O-Benzotriazol-1-yl-N,N,N,N-tetramethyluronium Hexafluorophosphate

[94790-37-1]  · C11H16F6N5OP  · O-Benzotriazol-1-yl-N,N,N,N-tetramethyluronium Hexafluorophosphate  · (MW 379.29)

(coupling reagent for peptide synthesis1)

Alternate Name: HBTU.

Physical Data: mp 206-207 °C (dec).

Solubility: sol DMF (0.5 mol L-1). With the tetrafluoroborate counterion the corresponding salt (TBTU) is slightly more soluble (0.6 mol L-1).

Form Supplied in: white solid; widely available.

Purification: by crystallization in a mixture of MeCN and CH2Cl2.

Handling, Storage, and Precautions: very stable, not hygroscopic, and can be stored indefinitely. Solutions in DMF (0.45 M) can be stored in an inert atmosphere for weeks. Syntheses of peptides carried out with freshly prepared, 6 and 13 week stored solutions show similar quality of the crude product.3 Violent decomposition can occur when dried at elevated temperature.4

Both O-benzotriazol-1-yl-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU) and tetrafluoroborate (TBTU) salts have been used as condensing reagents for the preparation of peptides in both solution and solid-phase strategies.1,4 In solution, reaction of a-amino protected amino acids or dipeptides with equimolar amounts of a-carboxyl protected amino acids or dipeptides and Diisopropylethylamine (DIEA) (2 equiv) in DMF proceeds cleanly for 15 min at 0 °C, yielding the corresponding peptides in good yields (80-96%) (eq 1).1,4

HBTU activation has been adapted for automated stepwise solid-phase peptide synthesis for both t-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc) strategies.5 For the former, a simple, effective protocol has been developed, which involves simultaneous in situ neutralization with coupling (extra equivalents of base are required). This protocol is particularly suitable for assembling complex peptides, arising from sequence-dependent peptide chain aggregation. Since aggregation occurs when protonated a-ammonium peptide-resin intermediates are neutralized, simultaneous neutralization and acylation can help to overcome this phenomenon.6 For Fmoc chemistry, efficient protocols are also available for both batch and continuous-flow systems.3,7

Various protected peptide segments have been coupled on solid-phase using a combination of TBTU, HOBt, and DIEA for the syntheses of several small proteins. Coupling yields, using threefold excess of peptide segment and coupling times of ~12 h, are greater than 95%.8

Racemization from uronium salt mediated couplings has been determined by analysis of the epimeric products by HPLC using different models. In all cases, racemization is similar or lower than that obtained with carbodiimide and benzotriazole based phosphonium salt methods.1,2,4,9

In the absence of the carboxylic component, HBTU reacts with amino groups leading to the formation of a Schiff base (eq 2).10 Thus in syntheses conducted in solution, the excess of both HBTU and amino component should be avoided. In both solution and solid-phase strategies the sequence of reagent addition is critical. HBTU should be delivered to the carboxylic component for preactivation, prior to the addition of the amine. The Schiff base formation can also occur during slow reactions, such as the preparation of cyclic peptides, where both amino and carboxylic components are in equimolar amounts and an excess of the uronium salt can block the amino group.11 For the synthesis of cyclic peptides the phosphonium derivatives Benzotriazol-1-yloxytris(dimethylamino)phosphonium Hexafluorophosphate (BOP)12 and benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP),13 can be more useful.

Uronium salts have been used for cyclization of linear peptides in both solution and solid-phase modes. Since this reaction is sequence dependent, there are no general conditions. Thus cyclization in solution with uronium salt methods give, for some peptides, better results than the classical reagent Diphenyl Phosphorazidate (DPPA),14 but for another case the converse is true.15 Furthermore, dehydration of C-terminal aspartylamide peptides during cyclization with HBTU has been described. This side-reaction can be prevented by the addition of one equivalent of HOBt.16 Although good results have been obtained in the solid-phase,17 guanidino formation side-reactions have also been reported.11

Recently, other uronium salts, such as O-(N-succinimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TSTU),4,18 O-(N-5-norbornene-endo-2,3-dicarboximidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TNTU),4,18 O-(2-oxo-1(2H)-pyridyl)-N,N,N,N-bis(pentamethylene)uronium tetrafluoroborate (TOPPipU),19 N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium Hexafluorophosphate N-Oxide (HATU),20 and O-(7-azabenzotriazol-1-yl)-N,N,N,N-bis(tetramethylene)uronium hexafluorophosphate (HAPyU)21 have been described and are commerically available. The uronium salts derived from 7-aza-1-hydroxybenzotriazole (HATU and HAPyU) have been shown to be superior to their benzotriazole analogs in terms of coupling efficiency,20,21 racemization,22 and cyclization,23 in both solution and solid-phase strategies.

Finally, an X-ray structure determination of HBTU revealed that the solid-state structure differs considerably from the formulation commonly presented in the literature. The solid-state structure is not the N,N,N,N-tetramethyluronium salt but rather the guanidinium N-oxide (3).24

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6. (a) Schnoelzer, M.; Alewood, P; Jones, A.; Alewood, D.; Kent, S. B. H. Int. J. Peptide Protein Res. 1992, 40, 180. (b) Reid, G. E.; Simpson, R. J. Anal. Biochem. 1992, 200, 301. (c) Sueiras-Diaz, J.; Horton, J. TL 1992, 33, 2721.
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13. Coste, J.; Le-Nguyen, D.; Castro, B. TL 1990, 31, 205.
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17. (a) Trzeciak, A.; Bannwarth, W. TL 1992, 33, 4557. (b) Neugebauer, W.; Willick, G. In Proceedings of the Twenty-Second European Peptide Symposium; Schneider, C. H.; Eberle, A. N. Eds.; ESCOM: Leiden, 1993; pp 393-394.
18. Bannwarth, W.; Schmidt, D.; Stallard, R. L.; Hornung, C.; Knorr, R.; Müller, F. HCA 1988, 71, 2085.
19. Henklein, P.; Beyermann, M.; Bienert, M.; Knorr, R. In Proceedings of the Twenty-First European Peptide Symposium; Giralt, E.; Andreu, D. Eds.; ESCOM: Leiden, 1991; pp 67-68 (CA 1991 115, 232 811m).
20. Carpino, L. A. JACS 1993, 115, 4397.
21. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. JCS (P1) 1994, 201.
22. Carpino, L. A.; E.-Faham, A. JOC 1994, 59, 695.
23. Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Carpino, L. A.; Bienert, M. TL 1993, 34, 4781.
24. Abdelmoty, I.; Albericio, F.; Carpino, L.A.; Foxman, B. M.; Kates, S. A. Lett. Peptide Sci. 1994, in press.

Fernando Albericio & Steven A. Kates

Millipore Corporation, Bedford, MA, USA

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