Synthesis of a C-Terminal Thioester Derivative of the Lipopeptide Pam₂CSKKKKG Using Fmoc SPPS

Abstract

Attempted preparation of the immunoadjuvant lipo­peptide Pam₂CysSKKKKG by Boc SPPS resulted in unexpected cleavage of the palmitoyl esters during HF-mediated cleavage from the resin. An alternative strategy using a combination of a sulfon­amide linker, Fmoc SPPS and milder deprotection conditions afford the requisite peptide with the palmitoyl groups intact.

Pam₂Cys - Boc SPPS - thioester - HF - Fmoc SPPS

References and Notes

1a Wiesmüller K.-H. Jung G. Hess G. Vaccine  1989,  7:  29 

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2 Jackson DC. Lau YK. Le T. Suhrbier A. Deliyannis G. Cheers C. Smith C. Zeng W. Brown LE. Proc. Natl. Acad. Sci. U.S.A.  2004,  101:  15440 

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5a Buwitt-Beckmann U. Heine H. Wiesmüller K.-H. Jung G. Brock R. Akira S. Ulmer AJ. Eur. J. Immunol.  2005,  35:  282 

5b Metzger JW. Beck-Sickinger AG. Loleit M. Eckert M. Bessler WG. Jung G. J. Pept. Sci.  1995,  3:  184 

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8 4-Sulfamylbutyryl aminomethyl polystyrene resin (Novabiochem) loading = 1.1 mmol/g (1.0 g, 1.1 mmol) was swelled in CH₂Cl₂ for 1 h. Fmoc-Gly (1.31 g, 4.4 mmol) was dissolved in CH₂Cl₂-DMF 4:1 (8 mL) and to this was added successively N-methylimidazole (0.361 g, 4.4 mmol), DIC (0.555 g, 4.4 mmol) and the solution added to the pre-swollen resin. After shirring for 1 h at r.t. the resin was drained, washed with DMF then CH₂Cl₂ and dried under nitrogen. The loading was determined as described in: Fmoc Solid Phase Synthesis, A Practical Approach   Chan WD. White PD. Oxford University Press; New York: 2000. 

9

Fmoc SPPS was carried out on a 0.182 mmol scale as described below. The N-terminal Fmoc group was removed with 2 successive treatments of 20% piperdine in DMF of 5 and 10 min. The protected amino acid (0.728 mmol) was dissolved in 0.5 M HOBt in N-methylpyrrolidine (NMP) (1.456 mL, 0.728 mmol) and added to the resin followed by 0.5 M HBTU in DMF (1.43 mL, 0.715 mmol) and DIPEA (0.986 mL, 5.28 mmol). The mixture was shaken for 45 min, drained and washed five times with DMF.

10a Metzger JW. Wiesmüller K.-H. Jung G. Int. J. Pept. Protein Res.  1991,  38:  545 

10b Hida T. Hayashi K. Yukishige K. Tanida S. Kawamura N. Harada S. J. Antibiot.  1995,  48:  589 

11

FmocPam₂CysOH (0.325 g, 0.364 mmol), PyBoP (0.189 g, 0.364 mmol), HOBt (0.057 g, 0.364 mmol) were dissolved in 5:1 DMF-CH₂Cl₂ (4.5 mL). DIPEA (0.129 mL, 0.728 mmol) was added, the solution added to the NH₂-peptide resin and the mixture shaken overnight. A quantitative ninhydrin test showed the reaction to be >98% complete. The N-terminal Fmoc was removed as described above and to the resin was added Boc₂O (0.4 g, 1.82 mmol) in 1:1 DMF-CH₂Cl₂ (4 mL). The mixture was shaken for 2 h and the procedure repeated.

12

The resin-bound lipopeptide was swelled in NMP for 1 h. The NMP was removed by filtration and replaced with fresh NMP (3 mL). To this was added IMeCN (0.264 mL, 0.182 mmol) that had been freshly filtered through basic alumina and DIPEA (0.123 mL, 0.728 mmol). The mixture was protected from light and stirred for 24 h after which time the reaction mixture was drained and washed with NMP (3×) and DMF (3×). To the activated resin was added benzylmercaptan (1.06 mL, 9.1 mmol) dissolved in DMF (4 mL) and the mixture stirred for 24 h. The peptide was recovered by filtration from the resin and the resin washed with DMF (3×) and CH₂Cl₂ (3×) and concentrated in vacuo to afford an oil. The side-chain protecting groups were removed using a mixture of TFA-PhOH-triisopropylsilane-H₂O (88:5:2:5, 10 mL) at r.t. for 2 h and the solvents concentrated in vacuo. The residue was dissolved in TFA (3-4 mL) and then added to ice cold Et₂O (40 mL) affording a white precipitate. The mixture was stored at 4 °C for 1 h, centrifuged and Et₂O decanted. This procedure was repeated once more. The recovered lipopeptide was dissolved in 50% aq MeCN and lyophilized.

13

Analytical HPLC was performed using a diphenyl column (4.6 mm × 150 mm, Vydac Cat # 219TP54) at a flow rate of 1 mL min^-1. A gradient of 10%B to 100% B over 35 min was used; buffer A = 0.1% TFA-H₂O, buffer B = 0.1% TFA-MeCN.

14

Purification of the crude lipopeptide was performed on a diphenyl semi-preparative column (10 mm × 250 mm, Vydac Cat #219TP1010) at a flow rate of 10 mL min^-1. A gradient of 10% B to 100% B over 45 min was used; buffer A = 0.1% TFA-H₂O, buffer B = 0.1% TFA-MeCN. The relevant fractions were pooled, lyophilized and identified by ESI-MS or MALDI-MS: m/z calcd or Pam₂CysSKKKKG-SBn [MH⁺]: 1435.0602; found: 1435.4746.

15 During the preparation of this manuscript the synthesis of Pam₃CysSKKKKG-SBn and its use in native chemical ligation was reported, see: Ingale S. Buskas T. Boons G.-J. Org. Lett.  2006,  8:  5785