The combination of 1M silicon tetrachloride and 3M phenol serves as an efficient N(α)-t-butoxycarbonyl deblocking agent in solid phase peptide synthesis, the duration of the cleavage being 10 min. This is demonstrated by the synthesis of the naturally occurring μ-receptor selective opioid heptapeptide, dermorphin

Gangadhar, B.P.

Sivanandaiah, K.M.

Sureshbabu, V.V.

ePrints@Bangalore University

Pergamon Tetrahedron Letters, Vol. 37, No. 33, pp. 5989-5990, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved PII: S0040-4039(96)01255-5 0040-4039/96 $15.00 +0.00 Si l icon Tetrachlor ide and Pheno l  as N~-t -Butoxycarbonyl  Group Deprotect ing  Agent  in Sol id Phase Pept ide Synthes is  Kadlebal M. Sivantndaiah', Vommina V. Suresh Babu and Beechanthalli P. Gangadhar Department of Studies in Chemistry, Central College, Bangalore University, Bangalore-560 001, India Abst ract  : The combination of IM silicon tetrachloride and 3M phenol serves as an efficient N a -t-butoxycarbonyl deblocking agent in solid phase peptide synthesis, the duration of the cleavage being 10 rain. This is demonstrated by the synthesis of the naturally occurring g-receptor selective opioid heptapeptide, dermorphin. Copyright © 1996 Elsevier Science Ltd t-Butoxycarbonyl (Boc) group is extensively used for temporary protection of the amino group in solid phase peptide synthesis (SPPS). Its subsequent removal is effected by acidolysis. A major concern of peptide chemists is the repetitive acidolysis that becomes mandatory in this approach and the consequent side reactions that result. In recent years, instead of acidolysis, some organo-silicon reagents have been tried. Lott et al. t have observed that iodotrimethylsilane can bring about the cleavage of Boc group from several protected amino acids and peptides. The cleavage was followed by NMR. However, its application for the synthesis of any specific peptide was not reported by them. We have earlier described the utility of iodotrichlorosilane, prepared from SiCI4 and NaI, for Boc deprotection during SPPS of oxytocin 2.Merrifield and co-workers 3'4 observed that 1M chlorotrimethylsilane d 3M phenol can be employed for Boc group deprotection i  SPPS. This communication utlines the use of the combination of 1M SiCI4 and 3M phenol in CH2C12 for efficient Boc group deprotection n SPPS. The reagent is inexpensive and is easily prepared by mixing freshly distilled SiCI4 and phenol in CHIC12. The cleavage of Boc group from Boc-Phe-OCH2-C6H4-resin was effected by combinations of 1M SiCI4 with 1M phenol, 2M phenol and 3M phenol and the duration of cleavage in these cases was found to be 45 rain, 30 min and 10 min respectively as estimated by the picrate method s. Subsequently further deprotection experiments were carried out employing 20 mmoles of SiCh and 60 mmoles of phenol in CH2C12 (20 ml) per gram of protected aminoacyl resin with an esterification level of about 0.5 mmole/g. The stability of different amino and carboxyl protecting roups to 1M SiCI4-3M phenol has been studied by the addition of the reagent to the amino acid erivative and stirring at room temperature. The reaction was monitored by thin layer chromatography. It was observed that Boc group was cleaved completely in 10 min, while the N~-9-fluorenylmethoxycarbonyl, l~-benzyloxycarbonyl, benzyl ester and benzyl ether groups were not cleaved to any noticeable extent even after 18 hr. The reagent does not also affect the side chain protecting roups such as nitro group of nitroarginine, thyl and methyl carboxylic esters after continuous treatment for 18 hr. 5989 5990 In order to determine the stability of the benzyl ester linkage between the C-terminal amino acid and the solid support, Boc-Pbe-OCH2-Cd-L-resin with an esterification level of 0.24 mmole/g was treated with the reagent for 40 hr. During this period, the reagent was replaced at intervals of 10 hr and the reaction was carried out under nitrogen atmosphere. A known quantity of the resin was withdrawn at intervals of 10 rain, 10 hr, 20 hr, 30 hr and 40 hr and the amino acid content on the resin was estimated by the picrate method s. No cleavage of the aminoacyl resin bond was observed in about 20 hr. After 40 hr treatment the cleavage was estimated to be about 2%. That the mixture of SiCh and phenol does not act as a source of HCI that causes Boc deprotection was shown by the slow introduction of one equivalent of 2% triethylamine in CH2C12 to the reaction mixture containing the reagent and Boc-Phe-OCH2-CrI-14-resin. It was observed that the deprotection, though slow, was not prevented as indicated by Kaiser's qualitative ninhydrin test 6. The reaction presumably involves an intermediate complex, (C13S.i---~)-CrHs), similar to the one proposed by Merrifield and co-workers 3't~! H The utility of this reagent for the synthesis of biologically active peptides is demonstrated by the solid phase synthesis of the g-receptor selective opioid heptapeptide, dermorphin 7 (Tyr-D-Ala-Phe- Gly-Tyr-Pro-Ser-NH2), using conventional Merrifield resin as solid support. Chain lengthening was carried out using appropriate Boc-amino acid (3 equiv.), N,N'-dicyclohexylcarbodiimide (3 equiv.), and 1-hydroxybenzotriazole (1 equiv.). After each coupling, Boc group was removed by 1M SiCh and 3M phenol in CH:CI2 in 10 rain. Ammonolysis of the protected heptapeptide-resin gave Boc-Tyr(Bzl)-D-Ala- Phe-Gly-Tyr(Bzl)-Pro-Ser(Bzl)-NH2 (overall yield 59% based on the amount of first amino acid esterified to the resin). Catalytic transfer hydrogenation ~ of this protected heptapeptide gave the free peptide (HPLC profile A) which was purified over Sephadex G-15 using 1N acetic acid in water. The purity of the peptide was satisfactory as indicated by the HPLC profile B shown in the figure. It had the same physical properties 9 as reported earlier TM and exhibited the same opioid agonistic activity when studied in vitro by guinea pig ileum assay as that of an authentic sample 7. -4r i i D 1Stain 30min A B Figure : Analytical RP-HPLC profile of dermorphin (A, crude sample; B, pure peptide). Waters Bondapak C-18 column (3.9 × 300 ram) was used. Elution was carried out with two solvents : A, 20% (v/v) acetonitrile in water ; B, 60% (v/v) acetonitrile in water both containing 4.5 mmoles trifluoroacetic acid and 4.9 mmoles triethylamine. Th  gradient programme used was linear gradient from 10% to 25% B in 15 min, 25% B for 5 min, linear gradient from 25% to 40  B in 10 rain at imi / min flow rate. UV, 280 rim. REFERENCES ANDNOTES 1. Lott, R. S.; Chauhan, V. S.; Stammer, C. H. J. Chem, Soc. Chem. Comm. 1979, 495-496. 2. Sivanandaiah, K. M.; Suresh Babu, V. V.; Shankaramma, S. C. Int. J. Peptide Protein. Res. 1995, 45,377-379. 3. Kaiser, E. Sr.; Tam, J. P.;Kubiak, T. M.; Merrifield, R. B. Tetrshedron Lett. 1988, 29,303-306. 4. Kaiser, E. Sr.;Picart, F.;Kubiak, T. M.;Tam, J.P.; Merrifield, R.B.J. Org. Chem. 1993,58,5167-5175. 5. Gism, B. F. Anal, Chim. Acta. 1972, 58,248-249. 6. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P.I.; Anal. Biochem. 1970, 34,595-598. 7. Montecucehi, P.C.; De Cartiglione, R.; Erspamer, V. Int. J. Peptide protein Res. 1981, 17, 316-321. 8. Sivanandaiah, K. M.; Gurusiddappa, S. J. Chem. Reds. (S), 1979, 108-109. 9. M.P. 156-158 ° ; [ot]~ 2 + 4.93 (el, 1N acetic acid) Reported 10 : M. P. 159-160 ° ; [or] 25 +4.95 ° (c I, IN acetic acid). Paper chromatography : Rf (n-butanol : acetic acid : water : pyridine :: 15 : 3 : 12 : 10, by volume) 0.66; R r (n-butanol : acetic acid : water :: 4 : 1 : 1, by volume) 0.60. Amino acid analysis : Amino acid (theory) found ; Ser(1) 1.03, Pro (1) 0.97, "l~yr (2) 2.04, Gly (1) 0.98, Phe (1) 1.01, Ala (1) 0.99. Elemental analysis : Anal. Caled. for C~ H50 N s Or0 (802.93) : C 59.78, H 6.22, N 13.95 %. Found : 59.67, H 6.4, N 13.86%. 10. Sivanandaiah, K. M.; Gurusiddappa, S.; Suresh Babu, V. V. Int. J. Peptide Protein Res. 1989, 33,463-467. (Received in UK 11 April 1996; revised 26 June 1996; accepted 28 June 1996) 

Silicon tetrachloride and phenol as N(α)-t-butoxycarbonyl group deprotecting agent in solid phase peptide synthesis

http://eprints-bangaloreuniversity.in/521/1/silicon.pdf

Silicon tetrachloride and phenol as N(α)-t-butoxycarbonyl group deprotecting agent in solid phase peptide synthesis

Abstract

Similar works

Full text

Available Versions

ePrints@Bangalore University