Modified Proteases for Peptide Synthesis in Organic Media

Article abstract of DOI:10.1023/A:1026061828077

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.

Full text of DOI:10.1023/A:1026061828077

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 5, 2003, pp. 496–501. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 5, 2003, pp. 544–550.
Original Russian Text Copyright © 2003 by Filippova, Lysogorskaya.
Modified Proteases for Peptide Synthesis
in Organic Media
1
I. Yu. Filippova and E. N. Lysogorskaya
Faculty of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia
Received October 31, 2002; in final form, December 6, 2002
Abstract—We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various
lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases
were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylform-
amide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate,
and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the nonco-
valent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for
the segment condensation of peptide fragments containing residues of trifunctional amino acids with unpro-
tected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.
Key words: enzymatic peptide synthesis, organic solvents, pepsin, subtilisin, thermolysin
1
INTRODUCTION
toward the product formation and the minimization of
undesired side reactions of the secondary hydrolysis
of target products. Polar organic solvents are the most
suitable for the enzymatic peptide synthesis, because
the majority of amino acids, their derivatives, and pep-
tides are insoluble in apolar media. However, it is
polar organic solvents that exert the largest inactivat-
ing effect on the enzymes by depriving them of the
hydrate cover necessary for the catalytic function in
polar organic media. Taking into account all these
facts, we studied the dependence of the synthase activ-
ity of the proteases of various classes in dependence
on the method of their modification and their ability to
adapt to the conditions of functioning in polar organic
Proteases are widely distributed in nature and play
2
an important role in life processes. The practical use
of proteases is mostly due to their ability to catalyze
the reactions of proteolysis (hydrolysis of peptide
bonds). Up to now, this function of the proteolytic
enzymes has deeply and comprehensively been stud-
ied. For a number of proteases of various classes, the
ability to catalyze the peptide bond formation has been
discovered but it has much less been studied [1, 3].
Evidently, the synthase properties of proteolysis
enzymes is very interesting because they can extend
the traditional knowledge on their properties as hydro-
lases. On the other hand, the ability of proteases to cat-
alyze the peptide bond formation is of a fundamental media.
interest for the development of new methods of enzy-
matic synthesis.
RESULTS AND DISCUSSION
One of the promising approaches to the in vitro
studies of proteases as synthases is the examination of
their enzymatic properties in the reactions of peptide
bond formation carried out in organic solvents. The
use of organic solvents as the medium for the enzy-
matic peptide synthesis ensures a significant shift of
the thermodynamic equilibrium of the reactions
As subjects for the study, we chose various catalytic
enzymes: pepsin (aspartyl protease, EC 3.4.23.1), ther-
molysin (metalloprotease, EC 3.4.24.4), and subtilisin
(
serine protease, EC 3.4.21.14). Pepsin and thermo-
lysin fulfil a general basic catalysis when substrate
forms a tetrahedral intermediate that is not covalently
bound to the enzyme. The two proteases can participate
only in the thermodynamically controlled synthesis of
peptide bond (Scheme 1, equation 1) [4]. In this case,
enzyme only accelerates the achievement of equilib-
rium in the synthesis–hydrolysis reaction.
1
Corresponding author; phone: +7 (095) 939-5529; e-mail: irfil-
ipp@genebee.msu.su
2
Abbreviations: cryoPVA, cryogel of polyvinyl alcohol; cryoPVA-
A, cryoPVA-E, and cryoPVA-VS, cryogels of polyvinyl alcohol
containing reactive aldehyde, epoxide, and vinyl sulfonic groups,
respectively. All amino acids were of the L-series.
1
068-1620/03/2905-0496$25.00 © 2003 MAIK “Nauka /Interperiodica”

MODIFIED PROTEASES FOR PEPTIDE SYNTHESIS IN ORGANIC MEDIA
497
(1)
(2)
RCOOH + H NR'
RCONHR' + H O
2
2
+
H NR''
2
RCOOR' + E
[RCOOR' ⋅ E]
[RCO] ⋅ E
RCONHR'' + E
+
H O
2
RCOOH + E
Scheme 1. The peptide bond formation catalyzed by the proteases: (1) the thermodynamically controlled synthesis and
2) the kinetically controlled synthesis.
(
Subtilisin is a typical representative of proteases that higher solubility in water than the reaction product
catalyze the substrate hydrolysis through the formation were more readily soluble in water and, therefore, were
of an intermediate covalent complex of the acyl frag- concentrated in aqueous “microphase” directly contact-
ment of substrate with the enzyme (so-called acylen- ing with the enzyme [8]. As a result, the local concen-
zyme). The functional group of the enzyme active site tration of reacting substances in the proximity to the
serves as a nucleophile (Scheme 1, equation 2). Subtil- enzyme active site increased and the synthesis pro-
isin can catalyze the peptide synthesis by both thermo- ceeded more efficiently under these conditions than in
dynamically controlled and kinetically controlled path- an aqueous organic medium. For example, hexapeptide
ways (Scheme 1, equations 1 and 2) [4].
(I) was synthesized in acetonitrile with the yield of
0%, whereas the yield of this compound was no higher
9
The enzymes were stabilized in nonaqueous sys-
tems by the known techniques of covalent and nonco-
valent modifications. In particular, pepsin was precipi-
tated on the surface of an inert inorganic support [5].
than 60% in 1 M acetate buffer (pH 4.6) containing
2
0 vol % of DMF.
We demonstrated in a special experiment that no
Subtilisin and thermolysin were modified by a covalent firm binding of the enzyme to none of the supports was
immobilization on a cryogel of polyvinyl alcohol [6]. observed. The protein was easily washed out even at the
Moreover, subtilisin was also studied in the form of suspending of the catalyst in 1 M acetate buffer
noncovalent hydrophobic ion complex with SDS solu- (pH 3.5). However, the support kept pepsin, which is
ble in organic medium [7].
insoluble in the organic phase, under the reaction con-
The special features of the action of the modified ditions. Such a fixation of the enzyme due to its precip-
proteases in the reactions of peptide synthesis in vari- itation on the surface of support allowed the utilization
ous organic media are discussed lower.
of one portion of pepsin precipitated on Celite in four
cycles of the hexapeptide (I) synthesis in acetonitrile.
The yields of the product decreased from cycle to cycle
and were 90, 86, 80, and 71%. The decrease in the yield
of the product could be explained by a partial inactiva-
Pepsin as a Catalyst of Peptide Synthesis
in Organic Medium
The enzyme was adapted to the action in organic tion of the enzyme by the organic solvent and by a grad-
solvents by the precipitation of its solution in 25 mM ual transition of water in organic phase.
citrate buffer (pH 4.5) on porous inert inorganic sup-
The efficiency of pepsin as a catalyst for the seg-
ports and drying to a constant mass [5]. Silochrom C-80,
ment condensation in organic media was demonstrated
Aminosilochrom, macroporous glass beads, and Celite
by the example of syntheses (in 70–90% yields) of dif-
were used as the supports for pepsin.
ferent tetra-, penta-, hexa-, octa-, and decapeptides con-
The synthase activity of pepsin was evaluated by the taining a Phe, Tyr, or Trp residue in position P and a
1
example of coupling two tripeptides, Z-Ala-Ala-Phe-
Phe, Tyr, Trp, Leu, or Phe(NO) residue in position P' .
2
1
OH and H-Leu-Ala-Ala-OMe, leading to Z-Ala-Ala-
Phe-Leu-Ala-Ala-OMe (I) according to Scheme 2. The
The specificity of pepsin catalyzing the peptide syn-
structure and the length of the starting fragments corre- thesis in organic solvents demonstrated no qualitative
sponded to the pepsin specificity that provided for their changes in this series of experiments. The Trp residue
effective binding to the enzyme active site.
in position P' was effectively bound to the enzyme
1
active site, which ensured a rather fast rate of the syn-
Z-Ala-Ala-Phe-OH + H-Leu-Ala-Ala-OMe
(
I) thesis (24 h) and a good yield (90%) of Z-Ala-Ala-
pepsin
3
Z-Ala-Ala-Phe-Leu-Ala-Ala-OMe
Phe*Trp-Ala-Ala-OMe. The Trp residue in position P
1
Scheme 2. The synthesis of model hexapeptide (I) cata-
lyzed by the pepsin precipitated on Celite in organic
medium.
worser corresponded to the pepsin specificity, and Z-
Ala-Ala-Trp*Leu-Ala-Ala-OMe
and
Z-Ala-Ala-
Trp*Phe-Ala-Ala-OMe were synthesized for 24 h in 20
and 50% yields, respectively. These results are in a
good agreement with the data on the specificity of
hydrolysis of peptide bonds in protein substrates [9, 10].
The synthesis was carried out in acetonitrile, meth-
ylene chloride, ethanol, and ethyl acetate. The use of
the enzyme precipitated on Celite in acetonitrile at
3
[
S]/[E] = 10 : 1 was optimal. The reacting peptides that
3
contained charged groups and had a lower length and
The formed bond is indicated by asterisk.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

4
98
FILIPPOVA, LYSOGORSKAYA
The formation of bonds between Tyr and Phe (Leu) or 65% of its starting activity after storage in acetonitrile
Phe (Leu) and Tyr proceeded with lower yields (50– for one year.
6
0%) for Z-Ala-Ala-Phe*Tyr-Ala-Ala-OMe, Z-Ala-
The synthase activity of subtilisin immobilized on
cryoPVA with the use of glutaraldehyde was demon-
strated in the coupling reactions of the obtaining of N-
acylated tetrapeptides of the general formula Z-Ala-
Ala-Xaa-Yaa-pNA (Xaa = Leu, Lys, or Glu and Yaa =
Phe or Asp) [6, 12] in the mixtures of DMF and aceto-
Ala-Tyr*Leu-Ala-Ala-OMe, and Z-Ala-Ala-Tyr*Phe-
Ala-Ala-OMe. However, a significant increase in the
reaction time (up to 72 h) resulted in the achievement
of equilibrium state and in the increased yields of the
described compounds to 85–90%. It was also possible
to introduce the Phe(NO ) residue in position P' of the nitrile with the content of DMF 60–95% in 90–98%
Z-Ala-Ala-Phe Phe(NO) -OMe peptide (the yield of
2
1
yields (Scheme 3).
*
2
7
0%). The possibility of effective coupling for the
Z-Ala-Ala-Xaa-OH + Yaa-pNA
longer hydrophobic peptides was demonstrated by the
examples of synthesis of the following peptides: Z-Ala-
Ala-Phe*Trp-Ala-Leu-Ala-Phe-OMe (80% yield),
Boc-Trp-Ala-Leu-Ala-Phe*Leu-Ala-Ala-OMe (80%
yield), and Boc-Trp-Ala-Leu-Ala-Phe*Trp-Ala-Leu-
Ala-Phe-OMe (70% yield).
cryoPVA-A–subtilisin
Z-Ala-Ala-Xaa-Yaa-pNA,
where Xaa = Leu, Lys, Glu; Yaa = Phe, Asp.
Scheme 3.
No activation of the carboxyl component and pro-
tection of side functions of trifunctional amino acid res-
idues were necessary for this enzymatic reaction.
The synthase activity of the immobilized thermo-
lysin was studied in the reaction illustrated in Scheme 4
in a 25 : 75 (v/v) DMF–acetonitrile medium:
Thus, our experiments demonstrated for the first
time that pepsin sorbed on the surface of inorganic sup-
ports exhibits a high catalytic activity in organic sol-
vents and is an effective catalyst for the synthesis of
hydrophobic hexa- and octapeptides, whose prepara-
tion is difficult or impossible in aqueous organic media.
Z-Ala-Ala-OH + H-Leu-pNÄ
immobilized thermolysin
Z-Ala-Ala-Leu-pNÄ
The Synthase Activity of Subtilisin and Thermolysin
Covalently Immobilized on a Cryogel
of Polyvinyl Alcohol
Scheme 4.
The yield of the product was 90% after 1-h-cou-
pling, in which the amino component and the carboxyl
component were taken at an equimolar ratio and the
One of the methods of enzyme adaptation to the
functioning in organic media is their covalent immobi-
lization on a solid carrier. CryoPVA was used as a
matrix for the immobilization of subtilisin and thermo-
lysin. This very macroporous carrier is prepared from
the aqueous solution of polyvinyl alcohol by the freez-
ing–thawing procedure [11]. Cryogels are character-
ized by a high hydrophilicity (water receptivity): they
can keep water inside the polymer support even in
anhydrous medium of polar organic solvents.
[
E]/[S] ratio was 1 : 750. The immobilized thermolysin
catalyzed the synthesis of Z-Ala-Ala-Leu-pNA in
organic solvents in 80% yield after twofold use in the
synthesis and six-months storage in a buffer (pH 7.8).
These results demonstrate a high efficiency of the
immobilized thermolysin as catalyst for the peptide
synthesis in organic medium.
SDS-Subtilisin in the Peptide Synthesis
in Organic Media
Subtilisin was immobilized on the cryoPVA-A,
cryoPVA-E [6], and CryoPVA-VS derivatives of the
PVA cryogel containing reactive aldehyde, epoxide,
and vinylsulfonic groups, respectively. The content of
subtilisin on the carrier depended on the immobiliza-
tion method and varied within the range from 0.1 (for
cryoPVA-E) to 13.7 (for cryoPVA-A) mg of the protein
per g of carrier. Thermolysin was covalently attached
only to the aldehyde carrier [6], and its content on the
carrier was 3.5 mg/g.
Homogenous systems in which an enzyme is
present in a soluble state are more suitable for the enzy-
matic reactions. One of the approaches to the solubili-
zation of an enzyme in organic media is the preparation
of an enzymic complex with an anionic detergent (e.g.,
SDS) at the detergent concentrations lower than its crit-
ical micelle-forming concentration. Formation of the
so-called hydrophobic ion pairs between the charged
groups on the enzyme surface and the molecules of sur-
face-active substance results in hydrophobization of the
enzyme surface, and the enzymes become soluble in
organic solvents [13].
The preparations of immobilized catalysts were
highly stable in a number of organic solvents. For
example, the immobilized subtilisin retained 60% of its
starting activity when being kept for two days in the
We prepared the subtilisin complex with SDS and
mixture of DMF and acetonitrile (90 : 10, v/v), whereas studied the synthase capacity of this complex cata-
the native subtilisin suspended in the same mixture of lyzing various reactions of the peptide bond formation
organic solvents was almost completely inactivated. in ethanol and isopropanol [14]. We found that subtili-
The preparation of immobilized thermolysin exhibited sin is rather effective catalyst of the peptide bond for-
an extremely high stability. This biocatalyst preserved mation [14, 15].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

MODIFIED PROTEASES FOR PEPTIDE SYNTHESIS IN ORGANIC MEDIA
499
Z-Protected amides and p-nitroanilides of tetra- and tripeptides of the general formula: A-Ala-Ala-Xaa-
pentapeptides containing the residues of hydrophobic OMe [where A is Z, Boc, or Dnp and Xaa is Leu or
amino acids (Gly, Ala, Leu, Met, Phe, Phe(NO ), Trp, Glu(OMe)] attached to Aminosilochrom according to
2
the (3 + 3 + 3) scheme.
and Tyr) in positions P - and P' were synthesized in
1
1
high yields. The acylating components with D-Leu,
Pro, and β-branched Ile in position P did not enter the
1
Special Features of Protease Behavior in Organic
Solvents (by the Example of Subtilisin)
reaction. A number of tri-, tetra-, and hexapeptides
simultaneously containing N- and C-terminal chro-
mogenic and fluorogenic groups (Ó-aminobenzoyl
group, p-nitroanilide group, or the residue of 2,4-dini-
trophenylethylenediamine) were also prepared by the
use of SDS-subtilisin.
A high synthetic potential of SDS-subtilisin made it
possible to develop a method of enzymatic coupling of
peptides attached to a solid support [16]. This method
We found some special features of the subtilisin
behavior in organic media in comparison with the
native enzyme on the basis of experimental peptide
syntheses catalyzed by subtilisin modified by various
methods.
The effect of the DMF content in medium and the
combines the advantages of the methodology of the structure of acylating component on the synthase activ-
chemical solid-phase peptide synthesis with the advan- ity of the native and covalently and noncovalently mod-
tages of the enzymatic formation of peptide bonds. We ified subtilisin were studied by the example of the reac-
demonstrated the basic possibility of such approach by tion of preparation of Z-Ala-Ala-Leu-Phe-pNA from
the example of the successive enzymatic coupling of Z-Ala-Ala-Leu-OMe and Phe-pNA (Scheme 5).
Z-Ala-Ala-Leu-OH
H O
2
Phe-pNa
Z-Ala-Ala-Leu-Oï + E
[Z-Ala-Ala-Leu-O-E]
ï = åÂ, Et, PhCl
Z-Ala-Ala-Leu-Phe-pNA
XOH
Scheme 5
We established that the curves of the product accu- mined in the reaction mixture along with the starting
mulation in the synthesis catalyzed by both subtilisin acylating component itself by the analysis of the reac-
immobilized on cryoPVA-A using glutaraldehyde and tion mixture after a short time from the beginning of the
the native subtilisin in 60% DMF varied little from one reaction.
another. The yield of Z-Ala-Ala-Leu-Phe-pNA
Z-Ala-Ala-Leu-Phe-pNA was also synthesized by
achieved maximum (90%) for 3 h and, then, remained
the treatment with subtilisin immobilized on the
practically unchanged. The situation was different at
cryoPVA-A in 60% DMF in acetonitrile. The yield of
the DMF content increased to 95%. The yield of the tar-
the product was 60% (in the case of Z-Ala-Ala-Leu-
get product in the reaction catalyzed by the native sub-
OMe) and 24% (in the case of Z-Ala-Ala-Leu-OH)
tilisin three times decreased, and the synthesis was
within 30 min. After two hours, the difference in the
stopped even after 3–5 h due to the loss of the enzyme
yields decreased to 10%, and after three h, the yields of
catalytic activity [17]. On the other hand, the synthesis
the product became practically identical independently
with cryoPVA-A-sublitisin proceeded even after 72 h in
of the structure of acylating component.
9
7
5% DMF, and the yield of the target product achieved
5% [6].
Thus, both esters of N-protected amino acids and
Z-Ala-Ala-Leu-Phe-pNA was synthesized starting their analogues with free carboxyl groups can be used
from Z-Ala-Ala-Leu-OR (R = H, Me, or PhCl) and as acylating agents with the same efficiency in the reac-
Phe-pNA in a ethanol–DMF mixture by the treatment tions of peptide bond formation with the use of modi-
with SDS-subtilisin at [S]/[E] ratio of 5000 : 1 [15]. In fied subtilisins. This result can probably be explained
all the cases, practically quantitative yields were by the fact that the acylating component is not charged
achieved even after 2 h, and no remarkable effect of the in the solvent mixtures with a low water content and
activation of carboxyl group of the acylating compo- effectively interacts with the enzyme active site. The
nent on the rate of the product formation was observed. same efficiency of the SDS-subtilisin toward the acylat-
It is probable that the reesterification of the acylating ing components with protected and free carboxyl
agent by ethanol was the first and the fastest stage of the groups in the ethanol-containing medium is possibly
reaction due to a large excess of ethanol in the reaction associated with the esterification reaction due to a large
mixture. Actually, the presence of ethyl ester was deter- ethanol excess that served as an additional nucleophile.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 5 2003

5
00
FILIPPOVA, LYSOGORSKAYA
'
1
The synthesis of p-nitroanilides of tetrapeptides containing Arg, Lys, Glu, and Asp residues in positions P and P catalyzed
1
by subtilisin immobilized on cryoPVA (experiments 1 and 2)* and by SDS-subtilisin (experiments 3–6)**
Experi-
ment no.
Acylating component
Amino component
Product
Time, h
Yield, %
1
2
3
4
5
6
Z-Ala-Ala-Lys-OH
Asp-pNA
Asp-pNA
Arg-pNA
"
Z-Ala-Ala-Lys-Asp-pNA
2
4
98
74
52
92
38
83
Z-Ala-Ala-Glu-OH
Z-Ala-Ala-Glu-Asp-pNA
Z-Ala-Ala-Glu-OH
Z-Ala-Ala-Glu-Arg-pNA
24
"
"
120
24
Z-Ala-Ala-Lys-OH
Glu-pNA
"
Z-Ala-Ala-Lys-Glu-pNA
"
"
120
*Concentrations of the acylating component and the amino component were 30 mM. The subtilisin content in the catalyst (80 mg) was
0.3 mg. The reaction was performed in the mixture of DMF and acetonitrile (60 : 40, v/v) at 20°C.
*
*Concentrations of the acylating component and the amino component were 31 mM. SDS-Subtilisin (6 µM) was dissolved in ethanol.
The reaction was performed at 20°C.
Evidently, kinetic studies are necessary for the com-
plete understanding of the processes.
Data on the syntheses of these derivatives are given
in the table. Subtilisin did not hydrolyzed peptide
bonds between the positively and negatively charged
The mentioned peculiarity of the catalysis of pep-
tide bond formation by subtilisin in the organic media
with a minimal water content was also demonstrated by
the example of the syntheses of N-acylated tetrapep-
tides containing unprotected basic or acidic amino acid
trifunctional amino acid residues in positions P and
1
P .
'
1
The composition of the synthesized compounds was
confirmed by amino acid analysis and, in some cases,
by mass spectrometry.
residues in position P [6, 12, 15] (Scheme 6).
1
Z-Ala-Ala-Xaa-OR + H-Phe-pNA
Z-Ala-Ala-Xaa-Phe-pNA,
where Xaa = Lys, Arg, His, Glu, or Asp and R = H or Me
Scheme 6.
Thus, our results substantially widen our knowledge
of the synthase capacity of proteases. The efficiency of
the enzymes as agents for the peptide synthesis is
known to be mainly associated with their stereo- and
regioselectivity. The absence of a universal peptidylli-
gase with a wide specificity is one of the main reasons
limiting the application of proteases to the synthesis of
peptides of various compositions. Therefore, a large
number of proteases are necessary for the purposeful
synthesis of peptides of the predetermined sequences.
The data we obtained during studying subtilisin as a
The tripeptides containing Arg or Lys on their
C-ends (in position P ) appeared to be very effective
1
acylating components in accordance with the literature
data. The yields of products achieved 70% even for 2 h
when both activated and nonactivated acylating compo-
nents were used. The reaction proceeded somewhat
slower in the case of Z-Ala-Ala-His-Phe-pNA.
The introduction of Glu in position P resulted in catalyst for the peptide bond formation suggest that its
1
8
4% yield of product for 6 h, and the formation of a sin- substrate specificity was extended in organic media.
gle product with free γ-carboxyl group was observed The synthetic potential of the enzyme can be enhanced
according to mass-spectrometric data. Both dimethyl by changing the conditions of its introduction in the
ester [Z-Ala-Ala-Glu(OMe)-OMe] and its analogue synthetic medium, and, therefore, effective methods of
with the free α- and γ-carboxyl groups exhibited an preparation of peptides of various structures could be
equally rapid reaction with H-Phe-pNA. The substitu- elaborated.
tion of Asp for Glu resulted in a decreased yield.
Components simultaneously containing unprotected
ACKNOWLEDGMENTS
basic or acidic side chains both in position P and in
1
This study was supported by the Russian Founda-
tion for Basic Research, project nos. 00-03-32803a and
position P' were condensed by the action of SDS-sub-
1
tilisin complex covalently immobilized on cryoPVA-A
6, 15] (Scheme 7).
0
0-04-48455.
[
Z-Ala-Ala-P -OR + H-P -pNA
1
1
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