Jeffrey D. Butler

Antimitochondrial antibody heterogeneity and the xenobiotic etiology of primary biliary cirrhosis

Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC‐E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC‐E2 with higher titers than native PDC‐E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8‐bis(acetylthio) octanoic acid (SAc)‐conjugated bovine serum albumin (BSA), recombinant PDC‐E2 (rPDC‐E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme‐linked immunosorbent assay (ELISA); SAc conjugate and rPDC‐E2‐specific affinity‐purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late‐stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC‐E2 in early‐stage versus late‐stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. Conclusion: Specific modifications of the disulfide bond within the lipoic‐acid‐conjugated PDC‐E2 moiety, i.e., by an electrophilic agent renders PDC‐E2 immunogenic in a genetically susceptible host. (HEPATOLOGY 2013)

Electrophile-modified lipoic derivatives of PDC-E2 elicits anti-mitochondrial antibody reactivity

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC.

Nucleophilic substitution of oxazino-/oxazolino-/benzoxazin [3,2-b]indazoles: an effective route to 1H-indazolones.

A variety of nucleophiles, thiolates, alkoxides, amines, iodide, and cyanide, react with oxazino-, oxazolino-, and benzoxazin[3,2-b]indazoles under microwave conditions to yield a diverse set of 2-substituted 1H-indazolones. The synthetic utility of these indazoles is further demonstrated by ANRORC (addition of the nucleophile, ring-opening, and ring closure) reactions to yield isomeric pyrazoloindazolones by a process wherein iodide acts first as a nucleophile and subsequently as a leaving group.

Orthogonally protected thiazole and isoxazole diamino acids: an efficient synthetic route

Heterocyclic and heteroaromatic amino acids (HAAs) are central to the motifs of peptide antibiotics, including microcin B17, nostocyclamide, telomestatin, and thiostrepton. aAmino acids undergo cyclization and oxidation to form heteroaromatic rings, notably, thiazoles, oxazoles, indoles, and pyridines, which give rise to well-documented antibiotic activity. Few of these targets have succumbed to total synthesis due, in large part, to the demand for orthogonally protected HAA building blocks. In contrast, commercial orthogonally protected natural amino acids, most commonly lysine and aspartic acid, are routinely used as the branch point in the synthesis of branched or cyclic peptide and oligosaccharide mimetics (Figure 1 a). Similarly, these agents see action in the ligation of imaging agents (Figure 1 b) and in diversity-oriented syntheses (e.g., I!II, Figure 1 c). However, the stringent orthogonal chemistry requirements, especially in solid-phase synthesis, make optimization at this branch-point region challenging. Surprisingly, methods to generate new heterocyclic nonnatural amino acids with an additional orthogonally protected amino group (e.g., diamino acids), are still rare. Nonnatural conformationally restrictive amino acids have potential in the discovery of new peptidomimetics and in efforts to improve the pharmacological and protease resistant properties of bioactive peptides. 9] There is demand for practical HAA syntheses that deliver orthogonally protected diamino acids compatible with the traditional solid and solution phase 9-fluorenylmethoxycarbonyl (Fmoc) protection strategy. Thus our focus herein is on the development of short, high yielding syntheses delivering heteroaromatic monoand diamino acids from readily available starting materials. Herein, we report an efficient synthesis yielding thiazoleand isoxazole-based HAAs from b-amino acids. This strategy allows for orthogonal carbamate protection that permits independent synthetic manipulation (Figure 1). Further, the viability of the synthesized HAAs as branch-point amino acids is demonstrated in the solid-phase synthesis of an inhibitor of two chorismate utilizing enzymes, anthranilate synthase (AS) and isochorismate synthase (IS). This inhibitor shows twoand threefold better activity than its lysine predecessor in the inhibition of AS and IS, respectively. A wide variety of b-amino acids are commercially available and considerable synthetic effort has been focused on producing novel optically active b-amino acids. This availability makes b-amino acids an attractive starting material for this work. As outlined in Figure 2, our synthetic method began by carbamate protection (Teoc, Boc, Cbz, and Alloc) of b-alanine following literature procedures. These protected acids were subjected to coupling conditions to install the Meldrum acid moiety in 94–98 % yield. Intramolecular cyclization of 1 a–d!2 a–d is accomplished quantitatively in EtOAc at reflux via a presumed ketene intermediate. In a modification of Suzuki s general method of cyclocondensa[a] Dr. J. D. Butler, K. C. Coffman, Dr. K. T. Ziebart, Prof. M. D. Toney, Prof. M. J. Kurth Department of Chemistry, University of California, Davis One Shields Avenue, Davis, CA 95616 (USA) Fax: (+1) 530-752-8995 E-mail : mjkurth@ucdavis.edu Homepage: http://chemgroups.ucdavis.edu/~kurth/ Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201001492. Figure 1. Common motifs and methods employing an orthogonally protected diamino acid, which include a) branched peptides, b) ligated imaging agents, and c) diversity-oriented methods.

Synthesis of substituted chromanones: an organocatalytic aldol/oxa-Michael reaction.

A diastereoselective organocatalytic aldol/oxa-Michael reaction has been developed to efficiently deliver medicinally relevant 2,3-ring-substituted chromanones. Development of this synthetic strategy revealed an unexpected kinetic anti-Saytzeff elimination; an origin for the observed selectivity is suggested on the basis of the results of quantum chemical calculations. This unusual kinetic selectivity necessitated an isomerization protocol that in turn led to the discovery of an intriguing Pd-mediated isomerization/intramolecular Friedel-Crafts-type alkylation.

Synthesis of Spiro-fused Pyrazolidoylisoxazolines

Routes to structurally unique spiro-fused pyrazolidoylisoxazolines are reported. These methods start with monosubstituted hydrazines or hydrazides and utilize the nitrile oxide 1,3-dipolar cycloaddition reaction to generate the targeted spiro-fused bis-heterocycles. Molecular shape space diversity analyses were performed on these pyrazolidoylisoxazolines showing that manipulation of the appended R groups significantly changes the molecular shape.