Benzotriazol-1-yloxytris(dimethylamino)phosphonium Hexafluorophosphate1

[566002-33-6]  · C12H22F6N6OP2  · Benzotriazol-1-yloxytris(dimethylamino)phosphonium Hexafluorophosphate  · (MW 442.29)

(peptide coupling agent providing high yield and low racemization levels;1 promotes peptide cyclization19 and other lactamization,23 especially b-lactam formation;24 used to effect various amidification reactions25-29 and selective esterification;31 reagent for nucleotidic coupling32)

Alternate Names: BOP; Castro's reagent.

Physical Data: mp 138 °C (dec.).

Solubility: insol H2O; sol THF, CH2Cl2, MeCN, acetone, DMF, NMP, DMSO.

Form Supplied in: white solid; commercially available. Purity 97-98%.

Analysis of Reagent Purity: 1H NMR (acetone-d6): 3.0 (d, 18H, JH-P = 10 Hz, NMe2), 7.9 (m, 4H, arom.). 31P NMR (CH2Cl2): +43.7 (s, P+), -144.2 (septet, PF6-). IR (KBr): 1010 (P-N), 840, 770, 560 (PF6-).

Preparative Methods: BOP reagent was initially obtained2a by reaction of Hexamethylphosphorous Triamide (tris(dimethylamino)phosphine, TDAP) with CCl4 in the presence of HOBt (1-Hydroxybenzotriazole) followed by precipitation of the phosphonium salt by anion exchange with aqueous KPF6 solution. It was then prepared2b,c by reaction of cheaper (Me2N)3PO (HMPA) with COCl2 (phosgene) to generate the chlorophosphonium chloride intermediate, with treatment of the latter with HOBt and base, and precipitation as below. COCl2 was further replaced by POCl3.2d,e The crude final product can, if necessary, be purified by recrystallization from a mixture of acetone-ether or CH2Cl2 (DCM)-ether.

Handling, Storage, and Precautions: irritant and harmful. Light sensitive. Incompatibility: strong oxidizing agents. Avoid breathing dust; avoid contact with eyes, skin, and clothing. Keep container closed and store in the dark. Refrigerate. Although BOP is widely used, it is important to stress that the carcinogenic HMPA is a side product and the reagent must be handled with caution. All operations should be carried out in a fume hood.

Peptide Coupling Reagent.

First prepared with the aim to associate both the coupling agent (phosphonium salt as dehydrating agent) and the racemization suppressor (HOBt additive) in the same compound, the BOP reagent has since then been widely developed, mainly in peptide synthesis, as well as in other fields. Mechanistic studies on the probable intermediates (acyloxyphosphonium salt, active ester, symmetrical anhydride, oxazolone, eq 1) during the activation step of carboxylic acids with BOP have been made. Early investigations indicating the intermediacy of a benzotriazolyl active ester,1a,2b,3 as in activation with the DCC/HOBt (see 1,3-Dicyclohexylcarbodiimide) reagent system, were later questioned1c in the light of better results obtained with BOP in a number of cases. After further studies, the direct formation of the acyloxyphosphonium salt, presumably through the cyclic complex (eq 1), was postulated as likely.1c

Unoptimized initial work in peptide coupling with BOP had already shown high yields and short reaction times, even with bulky amino acids (eq 2) and little if no side reactions with some sensitive amino acids bearing unprotected side chain functional groups.2

Unlike DCC, BOP reacts exclusively with carboxylate salts, with carboxylic acids remaining unchanged. The trifluoroacetate anion is not activated by BOP; therefore the trifluoroacetylation side reaction is never encountered.

In usual peptide synthesis (e.g. with the Boc/TFA deprotection strategy), a typical coupling step is conducted as follows: BOP is added to the mixture of the Boc-amino acid (or peptide acid segment) and the C-terminal-protected peptide salt (generally the TFA salt). The solvent can be THF, DCM, MeCN, DMF, or NMP, according to the solubility of the reaction partners, but using as high a concentration as possible. No reaction occurs until the tertiary base, N-methylmorpholine, Triethylamine, or preferably Diisopropylethylamine (DIEA), is added. In every case the reaction mixture has to be kept rather basic in order to ensure a fast coupling. Three equiv of base are necessary to neutralize the carboxylic acid, the amine salt, and the acidic HOBt. In such conditions the coupling rate is so high that racemization is negligible in urethane-protected amino acid coupling and fairly low, albeit not negligible, in segment coupling (see eq 3).4 The excess of acid and BOP is typically 1.1 mol equiv in solution synthesis.

Racemization in segment coupling has been studied with realistic models4a in solution; it appears to be very small when nonhindered amino acids (Ala, Leu, Lys…) are activated, but coupling of segments terminated with bulky residues (Val, Ile, Thr…) are to be avoided (eq 3).4b

Segment condensation with BOP is generally peculiarly fast compared with conventional use of the classical DCC/HOBt method, as a few hours are enough to achieve coupling of equimolar quantities of both partners, provided the highest concentration is used (eq 4).5

It is very characteristic that this procedure allows the skipping of a separate neutralization step, either in solution or in SPPS (solid phase peptide synthesis) using the Boc/TFA strategy. This is not only time-saving but also useful to minimize diketopiperazine formation during the third step of a synthesis.

The coupling reaction of Boc-Ile-OH with H-His-Pro-OMe . TFA is generally a very low yield reaction as the neutralization of the dipeptide salt is very readily followed by cyclization to cyclo(His-Pro) (eq 5). Using the BOP procedure allows the efficient in situ trapping of the dipeptide amine by the acylating reagent. This feature also makes strategies of segment coupling at a proline residue possible, which are very often restricted by the necessity of using prolyl t-butyl esters in order to avoid the cyclization reaction.

This specific ability of BOP for avoiding diketopiperazine formation is illustrated in liquid phase synthesis5a as well as in the SPPS.5c,d,6,7b

Another facility offered by BOP is the possibility to couple DCHA salts, due to the high rate ratio of amino acid coupling towards dicyclohexylamine (DCHA). This feature is especially useful in SPPS where the small amount of dicyclohexylamide formed is easily washed out. The main application of that procedure lies in the introduction of histidine as the cheap and easily acessible Boc-His(Boc)-OH both in liquid (eq 6)5a and solid7b-d phase synthesis.

Many peptides have been synthesized in solution,5a,b,8 stepwise or by segment coupling, using BOP as coupling agent.

In solid phase synthesis, BOP exhibits some distinct advantages, including the suppression of a separate neutralization step previously mentioned; indeed, after TFA treatment and washing, it is convenient to introduce the next Boc-amino acid and BOP as solids, with a minimal volume of solvent and, at last, DIEA. Coupling is generally completed after 15-30 min, with the pH maintained at 8 with excess of acylating reagents (1.5-3M). Finally, the water- and DCM-soluble byproducts, HMPA and HOBt, allow very easy washing of the reaction product, in contrast to DCC condensation where elimination of insoluble urea often remains a problem.

The use of BOP in SPPS, initially limited,7 has since been widely developed, with Boc/TFA,9,10 Boc/HF,11 and mainly Fmoc/piperidine12-15 deprotection systems. In most cases it compares favorably with other reagents such as DCC, DCC/HOBt, and symmetrical anhydride. As already observed in the liquid phase, some amino acids with side chain functional groups such as Tyr, Thr, Ser (hydroxy group),2a,9b or poorly soluble pGlu (g-lactam)7d,9c can be used without protecting groups; other functional groups such as CONH2 (Asn) can lead in some cases to partial dehydration (e.g. b-cyanoalanine)11,14b,c or cyclization (succinimide)9a,30 and need to be protected; such classical dehydration reactions, not observed by others,1a,2a,b are sometimes dependent on reactions conditions (e.g. repeated coupling cycles) and must be carefully examined.

Peptide Cyclization.

Various results were provided16-20 in peptide chain cyclization with BOP, arising either from the reaction conditions or from the ring size, and from the linear precursor conformation.

Better yields and purities are obtained16 with less powerful coupling agents such as DPPA (Diphenyl Phosphorazidate) and NDPP (norborn-5-ene-2,3-dicarboxamidodiphenyl phosphate) in cyclization of tetra- and pentapeptide sequences, despite the need for longer reaction times than with BOP or HBTU (O-Benzotriazol-1-yl-N,N,N,N-tetramethyluronium Hexafluorophosphate; 1,1,3,3-tetramethyluronium analog of BOP).

Comparable yields are given for Boc-g-D-Glu-Tyr-Nle-D-Lys-Trp-OMe cyclizations17 with BOP (NaHCO3, 4 h, 76%) and MTPA (dimethylphosphinothioyl azide, NEt3, 36 h, 79%), albeit in different reaction conditions; in this work the reported racemization associated with the use of BOP is not shown.

Cyclization, leading to Gly/Lys-containing peptide macrocycles,18 has been investigated with a number of coupling reagents. Among them DEPC (Diethyl Phosphorocyanidate), DPPA, and BOP are the most effective ones, giving near-quantitative yields.

Macro ring closure of a partly proteinogenic sequence in the synthesis19 of the cyclodepsipeptide Nordidemnin B was carried out using BOP in heterogeneous conditions (eq 7).

In SPPS, side chain to side chain cyclization by the BOP procedure not only proceeds more rapidly, but also gives a purer cyclic product than the DCC/HOBt procedure.20

The use of BOP has been reported in the final cyclization of cyclosporin,21b even if some racemization is observed. The very difficult coupling of N-methyl amino acids is achieved in the same work,21 as in the preceding syntheses.19 For the coupling of hindered N-methyl22a and a,a-dialkyl22b amino acids, new reagents22 such as PyBroP, PyCloP, or PyClU seem to be more promising.

Besides the peptide field, cyclization leading to macrocyclic polyether tetralactams (crown ethers) have been carried out with BOP.23

b-Lactam Cyclization.

Cyclization of b-amino acids to b-lactams is efficiently effected by treatment with BOP (eq 8).24a The present method appears to be limited to the formation of b-lactams from N-unsubstituted b-amino acids (R1 = R2 = R3 = R5 = H, R4 = Me, yield 10%).

In the cephem series, cyclization yields of 80% are obtained with BOP, in DCM at rt, with retention of the initial chirality.24b


A number of other various amidation reactions have been conducted using BOP. Such preparations include N,O-dimethyl hydroxamates of amino acids25a and peptides25b (precursors of chiral peptidyl aldehydes), heterocyclic amide fragments in the synthesis of macrolide26 and porphyrin models,27 dansylglycine anhydride as a mixed sulfonic-carboxylic imide by dehydration-cyclization of dansylglycine,28 and selective monoacylation of heterocyclic diamine in carbohydrate series (eq 9).29


Treatment of N-protected amino acids with phenol, NEt3, and BOP in the appropriate solvent (DCM, MeCN, DMF) affords the corresponding phenyl esters in good yield (eq 10).30

BOP in combination with Imidazole (ImH) as catalyst provides a very mild system for selective esterification of polyhydroxy compounds such as carbohydrates; thus trehalose is converted into mono-, di-, and tripalmitate mixtures, with the various quantities of each of them depending on the solvent and acid/alcohol ratio. Monoacylated compounds can be obtained selectively, albeit in moderate yield (31%).31

Nucleotidic Coupling and Related Reactions.

BOP has been used as a condensing agent to promote internucleotidic bond formation in phosphotriester oligodeoxyribonucleotide synthesis.32 31P NMR studies32a show that a benzotriazolyl phosphate derivative is involved as an active ester intermediate, as with carboxylic acids.3

The BOP procedure was applied to the preparation of undecanucleoside decaphosphate.32b The same type of activation with BOP is carried out for introducing a phosphonamide bond in dipeptide models33 with good efficiency (&egt;60%); coupling reactions by activation of phosphinic acid derivatives34 with BOP give poor yields.

1. (a) Le Nguyen, D.; Castro, B. Peptide Chemistry 1987; Protein Research Foundation: Osaka, 1988; p 231. (b) Kiso, Y.; Kimura, T. Yuki Gosei Kagaku Kyokai Shi 1990, 48, 1032 (CA 1991, 114, 164 722k). (c) Coste, J.; Dufour, M. N.; Le Nguyen, D.; Castro, B. In Peptides, Chem. Struct. Biol. ESCOM: Leiden, 1990; pp 885-888.
2. (a) Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. TL 1975, 1219. (b) Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Peptides 1976; University of Bruxelles: Brussels, 1976; p 79. (c) Castro, B.; Dormoy, J. R.; Dourtoglou, B.; Evin, G.; Selve, C.; Ziegler, J. C. S 1976, 751. (d) Dormoy, J. R.; Castro, B. TL 1979, 3321. (e) Dormoy, J. R.; Castro, B. T 1981, 37, 3699.
3. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. JCR(S) 1977, 182; JCR(M) 1977, 2118.
4. (a) Castro, B.; Dormoy, J. R.; Le Nguyen, D. TL 1978, 4419. (b) Castro, B.; Dormoy, J. R.; Le Nguyen, D. Peptides 1978; Wroclaw University Press, Poland, 1979; p 155. (c) Le Nguyen, D.; Dormoy, J. R.; Castro, B.; Prevot, D. T 1981, 37, 4229.
5. (a) Le Nguyen, D.; Seyer, R.; Heitz, A.; Castro, B. JCS(P1) 1985, 1025. (b) Fehrentz, J. A.; Seyer, R.; Heitz, A.; Fulcrand, P.; Castro, B.; Corvol, P. Int. J. Pept. Protein Res. 1986, 28, 620. (c) Seyer, R.; Aumelas, A.; Tence, M.; Marie, J.; Bonnafous, J. C.; Jard, S.; Castro, B. Int. J. Pept. Protein Res. 1989, 34, 235. (d) Seyer, R.; Aumelas, A.; Marie, J.; Bonnafous, J. C.; Jard, S.; Castro, B. HCA 1989, 72, 678.
6. Gairi, M.; Lloyd-Williams, P.; Albericio, F.; Giralt, E. T 1990, 31, 7363.
7. (a) Rivaille, P.; Gautron, J. P.; Castro, B.; Milhaud, G. T 1980, 36, 3413. (b) Le Nguyen, D.; Heitz, A.; Castro, B. JCS(P1) 1987, 1915. (c) Ratman, M.; Le Nguyen, D.; Rivier, J.; Sargent, P. B.; Lindstrom, J. B 1986, 25, 2633. (d) Seyer, R.; Aumelas, A.; Caraty, A.; Rivaille, P.; Castro, B. Int. J. Pept. Protein Res. 1990, 35, 465. (e) Evin, G.; Galen, F. X.; Carlson, W. D.; Handschumacher, M.; Novotny, J.; Bouhnik, J.; Menard, J.; Corvol, P.; Haber, E. B 1988, 27, 156. (f) Bouhnik, J.; Galen, F. X.; Menard, J.; Corvol, P.; Seyer, R.; Fehrentz, J. A.; Le Nguyen, D.; Fulcrand, P.; Castro, B. JBC 1987, 262, 2913. (g) Fehrentz, J. A.; Heitz, A.; Seyer, R.; Fulcrand, P.; Devilliers, R.; Castro, B.; Heitz, F.; Carelli, C. B 1988, 27, 4071. (h) Bonnafous, J. C.; Tence, M.; Seyer, R.; Marie, J.; Aumelas, A.; Jard, S. BJ 1988, 251, 873. (i) Liu, C. F.; Fehrentz, J. A.; Heitz, A.; Le Nguyen, D.; Castro, B.; Heitz, F.; Carelli, C.; Galen, F. X.; Corvol, P. T 1988, 44, 675.
8. (a) Eid, M.; Evin, G.; Castro, B.; Menard, J.; Corvol, P. BJ 1981, 197, 465. (b) Cumin, F.; Evin, G.; Fehrentz, J. A.; Seyer, R.; Castro, B.; Menard, J.; Corvol, P. JBC 1985, 260, 9154.
9. (a) Fournier, A.; Wang, C. T.; Felix, A. M. Int. J. Pept. Protein Res. 1988, 31, 86. (b) Fournier, A.; Danho, W.; Felix, A. M. Int. J. Pept. Protein Res. 1989, 33, 133. (c) Forest, M.; Fournier, A. Int. J. Pept. Protein Res. 1990, 35, 89.
10. Jarrett, J. T.; Landsbury, Jr, P. T. TL 1990, 31, 4561.
11. Jezek, J.; Houghten, R. A. Peptides 1990; ESCOM: Leiden, 1991; p 74.
12. Rule, W. K.; Shen, J. H.; Tregear, G. W.; Wade, J. D. Peptides 1988; de Gruyter: Berlin, 1989; p 238.
13. Hudson, D. JOC 1988, 53, 617.
14. (a) Frank, R. W.; Gausepohl, H. Modern Methods in Protein Chemistry; de Gruyter: Berlin, 1988; Vol. 3. p 41. (b) Gausepohl, H.; Kraft, M.; Frank, R. W. Int. J. Pept. Protein Res. 1989, 34, 287. (c) Gausepohl, H.; Kraft, M.; Frank, R. W. Peptides 1988; de Gruyter: Berlin, 1989; p 241.
15. Waki, M.; Nakahara, T.; Ohno, M. Peptides Chemistry 1990; Protein Research Foundation: Osaka, 1991; p 95.
16. Schmidt, R.; Neubert, K. Int. J. Pept. Protein Res. 1991, 37, 502.
17. Ueki, M.; Kato, T. see ref. 15, p 49.
18. Crusi, E.; Huerta, J. M.; Andreu, D.; Giralt, E. TL 1990, 31, 4191.
19. Jouin, P.; Poncet, J.; Dufour, M. N.; Pantaloni, A.; Castro, B. JOC 1989, 54, 617.
20. Felix, A. M.; Wang, C. T.; Heimer, E. P.; Fournier, A. Int. J. Pept. Protein Res. 1988, 31, 231.
21. (a) Wenger, R. M. HCA 1983, 66, 2672. (b) Wenger, R. M. HCA 1984, 67, 502.
22. (a) Coste, J.; Frerot, E.; Jouin, P.; Castro, B. TL 1991, 32, 1967. (b) Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M. N.; Jouin, P. T 1991, 47, 259.
23. Duriez, M. C.; Pigot, T.; Picard, C.; Cazaux, L.; Tisnes, P. T 1992, 48, 4347.
24. (a) Kim, S.; Lee, T. A. Bull. Kor. Chem. Soc. 1988, 9, 189. (b) Roze, J. C.; Pradere, J. P.; Duguay, G.; Guevel, A.; Quiniou, H.; Poignant, S. CJC 1983, 61, 1169.
25. (a) Fehrentz, J. A.; Castro, B. S 1983, 676. (b) Fehrentz, J. A.; Heitz, A.; Castro, B. Int. J. Pept. Protein Res. 1985, 26, 236.
26. Somers, P. K.; Wandless, T. J.; Schreiber, S. L. JACS 1991, 113, 8045.
27. Selve, C.; Niedercorn, F.; Nacro, M.; Castro, B.; Gabriel, M. T 1981, 37, 1903.
28. Weinhold, E. G.; Knowles, J. R. JACS 1992, 114, 9270.
29. Chapleur, Y.; Castro, B. JCS(P1) 1980, 2683.
30. Castro, B.; Evin, G.; Selve, C.; Seyer, R. S 1977, 413.
31. Chapleur, Y.; Castro, B.; Toubiana, R. JCS(P1) 1980, 1940.
32. (a) Molko, D.; Guy, A.; Teoule, R.; Castro, B.; Dormoy, J. R. NJC 1982, 6, 277. (b) Molko, D.; Guy, A.; Teoule, R. Nucleosides Nucleotides 1982, 1, 65.
33. Dumy, P.; Escale, R.; Vidal, J. P.; Girard, J. P.; Parello, J. CR(C) 1991, 312, 235.
34. Elhaddadi, M.; Jacquier, R.; Petrus, C.; Petrus, F. PS 1991, 63, 255.

Jean-Robert Dormoy & Bertrand Castro

Sanofi Chimie, Gentilly, France

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