PEPTIDE SYNTHESIS IN ORGANIC MEDIA
533
Severodonetsk, Ukraine); acetonitrile for HPLC of the (5–10 µl) were occasionally taken for an HPLC analy-
os. ch. (high purity) grade (Lekbiofarm, Russia) con- sis. The samples were diluted with the starting eluent to
taining no more than 0.01% of water; and calcium chlo-
ride (Sigma, United States), Tris (ICN Biomedicals,
United States), trifluoroacetic acid (Fluka Chemie AG,
Switzerland), Phe-pNA (Serva, Germany), and Leu-
pNA (Bachem Bioscience Inc., United States) of the
analytical grade quality were used in this study. Trieth-
ylamine and DMF of ch. d. a. (analytical grade) quality
the final concentration of 0.5–0.7 mM before the appli-
cation onto the column. The retention time of Z-Ala-
Ala-Leu-Phe-pNA was 19.0 min in gradient (B). The
amino acid analysis of Z-Ala-Ala-Leu-Phe-pNA: Ala
1
0.4 nmol, Leu 5.5 nmol, and Phe 5.2 nmol.
Condensation of Z-Ala-Ala-Leu-OMe or Z-Ala-
Ala-Leu-OH with Phe-pNA. The 400 mM solutions of
(Reakhim, Russia) were additionally purified according
to the procedure [16]. The Cam-esters of amino acids
and peptides were kindly presented by Yu.V. Mitin
Z-Ala-Ala-Leu-OCH (Z-Ala-Ala-Leu-OH) (125 µl)
3
and Phe-pNA (125 µl) in SMS were added to an immo-
(
Institute of Protein Research, Russian Academy of
bilized subtilisin (10 mg with the protein content of
Sciences, Pushchino). Other amino acid and peptide
derivatives were synthesized in our laboratory accord-
ing to standard procedures [17].
0
1
.054 mg) preliminarily washed with acetonitrile (1 ×
ml) and SMS (2 × 1 ml). The reaction mixture was
shaken on an orbital shaker at 20°ë, and samples (5 µl)
were taken for HPLC analysis at regular intervals. The
samples were diluted with the starting eluent (20%
solution of acetonitrile in water, 1.7 ml) before the
application onto the column.
The reactions were monitored and the peptides were
analyzed by HPLC on an Altex Model 110A liquid
chromatograph (United States) equipped with the fol-
lowing columns: a Microsorb-MV C (4.6 × 250 mm)
8
(
Rainin Instrument Company, Inc., United States) col-
umn [elution with a linear gradient of acetonitrile in
The peptides Z(Boc)-Xaa-Phe-pNA (Xaa = Leu
and Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu and Ala;
Yaa = Leu and Phe), Z-Ala-Ala-Arg-Phe-pNA, Z-
Ala-Ala-Gly-Phe-pNA, and Z-Ala-Ala-Leu-Gly-
pNA were similarly prepared.
0
2
2
.1% aqueous solution of trifluoroacetic acid (A) from
0 to 100% for 35 min or (B) from 10 to 70% for
6.2 min] and a Nucleosil C (4.6 × 250 mm, Biokhim-
1
8
mak, Russia) column [elution with a linear gradient of
acetonitrile in 0.1% trifluoroacetic acid (C) from 20 to
8
0% for 35 min or (D) from 10 to 80% for 42 min]. In
The coupling of Z-Ala-Ala-Arg-OMe with Glu-
pNA. A solution of Z-Ala-Ala-Arg-OMe (19.1 mg,
all the cases, flow rate was 1 ml/min, and the substances
were detected at 220 and 280 nm. The amino acid anal-
ysis was carried out on an automatic Hitachi-835 amino
acid analyzer (Japan) after the acidic hydrolysis of pep-
tides and proteins in 5.7 M HCl at 105°C in vacuum-
sealed ampoules for 24 and 48 h.
3
7.5 µmol) in SMS (100 µl) and a solution of HCl ×
Glu-pNA (37.5 µmol) and triethylamine (37.5 µmol) in
SMS (150 µl) were added to the immobilized subtilisin
(10 mg with the protein content of 0.05 mg) preliminar-
ily washed with MeCN (1 × 1 ml) and SMS (2 × 1 ml).
The reaction mixture was shaken at 20°ë on an orbital
shaker. The samples for HPLC (5 µl) were taken from
the reaction mixture at regular intervals. The retention
time of the Z-Ala-Ala-Arg-Glu-pNA product was
Preparation of the subtilisin immobilized on a
PVA cryogel (Sbt ). Preparation of the reactive alde-
im
hyde-containing PVA derivatives and covalent attach-
ment of the enzyme to them were carried out according
to the procedure [6]. The quantity of the immobilized
protein was determined by amino acid analysis and
proved to be 5.4 mg of protein per g of the carrier.
1
7.0 min in gradient C.
A preparative synthesis of Z-Ala-Ala-Leu-pNA
(
2
5). A solution (625 µl) of Z-Ala-Ala-OMe (88 mg,
50 µmol) and Leu-pNA (63 mg, 250 µmol) in SMS
Peptide Syntheses with the Use
of Immobilized Subtilisin
was added to the subtilisin immobilized on a PVA cryo-
gel (245 mg with the protein content of 1.3 mg). The
reaction mixture was shaken on an orbital shaker at
Determination of the concentration effect of the
starting reagents on the rate of synthesis. Solutions
of the starting components of 0.5–200 mM concentra-
tions were prepared by successive dilutions of a solu-
tion of Z-Ala-Ala-Leu-OMe (75 mg, 0.16 mmol) and
Phe-pNA (46 mg, 0.16 mmol) in a MeCN–DMF mix-
ture (40 : 60 v/v, SMS, 400 µl). A solution of Z-Al-Ala-
Leu-OMe and Phe-pNA (200 µl) of the corresponding
concentration was added to a preparation of immobi-
lized subtilisin (8 mg with the protein content of
3
7°C for one day. The samples for HPLC (5 µl) were
taken from the reaction mixture at regular intervals. The
biocatalyst was separated and washed with SMS (4 ×
0
.8 ml). The reaction mixture and all the washings were
combined and evaporated on a rotary evaporator. The
resulting solution was dropwise added to 0.1 M HCl
(
3 ml) upon a careful stirring and diluted with cool
water (10 ml). The precipitated solid was washed with
water on a glass filter and dried in a vacuum over
0
.043 mg) preliminarily washed with acetonitrile (1 ×
1
ml) and SMS (2 × 1 ml). The reaction mixture was NaOH. The yield of Z-Ala-Ala-Leu-pNA was 98 mg
shaken on an orbital shaker at 20–22°ë, and samples (74%); RT 16.8 min in gradient B.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005
Belyaeva
