Recent advances in the synthesis and application of fluorescent α-amino acids

Fluorescence spectroscopy has become a powerful technique for probing a range of complex biological processes including enzyme mechanisms and protein-protein interactions. While the application of this technique uses a number of strategies, many of these rely on the use of fluorescent α-amino acids. This review highlights the recent synthetic methods developed for the incorporation of highly conjugated chromophores into the side-chain of α-amino acids and the application of these compounds as probes for imaging in medicine and biology. In particular, the design and synthesis of α-amino acids bearing coumarin, flavone and polyaromatic derived chromophores is described

A Study of Bi-Directional Reflectance Distribution Functions and Their Effects on Infrared Signature Models

Since 2004, AFIT has been developing a trend-analysis tool to assess large commercial aircraft infrared (LCAIR) signatures. In many cases, this code predicted signatures to within 10% of measured data. However, other results indicated that the single-bounce, specular-reflection algorithm being used failed to adequately simulate interactions between aircraft parts where either the specular component is dominated by diffuse reflection or part-to-part multiple-bounce reflections contribute significantly to the signature. This research incorporates Bi-Directional Reflectance Distribution Functions (BRDF\u27s) and multiple-bounce calculations into the LCAIR model. A physical aircraft model was constructed from aluminum, and measurements were taken before and after a surface treatment in gloss black paint. The Sandford-Robertson model is used to parameterize the BRDF\u27s of both the bare aluminum and gloss black paint. Since the most efficient method of integrating a BRDF depends upon the reflectance distribution of the aircraft material, the sampling resolution of the BRDF integral is crucial to an accurate simulation. Additionally, care is taken to ensure that the integration of the hemispherical irradiance onto each facet of the computational model is sampled at a sufficient resolution to achieve convergence in the solution. Simulations in the mid-wave infrared (MWIR) and long-wave infrared (LWIR) bands validate both the previous specular reflectance simplification for the gloss black simulations and the failure of the previous algorithm for the highly reflective bare aluminum. The necessity of considering multiple bounces in the simulation is also demonstrated amongst part-to-part reflections near the wing root, where three or four bounces are required for the solution to converge. Finally, three scenarios simulating a man-portable air defense system (MAN-PADS) system engaging an Airbus A340-300 aircraft landing at a generic airport are performed

Access to 2,6-disubstituted 4-oxopiperidines using a 6-endo-trig cyclization: stereoselective synthesis of spruce alkaloid and (+)-241D

A synthetic route to cis-2-methyl-4-oxo-6-alkylpiperidines has been developed using a 6-endo-trig cyclization of E-enones. The base-mediated intramolecular cyclization was found to be general for both alkyl and aryl substituted enones, providing the corresponding 4-oxopiperidines in high yields (80–89%). Stereoselective reduction of the 2,6-cis-disubstituted 4-oxopiperidines then gave the 2,4,6-cis,cis-trisubstituted 4-hydroxypiperidines in high diastereoselectivity. The general nature of this approach was demonstrated with the synthesis of the natural products, spruce alkaloid and (+)-241D

Synthesis of novel fluorescent heterocyclic-derived α-amino acids and the total syntheses of piperidine natural products

During the course of this PhD, methodology for the synthesis of a series of novel, highly fluorescent pyridine-derived α-amino acids was developed. Enone-derived α-amino acids were subjected to an inverse electron demand hetero-Diels-Alder cycloaddition and aromatisation reaction, which led to a twelve-membered library of pyridine analogues. The optical properties of these compounds were analysed, with several exhibiting interesting fluorescent characteristics. One of the analogues was incorporated into a cell penetrating pentapeptide via solid phase peptide synthesis. The resulting hexapeptide was incubated with human fibroblast cells and fluorescence microscopy was used to show accumulation of the peptide in the cells. The total syntheses of piperidine containing natural products, spruce alkaloid and (+)-241D were also examined during this PhD. A short nine-step linear sequence was developed giving spruce alkaloid and (+)-241D in 21% and 19% overall yield, respectively. A base mediated 6-endo-trig cyclisation was employed as the key-step followed by stereoselective ketone reduction to complete the total syntheses. The scope of the cyclisation and reduction was examined with a range of enone side-chains resulting in a small library of novel 4-hydroxy-2,6-disubstituted piperidines

Stereoselective synthesis of 2,6-trans-4-oxopiperidines using an acid-mediated 6-endo-trig cyclisation

An acid-mediated 6-endo-trig cyclisation of amine-substituted enones has been developed for the stereoselective synthesis of trans-6-alkyl-2-methyl-4-oxopiperidines. Performed under conditions that prevent removal of the Boc-protecting group or acetal formation, the key cyclisation was found to generate cleanly the 4-oxopiperidine products in high overall yields from a wide range of alkyl substituted enones. The synthetic utility of the trans-6-alkyl-2-methyl-4-oxopiperidines formed from this process was demonstrated with the total synthesis of the quinolizidine alkaloid, (+)-myrtine and the piperidine alkaloid, (–)-solenopsin A

Synthesis and fluorescent properties of β-pyridyl α-amino acids

The preparation of a new class of β-pyridyl α-amino acid is described using a highly regioselective, ytterbium-catalyzed hetero Diels-Alder reaction of enones with vinyl ethers followed by a modified Knoevenagel-Stobbe reaction as the key heterocycle forming steps. Investigation of the properties and applications of these amino acids showed that they could be utilized in solid phase peptide synthesis for the preparation of a biologically relevant hexapeptide, while pyridines bearing electron-rich substituents exhibited strongly fluorescent properties with high quantum yields and MegaStokes shifts. A solvatochromic study with the most fluorogenic amino acid, a p-methoxyphenyl analogue revealed that this charge-transfer based chromophore is highly sensitive to solvent polarity with a bathochromic shift of 115 nm on changing from THF to phosphate-buffered saline

Synthesis and photophysical properties of benzotriazole-derived unnatural α -amino acids

The synthesis of a new class of benzotriazole-derived α-amino acid is described using a highly efficient nucleophilic aromatic substitution of ortho-fluoronitrobenzenes with l-3-aminoalanine and a polymer-supported nitrite reagent-mediated diazotization and cyclization of the subsequent 1,2-aryldiamines as the key steps. Further functionalization of the benzotriazole unit by preparation of halogenated analogues and Suzuki–Miyaura cross-coupling with aryl boronic acids allowed the synthesis of α-amino acids with conjugated side chains. Analysis of the photophysical properties of these α-amino acids revealed that incorporation of electron-rich substituents results in charge-transfer-based, fluorescent compounds with MegaStokes shifts

Diagnostic accuracy of the Enferplex Bovine TB antibody test using individual milk samples from cattle

Bovine tuberculosis is usually diagnosed using tuberculin skin tests or at post-mortem. Recently, we have developed a serological test for bovine tuberculosis in cattle which shows a high degree of accuracy using serum samples. Here, we have assessed the performance of the test using individual bovine milk samples. The diagnostic specificity estimate using the high sensitivity setting of the test was 99.7% (95% CI: 99.2-99.9). This estimate was not altered significantly by tuberculin boosting. The relative sensitivity estimates of the test using the high sensitivity setting in milk samples from comparative skin test positive animals was 90.8% (95% CI: 87.1-93.6) with boosting. In animals with lesions, the relative sensitivity was 96.0% (95% CI: 89.6-98.7). Analysis of paired serum and milk samples from skin test positive animals showed correlation coefficients ranging from 0.756-0.955 for individual antigens used in the test. Kappa analysis indicated almost perfect agreement between serum and milk results, while McNemar marginal homogeneity analysis showed no statistically significant differences between the two media. The positive and negative likelihood ratio were 347.8 (95% CI: 112.3-1077.5) and 0.092 (95% CI: 0.07-0.13) respectively for boosted samples from skin test positive animals. The results show that the test has high sensitivity and specificity in individual milk samples and thus milk samples could be used for the diagnosis of bovine tuberculosis.</p

Diagnostic accuracy of the Enferplex Bovine Tuberculosis antibody test in cattle sera

Bovine tuberculosis is a contagious bacterial disease of worldwide economic, zoonotic and welfare importance caused mainly by Mycobacterium bovis infection. Current regulatory diagnostic methods lack sensitivity and require improvement. We have developed a multiplex serological test for bovine tuberculosis and here we provide an estimate of the diagnostic accuracy of the test in cattle. Positive and negative reference serum samples were obtained from animals from Europe and the United States of America. The diagnostic specificity estimate was 98.4% and 99.7% using high sensitivity and high specificity settings of the test respectively. Tuberculin boosting did not affect the overall specificity estimate. The diagnostic sensitivity in samples from Mycobacterium bovis culture positive animals following tuberculin boosting was 93.9%.The relative sensitivity following boosting in tuberculin test positive, lesion positive animals and interferon gamma test positive, lesion positive animals was 97.2% and 96.9% respectively. In tuberculin test negative, lesion positive animals and in interferon gamma test negative, lesion positive animals, the relative sensitivity following tuberculin boosting was 88.2% and 83.6% respectively. The results show that the test has high diagnostic sensitivity and specificity and can detect infected animals that are missed by tuberculin and interferon gamma testing