A General Photocatalytic Strategy for Nucleophilic Amination of Primary and Secondary Benzylic C-H Bonds.

We report a visible-light photoredox-catalyzed method that enables nucleophilic amination of primary and secondary benzylic C(sp3)-H bonds. A novel amidyl radical precursor and organic photocatalyst operate in tandem to transform primary and secondary benzylic C(sp3)-H bonds into carbocations via sequential hydrogen atom transfer (HAT) and oxidative radical-polar crossover. The resulting carbocation can be intercepted by a variety of N-centered nucleophiles, including nitriles (Ritter reaction), amides, carbamates, sulfonamides, and azoles, for the construction of pharmaceutically relevant C(sp3)-N bonds under unified reaction conditions. Mechanistic studies indicate that HAT is amidyl radical-mediated and that the photocatalyst operates via a reductive quenching pathway. These findings establish a mild, metal-free, and modular protocol for the rapid diversification of C(sp3)-H bonds to a library of aminated products.

Decarboxylation with Carbon Monoxide: The Direct Conversion of Carboxylic Acids into Potent Acid Triflate Electrophiles.

We report a new strategy for the conversion of carboxylic acids into potent acid triflate electrophiles. The reaction involves oxidative carbonylation of carboxylic acids with I2 in the presence of AgOTf, and is postulated to proceed via acyl hypoiodites that react with CO to form acid triflates. Coupling this chemistry with subsequent trapping with arenes offers a mild, room temperature approach to generate ketones directly from broadly available carboxylic acids without the use of corrosive and reactive Lewis or Bronsted acid additives, and instead from compounds that are readily available, stable, and functional group compatible.

A metal-catalysed functional group metathesis approach to the carbon isotope labelling of carboxylic acids.

The distribution, metabolism and ultimate fate of molecules within the body is central to the activity of pharmaceuticals. However, the introduction of radioisotopes into the metabolically stable carbon sites on drugs to probe these features typically requires toxic, radioactive gases such as [14C]CO and [14C]CO2. Here we describe an approach to directly carbon-label carboxylic-acid-containing pharmaceuticals via a metal-catalysed functional group exchange reaction, forming 14C-labelled carboxylic-acid-containing drugs without radioactive gases, in one pot, using an easily available and handled carboxylic acid 14C source. To enable this process, a functional group metathesis of carbon-carbon covalent bonds in acid chloride functionalities is developed, exploiting the ability of nickel catalysts to both reversibly activate carbon-chloride bonds and exchange functionalities between organic molecules. The drug development applicability is illustrated by the direct incorporation of the 14C label or 13C label into an array of complex aryl, alkyl, vinyl and heterocyclic carboxylic acid drugs or drug candidates without gases or a special apparatus, at ambient conditions and without loss of the radiolabel.

Modeling the signatures of hydrides in metalloenzymes: ENDOR analysis of a Di-iron Fe(µ-NH)(µ-H)Fe core.

The application of 35 GHz pulsed EPR and ENDOR spectroscopies has established that the biomimetic model complex L(3)Fe(µ-NH)(µ-H)FeL(3) (L(3) = [PhB(CH(2)PPh(2))(3)](-)) complex, 3, is a novel S = (1)/(2) type-III mixed-valence di-iron II/III species, in which the unpaired electron is shared equally between the two iron centers. (1,2)H and (14,15)N ENDOR measurements of the bridging imide are consistent with an allyl radical molecular orbital model for the two bridging ligands. Both the (µ-H) and the proton of the (µ-NH) of the crystallographically characterized 3 show the proposed signature of a 'bridging' hydride that is essentially equidistant between two 'anchor' metal ions: a rhombic dipolar interaction tensor, T ≈ [T, -T, 0]. The point-dipole model for describing the anisotropic interaction of a bridging H as the sum of the point-dipole couplings to the 'anchor' metal ions reproduces this signature with high accuracy, as well as the axial tensor of a terminal hydride, T ≈ [-T, -T, 2T], thus validating both the model and the signatures. This validation in turn lends strong support to the assignment, based on such a point-dipole analysis, that the molybdenum-iron cofactor of nitrogenase contains two [Fe-H(-)-Fe] bridging-hydride fragments in the catalytic intermediate that has accumulated four reducing equivalents (E(4)). Analysis further reveals a complementary similarity between the isotropic hyperfine couplings for the bridging hydrides in 3 and E(4). This study provides a foundation for spectroscopic study of hydrides in a variety of reducing metalloenzymes in addition to nitrogenase.

A nonclassical dihydrogen adduct of S = ½ Fe(I).

We have exploited the capacity of the "(SiP(iPr)(3))Fe(I)" scaffold to accommodate additional axial ligands and characterized the mononuclear S = ½ H(2) adduct complex (SiP(iPr)(3))Fe(I)(H(2)). EPR and ENDOR data, in the context of X-ray structural results, revealed that this complex provides a highly unusual example of an open-shell metal complex that binds dihydrogen as a ligand. The H(2) ligand at 2 K dynamically reorients within the ligand-binding pocket, tunneling among the energy minima created by strong interactions with the three Fe-P bonds.

Protonation of the dinitrogen-reduction catalyst [HIPTN3N]Mo(III) investigated by ENDOR spectroscopy.

Dinitrogen is reduced to ammonia by the molybdenum complex of L = [HIPTN3N](3-) [Mo; HIPT = 3,5-(2,4,6-iPr3C6H2)2C6H3]. The mechanism by which this occurs involves the stepwise addition of proton/electron pairs, but how the first pair converts MoN2 to MoN ═ NH remains uncertain. The first proton of reduction might bind either at Nß of N2 or at one of the three amido nitrogen (N(am)) ligands. Treatment of MoCO with [2,4,6-Me3C5H3N]BAr'4 [Ar' = 2,3-(CF3)2C6H3] in the absence of reductant generates HMoCO(+), whose electron paramagnetic resonance spectrum has greatly reduced g anisotropy relative to MoCO. (2)H Mims pulsed electron nuclear double-resonance spectroscopy of (2)HMoCO(+) shows a signal that simulations show to have a hyperfine tensor with an isotropic coupling, aiso((2)H) = -0.22 MHz, and a roughly dipolar anisotropic interaction, T((2)H) = [-0.48, -0.93, 1.42] MHz. The simulations show that the deuteron is bound to N(am), near the Mo equatorial plane, not along the normal, and at a distance of 2.6 Å from Mo, which is nearly identical with the (Nam)(2)H(+)-Mo distance predicted by density functional theory computations.

The structure of formaldehyde-inhibited xanthine oxidase determined by 35 GHz 2H ENDOR spectroscopy.

The formaldehyde-inhibited Mo(V) state of xanthine oxidase (I) has been studied for four decades, yet it has not proven possible to distinguish unequivocally among the several structures proposed for this form. The uniquely large isotropic hyperfine coupling for (13)C from CH(2)O led to the intriguing suggestion of a direct Mo-C bond for the active site of I. This suggestion was supported by the recent crystal structures of glycol- and glycerol-inhibited forms of aldehyde oxidoreductase, a member of the xanthine oxidase family. (1)H and (2)H ENDOR spectra of I(C(1,2)H(2)O) in H(2)O/D(2)O buffer now have unambiguously revealed that the active-site structure of I contains a CH(2)O adduct of Mo(V) in the form of a four-membered ring with S and O linking the C to Mo and have ruled out a direct Mo-C bond. Density functional theory computations are consistent with this conclusion. We interpret the large (13)C coupling as resulting from a "transannular hyperfine interaction".

Formation of {[HIPTN(3)N]Mo(III)H}(-) by heterolytic cleavage of H(2) as established by EPR and ENDOR spectroscopy.

MoN(2) (Mo = [(HIPTNCH(2)CH(2))(3)N]Mo, where HIPT = 3,5-(2,4,6-i-Pr(3)C(6)H(2))(2)C(6)H(3)) is the first stage in the reduction of N(2) to NH(3) by Mo. Its reaction with dihydrogen in fluid solution yields "MoH(2)", a molybdenum-dihydrogen compound. In this report, we describe a comprehensive electron paramagnetic resonance (EPR) and (1/2)H/(14)N electron nuclear double resonance (ENDOR) study of the product of the reaction between MoN(2) and H(2) that is trapped in frozen solution, 1. EPR spectra of 1 show that it has a near-axial g tensor, g = [2.086, 1.961, 1.947], with dramatically reduced g anisotropy relative to MoN(2). Analysis of the g values reveal that this anion has the Mo(III), [d(xz), d(yz)](3) orbital configuration, as proposed for the parent MoN(2) complex, and that it undergoes a strong pseudo-Jahn-Teller (PJT) distortion. Simulations of the 2D 35 GHz (1)H ENDOR pattern comprised of spectra taken at multiple fields across the EPR envelope (2 K) show that 1 is the [MoH](-) anion. The 35 GHz Mims pulsed (2)H ENDOR spectra of 1 prepared with (2)H(2) show the corresponding (2)H(-) signal, with a substantial deuterium isotope effect in a(iso). Radiolytic reduction of a structural analogue, Mo(IV)H, at 77 K, confirms the assignment of 1. Analysis of the 2D (14)N ENDOR pattern for the ligand amine nitrogen further reveals the presence of a linear N(ax)-Mo-H(-) molecular axis that is parallel to the unique magnetic direction (g(1)). The ENDOR pattern of the three equatorial nitrogens is well-reproduced by a model in which the Mo-N(eq) plane has undergone a static, not dynamic, PJT distortion, leading to a range of hyperfine couplings for the three N(eq)'s. The finding of a nearly axial hyperfine coupling tensor for the terminal hydride bound Mo supports the earlier proposal that the two exchangeable hydrogenic species bound to the FeMo cofactor of the nitrogense turnover intermediate, which has accumulated four electrons/protons (E(4)), are hydrides that bridge two metal ions, not terminal hydrides.

A palladium-catalyzed C-H functionalization route to ketones via the oxidative coupling of arenes with carbon monoxide.

We describe the development of a new palladium-catalyzed method to generate ketones via the oxidative coupling of two arenes and CO. This transformation is catalyzed by simple palladium salts, and is postulated to proceed via the conversion of arenes into high energy aroyl triflate electrophiles. Exploiting the latter can also allow the synthesis of unsymmetrical ketones from two different arenes.

A general approach to intermolecular carbonylation of arene C-H bonds to ketones through catalytic aroyl triflate formation.

The development of metal-catalysed methods to functionalize inert C-H bonds has become a dominant research theme in the past decade as an approach to efficient synthesis. However, the incorporation of carbon monoxide into such reactions to form valuable ketones has to date proved a challenge, despite its potential as a straightforward and green alternative to Friedel-Crafts reactions. Here we describe a new approach to palladium-catalysed C-H bond functionalization in which carbon monoxide is used to drive the generation of high-energy electrophiles. This offers a method to couple the useful features of metal-catalysed C-H functionalization (stable and available reagents) and electrophilic acylations (broad scope and selectivity), and synthesize ketones simply from aryl iodides, CO and arenes. Notably, the reaction proceeds in an intermolecular fashion, without directing groups and at very low palladium-catalyst loadings. Mechanistic studies show that the reaction proceeds through the catalytic build-up of potent aroyl triflate electrophiles.

Osteoinductivity of demineralized bone matrix in immunocompromised mice and rats is decreased by ovariectomy and restored by estrogen replacement.

The osteoinduction potential of human demineralized bone matrix (DBM) in females with low estrogen (E2) is unknown. Moreover, the osteoinductivity of commercial human DBM is tested in male athymic rats and mice, but DBM performance in these animals may not reflect performance in female animals or provide information on E2's role in the process. To gain insight, human DBM was implanted bilaterally in the gastrocnemius of twenty-four athymic female mice (10 mg/implant) and twenty-four athymic female rats (15 mg/implant). Eight animals in each group were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized with E2-replacement (OVX+E2) via subcutaneous slow release capsules of 17beta-estradiol. OVX and OVX+E2 animals were pair-fed to SHAM animals. Four animals from each group were euthanized at 35 days and four at 56 days. Animal weight, uterine weight, and blood estrogen levels confirmed that pair feeding, ovariectomy, and E2 replacement were successful. Histological sections of implanted tissues were evaluated qualitatively for absence or presence of DBM, ossicle formation, and new bone or cartilage using a previously developed qualitative scoring system (QS) and by histomorphometry to obtain a quantitative assessment of osteoinduction. OVX mice had a small but significant QS decrease at 35 days compared to SHAM mice, confirmed by quantitative measurement of ossicle, marrow space, and new bone areas. The QS in rats was not affected by OVX but histomorphometry showed decreased new bone in OVX rats, which was restored by E2. The QS indicated that the number of new bone sites was not reduced by OVX in rats or mice at 56 days, but the relative amount of new bone v. marrow space was affected and differed with animal species. Residual DBM was less in OVX animals, indicating that DBM resorption was affected. Cartilage was present in rats but not in mice, suggesting that endochondral ossification was slower and indicating that bone graft studies in these species are not necessarily comparable. These results show the importance of E2 in human DBM-induced bone formation and suggest that E2 may be needed for clinical effectiveness in post-menopausal women.

Multi-generational genome wide association studies identify chromosomal regions associated with ascites phenotype.

Ascites is a multi-faceted disease commonly observed in fast growing broilers, which is initiated when the body is insufficiently oxygenated. A series of events follow, including an increase in pulmonary artery pressure, right ventricle hypertrophy, and accumulation of fluid in the abdominal cavity and pericardium. Advances in management practices along with improved selection programs have decreased ascites incidence in modern broilers. However, ascites syndrome remains an economically important disease throughout the world, causing estimated losses of $100 million per year. In this study, a 60 K Illumina SNP BeadChip was used to perform a series of genome wide association studies (GWAS) on the 16th and 18th generation of our relaxed (REL) line descended from a commercial elite broiler line beginning in 1995. Regions significantly associated with ascites incidence were identified on chromosome 2 around 70 megabase pairs (Mbp) and on chromosome Z around 60 Mbp. Five candidate single nucleotide polymorphisms (SNP) were evaluated as indicators for these 2 regions in order to identify association with ascites and right ventricle to total ventricle weight (RVTV) ratios. Chromosome 2 SNP showed an association with RVTV ratios in males phenotyped as ascites resistant and ascites susceptible (P = 0.02 and P = 0.03, respectively). The chromosome Z region also indicates an association with resistant female RVTV values (P = 0.02). Regions of significance identified on chromosomes 2 and Z described in this study will be used as proposed candidate regions for further investigation into the genetics of ascites. This information will lead to a better understanding of the underlying genetics and gene networks contributing to ascites, and thus advances in ascites reduction through commercial breeding schemes.

Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer.

Previous studies have suggested that overexpression of HER-2/neu oncogene occurs in 15-40% of breast cancers and that overexpression is associated with poor prognosis. In the present report, we have used an immunohistochemical technique involving a monoclonal antibody specifically reactive with the external domain of HER-2/neu to study expression of HER-2/neu in frozen sections of normal ovary and advanced epithelial ovarian cancer. The intensity of staining for HER-2neu was always moderate or less (0-2+) in normal ovarian epithelium. Among 73 ovarian cancers, 50 (68%) had staining similar to that for normal ovarian epithelium (0-2+) while 23 (32%) stained heavily (3+). Survival of the 23 patients with high HER-2/neu expression (median, 15.7 months) was significantly worse (P = 0.001) than that of the 50 patients (median, 32.8 months) with normal HER-2/neu expression. In addition, patients whose tumors had high HER-2/neu expression were significantly less likely to have a complete response to primary therapy (P less than 0.05) or have a negative second-look laparotomy when serum CA 125 levels were normal preoperatively (P less than 0.05). These findings suggest that HER-2/neu deserves further evaluation as a prognostic marker in epithelial ovarian cancer.

Immunohistochemical expression of CA 125 in endometrial adenocarcinoma: correlation of antigen expression with metastatic potential.

Immunohistochemical localization of CA 125 using murine monoclonal antibody OC 125 was performed on fresh frozen tissue from 44 endometrial adenocarcinomas and 26 benign endometria. Immunohistochemical evaluation incorporated both intensity and distribution of staining (CA 125 HSCORE). Thirty-seven cancers (84%) and 23 benign endometria (88%) expressed immunohistochemically detectable CA 125. Staining was confined to epithelial cells and was present both on the cell membrane and in the cytoplasm. Among the 44 endometrial cancers, CA 125 HSCORE did not correlate with histological grade, depth of myometrial invasion or estrogen/progesterone receptor levels. Following surgical staging, 13 patients (30%) were found to have extrauterine metastatic disease. The median CA 125 HSCORE of patients with metastatic disease (2.25) was significantly higher than that of patients with disease confined to the uterus (0.6) (P less than 0.001). In addition, high CA 125 HSCORE also correlated with the presence of lymph node metastasis (P less than 0.001). The results of this study suggest that high CA 125 expression by endometrial adenocarcinomas is associated with increased metastatic potential.

DNA image cytometry of keratoacanthoma and squamous cell carcinoma.

The distinction between the keratoacanthoma (KA) and the squamous cell carcinoma (SCC) can sometimes be difficult on the basis of histologic and clinical criteria. The possible diagnostic significance of DNA ploidy initiated the present study evaluating the DNA ploidy in paraffin-embedded tissue sections of 7 KA and 15 SCC, and fresh frozen tissue touch preparations of 15 of the same cases using the CAS 200 Image Analyzer. In paraffin-embedded tissue sections the main peak DNA index was based on normal epidermis, and ranged from 1.03 to 1.59 in KA and from 1.47-2.71 in SCC. The DNA Index (DI) discriminated KA from SCC in 17 of 22 cases (p less than 0.0007). The highest DNA content of single nuclei ranged from 9.0-18.0 picograms (pg) (DI 2.9-6.03) in KA and 14.8-38.6 pg (DI 4.0-11.03) in SCC. The highest DNA content discriminated KA from SCC in 16 of 22 cases (p less than 0.003). In fresh frozen tissue touch preparations from 15 of the same lesions, there was considerable overlap in DNA indices of KA (0.534-1.39) and SCC (0.464-1.41). Abnormal DNA peaks seen in histograms from three SCC in paraffin-embedded tissue sections were lost in the touch preparation histograms, probably due to inadequate sampling. Therefore, image analysis of paraffin-embedded tissue sections is better able to distinguish KA from SCC than touch preparations.

c-erbB-2 expression in breast cancer detected by immunoblotting and immunohistochemistry.

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex.

The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.

DNA ploidy and proliferation index of soft tissue sarcomas determined by image cytometry of fresh frozen tissue.

The DNA content of Feulgen-stained monolayer imprint preparations from 30 fresh frozen soft tissue and bone sarcomas was analyzed using image cytometry. DNA aneuploidy showed a significant association with increasing tumor grade. Of the grade III tumors, 83.3% (15 of 18) were aneuploid; of the grade I and II sarcomas, 33.3% (4 of 12) were aneuploid (P less than 0.00861). The proliferation index (PI) was determined by the percentage of tumor nuclear area staining with the monoclonal antibody Ki-67. The PI was significantly associated with ploidy and tumor grade. Ki-67 PI was elevated (greater than 2.0%) in 73.7% (14 of 19) of the aneuploid tumors, compared with 30% (3 of 10) of the euploid tumors (P less than 0.04597). Ki-67 PI was greater than 2.0% in 77.8% (14 of 18) of the grade III tumors, compared with 27.2% (3 of 11) of the grade I and II tumors (P less than 0.01773). These findings suggest that DNA ploidy and Ki-67 PI as determined by image analysis may be a useful supplement to tumor grading. The literature examining previous studies of sarcoma DNA ploidy is reviewed.

Determination of proliferation index in advanced ovarian cancer using quantitative image analysis.

It has been shown that the monoclonal antibody Ki-67 reacts with a nuclear antigen that is expressed only by proliferating cells. The feasibility of using image analysis to quantitate Ki-67 staining (proliferation index [PI]) of epithelial ovarian cancers was investigated. The PI was determined in 50 advanced-stage primary ovarian cancers. Frozen sections were immunostained with the Ki-67 monoclonal antibody, and the PI was calculated using static image analysis. Among 35 stage III ovarian carcinomas, the median PI was 8.9%, compared with 17.7% in 15 stage IV cancers (P = 0.06). There was no relationship between PI and histologic grade. The median survival time of 32 patients whose cancers had a high Ki-67 expression (> or = 7.5%) was 16.8 months, which differed significantly (P < 0.01) from the median survival of 31.5 months observed in patients whose tumors demonstrated low Ki-67 expression (< 7.5%). Quantitative image analysis of Ki-67-stained fresh-frozen ovarian cancers may provide useful prognostic information. Further studies are warranted to investigate the relationship between Ki-67 expression and other known clinicopathologic and genetic features of ovarian cancer.