Labeled oxytocin and method of manufacture and use
Disclosed are 13 N-oxytocin molecules, methods of manufacture of 13 N-oxytocin molecules and methods of use of 13 N-oxytocin molecules in the determination of the distribution and kinetics of 13 N-oxytocin molecules after craniofacial or other application methods.
1 . A method of manufacturing a compound of Formula IV, wherein the compound is a 13 N-labeled oxytocin peptide comprising the amino acid sequence of SEQ ID NO: 1, the method comprising:
a) converting a compound of Formula (I):
into a compound of Formula (II):
using a R-LG reagent, wherein LG is a leaving group selected from a group comprising CN, OH, O-Acyl, OAt, OBt, Cl, 1-imidazolyl; and
wherein R is P(═O)(OR 1 )(OR 2 );
wherein R 1 and R 2 are independently C 1 -C 6 alkyl or C 6 -C 14 aryl, wherein the C 1 -C 6 alkyl and C 6 -C 14 aryl are independently optionally substituted by halogen, cyano, or C 1 -C 6 alkyl; and
wherein PG1 is an amine protecting group;
b) reacting a compound of Formula (II) with gaseous 13 NH 3 to provide a compound of Formula (III):
c) deprotecting a compound of Formula (III) to provide the compound of Formula IV:
and
d) purifying the compound of Formula IV.
2 . The method of claim 1 , wherein converting a compound of Formula (I) to a compound of Formula (II) is carried out using the R-LG reagent in the presence of dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and pentamethylpiperidine (PMP), wherein R-LG is diethyl cyanophosphonate (DECP).
3 . The method of claim 2 , wherein the amount of DECP is between about 0.7 and about 1.1 equivalents.
4 . The method of claim 2 , wherein the ratio of DMSO to THF is between about 1:7 and about 1:11.
5 . The method of claim 1 , wherein the deprotecting comprises reacting a compound of Formula (III) with HCl in dioxane.
6 . The method of claim 1 , wherein the purifying comprises purification by solid-phase extraction (SPE).
7 . The method of claim 6 , wherein purification by SPE comprises:
a) applying the compound of Formula IV to a first hydrophobic SPE column, such that the compound of Formula IV is retained on the first hydrophobic SPE column;
b) applying a solution comprising an aqueous ion-pairing reagent to the first hydrophobic SPE column, such that the compound of Formula IV is eluted in a first eluate;
c) applying the first eluate to a second hydrophobic SPE column, such that the compound of Formula IV of the first eluate is retained on the second hydrophobic SPE column; and
d) eluting the compound of Formula IV in a second eluate.
8 . The method of claim 7 , wherein the first and/or second hydrophobic SPE columns comprise a silica-based bonded phase with strong hydrophobicity.
9 . The method of claim 7 , wherein the solution comprising an aqueous ion-pairing reagent comprises between about 15% and about 25% acetonitrile.