Tailoring surface properties to build colloidal diagnostic devices: Controlling interparticle associations

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (VIES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.

Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca2+-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (∼1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability. © 2002 Elsevier Science B.V. All rights reserved.

Symmorphosis through dietary regulation: a combinatorial role for proteolysis, autophagy and protein synthesis in normalising muscle metabolism and function of hypertrophic mice after acute starvation

Animals are imbued with adaptive mechanisms spanning from the tissue/organ to the cellular scale which insure that processes of homeostasis are preserved in the landscape of size change. However we and others have postulated that the degree of adaptation is limited and that once outside the normal levels of size fluctuations, cells and tissues function in an aberant manner. In this study we examine the function of muscle in the myostatin null mouse which is an excellent model for hypertrophy beyond levels of normal growth and consequeces of acute starvation to restore mass. We show that muscle growth is sustained through protein synthesis driven by Serum/Glucocorticoid Kinase 1 (SGK1) rather than Akt1. Furthermore our metabonomic profiling of hypertrophic muscle shows that carbon from nutrient sources is being channelled for the production of biomass rather than ATP production. However the muscle displays elevated levels of autophagy and decreased levels of muscle tension. We demonstrate the myostatin null muscle is acutely sensitive to changes in diet and activates both the proteolytic and autophagy programmes and shutting down protein synthesis more extensively than is the case for wild-types. Poignantly we show that acute starvation which is detrimental to wild-type animals is beneficial in terms of metabolism and muscle function in the myostatin null mice by normalising tension production.

Novel Photochemical Methodologies For Diversity Oriented Synthesis And Screening Of Combinatorial Libraries

The main goal of this project was to develop an efficient methodology allowing rapid access to structurally diverse scaffolds decorated with various functional groups. Initially, we discovered and subsequently developed an experimentally straightforward, high-yielding photoinduced conversion of readily accessible diverse starting materials into polycyclic aldehydes and their (hemi)acetals decorated by various pendants. The two step sequence, involving the Diels-Alder addition of heterocyclic chalcones and other benzoyl ethylenes to a variety of dienes, followed by the Paternò-Büchi reaction, was described as an alkene-carbonyl oxametathesis. This methodology offers a rapid increase in molecular complexity and diversity of the target scaffolds. To develop this novel methodology further and explore its generality, we directed our attention to the Diels-Alder adducts based on various chromones. We discovered that the Diels-Alder adducts of chromones are capable of photoinduced alkene-arene [2+2] cycloaddition producing different dienes, which can either dimerize or be introduced into a double-tandem [4π+2π]·[2π+2π]·[4π+2π]·[2π+2π] synthetic sequence, followed by an acid-catalyzed oxametathesis, leading to a rapid expansion of molecular complexity over a few experimentally simple steps. In view of the fact that oxametathesis previously was primarily observed in aromatic oxetanes, we decided to prepare model aliphatic oxetanes with a conformationally unconstrained or "flexible" methyl group based on the Diels-Alder adducts of cyclohexadiene or cyclopentadiene with methyl vinyl ketone. Upon addition of an acid, the expected oxametathesis occurred with results similar to those observed in the aromatic series proving the generality of this approach. Also we synthesized polycyclic oxetanes resulting from the Diels-Alder adducts of cyclic ketones. This not only gave us access to remarkably strained oxetane systems, but also the mechanism for their protolytic ring opening provided a great deal of insight to how the strain affects the reactivity. Additionally, we discovered that although the model Hetero-Diels-Alder adducts did not undergo [2+2] cycloaddition, both exo- and endo-Sulfa-Diels-Alder products, nonetheless, were photochemically active and various products with defined stereochemistry could be produced upon photolysis. In conclusion, we have developed an approach to the encoding and screening of solution phase libraries based on the photorelease of externally sensitized photolabile tags. The encoding tags can be released into solution only when a binding event occurs between the ligand and the receptor, equipped with an electron transfer sensitizer. The released tags are analyzed in solution revealing the identity of the lead ligand or narrowing the range of potential leads.

Combinatorial solid phase synthesis of multiply substituted 1,4-benzodiazepines and affinity studies on the CCK2 receptor (Part 1)

One hundred sixty-eight multiply substituted 1,4-benzodiazepines have been prepared by a five-step solid-phase combinatorial approach using syn-phase crowns as a solid support and a hydroxymethyl-phenoxy-acetamido linkage (Wang linker). The substituents of the 1,4-benzodiazepine scaffold have been varied in the -3, -5, -7, and 8-positions and the combinatorial library was evaluated in a cholecystokinin (CCK) radioligand binding assay. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCK-B (CCK2) receptor have been optimized on the lipophilic side chain, the ketone moiety, and the stereochemistry at the 3-position. Various novel 3-alkylated compounds were synthesized and [S]3-propyl-5-phenyl-1,4-benzodiazepin-2-one, [S]NV-A, has shown a CCK-B selective binding at about 180 nM. Fifty-eight compounds of this combinatorial library were purified by preparative TLC and 25 compounds were isolated and fully characterized by TLC, IR, APCI-MS, and 1H/13C-NMR spectroscopy.

A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

Novel anti-bacterials against MRSA:Synthesis of focussed combinatorial libraries of tri-substituted 2(5H)-furanones

Mucobromic and mucochloric acid were used as building blocks for the construction of a chemical combinatorial library of 3,4,5-trisubstituted 2(5H)-furanones. With these 2 butenolide building blocks, and eight alcohols a sublibrary of 16 dihalogenated 5-alkoxy-2(5H)-furanones was prepared. This sublibrary of 5-alkoxylated furanones was reacted with 16 amines generating a full size focussed combinatorial library of 256 individual compounds. This three dimensional combinatorial library of 3-halogen-4-amino-5-alkoxy-2(5H)-furanones was prepared around the benzimidazolyl furanone lead structure by applying a solution phase combinatorial chemistry concept. Typical representatives of the library were purified and fully characterized and one x-ray structures was recorded, additionally. The 3-bromo-4-benzimizazolyl-5-methoxy-2(5H)furanone, Br-A-l, showed an MIC of 8 μg/ml against the multiresistant Staphylococcus aureus ( MRSA). © 2006 Bentham Science Publishers Ltd.

Novel and facile solution-phase synthesis of 2,5-diketopiperazines and O-glycosylated analogs

This work describes the synthesis in Solution of a series of related diketopiperazines with potential biological activities: cyclo(L-Pro-L-Ser), cyclo(L-Phe-L-Ser), cyclo(D-Phe-L-Ser) and the corresponding glycosylated analogs of the latter, cyclo[D-Phe-L-Ser(alpha GlcNAc)] and cyclo[D-Phe-L-Ser(beta GlcNAc)]. The synthetic approach involved coupling reactions of -OH or O-glycosylated serine benzyl esters with NFmoc-protected amino acids (Pro or Phe), followed by one-pot deprotection-cyclization reaction in the presence of 20% piperidine in DMF. (C) 2009 Elsevier Ltd. All rights reserved.

Encoding combinatorial libraries: A novel application of fluorescent silica colloids

A major challenge associated with using large chemical libraries synthesized on microscopic solid support beads is the rapid discrimination of individual compounds in these libraries. This challenge can be overcome by encoding the beads with 1 mum silica colloidal particles (reporters) that contain specific and identifiable combinations of fluorescent byes. The colored bar code generated on support beads during combinatorial library synthesis can be easily, rapidly, and inexpensively decoded through the use of fluorescence microscopy. All reporters are precoated with polyelectrolytes [poly(acrylic acid), PAA, poly(sodium 4-styrenesulfonate PSSS, polyethylenimine, PEI, and/or poly(diallyldimethylammonium chloride), PDADMAC] with the aim of enhancing surface charge, promoting electrostatic attraction to the bead, and facilitating polymer bridging between the bead and reporter for permanent adhesion. As shown in this article, reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to quantity of attached reporters per bead (54 +/- 23 in 2500 mum(2) area for PEI/PAA coated and 11 +/- 6 for uncoated reporters) and minimization of cross-contamination (1 red reporter in 2500 mum(2) area of green-labeled bead for PEI/PAA coated and 26 +/- 15 red reporters on green-labeled beads for uncoated reporters after 10 days). Examination of various polyelectrolyte systems shows that the magnitude of the xi -potential of polyelectrolyte-coated reporters (-64 mV for PDADMAC/PSSS and -42 mV for PEI/PAA-coated reporters) has no correlation with the number of reporters that adhere to the solid support beads (21 +/- 16 in 2500 mum(2) area for PDADMAC/PSSS and 54 +/- 23 for PEI/PAA-coated reporters). The contribution of polymer bridging to the adhesion has a far greater influence than electrostatic attraction and is demonstrated by modification of the polyelectrolyte multilayers using gamma irradiation of precoated reporters either in aqueous solution or in polyelectrolyte solution.

Multi-fluorescent silica colloids for encoding large combinatorial libraries

Large chemical libraries can be synthesized on solid-support beads by the combinatorial split-and-mix method. A major challenge associated with this type of library synthesis is distinguishing between the beads and their attached compounds. A new method of encoding these solid-support beads, 'colloidal bar-coding', involves attaching fluorescent silica colloids ('reporters') to the beads as they pass through the compound synthesis, thereby creating a fluorescent bar code on each bead. In order to obtain sufficient reporter varieties to bar code extremely large libraries, many of the reporters must contain multiple fluorescent dyes. We describe here the synthesis and spectroscopic analysis of various mono- and multi-fluorescent silica particles for this purpose. It was found that by increasing the amount of a single dye introduced into the particle reaction mixture, mono- fluorescent silica particles of increasing intensities could be prepared. This increase was highly reproducible and was observed for six different fluorescent dyes. Multi-fluorescent silica particles containing up to six fluorescent dyes were also prepared. The resultant emission intensity of each dye in the multi-fluorescent particles was found to be dependent upon a number of factors; the hydrolysis rate of each silane-dye conjugate, the magnitude of the inherent emission intensity of each dye within the silica matrix, and energy transfer effects between dyes. We show that by varying the relative concentration of each silane-dye conjugate in the synthesis of multi-fluorescent particles, it is possible to change and optimize the resultant emission intensity of each dye to enable viewing in a fluorescence detection instrument.

A subset-oriented algorithm for minimizing the number of steps required for synthesis of cyclic-peptide libraries

Libraries of cyclic peptides are being synthesized using combinatorial chemistry for high throughput screening in the drug discovery process. This paper describes the min_syn_steps.cpp program (available at http://www.imb.uq.edu.au/groups/smythe/tran), which after inputting a list of cyclic peptides to be synthesized, removes cyclic redundant sequences and calculates synthetic strategies which minimize the synthetic steps as well as the reagent requirements. The synthetic steps and reagent requirements could be minimized by finding common subsets within the sequences for block synthesis. Since a brute-force approach to search for optimum synthetic strategies is impractically large, a subset-orientated approach is utilized here to limit the size of the search. (C) 2002 Elsevier Science Ltd. All rights reserved.

The synthesis and X-ray crystal structure of 9-carboxyhexahydro-7-methoxy-4a,7-ethano-benzopyran-5-en-1-one

9-Carboxyhexahydro-7-methoxy-4a,7-ethano-benzopyran-5-en-1-one (1) was prepared and examined by X-ray crystallography to probe its potential as a new peptide scaffold/template. The crystal structure of the anhydride precursor 7-(2-acetoxyethyl)-4-methoxy-3a,4,7,7a-tetrahydro-4,7-ethanoisobenzofuran-1,3-dione (6) is also reported.

A new and efficient synthesis of N3-cycloalkylamino-6,8-diaminopurines

[Excerpt] The purine core is a privileged scaffold in medicinal chemistry and the biological relevance of purine derivatives makes them attractive targets in the preparation of combinatorial libraries.1,2 In particular, there is a great interest in the synthesis of 8-substituted purines due to their important potential as antiviral and anticancer agents.3 Reports on 8-aminopurines are limited and general methods to obtain these purine derivatives are still needed.4 Cyclic amines and hydrazines are key structural motifs in various bioactive agents.5 Here we report a novel, efficient and inexpensive method for the synthesis of 6,8-diaminopurines 4 incorporating cycloalkylamino substituents at N3position of the purine ring. (...)

High Throughput Purification of Combinatorial Libraries by Preparative LC/MS

Viime vuosien nopea kehitys on kiihdyttänyt uusien lääkkeiden kehittämisprosessia. Kombinatorinen kemia on tehnyt mahdolliseksi syntetisoida suuria kokoelmia rakenteeltaan toisistaan poikkeavia molekyylejä, nk. kombinatorisia kirjastoja, biologista seulontaa varten. Siinä molekyylien rakenteeseen liittyvä aktiivisuus tutkitaan useilla erilaisilla biologisilla testeillä mahdollisten "osumien" löytämiseksi, joista osasta saatetaan myöhemmin kehittää uusia lääkeaineita. Jotta biologisten tutkimusten tulokset olisivat luotettavia, on syntetisoitujen komponenttien oltava mahdollisimman puhtaita. Tämän vuoksi tarvitaan HTP-puhdistusta korkealaatuisten komponenttien ja luotettavan biologisen tiedon takaamiseksi. Jatkuvasti kasvavat tuotantovaatimukset ovat johtaneet näiden puhdistustekniikoiden automatisointiin ja rinnakkaistamiseen. Preparatiivinen LC/MS soveltuu kombinatoristen kirjastojen nopeaan ja tehokkaaseen puhdistamiseen. Monet tekijät, esimerkiksi erotuskolonnin ominaisuudet sekä virtausgradientti, vaikuttavat preparatiivisen LC/MS puhdistusprosessin tehokkuuteen. Nämä parametrit on optimoitava parhaan tuloksen saamiseksi. Tässä työssä tutkittiin emäksisiä komponentteja erilaisissa virtausolosuhteissa. Menetelmä kombinatoristen kirjastojen puhtaustason määrittämiseksi LC/MS-puhdistuksen jälkeen optimoitiin ja määritettiin puhtaus joillekin komponenteille eri kirjastoista ennen puhdistusta.