Protease-catalysed synthesis of peptides containing histidine and lysine

Article abstract of DOI:10.1016/S0957-4166(98)00134-7

The kinetically controlled α-chymotrypsin- and trypsin-catalysed syntheses of peptides starting from simple acyl donor esters containing histidine at the P₁-position (nomenclature according to Schechter and Berger) and lysine derivatives as amino components were examined on the basis of their kinetic parameters. Despite higher specificity constants (k(cat)/K(M)) of trypsin-catalysed ester hydrolysis, α-chymotrypsin- catalysed acyl transfer to N(ε)unprotected lysine derivatives gave higher peptide yields as compared to trypsin-catalysed reactions, whereas in acyl transfer to N(ε)-protected lysine derivatives the trypsin-catalysed reaction gave higher yields. α-Chymotrypsin-catalysed acyl transfer reactions in frozen systems demonstrated the yield-enhancing effect of freezing. Using specific ester leaving groups, both the amount of enzyme and the reaction time can be reduced. In frozen systems the ε-amino function of H-Lys-OH acts as an acyl acceptor at pH ≤9.

Full text of DOI:10.1016/S0957-4166(98)00134-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1505–1518
Protease-catalysed synthesis of peptides containing histidine and
lysine
Karin Beck-Piotraschke and Hans-Dieter Jakubke ∗
Universität Leipzig, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Institut für Biochemie, Talstr. 33,
D-04103 Leipzig, Germany
Received 3 February 1998; accepted 30 March 1998
Abstract
The kinetically controlled α-chymotrypsin- and trypsin-catalysed syntheses of peptides starting from simple
acyl donor esters containing histidine at the P1-position (nomenclature according to Schechter and Berger1) and
lysine derivatives as amino components were examined on the basis of their kinetic parameters. Despite higher
specificity constants (kcat/KM) of trypsin-catalysed ester hydrolysis, α-chymotrypsin-catalysed acyl transfer to Nε-
unprotected lysine derivatives gave higher peptide yields as compared to trypsin-catalysed reactions, whereas in
acyl transfer to Nε-protected lysine derivatives the trypsin-catalysed reaction gave higher yields. α-Chymotrypsin-
catalysed acyl transfer reactions in frozen systems demonstrated the yield-enhancing effect of freezing. Using
specific ester leaving groups, both the amount of enzyme and the reaction time can be reduced. In frozen systems
the ε-amino function of H–Lys–OH acts as an acyl acceptor at pH ≥9. © 1998 Published by Elsevier Science Ltd.
All rights reserved.
1. Introduction
The biological activity of peptides containing His-Lys sequences is based on their function as a carrier
for ionic copper(II).2 In particular, the tripeptide H–Gly-His-Lys–OH has been reported to have broad
biological activity. This tripeptide is believed to influence the growth of a variety of differentiated cells
and is suggested to have an important role in several physiological processes, e.g. wound healing, tissue
repair or immunostimulation.3
Due to the stereo- and regiospecificity of enzymes there is no need for side chain protection and,
furthermore, the danger of racemisation is excluded. The main goals in thermodynamically and kinet-
ically controlled syntheses are the equilibrium shift towards the peptide products, and the prevention
of undesired hydrolysis of the acyl donor and proteolysis of peptide products, respectively. A broad
∗
Corresponding author. E-mail: bordusa@rz.uni-leipzig.de
0957-4166/98/$19.00 © 1998 Published by Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00134-7

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K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
methodological repertoire of media-, substrate- and enzyme-engineering4–11 has been published. In
kinetically controlled processes, which are limited to serine and cysteine proteinases, less enzyme and
shorter reaction times are required. Studies of the catalytic mechanism of serine and cysteine proteinases
have revealed that the first step of an acyl transfer reaction is the acylation of the enzyme by a weakly
activated amino acid or peptide ester, respectively. In the presence of a nucleophilic amino component,
the acyl-enzyme intermediate is deacylated competitively by water and the added nucleophile, as shown
in Scheme 1.12
Scheme 1. Kinetic scheme of protease-catalysed acyl transfer reactions: EH=free enzyme; Ac-X=acyl donor;
E· · ·Ac-X=Michaelis complex; HX=leaving group; AcOH=hydrolysis product; Ac-N=aminolysis product; HN=nucleophile;
Ac-E=acyl-enzyme; Ac-E· · ·HN=acyl-enzyme–nucleophile complex
The properties of acyl donors can be expressed by the kinetic parameters of enzymatic hydrolysis.13
The efficiency of the nucleophilic amino component to an acyl-enzyme is expressed by the partition
values (p). The p value was introduced as a parameter for estimating nucleophile reactivity and character-
izing the S0 binding side of an enzyme. The partition value corresponds to the nucleophile concentration
at which aminolysis and hydrolysis proceed with the same velocity.14,15
In the present paper we report on the results of α-chymotrypsin- and trypsin-catalysed formation of
His-Lys bonds both in aqueous and frozen aqueous systems.
2. Results and discussion
2.1. α-Chymotrypsin- and trypsin-catalysed hydrolysis of acyl donor esters containing histidine in the
P1-position
Firstly, the α-chymotrypsin- and trypsin-catalysed hydrolysis of a series of acyl donors containing
histidine in the P1 position and various protecting and leaving groups was examined. The kinetic
parameters of hydrolysis are compiled in Table 1. Comparing both enzymes, the acyl donor esters
used were slightly better substrates for trypsin in comparison with α-chymotrypsin. In trypsin-catalysed
hydrolysis, the specificity constants (kcat/KM) were about one order of magnitude higher compared with
the corresponding data of α-chymotrypsin-catalysed reactions. The use of the 4-nitrobenzyl ester leaving
group instead of the methyl ester moiety led to higher values of kcat/KM for both α-chymotrypsin- and
trypsin-catalysed hydrolysis. These results are in agreement with literature data.13 In the case of α-
chymotrypsin-catalysed hydrolysis of Y–His–OR (Y=Mal, Z–Gly; R=Me, Nb),† KM decreases with
increasing hydrophobicity of the leaving group. On the other hand, there are increasing kcat values.
†
Abbreviations: ACN, acetonitrile; Boc–, tert-butyloxycarbonyl; BzSO3H, benzylsulfonic acid; DTE, dithioerythritol; ED-
TA, ethylenediaminetetraacetic acid; HEPES, N-(2-hydroxyethyl)piperazine-N0-(2-ethanesulfonic) acid; Mal–, maleyl; Mca–,

K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
1507
Table 1
Kinetic constants of α-chymotrypsin- and trypsin-catalyzed hydolysis
From these data an improved suitability of histidine subtrates bearing hydrophobic leaving groups can be
concluded.
2.2. α-Chymotrypsin- and trypsin-catalysed acyl transfer of histidine esters to lysine derivatives
In order to compare both enzymes, we examined the α-chymotrypsin- and trypsin-catalysed acyl
transfer of histidine esters (Mal–His–ONb, Z–His–OMe, Z–Gly-His–ONb) to H–Lys–NH2, H–Lys–OMe
and H–Lys(Z)0–NH2. The results are summarized in Table 2. From these data it can be established that
due to the S1 -specificity of α-chymotrypsin for basic amino acids15, α-chymotrypsin favoured the
Nε-unprotected nucleophiles H–Lys–NH2 and H–Lys–OMe, whereas trypsin-catalyzed acyl transfer to
H–Lys(Z)–NH2 provided the highest efficiency.
The results of chymotrypsin- and trypsin-catalysed syntheses of Mal–His-Lys–NH2, Mal–His-
Lys(Z)–NH2 and Mal–His-Lys–OMe were reflected by their partition values for the corresponding acyl
transfer reaction, as shown in Table 3.
0
According to the S1 specificity of α-chymotrypsin, H–Lys–NH2 acts as an efficient acyl acceptor
whereas H–Lys(Z)–NH2 is a less efficient nucleophile followed by H–Lys–OMe. The lower partition
value for H–Lys–NH2 is well documented in the literature15 and is caused mainly by a hydrogen bond
0
formed between the amide group at the P2 -position and Phe-41 of α-chymotrypsin, which plays an
important role in the orientation of the nucleophile.16,17 Besides favourable electrostatic interactions,15
a second hydrogen bond is formed between the side chain amino group of lysine and Cys-58 of the
enzyme in α-chymotrypsin-catalysed reactions.18 We propose that in our model reactions H–Lys–NH2
monochloracetyl; –NH2, amide; Nps–, 2-nitrophenylsulfenyl; –OCam, carboxamido methyl ester; –OMe, methyl ester; –ONb,
4-nitrobenzyl ester; TFA, trifluoroacetate; TPCK, N-tosyl-L-phenylalanine chloromethylketone; Z–, benzyloxycarbonyl.

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K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
Table 2
Protease-catalysed condensation of histidine esters with lysine derivatives
Table 3
p0 -Values of α-chymotrypsin- and trypsin-catalysed acyl transfer of Mal–His–ONb to lysine deriva-
tives
and H–Lys(Z)–NH2 have favourable orientations as a consequence of forming both of these hydrogen
bonds. The comparatively low partition value of H–Lys–OMe, which is not typical for nucleophilic ester
0
substrates, is based on these favourable interactions in the S1 binding region. The partition value obtained
for the α-chymotrypsin-catalysed acyl transfer of Mal–His–ONb to H–Lys–NH2 corresponds to acyl
transfer data of Mal–Phe–OMe to H–Arg–OMe.17
The lower efficiency of the Nε-unsubstituted lysine derivatives H–Lys–NH2 and H–Lys–OMe in
trypsin-catalysed acyl transfer leads to higher partition values compared with α-chymotrypsin-catalysed
reactions. Comparing both enzymes, the affinity of trypsin for H–Lys–NH2 is 6.7 times less efficient
than for α-chymotrypsin, while H–Lys–OMe differs by about one order of magnitude. The efficiency
of H–Lys(Z)–NH2 indicated for trypsin-catalysed reactions is higher than for unprotected H–Lys–NH2
which corresponds to the partition values found in trypsin-catalysed acyl transfer of Nps–Arg–OMe to
H–Lys-Leu–NH2 and H–Lys(Ac)-Leu–NH2,19 respectively. According to published data17 for nucle-

K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
1509
Table 4
α-Chymotrypsin-catalysed dipeptide synthesis using Mal–His–OR (R=Nb, Me) and lysine derivatives
as nucleophiles at 25°C compared with −25°C
ophilic ester substrates, the absence of this important hydrogen bond mentioned above in the case of
H–Lys–OMe causes the drastic increase of the partition value.
2.3. The effect of freezing in chymotrypsin-catalysed condensations of Mal–His–ONb with lysine deriva-
tives
It has been shown previously that freezing the reaction mixture can increase the peptide yield in
protease-catalysed peptide syntheses also in the case of less efficient nucleophiles, e.g. free amino acids
and amino acid esters (for a review, see the literature20).
We investigated the α-chymotrypsin-catalysed acyl transfer of Mal–His–OR (R: Me, Nb) to dif-
ferent lysine derivatives (H–Lys–NH2, H–Lys(Z)–NH2, H–Lys–OMe, H–Lys–OH). The results of α-
chymotrypsin-catalysed reactions at room temperature compared with those in frozen aqueous systems
are shown in Table 4. These data clearly demonstrate that freezing could also improve the peptide yield
when less efficient nucleophiles were used. We found a slight increase of peptide yield when using a
more specific acyl donor, Mal–His–ONb at room temperature compared with Mal–His–OMe. In frozen
systems, the synthesis rate can be increased by the application of specific leaving groups. This effect is
very important for practical purposes.
2.4. Regiospecificity of chymotrypsin-catalysed coupling of Mal–His–ONb with Nε-unprotected lysine
derivatives
It is well established that freezing can induce a dramatic change in the specificity of enzymes.20 Due
to this shift of specificity, we investigated the effect of freezing and pH on α-chymotrypsin-catalysed
coupling of Mal–His–ONb with Nα,Nε-unprotected lysine derivatives H–Lys–NH2 and H–Lys–OH.
As shown in Fig. 1, we found that α-chymotrypsin-catalysed synthesis of Mal–His-Lys–NH2 gave
one product in high yield independent of the temperature and pH. In the acyl transfer reaction from

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K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
Fig. 1. Dependence of regiospecificity of acyl transfer from Mal–His–ONb to H–Lys–NH2 and H–Lys–OH on temperature and
pH. Reaction conditions: [Mal–His–ONb]=2 mM, [nucleophile]=50 M, 10% (v/v) DMSO; pH 9; (+25°C: 0.1 M carbonate
buffer; −25°C: adjusted with 1 N NaOH before freezing); [α-chymotrypsin]=9.27 µM
Table 5
1 H-NMR chemical shift data of α-methine and ε-methylene protons in lysine (solvent D2O)
Mal–His–ONb to H–Lys–OH, no product was detected at room temperature. In contrast, we observed
the formation of two products at pH ≥9 in frozen reaction mixtures.
1
Comparing the H-NMR chemical shift data of α-methine and ε-methylene protons of the lysine
moiety of isolated products with those of lysine compounds, a significant downfield shift was observed
for signals of α-methine protons of the product of acyl transfer to lysine amide and of the main product
of acyl transfer to lysine, while chemical shifts of ε-methylene protons showed small differences. The
proton signals of the α-methine and ε-methylene groups of the by-product in acyl transfer reactions to
lysine indicated a strong downfield shift of ε-methylene protons and no significant change in α-methine
signals (see Table 5).

K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
1511
These data verify the formation of α-peptide bonds using H–Lys–NH2 as the amino component. The
main product of acyl transfer reactions to H–Lys–OH in a frozen system was Mal–His-Lys–OH. The
formation of the ε-isomer H–Lys(Mal–His)–OH as a by-product was observed at pH ≥9. The amount
of the ε-isomer in the reaction mixture increased with increasing pH and deprotonation of the ε-amino
group, respectively. We assume that this undesired side reaction is caused by an unfavourable orientation
of H–Lys–OH in the active site since no hydrogen bond can be formed between this substrate and Phe-41
of α-chymotrypsin. In addition, unfavourable ionic interactions between the carboxyl group of lysine
and the S0 subsite of α-chymotrypsin may occur.
3. Conclusions
The results of the present study show that the stereo- and regiospecificity of proteases can be
favourably exploited for the synthesis of peptides containing His-Lys sequences. α-Chymotrypsin
and trypsin are capable of hydrolysing esters containing a histidine moiety at the P1-position and
catalyse acyl transfer to Nε-protected and Nε-unprotected lysine amides. In frozen aqueous reaction
mixtures even inefficient amino components, such as H–Lys–OMe and H–Lys–OH, were accepted by
α-chymotrypsin, demonstrating the yield-enhancing effect of freezing. Rates of peptide synthesis could
be markedly increased using specific hydrophobic leaving groups. This finding is very important for
practical purposes, since side reactions can be suppressed and the reaction time can be dramatically
shortened. In frozen aqueous systems, unprotected Nε-amino functions of inefficient lysine derivatives
provide ε-isomers at pH ≥9 as by-products.
4. Experimental
4.1. Apparatus, materials and methods
Melting points were determined on a Boetius heating block and are uncorrected. The NMR spectra
were obtained with a Varian Genius 300 spectrometer using CDCl3, D2O or DMSO-d6 as internal
standards. Mass spectra were obtained with a Hewlett–Packard VG ZAB-HSQ (FAB method), a 5989
A (thermospray method) and a Kratos Kompakt MALDI 5 V5.1.2 (MALDI method), respectively.
Elementary analyses were performed on a Heraeos Analyser CHN-O Rapid.
4.2. Chemicals and enzymes
α-Chymotrypsin (EC 3.4.21.1) was a product of Serva (Germany), trypsin (EC 3.4.21.2, TLCK-
treated) was purchased from Fluka (Germany). Both enzymes were used without further purification.
Normalities of enzyme-stock solutions were determined by active site titration according to general pro-
cedures described to be 61% (α-chymotrypsin, 4-nitrophenylacetate) and 50% (trypsin, 4-nitrophenyl-
40-guanidinobenzoate), respectively. Amino acids, amino acid amides and Boc–His–OMe were used
from Bachem (Switzerland). H–Lys(Z)–OH was a generous gift from Degussa AG Hanau (Germany).
Z–His–OH was a product of Rexim (France). All other substances were synthesized as described below.

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K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
4.3. Specificity studies
The specificity constants of substrates were determined potentiometrically using a pH-stat video
titrator VIT 90 (Radiometer, Copenhagen). The reactions were carried out at 25°C and pH 7.5. The
total volume was adjusted to 2 ml, containing 0.2 M NaCl and 10% (v/v) DMF. Substrate concentrations
were varied from 0.2 KM to 5 KM. Reactions were started by addition of the enzyme solution. A final pH
of 7.5 was adjusted by addition of 0.02 N NaOH. The kinetic parameters KM and Vmax were obtained
by nonlinear curve fitting of the data (ENZFITTER, Elsevier Biosoft, Cambridge, UK). The kcat values
were determined as a value of Vmax/[E0].
4.4. HPLC
The samples were analysed by RP-HPLC. Analyses were performed using a gradient system (Shi-
madzu LC 6A, Shimadzu, Japan) equipped with an autoinjector SIL-6A, 2 pumps LC-6A, UV-detector
SPD-6AV, a system controller SCL-6A and an integrator S-R4A chromatopac. A Lichrosorb RP-18
column with 7 µm particle size (250×4 mm2; E. Merck, Germany) and an Impaq RP 18 column
with 10 µm particle size (250×4 mm2; Bischoff, Germany) were used. Samples were eluted at a flow
rate of 1 ml/min by a gradient elution system using mixures of 2–60% acetonitrile in 0.1% aqueous
trifluoroacetic acid (Mal–His-Lys(Z)–NH2) and 2–35% methanol in 0.1% aqueous trifluoroacetic acid
(all other peptides) respectively. The substrate and peptide ratio was monitored at 254 nm. When
the hydrolysis and aminolysis products contained the same chromophoric group, the molar extinction
coefficients were assumed to be equal. When the acyl donor contained the 4-nitrobenzyl leaving group,
the ratio of absorbtion coefficients was determined.
Preparative RP-HPLC was perfomed on the HPLC system described above using an Impaq RG 1010
C-18 column with 10 µm particle size (250×12.5 mm2; Bischoff, Germany) with a precolumn at a flow
rate of 2.5–5 ml/min. Absorbance was monitored at 254 nm.
4.5. Peptide syntheses on an analytical scale
Peptide synthesis reactions at −25°C were performed in 1.5 ml polypropylene tubes at a total sample
volume of 0.1 ml. The nucleophile (5 µmol) dissolved in 80 µl of water, was adjusted with 1 N NaOH to
give final pHs of 7.5, 8.0, 9.0, 10.0 and 11.0, respectively. After addition of the reaction components in the
order enzyme (10 µl; concentrations see tables) and acyl donor (0.2 µmol of acyl donor dissolved in 10 µl
of DMSO), the tubes were shaken and placed in liquid nitrogen for 20 s to achieve shock-freezing. Then
they were transferred for the time of reaction into a constant temperature cryostat (HAAKE, Germany).
After a predefined time, the reactions were stopped by adding 0.8 ml of 0.7 N acetic acid in water. The
reactions at room temperature were performed in the same manner as described without freezing using a
total volume of 1 ml, containing 0.1 M phosphate buffer and carbonate buffer, respectively.
4.6. Determination of partition values
Acyl transfer reactions were carried out at 25°C in 1 ml of solution containing 0.1 M carbonate buffer
(pH 9.5) and 0.25 M NaCl. The stock solutions of acyl donor (20 mM in DMSO), nucleophile (100 mM in
water) and enzyme (46 µM α-chymotrypsin and 20 µM trypsin, respectively) were prepared daily. After
mixing the stock solutions of the nucleophile (5–250 µl) and enzyme (10 µl), the reactions were started
by addition of 100 µl of acyl donor stock solution to give a final acyl donor concentration of 2 mM and

K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
1513
nucleophile concentrations of 2 mM, 4 mM, 10 mM, 20 mM, 50 mM and 100 mM, respectively. When
H–Lys–OMe was used as nucleophile, the reaction was started by addition of 10 µl of enzyme stock
solution. After 5–15% ester consumption, a sample was diluted in 0.7 N acetic acid and analysed by
RP-HPLC as described above. In order to determine non-enzymatic ester hydrolysis, a blank experiment
without the enzyme was perfomed. Three independent reactions with equal starting concentrations were
carried out. Calculation of partition values p0 was perfomed using the method of Schellenberger et al.21
4.7. Mal–His–OMe
Gaseous NH3 (dried over NaOH) was bubbled through a suspension of 6.03 g (25 mmol) of H–His-
OMe·2HCl in 145 ml of dry CHCl3 for 15 min at 0°C. The precipitated NH4Cl was filtered off and
washed three times with 10 ml of dry CHCl3. The filtrate was evaporated, the obtained oily residue was
dissolved in 10 ml of dry methanol and evaporated again to remove all NH3. The clear oil was dissolved
in 50 ml of dry CHCl3, then a solution of 2.5 g (25 mmol) of maleic anhydride in 20 ml of dry ether
was added. The reaction mixture was stirred for 2.5 h at room temperature, then 70 ml of ether was
added and the precipitate formed was filtered off. The product was washed twice with 10 ml of ether,
dried over KOH/P2O5 and crystallized twice from methanol containing 1% water, C11H13N3O5=267.24.
Yield: 4.27 g (64%); m.p.: 136–138°C; m/z (FAB): 268 (M+H)+; 1H-NMR (400 MHz; D2O): 3.12–3.26
(m, 2H, His–CβH2), 3.67 (s, 3H, O–CH3), 4.70–4.76 (m, 1H, His–CαH), 5.87 (d, 1H, HOOC–CH_),
6.25 (d, 1H, _CH–CO–NH–), 7.19 (s, 1H, >C_CH–NH–), 8.47 (s, 1H –N_CH–NH–). Anal. calcd: C,
49.44; H, 4.90; N, 15.72; found: C, 48.83; H, 4.99; N, 15.32.
4.8. Mal–His–ONb
The preparation of Mal–His–ONb was performed analogously to the procedure described for
Mal–His–OMe starting from 6.06 g (10 mmol) of H–His–ONb·2BzSO3H and 1.0 g (25 mmol) of maleic
anhydride to give Mal–His–ONb. The product was crystallized from methanol, C17H16N4O7=388.34.
1
Yield: 2.82 g (67%); m.p.: 119–121°C; m/z (FAB): 389 (M+H)+; H-NMR (400 MHz; DMSO-
d6): 2.93–3.08 (m, 2H, His–CβH2), 4.68 (t, 1H, His–CαH), 5.25 (s, 2H, –OCH2–), 6.28 (s, 2H,
HOOC–CH_CH–CONH–), 6.89 (s, 1H, >C_CH–NH–), 7.53 (d, 2H, _CH–C(CH2–)_CH–), 7.68 (s,
1H –N_CH–NH–), 8.21 (d, 2H, _CH–C(NO2)_CH–), 9.50 (1H, brs, –NH–). Anal. calcd: C, 50.25; H,
4.64; N, 13.75; found: C, 50.94; H, 4.58; N, 14.09.
4.9. Mca–His–ONb
The synthesis of Mca–His–ONb was similar to the preparation of Mal–His–OMe starting from
3.03 g (5 mmol) of H–His–ONb·2BzSO3H and 0.85 g (5 mmol) of monochloracetic acid anhydride
to give Mca–His–ONb. The crude product was crystallized from acetyl acetate/methanol/n-hexane,
1
C15H15N4O5Cl=366.45. Yield: 0.68 g (42%); m.p.: 120–124°C; m/z (TS-POS): 367 (M+H)+; H-
NMR (300 MHz; DMSO-d6): 3.00 (d, 2H, His–CβH), 4.13 (s, 2H, Cl–CH2–), 4.65 (q, 1H, His–CαH),
5.25 (s, 2H, –OCH2–), 6.87 (s, 1H, >C_CH–NH–), 7.53 (d, 2H, _CH–C(CH2–)_CH–), 7.67 (s, 1H
–N_CH–NH–), 8.21 (d, 2H, _CH–C(NO2)_CH–), 8.78 (1H, d, –NH–). Anal. calcd: C, 49.12; H, 4.09;
N, 15.28; found: C, 48.5; H, 4.33; N, 14.56.

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K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
4.10. Z–His–OMe
Z–His–OH (1 g, 3.46 mmol) was suspended in 50 ml of dry methanol at 0°C. Then 0.93 ml (12.7
mmol) of SOCl2 were added slowly. The reaction mixture was incubated under stirring for 1 h at 0°C,
then 20 h at room temperature. The solvent was evaporated, the oily residue was dissolved in 5 ml of
methanol and the pH was adjusted to 7 with triethylamine. After adding 150 ml of dry ether, triethylamine
hydrochloride precipitated. The salt was filtered off and washed twice with 10 ml of dry ether. The filtrate
and the washings were evaporated and the oily residue was washed several times with ether containing
n-hexane to give solid Z–His–OMe, C15H17N3O4=303.32. Yield: 0.84 g (42%); m.p.: 72–73°C; m/z
1
(TS-POS): 304 (M+H)+; H-NMR (300 MHz; DMSO-d6): 2.80–8.98 (m, 2H, His–CβH), 3.60 (s, 3H,
O–CH3), 4.28–4.38 (dd, 1H, His–CαH), 5.01 (s, 2H, –OCH2–), 6.83 (s, 1H, >C_CH–NH–), 7.28–7.41
(m, 5H, –C6H5), 7.59 (s, 1H –N_CH–NH–), 7.67 (d, 1H, –NH–). Anal. calcd: C, 59.40; H, 5.65; N,
13.85; found: C, 59.22; H, 5.07; N, 14.09.
4.11. Z–Gly-His–OMe
Z–Gly–OCam (0.222 g, 0.836 mmol; prepared using a modified procedure according to Martinez et
al.22 by reaction of Nα-benzyloxycarbonylglycine with iodoacetamide in the presence of dicyclohexyl-
amine in dry dioxan) was suspended in 125 µl of HEPES/Na+-buffer (1 M, pH 7.9), containing 0.202
g (0.835 mmol) of H–His–OMe·2HCl. Concentrated aqueous ammonium hydroxide solution (225 µl)
was used to release the nucleophile from its salt and to adjust the pH. After addition of 12 µl of Triton
X-100 the reaction was started by addition of a solution of 40 mg papain and 8 mg DTE in 125 µl
HEPES/Na+ (1 M, pH 7.9) and 18 µl EDTA (4.67 mM). The reaction mixture was intensively mixed.
After complete ester consumption, the reaction mixture was dissolved in 20 ml of methanol and heated
to inactivate the enzyme. After filtration, the product was isolated by column chromatography (silica gel
60, 70–230 mesh ASTM; E. Merck, Germany) using CHCl3 containing 5–15% (v/v) methanol as eluents
to give Z–Gly-His–OMe, C17H20N4O5=360.35. Yield: 0.178 g (59%); m/z (MALDI): 361.89 (M+H)+;
1H-NMR (300 MHz; CDCl3): 3.05 (d, 2H, His–CβH), 3.62 (s, 3H, O–CH3), 3.81 (d, 2H, Gly–CH2), 4.74
(dd, 1H, His–CαH), 5.06 (s, 2H, –OCH2–), 6.28 (brs, 1H, –NH–), 6.72 (s, 1H, >C_CH–NH–), 7.21–7.37
(brs, 5H, –C6H5), 7.21–7.37 (d, 1H, –N_CH–NH–), 7.75 (d, 1H, –NH–), 8.29 (brs, 1H, NH).
4.12. Z–Gly-His–ONb
The dipeptide was prepared as described for Z–Gly-His–OMe starting from 400.8 mg (1.67 mmol) of
Z–Gly–OCam, 1.063 g (1.67 mmol) of H–His–ONb·2BzSO3H and 80 mg of papain. After filtration of
denaturated enzyme, the product was precipitated by addition of three volumes of distilled water. After
completing the precipitation at 4°C overnight, the peptide product was filtered off and dried over P2O5,
1
C23H23N5O7=481.47. Yield: 1.26 g (78%); m.p.: 125–130°C; m/z (TS-POS): 482 (M+H)+; H-NMR
(300 MHz; DMSO-d6): 2.98 (d, 2H, His–CβH2), 3.67 (d, 2H, Gly–CH2), 4.58–4.67 (m, 1H, His–CαH),
5.05 (s, 2H, O–CH2–CO–NH–), 5.22 (s, 2H, O–CH2–C6H4–NO2), 6.83 (s, 1H, >C_CH–NH–),
7.22–7.43 (m, 5H, –C6H5), 7.44–7.60 (brd, 4H, _CH–CH_C(NO2)–CH_CH–, –N_CH–NH– and
–NH–), 8.20 (d, 2H, –CH_C(NO2)–CH), 8.44 (d, 1H, –NH–). Anal. calcd: C, 57.38; H, 4.82; N, 14.55;
O, 23.26; found: C, 57.69; H, 5.14; N, 15.37; O, 22.90.

K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
1515
4.13. H–Lys(Z)–NH2·HCl
H–Lys(Z)–OH (1.4 g, 5 mmol) in 15 ml of dry methanol was reacted with 1.3 ml of SOCl2 as described
for Z–His–OMe. After removal of methanol and SOCl2, the product was fractionally crystallized from
1
methanol/ether. Yield: 1.15 g (77%) H–Lys(Z)–OMe·HCl [m.p.: 110–113°C, H-NMR (400 MHz,
DMSO): 1.22–1.32 (m, 2H, Lys–CγH2), 1.32–1.46 (m, 2H, Lys–CδH2), 1.78 (q, 2H, Lys–CβH2), 2.97
(q, 2H, Lys–CεH2), 3.72 (s, 3H, –OCH3), 3.96 (t, 1H, Lys–CαH), 4.99 (s, 2H, –CH2–O–CO–NH–),
7.26–7.38 (m, 5H, –C6H5), 8.62 (brs, 2H, –NH2)]. H–Lys(Z)–OMe·HCl (1 g, 3.45 mmol) was dissolved
in 50 ml of methanol (saturated at 0°C with dry NH3) and the mixture was allowed to stand for 8 days
at room temperature. After removal of methanol and NH3 the solid product was crystallized twice from
methanol:ether (1:5), C14H22N3O3Cl=315.78. Yield: 0.75 g (77%); m.p.=144–152°C, m/z (FAB): 280
1
(M+1)+, H-NMR (300 MHz, DMSO): 1.25–1.35 (m, 2H, Lys–CγH2), 1.35–1.50 (m, 2H, Lys–CδH2),
1.67–1.78 (q, 2H, Lys–CβH2), 2.92–3.04 (q, 2H, Lys–CεH2), 3.68 (t, 1H, Lys–CαH), 5.00 (s, 2H,
–CH2–O–CO–NH–), 7.23–7.41 (m, 5H, –C6H5), 7.50 (s, 1H, –NH–), 7.93 (s, 2H, –NH2), 8.1 (brs, 2H,
–NH2).
4.14. Mal–His-Lys–NH2
H–Lys–NH2 (109 mg, 0.5 mmol) and 4.7 mg of α-chymotrypsin were dissolved in 2.5 ml of carbonate
buffer (0.2 M, pH 9). The reaction was started by addition of 101.5 mg (0.25 mmol) of Mal–His–ONb in
1 ml DMSO. The mixture was stirred for 1 h at 25°C. After complete ester consumption, 7 ml of 0.7 N
acetic acid were added to inactivate the enzyme. The enzyme was filtered through a polyester membrane
(pore size 0.4 µm, Reichelt Chemietechnik, Germany) and the filtrate was freeze-dried. Elution from
silica gel 60 (70–230 mesh ASTM; E. Merck, Germany) using methanol:CHCl3:8% aqueous NH3
(5.1:3.4:1.5) provided the pure dipeptide, C16H24N6O5=380.39. Yield: 65 mg (68%); m.p.: 290°C
1
(decomp.); m/z (TS-POS): 381 (M+H)+; H-NMR (300 MHz; D2O): 1.32–1.60 (m, 2H, Lys–CγH2),
1.64–1.78 (m, 2H, Lys–CδH2), 1.78–2.02 (m, 2H, Lys–CβH2), 3.02 (t, 2H, Lys–CεH2), 3.13–3.37
(brm, 2H, His–CβH), 4.30 (dd, 1H, Lys–CαH), 4.71 (t, 1H, His–CαH), 6.02 (d, 1H, HOOC–CH_),
6.35 (d, 1H _CH–CO–NH), 7.26 (s, 1H, >C_CH–NH–), 7.99 (s, 1H, –N_CH–NH–). Preparation of
dihydrochloride was carried out by solvation of 10 mg of Mal–His-Lys–NH2 in 0.2 ml of 0.4 N HCl and
1
freeze-drying. H-NMR (300 MHz; D2O): 1.38–1.61 (m, 2H, Lys–CγH2), 1.65–1.97 (m, 4H, Lys–CδH2
and Lys–CβH2), 3.05 (t, 2H, Lys–CεH2), 3.10–3.42 (brm, 2H, His–CβH), 4.34 (dd, 1H, Lys–CαH), 4.71
(His–CαH), 6.33 (d, 1H, HOOC–CH_), 6.41 (d, 1H, _CH–CO–NH), 7.36 (s, 1H, >C_CH–NH–), 8.68
(s, 1H, –N_CH–NH–).
4.15. Mal–His-Lys(Z)–NH2
Preparation of Mal–His-Lys(Z)–NH2 was carried out as described for Mal–His-Lys–NH2 starting
from 40.6 mg (0.1 mmol) of Mal–His–ONb and 116.6 mg (0.4 mmol) of H–Lys(Z)–NH2 and 2.5
mg of α-chymotrypsin. The crude material was separated via RP-HPLC by gradient elution using
10–25% ACN in 0.1% trifluoroacetic acid as eluent. The product was obtained as the trifluoroacetate,
C24H30N6O7=514.52. Yield: 56.7 mg (91%); m/z (TS-POS): 515 (M+H)+; 1H-NMR (300 MHz; D2O):
1.30–1.47 (m, 2H, Lys–CγH2), 1.47–1.60 (m, 2H, Lys–CδH2), 1.73–1.89 (m, 2H, Lys–CβH2), 3.12–3.21
(m, 2H, Lys–CεH2), 3.21–3.40 (brm, 2H, His–CβH), 4.26–4.38 (m, 1H, Lys–CαH), 4.71–4.75 (m, 1H,
His–CαH), 5.14 (s, 2H, O–CH2), 6.31 (d, 1H, HOOC–CH_), 6.35 (d, 1H, _CH–CO–NH), 7,32 (s, 1H,
>C_CH–NH–), 7.39–7.53 (m, 5H, –C6H5), 8.65 (s, 1H, –N_CH–NH–).

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K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
4.16. Mal–His-Lys–OMe
H–Lys–OMe·2HCl (233 mg, 1 mmol) and 9.5 mg of α-chymotrypsin were dissolved in 3.58 ml water.
A solution of 101.5 mg (0.25 mmol) of Mal–His–ONb in 1 ml DMSO was added and the reaction was
started by addition of 0.42 ml of 5 N KOH. The mixture was quickly shaken and placed in liquid nitrogen
to achieve shock-freezing. Then it was incubated in a constant temperature thermostat at −25°C for three
days. After complete ester consumption, 5 ml of 3 N acetic acid was added and the enzyme was removed
by filtering through a polyester membrane (pore size 0.4 µm, Reichelt Chemietechnik, Germany).
The filtrate was freeze-dryed and seperated via isocratic preparative RP-HPLC using 2.5% acetonitrile
in 0.1% aqueous trifluoroacetic acid as eluent. The dipeptide was obtained as the ditrifluoroacetate,
1
C17H25N5O6=365.40. Yield: 43 mg (28%); m/z (TS-POS): 396 (M+H)+; H-NMR (300 MHz; D2O):
1.28–1.51 (m, 2H, Lys–CγH2), 1.55–1.77 (m, 2H, Lys–CδH2), 1.77–1.89 (m, 2H, Lys–CβH2), 2.89–3.02
(m, 2H, Lys–CεH2), 3.02–3.44 (brm, 2H, His–CβH), 3.71 (s, 3H, –OCH3), 4.28–4.48 (m, 1H, Lys–CαH),
4.63–4.71 (m, 1H, His–CαH), 6.30 (d, 1H, HOOC–CH_), 6.44 (d, 1H, _CH–CO–NH), 7.30 (s, 1H,
>C_CH–NH–), 8.62 (s, 1H, –N_CH–NH–).
4.17. Mal–His-Lys–OH (Mal–His-αLys–OH) and H–Lys(Mal–His)–OH (Mal–His-εLys–OH)
The preparation of Mal–His-Lys–OH and H–Lys(Mal–His)–OH was carried out as described for
Mal–His-Lys–OMe starting from 365 mg (2 mmol) of H–Lys–OH·HCl, 203 mg (0.5 mmol) of
Mal–His–ONb and 19 mg of α-chymotrypsin at pH 11. The freeze-dried residue was eluted from silica
gel 60 column (70–230 mesh ASTM; E. Merck, Germany) using methanol:CHCl3:8% aqueous NH3
(5.1:3.4:1.5) as eluent. The first fraction contained a mixure of Mal–His–OH and H–Lys(Mal–His)–OH,
which was further purified by isocratic elution via RP-HPLC using 2% methanol in 0.1% aqueous
tri-fluoroacetic acid as eluent to give H–Lys(Mal–His)–OH·2TFA, C16H23N5O6=381.37. Yield: 34
1
mg (12%); m/z (TS-POS): 382 (M+H)+; H-NMR (300 MHz; D2O): 1.36–1.53 (m, 2H, Lys–CγH2),
1.53–1.66 (m, 2H, Lys–CδH2), 1.89–2.04 (m, 2H, Lys–CβH2), 3.20–3.32 (m, 2H, Lys–CεH2), 3.32–3.42
(brm, 2H, His–CβH), 4.00 (t, 1H, Lys–CαH), 4.68–4.78 (m, 1H, His–CαH), 6.37 (d, 1H, HOOC–CH_),
6.53 (d, 1H, _CH–CO–NH), 7.36 (s, 1H, >C_CH–NH–), 8.68 (s, 1H, –N_CH–NH–). The second
fraction of column chromatography contained Mal–His-Lys–OH. Yield: 72 mg (38%); m.p.: 134–145°C;
1
m/z (TS-POS): 382 (M+H)+; H-NMR (300 MHz; D2O): 1.36–1.55 (m, 2H, Lys–CγH2), 1.64–1.84
(m, 2H, Lys–CδH2), 1.84–1.95 (m, 2H, Lys–CβH2), 3.03 (t, 2H Lys–CεH2), 3.12–3.40 (brm, 2H,
His–CβH), 4.13–4.22 (m, 1H, Lys–CαH), 4.71 (t, 1H, His–CαH), 5.97 (d, 1H, HOOC–CH_), 6.40
(d, 1H, _CH–CO–NH), 7.20 (s, 1H, >C_CH–NH–), 7.26 (s, 1H, –N_CH–NH–). Preparation of the
1
dihydrochloride of Mal–His-Lys–OH was carried out as described for Mal–His-Lys–NH2. H-NMR
(300 MHz; D2O): 1.38–1.58 (m, 2H, Lys–CγH2), 1.68–1.78 (m, 2H, Lys–CδH2), 1.78–2.04 (m, 2H,
Lys–CβH2), 3.04 t, 2H, Lys–CεH2), 3.19–3.51 (brm, 2H, His–CβH), 4.36–4.49 (m, 1H, Lys–CαH),
6.38 (d, 1H, HOOC–CH_), 6.54 (d, 1H, _CH–CO–NH), 7.37 (s, 1H, >C_CH–NH–), 8.67 (s, 1H,
–N_CH–NH–).
4.18. Z–His-Lys–NH2
The preparation of Z–His-Lys–NH2 was carried out as described for Mal–His-Lys–NH2 starting from
218 mg (1 mmol) of H–Lys–NH2, 76 mg (0.25 mmol) of Z–His–OMe and 9.5 mg of α-chymotrypsin.
The product was separated by isocratic RP-HPLC using 10% acetonitrile in 0.1% aqueous trifluoroacetic
acid as eluent to give Z–His-Lys–NH2·2TFA, C20H28N6O4=416.46. Yield: 98 mg (61%); MS (TS-POS):

K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
1517
417 (M+H)+; 1H-NMR (300 MHz; D2O): 1.28–1.52 (m, 2H, Lys–CγH2), 1.54–1.72 (m, 2H, Lys–CδH2),
1.72–1.86 (m, 2H, Lys–CβH2), 2.99 (t, 2H, Lys–CεH2), 3.09–3.36 (brm, 2H, His–CβH), 4.30–4.40 (m,
1H, Lys–CαH), 4.50–4.58 (dd, 1H, His–CαH), 5.15 (s, 2H, O–CH2–), 7.28 (s, 1H, >C_CH–NH–), 7.40
(d, 2H, _CH–C(CH2)–CH–), 7.45–7.54 (m, 3H, _CH–CH_CH–), 8.57 (s, 1H, –N_CH–NH–).
4.19. Z–Gly-His-Lys–NH2
The synthesis of Z–Gly-His-Lys–NH2 was carried out as described for Mal–His-Lys–NH2 starting
from 350 mg (0.25 mmol) of Z–Gly-His–ONb, 218 mg (1 mmol) of H–Lys–NH2 and 0.47 mg of α-
chymotrypsin. The product was isolated from a silica gel 60 column (70–230 mesh ASTM; E. Merck,
Germany) using methanol:CHCl3:8% aqueous NH3 (5.1:3.4:1.5) as eluent, C22H31N7O5=473.51. Yield:
93 mg (79%); MS (TS-POS): 474 (M+H)+; 1H-NMR (300 MHz; D2O): 1.36–1.54 (m, 2H, Lys–CγH2),
1.65–1.78 (m, 2H, Lys–CδH2), 1.78–1.93 (m, 2H, Lys–CβH2), 3.02 (t, 2H, Lys–CεH2), 3.10–3.35 (brm,
2H, His–CβH), 3.89 (s, 2H, Gly–CH2), 4.23–4.35 (m, 1H, Lys–CαH), 4.67–4.75 (m, 1H, His–CαH),
5.20 (s, 2H, O–CH2–), 7.19 (s, 1H, >C_CH–NH–), 7.48 (brs, 5H, C6H5), 8.29 (s, 1H, –N_CH–NH–).
The dihydrochloride was obtained as described for Mal–His-Lys–NH2. MS (TS-POS): 474 (M+H)+;
1H-NMR (300 MHz; D2O): 1.38–1.58 (m, 2H, Lys–CγH2), 1.63–1.78 (m, 2H, Lys–CδH2), 1.78–1.94
(m, 2H, Lys–CβH2), 3.02 (t, 2H, Lys–CεH2), 3.16–3.40 (brm, 2H, His–CβH2), 3.89 (s, 2H, Gly–CαH2),
4.29–4.38 (m, 1H, Lys–CαH), 4.70–4.75 (m, 1H, His–CαH), 5.21 (s, 2H, O–CH2–CO–NH–), 7.30 (s,
1H, >C_CH–NH), 7.49 (brs, 5H, C6H5), 8.63 (s, 1H, –N_CH–NH–).
Acknowledgements
We gratefully acknowledge the financial support by the Bundesministerium für Wissenschaft und
Technik (BMFT-project 03190007 C), by the Sächsisches Staatsministerium für Wissenschaft und Kunst
and by the Deutsche Forschungsgemeinschaft (INK23-A1). We wish to thank Mr. St. Theophilou
(London) for reading and correcting the manuscript.
References
1. Schechter, I., Berger, A. Biochem. Biophys. Res. Commun. 1968, 32, 888–902.
2. Pickart, L. Lymphokines 1993, 8, 425–446.
3. Pickart, L. In Vitro 1981, 17(6), 459–466.
4. Jakubke, H.-D. Peptide: Chemie und Biologie, Spectrum Akademischer Verlag, Heidelberg, Berlin, Oxford, 1996.
5. Jakubke, H.-D. J. Chin. Chem. Soc. 1994, 41, 335–370.
6. Jakubke, H.-D., Kuhl, P., Könnecke, A. Angew. Chem. Int. Ed. Engl. 1985, 24, 85–93.
7. Morihara, K. Trends Biotechnol. 1987, 5, 164–170.
8. Jakubke, H.-D.: Angew. Chem. Int. Ed. Engl. 1985, 34(2), 175–177.
9. Wong, C.-H., Wang, K. T. Experientia 1991, 47, 1123–1129.
10. Bongers, I., Heimers, E. P. Peptides 1994, 15, 183–193.
11. Jakubke, H.-D., Eichhorn, U., Haensler, M., Ullmann, D. Biol. Chem. 1996, 377(7/8), 455–464.
12. Brubacher, L. J., Bender M. L. J. Am. Chem. Soc. 1966, 88, 5871–5899.
13. Schellenberger, V., Schellenberger, U., Jakubke, H.-D. Collect. Czech. Chem. Commun. 1988, 53, 2884–2889.
14. Könnecke, A., Schellenberger, V., Hofmann, H.-J., Jakubke, H.-D. Pharmazie 1984, 39, 758–786.
15. Schellenberger, V., Schellenberger, U., Mitin, Y. V., Jakubke, H.-D. Eur. J. Biochem. 1980, 187, 163–167.
16. Bizzozero, S. A., Baumann, W. K., Duttler, H. Eur. J. Biochem. 1982, 122, 251–258.
17. Schellenberger, V., Jakubke, H.-D. Biochim. Biophys. Acta 1986, 869, 54–60.

1518
K. Beck-Piotraschke, H.-D. Jakubke / Tetrahedron: Asymmetry 9 (1998) 1505–1518
18. Crout, D. H. G., Davies, S., Heath, R. J., Miles, C. O., Rathbone, D. R., Swoboda, B. E. P. Biocatalysis 1994, 9, 1–30.
19. Hanisch, U.-K., Könnecke, A., Schellenberger, V., Jakubke, H.-D. Biocatalysis 1987, 1, 129–136.
20. Hänsler, M., Jakubke, H.-D. J. Pept. Sci. 1996, 2, 279–289.
21. Schellenberger, V., Schuster, M., Jakubke, H.-D. Biocatalysis 1990, 4, 105–111.
22. Martinez, J., Laur, J., Castro, B. Tetrahedron Lett. 1983, 24, 5219–5222.