Monofunctional Derivatives of Coproporphyrins for Phosphorescent Labeling of Proteins and Binding Assays

Tomás C. O'Riordan; Aleksi E. Soini; Dmitri B. Papkovsky

doi:10.1006/abio.2001.4989

Monofunctional Derivatives of Coproporphyrins for Phosphorescent Labeling of Proteins and Binding Assays

Analytical Biochemistry ◽

10.1006/abio.2001.4989 ◽

2001 ◽

Vol 290 (2) ◽

pp. 366-375 ◽

Cited By ~ 36

Author(s):

Tomás C. O'Riordan ◽

Aleksi E. Soini ◽

Dmitri B. Papkovsky

Keyword(s):

Binding Assays ◽

Derivatives Of

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Studies toward bivalent κ opioids derived from salvinorin A: heteromethylation of the furan ring reduces affinity

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.9.328 ◽

2013 ◽

Vol 9 ◽

pp. 2916-2924 ◽

Cited By ~ 6

Author(s):

Thomas A Munro ◽

Wei Xu ◽

Douglas M Ho ◽

Lee-Yuan Liu-Chen ◽

Bruce M Cohen

Keyword(s):

Furan Ring ◽

Allosteric Modulation ◽

Salvinorin A ◽

Allosteric Modulators ◽

Design Strategies ◽

Binding Assays ◽

Bivalent Ligand ◽

Basic Nitrogen ◽

Derivatives Of ◽

Aspartate Residue

The recent crystal structure of the κ-opioid receptor (κ-OR) revealed, unexpectedly, that the antagonist JDTic is a bivalent ligand: in addition to the orthosteric pocket occupied by morphinans, JDTic also occupies a distinct (allotopic) pocket. Mutagenesis data suggest that salvinorin A (1) also binds to this allotopic pocket, adjacent to the aspartate residue that anchors the basic nitrogen atom of classical opiates (Asp138). It has been suggested that an H-bond donor appended to 1 might interact with Asp138, increasing affinity. Such a bivalent ligand might also possess altered functional selectivity. Based on modeling and known N-furanylmethyl opioid antagonists, we appended H-bond donors to the furan ring of 1. (Dimethylamino)methyl groups at C-15 or C-16 abolished affinity for κ-OR. Hydroxymethylation at C-16 was tolerated, but 15,16-bis-hydroxymethylation was not. Since allosteric modulators may go undetected in binding assays, we also tested these and other low-affinity derivatives of 1 for allosteric modulation of dynorphin A in the [35S]GTPγS assay. No modulation was detected. As an alternative attachment point for bivalent derivatives, we prepared the 2-(hydroxyethoxy)methyl ether, which retained high affinity for κ-OR. We discuss alternative design strategies for linked, fused or merged bivalent derivatives of 1.

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THE STRANGE CROSS-REACTION OF MENADIONE (VITAMIN K3) AND 2,4-DINITROPHENYL LIGANDS WITH A MYELOMA PROTEIN AND SOME CONVENTIONAL ANTIBODIES

Journal of Experimental Medicine ◽

10.1084/jem.140.3.687 ◽

1974 ◽

Vol 140 (3) ◽

pp. 687-702 ◽

Cited By ~ 33

Author(s):

Maria C. Michaelides ◽

Herman N. Eisen

Keyword(s):

Equilibrium Dialysis ◽

Tryptophan Fluorescence ◽

Structural Features ◽

Cross Reaction ◽

Binding Assays ◽

Vitamin K3 ◽

Myeloma Protein ◽

Direct Binding ◽

Antibody Preparations ◽

Derivatives Of

To explore the possibility that the affinity of some myeloma proteins for 2,4-dinitrophenyl (DNP) ligands is the consequence of a "strange" (i.e., unexpected) cross-reaction for more natural ligands, a variety of substances (primarily derivatives of purines, pyrimidines, naphthaquinone) were tested for ability to block the binding of [3H]-ϵ-DNP-L-lysine by protein 315, an IgA mouse myeloma protein with high affinity for DNP ligands. The most impressive inhibiting activity was observed with 2-methyl-1,4-napthaquinone (menadione, vitamin K3). The affinity (intrinsic association constant) of protein 315 for menadione was 5 x 105 L/M (at 4°C). Because the same affinity was measured in direct-binding assays (e.g., equilibrium dialysis) and in an indirect one based on the assumption of competitive binding with DNP-lysine, it is likely that menadione and DNP bind at overlapping sites in the protein's combining region. This conclusion is supported by molecular models which reveal some common structural features in these ligands. Hence it is not surprising that antinitrophenyl antibody preparations, raised by conventional immunization procedures (anti-2,4-DNP; anti-2,6-DNP; anti-2,4,6-TNP) also bind menadione with considerable affinity. As with DNP ligands, when menadione binds to protein 315 or to conventional antinitrophenyl antibodies, some of the protein's tryptophan fluorescence is quenched, there is a change in the ligand's absorption spectrum (hypochromia and/or red shift), and the binding is temperature-dependent (exothermal).

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