반응 #1355836

반응 방정식

TFA

HATU

HOAt

iPr2EtN

2,2,2-trifluoroethanol

→

NEM

용매

CH2Cl2

DMF

ammonium acetate

AcOH

H2O

반응 조건

후처리

1. 1
   농축After concentration in vacuo the peptide
2. 2
   기타was precipitated with diethyl ether
3. 3
   기타purified by reverse-phase HPLC (C18 column)
4. 4
   기타over 15 min
5. 5
   세척After washing the resin with DMF (4×) and CH2C12(4×) the peptide conjugate
6. 6
   농축The combined organic fractions were concentrated in vacuo
7. 7
   workup.DISSOLUTIONthe conjugate was redissolved in TFA
8. 8
   기타The TFA was then removed in vacuo and AMA49-L1was
9. 9
   기타precipitated with iPr2O
10. 10
    기타purified by HPLC on a C4 column
11. 11
    기타by reaction with excess N-ethylmaleimide (NEM) and detection of the bis-NEM derivative by HPLC-MS
12. 12
    기타AMA49-C1 was prepared in the same way except that sPE-succ-OH
13. 13
    기타was purified by HPLC as above for AMA49-L1

실험 절차

This example shows the design and synthesis of a linear peptide based on loop 1 of domain III of AMA-1. A 49 residue peptide comprising residues AMA-1446–490, with three additional amino acids (GGC) at the N-terminus, and one additional G residue at the C-terminus (FIG. 1), was prepared by solid phase peptide synthesis on Sieber amide resin (0.5 mmol/g) using Fmoc chemistry and an ABI433A peptide synthesizer. The pseudoproline unit Fmoc-IleSer(ψMe,Mepro)-OH was used in place of Ile39 and Ser40. After assembly, the peptide was cleaved from a portion of the resin using TFA:H2O:EDT:TIS (92.5:2.5:2.5:2.5) for 3.5 h at room temperature. After concentration in vacuo the peptide was precipitated with diethyl ether, and purified by reverse-phase HPLC (C18 column) using a gradient of MeCN in water (25% to 50%) with 0.1% TFA over 15 min. The purity was >95% by analytical HPLC. The constitution was confirmed by amino acid analysis and electrospray mass spectrometry (Table-1). Another portion of the resin carrying the intact peptide chain (ca. 60 μmol) was treated with PE-succ-OH (130 mg, 160 μmol) (FIG. 1), HATU (61 mg, 160 μmol), HOAt (22 mg, 160 μmol), and iPr2EtN (82 μl) in DMF:CH2Cl2 (4 ml, 1:2) for 18 h at room temp. After washing the resin with DMF (4×) and CH2C12(4×) the peptide conjugate was cleaved from the resin with 1% TFA in CH2Cl2 (4×4 ml). The combined organic fractions were concentrated in vacuo and the conjugate was redissolved in TFA:H2O:EDT:TIS (92.5:2.5:2.5:2.5) for 4 h at room temp. The TFA was then removed in vacuo and AMA49-L1was precipitated with iPr2O and then purified by HPLC on a C4 column using a gradient of 10 to 100% MeCN/water with 0.1% TFA. The purity was >95% by analytical HPLC. The constitution was confirmed by amino acid analysis and electrospray mass spectrometry (Table-1). The presence of two free thiols was proven by an Ellman test, and by reaction with excess N-ethylmaleimide (NEM) and detection of the bis-NEM derivative by HPLC-MS. AMA49-C1 was prepared in the same way except that sPE-succ-OH was used (FIG. 1), and in the final step the conjugate (4 mg) was oxidized in ammonium acetate buffer (50 mM, pH 8) and 2,2,2-trifluoroethanol (TFE) (1:1, 100 ml) for 4 days at room temperature. After addition of AcOH (100 μl) and lyophilization, AMA49-C1 was purified by HPLC as above for AMA49-L1. The formation of the disulfide bridge was confirmed by a negative Ellman test and by attempted derivatization with NEM, which gave no bis NEM-derivative by HPLC-MS. For the synthesis of AMA49-L2, the dithiol (6 mg) was alkylated with iodoacetamide (43μmol) in a mixture (5:4) of phosphate buffer (0.1 M, pH 7.5) and TFE. The AMA49-L2 was purified by HPLC on a C4 column using a gradient of 10 to 100% MeCN/water with 0.1% TFA. The purity was >95% by analytical HPLC. Electrospray mass spectrometry showed the expected mass (Table 1).

출처