Baláspiri Lajos

Kovács Kálmán

Pallai P.

Penke Botond

English

University of Szeged

A P P L I C A T I O N O F P E N T A F L U O R O P H E N Y L E S T E R S O F B O C - A M I N O A C I D S I N S O L I D P H A S E P E P T I D E S Y N T H E S I S By B. PENKE, L. BALÁSPIRÍ, P. PALLAI and K. KOVÁCS Institute of Organic Chemistry, Attila József University, Szeged (Received October 16, 1974) Owing to their high reactivity, pentafluorophenyl esters of BOC-amino acids can be well applied in solid phase peptide synthesis. With their application we synthesized the protected tetra-peptide A C T H 11—14. The synthesis is compared with other methods. In the first years of the application of solid phase peptide synthesis DCC was almost exclusively used as condensing agent in the peptide coupling reactions; only the coupling of N-acylglutamine and asparagine was performed by active esters. When the numerous disadvantages of the DCC-method had become evident, various active esters, mostly p-nitrophenyl esters (/?-ONP), were often successfully applied in solid phase peptide synthesis [1—3]. A great advantage of the active ester coupling is to give generally purer products than DCC-condensation, furthermore the excess active ester can be regenerated. However, the use of />-ONP has also shortcomings, because, according to KARLSSON [4] and LOSSE [5], quantitative reaction cannot be reached even after 2X16 hours coupling. BODANSZKY [6] found, that in solid phase peptide synthesis o-nitrophenyl esters (o-ONP) are more reactive than /7-nitrophenyl- and pentachlorophenyl esters, probably owing to steric effects. As the active esters applied hitherto in solid phase synthesis were not sufficiently reactive, we tried to use for solid phase peptide synthesis the pentafluorophenyl esters of high reactivity, successfully applied in classical peptide synthesis by KISFALUDY and KOVÁCS [7]. First, we prepared o- and /7-nitrophenyl esters as well as penta-fluorophenyl esters of several BOC-amino acids (see Table I) then we stated [8] by reaction kinetic measurements that the pentafluorophenyl esters of BOC-amino acids are much more reactive in solid phase peptide synthesis, than o-ONP and J3-ONP. In our kinetic measurements we always used the concentration generally applied in solid phase peptide synthesis, because only results obtained in this way can be utilized in synthetic work. For comparing the reactivity of different active esters, two data were used: (a) the time (t1/2) necessary for 50% reaction, (b) the degree of coupling reached after 16 hours reaction time with 3fold excess of the active ester. The results are given in Table II. According to Table II, the difference between the reactivities of o- and />-nitro-phenyl esters is not high, and in some cases the /?-nitrophenyl esters prove to be more reactive. The deviation of these results from the measurements of BODANSZKY [6] 472 B. PENKE, L. BALÁSPIRI, P. PALLAI AND K . KOVÁCS Table I Physical properties and analytical data of active esters of BOC-amino acids Active.ester Solvent for recrystallization Melting point °C Formula; (MW) Elemental analysis Caled. Found BOC-Phe-0-ONP ethanol 143—144 c 62.2 62.5 (386.4) H 5.7 6.0 BOC-Phe-p-ONP ethanol 125—126 c 62.2 62.0 (386.4) H 5.7 5.9 BOC-Phe-OPFP ethyl acetate— 108—109 C20HL8O4NF5 c 55.7 55.5 petroleum- (431.3) H 4.2 4.4 ether BOC-Leu-o-ONP ether—hexane 52—54 C17H24O6N2 c 57.9 .57.8 (352.4) H 6.9 7.1 BOC-Leu-/>-ONP ethanol—water 85—86 C17H„406N2 c 57.9 58.1 (352.4) H 6.9 6.7 BOC-Leu-OPFP oil oil C17H20O4NF5 c 51.5 51.9 (397.3) H 5.1 5.4 BOC-Asn-o-ONP DMF—water 143—145 C15H19O,N3 • c 51.0 51.3 (353.3) H 5.4 • 5:6 BOC-Asn-/)-ONP ethanol 162—163 CUH1 8O,N3 c 51.0 51.4 (353.3) H 5.4 5.6 BOC-Asn-OPFP ether—hexane 119—121 C15H15O6N2F6 c 45.5 46.1 (397.3) H 3.8 4.2 BOC-Asn(Xa)-o-ONP DMF—water 139—141 C28H27O8N3 c 63.0 63.4 (533.6) H 5.2 5.4 BOC-Asn(Xa)-p-ONP DMF—water 149—152 C28H2708N3 c 63.0 63.7 (533.6) H 5.2 5.3 BOC-Asn(Xa)-OPFP ethyl acetate— 160—162 C28H23O6N2F5 c 58.2 58.4 hexane (579.2) H 4.0 4.0 BOC-Gln(Xa)-o-ONP DMF—water 115—117 C29H29O8N3 c 63.5 64.0 (547.6) H 5.3 5.5 BOC-Gln(Xa)-p-ONP DMF—water 109—111 C29H09OJN3 c 63.6 64.1 (547.6) H 5.3 5.7 BOC-Gln(Xa)-OPFP ethyl acetate— 149—150 C29H25O6N2F5 c 58.8 59.2 hexane (593.2) H 4.2 4.4 BOC-Gly-o-ONP ethanol 97—98 C1 3H1 606N, c 52.7 53.1 (296.3) H 5.4 5.6 . BOC-Gly-p-ONP ethanol—water 78—80 C13H16O6N2 c 52.7 53.0 (296.3) H 5.4 5.5 BOC-Gly-OPFP ethyl acetate— 75—76 C13H12O4NF5 C 45.7 45.8 hexane (341.3) H 3.5 3.8 BOC-Tyr(Bzl) OPFP ethyl acetate— 121—123 C27H24O5NF5 c 60.4 60.8 hexane . (537.5) ' H 4.5 4.6 BOC-Pro-OPFP hexane 48—51 C16H16O4NF6 c 50.4 50.3 (381.3) H 4.2 4.1 BOC-Val-OPFP hexane 58—60 C 1 6 H l e 0 4 NF 5 c 50.1 50.4 (383.3) H 4.7 4.9 Z-Lys(BOC)-OPFP ether—hexane 68—70 C25H27O6N2F5 c 55.0 55.0 (546.5) H 5.0 4.8 PENTAFLUOROPHENYL ESTERS OF BOC-AMINO ACIDS IN SOLID PHASE PEPTIDE SYNTHESIS 473-Table II Results of kinetic measurements . Amino acid Active ester in equivalents t1/2 (hours) Coupling (%) after 16 hrs reaction p-ONP o-ONP OPFP p-ONP o-ONP OPFP BOC-Phe 1 11.5 3.2 0.4 3 1.0 0.5 0.3 68 80 96 BOC-Leu 1 23.0 18.0 — . — — — 3 . 2.5 1.5 1.0 54 82 97 BOC-Asn 1 4.5 6.0 — • — — — 3 1.5 1.0 . 0.5 67 75 88 BOC-Val 3 — — 1.5 — — . 92 BOC-Gly 1 24.0 9.3 — . — — — 3 2.8 1.4 1.1 55 70 95 BOC-Ile 3 — — 2.4 . _ — . 77 BOC-Pro • 3 — — . 0.3 — • — . 98 BOC-Met 3 — — 2.1 — — 82 BOC-Ala 3 . — — 1.2 — . . — 94 BOC-Cys(Bzl) 3 • — • 0.6 - — • — 96 BOC-Tyr(Bzl) - 3 2.0 2.1 - 1.4 63 60 91 BOC-Asn(Xa) 3 2.6 2.8 1.7 65 58 86 BOC-Gln(Xa) 3 6.0 2.9 1.5 52 60 88 may be explained by the much higher concentrations applied in our method, and-perhaps by the difference of polymers. It is worth mentioning that the t1/2 values of pentafluorophenyl esters exceed 1 hour only in few cases. The difference in reacti-vities of active esters is even more apparent from the comparison of the percentage of coupling after 16 hours. This value often does not reach 60%, and never exceeds-90% for nitrophenyl esters, while for pentafluorophenyl esters it is never lower than 80% and often as high as 95—97%. On the basis of these data it seemed probable that pentafluorophenyl esters-can be succesfully used in solid phase synthesis; we proved their applicability by the synthesis of the protected tetrapeptide Z-Lys(BOC)-Pro-Val-GlyOMe (ACTH 11—14). In the synthesis the reaction of the active esters BOC-Val-OPFP (in. 6x excess), BOC-Pro-OPFP (in 3x excess), Z-Leu(BOC)-OPFP (in 6x excess) with the glycyl-polymer was successively used. We obtained 95—97% coupling af ter 10—12 hours in all cases. The tetrapeptide was split off from the resin in methyl-ester form, yielding ~ 80% rough product, which, purified by crystallization, gave: 70% pure, chromatographically homogeneous protected tetrapeptide. * Our method was compared with other methods of solid phase peptide synthesis-and with the repetitive excess mixed anhydride (REMA) method. We synthesized the ACTH 11—14 sequence mentioned above according to MERRIFIELD'S original' description, using 3x excess BOC-amino acids and DCC for.each coupling. The rough product contained more impurities than that obtained with our method; it could be, however, comparatively easily purified by extraction. Compared with the active: 4 7 4 B. PENKE, L. BALASPrRI, P. PALLAI A N D K. KOVACS ester method the synthesis of this tetrapeptide by simple DCC-condensation gave a lower yield and required longer purification. However, applying 3-nitrophthalic anhydride for preventing the formation of failure sequences [9], the DCC-condens-ation could also be performed with good yield, giving a relatively pure end-prod-uct. Details of this synthesis will be described elsewhere [10]. For comparison, we tried also to apply the REMA-method [11] in the synthesis •of the ACTH 11—14. As the sequence contains valine and proline, this method did not prove to be suitable for the synthesis of this tetrapeptide. Summarizing the results of kinetic measurements and the methods of peptide sysntheses described, it can bfe stated that the BOC-amino acid pentafluorophenyl esters, owing to their high reactivity, can be well applied in solid phase peptide synthesis. The synthesis of ACTH 11—14 can be performed also with DCC method, the active ester method gives, however, better results. Experimental Pentafluorophenyl esters of BOC-amino acids 10 mmole BOC-amino acid and lOmmole pentafluorophenol were dissolved in 25 ml ethyl acetate, (in the case of derivatives of glutamine and asparagine with addition of some dimethylformamide). The solution was cooled to 0°C, then 10 mmole DCC solved in 5 ml ethyl acetate was added, the solution was stirred at 0°C for 1 hr. The DCU, precipitated after standing at — 10°C for 30min, was filtered off and the solution evaporated in vacuum. The rough product was triturated with n-hexane and purified by recrystallization (see Table I). •o- and p-Nitrophenyl esters of BOC-amino acids 5 mmole BOC-amino acid and 6 mmole nitrophenol was dissolved in 10 ml •ethyl acetate (in the case of derivatives of glutamine and asparagine in DMF— tetrahydrofuran mixture). Further process was as for pentafluorophenyl esters. Kinetic measurements The first charge of the glycyl-polymer prepared from MERRIFIELD-polymer '(FLUKA 63 871) by the method described in [12] was 0.15 mmole/g. The BOC protecting group was split off with 25 % trifluoroacetic acid (TFA) in CH2C12. The active esters were used in two different quantities (see Table II). The rate of the •coupling reaction was followed by amino-acid analysis, as the measurement of the liberated phenol reagent did not give reliable results. To 20 mg (3 nmole) glycyl-polymer 3 nmole= 1 equivalent or 9 nmole=3 equi-valents active ester of BOC-amino acid dissolved in 0.2 ml D M F was added. After 2, 10, 30 min, and 1, 2, 4, 8, 16, 24 hours the suspension was filtered, the unreacted active ester was washed off 3 x with DMF, 2X with CH 2 C1 2 ,2x with methanol and 1 X with ether. The dipeptidyl-resin was hydrolysed in vacuum in a sealed tube with a mixture of 0.5 ml cc. HC1 and 0.5 ml acetic acid for 8 hrs or with 3M mercapto-PENTAFLUOROPHENYL ESTERS OF BOC-AMINO ACIDS IN SOLID PHASE PEPTIDE SYNTHESIS 4 7 5 ethanesulfonic acid (MES) in acetic acid. The hydrólysate was filtered, and condensed. (When applying MES the pH was adjusted to 2.20 by 4/VNaOH). The residue ' was dissolved in 1.00 ml buffer (pH 2.20) and the amino-acids were analyzed with an automatic amino-acid analyser Type Hd-1200E. Solid phase synthesis of Z-Lys(BOC)-Pro-Val-Gly-OMe (a) By DCC-condensation. The synthesis was performed with12 g ( = 4.8 mmole) glycyl-resin, using subsequently the BOC-valine (in 4 X excess), BOC-proline and Z-Lys(BOC)OH (both in 3 X excess). CH2C12 was used as solvent for coupling and subsequent washing. The BOC protecting groups were split off by 25% TFA in CH2C12. The coupling reactions were checked by amino-acid analysis. (Repetition of the coupling reaction proved not necessary in any case). The peptide was split off from the resin with BEYERMAN'S method [13]: the polymer was suspended in 150 ml methanol, and 30 ml diisopropylethylamine was added. After 20 hrs. shaking the resin was filtered off, washed with 50 ml methanol and the methanol distilled off in vacuum. The oily residue obtained was triturated with ether, yielding 2.8 g (87%) amorphous material, which was chromatographycally inhomogeneous. Further purification (boiling in ether, crystallization from ethyl acetate—petroleum ether) gave 1.76 g (55%) pure product, m.p: 109—111 °C. Amino-acid analysis: Gly 1.00; Pro 1.1 ;Val0.96; Lys 0.95. C32H4909N5(674.75); Calc.: C 59.33; H7.62; N 10.79. Found: C 58.90; H 7.80; N 10.65%. (b) By application of pentafluorophenyl esters of BOC-amino acids. The synthesis was made with 5 g ( = 2 mmole) glycyl-resin using subsequently BOC-Val-OPFP (in 6 X excess), BOC-Pro-OPFP (in 3 X excess) and Z-Lys(BOC)-OPFP (in 6 Xexcess). A 2:1 mixture of CH2C12 and D M F was used as solvent for coupling and subsequent washing. Coupling time was 10—12. hrs, the reaction was checked by the semiquantitative K A I S E R test [ 1 4 ] and by amino-acid analysis. Repeated coupling was not necessary. The BOC-groups were split off by 25% TFA in CH2C12. The tetrapeptide was cleaved from the resin with BEYERMAN'S method (see preceding paragraph). 1.1 g (80%) rough material was'obtained and purified by crystallization from ethyl acetate—petroleum ether, yielding 0.94 g (70%) chromatographically pure tetrapeptide, m.p. 110—113 °C. Amino-acid analysis: Gly 1.00; Pro 1.08; Val 0.92; Lys 1.05. C32H4909N5 (674.75); Calc.: C 59.33; H 7.62; N 10.79. Found: C 59.05; II 7.80; N 10.52%. References [1] Bodanszky, M., J. T. Sheehan; Ghem. and Ind. (London) 1966,1597. [2] Hörnle, S.: Z. Physiol. Chem. 348, 1355 (1967). [3] Johnson, B. J., W. P. May: J. Pharm. Sci. 58, 1568 (1969). [4] Karlsson, S., G. Lindeberg, U. Ragnarsson; Acta Chem. Scand. 24, 337 (1970). [5] Losse G., R. Ulbrich: 12th European Peptide Symposium. See: Peptides 1972. (North Holland . Publishing Company, Amsterdam 1973), p. 152: [6] Bodanszky, M., R. I. Bath: Chem. Commun. 1969, 1259. [7] Kisfaludy, L., M. Q. Ceprini, F. Rákóczy, J. Kovács: Peptides 1967. (North Holland Publishing Company, Amsterdam 1967), p. 23. [8] Kovács, K., B. Penke: 12th European Peptide Symposium. See: Peptides 1972. (North Holland Publishing Company, Amsterdam 1973). p. 187. / 4 7 6 В. PENKE, L. PALÁSPIRI, P. PALL АД A N D К. KOVÁCS [9] Wieland, Т., С. Birr, H. Wissenbach: Angew. Chem. 81, 782 (1969). [10] Репке, В., С. Birr; Annalen, in preparation. [11] Beyerman, H. С., E. W. F. De Leer, J. Floor: Ree. Trav. Chim. Pays-Bas 92, 481 (1973). [12] Loffet, A.: Z. Phys. Chem. 352, 4 (1971). [13] Beyerman, H. C., H. Hindriks, E. W. F. De Leer: Chem. Commun. 1968, 1668. [14] Kaiser, E., R. L. Colescott, C. D. Bossinger, P. I. Cook: Anal. Biochem. 34, 595 (1970). ПРИМЕНЕНИЕ ПЕНТАФТОРФЕНИЛОВЫХ ЭФИРОВ ВОС-АМИНОКИСЛОТ В СИНТЕЗЕ ПЕПТИДОВ В ТВЁРДОЙ ФАЗЕ Б. Пэнкэ, Л. Балашпири, П. Паллаи , К. Ковач Пентафторфениловые эфиры ВОС-аминокислот вследствие их большой реактивности с успехом употребляются в синтезе пептидов в твёрдой фазе. С применением этих соединений нами был синтезирован защитный тетрапептид А CT H 11—-14. Этот синтез сравнивался с другими методами синтеза. 

Application of pentafluorolphenyl esters of boc-amino acids in solid phase peptide synthesis

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Application of pentafluorolphenyl esters of boc-amino acids in solid phase peptide synthesis

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University of Szeged