1-ethylpyrrole-2,5-dione

Conditions: temp:15.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 260 g portion of 3,4-dichloroaniline was dissolved in a mixture of 400 ml of concentrated hydrochloric acid and 1.5 liters of water, cooled to 15° C. in an ice-salt bath. A solution of 111 g of sodium nitrite in 250 ml of water was added dropwise, with stirring, over a 90 minute period. Then 40 g of cupric chloride dihydrate was added followed by 200 g of N-ethylmaleimide in one liter of warm acetone. The reaction mixture was cooled to 10° C. and sodium bicarbonate was added in portions over 2 hours to adjust the pH to 3.5. The mixture was allowed to stand for 48 hours. The precipitate formed was collected, air dried, and taken up in 2.5 liters of isopropanol, then 185 ml of 2,6-lutidine was added and the mixture was heated to boiling, then poured into water and allowed to stand. The solid was collected and dried. Recrystallization from acetone gave 187.1 g of 3-(3,4-dichlorophenyl)-1-ethyl-1H-pyrrole-2,5-dione as a yellow solid, mp 138°-141° C. The reaction mixture was cooled to 10° C. to stand for 48 hours The precipitate formed was collected air dried 185 ml of 2,6-lutidine was added the mixture was heated poured into water The solid was collected dried Recrystallization from acetone

Conditions: temp:-30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution in toluene of N-ethylmaleimide, HN3 (excess relative to the maleimide), and the (salen)AlN3 catalyst depicted above (10 mol % relative to the maleimide) was maintained at −30° C. for 18 h. After a standard quench and work-up of the reaction mixture, the crude material was purified to yield 2-azido-N-ethylsuccinimide (93%, 93% ee). After a standard quench work-up of the reaction mixture, the crude material was purified

Conditions: temp:-30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution in toluene of N-ethylmaleimide, HN3 (excess relative to the maleimide), and the (salen)AlN3 catalyst depicted above (10 mol % relative to the maleimide) was maintained at −30° C. for 18 h. After a standard quench and work-up of the reaction mixture, the crude material was purified to yield 2-azido-N-ethylsuccinimide (93%, 93% ee). After a standard quench work-up of the reaction mixture, the crude material was purified

Conditions: temp:15.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 260 g portion of 3,4-dichloroaniline was dissolved in a mixture of 400 ml of concentrated hydrochloric acid and 1.5 liters of water, cooled to 15° C. in an ice-salt bath. A solution of 111 g of sodium nitrite in 250 ml of water was added dropwise, with stirring, over a 90 minute period. Then 40 g of cupric chloride dihydrate was added followed by 200 g of N-ethylmaleimide in one liter of warm acetone. The reaction mixture was cooled to 10° C. and sodium bicarbonate was added in portions over 2 hours to adjust the pH to 3.5. The mixture was allowed to stand for 48 hours. The precipitate formed was collected, air dried, and taken up in 2.5 liters of isopropanol, then 185 ml of 2,6-lutidine was added and the mixture was heated to boiling, then poured into water and allowed to stand. The solid was collected and dried. Recrystallization from acetone gave 187.1 g of 3-(3,4-dichlorophenyl)-1-ethyl-1H-pyrrole-2,5-dione as a yellow solid, mp 138°-141° C. The reaction mixture was cooled to 10° C. to stand for 48 hours The precipitate formed was collected air dried 185 ml of 2,6-lutidine was added the mixture was heated poured into water The solid was collected dried Recrystallization from acetone

Conditions: temp:15.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 260 g portion of 3,4-dichloroaniline was dissolved in a mixture of 400 ml of concentrated hydrochloric acid and 1.5 liters of water, cooled to 15° C. in an ice-salt bath. A solution of 111 g of sodium nitrite in 250 ml of water was added dropwise, with stirring, over a 90 minute period. Then 40 g of cupric chloride dihydrate was added followed by 200 g of N-ethylmaleimide in one liter of warm acetone. The reaction mixture was cooled to 10° C. and sodium bicarbonate was added in portions over 2 hours to adjust the pH to 3.5. The mixture was allowed to stand for 48 hours. The precipitate formed was collected, air dried, and taken up in 2.5 liters of isopropanol, then 185 ml of 2,6-lutidine was added and the mixture was heated to boiling, then poured into water and allowed to stand. The solid was collected and dried. Recrystallization from acetone gave 187.1 g of 3-(3,4-dichlorophenyl)-1-ethyl-1H-pyrrole-2,5-dione as a yellow solid, mp 138°-141° C. The reaction mixture was cooled to 10° C. to stand for 48 hours The precipitate formed was collected air dried 185 ml of 2,6-lutidine was added the mixture was heated poured into water The solid was collected dried Recrystallization from acetone

Conditions: temp:-30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution in toluene of N-ethylmaleimide, HN3 (excess relative to the maleimide), and the (salen)AlN3 catalyst depicted above (10 mol % relative to the maleimide) was maintained at −30° C. for 18 h. After a standard quench and work-up of the reaction mixture, the crude material was purified to yield 2-azido-N-ethylsuccinimide (93%, 93% ee). After a standard quench work-up of the reaction mixture, the crude material was purified

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 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). After concentration in vacuo the peptide was precipitated with diethyl ether purified by reverse-phase HPLC (C18 column) over 15 min After washing the resin with DMF (4×) and CH2C12(4×) the peptide conjugate The combined organic fractions were concentrated in vacuo the conjugate was redissolved in TFA The TFA was then removed in vacuo and AMA49-L1was precipitated with iPr2O purified by HPLC on a C4 column 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 purified by HPLC as above for AMA49-L1

Conditions: temp:-30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution in toluene of N-ethylmaleimide, HN3 (excess relative to the maleimide), and the (salen)AlN3 catalyst depicted above (10 mol % relative to the maleimide) was maintained at −30° C. for 18 h. After a standard quench and work-up of the reaction mixture, the crude material was purified to yield 2-azido-N-ethylsuccinimide (93%, 93% ee). After a standard quench work-up of the reaction mixture, the crude material was purified

Conditions: temp:15.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A 260 g portion of 3,4-dichloroaniline was dissolved in a mixture of 400 ml of concentrated hydrochloric acid and 1.5 liters of water, cooled to 15° C. in an ice-salt bath. A solution of 111 g of sodium nitrite in 250 ml of water was added dropwise, with stirring, over a 90 minute period. Then 40 g of cupric chloride dihydrate was added followed by 200 g of N-ethylmaleimide in one liter of warm acetone. The reaction mixture was cooled to 10° C. and sodium bicarbonate was added in portions over 2 hours to adjust the pH to 3.5. The mixture was allowed to stand for 48 hours. The precipitate formed was collected, air dried, and taken up in 2.5 liters of isopropanol, then 185 ml of 2,6-lutidine was added and the mixture was heated to boiling, then poured into water and allowed to stand. The solid was collected and dried. Recrystallization from acetone gave 187.1 g of 3-(3,4-dichlorophenyl)-1-ethyl-1H-pyrrole-2,5-dione as a yellow solid, mp 138°-141° C. The reaction mixture was cooled to 10° C. to stand for 48 hours The precipitate formed was collected air dried 185 ml of 2,6-lutidine was added the mixture was heated poured into water The solid was collected dried Recrystallization from acetone