| dc.description.abstract | 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. | en |
