[2592-95-2]  · C6H5N3O  · 1-Hydroxybenzotriazole  · (MW 135.14) (hydrate)


(peptide synthesis; nucleotide synthesis)

Alternate Names: HOBT; HOBt.

Physical Data: mp 155-160 °C; exact melting point depends on the amount of water of hydration present.

Form Supplied in: white solid usually containing 12-17% water of hydration; widely available commercially.

Analysis of Reagent Purity: should be a white solid; if it becomes discolored, it is advisable to purify.

Purification: recrystallize from either water or aqueous ethanol.

Handling, Storage, and Precautions: should be stored in the dark; avoid contact with strong acids, oxidizing agents, and reducing agents; heating above 180 °C causes rapid exothermic decomposition; toxicity not fully investigated so should be treated with caution.

Peptide Coupling.

1-Hydroxybenzotriazole is most widely used in reactions involving the coupling of amino acid units to give peptides.1 In this context it has been used mainly as an additive to a coupling reaction, although there are also examples of HOBT being incorporated into the coupling reagent itself. The most common use of 1-hydroxybenzotriazole in peptide synthesis is in conjunction with a carbodiimide such as 1,3-Dicyclohexylcarbodiimide (DCC). Although it is quite possible to couple amino acids using DCC alone, it is found that the addition of 1-hydroxybenzotriazole to the reaction system results in improved reaction rates and suppressed epimerization of the chiral centres present in the peptide (eq 1).2 This reaction appears to proceed via DCC-mediated formation of a hydroxybenzotriazole ester intermediate which then reacts with the amino function of a second amino acid to give the coupled product.

This coupling protocol is not limited to the synthesis of small peptides in solution, but can also be used in the solid-phase preparation of larger peptides, and for the coupling of larger peptide fragments. It should be noted, however, that there are some drawbacks to this method; most notably, it is sometimes difficult to purify the peptide due to contamination with dicyclohexylurea and the method is unsatisfactory for the coupling of N-methylated amino acids.3 The problem of purification can often be overcome by using an alternative carbodiimide, e.g. ones that give water-soluble byproducts can be used, such as EDC.MeI (see 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride).4 Polymer-bound variants of HOBT have also been used to overcome purification problems,5 since the urea byproduct can be removed from the polymer-bound active ester intermediate (1) by washing with chloroform-isopropyl alcohol mixtures. Once the urea has been removed, the active ester can be reacted with the second peptide fragment to give the product (eq 2), regenerating the polymer-bound HOBT. While this method appears quite successful for the preparation of small peptide fragments, it is clearly unsuitable for the preparation of larger peptides by solid-phase synthesis.

Although the addition of HOBT to the carbodiimide-mediated coupling of amino acids has been shown to suppress epimerization of the chiral centres present, there are still a number of cases where the level of epimerization is unsatisfactory. One method for further suppressing epimerization is to add Copper(II) Chloride to the reaction system.6 In this case it is important that the correct stochiometry is determined, since the copper(II) chloride not only suppresses epimerization, but it also slows down the reaction rate and reduces the overall yield (eq 3).

The problem of epimerization during carbodiimide-mediated coupling of amino acids can also be overcome by the use of an alternative coupling agent. Once such system that still employs HOBT as an additive involves the use of bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) and N-methylmorpholine (NMM) (eq 4).7 See also Bis(2-oxo-3-oxazolidinyl)phosphinic Chloride.

HOBT has also been used as an additive in other reactions involving amino acid derivatives.8 It would appear that most reaction systems that are capable of generating a hydroxybenzotriazole ester intermediate will give successful coupling. One such system involves the use of amino acid derived trichlorophenyl esters.9 These can be readily reacted with a second amino acid unit in the presence of HOBT to give the coupled product with little or no racemization (eq 5). p-Nitrophenyl and pentachlorophenyl esters can also be employed with similar success. This reaction again is thought to proceed via the hydroxybenzotriazole ester intermediate and it has been shown that use of the potassium salt of HOBT in conjunction with a crown ether can lead to substantial rate increase,10 presumably by enhancing the rate of formation of the active ester intermediate.

HOBT has also been used in conjunction with amino acid chlorides to facilitate peptide bond formation. This has proved of particular use in the case of FMOC-protected amino acid chlorides in solid-phase peptide synthesis where direct coupling is rather slow and suffers from competing oxazolone formation.11

As mentioned earlier, HOBT has also been incorporated into the peptide coupling reagent itself and one example of such a reagent is benzotriazol-1-yloxytris(dimethylamino)phosphonium chloride (BOP).12 BOP is prepared by the reaction of HOBT with Hexamethylphosphorous Triamide in the presence of carbon tetrachloride, and is very effective in the coupling of amino acids (eq 6). See also Benzotriazol-1-yloxytris(dimethylamino)phosphonium Hexafluorophosphate (also known as BOP).

A second peptide coupling reagent that incorporates the HOBT unit is O-Benzotriazol-1-yl-N,N,N,N-tetramethyluronium Hexafluorophosphate (HBTU),13 which can be prepared by the reaction of tetramethylurea with oxalyl chloride followed by treatment with HOBT and Potassium Hexafluorophosphate. HBTU can then be used as a direct coupling agent as outlined in eq 7.

Nucleotide Synthesis.

Phosphotriester derivatives of HOBT have been employed in the synthesis of nucleotides.14 The phosphotriesters are readily formed by reaction of HOBT with the corresponding aryl phosphorodichloridates in the presence of pyridine and can then be used to couple nucleosides as outlined in eq 8. This reaction takes advantage of the fact that a differentially protected nucleoside will react rapidly with the reagent, displacing one molecule of HOBT to form an intermediate phosphotriester (2), but at this stage reaction with a second nucleoside molecule is extremely slow due to steric hindrance. Consequently the intermediate can then be reacted with a second nucleoside, the more reactive primary hydroxy displacing the second molecule of HOBT and giving the coupled product. This method for coupling nucleosides has been applied to both solution and solid-phase synthesis of a variety of RNA fragments.

Other Applications.

HOBT has also been employed as a catalyst for the conversion of isoamides into maleimides (eq 9).

1. For recent general reviews on peptide synthesis involving the use of HOBT, see: Bodanszki, M. Int. J. Pept. Protein Res. 1985, 25, 449; Kent, S. B. H.; Annu. Rev. Biochem. 1988, 57, 957; Bodanszky, M. J. Protein Chem. 1989, 8, 461; Fields, G. B.; Nobel, R. L. Int. J. Pept. Protein Res. 1990, 35, 161.
2. König, W.; Geiger, R. CB 1970, 103, 788; König, W.; Geiger, R. CB 1970, 103, 2024; König, W.; Geiger, R. CB 1970, 103, 2034; Windridge, G. C.; Jorgensen, E. C. JACS 1971, 93, 6318; Nagaraj, R.; Balaram, P. T 1981, 37, 2001; Benoiton, N. L.; Kuroda, K. Int. J. Pept. Protein Res. 1981, 17, 197; Chen, S. T.; Wu, S. H.; Wang, K. T. S 1989, 37; Dardoize, F.; Goasdoué, C.; Goasdoué, N.; Laborit, H. M.; Topall, G. T 1989, 45, 7783; Bennoiton, N. L.; Lee, Y. C.; Steinaur, R.; Chen, F. M. F. Int. J. Pept. Protein Res. 1992, 40, 559; Bennoiton, N. L.; Lee, Y. C.; Chen, F. M. F. Int. J. Pept. Protein Res. 1993, 41, 587.
3. Coste, J.; Frérot, E.; Jouin, P.; Castro, B. TL 1991, 32, 1967.
4. Kimura, T.; Takai, M.; Masui, Y.; Morikawa, T.; Sakakibara, S. Biopolymers 1981, 20, 1823; Hagiwara, D.; Neya, M.; Miyazaki, Y.; Hemmi, K.; Hashimoto, M. CC 1984, 1676.
5. Berrada, A.; Cavelier, F.; Jacquier, R.; Verducci, J. BSF(1) 1989, 511; Grigor'ev, E. I.; Zhil'tsov, O. S. JOU 1989, 25, 1774; Chen, S. T.; Chang, C. H.; Wang, K. T. JCR(S) 1991, 206.
6. Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamada, T.; Kuwata, S. CC 1988, 419; Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamada, T.; Kuwata, S. Int. J. Pept. Protein Res. 1992, 39, 237; Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamada, T.; Kuwata, S. Int. J. Pept. Protein Res. 1992, 39, 308.
7. van der Auwera, C.; van Damme, S.; Anteunis, M. J. O. Int. J. Pept. Protein Res. 1987, 29, 464.
8. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. TL 1989, 30, 1927.
9. König, W.; Geiger, R. CB 1973, 106, 3626.
10. Horiki, K.; Murakami, A. H 1989, 28, 615.
11. Carpino, L. A.; Chao, H. G.; Beyermann, M.; Bienert, M. JOC 1991, 56, 2635; Sivanandaiah, K. M.; Babu, V. V. S.; Renukeshwar, C. Int. J. Pept. Protein Res. 1992, 39, 201.
12. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. TL 1975, 1219.
13. Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Zioudrou, C. S 1984, 572.
14. van der Marel, G.; van Boeckel, C. A. A.; Wille, G.; van Boom, J. H. TL 1981, 22, 3887; Marugg, J. E.; Tromp, M.; Jhurani, P.; Hoyng, C. F.; van der Marel, G. A.; van Boom, J. H. T 1984, 40, 73; Gottikh, M.; Ivanovskaya, M.; Shabarova, Z. Bioorg. Khim. 1988, 14, 500; Hirao, I.; Miura, K. CL 1989, 1799; Colonna, F. P.; Scremin, C. L.; Bonora, G. M. TL 1991, 32, 3251.

Barry Lygo

Salford University, UK

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