Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Article abstract of DOI:10.1016/S0968-0896(99)00237-0

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and α-chymotrypsin, were determined. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00237-0

Bioorganic & Medicinal Chemistry 7 (1999) 2953±2959
Synthesis of Peptide Aldehydes via Enzymatic
Acylation of Amino Aldehyde Derivatives
Tatiana L. Voyushina,* Joanna V. Potetinova, Ekaterina I. Milgotina and
Valentin M. Stepanovy
V.M. Stepanov's Laboratory of Protein Chemistry, Institute of Genetics and Selection of Industrial Microorganisms,
1 Dorozhny pr., 1, 113545, Moscow, Russia
Received 4 November 1998; accepted 2 August 1999
AbstractÐTwo ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino
alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride
mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72,
serine proteinase with a broad speci®city, distributed over macroporous silica, was used as a catalyst in both cases. Due to the
practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative.
The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less
valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leu-
cinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of
corresponding chromogenic substrates for subtilisin and a-chymotrypsin, were determined. # 1999 Elsevier Science Ltd. All rights
reserved.
Introduction
isosteres, inhibitors for HIV proteinase, ligands for a-
nity chromatography of proteins etc.6±9
Peptide aldehydesÐpeptide derivatives terminated with
a-amino aldehydesÐhave been shown to be very strong
competitive inhibitors of serine and thiol proteinases.
These compounds were characterized as transition state
analogues, based on their ability to form extremely
stable tetrahedral adduct with the enzyme. The stability
of such an adduct has been attributed to the formation
of covalent hemiacetal bond between peptide aldehyde
and serine or cysteine of the active center.1±5 Peptide
aldehydes are interesting, also, as intermediates in the
synthesis of di€erent peptide derivatives such as peptide
Chemical coupling of amino aldehyde derivatives (semi-
carbazone, acetals etc.) with peptides have been usually
applied to prepare peptide aldehydes.9±11 Occasionally
peptide alcohols were oxidized to peptide aldehydes.12,13
Recently we reported a convenient method for these deri-
vatives preparation which consists in enzymatic acylation
of amino alcohols with acylpeptide esters followed by
chemical oxidation of the resulted peptide alcohols.14
Subtilisin 72, a serine proteinase similar to Subtilisin
Carlsberg, sorbed on macroporous silica, served as a cat-
alyst for amino alcohols acylation. The yields of the
enzymatic reactions were 70±99%, but in the stage of
chemical oxidation a signi®cant loss of the products took
place. We decided to reverse the sequence of the reactions,
so that the amino alcohol which is of less value than a
peptide derivative was submitted to oxidation. Sub-
sequent reactionÐenzymatic acylation of amino aldehyde
derivatives (e.g. of semicarbazone) with acyl peptide esters
leads to the peptide aldehydes.
Key words: Peptide aldehydes; enzymatic synthesis: peptide inhibitors;
serine proteinases.
Abbreviations: Z-, benzyloxycarbonyl-; PHT-, phthalyl-; -ONSu, suc-
cinimidooxy-; -Sem, semicarbazone; -pNA, p-nitroanilide; DMSO,
dimethylsulfoxide; Ac2O, acetic anhydride; Py, pyridin; DMF, dime-
thylformamide; pheol, amino alcohol phenylalaninol; pheal, amino
aldehyde phenylalaninal; Tris, Tris(hydroxymethyl)amino-methane.
The notation of enzyme binding subsites corresponds to the Schechter
and Berger nomenclature. Standard three abbreviations are used for
amino acid residues, which belong to l-series.
Here we present the peptide aldehyde synthesis follow-
ing this ``improved'' scheme in comparison with the one
discussed in our previous paper.14
* Corresponding author. Tel.:+7-095-315-3738; fax:+7-095-315-05-
01; e-mail: ost@vnigen.msk.su
y
Deceased.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00237-0

2954
T. L. Voyushina et al. / Bioorg. Med. Chem. 7 (1999) 2953±2959
Results
tion was still unsatisfactory. Thus the complex CrO3ÂPy
is the most convenient oxidant for N-acylated amino
alcohols, whenever the DMSO±Ac2O mixture seems to be
the more e€ective one for peptide alcohols. Note that the
aldehyde product must be puri®ed in both cases.
Oxidation of amino alcohols
Although the oxidation of alcohols is well known reac-
tion, the oxidation of peptide and amino alcohols needs
some comments.
It is known that acyl amino aldehyde is the rather
unstable compound which easily su€ers racemization.
The racemization is especially intensive when acyl
amino or peptide aldehydes are puri®ed on silica,
whereas aldehyde derivatives, e.g. semicarbazone, are
stereo stable.17,20,21 Therefore, it would be reasonable to
convert the peptide aldehydes after oxidation in corre-
sponding semicarbazones before their puri®cation. After
removing the Z-protecting group semicarbazones of
amino aldehydes, were used as nucleophiles in enzymatic
acylation.
There are many reagents for oxidation of alcohol
groups. The problem is to choose the reaction which is
free from overoxidation danger, give optically pure
product and proceed with a good yield. Among the
oxidants which we tried, the most convenient turned out
to be the mixture of DMSO and Ac2O.15 Using this
method some peptide and acyl amino aldehydes were
obtained (Table 1).
In the course of the oxidation the aliquots of the reac-
tion mixture were examined by HPLC method. A broad
pick of the aldehyde product was observed (Fig. 1 ). To
all appearance such an unusual form of the pick may be
explained by reversible hydration of the aldehyde func-
1
tion. Examination of HNMR spectra of the products
con®rmed the presence of the aldehyde group in the
peptide derivatives. Optical rotation of Z-Pheal, thus
obtained, was fully identical to the literature data, so
racemization does not occur during this oxidation.
The mixture of DMSO and Ac2O gave satisfactory
results, with some inconvenience:
1. both DMSO and Ac2O should be freshly distilled;
2. the yield of the product does not exceed 70%.
Moreover acylation of the alcohol group with the
excess of Ac2O could decrease the yield
3. the reaction mixture should be freeze-dried to
remove the excess of DMSO which is the oxidant
and the solvent simultaneously.
A number of reactions in which di€erent derivatives of
CrO3 are used as the oxidant were described.16±19
Among which the complex CrO3 *Py seems to be the
most attractive one. The non-polar solvents, like
CH2Cl2 or CHCl3, are usually applied. We found the
optimal conditions for oxidation of Z-Leuol and Z-
Valol with complex CrO3ÂPy in the presence of Ac2O
(Table 2).
Figure 1. Chromatograms of the reaction mixtures. (a) Oxidation of
Z-Gly-Pro-Phe-Leuol with DMSO-Ac2O mixture. Condition applied:
Z-Gly-Pro-Phe-Leuol 18.7 mmol, DMSO 330 ml, Ac2O 35 ml, 24 h,
16ꢀC. (b) Oxidation of Z-Valol with CrO3 *Py complex. For details see
Experimental.
Contrary to N-acylated amino alcohols, peptide alcohols
are practically insoluble in CH2Cl2 and CHCl3. In the
polar solvents e.g. DMF, DMSO the result of the oxida-
Table 1. Oxidation of amino and peptide alcohols (RCH2OH) to the
corresponding aldehydes with the mixture of DMSO and Ac2O
Table 2. Oxidation of N-acylated amino alcohols (RCH2OH) to the
corresponding amino aldehydes with the complex CrO3ÂPy and Ac2O
at 16ꢀC
Starting alcohol
Reagents molar Time Temperature
(Cꢀ)
Yield
assessed
HPLC (%)
ratio RCH2OH: (H)
Ac2O
Z-Pheol
CHO-Pheol
PHT-Pheol
Z-Ala-Ala-Pheol
Z-Ala-Ala-Valol
Z-Ala-Ala-Leu-Pheol
Z-Gly-Pro-Phe-Leuol
1:10
1:10
1:10
1:20
1:10
1:20
1:20
3
2
20
37
37
37
37
16
16
16
81
49
51
67
62
45
62
Starting
amino
alcohol
Reagents
molar ratio
Time
(min)
Yield
assessed
HPLC (%)
RCH2OH:CrO3Â
2.5
Py:Ac2O
5
24
24
Z-Leuol
Z-Valol
1:1:4
1:1:3
18
20
81
88

T. L. Voyushina et al. / Bioorg. Med. Chem. 7 (1999) 2953±2959
2955
Enzymatic reactions
demonstrated by the direct experiment (Fig. 3). Thus,
the yield of the tripeptidyl amino alcohol is the result of
the following reactions:
Since subtilisin 72 possesses a rather broad speci®city,
its S10-subsite is capable of binding di€erent amino com-
ponents, e.g. amino alcohols. On the other hand b-
amino alcohol has no carbonyl group, therefore, its
interaction with the enzyme S10-subsite is handicapped.
To compensate for this shortcoming, the 10-fold excess
of the amino component needed to ensure a good yield
of the peptide alcohol. Although enzymatic acylation of
amino alcohols has been described earler,14 some details
of this reaction should be discussed brie¯y.
Z-Ala-Ala-Leu-OCH3 ‡ H-Pheol !
ꢀ1†
Z-Ala-Ala-Leu-Pheol ‡ CH3OH
Z-Ala-Ala-Leu-COOH ‡ H-Pheol !
ꢀ2†
Z-Ala-Ala-Leu-Pheol ‡ H2O
The amino alcohol acylation with N-protected dipeptide
esters proceeds in a similar way (Fig. 4), but in this case
the reaction
When amino alcohol is acylated with acyl tripeptide
esters (e.g. Z-Gly-Pro-Phe-OCH3 or Z-Ala-Ala-Leu-
OCH3), the latter become exhausted rather early in the
course of the reaction (Fig. 2). Although the 5% DMF
in acetonitrile was used as a solvent and no water was
introduced in it, the hydrolysis of the starting peptide
esters occurs, which led to the formation of Z-Gly-Pro-
Phe-COOH or Z-Ala-Ala-Leu-COOH. Perhaps this is
to be attributed to the water traces present in the solvent
and/or bound by this hydrophilic silica support. The
peptide, thus formed, is still capable to act as an acy-
lating agent under equilibrium conditions. This was
Z-Ala-Ala-COOH ‡ H-Pheol !
ꢀ3†
Z-Ala-Ala-Pheol ‡ H2O
Figure 3. Time course of the reactions observed during peptide synthesis
catalyzed by sorbed subtilisin (Z-Ala-Ala-Leu-COOH was used as the
acylating agent). Conditions applied: DMF-5%, CH3CN-95%, (v/v),
20ꢀC. The total volume of the reaction mixture was 600mL. [Z-Ala-Ala-
Leu-COOH]o=16.7 mM, [Pheol]o=166.7 mM, subtilisin 72±0.23 mM.
(*) - Z-Ala-Ala-Leu-COOH, (~) - Z-Ala-Ala-Leu-Pheol.
Figure 2. Time course of the reactions observed during tripeptide
alcohol synthesis catalyzed by sorbed subtilisin (Acyl tripeptide esters
were used as the acylating agents). Conditions applied: DMF-5%,
CH3CN-95%, (v/v), 16ꢀC. The total volume of the reaction mixture
was 600 mL, subtilisin 72±0.23 mM (a) [Z-Gly-Pro-Phe-OCH3]o=
16.7 mM, [Leuol]o=166.7 mM; (&) Z-Gly-Pro-Phe-OCH3, (*) - Z-
Gly-Pro-Phe-COOH; (~) - Z-Gly-Pro-Phe-Leuol. (b) [Z-Ala-Ala-Phe-
Figure 4. Time course of the reactions observed during dipeptide
alcohol synthesis catalyzed by sorbed subtilisin (Acyl dipeptide methyl
ester was used as the carboxyl component). Conditions applied: DMF-
5%, CH3CN-95%, (v/v), 20ꢀC. The total volume of the reaction mix-
ture was 600 mL. [Z-Ala-Ala-OCH3]o=16.7 mM, [Pheol]o=166.7 mM,
subtilisin 72±0.46 mM. (&) - Z-Ala-Ala-OCH3, (*) - Z-Ala-Ala-OH,
(~) - Z-Ala-Ala-Pheol.
OCH3]o=16.7 mM, [Pheol]o=166.7 mM; (&)
- Z-Ala-Ala-Leu-
OCH3, (*) - Z-Ala-Ala-Leu-COOH, (~) - Z-Ala-Ala-Leu-Pheol.

2956
T. L. Voyushina et al. / Bioorg. Med. Chem. 7 (1999) 2953±2959
is not signi®cant. (Fig. 5). Probably this is because the
N-protected dipeptide with a free COOH-group satis®es
the speci®city requirements of subtilisin, an enzyme that
binds, preferably, acylated tri- and longer peptides, only
marginally. Therefore, the direct acylation of the amino
alcohol with dipeptide (reaction 3) plays only a limited
role under these circumstances.
Enzymatic acylation of amino aldehyde semicarbazones
was performed analogously, but there are some di€er-
ences in the course of the reactions.
On Fig. 6 the HPLC typical chromatogram of the reaction
mixture (Z-Ala-Ala-Leual-Sem synthesis) is presented.
The amino componentÐleucinal semicacarbazone
(Leual-Sem)Ðbeing very hydrophilic, was eluted ®rst
and was masked by the broad peak of DMSO, so the
yield of the peptide product could be evaluated through
the conversion of the carboxyl component, taken in a
double excess. Therefore the yield of the peptide asses-
sed by the HPLC method could not exceed 50% (Fig.
7). The twofold excess of the carboxyl component leads
to the accumulation of Z-Ala-Ala-COOH in the course
of the reaction, as the result of enzymatic hydrolysis of
the starting ester. It seems that dipeptide Z-Ala-Ala-
COOH does not take part in the enzymatic acylation of
the semicarbazone.
The structure of amino aldehyde semicarbazone resem-
bles one of azapeptide:
In the Z-Ala-Ala-Leu-Pheal-Sem synthesis, the double
excess of the amino component was used. The enzy-
It seems, that due to the presence of CˆO and NH-
groups, semicarbazone become rather easily bound by
the S'1 -subsite of subtilisin which assists the enzymatic
acylation. There is no need for an excess of the amino
component and the yields of the reactions in most cases
are practically quantitative (Table 3).
Table 3. Enzymatic acylation of amino aldehyde semicarbazones
(NH2B) with acyl peptide esters (RCOOCH3) catalyzed by subtilisin
72
Reagents
Reagents
molar ratio
RCOOCH3:NH2B
Time Yield
(H) assessed
HPLC (%)
Carboxyl
components
Amino
components
Z-Ala-Ala-OCH3
Z-Ala-Ala-OCH3
Z-Ala-Ala-OCH3
Z-Ala-Ala-OCH3
Pheal-Sem
Alaal-Sem
Leual-Sem
Valal-Sem
2:1
2:1
2:1
2:1
1:2
22
19
8
5
26
91
84
80
92
99
Z-Ala-Ala-Leu-OCH3 Pheal-Sem
Figure 6. Chromatogram of the reaction mixture during Z-Ala-Ala-
Leual-Sem synthesis. HPLC in System B. Condition applied see Figure 7.
Figure 5. Time course of the reactions observed during dipeptide alcohol
synthesis catalyzed by sorbed subtilisin (Z-Ala-Ala-COOH was used as
the carboxyl component). Conditions applied: DMF-5%, CH3CN-95%,
(v/v), 20ꢀC. The total volume of the reaction mixture was 600 mL. [Z-Ala-
Ala-COOH]o=16.7 mM, [Pheol]o=166.7 mM, subtilisin 72±0.46 mM.
(*) - Z-Ala-Ala-COOH, (~) - Z-Ala-Ala-Pheol.
Figure 7. Time course of the reactions observed during enzymatic acy-
lation of amino aldehyde semicarbazone with acyl dipeptide ester. Con-
ditions applied: DMSO-8%, CH3CN-92%, (v/v), 20ꢀC. The total
volume of the reaction mixture was 600mL. [Z-Ala-Ala-
OCH3]o=50 mM, [Leual-Sem]o=26 mM, subtilisin 72±0.46 mM. (&) -
Z-Ala-Ala-OCH3, (*) - Z-Ala-Ala-COOH, (~) - Z-Ala-Ala-Leual-Sem.

T. L. Voyushina et al. / Bioorg. Med. Chem. 7 (1999) 2953±2959
2957
Figure 8. Time course of the reactions observed during enzymatic
acylation of amino aldehyde semicarbazone with acyl tripeptide ester.
Conditions applied: DMF-5%, CH3CN-95%, (v/v), 20ꢀC. The total
volume of the reaction mixture was 600 mL. [Z-Ala-Ala-Leu-
OCH3]o=16.7 mM, [Pheal-Sem]o=33.4 mM, subtilisin 72±0.23 mM.
(&) - Z-Ala-Ala-Leu-OCH3, (*) - Z-Ala-Ala-Leu-COOH, (~) - Z-Ala-
Ala-Leu-Pheal-Sem.
Scheme 1. General scheme of semi-enzymatic peptide aldehyde synthesis.
rather fast and could be useful for preliminary investi-
gation of inhibition of the enzymes.
The second wayÐenzymatic acylation of amino alde-
hyde semicarbazones which were preliminarily obtained
by the chemical route. The latter way is longer than the
®rst one, but all chemical stages were carried out with
amino acid derivatives which are more simple and less
valuable than peptide ones. If the reaction is scaled-up,
the loss of the product could be diminished. Amino
aldehyde semicarbazones are very stable and could be
stored for a long time without any loss of their optical
purity. Moreover, this way allows us to obtain a family
of peptide derivatives containing the same amino alde-
hyde residue but with di€erent peptide moieties which
would be useful when speci®city requirements of the
enzymes must be examined.
Table 4. Inhibition of subtilisin and a-chymotrypsin with peptide
aldehydes
Enzyme (substrate)
Ki(M)
Z-Ala-
Ala-Leual
Z-Ala-
Ala-Pheal
Z-Ala-
Ala-Valal
7
7
6
Subtilisin 72
2.23Â10
4.0Â10
2.9Â10
(Z-Ala-Ala-Leu-pNA)
a-Chymotrypsin
(Pyr-Phe-pNA)
5
6
4
9.05Â10
2.2Â10
4.9Â10
matic hydrolysis of starting tripeptide ester was negli-
gible and conversion of the reaction to the equilibrium
synthesis in this case was not observed (Fig. 8).
Experimental
General
After removal of the protecting group with acetyl-
acetone,22 the peptides with a free aldehyde group were
tested as proteinase inhibitors.
Amino acid and peptide esters have been prepared by
conventional routes. Phenylalaninol was obtained as
described.23 l-Leuol and a-chymotrypsin were pur-
chased from Sigma.
Inhibitory tests
Peptide aldehydes obtained in a such way was shown to
be potent inhibitors of subtilisin and a-chymotrypsin.
Kinetic parameters of inhibition were determined in the
hydrolysis of the corresponding chromogenic substrates
for every enzyme (Table 4)
Subtilisin 72, puri®ed in this laboratory has been dis-
tributed over the surface of the silochrome as describe
earlier.24
Reverse-phase HPLC was performed on a Gilson 704
chromatograph using Becman Ultrasphere ODS column
(4.6Â250 mm). Solutions A and B contained 0.05%
CF3COOH and 0.05% (C2H5)3N respectively. Solutions
AÐ5% CH3CN, solution BÐ90% CH3CN. A linear
gradient of B from 30 to 78% in 40 min (System A) and
linear gradient of B from 25 to 40% in 25 min (System
Discussion
There are two ways of semi-enzymatic synthesis of the
peptide aldehydes following our scheme Scheme 1. The
®rst oneÐthe enzymatic acylation of amino alcohols
and subsequent oxidation of the resulted peptide alco-
hols. Synthesis of peptide aldehydes via peptide alcohols
oxidation have some inconveniences, which are men-
tioned above (large excess of amino alcohol, freshly
distilled reagents for oxidation, loss of the peptide pro-
duct, possibility of the racemization during puri®cation
of the peptide aldehyde after oxidation). However, it is
1
B) with 1.5 mL min was used. Detection was per-
formed at 220 nm.
Optical rotation was measured with Jasco DIP-360
1
polarimeter. H NMR Spectra were recorded on Bruker
AC-200 spectrometer.

2958
T. L. Voyushina et al. / Bioorg. Med. Chem. 7 (1999) 2953±2959
Reduction of the amino acid esters
Yellow oil of Z-Pheal was dissolved in C2H5OH
(6.3 mL) and H2O (2.7 mL), then semicarbazide hydro-
chloride (330 mg) and CH3COONa (270 mg) was
added. The reaction mixture was stirred for 5 min at
80ꢀC, then the solvent was evaporated. Z-Pheal-Sem
was puri®ed by ¯ash-chromatography in chloroform:
ethyl acetate (7:3) and in ethyl acetate on silica. The yield
of the productÐ670 mg (66%), HPLC retention timeÐ
11.7 min (System A). Semicarbazones of other amino
and peptide aldehydes were prepared analogously.
Z-Leuol. To the solution of Z-Leu-ONSu (60 mg;
160 mmol) in 700 ml of dioxane sodium borohydride
(50 mg; 1.4 mmol) and methyl alcohol (110 ml;
2.75 mmol) subsequently were added, then the solution
of acetic acid (80 ml) in dioxane (500 ml) was added
dropwise at 12ꢀC. The mixture was stirred at 75ꢀC for
1.5 h. The solvent was evaporated and the residue was
distributed between water (10 mL) and chloroform
(20 mL). The water layer was washed with chloroform
(3Â5 mL). The combined chloroform fractions were
washed with water (1Â5 mL), and with saturated aqueous
NaCl. Then the chloroform solution was dried over
Na2SO4 and was evaporated. Flash chromatography on
silica gel using chloroform:ethyl acetate (6:1) v/v gave
350 mg (88% yield) of Z-Leuol. [a]2D0= 28 (c=1, etha-
nol) HPLC retention time of Z-Leuol was 13.0 min (Sys-
tem A); conversion of Z-Leu-ONSu to Z-Leuol was 96%.
Pheal-Sem. Z-Pheal-Sem (210 mg, 0.618 mmol) was dis-
solved in CH3OH (20.8 mL) and H2O (5.2 mL), then 0.1
M HCOONH4 (6.5 mL) and Pd-black was added, pH of
reaction mixture was be 4. After stirring for 2 h at 20ꢀC
the catalyst was ®ltered o€, the ®ltrate evaporated in
vacuo. The yield of Pheal-Sem was 150 mg (93%).
[a]2D0=-51 (c=1, MeOH), HPLC retention time - 2.79
min (System A)
1H NMR (CDCl3, 200 MHz, d ppm): 0.88(d, 6H,
-CH(CH3)2); 1.28(m, 2H, -CH2CH(CH3)2); 1.6(m, 1H,
-CH2 CH(CH3)2; 3.44(m, 2H, -CH2OH); 3.6(q, 1H, CH2
OH); 3.74(m, 1H,>CHCH2CH(CH3)2; 5.04(q, 2H, C6
H5CH2O-); 5.19(t, 1H, -CONH-); 7.29(s, 5H, C6H5-). Z-
Pheol and Z-Valol were obtained analogously.
1H NMR (CDCl3, 200 MHz, d ppm): 2.85(d, 2H,
J=7 Hz, -CH2C6H5); 4.08±4.13(m, 1H, (CHCH2-); 6.15
(2H, broad, -NHCONH2); 7.1±7.38(m, 6H, -C6H5 and
-CH=); 8.3(d, 2H, J=7 Hz, NH2CH<); 9.85(1H,
broad, -NHCO-)
Valal-Sem. This was obtained analogously. The yield
was 198 mg (98%). [a]2D0=20 (c=1, MeOH), HPLC
retention time Ð 2.01 min (System A).
Z-Valol. (CDCl3, 200 MHz, d ppm): 0.91(t, 6H, -CH
(CH3)2); 1.82(m, 1H, -CH(CH3)2); 2.55(s, 1H, -CH2
OH); 3.44(m, 1H, >CHCH(CH3)2); 3.62(m, 2H, -CH2
OH); 5.0(s, 1H, -CONH-); 5.04(q, 2H, C6H5CH2-); 7.32
(m, 5H, C6H5-)
1H NMR (CDCl3, 200 MHz, d ppm): 0.9(t, 6H, -CH
(CH3)2); 1.85(m, 1H, -CH(CH3)2; 3.43(m, 1H, (CHCH
(CH3)2; 6.3(s, 2H, -CONH2); 7.15(d, 1H, -CH=N-);
8.35(s, 2H, NH2CH<); 9.9(s, 1H,=N-NHCO-).
Z-Pheol. (CDCl3, 200 MHz, d ppm): 2.9(d, 2H, J=7 Hz,
C6H5CH2CH-); 3.5±3.8(m, 3H, C6H5CH2CH- and -CH2
OH); 3.95(1H, broad, -OH); 5.0(1H, broad, -NH); 5.2(s,
2H, C6H5CH2O-); 7.12±7.45(m, 10H, 2C6H5-)
Oxidation of peptidyl amino alcohols with DMSO:Ac2O
mixture
Z-Ala-Ala-Pheal. Peptide alcohol Z-Ala-Ala-Pheol
(114.5 mg, 269 mmol) was dissolved in DMSO (4.5 mL),
then freshly distilled (CH3CO)2O (508 mL 5.38 mmol)
was added. The reaction mixture was shaken for 3 h at
37 ꢀC, then diluted with 8 ml of water and freeze-dried.
The total yield of Z-Ala-Ala-Pheal according to the
HPLC data was equal to 83%. HPLC retention time
9.82 min (SystemA). The resulted peptide aldehyde was
converted into its semicarbazone without puri®cation.
The yield of Z-Ala-Ala-Pheal-Sem after puri®cation by
¯ash-chromatography in chloroform:ethyl acetate (7:3)
was 62 mg (48%).
Oxidation of amino alcohols with CrO3 *Py complex in
the presence of Ac2O
Z-Valal. PyÂCrO3 (805 mg; 4.5 mmol) was suspended
in dichloromethane (6.5 ml) and then Z-Valol (483 mg;
2 mmol) and acetic anhydride (1.63 ml; 17.4 mmol) were
added. The mixture was stirred for a period of 18 min at
16ꢀC. Conversion of Z-Valol to Z-Valal according to
the HPLC data was 80%, retention time of Z-Valal was
12.9 min (System A). The reaction mixture was supplied
on silica column (3Â3.5sm) and eluted with ethyl ace-
tate (20 ml). The solvent was evaporated. The resulted
aldehyde was converted into its semicarbazone without
puri®cation. (See below). The yield of Z-Valal-Sem after
puri®cation by ¯ash-chromatography in chloroform:
ethyl acetate (7:3) was 375 mg (63%)
Enzymatic synthesis
Z-Ala-Ala-Pheal-Sem. Z-Ala-Ala-OCH3 (154 mg, 0.5 mmol)
and Pheal-Sem (51.5 mg, 0.25 mmol) were dissolved in
DMF (0.75 mL) and CH3CN (14.25 mL), then silo-
chrom (2.5 g) containing subtilisin 72 (200 mg) was
added. After shaking for 20 h at 20ꢀC the catalyst was
®ltered o€, rinsed with a mixture of DMF and
CH3CN, the ®ltrates combined and evaporated in
vacuo. The yield of the product according to the HPLC
data was 91% (System B, retention timeÐ16.8 min).
The resulted peptidyl amino aldehyde semicarbazone
Preparation of semicarbazones
Z-Pheal-Sem. Z-Pheol (855 mg, 3 mmol) was dissolved
in DMSO (25 mL), then freshly distilled (CH3CO)2O
(2.8 mL, 30 mmol) was added. The reaction mixture was
shaken for 3.5 h at 37ꢀC, then diluted with 50 ml of water
and freeze-dried. The resulting acyl amino aldehyde was
converted into its semicarbazone without puri®cation.

T. L. Voyushina et al. / Bioorg. Med. Chem. 7 (1999) 2953±2959
2959
was puri®ed by ¯ash-chromatography in chloroform:
ethyl acetate (7:3), then eluted with C2H5OH. The yield
of Z-Ala-Ala-Pheal-Sem was 90 mg (75%), [a]2D0=-40
(c=1, methanol).
Russian Foundation for Fundamental Research (Grant
97-04-48485, Grant 97-03-33039)
References
1H NMR (CDCl3, 200 MHz, d ppm): 1.25(d, 9H,
J=7 Hz, >CHCH3); 4.02±4.12(m, 1H, >CHCH=);
4.18±4.32(m, 2H, >CHCO-); 5.05(s, 2H, -OCH2C6H5);
6.2(2H, broad, -CONH2); 7.12(s, 1H, -CH=); 7.32(s, 5H,
-C6H5); 7.75±8.0(m, 3H, -CONHCH>); 9.9(1H, broad,
-NHCO-)
1. Tompson, R. Biochem. 1973, 12, 47.
2. Westerik, J.; Wolfenden, R. J. Biol. Chem. 1972, 25, 8195.
3. Brayer, G.; Delbaere, L.; James, M.; Bauer, C.-A.; Thomp-
son, R. Proc. Natl. Acad. Sci. 1979, 76, 96.
4. Bajusz, S.; Szell, E.; Bagdy, D.; et al. J. Med. Chem. 1990,
33, 1729.
5. Bullock, T.; Breddam, K.; Remington, J. J. Mol. Biol. 1996,
255, 714.
Removal of protecting group
6. Kammermeier, B.; Beck, G.; Jakobi, D.; Jendrella, H.
Angew. Chem., Int. Ed. Engl. 1994, 33, 685.
7. Damodaran, A.; Harris, R. B. J.Prot.Chem. 1995, 14, 431.
8. Someno, T.; Saino, T.; Katon, K.; Miyazaki, H.; Ishii, S. J.
Biochem. 1985, 97, 1493.
9. Nishikata, M.; Kasai, K.; Ishii, S. Biochim. Biophys. Acta
1981, 660, 256.
10. Yokosawa, H.; Nishikata, M.; Ishii, S. J.Biochem 1984, 95,
1819.
Z-Ala-Ala-Pheal-Sem (12 mg, 25 mmol) was dissolved in
CH3OH (1.6 mL) and) 0.5 N HCl (850 mL), then
(CH3CO)CH2 (51 mL, 0.5 mmol) was added. The reac-
tion mixture was shaken for 4.5 h at 20ꢀC. The solvent
was evaporated and the resulted peptide aldehyde was
puri®ed by ¯ash-chromatography in chloroform:ethyl
acetate (7:3) on silica. The yield of Z-Ala-Ala-Pheal was
8 mg (76%). HPLC retention time 10.43 min (System
A). [a]2D0=-63 (c=1, methanol).
11. McConell, R.; Barnes, G.; Hyong, C.; Gunn, M. J. Med.
Chem. 1990, 33, 86.
12. Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1982, 30,
1924.
13. Vinitsky, A.; Cardozo, C.; Sepp-Lorenzino, L.; Michaud,
C.; Orlowsky, M. J. Biol. Chem. 1994, 269, 29860.
14. Potetinova, J.; Voyushina, T.; Stepanov, V. Bioorg. Med.
Chem. Letts. 1997, 7, 705.
1H NMR (CDCl3, 200 MHz, d ppm): 1,98 (d, 6H, J=7 Hz,
(CHCH3); 3,42 (d, 2H, J=6 Hz, C6H5CH2CH>); 4.08±
4.25 (m, 2H, >CHCH3); 4.32±4.62 (m, 1H, C6H5CH2
CH<); 5.12 (s, 2H, C6H5CH2CO-); 6.45 (2H, broad, NH);
7.08±7.5(m, 11H, 2C6H5- and NH); 9.6(s 1H, -CHO).
15. Albright, J. D.; Goldman, L. J. Am. Chem. Soc. 1967, 89,
2416.
16. Stan®eld, G.; Parker, J.; Kanellis, P. J. Org. Chem. 1981,
46, 4797.
17. Rittle, K.; Homnick, C.; Ponticello, G.; Evans, B. J. Org.
Chem. 1982, 47, 3016.
18. Cainelli, G.; Casdillo, G.; Orena, U.; Sandri, S. J. Am.
Chem. Soc. 1979, 98, 6737.
19. Garegg, G.; Samuelsson, B. Carbohydrate Res. 1978, 67,
267.
20. Ito, A.; Takahashi, K.; Miura, C.; Baba, Y. Chem. Pharm.
Bull. 1973, 23, 3106.
21. Ito, A.; Takahashi, K.; Baba, Y. Chem. Pharm. Bull. 1975,
23, 3081.
Inhibitory tests
The kinetic parameters for subtilisin-or a-chymotrypsin-
catalyzed hydrolysis of the corresponding chromogenic
substrates were determined under the condition of
initial rate measurements from double reciprocal plots
1
1
of v versus [S] both in the presence and in the
absence of peptide aldehydes. Conversion of the sub-
strates did not exceed 10% for 10±15 min. Each experi-
mental set was performed in triplicate.
22. Ried, W.; Muhle, G. J. Lieb. Ann. Chem. 1962, 656, 119.
23. Seki, H.; Koga, K.; Matsuo, H. Chem. Pharm. Bull. 1965,
13, 995.
Acknowledgements
This work was partially supported by National Program
``Protein Engineering'' (Grant 03.003H-333) and by
24. Stepanov, V.; Terent'eva, E.; Voyushina, T.; Gololobov,
M. Bioorg. Med. Chem. 1995, 3, 479.