To broaden the scope of Alcalase-catalysed peptide synthesis even further, OCam and OTfe esters were introduced as acyl donors.[7] These moieties are known to be specifically recognised by some enzymes,[8] thereby overcoming their primary specificity. The coupling of challenging substrates such as sterically demanding amino acids as acyl donors (valine, isoleucine, threonine), notoriously weak nucleophiles (proline), or D- and other non-
proteinogenic amino acid residues, became well feasible in high yields without any hydrolytic side reactions. However, coupling reactions sometimes remained rather slow and a relatively large amount of enzyme was required, especially when longer peptide fragments were to be coupled. Clearly, there is room for improvement of the acyl donor ester in Alcalase catalysed peptide synthesis.
6.2
Results & Discussion
The available library of Z-Gly-Act esters was employed to test the coupling efficiencies in Alcalase-catalysed dipeptide synthesis, with H-Phe-NH2 functioning as the nucleophile. A solvent mixture of DMF/THF (1:9 (v/v)) ensured good solubility of all starting materials. Molecular sieves were added to remove water from the reaction mixture, preventing any hydrolytic side reactions and Alcalase-CLEA facilitated convenient handling and workup. The results of the screening are given in Table 6.1.
Table 6.1 Relative activity of various Z-Gly-Act esters[a]
Entry Act Structure Relative activity (%)
1 OGp 100
2 OTmap 95
3 ODmap 70
4 OTfe 68
6 ONb 34 7 OGb 31 8 OTmape 30 9 OAb 28 10 O4G 23 11 O4Cam 11 12 O3Cam 9 13 O4A 8 14 OBn 8 15 O3G 7 16 O5G 4 17 O5A 4
[a] Conditions: 2 mM Z-Gly-Act, 0.33 M H-Phe-NH2, DMF/THF, crushed 3Å mol sieves, 50 °C, 60 min
Clearly, the substituted phenyl esters (entries 1-3) are the most active species for the Alcalase-CLEA catalysed peptide coupling. Especially for entries 1 and 2 this is remarkable, since Alcalase has a preference for large uncharged hydrophobic residues in both the P1 and the P1’ positions.[9] The OTfe and OCam ester derivatives follow closely (entries 4 and 5). It is believed that the amide group of the OCam ester moiety (Figure 6.1 B) binds to the enzyme via a hydrogen bond in the same fashion as an amide of a peptide backbone (Figure 6.1 A) binds when it is recognised and cleaved by an endoprotease.
Figure 6.1 Similarity of the natural peptide at the cleavage site of an endoprotease and the OCam and OCam-XAA-NH2 ester.
The relative activities of the substituted benzyl (entries 6, 7 and 9) and phenethyl (entry 8) esters are mutually comparable, yet significantly decreased compared to entries 1-5. An exception is the benzyl ester (entry 14), which clusters with the aliphatic esters (entries 11-13 and 15-17) in the low activity range. This contrasts with the high activity of the benzyl ester in combination with the enzyme papain.[10] Apparently, the length of the carbon linker is important, as demonstrated by the O4G ester (entry 10), which clearly outperformed both the O3G and O5G ester (entries 15 and 16, respectively). Generally, it is noticeable that high activity seems to coincide with strong electron-withdrawing properties of the esters, which render the carbonyl more susceptible to nucleophilic attack by the active site serine of the protease and the corresponding alcohol a better leaving group.
An important drawback of phenyl esters is that, due to their high activation level, they are relatively difficult to synthesise, while both the chemical[11] and enzymatic[7] synthesis of the OCam ester (entry 5) is well feasible. Analogous to the strategy followed by Wells et al., the OCam ester can presumably be improved by elongating it with an amino acid amide, thereby creating additional binding interactions with the enzyme (Figure 6.1 C).[12] To this end, a library of Fmoc-Val-Ala-OGlyc-XAA-NH2 esters was evaluated, where XAA stands for all 20 proteinogenic amino acids, with either a protected or an unprotected side chain functionality. The best results were obtained with the OGlyc-Phe-NH2 ester, resulting in a two-fold enhancement.[13]
This optimal OGlyc-Phe-NH2 ester was compared to the most active substituted phenol esters from the first Z-Gly-Act screening (Table 6.1, entries 1, 2). To clearly discern the intrinsic reactivities, the challenging substrate Z-D-Phe-OH was chosen as the acyl donor
with H-Phe-NH2 as the nucleophile (Table 6.2). D-Amino acids are notoriously difficult substrates for Alcalase, in fact, Chen et al. reported that no peptide product was obtained at all using Alcalase and Z-D-Phe as acyl donor.[2]
Table 6.2 Relative activity of various Z-D-Phe-Act esters[a]
Entry Act Structure Relative activity (%)
1 OTmap 100
2 OGp 98
3 OGlyc-Phe-NH2 90
4 OCam 54
[a] Conditions: 2 mM Z-Gly-Act, 0.33 M H-Phe-NH2, DMF/THF, crushed 3Å mol sieves, 50 °C, 60 min
As is evident from Table 6.2, the elongated OCam ester (entry 3) shows a comparable reactivity as the substituted phenyl esters (entry 1, 2). An equally active but more conveniently accessible ester was thus developed for Alcalase-CLEA catalysed peptide synthesis. Another advantage is that no racemisation occurred on the activated amino acid ester, i.e. D-Phe, using the Z-D-Phe-OGlyc-Phe-NH2 ester (entry 3, e.e. of D-Phe >99.5), this is in contrast with the Cbz-D-Phe-OTmap ester (entry 1, e.e. of D-Phe 85.8%).
6.3 Conclusion
Summarising, our set of activated esters was subjected to the enzyme Alcalase in anhydrous organic solvents. With this system developed by DSM, it became possible to shift the Alcalase activity entirely from hydrolysis in aqueous buffers to synthesis in the absence of water. To our surprise, it appeared that positively charged phenolic esters were most active. It was also demonstrated that the activity of OCam esters can be increased to the level of the most active phenolic esters by elongation with (apolar) amino acids and amino acid amides.
6.4 Acknowledgements
T. Nuijens, L. Wiermans and dr. P. J. L. M. Quaedflieg (DSM Innovative Synthesis B.V.) are kindly acknowledged for the fruitful collaboration.
6.5 Experimental Section
General remarks:
Before use, 3 g Alcalase-CLEA (Type OM, CLEA-Technologies, 580 U/g) was suspended in 100 mL tBuOH and crushed with a spatula. After filtration, the enzyme was resuspended in 50 mL
MTBE followed by filtration. Large enzyme particles were removed by a sieve (0.5 mm pore size). Analytical HPLC chromatograms were recorded on an HP1090 Liquid Chromatograph, using a reversed-phase column (Phenomenex, C18, 5 m particle size, 150 × 4.6 mm) at 40°C. The gradient program was: 0-25 min linear gradient ramp from 5% to 98% eluent B and from 25.1-30 min with 5% eluent B (eluent A: 0.5 mL/L methane sulfonic acid (MSA) in H2O, eluent B 0.5 mL/L MSA in acetonitrile). The flow was 1 mL/min from 0-25.1 min and 2 mL/min from 25.2-29.8 min, then back to 1 mL/min until stop at 30 min. Injection volumes were 20 μL. The 3 Å molecular sieves (Acros, 8 to 12 mesh) were activated (200°C under vacuum overnight), crushed and sieved (0.5 mm pore size) to remove large particles. To determine the e.e. of Phe the samples concentrated in vacuo and the residue suspended in excess 6 N HCl and refluxed overnight. Chiral HPLC was performed on a crownether (+) column (150 mm length, 4.0 mm internal diameter, 5 μm particle size) at 25 °C with 30 mM aqueous HClO4 (pH = 2.0) as the eluent. UV detection was performed at 210 nm using a UV-VIS linear spectrometer. The flow was 1 mL/min. Injection volumes were 5 μL. Rt (D-Phe) = 6.90 min, Rt (L-Phe) = 8.82 min.
General procedure for the relative activity determination of Alcalase-CLEA catalysed peptide coupling in organic media:
To a suspension of Alcalase-CLEA (4.5 mg), H-Phe-NH2 (0.54 mg) and crushed 3 Å molecular sieves (4.5 mL) in THF (900 μL), was added amino acid or peptide ester stock solution in DMF (20 mM, 100 μL). The reaction mixture was shaken at 50 °C with 200 rpm for 60 min. Afterwards, the reaction mixtures were filtrated and analysed by analytical HPLC by integrating the peptide coupling product peak. Integration areas of different reactions were compared to determine the relative activity (ester which gave the highest peptide product intergration area = 100%).
6.6 References
[1] (a) S.-T. Chen, K.-T. Wang, C.-H. Wong, J. Chem. Soc., Chem. Commun. 1986, 20, 1514- 1517; (b) R. J. Siezen, J. A. M. Leunissen, Protein Sci. 1997, 6, 501-523; (c) E. I. Smith, R. J. DeLange, W. H. Evans, M. Landon, F. S. Markland, J. Biol. Chem. 1968, 243, 2184-2191. [2] S.-T. Chen, S.-Y. Chen, K.-T. Wang, J. Org. Chem. 1992, 57, 6960-6965.
[3] T. Nuijens, C. Cusan, J. A. W. Kruijtzer, D. T. S. Rijkers, R. M. J. Liskamp, P. J. L. M. Quaedflieg, Synthesis 2009, 5, 809–814.
[4] R. A. Sheldon, Biochem. Soc. Trans. 2007, 35, 1583-.
[5] T. Nuijens, C. Cusan, T. J. G. M. van Dooren, H. M. Moody, R. Merkx, J. A. W. Kruijtzer, D. T. S. Rijkers, R. M. J. Liskamp, P. J. L. M. Quaedflieg, Adv. Synth. Catal. 2010, 325, 2399- 2404.
[6] T. Nuijens, E. Piva, J. A. W. Kruijtzer, D. T. S. Rijkers, R. M. J. Liskamp, P. J. L. M. Quaedflieg, Adv. Synth. Catal. 2011, 353, 1039-1044.
[8] (a) T. Miyazawa, S. Nakajo, M. Nishikawa, K. Hamahara, K. Imagawa, E. Ensatsu, R. Yanagihara, T. Yamada, J. Chem. Soc. Perkin Trans. 1 2001, 82–86; (b) T. Miyazawa, K. Tanaka, E. Ensatsu, R. Yanagihari, T. Yamada, J. Chem. Soc. Perkin Trans. 1 2001, 87-93; (c) S. M. A. Salam, K. Kagawa, T. Matsubara, K. Kawashiro, Enzyme Microb. Technol.
2008, 43, 537–543.
[9] D. F. Tai, Curr. Org. Chem. 2003, 7, 515-554.
[10] R. J. A. C. de Beer, B. Zarzycka, M. Mariman, H. I. V. Amatdjais-Groenen, M. J. Mulders, P. J. L. M. Quaedflieg, F. L. van Delft, S. B. Nabuurs, F. P. J. T. Rutjes, ChemBioChem 2012,
13, 1319-1326 .
[11] T. Miyazawa, E. Ensatsu, M. Hiramatsu, R. Yanagihara, T. Yamada, J. Chem. Soc. Perkin
Trans. 1 2002, 396-401.
[12] L. Abrahmsén, J. Tom, J. Burnier, K. A. Butcher, A. Kossiakoff, J. A. Wells, Biochemistry
1991, 30, 4151-4159.
[13] R. J. A. C. de Beer, T. Nuijens, L. Wiermans, P. J. L. M. Quaedflieg, F. P. J. T. Rutjes, Org.