Backbone amide linker (BAL) strategy for N(α)-9- fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of unprotected peptide p-nitroanilides and thioesters

Journal of Organic Chemistry

Volumn 64, Issue 24, 1999, Pages 8761-8769

  Alsina, Jordi ^a   Yokum, T Scott ^a   Albericio, Fernando ^b   Barany, George ^a  

^a UNIVERSITY OF MINNESOTA   (United States)

^b UNIVERSITY OF BARCELONA   (Spain)

Author keywords

[No Author keywords available]

Indexed keywords

ANILIDE; PEPTIDE DERIVATIVE; THIOESTER;

ARTICLE; CHEMICAL ANALYSIS; CHEMICAL REACTION; CHEMICAL STRUCTURE; ORGANIC CHEMISTRY; PEPTIDE ANALYSIS; PEPTIDE SYNTHESIS;

EID: 0033607759     PISSN: 00223263     EISSN: None     Source Type: Journal    
DOI: 10.1021/jo990629o     Document Type: Article

References (53)

1. Abbreviations used for amino acids and the designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247, 977-983. The following additional abbreviations are used: BAL, Backbone Amide Linker; Bzl, benzyl; Ddz, 2-(3,5-dimethyloxyphenyl)propyl(2)oxycarbonyl; DIEA, N,N-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; EDC, 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride; ESMS, electrospray mass spectrometry; FABMS, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, N-[(dimethylanuno)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-yhnethylene]-N- methylmethanaminium hexafluorophosphate N-oxide; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HOAt, 1-hydroxy-7-azabenzotriazole (3-hydroxy-3H-1,2,3-triazolo-[4,5-b]pyridine); HOBt, 1-hydroxybenzotriazole; IRAA, quot;internal reference" amino acid (ref 19); NMM, N-methylmorpholine; op-PALdehyde, mixture of 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid and 4-(2-formyl-3,5-dimethoxyphenoxy)butyric acid; PEG-PS, poly(ethylene glycol)-polystyrene (graft resin support); Ph, phenyl; pNA, p-nitroanilide; Py, pyrrolidino; PyAOP, 7-azabenzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium hexafluorophosphate; PyBOP, benzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium hexafluorophosphate; SPS, solid-phase synthesis; TFFH, 1,1,3,3-tetramethyl-2-fluoroformamidinium hexafluorophosphate; TMP, 2,4,6-trimethylpyridine (collidine). Amino acid symbols denote the L-configuration unless stated otherwise. All solvent ratios are volume/volume unless stated otherwise.
2. (a) University of Minnesota. (b) University of Barcelona.
3. Jensen, K. J.; Alsina, J.; Songster, M. F.; Vágner, J.; Albericio, F.; Barany, G. J. Am. Chem. Soc. 1998, 120, 5441-5452 and references therein.
4. As described to account for racemization in the coupling of W-acyl-N-methylamino acids; see: (a) McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51, 2562-2570. (b) Davies, J. S.; Mohammed, A. K. J. Chem. Soc., Perkin Trans. 1 1981, 2982-2990.
5. As described to account for racemization in the coupling of W-acyl-N-methylamino acids; see: (a) McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51, 2562-2570. (b) Davies, J. S.; Mohammed, A. K. J. Chem. Soc., Perkin Trans. 1 1981, 2982-2990.
6. Compare to discussions of the mechanism of racemization via 5(4H)-oxazolone mechanisms upon activation of N-acylamino acids. Reviews: (a) Kemp, D. S. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1979; Vol. 1, pp 315-383. (b) Barany, G.; Merrifield, R. B. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1980; Vol, 2, pp 1-284, especially pp 122-123. (c) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997; pp 116-119.
7. Compare to discussions of the mechanism of racemization via 5(4H)-oxazolone mechanisms upon activation of N-acylamino acids. Reviews: (a) Kemp, D. S. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1979; Vol. 1, pp 315-383. (b) Barany, G.; Merrifield, R. B. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1980; Vol, 2, pp 1-284, especially pp 122-123. (c) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997; pp 116-119.
8. Compare to discussions of the mechanism of racemization via 5(4H)-oxazolone mechanisms upon activation of N-acylamino acids. Reviews: (a) Kemp, D. S. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1979; Vol. 1, pp 315-383. (b) Barany, G.; Merrifield, R. B. In The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1980; Vol, 2, pp 1-284, especially pp 122-123. (c) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997; pp 116-119.
9. It is precisely for this reason that stepwise SPPS is rarely carried out in the N → C direction, as has been reviewed in ref 5b. For a few examples of N → C SPPS, see: (a) Letsinger, R. L.; Kornet, M. J. J. Am. Chem. Soc. 1963, 85, 3045-3046. (b) Felix, A. M.; Merrifield, R. B. J. Am. Chem. Soc. 1970, 92, 1385-1391. (c) Henkel, B.; Zhang, L.; Bayer, E. Liebigs Ann. Recl. 1997, 2161-2168. (d) Léger, R.; Yen, R.; She, M. W.; Lee, V. J.; Hecker, S. J. Tetrahedron Lett. 1998, 39, 4171- 4174.
10. It is precisely for this reason that stepwise SPPS is rarely carried out in the N → C direction, as has been reviewed in ref 5b. For a few examples of N → C SPPS, see: (a) Letsinger, R. L.; Kornet, M. J. J. Am. Chem. Soc. 1963, 85, 3045-3046. (b) Felix, A. M.; Merrifield, R. B. J. Am. Chem. Soc. 1970, 92, 1385-1391. (c) Henkel, B.; Zhang, L.; Bayer, E. Liebigs Ann. Recl. 1997, 2161-2168. (d) Léger, R.; Yen, R.; She, M. W.; Lee, V. J.; Hecker, S. J. Tetrahedron Lett. 1998, 39, 4171- 4174.
11. It is precisely for this reason that stepwise SPPS is rarely carried out in the N → C direction, as has been reviewed in ref 5b. For a few examples of N → C SPPS, see: (a) Letsinger, R. L.; Kornet, M. J. J. Am. Chem. Soc. 1963, 85, 3045-3046. (b) Felix, A. M.; Merrifield, R. B. J. Am. Chem. Soc. 1970, 92, 1385-1391. (c) Henkel, B.; Zhang, L.; Bayer, E. Liebigs Ann. Recl. 1997, 2161-2168. (d) Léger, R.; Yen, R.; She, M. W.; Lee, V. J.; Hecker, S. J. Tetrahedron Lett. 1998, 39, 4171- 4174.
12. It is precisely for this reason that stepwise SPPS is rarely carried out in the N → C direction, as has been reviewed in ref 5b. For a few examples of N → C SPPS, see: (a) Letsinger, R. L.; Kornet, M. J. J. Am. Chem. Soc. 1963, 85, 3045-3046. (b) Felix, A. M.; Merrifield, R. B. J. Am. Chem. Soc. 1970, 92, 1385-1391. (c) Henkel, B.; Zhang, L.; Bayer, E. Liebigs Ann. Recl. 1997, 2161-2168. (d) Léger, R.; Yen, R.; She, M. W.; Lee, V. J.; Hecker, S. J. Tetrahedron Lett. 1998, 39, 4171-4174.
13. Amino acid and peptide p-nitroanilides are needed as chromogenic substrates to monitor the activity of numerous proteolytic enzymes. See: Handbook of Synthetic Substrates for the Coagulation and Fibrinolytic System; Hemker, H. C., Ed.; Martinus Nijhof Publishers: Boston, 1983.
14. Chemical ligation of unprotected peptides in aqueous solution has emerged as an extremely powerful method of peptide/protein assembly. Thioesters are often the reactive C-terminal functionality used in these chemical ligations. See: (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779. (b) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485-12489. (c) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363-2370. Review: (d) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289, 266-298.
15. Chemical ligation of unprotected peptides in aqueous solution has emerged as an extremely powerful method of peptide/protein assembly. Thioesters are often the reactive C-terminal functionality used in these chemical ligations. See: (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779. (b) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485-12489. (c) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363-2370. Review: (d) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289, 266-298.
16. Chemical ligation of unprotected peptides in aqueous solution has emerged as an extremely powerful method of peptide/protein assembly. Thioesters are often the reactive C-terminal functionality used in these chemical ligations. See: (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779. (b) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485-12489. (c) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363-2370. Review: (d) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289, 266-298.
17. Chemical ligation of unprotected peptides in aqueous solution has emerged as an extremely powerful method of peptide/protein assembly. Thioesters are often the reactive C-terminal functionality used in these chemical ligations. See: (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776-779. (b) Tam, J. P.; Lu, Y.-A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485-12489. (c) Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363-2370. Review: (d) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289, 266-298.
18. 2, although the reductive animation step gave the expected N-PAL-p-nitroaniline intermediate, this compound could not be acylated further. See: Songster, M. F.; Vágner, J.; Barany, G. Lett. Pept. Sci. 1996, 2, 265-270.
19. An indirect Boc SPS route to p-nitroanilides involves a urethanelinked p-aminoanilide resin, and a late-stage solution oxidation step to convert an aromatic amine to a nitro group. See: Burdick, D. J.; Struble, M. E.; Burnier, J. P. Tetrahedron Lett. 1993, 34, 2589-2592.
20. Another Boc SPS chemistry route involves oxime ester anchoring, but suffers from low yields during the cleavage step using p-nitroaniline as the attacking nucleophile. See: Voyer, N.; Lavoie, A.; Pinette, M.; Bernier, J. Tetrahedron Lett. 1994, 35, 355-358.
21. (a) Kaspari, A.; Schierhorn, A.; Schutkowski, M. Int. J. Pept. Protein Res. 1996, 48, 486-494.
22. (b) Bernhardt, A.; Drewello, M.; Schutkowski, M. J. Pept. Res. 1997, 50, 143-152.
23. Such instability is reported in ref 12a, which states that about half of the p-nitroanilide moieties are lost after 1 h of treatment of Fmoc-Glu-pNA with piperidine-NMP (1:4). However, this result is in contrast to a claim that Boc-Ala-pNA is completely stable toward piperidine-DMF (1:1) and pyrrolidine-DMF (1:3); see: Rijkers, D. T. S.; Adams, H. P. H. M.; Hemker, H. C.; Tesser, G. I. Tetrahedron 1995, 51, 11235-11250.
24. On the other hand, thioester intermediates required for chemical ligation are readily prepared by Boc chemistry, as reviewed in ref 8d. See also: (a) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4055-4058. (b) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, S.; Tanaka, I.; Hikichi, K.; Aimoto, S. Bull. Chem. Soc. Jpn. 1993, 66, 2700-2706. (c) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995, 36, 1217-1220.
25. On the other hand, thioester intermediates required for chemical ligation are readily prepared by Boc chemistry, as reviewed in ref 8d. See also: (a) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4055-4058. (b) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, S.; Tanaka, I.; Hikichi, K.; Aimoto, S. Bull. Chem. Soc. Jpn. 1993, 66, 2700-2706. (c) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995, 36, 1217-1220.
26. On the other hand, thioester intermediates required for chemical ligation are readily prepared by Boc chemistry, as reviewed in ref 8d. See also: (a) Blake, J.; Li, C. H. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4055-4058. (b) Hojo, H.; Kwon, Y.; Kakuta, Y.; Tsuda, S.; Tanaka, I.; Hikichi, K.; Aimoto, S. Bull. Chem. Soc. Jpn. 1993, 66, 2700-2706. (c) Canne, L. E.; Walker, S. M.; Kent, S. B. H. Tetrahedron Lett. 1995, 36, 1217-1220.
27. Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669-8672.
28. Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J. Pept. Protein Res. 1991, 37, 513-520.
29. Futaki, S.; Sogawa, K.; Maruyama, J.; Asahara, T.; Niwa, M.; Hojo, H. Tetrahedron Lett. 1997, 38, 6237-6240.
30. For all structural formulas in this paper, BAL handles are depicted as the isomer in which the aminomethyl group is para to the valeryl linking group. However, many of the experiments described herein start with a mixture (1:2) of the ortho and para isomers of PALdehyde, i.e., o,p-PALdehyde; furthermore, the side-chain moiety had one fewer carbon (substitution of butyryl for valeryl). The latter compound is commercially available from PE Biosystems (Framingham, MA).
31. (a) Atherton, E.; Clive, D. L.; Sheppard, R. C. J. Am. Chem. Soc. 1975, 97, 6584-6585.
32. (b) Matsueda, G. R.; Haber, E. Anal. Biochem. 1980, 104, 215-227.
33. (c) Albericio, F.; Barany, G. Int. J. Pept. Protein Res. 1993, 41, 307-312.
34. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem. Soc., Chem. Commun. 1994, 201-203.
35. Built into the BAL approach (see structure in Scheme 1), the anchored first amino acid allyl ester is N-alkylated, and therefore more likely to form a diketopiperazine at the dipeptidyl ester stage.
36. Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.; Kates, S. A. Tetrahedron Lett. 1997, 38, 4853-4856.
37. For longer peptides, the strategy from this point on would be to use standard cycles of Fmoc chemistry to achieve chain growth in the C → N direction. The second phase of this study uses a longer model peptide-resin.
38. Kates, S. A.; Daniels, S. B.; Albericio, F. Anal. Biochem. 1993, 212, 303-310.
39. (a) Dourtoglou, V.; Ziegler, J.-C.; Gross, B. Tetrahedron Lett. 1978, 19, 1269-1272.
40. (b) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927-1930.
41. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205-208.
42. Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995, 117, 5401-5402.
43. From these experiments, both diastereomers (L-Ala and D-Ala at penultimate position) were formed, isolated, and characterized.
44. +), The formation of this carboxylcapped derivative is attributed to the presence of small amounts (e.g., 0.4%, w/w) of pyrrolidine as a contaminant to commercial phosphonium salts. For more on this side reaction, which is avoided entirely by using purified coupling reagent, see: Alsina, J.; Barany, G.; Albericio, P.; Kates, S. A. Lett. Pept. Sci. 1999, 6, 243-245.
45. Formation of the guanidino side product consumes the nucleophilic moiety, and as a result, it is more difficult to carry out the desired amide formation to completion. Therefore, in the solid-phase mode, the side reaction leads to lower yields (unreacted starting material observed), although the purity of the product on the support is not affected. Interestingly, the side reaction was not observed when aminium/uronium salts tased on HOXt were used.
46. Preparation of this model peptide-resin was along the same lines already described (see the text and ref 23), as detailed in the Experimental Section.
47. The required ammo acid thioester building blocks were synthesized by EDC (1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride)/HOBt-mediated coupling of RSH to the Boc-AA-OH, followed by Boc removal with 4 N HCl-dioxane. The products were obtained in 63-71% yields, and suitable for direct use, as detailed in Experimental Section.
48. When TFFH was used to mediate couplings, the desired peptide thioesters were formed with little racemization or other byproducts, but due to the guanidino side reaction already discussed (ref 30), reactions seldom went to completion and unreacted starting material was observed. See Table 3 in the Supporting Information.
49. As was precedented in the p-nitroanilide system, substantial formation of the diastereomer occurred when a preactivation protocol was used. Therefore, when such a protocol was carried out intentionally, sufficient levels of the racemized species formed to allow their characterization.
50. These experiments are described in the Supporting Information.
51. Solé, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403.
52. Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem. 1990, 55, 3730-3743.
53. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Anal. Biochem. 1970, 34, 595-598.