Protease-catalysed synthesis of Z-L-aminoacyl-L-caprolactam amides from Z-protected amino acid esters and DL-α-amino-e-caprolactam

Article abstract of DOI:10.1016/j.tetlet.2010.05.048

The enzymatic synthesis of Z-L-aminoacyl-L-caprolactam amides from Z-protected amino acid esters and DL-a-amino-e-caprolactam (ACL) was accomplished by the thiol proteases papain, bromelain and ficin in aqueous-organic media. Product yields of 96% and 87% for Z-Gly-L-ACL and Z-Ala-L-ACL, respectively, could be obtained. The products were purified and characterised by polarimetry, NMR and LC-MS. The suitability to accept a bulky 1,2-amino ketone as a nucleophile expands the general knowledge of thiol proteases and their catalytic potential.

Full text of DOI:10.1016/j.tetlet.2010.05.048

Tetrahedron Letters 51 (2010) 3779–3781
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Tetrahedron Letters
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Protease-catalysed synthesis of Z-L-aminoacyl-L-caprolactam amides
from Z-protected amino acid esters and DL-a-amino-e-caprolactam
*
Alexander Lang , Peter Kuhl
Lehrstuhl für Biochemie/Naturstoffchemie, Fachrichtung Chemie und Lebensmittelchemie, Technische Universität Dresden, Bergstraße 66, D-01069 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The enzymatic synthesis of Z-L-aminoacyl-L-caprolactam amides from Z-protected amino acid esters and
Received 18 February 2010
Revised 11 May 2010
Accepted 12 May 2010
Available online 20 May 2010
DL
-
a
-amino-
e
-caprolactam (ACL) was accomplished by the thiol proteases papain, bromelain and ficin in
-ACL and Z-Ala- -ACL, respectively,
aqueous–organic media. Product yields of 96% and 87% for Z-Gly-
L
L
could be obtained. The products were purified and characterised by polarimetry, NMR and LC–MS. The
suitability to accept a bulky 1,2-amino ketone as a nucleophile expands the general knowledge of thiol
proteases and their catalytic potential.
Keywords:
Amidation
Ó 2010 Elsevier Ltd. All rights reserved.
a-Amino-e-caprolactam
Enzymatic synthesis
Thiol proteases
Proteases as biocatalysts for the specific formation of peptide
bonds are currently well investigated, established and applied in
biotechnological processes.1–3 The hypothesis of enzymatic pep-
tide bond formation by reversal of the hydrolytic reaction of prote-
ases dates back to 1898 and was supposed by van’t Hoff.4
The advantage of protease-catalysed coupling reactions consists
in environmentally friendly reaction conditions, avoiding extensive
use of organic solvents and toxic auxiliary agents. Besides this, the
conservation of stereospecificity and regiospecificity is an intrinsic
attribute of proteases to favour enzymatic syntheses compared to
conventional chemical conversions without catalysts.
with AAP and whether there are further 1,2-amino ketones to be
accepted by proteases.
In an extensive study the thiol proteases papain, bromelain and
ficin, the serine endopeptidase
a-chymotrypsin and the metallo-
proteases thermolysin and pronase were singled out to find an-
swers to the above question. In the course of that work several
new antipyrine amides with N-protected
L-amino acid and
L-peptide derivatives have been synthesised enzymatically. For
the first time also Boc-protected amino acids and dipeptide
derivatives were covalently attached to AAP resulting in the
corresponding Boc-L-Xaa- and Z-L
-Xaa-Ala-antipyrine amides.8 It
As a matter of common knowledge the catalytic potential of
proteases is not restricted to the formation of peptide bonds only.
There is evidence of coupling reactions catalysed by papain be-
tween N-protected amino acids or esters and glycerine5 or fatty
alcohols.6
In addition, the thiol protease papain catalyses the reaction of
Z-protected amino acid esters with the heterocyclic 1,2-amino
ketone 4-aminoantipyrine (AAP).7
The free amino group and the modified carboxamide structure
mimicking a substituted amino acid amide qualify 4-aminoantipy-
rine (Fig. 1) as an accepted nucleophile in enzymatic synthesis with
papain. The aromaticity of the heterocyclic ring and the phenyl
moiety makes AAP almost planar to approach the active site of
papain more easily than a bulky molecule. The question arose,
whether papain is the only protease to catalyse coupling reactions
has been found that besides papain, also bromelain and ficin are
able to couple amino acid or peptide derivatives to AAP. The ability
of the serine protease a-chymotrypsin to aminoacylate the hetero-
cycle resulted in far lower yields compared to cysteine proteases.
Metalloproteases catalysed the described reaction with negligible
yields lower than 10%.
The effort to understand why thiol proteases are so privileged to
accept AAP brings substrate and nucleophile specificity into focus.
Papain shows a primary specificity for aromatic and bulky aliphatic
moieties in the P2 position9,10 which are provided by common
* Corresponding author. Tel.: +49 351 463 33805; fax: +49 351 463 35506.
E-mail address: alexander.lang@chemie.tu-dresden.de (A. Lang).
Figure 1. 4-Aminoantipyrine (left) and DL
-a-amino-e-caprolactam (right) with a
partial structure of an -amino acid amide highlighted in bold.
a
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.05.048

3780
A. Lang, P. Kuhl / Tetrahedron Letters 51 (2010) 3779–3781
protecting groups such as Z and Boc, whereas small amino acids are
favoured without any distinctive specificity in the P1 position.11
Hydrophobic amino acid derivatives are the most accepted nucle-
ophiles to bind to the S01 subsite of papain.12 Bromelain’s and ficin’s
specificity are similar as stated in former investigations.3,8,13
Thus, cysteine proteases are so far the most suited proteases for
coupling reactions between amino acid-based acyl donors and AAP
and possibly could be a good choice for probing on other 1,2-amino
acyl-L-caprolactam amides of alanine and glycine were synthesised
according to classical peptide chemistry by applying Z-Gly-OH,
Z-Ala-OH,
L
-ACLꢁHCl, K2CO3, N,N0-dicyclohexyl-carbodiimide and
1-hydroxytriazole in tetrahydrofuran. The purified reaction products
gave ½a 2D0
ꢂ
8.3 (c 0.5, MeOH) and ½a D20
ꢃ14.2 (c 0.5, MeOH) for
ꢂ
Z-Gly-L-ACL and Z-Ala-L-ACL, respectively. These data are in accor-
dance with the values determined for enzymatically synthesised
products.15,16 This finding points out that indeed there is a prefer-
ketones. One of these substances is
a-amino-e-caprolactam (ACL)
ence for the L-isomer of a-amino-e-caprolactam by thiol proteases.
which also has a carboxamide structure with a free amino group
(Fig. 1). Compared to 4-aminoantipyrine this compound is not pla-
nar, but bulky, analogous to suberone, although there are no large
substituents. Therefore, it is not apparent whether it fits to the ac-
tive site of thiol proteases.
The experimental approach allows due to the esterase activity
of thiol proteases both the thermodynamically and the kinetically
controlled conversion. Utilising an ester substrate results in fast
formation of an acylated enzyme intermediate, which subse-
quently acylates the amino component, thereby often yielding
temporary higher product concentrations than in equilibrium-
controlled synthesis. Only when the donor ester is completely
consumed, secondary hydrolysis occurs because of the enzyme’s
greater acceptance for ester than for peptide bonds.1
In the beginning the enzymatic conversions were carried out
under alkaline conditions (0.2 M KH2PO4; pH 8.6; 20% MeOH) to
obtain a higher concentration of ACL with unprotonated amino
group. The organic solvent has the role of solubilising the ester
substrate but the concentration is limited due to enzyme’s
denaturation. The reactions were conducted at 40 °C for 2 h
and 24 h with ratios of acyl donor to nucleophile of 1:2 and 1:5,
respectively. Results obtained are presented in Tables 1 and 2.
Considering the results of chemical and enzymatic synthesis
that just L-configurated isomers are utilised by the thiol proteases,
the effective acyl donor–nucleophile ratio is reduced to 1:1 and
1:2.5, respectively. According to the law of mass action, the yields
of Z-Xaa-
L
-ACL increased with a higher supply of DL-ACL and could
-ACL
be raised to nearly complete conversion in the case of Z-Gly-
L
In this Letter we present the first results of thiol protease-cata-
with papain and ficin. Although papain and ficin generate the
lysed reactions of Z-protected amino acid esters and DL
-
a-amino-e-
caprolactam (ACL) which result in Z-L-aminoacyl-L-caprolactam
amides.
Table 1
Protease-catalysed synthesis of Z-Gly-L-ACL (3a) in a basic aqueous–organic mediuma
The syntheses (Scheme 1) were performed in cylindrical glass
vessels of 5 mL volume with a conical bottom placed in a magnetic
stirring bath. Papain (Merck, 30,000 USP-U/mg), bromelain (Sigma,
2480 U/g) and ficin (Serva, 0.8 DMC-U/mg) were activated with
2-3 mg cysteine hydrochloride in 2 mL buffer for about 15 min.
After addition of DL-ACL while stirring with 700 rpm for additional
5 min, the acyl donor ester dissolved in 0.5 mL methanol was
added to start the reaction. Finally, the enzyme was inactivated
by heating up to 70 °C for 10 min and the solvent was evaporated
under vacuum. The reaction mixture was then dissolved in 5 mL
acetonitrile, filtered and analysed by RP-HPLC (29% acetonitrile;
71% water; 0.1% TFA; v/v; flow 1 mL/min; Nucleosil 100, C18,
Acyl donor–
nucleophile-ratio
Reaction time (h)
Yield of Z-Gly-L-ACL (%)
Papain
Bromelain
Ficin
1:2
1:5
2
24
72
74
49
62
91
93
2
24
94
92
44
89
96
96
a
20 mg protease, 40 °C, 2 mL buffer (0.2 M KH2PO4, pH 8.6); 0.5 mL methanol;
0.05 M Z-Gly-OMe (1a, acyl donor).
5
l
m, 250 ꢀ 4 mm) against authentic samples. Without thiol pro-
Table 2
Protease-catalysed synthesis of Z-Ala-L-ACL (3b) in a basic aqueous–organic mediuma
teases we could not observe the formation of Z- -aminoacyl- -cap-
L
L
rolactam amides. All given yields in this work are obtained from
triplicate determination. The average values have an error of 3%.
To acquire characteristic data of the synthesised compounds,
Acyl donor–
nucleophile-ratio
Reaction time (h)
Yield of Z-Ala-L-ACL (%)
Papain
Bromelain
Ficin
the Z-L-aminoacyl-L
-caprolactam amides were purified14 and char-
1:2
1:5
2
24
23
9
20
16
53
45
acterised by polarimetry, LC–MS, 1H NMR and 13C NMR.15,16
By polarimetry an optical activity even for Z-Gly-ACL could be
determined. Thus, the proteases seem to use only one enantiomer
of the racemate DL-ACL. Usually, but not exclusively, proteases pre-
2
24
70
50
56
29
87
83
a
20 mg protease, 40 °C, 2 mL buffer (0.2 M KH2PO4, pH 8.6); 0.5 mL methanol;
0.05 M Z-Ala-OMe (1b, acyl donor).
fer L-configured isomers. To prove that assumption, the Z-L-amino-
Scheme 1. Synthesis of Z-L-aminoacyl-L-caprolactam amides (3a–b) from Z-L-amino acid esters (1a–b) and DL-a-amino-e-caprolactam (2).

A. Lang, P. Kuhl / Tetrahedron Letters 51 (2010) 3779–3781
3781
Table 3
interesting properties, which can easily be prepared by protease-
catalysed synthesis.
Protease-catalysed synthesis of Z-Ala-L-ACL (3b) in an acidic aqueous–organic
mediuma
Acyl donor–
nucleophile-ratio
Reaction time (h)
Yield of Z-Ala-L-ACL (%)
Acknowledgement
Papain
Bromelain
Ficin
The authors thank Ms A. Kühnel for some experimental
contributions.
1:5
2
24
47
43
31
23
82
69
a
20 mg protease, 40 °C, 2 mL buffer (0.1 M sodium citrate buffer, pH 5.0); 0.5 mL
References and notes
methanol; 0.05 M Z-Ala-OMe (1b, acyl donor).
1. Jakubke, H.-D. Enzyme Catalysis in Organic Synthesis; VCH Verlagsgesellschaft
mbH: Weinheim, 1995.
2. Bordusa, F. Chem. Rev. 2002, 102, 4817–4867.
3. Tai, D.-F. Curr. Org. Chem. 2003, 7, 515–554.
4. van’t Hoff, J. H. Z. Anorg. Chem. 1898, 18, 1–13.
highest yields, with Z-Ala-OMe the proteases show rather less
conversion than with Z-Gly-OMe. For papain this is surprising be-
cause of its known far wider substrate acceptance than that of ficin.
5. Mitin, Y. V.; Braun, K.; Kuhl, P. Biotechnol. Bioeng. 1997, 54, 287–290.
6. Clapés, P.; Infante, M. R. Biocatal. Biotransform. 2002, 20, 215–233.
7. Lang, A.; Hatscher, C.; Kuhl, P. Tetrahedron Lett. 2007, 48, 3371–3374.
8. Lang, A.; Hatscher, C.; Wiegert, C.; Kuhl, P. Amino Acids 2009, 36, 333–340.
9. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157–167.
10. Barbas, C. F.; Wong, C. J. Chem. Soc., Chem. Commun. 1987, 533–534.
11. Kuhl, P.; Jakubke, H.-D. Pharmazie 1990, 45, 393–400.
12. Mitin, Y. V.; Zapevalova, N. P.; Gorbunova, E. Y. Int. J. Peptide Protein Res. 1984,
23, 528–534.
The formation of Z-Gly-L-ACL catalysed by bromelain seems to be
the slowest process. After 2 h there is the lowest yield compared
to the other proteases but it increases during the ongoing reaction
until 24 h, whereas for papain and ficin the gain ratios remain
nearly constant. The reaction time of 2 h is rather short and reason-
able and gives for Z-Ala-L-ACL better conversion than in reactions
over 24 h, without much doubt due to partial secondary hydrolysis
in the latter case.
In additional experiments an aqueous–organic medium has
been tested for the synthesis of Z-Ala-L-ACL consisting of 20%
methanol and 80% buffer with a pH of 5.0 near the pH optimum
of thiol proteases. The results are presented in Table 3.
Compared to the product yields obtained in basic buffer this
medium can just compete in ficin-catalysed reactions. Papain-
and bromelain-catalysed conversions differ from the results under
basic conditions. In that acidic medium also secondary hydrolysis
occurs. This is apparent from the yields after 24 h. All tested buf-
fer-methanol compositions displayed this characteristic.
Summing up all reactions, ficin seems to be the best thiol
protease for these types of substrates and this nucleophile. The
difference with the three thiol proteases is significant in the case
13. Tai, D.-F.; Fu, S.-L. J. Chin. Chem. Soc. 2003, 50, 179–183.
14. Several reaction batches were pooled to gain an amount of about 0.5 mmol
Z-Xaa-L-ACL. The mixture was evaporated to dryness under vacuum. The crude
product was dissolved in 20 mL CHCl3. The solution was washed thrice with
5 mL saturated sodium bicarbonate solution, and the two phases were
separated. The aqueous phase was discarded to waste, and from the organic
layer ACL was extracted twice with 5 mL 1 M HCl. Then, the organic phase was
washed twice with 2 mL distilled water and dried afterwards with sodium
sulfate, filtered and evaporated. The obtained product was desiccated
overnight at 40 °C. 1H and 13C NMR spectra were recorded with DRX 500,
Bruker. Polarimetry was performed with Model 341 LC, Perkin Elmer
Instruments and ESI/APCI-MS data were acquired by LC/MS equipment HP
1100—Bruker Esquire Ion Trap.
15. Z-Gly-
L
-ACL: white solid; ½a D20
ꢂ
8.3 (c 0.4, MeOH); 1H NMR [500 MHz, CDCl3] d
(in ppm) 1.37–1.47 (m, 1H, CH2 (ACL)), 1.47–1.52 (m, 1H, CH2 (ACL)), 1.79–1.82
(m, 1H, CH2 (ACL)), 1.82–1.86 (m, 1H, CH2 (ACL)), 1.98–2.01 (m, 1H, CH2 (ACL)),
2.05–2.08 (m, 1H, CH2 (ACL)), 3.23–3.28 (m, 2H, CH2 (ACL)), 3.86–3.97 (dd, 2H,
CH2 (Gly)), 4.50–4.53 (q, 1H, CH (ACL)), 5.12 (s, 2H, CH2 (Z)), 5.41 (t, 1H, NH
(Gly)), 6.01 (t, 1H, NH (ACL)), 7.22 (d, 1H, NH (ACL-Ala)), 7.29–7.33 (m, 3H,
C6H5 (Z)), 7.35 (d, 3J = 4.4 Hz, 2H, C6H5 (Z)); 13C NMR [125.75 MHz, CDCl3] d (in
ppm) 27.86 (CH2, ACL), 28.82 (CH2, ACL), 31.42 (CH2, ACL), 42.16 (CH2, ACL),
44.33 (CH2, Gly), 52.25 (CH, ACL), 67.14 (CH2, Z), 128.11, 128.17, 128.53, 136.18
(C6H5, Z), 156.39 (CO, Z), 167.77 (CO, Gly), 175.00 (CO, ACL); ESI-MS m/z 320.1
[M+H+], m/z 342.1 [M+Na+], C16H21N3O4 requires 319.4
of Z-Ala-L-ACL, but more balanced in the formation of Z-Gly-L-ACL.
In summary, this work emphasises the far wider synthetic
potential of proteases than that known so far. It gives proof of
introducing another 1,2-amino ketone besides AAP as the nucleo-
phile in the amidation of amino acid derivatives. For the first time
16. Z-Ala-
L
-ACL: white solid; ½a D20
ꢂ
ꢃ13.7 (c 0.4, MeOH); 1H NMR [500 MHz, DMSO-
d6] d (in ppm) 1.20 (d, 3J = 12.6 Hz, 3H, CH3 (Ala)), 1.13–1.24 (m, 2H, CH2 (ACL)),
1.71–1.81 (m, 2H, CH2 (ACL)), 1.85–1.88 (m, 2H, CH2 (ACL)), 3.03–3.07 (m, 2H,
CH2 (ACL)), 4.02–4.06 (quin, 1H, CH (Ala)), 4.31–4.35 (q, 1H, CH (ACL)), 5.02 (s,
2H, CH2 (Z)), 7.30–7.33 (m, 1H, C6H5 (Z)), 7.33–7.38 (m, 4H, C6H5 (Z)), 7.63 (d,
3J = 7.6 Hz, 1H, NH (ACL-Ala)), 7.78 (d, 3J = 6.5 Hz, 1H, NH (Ala)), 7.88 (t, 1H, NH
(ACL)); 13C NMR [125.75 MHz, DMSO-d6] d (in ppm) 18.06 (CH3, Ala), 27.68
(CH2, ACL), 28.87 (CH2, ACL), 31.11 (CH2, ACL), 40.65 (CH2, ACL), 50.40 (CH,
Ala), 51.38 (CH, ACL), 65.44 (CH2, Z), 127.71, 127.83, 128.43, 137.07 (C6H5, Z),
155.74 (CO, Z), 171.49 (CO, ACL), 174.14 (CO, Ala); ESI-MS m/z 334.2 [M+H+],
m/z 356.2 [M+Na+], C17H23N3O4 requires 333.4
Z-L-aminoacyl-L-caprolactam amides could be synthesised by using
thiol proteases as catalysts, because there is nothing reported so far
about these compounds neither in enzymatic nor in chemical syn-
thesis. The findings accentuate the stereospecificity of proteases to
prefer the
L-configured isomer of a DL-ACL racemate. These results
encourage the idea of finding new substances with possibly