2 hrs after this time the catalyst was removed by filtration and the filtrate concentrated to dryness two additional concentrations each from 1 ml of methanol effectively removed labile deuterium and the resulting product 6 3 mg was shown by amino acid analysis to be free of u03b4 3 proline and to contain the expected constituent amino acids in the proper ratio a solution of l pyroglutamyl l histidine hydrazide 1 123 g 3 97 mmol in dmf 16 7 ml and dmso 13 9 ml was cooled to 20 u00b0 for 30 min triethylamine 3 314 ml 23 82 mmol was added at 25 u00b0 a mixture of l 3 4 dehydroprolinamide hydrochloride 0 707 g 4 76 mmol in dmf 1 19 ml and dmso 1 19 ml pretreated with triethylamine 0 663 ml 4 76 mmol was added to the reaction mixture at 20 u00b0 stirring proceeded at 20 u00b0 for 30 min 0 u00b0 for 30 min and 4 u00b0 for 48 hrs the reaction mixture was filtered and the filtrate evaporated to dryness and precipitated from methanol thf ether the product was purified by chromatography on sephadex g10 using 0 2m acetic acid as eluent further purification was achieved by chromatography on silica gel using chloroform methanol 1 2 as eluent there was obtained 718 mg 50 2 of u03b4 3 pro 3 trf as a white solid m p 155 u00b0 162 u00b0 dec u03b1 d 25 146 12 u00b0 c 1 meoh anal calcd for c 16 h 20 n 6 o 3 1 2ch 3 cooh1 2 hcl an 11 1 mg sample of.