Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.

Organic solvent-soluble α-chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of kcatKm for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH2 to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH2 that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of α-chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, α-chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using α-chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.

A new technique for the purification of proteins has been developed which combines the high selectivity of affinity interaction with the scalability and ease of operation of liquid-liquid extraction. The approach is called affinity-based reverse micellar extraction and separation (ARMES). The salient features of ARMES include the following: (1) intraphasic interaction between the ligand and ligate which provides for high ligand utilization; (2) no chemical modification of the ligand is needed; and (3) ease of operation and inherent scalability due to the use of liquid-liquid extraction. This technique has been used to purify the peroxidase from soybean hulls using the lectin concanavalin A (con A) as a sugar-binding affinity ligand. A purification factor of 30 is achieved to provide a nearly pure peroxidase solution (as determined by HPLC and SDS-PAGE) with nearly complete regeneration of the con A ligand. We propose that ARMES will be useful in the facile purification of complex biomolecules such as glycoform protein variants using lectins as affinity ligands and proteins of therapeutic importance using antibodies as affinity ligands.

A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.

A regioselective synthesis of 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (LY134046, 10) and its 3-methyl analogue 26 from 6,7-dichloro-3-hydroxyphthalide (16) is described. The key step involved 1,4-hydride addition to the alpha,beta-unsaturated nitrile 17 to give the saturated nitrile 18 using sodium borohydride in 2-propanol. In the preparation of LY134046 10, the COOH group in 18 was first esterified and then the nitrile function was selectively reduced with borane to yield the aminoester 20. The aminoester 20 was then cyclized to the azepinone 21 which on reduction with borane provided LY134046 10 in an overall yield of 22%. The route is adaptable to the preparation of hitherto unknown 3-substituted-2-benzazepines as demonstrated by the preparation of the 3-methyl analogue 26. In this case the nitrile 18 was reacted with an excess methylmagnesium iodide to give the ketoacid 22. Esterification of 22 followed by reductive amination with sodium cyanoborohydride and ammonium acetate provided the aminoester 24, which was then converted to the target benzazepine 26 as described earlier for the title compound. The reaction conditions and the reagents used throughout the sequence are fairly mild and many functional groups may be tolerated. The only limitation to this procedure is the availability of the corresponding hydroxyphthalide. A variation in the choice of reagent in the Grignard reaction of 18 should provide an access to a variety of 3-substituted-2-benzazepines.

A regioselective synthesis of 2,3,4,5-tetrahydro-1H-2-benzazepines substituted on the aryl and/or azepine rings is presented and used for the preparation of the title compound in overall yield of 22% from intermediate 1.

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AbstractDie Grundkörper (Va) und (Xa) bzw. die stereoisomeren Trifluormethyl‐benzobicycloheptene (Ia) bis (IVa) bzw. (VIa)‐(lXa) werden mit Phthalimid in Gegenwart von Triphenylphos‐ phin und Azodicarbonsäureester zu den Phthalimiden (Ib)‐(Xb) umgesetzt, wobei allerdings nur teilweise eine stereospezifische Umsetzung erfolgt.

AbstractIn Abhängigkeit von Lösungsmittel erhält man aus dem ungesättigten Amid (I) durch Belichtung das trans‐ (II) bzw. das cis‐Addukt (III), von denen jedes bei der Behandlung mit Kaliumhydroxid zu einem 30z70‐Gemisch von (II) und (III) führt.