Papain-catalysed synthesis of Z-l-aminoacyl-antipyrine amides from Z-protected amino acid esters and 4-aminoantipyrine

Article abstract of DOI:10.1016/j.tetlet.2007.03.067

The enzymatic synthesis of Z-l-aminoacyl-antipyrine amides from Z-protected amino acid esters and 4-aminoantipyrine (AAP) was accomplished by papain in aqueous-organic and biphasic media as well as in suspension. Product yields of 80% and 68% for Z-Gly-AA

Full text of DOI:10.1016/j.tetlet.2007.03.067

Tetrahedron Letters 48 (2007) 3371–3374
Papain-catalysed synthesis of Z-L-aminoacyl-antipyrine
amides from Z-protected amino acid esters and 4-aminoantipyrine
Alexander Lang, Catharina Hatscher and Peter Kuhl*
Lehrstuhl fu¨r Biochemie/Naturstoffchemie, Fachrichtung Chemie und Lebensmittelchemie, Technische Universita¨t Dresden,
Bergstraße 66, D-01062 Dresden, Germany
Received 22 December 2006; revised 9 March 2007; accepted 13 March 2007
Available online 15 March 2007
Abstract—The enzymatic synthesis of Z-L-aminoacyl-antipyrine amides from Z-protected amino acid esters and 4-aminoantipyrine
(AAP) was accomplished by papain in aqueous-organic and biphasic media as well as in suspension. Product yields of 80% and 68%
for Z-Gly-AAP and Z-Ala-AAP, respectively, could be obtained. The products were purified and characterised by polarimetry and
NMR. The following results expand our knowledge of the catalytic potential of proteases, in particular the suitability of papain to
accept as nucleophile the 1,2-amino ketone moiety of appropriate heterocyclic compounds.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The advantageous use of proteases as biocatalysts for
specific formation of peptide bonds is currently well
established. Protease-catalysed coupling reactions with
protected amino acid or peptide derivatives may be car-
ried out under mild conditions like room temperature
and neutral pH value. This in combination with inherent
properties of proteases such as stereospecificity and
regiospecificity gives the chemist an instrument for faster
and more economic syntheses compared to chemical
conversions without catalyst.1,2
plished by using common protecting groups such as Z
or Boc.5 In the P1 position small amino acids are
favoured without any direct specificity.6 Hydrophobic
amino acid derivatives are the best nucleophiles to bind
to the S01 position of papain.4,7
The following experiments demonstrate that the cata-
lytic potential of papain with regard to N-components
is broader than known so far. Thus, this protease is able
to catalyse the reaction of N-protected amino acid esters
with the heterocyclic 1,2-amino ketone 4-aminoanti-
pyrine (AAP). There are so far no examples in literature
for application of AAP in enzymatic synthesis.
The esterase activity of cysteine (thiol-) proteases allows
the kinetically-controlled approach starting with an
ester substrate and the fast formation of an acylated
enzyme as intermediate. This reacts in the second step
with the nucleophilic component and often gives rise
to temporary higher product concentrations than in
thermodynamically-controlled synthesis. Secondary
hydrolysis does not start until the donor ester is com-
pletely consumed because of the greater acceptance of
the enzyme for ester than for peptide bonds.3
AAP (CAS No. 83-07-8) is a metabolite of metamizole
and has also analgesic, antipyretic and antiphlogistic
effects with an activity lower than that of metamizole.8
4-Aminoantipyrine has already been chemically coupled
to amino acid derivatives via the methods of clas-
sical peptide synthesis utilising N,N0-dicyclohexyl-
carbodiimide.9
The thiol-endopeptidase papain is a natural product of
Carica papaya and shows a primary specificity for aro-
matic or bulky aliphatic moieties in the P2 position.4
For amino acid derivatives this requirement is accom-
On closer examination of the AAP structure we got the
idea that some parts of it suggest a certain suitability for
mimicking a substituted amino acid amide with a free
a-amino group (Fig. 1). The side chain is part of a
ring structure. Its aromaticity and that of the phenyl
moiety make AAP almost planar. The free amino group
and the protected carboxamide structure qualify 4-amino-
antipyrine as a potential nucleophile in enzymatic
synthesis.
Keywords: Papain; 4-Aminoantipyrine; Enzymatic synthesis.
*
Corresponding author. Tel.: +49 351 463 33806; fax: +49 351 463
35506; e-mail: peter.kuhl@chemie.tu-dresden.de
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.067

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A. Lang et al. / Tetrahedron Letters 48 (2007) 3371–3374
Table 1. Papain-catalysed synthesis of Z-L-aminoacyl-antipyrine
amides in aqueous-organic medium and suspensiona
Acyl donor
Product
Yield of Z-Xaa-AAP (%)
Aqueous-organicb Suspensionc
1a Z-Gly–OMe 3a Z-Gly-AAP 45
1b Z-Ala–OMe 3b Z-Ala-AAP 62
1c Z-Ser–OMe 3c Z-Ser-AAP 16
80
68
25
Figure 1. 4-Aminoantipyrine with a partial structure of an a-amino
acid amide standing out in bold.
a 20 mg papain, 40 ꢁC, reaction time: 1 h.
b 2 mL 0.1 M sodium citrate buffer (pH 5.0); 0.5 mL methanol; 0.1 M
acyl donor; 0.2 M AAP.
In this Letter we present the successful papain-catalysed
synthesis of Z-L-aminoacyl-antipyrine amides utilising
different strategies of medium engineering. Aqueous-
organic and biphasic media as well as suspensious were
applied as described elsewhere6,7,10 to optimise product
yield.
c 2 mL 0.1 M sodium citrate buffer (pH 5.0); 0.5 mL methanol; 0.5 M
acyl donor; 1 M AAP.
solved 1a and product 3a precipitated. Products 3b
and 3c remained dissolved in a clear or slightly turbid
solution. In the case of the reaction in suspension none
of the starting substances and products was completely
dissolved. The suspension was made of the same solvent
but with a 5-fold concentration of substrates and nucleo-
phile. Working with a suspension led to higher product
yields. Such reaction systems frequently approved to be
advantageous because of reduced water activity result-
ing in lower hydrolysis of the acyl donor and secondary
product cleavage.10,11
The reactions (Scheme 1) were performed in conus ves-
sels of 5 mL volume placed in a magnetic stirring bath.
Papain (Merck, 30000 USP-U/mg) was activated with
2–3 mg cysteine hydrochloride in 2 mL buffer for about
15 min. After addition of AAP while stirring with
640 rpm for additional 5 min, the acyl donor ester dis-
solved in 0.5 mL methanol was added to start the reac-
tion. Finally, the enzyme was inactivated by heating up
to 70 ꢁC for 10 min. We did not verify whether papain
was totally deactivated, but the yields were slightly low-
er and absolutely reproducible compared to reactions
without inactivating papain. After deactivation of the
enzyme the solvent was evaporated under vacuum.
The dried up reaction mixture was dissolved in 5 mL
acetonitrile and analysed by RP-HPLC (29% aceto-
nitrile; 71% water; 0.1% TFA; v/v; flow 1 mL/min;
Nucleosil 100, C18, 5 lm, 250 · 4 mm) against authentic
samples. Without papain we could not observe the
formation of Z-L-aminoacyl-antipyrine amides.
According to the substrate specificity, papain accepted
1a and 1b far better than 1c. The side chain of 1c is more
bulky than that of the two other substrates.
In order to explore whether there is a kinetic maximum
in the formation of 3b, the time course of the reaction
was followed and is shown in Figure 2.
As shown in Figure 2, 1b reacts very fast and already
after 5 min 35% yield of 3b are formed. After approx.
4 h the kinetic maximum is reached and secondary
hydrolysis starts.
In first experiments we used an aqueous-organic
medium consisting of 20% methanol and 80% buffer
with a pH of 5.0 near pH optimum of papain wherein
all starting compounds were soluble. The organic
solvent has the function of solubilising the ester sub-
strate but the concentration is limited due to enzyme’s
denaturation. The reactions were conducted at 40 ꢁC
for 1 h with a ratio of acyl donor to nucleophile of
1:2. The results are presented in Table 1.
To optimise the conditions from an economical point of
view, we reduced the reaction time to 20 min and the
enzyme amount to 10 mg papain. In addition, the
following conversions were carried out under alkaline
conditions (0.2 M KH2PO4; 0.2 M EDTA; pH 8.6) in
order to obtain a higher concentration of AAP with
unprotonated amino group. The results are presented
in Table 2.
The reaction solution of the aqueous-organic medium
became turbid immediately after addition of the dis-
Scheme 1. Papain-catalysed synthesis of Z-L-aminoacyl-antipyrine amides (3a–c) from Z-L-amino acid esters (1a–c) and 4-aminoantipyrine (2).

A. Lang et al. / Tetrahedron Letters 48 (2007) 3371–3374
3373
For the first time Z-L
-aminoacyl-antipyrine amides
could be synthesised by using papain as catalyst.
Thus, one could be keen on the answer of the question:
Is papain the only protease to accept 1,2-amino
ketones in enzymatic synthesis? Further experiments
in this line are currently under investigation in our
group.
Acknowledgements
We are grateful to Professor Yu. V. Mitin for his hint
encouraging this work. The authors thank Ms I. Krie-
nitz-Albrecht for some experimental contributions.
References and notes
Figure 2. Time-dependent formation of Z-Ala-AAP from Z-Ala–OMe
and AAP catalysed by papain (2 mL 0.1 M sodium citrate buffer (pH
5.0); 0.5 mL methanol; 20 mg papain; 40 ꢁC; 0.1 M Z-Ala–OMe; 0.2 M
AAP).
1. Bordusa, F. Chem. Rev. 2002, 102, 4817–4867.
2. Jakubke, H.-D. Angew. Chem. 1995, 107, 189–191.
3. Jakubke, H.-D. Enzyme Catalysis in Organic Synthesis;
Weinheim: VCH Verlagsgesellschaft mbH, 1995.
4. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun.
1967, 27, 157–167.
Table 2. Papain-catalysed synthesis of Z-L-aminoacyl-antipyrine
amides in aqueous-organic and biphasic mediuma
5. Barbas, C. F.; Wong, C. J. Chem. Soc., Chem. Commun.
1987, 533–534.
6. Kuhl, P.; Jakubke, H.-D. Pharmazie 1990, 45, 393–400.
7. Mitin, Y. V.; Zapevalova, N. P.; Gorbunova, E. Y. Int. J.
Pept. Protein Res. 1984, 23, 528–534.
Acyl donor
Acyl donor–
nucleophile ratio
Yield of Z-Xaa-AAP (%)
Aqueous-organicb Biphasicc
8. The European Agency for the Evaluation of Medicinal
Products, Veterinary Medicines Evaluation Unit, EMEA/
MRL/529/98-Final-Corrigendum, 1999 and EMEA/
MRL/878/03-Final, 2003.
9. Kwapiszewski, W. Acta Polon. Pharm. 1971, 28, 285–
289.
10. Erbeldinger, M.; Ni, X.; Halling, P. J. Enzyme Microb.
Technol. 1998, 23, 141–148.
11. Kuhl, P.; Halling, P. J.; Jakubke, H.-D. Tetrahedron Lett.
1990, 31, 5213–5216.
1a Z-Gly–OMe 1:1
33
66
58
67
1:2
1b Z-Ala–OMe 1:1
27
36
57
50
1:2
1c Z-Ser–OMe 1:1
12
13
30
34
1:2
a 10 mg papain, 40 ꢁC, reaction time: 20 min.
b 2 mL buffer (0.2 M KH2PO4, 0.2 M EDTA, pH 8.6); 0.5 mL meth-
anol; 0.1 M acyl donor.
12. Two batches of about 0.5 mmol product were evaporated
under vacuum to dryness and then dissolved in 20 mL
CHCl3. The solution was washed twice with saturated
sodium bicarbonate solution, and the two phases were
separated. AAP was extracted with 1 M HCl from the
organic phase as long as the reaction of the aqueous phase
with Ehrlich reagent turned out positive. Then the organic
layer was dried with sodium sulfate, filtered and evapo-
rated. The obtained product was desiccated overnight at
40 ꢁC.
c 0.25 mL buffer (0.2 M KH2PO4, 0.2 M EDTA, pH 8.6); 2.25 mL ethyl
acetate; 0.1 M acyl donor.
In an aqueous-organic medium the higher pH value
favoured remarkably the formation of 3a, even at a
considerably diminished amount of papain and at
shorter reaction time. Scarcely influenced remained the
synthesis of 3c.
20
13. Z-Ala-AAP: slightly yellow solid; ½aꢀD ꢁ31.7 (c 0.3,
MeOH); 1H NMR [500 MHz, CDCl3] d (in ppm) 1.35
Using a biphasic reaction medium proved to be advan-
tageous in comparison to an aqueous-organic reaction
mixture. The yield of 3c reached an acceptable value.
3
(d, J = 6.9 Hz, 3H), 2.16 (s, 3H), 3.05 (s, 3H), 4.38–4.41
(m, 1H), 5.05, 5.15 (2J = 12.2 Hz, 2H), 5.91 (d,
3J = 7.5 Hz, 1H), 7.24–7.40 (m, 10H), 8.79 (s, 1H); 13C
NMR [125.75 MHz, CDCl3] d (in ppm) 12.23 (CH3), 18.93
(CH3), 35.85 (CH3), 51.03 (CH), 66.72 (CH2), 107.95 (C),
124.45, 127.10, 127.98, 128.00, 128.44, 129.25 (C6H5),
134.28 (C), 136.52 (C6H5), 150.06 (C6H5), 155.79 (CO),
161.61 (CO), 172.19 (CO).
We consider a suspension of educts in buffer with slight
addition of methanol as the most suited medium. The
product yields obtained here are the highest so far for
Z-Gly-AAP (80%) and Z-Ala-AAP (68%) (Table 1).
14. Z-Gly-AAP: slightly yellow solid; 1H NMR [500 MHz,
DMSO-d6] d (in ppm) 2.10 (s, 3H), 3.04 (s, 3H), 3.79 (d,
3J = 6.2 Hz, 2H), 5.05 (s, 2H), 7.30–7.54 (m, 10H), 9.13 (s,
1H); 13C NMR [125.75 MHz, DMSO-d6] d (in ppm) 11.20
(CH3), 36.03 (CH3), 43.40 (CH2), 65.45 (CH2), 107.24 (C),
123.46, 126.21, 127.71, 127.79, 128.35, 129.10 (C6H5),
135.03 (C), 137.07 (C6H5), 152.31 (C6H5), 156.52 (CO),
161.70 (CO), 168.65 (CO).
All the synthesised Z-L-aminoacyl-antipyrine amides
were purified12 and characterised by polarimetry, LC–
1
13
MS, H NMR and C NMR.13–15
In conclusion, this work emphasises the far wider syn-
thesis potential of proteases than that known so far.

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A. Lang et al. / Tetrahedron Letters 48 (2007) 3371–3374
20
1
15. Z-Ser-AAP: yellow solid; ½aꢀD ꢁ23.4 (c 0.3, MeOH); H
NMR [500 MHz, CDCl3] d (in ppm) 2.15 (s, 3H), 3.07 (s,
3H), 3.71–3.78 (m, 2H), 3.95–4.02 (m, 1H), 4.42–4.44 (m,
CDCl3] d (in ppm) 11.69 (CH3), 35.43 (CH3), 56.89 (CH),
62.73 (CH2), 66.89 (CH2), 107.15 (C), 124.82, 127.58,
127.94, 128.08, 128.41, 129.32 (C6H5), 133.86 (C), 136.41
(C6H5), 150.65 (C6H5), 156.40 (CO), 161.69 (CO), 171.15
(CO).
3
1H), 5.05–5.22 (m, 2H), 6.54 (d, J = 7.9 Hz, 1H), 7.25–
7.46 (m, 10H), 8.57 (s, 1H); 13C NMR [125.75 MHz,