Solid-phase peptide syntheses of oxytocin, oxytocin analogs and interferon short chain with the amide side-chain functionality of asparagine protected with 1-tetralinyl group
Amir, Okeyo Y
MetadataShow full item record
The aim of the project was to transform ketones to the amines, which were then used as precursors in the carboxamide protection of asparagine and glutamine side chains. Peptide resins of oxytocin, oxytocin analogs and interferon short chain were then synthesised by solid-phase peptide synthesis (SPPS) via Boc-strategy, with asparagine and glutamine side chains protected with l-tetralinyl and benzhydryl groups respectively. Conversion of l-tetralone and benzophenone to N-l-tetraliny lformamide (1) and N-benzhydrylformamide (2) respectively were done by Leuckart Reaction at l70-180aC for three hours. The fonnamides were then hydrolysed to the amines. N-Tetralinylformamide was hydrolysed with 10% aqueous sodium hydroxide to give l-aminotetralin (3) (75%), while concentrated hydrochloric acid hydrolysis of N-benzhydrylformamide yielded an amine salt (75 %). This salt was neutralized with 10% aqueous sodium hydroxide to give benzhydrylamine (4) (57%). These amines were then used as precursors in the carboxamide protection of asparagine and glutamine amide side chains. 1- Aminotetralin was reacted with Boc-Asp-a-OBzl in the presence of DCC to give carboxamide protected derivative, Boc-Asn(Tet)-a-OBzl (5) . Boc- Gln(Bzh)-a-OBzl (6) was formed by coupling benzhydrylamine and BocGlu- a-OBzl. The two fully protected amino a.cids were then hydrogenolyzed with ~ hydrogen at atmospheric pressure in the presence of Pd/C for eight hours. This led to the removal of benzyl group from the fully protected amino acids, giving rise to Boc-Gln(Bzh)-OH (7) (77%) and Boc-Asn(Tet)-OH (8) (63%), which were then used to- introduce glutamine and asparagine residues in a growing peptide chain by solid-phase peptide synthesis (SPPS). Peptide resins of oxytocin (43%), isotocin ( 97%), lysine-vasopressin ( 89%), arginine-vasopressin ( 85 %), mesotocin ( 79%), glumitocin ( 91%) and an octapeptide short chain of interferon ( 96%) were then prepared by solid-phase peptide synthesis via Bee-strategy. The solid support used was benzhydrylamine resin (substitution of 0.9 mmol/g). In releasing the peptide from the resin, a cleavage cocktail of trifluoromethanesulfonic acid (TFMSA)- trifluoroacetic acid (TFA)-thioanisole-l,2-ethanedithiol (EDT) (2:20:2: 1v/v) was used at different reaction conditions: (1) room temperature for two hours (2) 40°C for half an hour (3) 40°C for two hours. Reaction (1) gave crude peptide of oxytocin ( 84.095%), isotocin (78.53%), lysine-vasopressin ( 59.94 %), arginine-vasopressin ( 81. 96%), mesotocin ( 89.45 %), glumitocin ( 83.2%) and interferon short chain ( 88.75%). Reaction (2) gave peptide derivatives of oxytocin ( 87.54%), isotocin ( 85.92%), lysine-vasopressin (90.08%), arginine-vasopressin ( 93.23%), mesotocinJ 64.2%), glumitocin (60.9%). The last cleavage condition gave crude peptide of oxytocin (96.64%), isotocin (95.48 %), lysine-vasopressin (94.63 %), arginine-vasopressin (96.25%), mesotocin ( 97.28%), glumitocin ( 95.25%) and interferon short chain (96.19%). In all the three cases, TFMSA was able to cleave the peptide from the resin. When crude peptides given by the three conditions were analyzed by ion electrospray mass spectroscopy (ESMS), they revealed the presence of the target peptide (i.e. the cyclic moieties of oxytocin, isotocin, lysine-vasopressin, arginine-vasopressin, mesotocin and glumitocin) when cleavage was done 'at 40°(: for two hours . Linear interferon chain was also formed. This reaction condition wasi-therefore suitable in deprotecting all groups from the peptide and subsequent oxidation of free mercapto groups on cysteine to cyclic nonapeptides. 10 mg of each of the crude peptides from reaction condition (3) were then used in separation of the pure peptides by semipreparative H?JLC. Elution was done at 3.5 mL/min using 0.1 % aqueous TFA and 0.1% TFA in acetonitrile (90: 10 to 30:70 over 45 minutes). These pure peptides (9,10,11,12,13,14 and 15) gave expected results when analyzed by amino acid analysis, ESMS and sequence analysis.