Investigation of the 'n-1' impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase beta-cyanoethyl phosphoramidite method using stepwise sulfurization.

Electrospray ionization mass spectrometry (ESI-MS) of reversed-phase HPLC-purified phosphorothioate oligodeoxynucleotides (S-ODNs), and the single-('n - 1') and double-nucleotide deletion ('n - 2') impurities subsequently isolated from them by preparative polyacrylamide gel electrophoresis (PAGE), has provided direct analytical data for the identification of both S-ODN products and their major oligomeric impurities. The 'n - 1' impurity seen by PAGE consists of a mixture of all possible single deletion sequences relative to the parent S-ODN (n-mer) and results from repetitive, though minor, imperfections in the synthesis cycle, such as incomplete detritylation, or incomplete coupling followed by incomplete capping or incomplete sulfurization. Therefore each possible 'n - 1', 'n - 2', and other short-mer sequence is present only in very low abundance. The conversion of the gel-isolated 'n - 1' impurity from phosphorothioate to phosphodiester followed by base composition-dependent anion-exchange chromatography allowed for independent confirmation of its heterogeneity and quantitation of its various components. ESI-MS of both S-ODN products and their gel-isolated impurities allowed for this first molecular identification of 'n - 1', 'n - 2' and other oligomeric impurities in S-ODNs obtained from state-of-the-art solid-phase synthesis and reversed-phase HPLC purification methods.

Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.

The purification of oligonucleotides by ion-exchange displacement chromatography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.

Pheromone biosynthetic pathways: Conversion of ipsdienone to (-)-ipsdienol, a mechanism for enantioselective reduction in the male bark beetle,Ips paraconfusus.

The enantiomeric composition of the pheromone components (+)-ipsdienoI, e.e. 87.6%, and (-)-ipsenol, e.e. 93.8%, produced by the male bark beetleIps paraconfusus (Scolytidae) under natural conditions was determined by HPLC separation of their diastereomeric ester derivatives. Males confined in an atmosphere of ipsdienone produced (-)-ipsdienol, e.e. 28%, and (-)-ipsenol, e.e. 86%, indicating an enantiomeric selectivity in the conversion of the ketone to the alcohols. These findings demonstrate an enantioselective conversion mechanism in the biosynthetic pathway to the pheromones from myrcene, a host-plant terpene.

JH Zero: New Naturally Occurring Insect Juvenile Hormone from Developing Embryos of the Tobacco Hornworm.

A new insect juvenile hormone was isolated from developing embryos of the tobacco hornworm moth, Manduca sexta. The new hormone was found with juvenile hormone I and is a 1-carbon homolog of this substance. The assigned structure is methyl (2E,6E,10-cis)-10,11-epoxy-3,7-diethyl-11-methyl-2,6-tridecadienoate, which constitutes a trishomosesquiterpenoid skeleton. This is the first chemical idetification of any juvenile hormone from insect eggs.

Biosynthesis of the Juvenile Hormones of Manduca sexta: Labeling Pattern from Mevalonate, Propionate, and Acetate.

Using organ culture, high-resolution liquid chromatography, and microchemical techniques, we demonstrated the efficient incorporation in vitro of several radiolabeled precursors into the two juvenile hormones of Manduca sexta. JH II, a homosesquiterpene hormone, reported from M. sexta as well as several other insects, incorporates radiolabel from acetate, mevalonate, and propionate. JH III, a sesquiterpene hormone recently reported as a natural product of M. sexta, incorporates label from acetate and mevalonate, but not from propionate. Based on the position of the labeled atoms in the precursors and upon the position of incorporation obtained from label-distribution data, a scheme for juvenile hormone biosynthesis is advanced.

Isolation, Structure, and Absolute Configuration of a New Natural Insect Juvenile Hormone from Manduca sexta.

Two juvenile hormones are isolated from organ cultures of corpora allata of the tobacco hornworm moth, Manduca sexta Johannson, and are purified by high-resolution liquid chromatography. These are identified as methyl (2E, 6E)-(10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate, a new natural hormone, and methyl (2E,6E) - (10R,11S) - 10,11 - epoxy - 3,7,11 - trimethyl-2,6-tridecadienoate. [(14)C]Methionine is incorporated only into their methoxycarbonyl group. Details of the in vitro techniques and the chemical proof of structures are presented. The significance of the occurrence of a new juvenile hormone and the new techniques used for production, isolation, and identification are discussed.