On the basis of the Isolndole formation mechanism In the o-phthalaldehyde/2-mercaptoethanol (OPA/2-ME) derivatization of primary amines and the structure-stability relationships for Isolndoles, an Improved fluorogenic reagent, naphthalene-2,3-dlcarboxaldehyde (NDA) In the presence of cyanide ion (CN ), has been developed. Reaction of NDA/ CN' with primary amines In aqueous media results In the formation of -substKuted 1-cyanobenz[/]lsoindole (CBI) derivatives which have significantly Improved stability compared to the corresponding OPA/2-ME derivatives (for glycine greater than 50-fold Improvement was realized) and have high quantum efficiencies for fluorescence ( , = 0.54 In 60% aqueous acetonitrile for the CBI--propylamine derivative) In solvent systems commonly used In liquid chromatography. Parameters In the NDA/CN' derivatization of alanine are defined (l.e., pH and the reagent component concentrations) and used In the development of a labeling procedure for amino acid mixtures. Gradient elution fractionation of 18 CBI-amino acid derivatives was accomplished In 60 min and permitted detection limits of less than 200 fmol Injected (excitation 246 nm) or 3 pmol Injected (excitation 420 nm). The utility of the reagent In assaying amino acid mixtures resulting from the enzymatic hydrolysis of the peptides Met-enkephalln and glucagon Is demonstrated.

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2,3-Naphthalenedicarboxaldehyde (NDA) is shown to be a very useful reagent for the derivatization of primary amines, amino acids, and small peptides. The reaction of these amino compounds with NDA and cyanide ion produces highly fluorescent 2-substituted 1 -cyanobenz [/]isoindoles that are relatively stable. The physical and fluorescent properties of a variety of 1,2-disubstituted benz[/] isoindoles are presented.

A study of the cycloadditions of cyanoketenes [tert-butylcyanoketene (TBCK), chlorocyanoketene (CCK), and hexynylcyanoketene (HCK)] to cinnamylideneamines and benzylideneamines is presented. The size and degree of unsaturation of the imine substitutents were varied in order to probe those factors that influence both stereo-and periselectivity of the cycloadditions. In the cinnamylidene series, it was generally found that 2-azetidinone formation is enhanced when the N-substituent of the imine is small. In general, the stereochemistry of the 2-azetidinone can be controlled by the bulk of this substituent as well as the substitution pattern of the imine. Within the benzylidene series, the cycloadditions are stereospecific when the N-substituent is an aryl group; only 3-cyano-2-azetidinones having a trans relationship between the 3-cyano group and the proton at position * 86(204-205) * 89(193-194) * 93 (182-183) * 82 (I 19-120) * 94(154-155

We have evaluated the in vivo efficacy of anti-CD3-CRM9, a holo-immunotoxin constructed with a diphtheria toxin binding-site mutant. Eighty percent of established human T-cell subcutaneous tumors in nude mice completely regressed following intraperitoneal ih'ection of immunotoxin at a dose set at half the minimum lethal dose assayed in toxin-sensitive animals. Similar regressions produced by a "7Cs source required a dose in excess of 500 cGy. The high degree of in vivo T-cell ablation produced by this immunotoxin is apparently due to maintenance of the toxin translocation function provided by CRM9 and a necessary intracellular routing function supplied by CD3. This immunotoxin may be useful in treating conditions caused by pathologic oligoclonal T-cell expansion such as graft-versus-host disease, autoimmune diseases, and possibly AIDS.The protein toxins, of which diphtheria toxin (DT) may be considered the prototype, have their effector or enzymatic functions on domains separate from the receptor-mediated entry functions (1)(2)(3)(4). By mixing and splicing toxin domains with binding domains derived from growth factors or monoclonal antibodies, a wide variety of immunotoxins, toxins with altered cellular specificity, have been created (5, 6). The goal has been to maximize in vivo targeted cell killing for therapeutic and experimental purposes (5).Recently, Youle and coworkers (7-9) have introduced highly efficacious holoimmunotoxins based on DT binding mutants. These immunotoxins were equal in potency to immunotoxins made with wild-type DT when directed at the human transferrin receptor or a component of the human T-cell receptor complex, CD3. Because the binding of the mutants was only 1/100th to 1/1000th that of native DT, non-target cell toxicity was similarly reduced. We showed that intracellular routing of DT-based immunotoxins via intracellular DT receptors or alternative receptors is an important determinant of their efficacy (10). CD3 and the transferrin receptor apparently are routed along the same path as the DT receptor. Consequently, CRM9 immunotoxins, which lack the DT binding site, are routed appropriately when directed at CD3 and the transferrin receptor (10). Because CD3 is highly restricted to mature T cells, immunotoxins directed toward this epitope should be useful in producing pan-T-cell ablation in vivo. We have evaluated anti-CD3-CRM9 for this purpose.