112 mg, 0.14 mmol) was suspended in H2O (10ml), and the pH was adjusted to 7.6 with 0.1 m
NaOH. After addition of PGA/Eupergit C (350mg), the mixture was stirred at 35 8 for 30h. The reaction
was monitored with HPLC system v. To extract the product, the mixture was diluted with 80% EtOH
(20ml), sonicated, and filtered. The filtrate was evaporated to dryness. The residue was dissolved in 40%
aq. MeOH and separated by MPLC. The pooled fractions were lyophilized twice yielding a white powder
(15 mg, 16.1%). tR 3.85 min (HPLC system iv). FAB-MS (C32H41N7O7S; calc. 667.3): 668.2 ([M þ H]þ).
Synthesis of Bz-Arg-Asp(OEt)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2. Bz-Arg-Asp(OEt)-Tyr-
Met-OAl (46 mg, 0.06 mmol) and Gly-Trp-Met-Asp(OMe)-Phe-NH2 (21 mg, 0.03 mmol) were dissolved
in MeCN (4 ml) containing Tris · HCl buffer (0.05m, 18 ml, pH 8.0) and Et3N (18 ml). To this mixture, a-
chymotrypsin/Eupergit C (70mg) was added, and the mixture was stirred at r.t. The reaction was
monitored with HPLC system v. After 118 h, the reaction was stopped, and the mixture was filtered to
remove the immobilized enzyme. The filter cake was washed with 80% MeCN in which the solubility of
reactants and product was higher in comparison to other solvents like MeOH, EtOH, or AcOEt. The
combined filtrates were dried in vacuo, and the residue was dissolved in 50% MeOH and separated by
MPLC. The pooled fractions were lyophilized twice yielding a white solid (7 mg, 17.1%). tR 11.02 min
(HPLC system v). FAB-MS (C65H84N14O15S2; calc. 1364.6): 1365.4 ([M þ H]þ).
Cleavage of Bz-Arg from Bz-Arg-Asp(OEt)-Tyr-Met-Gly-Trp-Met-Asp(OMe)-Phe-NH2. The Bz-
Arg-protected octapeptide (15 mg, 0.01 mmol) was suspended in H2O (3.5 ml), and pH was adjusted to
8.5 with 0.1m NaOH. TPCK-Treated trypsin (2 mg) was added followed by stirring at r.t. for 4 h. The
reaction was monitored with HPLC system iv. The reaction was stopped by adjusting the pH to 6.0with a
few drops of AcOH. The mixture was evaporated to dryness in vacuo, and the residue was dissolved in
50% MeCN and separated isocratically by prep. HPLC with 52% B of the MeCN system. The pooled
fractions were lyophilized yielding a white powder (2 mg, 16.5%). tR 10.09 min (HPLC system iv). ESI-
MS (C52H68N10O13S2; calc. 1104.4): 1105.2 ([M þ H]þ), 1127.4 ([M þ Na]þ).
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Received January 21, 2008