Enzymatic C-terminal amidation of amino acids and peptides

Tetrahedron Letters 53 (2012) 3777–3779

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Enzymatic C-terminal amidation of amino acids and peptides Timo Nuijens a,b, Elena Piva a, John A. W. Kruijtzer b, Dirk T. S. Rijkers b, Rob M. J. Liskamp b, Peter J. L. M. Quaedflieg a,⇑ a b

DSM Innovative Synthesis B.V., PO Box 18, NL-6160 MD Geleen, The Netherlands Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, NL-3508 TB Utrecht, The Netherlands

a r t i c l e

i n f o

Article history: Received 9 March 2012 Revised 2 May 2012 Accepted 10 May 2012 Available online 15 May 2012 Keywords: Enzymes Amino acids Peptides Amidation

a b s t r a c t Herein, we describe two versatile and high yielding enzymatic approaches for the conversion of semiprotected amino acid and peptidyl C-terminal a-carboxylic acids into their corresponding amides. In the first approach, the lipase Candida antarctica lipase-B (Cal-B), and in the second approach, the protease Subtilisin A, are used, respectively. We found that by using the ammonium salt of the a-carboxylic acid instead of separate ammonia sources, the enzymatic amidation reactions proceeded much faster without side reactions and gave near to quantitative yields of products. Ó 2012 Elsevier Ltd. All rights reserved.

Many peptide hormones and pharmaceutically relevant peptides contain a C-terminal primary amide functionality, which is not only important for their biological activity, but also increases their stability against exoproteases.1 Furthermore, the primary amide functionality is often used as a (temporary) protecting group in chemical and enzymatic peptide synthesis; its cleavage can be achieved by using peptide amidases such as the peptidase from the flavedo of oranges.2 Solution phase synthesis of peptides bearing a C-terminal primary amide functionality usually starts from the C-terminal amino acid carboxamide and the desired peptide sequence is assembled stepwise in the C?N direction using N-carbamate protected amino acid building blocks to minimize racemization. However, the generally limited solubility of amino acid and peptide C-terminal amides makes the synthesis of longer peptide amides rather laborious by the solution phase methodology. Therefore, peptide amides are mostly synthesized by solid phase peptide synthesis (SPPS) using, among others, Rink Amide resin or 4-methylbenzhydrylamine (MBHA) resin in Fmoc- or Boc-SPPS, respectively.3 However, these resins are expensive and not attractive for large scale use. On the other hand the solid-phase synthesis of peptide C-terminal carboxylic acids is much more cost-efficient since cheap and industrially available resins can be used (such as the 2-chlorotritylchloride resin). Alternatively, several peptide Cterminal carboxylic acids are accessible via fermentation routes, while their C-terminal amides are the desired end product. Therefore, several chemical approaches have been developed to convert ⇑ Corresponding author. Tel.: +31 46 4761592. E-mail address: peter.quaedfl[email protected] (P.J.L.M. Quaedflieg). 0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2012.05.039

peptide C-terminal carboxylic acids into the corresponding amides. Unfortunately, these methods lack selectivity toward the C-terminus and usually cause racemization or prove incompatible with labile substrates or other protecting groups.4 In contrast to chemical approaches for modification of amino acids and peptides, enzymatic conversions proceed under mild conditions and are often chemo-, regio-, and stereoselective. Several enzymes are candidates to be used for the amidation of amino acids and peptidyl C-terminal carboxylic acids. For instance, a few peptide amidases have been described in the literature, but they tend to be selective for a limited number of C-terminal amino acids and cannot be produced on large scale.2 Lipases, such as Candida antarctica lipase-B (Cal-B), have been used to perform esterification and subsequent amidation of aliphatic carboxylic acids, but amidation of peptides by Cal-B has so far not been reported.5 The C-terminal amidation of amino acid esters and peptidyl esters has been reported using the protease Subtilisin A.6 Subtilisin A is a serine endoprotease produced from Bacillus licheniformis and commercially available as Alcalase in which it is the main constituent. This amidation reaction has recently been optimized using C-terminal peptide methyl esters and ammonium carbamate as the amine source.7 However, the chemical synthesis of the C-terminal peptide (methyl) esters that are required for the enzymatic reaction encounters the same disadvantages as the direct chemical amidation, that is lack of C-terminal selectivity, racemization issues, and incompatibility with labile substrates or other protecting groups. Herein, we describe, for the first time, the direct and C-terminally selective enzymatic amidation of side-chain protected as well as unprotected amino acids and

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T. Nuijens et al. / Tetrahedron Letters 53 (2012) 3777–3779

Table 1 C-terminal amidation of amino acids and peptides with Cal-B using ammonium benzoate or NH3 gas in toluenea,9 Entry

Amide product

Conversion (%) into amide using ammonium benzoate

Conversion (%) into amide using ammonia gas

1 2 3 4 5 6 7 8 9

Cbz-Pro-NH2 Cbz-Ala-NH210 Cbz-Gly-NH2 Cbz-Met-NH2 Cbz-Val-Pro-Pro-NH2 Cbz-Gly-Leu-Ala-NH2 Cbz-Gly-Phe-Ala-NH2 Cbz-Gly-Ile-Ala-NH2 Cbz-Val-Val-Pro-NH2

99 95 80 60 90 65 60 52 12

ndb nd nd nd nd nd 82 85 56

a Conversions were determined by HPLC relative to the carboxylic acid starting material. The amide products were identified by LC-MS analysis and compared with commercially available reference compounds. b Experiment not done.

peptides in high yield starting from the corresponding carboxylic acids using Cal-B and Subtilisin A. The direct amidation of N-protected amino acids and peptides is energetically unfavorable and should therefore be conducted in dry organic solvents with removal of the formed water to drive the equilibrium to the amide product. Additionally, the activation energy of the amidation reaction starting from the carboxylic acids is much higher than starting from the ester congeners. Therefore, longer reaction times are required and side reactions, such as hydrolysis or transamidation of the peptide backbone, can be expected. In fact, when we applied the reported optimized amidation conditions with Alcalase7 on Cbz-Phe-Leu-Ala-OH as the substrate, we observed 81% conversion of the starting material into the desired product, but also formation of a significant amount (15%) of side products caused by transamidation and hydrolysis.8 Therefore, we first tested the versatility of lipases for the direct amidation, since these enzymes virtually lack the ability to hydrolyze or transamidate peptide bonds. Cal-B, a commercially available lipase active in organic solvents, proved to be the most promising candidate (data of lipase screening not shown). Gratifyingly, in the case of Cal-B we did not observe any side products during the direct amidation of a number of amino acid and peptide acids, as shown in Table 1. The Cal-B catalyzed amidations proceeded smoothly in toluene using 10 equiv of ammonium benzoate. Up to quantitative yields were obtained in the presence of molecular sieves to absorb the liberated water during amide formation (Table 1, entries 1–5). Although no peptide-based side products were observed, the amidation of benzoic acid turned out to be a serious problem, especially when more challenging peptide substrates were used (Table 1, entries 6–9). Therefore, we investigated the possibility of using a direct NH3 source (e.g., NH3 in 1,4-dioxane) or the ammonium salt of the corresponding amino acid or peptide acid. Gratifyingly, the amidation reaction with Cal-B in the presence of only one equivalent of NH3 gas or NH3/1,4-dioxane (via the amino acid or peptide ammonium salt) resulted in a much higher yield and proceeded much faster than in the presence of ammonium benzoate (Table 1, entries 7–9).9 However, Cal-B appeared rather restricted in accepting bulky side-chains in its active site, so that phenylalanine, tyrosine, tryptophan, or arginine and peptides containing these amino acids at the C-terminus, were not converted into the corresponding amides (data not shown). Hence, we investigated the use of Subtilisin A in the direct amidation reaction of amino acid and peptide C-terminal carboxylic ammonium salts under anhydrous conditions. It is known11 that this serine endoprotease has a very broad substrate tolerance

Table 2 C-terminal amidation of peptide ammonium salts using Alcalase-CLEA in tBuOH/DMF (82.5/17.5, v/v)a,13 Entry

Amide product

Conversion into amide (%)

Transamidation (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Cbz-Phe-Leu-Ala-NH2 Cbz-Gly-Gly-Ala-NH2 Cbz-Val-Ala-NH2 Cbz-Ile-Met-NH2 Cbz-Gly-Leu-Ala-NH2 Cbz-Ala-Leu-NH2 Cbz-Ala-Phe-NH2 Cbz-Ala-Ala-NH2 Cbz-Val-Met-NH2 Cbz-Gly-Pro-Ala-NH2 Cbz-Ala-Met-NH2 Cbz-Tyr-Leu-NH2 Cbz-Gly-Phe-NH2 Cbz-Val-Phe-NH2 Cbz-Leu-Phe-NH2 Cbz-Gly-Ile-Ala-NH2 Cbz-Phe-Leu-NH2 Cbz-Ala-Pro-Leu-NH2 Cbz-Pro-Leu-Gly-NH2 Cbz-Gly-Tyr-NH2 Cbz-Val-Gly-NH2

94 92 9114 89 89 87 87 86 85 85 81 81 81 80 79 78 77 68 53 46 30

1 — — — 1 — — — — — — — — — — — — — 4 — —

a Conversions were determined by HPLC relative to the carboxylic acid starting material. Products were identified by LC-MS analysis and compared with commercially available reference compounds when available.

Table 3 a-Selective amidation of amino acid ammonium salts using Alcalase-CLEA in tBuOH/ DMF (82.5/17.5, v/v)a,13 Entry

Amide product

Conversion (%)

1 2 3 4 5

Cbz-Gln-NH2 Cbz-Ser-NH2 Cbz-Asn-NH2 Cbz-Glu-NH2b Cbz-Asp-NH2b

96 87 70 67 57

a Conversions were determined by HPLC relative to the carboxylic acid starting material. The amide products were compared with commercially available reference compounds and identified by LC-MS analysis. b Using 2.1 equiv of NH3 in 1,4-dioxane.

and is also active in organic solvents. We applied this biocatalyst in the form of cross-linked enzyme aggregates (Alcalase-CLEA, from CLEA Technologies)12 to achieve maximum stability and activity in the anhydrous organic solvent applied. As shown in Table 2, a large number of peptide ammonium salts could be converted into their corresponding amides using Alcalase. Surprisingly, transamidation did not occur at all in most substrates, or to a very limited extent in a few. High conversions were obtained for almost all the substrates and by increasing the reaction time or the amount of enzyme, even the amidation of more challenging substrates, such as Cbz-Val-Gly-NH2 (entry 21), could be brought to full completion. To avoid any hydrolysis, the amidation reactions were run in the presence of molecular sieves. To show the versatility of the amidation reaction, and to demonstrate the complete specificity toward a-carboxylic acids, Alcalase-CLEA was also applied to amino acids containing reactive side-chain functionalities, as shown in Table 3. The amidation of amino acids turned out to be slower than the amidation of peptide acids. Nevertheless, no side reactions occurred and full a-selectivity was observed when b- or c-carboxylic acids were present (entries 4 and 5). Finally, we applied the direct amidation approach to the tetramer fragment Tyr-Met-Asp-Phe with several degrees of protection (Table 4).

T. Nuijens et al. / Tetrahedron Letters 53 (2012) 3777–3779 Table 4 C-terminal amidation of Tyr-Met-Asp-Phe using Alcalase-CLEA in tBuOH/DMF (82.5/ 17.5, v/v)a,13

a b

Entry

Amide product

Yield (%)

1 2 3

Ac-Trp(Boc)-Met-Asp(tBu)-Phe-NH2 H-Trp(Boc)-Met-Asp(tBu)-Phe-NH2 H-Trp-Met-Asp-Phe-NH2b

77 68 63

Yield after purification by preparative HPLC.15 Using 2.1 equiv of NH3 in 1,4-dioxane.

Both diagnostic peptides Sincalide and Pentagastrine contain the C-terminal sequence Tyr-Met-Asp-Phe-NH216 which is a general motif of gastrin-related peptides. As a first approach we examined the amidation of the fully protected peptide (Table 4, entry 1). Its carboxylic acid congener is easily accessible by SPPS and is highly soluble in anhydrous organic solvents. Despite the presence of two bulky side-chain protecting groups, the peptide acid was well recognized by alcalase-CLEA and smoothly converted into its corresponding amide. The same peptide sequence, with a free Nterminal amine (entry 2), was also amidated without side reactions. This result is important, since side-chain protected peptides with a C-terminal primary amide and an unprotected N-terminus are very useful building blocks for chemical peptide fragment condensation, for example in the synthesis of the pharmaceutical peptide products Fuzeon and Exenatide.17 Finally, the unprotected tetrapeptide (entry 3) was also amidated in a good yield, without amidation of the aspartic acid side-chain. In conclusion, we have shown for the first time, that amino acids and peptide acids can be enzymatically converted into their C-terminal amide congeners directly using Cal-B or Subtilisin A. The amidation is high yielding and competitive with other functional groups and is independent of the presence of protecting groups.

6. 7. 8.

9.

10.

11.

12. 13.

14.

15.

Acknowledgements We would like to thank Mr. Math Boesten B.Sc. for his analytical support and Dr. Claudia Cusan for fruitful discussions. References and notes 1. Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. J. Am. Chem. Soc. 2009, 131, 7293–7303. , V.; Kula, M. R. Angew. Chem. Int. Ed. 1998, 37, 1885–1887; (b) 2. (a) Cˇerˇovsky ˇ erˇovsky , V.; Kula, M. R. Biotechnol. Appl. Biochem. 2001, 33, 181–187. C 3. Bernatowicz, M. S.; Daniels, S. B.; Köster, H. Tetrahedron Lett. 1989, 30, 4645– 4648. 4. Bailén, M. A.; Chinchilla, R.; Dodsworth, D. J.; Nájera, C. Tetrahedron Lett. 2000, 41, 9809–9813. 5. (a) Litjens, M. J. J.; Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Chem. Commun. 1999, 1255–1256; (b) De Zoete, M. C.; Kock-van Dalen, A. C.; van Rantwijk, F.; Sheldon, R. A. J. Mol. Catal. B: Enzym. 1999, 2, 19–25; (c) Du, W.; Zong, M.; Guo, Y.; Liu, D. Biotechnol. Appl. Biochem. 2003, 38, 107–110; (d) Litjens, M. J. J.;

16.

17.

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Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Tetrahedron 1999, 55, 12411– 12418. Chen, S. T.; Jang, M. K.; Wang, K. T. Synthesis 1993, 858–860. Boeriu, C. G.; Frissen, A. E.; Boer, E.; Kekem, K.; van Zoelen, D.-J.; Eggen, I. F. J. Mol. Catal. B: Enzym. 2010, 66, 33–42. The reaction mixture was analyzed by HPLC-MS and all resulting peaks could be identified. Area distribution: 4% Cbz-Phe-Leu-Ala-OH (starting material), 81% Cbz-Phe-Leu-Ala-NH2 (product), 9% Cbz-Phe-Leu-NH2, 4% Cbz-Phe-Leu-OH and 2% Cbz-Phe-NH2. Using ammonium benzoate: Cal-B (100 mg) was added to a mixture of Cbz-XaaOH (50 mg), ammonium benzoate (10 equiv) and 3 Å MS (200 mg) in toluene (6 mL). The mixture was shaken at 50 °C, 150 rpm for 16 h. Using NH3 gas: NH3 gas was bubbled through a solution of tripeptide (50 mg) in toluene (6 mL) for 30 s. Subsequently, Cal-B (100 mg) and 3 Å MS (200 mg) were added. The mixture was shaken at 50 °C, 150 rpm for 16 h. After the enzymatic conversion of Cbz-Ala-OH the reaction mixture was filtered and the Cal-B enzyme particles resuspended in MeOH followed by filtration (3  5 mL). The combined organic layers were concentrated in vacuo. The crude product was purified by column chromatography on silica gel using MeOH/CH2Cl2 (7/93 v/v) as the eluent. The pure fractions were combined, concentrated in vacuo and the volatiles co-evaporated with toluene (2) and CHCl3 (2). Cbz-Ala-NH2 was obtained in 87% yield. (a) Nuijens, T.; Cusan, C.; Kruijtzer, J. A. W.; Rijkers, D. T. S.; Liskamp, R. M. J.; Quaedflieg, P. J. L. M. Synthesis 2009, 809–814; (b) Nuijens, T.; Kruijtzer, J. A. W.; Cusan, C.; Rijkers, D. T. S.; Liskamp, R. M. J.; Quaedflieg, P. J. L. M. Tetrahedron Lett. 2009, 50, 2719–2721; (c) Nuijens, T.; Cusan, C.; Kruijtzer, J. A. W.; Rijkers, D. T. S.; Liskamp, R. M. J.; Quaedflieg, P. J. L. M. J. Org. Chem. 2009, 74, 5145–5150. Sheldon, R. A. Biochem. Soc. Trans. 2007, 35, 1583–1587. Alcalase-CLEA (50 mg, washed once with dry tBuOH and once with MTBE) was added to a mixture of peptide (0.10 mmol) and 2 N NH3 in 1,4-dioxane (0.11 mmol) in tBuOH/DMF (5 mL, 82.5/17.5 v/v). The mixture was shaken at 50 °C, 150 rpm for 16 h. When twice the amount of enzyme was used for the amidation of Cbz-Val-AlaOH, the HPLC conversion was 98%. The reaction mixture was filtered and the solid enzyme particles were resuspended in MeOH (3  5 mL) and in EtOAc (25 mL) followed by filtration. The combined organic layers were washed with saturated aqueous NaHCO3 (50 mL), brine (50 mL), dried over Na2SO4, concentrated in vacuo and the volatiles co-evaporated with toluene (2  50 mL) and CHCl3 (2  50 mL). Cbz-Val-Ala-NH2 was obtained in a yield of 89%. Ac-Trp(Boc)-Met-Asp(OtBu)-Phe-NH2: 1H NMR (DMSO-d6, 300 MHz): d = 1.35 (s, 9H), 1.62 (s, 9H), 1.77–1.94 (m, 5H), 2.01 (s, 3H), 2.28–2.45 (m, 2H), 2.61–2.68 (m, 1H), 2.79–3.09 (m, 4H), 4.26–4.41 (m, 2H), 4.50–4.64 (m, 2H), 7.14–7.33 (m, 9H), 7.53 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 8.17–8.24 (m, 3H); 13C NMR (DMSO-d6, 75 MHz): d = 14.5, 27.6, 29.2, 32.5, 37.2, 49.4, 51.6, 51.8, 53.7, 80.2, 83.6, 113.3, 114.7, 119.4, 122.3, 124.4, 125.6, 126.2, 127.9, 129.0, 129.8, 134.8, 137.6, 148.9, 168.0, 169.2, 169.7, 170.1, 172.4; TFA.H-Trp(Boc)-Met-Asp(OtBu)-Phe-NH2: 1H NMR (DMSO-d6, 300 MHz): d = 1.36 (s, 9H), 1.63 (s, 9H), 1.68–1.95 (m, 2H), 2.03 (s, 3H), 2.64– 2.81 (m, 2H), 2.91–3.05 (m, 2H), 3.11–3.21 (m, 1H), 4.10–4.21 (m, 1H), 4.36– 4.48 (m, 2H), 4.55–4.63 (m, 1H), 7.14–7.37 (m, 9H), 7.63 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 8.00–8.10 (m, 5H), 8.40 (d, J = 7.8 Hz, 1H), 8.90 (d, J = 7.5 Hz, 1H); 13C NMR (DMSO-d6, 75 MHz): d = 14.5, 22.4, 26.9, 27.6, 29.3, 31.7, 37.3, 49.5, 52.0, 52.5, 53.8, 80.2, 83.4, 114.5, 116.6, 119.4, 122.3, 123.8, 124.2, 126.1, 128.0, 129.0, 130.2, 134.5, 137.7, 149.0, 169.3, 169.4, 169.7, 170.8, 171.5, 172.6; TFA.H-Trp-Met-Asp-Phe-NH2: 1H NMR (DMSO-d6, 300 MHz): d = 1.68–1.90 (m, 2H), 1.96 (s, 3H), 2.80–2.92 (m, 2H), 3.01–3.13 (m, 2H), 3.14–3.20 (m, 1H), 3.94–4.05 (m, 1H), 4.28–4.40 (m, 2H), 4.46–4.53 (m, 1H), 6.95–7.32 (m, 11H), 7.62 (d, J = 7.8 Hz, 1H), 7.88–7.94 (m, 4H), 8.32 (d, J = 7.5 Hz, 1H), 8.75 (d, J = 8.1 Hz, 1H), 10.9 (s, 1H), 12.3 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): d = 14.5, 16.8, 27.3, 29.3, 32.3, 35.9, 36.4, 37.3, 49.5, 51.8, 52.4, 53.8, 106.7, 111.4, 118.4, 121.0, 125.0, 126.1, 127.0, 128.0, 129.0, 136.2, 137.7, 168.4, 167.0, 170.3, 171.9, 172.5. (a) Ziessman, H. A.; Tulchinsky, M.; Lavely, W. C.; Gaughan, J. P.; Allen, T. W.; Maru, A.; Parkman, H. P.; Maurer, A. H. J. Nucl. Med. 2010, 51, 277–281; (b) Bergant, D.; Hocevar, M. Acta Chir. Iugosl. 2003, 50, 121–124. Góngora-Benítez, M.; Cristau, M.; Giraud, M.; Tulla-Puche, J.; Albericio, F. Chem. Commun. 2012, 48, 2313–2315.