[39968-33-7]  · C5H4N4O  · 3-Hydroxy-3H-1,2,3-triazolo[4,5-b]pyridine  · (MW 136.13)

(coupling additive for peptide synthesis1-4)

Alternate Names: HOAt; 7-aza-1-hydroxybenzotriazole.

Physical Data: mp: sample decomposes at temperatures above 205 °C.

Solubility: sol DMF.

Form Supplied in: white solid; commercially available.

Purification: by crystallization from ethanol or water.

Handling, Storage, and Precautions: is very stable, not hygroscopic, and can be stored indefinitely. HOAt solutions in DMF (0.3 M) can be stored in an inert atmosphere for weeks. Syntheses of peptides carried out with freshly prepared and 4-week old solutions show similar quality for the crude product.5 Violent decomposition may occur when dried at elevated temperatures.

General Considerations.

HOAt is the 7-aza derivative of 1-Hydroxybenzotriazole (HOBt), the most common peptide coupling additive.6,7 These reagents are employed to decrease racemization, inhibit side reactions such as formation of N-acylurea, and increase yield and purity during peptide assembly. These compounds are used in conjunction with carbodiimides or active esters such as the pentafluorophenyl ester or built into stand-alone reagents such as N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium Hexafluorophosphate N-Oxide (HATU) and 7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP). Although known for many years, HOAt8-12 has only recently been applied as an additive in peptide synthesis. The greater effectiveness of HOAt relative to HOBt is shown by its use in the coupling of 2-phenylpropionic acid with the highly hindered secondary amine (1) (eq 1), a reaction difficult to achieve in the presence of HOBt.1

The extent of racemization during stepwise and segment condensation methods for the assembly of peptides in solution via 1,3-Dicyclohexylcarbodiimide (DCC) or 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride (EDC) in the presence of HOAt and HOBt has been examined. When the amino acid ester is used as a salt, various bases such as Diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), and 1,8-Bis(dimethylamino)naphthalene (Proton Sponge, PS) were also needed (eq 2) (Table 1). In all examples, racemization was lowered when HOBt was replaced with HOAt (run 1 vs. 2, 3 vs. 4, 5 vs. 6, 8 vs. 9). The level of epimerization was further lowered when methylene chloride (DCM) was substituted for DMF as the solvent (runs 8 vs. 10).1

HOAt has also been applied to automated stepwise solid-phase synthesis via the Fmoc/t-Bu strategy.2,3,5 The effectiveness of HOAt-based reagents was demonstrated in the assembly of the acyl carrier protein fragment 65-74 (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Gln-Gly-NH2). Coupling times and excesses of reagents (amino acid and activator) were reduced in order to exaggerate poor couplings. In N,N-diisopropylcarbodiimide and pentafluorophenyl-mediated couplings, the superiority of HOAt over HOBt was clearly shown in terms of greatly enhanced purity. The extent of amino acid deletions depended on the exact conditions. Addition of HOXt in the case of guanidinium or phosphonium salt-based couplings did not significantly improve the coupling yield.

Solid-phase segment couplings were also aided by the use of HOAt. In a model study, Fmoc-Phe-Ser(t-Bu)-OH was coupled to H-Pro-PAL-PEG-PS-resin.3,13 With HOAt in the presence of DCC, the extent of racemization was lowered dramatically relative to HOBt. A more complex example involved synthesis of the N-terminal domain of g-zein protein by eight repetitive couplings of the appropriate protected hexapeptide.14

N-Methyl amino acids are often very difficult to couple cleanly and in good yield, especially by solid-phase techniques. Excellent results were obtained for DIPCDI/HOAt-mediated couplings, leading to various cyclosporin (CsA) segments, including H-MeLeu-MeLeu-Ala-NH2, H-Val-MeLeu-Ala-NH2, and H-MeLeu-MeVal-Ala-NH2, all of which were obtained in >99% yield.15

The HOAt-based uronium salt 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU) was employed in solution for the efficient cyclization of a linear hexapeptide constructed exclusively from L-amino acids. Such systems are difficult to cyclize without C-terminal racemization.16

Significant improvements in the synthesis of phosphonamidate peptides were achieved via the HOAt esters of the phosphonochloridates (eq 3).17

1. Carpino, L. A. JACS 1993, 115, 4397.
2. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. JCS(D) 1994, 201.
3. Carpino, L. A.; El-Faham, A.; Albericio, F. TL 1994, 35, 2279.
4. Carpino, L. A.; El-Faham, A. JOC 1994, 59, 695.
5. Kates, S. A.; Minor, C. A.; Shroff, H.; Haaseth, R. C.; Triolo, S. A.; El-Faham, A.; Carpino, L. A.; Albericio, F. In Peptides 1994: Proceedings of the Twenty-Third European Peptide Symposium; Maia, H. L. S., Ed.; ESCOM: Leiden, 1995; pp 248-249.
6. König, W.; Geiger, R. CB 1970, 103, 788.
7. König, W.; Geiger, R. CB 1970, 103, 2024, 2034.
8. Azev, Y.; Mokrushina, G. A.; Postovoskii, I. Ya.; Sheinker, Yu. N.; Anisimova, O. S. Chem. Heterocycl. Compd. 1976, 1172.
9. Mokrushina, G. A.; Azev, Y.; Postovoskii, I. Ya.; Sheinker, Yu. N.; Anisimova, O. S. Chem. Heterocycl. Compd. 1975, 880.
10. Azev, Y.; Mokrushina, G. A.; Postovoskii, I. Ya. Chem. Heterocycl. Compd. 1974, 687.
11. Sacher, R. M.; Alt, G. H.; Darlington, W. A. J. Agric. Food Chem. 1973, 21, 132.
12. Yutilov, Y. M.; Ignaatenko, A. G. Khim. Prom-st., Reakt. Osobo Chist. Veshchestva 1981, 27 (CA 1982, 96, 68 883s).
13. (a) Barany, G.; Albericio, F.; Solé, N. A.; Griffin, G. W.; Kates, S. A.; Hudson, D. In Peptides 1992: Proceedings of the Twenty-Second European Peptide Symposium; Schneider, C. H.; Eberle, A. N., Eds.; ESCOM: Leiden, 1993; pp 267-268. (b) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.; Hudson, D.; Barany, G. JOC 1990, 55, 3730.
14. Dalcol, I.; Rabanal, F.; Albericio, F.; Pons, M.; Geli, M.; Torrent, M.; Ludevid, M. D.; Giralt, E. In Peptides 1994: Proceedings of the Twenty-Third European Peptide Symposium; Maia, H. L. S., Ed.; ESCOM: Leiden, 1995; pp 60-61.
15. Angell, Y. M.; García-Echeverría, C.; Rich, D. H. TL 1994, 35, 5981.
16. Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Carpino, L. A.; Bienert, M. TL 1993, 34, 4781.
17. Musiol, H.-J.; Grams, F.; Rudolph-Bohner, S.; Moroder, L. JOC 1994, 59, 6144.

Steven A. Kates

PerSeptive Biosystems, Framingham, MA, USA

Fernando Albericio

University of Barcelona, Spain

Louis A. Carpino

University of Massachusetts, Amherst, MA, USA

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