N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium Hexafluorophosphate N-Oxide

[148893-10-1]  · C10H15F6N6OP  · N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium Hexafluorophosphate N-Oxide  · (MW 380.28)

(coupling reagent for peptide synthesis1-4)

Alternate Name: HATU.

Physical Data: mp: sample decomposes at temperatures above 180 °C.

Solubility: sol DMF (0.6 mol L-1).

Form Supplied in: white solid; commercially available.

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

Handling, Storage, and Precautions: is very stable, not hygroscopic, and can be stored indefinitely at 0 °C. HATU solutions in DMF (0.5 M) can be stored in an inert atmosphere for weeks (after 4 weeks, purity is 85 %). Syntheses of peptides carried out with freshly prepared and 3-week old solutions show crude products of similar quality.5 Violent decomposition can occur when dried at elevated temperature.

General Considerations.

HATU is the 7-azabenzotriazolyl analog of O-Benzotriazol-1-yl-N,N,N,N-tetramethyluronium Hexafluorophosphate (HBTU).6 X-ray structure determinations of both HATU and HBTU have revealed that the solid-state structures differ from the formulations commonly presented in the literature.7 The solid-state structure is not the N,N,N,N-tetramethyluronium salt but rather the guanidinium N-oxide.

HATU and its pyrrolidinium derivative, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU), have been used as condensing reagents for the preparation of peptides by both solution and solid-phase techniques.1-3 In solution, reaction of a-amino protected amino acids or dipeptides with equimolar amounts of a-carboxyl protected amino acid hydrochlorides or amino amides, and base (2 equiv) [Diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), 2,4,6-Collidine, or 1,8-Bis(dimethylamino)naphthalene (Proton Sponge, PS)] in DMF proceeds cleanly, yielding the corresponding peptides in good yields. The level of epimerization in HATU-mediated couplings was reduced compared to HBTU-promoted acylations (eq 1) (Table 1).1,4,8

The results, collected in Table 1, clearly indicated that (i) HATU is superior to HBTU (entries 1 vs. 2, 3 vs. 4, 5 vs. 6, 7 vs. 9, and 12 vs. 13); (ii) HAPyU is similar to HATU, although in some cases HAPyU can give slightly better results (entries 7 vs. 10); and (iii) the use of collidine considerably reduces the level of epimerization (entries 7 vs. 8, 12 vs. 14, 13 vs. 15, and 16 vs. 17).

HATU activation has been adapted for automated stepwise solid-phase peptide synthesis for both fluorenylmethyloxycarbonyl (Fmoc) and t-butyloxycarbonyl (Boc) strategies. For the former, the fragment 65-74 (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH2) of the acyl carrier protein was chosen as a model to compare the performance of HATU and HBTU. Results from these studies indicated that uronium salt coupling is superior to carbodiimide and pentafluorophenyl ester-mediated methods, and the aza derivatives are more efficient than HBTU.3

HATU has shown special utility for the synthesis of peptides containing hindered amino acids. Thus the solid-phase assembly of the model pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH2 was compared via three different coupling methods, HATU, HBTU, and Fmoc-amino acid fluorides, the last named being known as one of the most efficient reagents for couplings involving consecutive Aib units.9 After 2 h coupling using 4 equiv of both Fmoc-Aib-OH and Fmoc-Tyr(t-Bu)-OH, as well as HXTU plus 8 equiv of DIPEA (all remaining couplings were carried out for 30 min), the pentapeptide was obtained with a purity of 94% for the HATU synthesis, and only 43% for HBTU. The synthesis conducted under similar conditions with Fmoc-Aib-F gave a purity of 96%, indicating that the reactivity of HATU is much closer to that of fluorides than to that of HBTU.3

The use of HATU for the solid-phase coupling of N-methyl amino acids has also been shown to be beneficial if compared with HBTU, as found during the syntheses of fragments of cyclosporin (CsA): H-Val-MeLeu-Ala-NH2 (HATU, >99%; HBTU, 87%);10 H-MeLeu-MeLeu-Ala-NH2 (HATU, >99% HBTU, 34%);10 H-D-Ala-MeLeu-MeLeu-MeVal-Phe-Val-OH (HATU, 85%; HBTU, 8%).3

Various protected peptide segments have been coupled by solid-phase techniques using HATU-mediated coupling methods.11 Thus the synthesis of the N-terminal domain of g-zein protein has been reported in good yield by eight successive segment couplings of the same protected hexapeptide to give the desired sequence using HATU and 3-Hydroxy-3H-1,2,3-triazolo[4,5-b]pyridine (HOAt) in the presence of DIPEA.12

Less racemization is observed for solid-phase segment condensations in the presence of HATU relative to HBTU. The coupling of Fmoc-Phe-Ser(t-Bu)-OH onto H-Pro-resin was accompanied by 5% and 15% racemization, respectively, with HATU and HBTU in the presence of DIPEA. Replacement of DIPEA with collidine reduced the racemization level to 3%, confirming the effects observed for coupling reactions carried out in solution.4

HATU and HAPyU have been used for the cyclization of linear peptides in both solution and solid-phase modes. Cyclization in solution with HAPyU of a linear hexapeptide constructed exclusively from L-amino acids gave a better yield and less racemization at the C-terminal residue compared to other uronium or phosphonium salts.13 HATU has also been shown to be the method of choice for cyclizations conducted while the peptide still remains anchored to the polymeric support. In the preparation of a library of 1296 cyclic pentapeptides, cyclization was complete after 1 h as judged by the ninhydrin test.14

In the absence of the carboxylic component, HATU, as is the case for HBTU, reacts with amino groups leading to the formation of tetramethylguanidinium derivatives.5,15 In syntheses conducted in solution, an excess of either HATU or amino component should be avoided. In both solution and solid-phase strategies, the order of addition of reagents is critical. HATU should be delivered to the carboxylic component for preactivation prior to addition of the amine.

In addition, formation of the guanidinium derivative can also occur during slow reactions, such as the preparation of cyclic peptides, where both amino and carboxylic components are of necessity present together in equimolar amounts. An excess of the uronium salt can block the amino group.16 For the synthesis of cyclic peptides, the phosphonium derivatives 7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) and 7-azabenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (AOP), which are also commercially available, may be more useful.

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. JOC 1994, 59, 695.
4. Carpino, L. A.; El-Faham, A.; Albericio, F. TL 1994, 35, 2279.
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. (a) Dourtoglou, V.; Ziegler, J. C.; Gross, B. TL 1978, 1269. (b) Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Zioudrou, C. S 1984, 572. (c) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. TL 1989, 30, 1927.
7. Abdelmoty, I.; Albericio, F.; Carpino, L. A.; Foxman, B. F.; Kates, S. A. Lett. Peptide Sci. 1994, 1, 57.
8. Albericio, F.; Abdelmoty, I.; Bofill, J. M.; Carpino, L. A.; El-Faham, A.; Foxman, B. F.; Gairí, M.; Griffin, G. W.; Kates, S. A.; Lloyd-Williams, P.; Scarmoutzos, L. M.; Shroff, H.; Triolo, S. A.; Wenschuh, H. In Peptides 1994: Proceedings of the Twenty-Third European Peptide Symposium; Maia, H. L. S., Ed.; ESCOM: Leiden, 1995; pp 23-25.
9. Wenschuh, H.; Beyermann, M.; Krause, E.; Brudel, M.; Winter, R.; Schümann, M.; Carpino, L. A.; Bienert, M. JOC 1994, 59, 3275.
10. Angell, Y. M.; García-Echeverría, C.; Rich, D. H. TL 1994, 35, 5981.
11. Giralt, E.; Albericio, F.; Dalcol, I.; Gairí, M.; Lloyd-Williams, P.; Rabanal, F. In Innovation and Perspectives in Solid-Phase Peptide Synthesis: Biological & Biomedical Applications; Epton, R., Ed.; Mayflower Worldwide: West Midlands, UK, 1994; pp 51-60.
12. 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.
13. Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Carpino, L. A.; Bienert, M. TL 1993, 34, 4781.
14. Darlak, K.; Romanovskis, P.; Spatola, A. F. In Proceedings of the Thirteenth American Peptide Symposium; Hodges, R. S., Smith, J. A., Eds.; ESCOM: Leiden, 1994; pp 981-983.
15. Gausepohl, H.; Pieles, U.; Frank, R. W. In Proceedings of the Twelth American Peptide Symposium; Smith, J. A.; Rivier, J. E., Eds.; ESCOM: Leiden, 1992; pp 523-524 (CA 1992, 117, 171 981j).
16. Arttamangkul, S.; Aldrich, J. V. Int. J. Peptide Protein Res. 1995, in press.

Fernando Albericio

University of Barcelona, Spain

Steven A. Kates

PerSeptive Biosystems, Framingham, MA, USA

Louis A. Carpino

University of Massachusetts, Amherst, MA, USA

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