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categories: method development, biomaterials, high-throughput screening, silk-elastin-like proteins

 

Qin Wang, Xiaoxia Xia, Wenwen Huang, Yinan Lin, Qiaobing Xu,* and David L. Kaplan*

Department of Biomedical Engineering, Tufts University, Massachusetts, USA and State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

Advanced Functional Materials, 2014, in press, doi: 10.1002/adfm.201304106

Highlight by: Dr. Alessandro Poma, The Open University

 

This study describes for the first time the construction and screening of a Silk-Elastin-Like Protein (SELP) library in which different amino acids were tested at the “X” position of the elastin domain (thus obtaining a broader range of functional material properties) and systematically compared in terms of dynamic properties (thermal, electrochemical, mechanical and adhesive) using high-throughput methods. The aim of the authors was to improve the fundamental understanding of the stimuli-responsive properties of silk and elastin, together with the fulfillment of the need for more elastic polymeric systems in many biomaterial applications.

 

Central to the work is the generation of 12 monomeric genes and subsequent engineering of plasmids and transformation to encode SELP polymers in E. coli host cells. The colonies containing plasmids carrying SELP genes in various lengths or empty plasmids were randomly cultured in 96 well plates and the expressed protein polymer library was purified in situ through inverse temperature transition cycling and screened by optical response, thus selecting 64 SELPs from the initial screening of over 2000 colonies. The selected proteins (ranging from 20 to 130 kDa in molecular weight) were further characterized and selected for specific functions by screening for optical, mechanical or adhesive properties in dependence of which amino acid was present at the “X” position of the elastin domain or in dependence of the “elastin-to-silk” ratio in the polymer. The results obtained suggest that the hydrophobicity or hydrophilicity of the amino acid in position X greatly influences the thermal response of the polymer, as well as its adhesive properties, especially for high molecular weights, while its elastic module is more affected by the “elastin-to-silk” ratio.

 

This work directly addresses the lack of diversity of current protein polymers as biomaterials due to the low efficiency of synthesis and limited nature of screening procedures currently used. The combinatorial methods described here can be extended to construct libraries for other protein motifs (e.g., collagens, keratins, binding domains).

 

Recommended reading:

- Peptide-based biopolymers reviews:  (a) “Peptide-based Biopolymers in Biomedicine and Biotechnology” Chow, D.; Nunalee, M.L.; Lim, D.W.; Simnick, A.J.; Chilkoti, A., Mater. Sci. Eng. R. Rep. 2008, 62, 125-155. (b) “Peptide-based fibrous biomaterials: some things old, new and borrowed” Woolfson, D.N.; Ryadnov, M.G., Curr. Opin. Chem. Biol. 2006, 10, 559-567.

- Silk-elastin-like proteins: (a) “Genetic engineering of stimuli-sensitive silkelastin-like protein block copolymers.” Nagarsekar, A.; Crissman, J.; Crissman, M.; Ferrari, F.; Cappello, J.;, Ghandehari, H. Biomacromolecules 2003, 4, 602-607. (b) “A Complete Recombinant Silk-Elastinlike Protein-Based Tissue Scaffold” Qiu, W.; Huang, Y.; Teng, W.; Cohn, C.M.; Cappello, J.; Wu, X., Biomacromolecules 2010, 11, 3219-3227.

categories: catalyst screening, asymmetric synthesis, solid supported chemistry, peptides

Screening Rhodium Metallopeptide Libraries “On Bead”: Asymmetric Cyclopropanation and a Solution to the Enantiomer Problem

Ramya Sambasivan and Zachary Ball*

Rice University Department of Chemistry

Angewandte Chemie, International Edition 2012, 51, 8568-8572, doi: 10.1002/anie.201202512

Highlight by: Dr. Thomas O. Painter, University of Kansas Center for Chemical Methodologies and Library Development

 

  1. In this article, the authors confront the well known “enantiomer problem” that results when naturally occurring compounds are used for asymmetric induction, i.e. one product enantiomer is easily accessible, but the other is not.  The article details a method for quickly generating libraries of peptide-based rhodium(II)-metallopeptides to identify catalysts of a cyclopropanation between simple alkenes and a-diazoesters.

 

To begin, the authors cite their prior work to develop a Rh(II)-metallopeptide catalyst consisting of the metal and two nonapeptide ligands.  This catalyst provides Re selectivity in the cyclopropanation reaction and served as the starting point for the study.  To obtain Si products, peptide ligands derived from D-amino acids could be used, however this strategy is expensive.  In the present paper, the authors took an alternative approach whereby a high-throughput ligand/catalyst screen was carried out.  The screen surveyed the efficacy of a rhodium catalyst featuring a single nonopeptide “on bead” as a faster alternative to solution-phase screening of bis-peptide catalysts, which cannot be assembled on solid support.  Because the monopeptide-derived catalysts are known to give lower enantioselectivities than bis-peptide-derived catalysts, the best ligands found in the screen would be further used in a bis-peptide format to provide optimal selectivity.  The screened nonapeptides consisted of natural amino acid residues and featured carboxyl groups in the i and i+4 positions, which would induce helicity upon metal binding. 

 

Two rounds of iterative library screening identified monopeptide catalysts that provided Si selectivity in ca. 45% ee.  The best peptide ligands were then used in bis-peptide catalysts to produce Si cyclopropanation products in up to 97% ee. Finally, a series of reactions was performed to compare the original ligand that provides the “normal” Re addition products to the newly discovered ligand that provides “opposite” Si enantiomers.

 

The most interesting aspect of this study is the difference in residues found in the two catalyst ligands.  The ligand that provides Re selectivity consists mostly of bulkier residues such as leucine, isoleucine, and phenylalanine at the i-1 and i+3 positions.  In contrast, the ligand that provides good Si selectivity contains residues possessing polar side chains such as glutamine and asparagine in the same positions. The authors suggest that each catalyst system is operating through a different mechanism of asymmetric induction.

 

The power of high-throughput techniques coupled with modular design is showcased well in this article.  An elegant method for tackling the “enantiomer problem” has been demonstrated without recourse to peptides derived from rare D-amino acids.

 

Recommended reading:

- Recent coverage on peptide synthesis:  Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 3: Building Blocks, Catalysis and Coupling Chemistry; Hughes, A. B., WILEY-VCH Verlag & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany, 2011.

- Selected articles on high-throughput catalyst screening: (a) “Evolutionary Catalyst Screening: Iridium-Catalyzed Imine Hydrogenation” Kluwer, A. M.; Detz, R. J.; Abiri, Z.; van der Burg, A. M.; Reek, J. N. H., Adv. Synth. Catal. 2012, 354, 89-95. (b) “From High-Throughput Catalyst Screening to Reaction Optimization: Detailed Investigation of Regioselective Suzuki Coupling of 1,6-Naphthyridone Dichloride” Cai, C.; Chung, J. Y. L.; McWilliams, J. C.; Sun, Y.; Shultz, C. S.; Palucki, M., Org. Proc. Res. Dev. 2007, 11, 328-335. (c) “New Catalysts and Conditions for a C-H Insertion Reaction Identified by High Throughput Catalyst Screening” Burgess, K.; Lim, H. -J.; Porte, A. M.; Sulikowski, G. A., Angew. Chem., Int. Ed. Engl. 1996, 35, 220-222.

-An instructive review on the asymmetric cyclopropanation reaction using rhodium(II)-catalysis: Davies, H. M. L.; Walji, A. M. “Rhodium(II)-Stabilized Carbenoids Containing Both Donor and Acceptor Substituents.”  In Modern Rhodium-Catalyzed Organic Reactions, Evans, P. A., Ed.; WILEY-VCH Verlag & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany, 2005; pp 301-340.

 

 

Yollete V. Guillen Schlippe , Matthew C. T. Hartman , Kristopher Josephson , and Jack W. Szostak *

Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital

Journal of the American Chemical Society 2012, 134 (25) pp 10469-10477, doi:  10.1021/ja301017y

Highlight by: Ms. Zinaida Polonskaya, The Scripps Research Institute

 

Peptide-based drugs have the potential to combine the advantages of small molecule drugs (size, low production costs, ease of storage and handling) with those of biologics (high selectivity and specificity, low toxicity). Large peptide libraries (>106 members) can be screened using molecular evolution techniques, which facilitates lead identification. But until recently peptides have been considered relatively poor drug candidates due to their susceptibility to protease degradation and low bioavailability. While inclusion of unnatural amino acids and cyclization of the peptide backbone can increase both peptide stability and affinity, such modifications are not readily incorporated using standard ribosomal peptide synthesis.

 

The authors of this paper combined two previously reported techniques to perform in vitro selection with unnatural amino acid-containing peptides. They used mRNA display technology to chemically attach peptides to the mRNA that encodes them, effectively linking “genotype” to “phenotype” and enabling selection. To diversify the pool of amino acids the authors applied Protein Synthesis Using Recombinant Elements (PURE) system that reconstitutes translational machinery of the cell in vitro. In the absence of natural substrates some E. coli aminoacyl-tRNA synthetases will charge tRNAs with certain structurally similar unnatural amino acids. 12 out of 20 natural amino acids were replaced with unusual amino acids displaying various functional groups not found in standard proteins, such as alkynes, aryl halides, etc. A DNA library encoding 10 random amino acids flanked by two cysteines (to enable cyclization) was designed, transcribed and translated with unnatural amino acids to yield ~1013 unique members. A control library was made using only natural amino acids. Both libraries were subjected to in vitro selection for members that bind to the protein thrombin. After 7 selection cycles the “natural library” converged on a single motif that was previously found in a similar screen. In contrast, the “unnatural library” yielded a diverse set of winners after 10 cycles, all of them having 4 to 7 unnatural amino acids. 2 representative unnatural peptides had binding affinities of 4.5 and 20 nM, and inhibited thrombin activity at 23 and 35 nM, respectively. These parameters are comparable to those of the natural peptide (affinity of 1.5 nM, inhibition at 6.3 nM), however,  “unnatural” peptides may have other beneficial characteristics, such as improved stability.

 

This work demonstrates that in vitro selection techniques can be successfully applied to highly modified peptides, putting the powerful tools of molecular biology in hands of peptide designers. It is a great first step towards generating more chemically diverse peptides with higher stability and bioavailability and, ultimately, better drug-like properties.

 

Recommended reading:

- mRNA display:  (a) “RNA-peptide fusions for the in vitro selection of peptides and proteins” Roberts R.W.; Szostak, J.W., PNAS 1997, 94(23), 12297-12302. (b) “In-vitro protein evolution by ribosome display and mRNA display” Lipovsek, D.; Plückthun, A., J. Immunol. Meth. 2004, 290, 51-67.

- PURE: (a) “Cell-free translation reconstituted with purified components” Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T., Nat. Biotechnol. 2001, 19, 751 -755. (b) “Ribosomal Synthesis of Unnatural Peptides”  Josephson, K.; Hartman, M. C. T.; Szostak, J. W. J. Am. Chem. Soc. 2005, 127, 11727-11735.

Seong-Wook Yun, Cheryl Leong, Duanting Zhai, Yee Ling Tan, Linda Lim, Xuezhi Bi, Jae-Jung Lee, Han Jo Kim, Nam-Young Kang, Shin Hui Ng, Lawrence W. Stanton, and Young-Tae Chang*

National University of Singapore

Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 10214-10217, doi: 10.1073/pnas.1200817109

Highlight by: Dr. Eunha Kim, Massachusetts General Hospital / Harvard Medical School

 

Molecular imaging is essential tool for current biomedical research fields. Therefore there is explosive increase on demands for the imaging tools and bio-probes for the visualization of cellular function and molecular process in living organism without perturbing their system. Chang and coworkers recently described a discovery of first Neural Stem Cell (NSC)-specific small molecular weight fluorescent chemical compound utilizing combinatorial concept, and they identified the binding target of the compound as intracellular NSC marker Fatty Acid Binding Protein 7 (FABP7) by proteomic analysis. This result clearly shows combinatorial science accelerating the discovery of new novel fluorescent bio-probes.

 

Continuing their excellent ideas of Diversity Oriented Fluorescence Library (DOFL) strategies, emission intensity based high throughput/content screening of 3,160 fluorescent compounds narrows down the candidates and follow-up further validation allows the authors to find a first NSC specific staining fluorescent compound, CDr3 (λex/em = 579/604 nm, εabs = 1.02 × 106M-1 cm-1, Φem = 0.77), without inhibition of proliferation within 48hrs. In addition, subsequent target identification of the compound by 2D-SDS-PAGE and following MALDI-TOF/TOF MS and MS/MS analysis is convinced them that FABP7 is the binding target of CDr3. It is remarkable that they don’t have to modify the compound for target identification because of their unique strategy. Based on the result, they found that CDr3 selectively stained ReNcell VM human NSC line, having 540-fold higher FABP7 expression level than H1 human ESC. Moreover they can isolate the NSC from heterogeneous cell population, generated from random differentiation of ESC, based on staining pattern of the cells with CDr3.

 

This interesting result shows that fluorescent compounds are not the simple twinkling tags for labeling certain molecules in cellular environment but it is also the compounds, which can specifically bind with the cellular biomolecules.

 

Recommended reading:

  - Combinatorial developments of fluorescent library:  (a) “Single-Compound Libraries of Organic Materials: Parallel Synthesis and Screening of Fluorescent Dyes” Schiedel, M. S.; Briehn, C. A.; Bäuerle, P., Angew. Chem., Int. Ed. 2001, 40, 4677-4680. (b) “Combinatorial Approach to Organelle-Targeted Fluorescent Library Based on the Styryl Scaffold” Rosania, G. R.; Lee, J. W.; Ding, L.; Yoon, H. –S.; Chang, Y. –T., J. Am. Chem. Soc. 2003, 125, 1130-1131. (c) “Emission Wavelength Prediction of a Full-Color-Tunable Fluorescent Core Skeleton, 9-Aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one” Kim, E.; Koh, M.; Lim, B. J.; Park, S. B., J. Am. Chem. Soc. 2011, 133, 6642-6649. (d) “Combinatorial Discovery of Fluorescent Pharmacophores by Multicomponent Reactions in Droplet Arrays” Burchak, O. N.; Mugherli, L.; Ostuni, M.; Lacapère, J. J.; Balakirev, M. Y., J. Am. Chem. Soc. 2011, 133, 10058-10061. (e) “Combinatorial Strategies in Fluorescent Probe Development” Vendrell, M.; Zhai, D.; Er, J. C.; Chang, Y. –T. Chem. Rev. 2012, 112, 4391-4420

Miloslav Sailer and Christopher J. Barrett*

McGill University

Macromolecules 2012, 45, 5704-5711, doi: 10.1021/ma300635n

Highlight by: Dr. Julie Albert, North Carolina State University

 

The authors describe a fabrication method for assembling gradient layer-by-layer (LbL) films from weak polyelectrolytes in which the assembly pH of the first component [poly(allylamine hydrochloride) (PAH)] is varied in one direction and the assembly pH of the second component [poly(acrylic acide) (PAA)] is varied orthogonally to the first.  The resultant 2D combinatorial film provides access to thousands of assembly conditions on a single surface. 

 

The key value of this work lies in its application for the first time of a common gradient fabrication technique to LbL assembly.  The authors also complement the method development aspect with an elegant demonstration of the new gradient library as a tool for screening cell viability on LbL surfaces as a function of film assembly conditions.  The LbL film libraries were prepared by pumping the polyelectrolyte stock solution into an initially empty reservoir containing the silicon substrate while simultaneously adding HCl or NaOH to the stock solution to vary the pH.  Thus, as the liquid level in the reservoir rose, the pH of the deposition solution continually increased or decreased.  PAH was deposited first, the sample was rinsed and rotated 90°, and the process was repeated for PAA.  This cycle was repeated up to 5 times to fabricate multilayer films.  The authors also examined gradients in salt concentration by charging the reservoir with a small volume of highly concentrated solution, which was diluted by the addition of the polyelectrolyte solution.  The most noticeable effect of assembly pH on LbL assembly is on the film thickness, though differences in density, surface energy, and modulus properties were also noted.  By mapping the viability of human embryonic kidney cells (HEK 293) on the combinatorial surface, the authors clearly identified the assembly pH conditions that provided optimal cell growth (≈pH 4-6 for PAH and ≈pH 4 for PAA). 

 

The ability to easily generate thousands of LbL assembly conditions on a single sample opens doors not only for combinatorial bioscience, as the authors suggest, but also for in-depth characterization of LbL film properties as a function of assembly conditions (e.g., film thickness, interpenetration of polymer chains, wettability, mechanical properties) and for other applications that would benefit from combinatorial screening approaches, such as optical coatings and anti-fouling surfaces.

 

Recommended reading:

- On layer-by-layer assembly:  (a) “Form and Function in Multilayer Assembly: New Applications at the Nanoscale” Hammond, P. T., Adv. Mater. 2004, 16, 1271-1293. (b) “Biomedical Applications of Layer-by-Layer Assembly: From Biomimetics to Tissue Engineering” Tang, Z.; Wang, P.; Podsiadlo, P.; Kotov, N. A., Adv. Mater. 2006, 18, 3203-3224.

- On polymer surface gradients: (a) “Surface Bound Soft Matter Gradients” Genzer, J. and Bhat, R. R., Langmuir. 2008, 24, 2294-2317.  (b) “Solution and Surface Composition Gradients via Microfluidic Confinement: Fabrication of a Statistical-Copolymer-Brush Composition Gradient” Xu, C.; Barnes, S.E.; Wu, T.; Fischer, D. A.; DeLongchamp, D.M.; Batteas, J.D.; Beers, K. L; Adv. Mater. 2006, 18, 1427-1430.

Patrick S. Fier and John F. Hartwig

University of California

Journal of the American Chemical Society, 2012, 134, 10795-10798.

Highlight by: Jiang Wang, Shanghai Institute of Materia Medica

 

This report describes an operationally simple and high-yielding route to synthesize fluorine-substituted aromatic rings, providing access to molecules that could impact agrochemistry, medicinal chemistry, and other fields. Starting with iodine-substituted aryl rings, the researchers apply a combination of the copper complex ((tBuCN)2CuOTf) and AgF for the synthesize aryl fluoride. Sensitive functional groups, including esters, amides, aldehydes, ketones, and indole heterocycles are stable under the reaction conditions. The researchers also proposed the mechanism of this reaction, as the authors pointed out, “We propose that this reaction occurs by oxidative addition to form a Cu(III) intermediate and C−F reductive elimination from an arylcopper(III) fluoride.

 

Modern fluorine-organic chemistry has dramatically widened the synthetic repertoire for the specific introduction of fluorine into organic molecules. This work improved the method for last-stage aromatic fluorination, so it is very important for diversification in agrochemistry, medicinal chemistry, and other fields. Fluorinated compounds comprise a substantial proportion in the agrochemicals and therapeutic drugs. It is an important strategy to introduce fluorine and fluorinated substitutes in the small molecule for structure-based medicinal chemistry. Fluorine can modulate the physicochemical and pharmacokinetic properties to improve bioavailability, alter the conformation of a molecule to enhance the selectivities and binding affinity to target proteins, and block metabolically labile sites to increase the metabolic stability of drugs. Hartwig and Fier’s new method to a wide range of aryl fluorides may provide a straightforward way of synthesizing new molecules containing fluorine. The wider range of accessible fluorinated compounds will, in turn, allow for further applications in agrochemistry and medicinal chemistry will emerge.

 

Recommended reading:

- C-F Bond Formation in organic synthesis:  (a) “Palladium-catalyzed allylic fluorination.” Hollingworth, C.; Hazari, A.; Hopkinson, M. N.; Tredwell, M.; Benedetto, E.; Huiban, M.; Gee, A. D.; Brown, J. M.; Gouverneur, V., Angew. Chem., Int. Ed. Engl. 2011, 50, 2613-2617. (b) “Silver-catalyzed late-stage fluorination.” Tang, P.; Furuya, T.; Ritter, T., J. Am. Chem. Soc. 2010, 132, 12150-12154.

- Fluorine in medicinal chemistry: (a) “Fluorine in pharmaceuticals: Looking beyond intuition” Müller, K.; Faeh, C.; Diederich, F., Science 2007, 317, 1881-1886. (b) “Fluorine in medicinal chemistry”  Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V., Chem. Soc. Rev. 2008, 37, 320-330. (c) “Fluorine in Medicinal Chemistry” Böhm, H.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Obst-Sander, U.; Stahl, Martin. Chem. Bio. Chem. 2004, 5, 637-643.