96 Matching Annotations
  1. Aug 2021
    1. Z. J. Wang, N. E. Peck, H. Renata, F. H. Arnold, Chem. Sci. 5, 598–601 (2014).

      Professor Arnold's team demonstrates the first enzyme catalyzed carbenoid insertion into N-H bonds. The reaction proceeds in water with moderate yield.

    2. P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Science

      This paper shows how directed evolution can be used to modify existing enzymes to carry out synthetically useful reactions. P450 BM3 enzymes were engineered to catalyze cyclopropanation of styrenes with very high diastereoselectivity and enantioselectivity.

    3. U. T. Bornscheuer et al., Nature 485, 185–194 (2012).

      Bornscheuer discusses the various applications of biocatalysis. Biocatalysts offer a more practical and a green route to synthesis when compared to organometallic catalysts. Professor Arnold's work features the use of biocatalysis as a key step in C-Si bond formation.

    4. Y.-Z. Zhang, S.-F. Zhu, L.-X. Wang, Q.-L. Zhou, Angew. Chem. Int. Ed. 47, 8496–8498 (2008).

      This paper describes how Cu(OTf)2 can be used to catalyze asymmetric carbenoid insertion into a Si-H bond. 22 reactions were run and the product, alpha-silyl esters, was formed in high yields and up to 99% enantiomeric excess. When Professor Arnold's group ran the desired reaction with Cu(OTf)2, a complex mixture of products from Si-H, O-H and N-H insertion reactions was observed.

    5. 10. T. Lee, J. F. Hartwig, Angew. Chem. Int. Ed. 55, 8723–8727 (2016) and references therein.

      This paper discusses the use of rhodium catalysts in the asymmetric, intramolecular silylation reaction of cyclopropyl C-H bonds. The reaction proceeds with high enantiomeric excess. However, when Professor Arnold's group used Rh2(OAc)4 to catalyze C-Si bond formation, they observed that O-H and N-H insertions often dominated over the preferred silylation reactions.

    6. A. K. Franz, S. O. Wilson, J. Med. Chem. 56, 388–405 (2013).

      Silicon, an isostere of carbon, has unique properties. Properties of silicon and application of organosilicon molecules in drug release technology, etc., are discussed. These properties can be used to enhance drug potency and improve pharmacological action.

    7. Indeed, when the same reactants were subjected to rhodium catalysis [1 mol % Rh2(OAc)4], O–H and N–H insertions were the predominant reaction pathways, and copper catalysis [10 mol % Cu(OTf)2] gave complex mixtures of products (table S7).

      Reactions catalyzed by Rma cyt c have distinct advantages such as high chemoselectivity and enantiospecificity over reactions catalyzed by organometallic catalysts.

    8. free alcohols and primary amines

      Free alcohols and primary amines are reactive functional groups that do not interfere with this enzymatic carbene-transfer reaction.

    9. chemoselectivity

      Rma cyt c exhibits preference for carbon-silicon bond formation over other competing side reactions.

    10. site-saturation mutagenesis

      M100 is the specific amino acid residue within the protein sequence that has been identified to be critical for the protein’s function. it is very important to determine the ideal amino acid residue for this position. Site saturation mutagenesis is employed.<br> Site-saturation mutagenesis is a form of random mutagenesis, allowing the substitution of a specific amino acid site with one of 20 possible amino acids in a single experiment. In this study, this technique is employed to generate a series of enzymes with enhanced activity and enantiospecificity.

    11. His

      The amino acid, histidine

    12. We thus chose Rmacyt c as the platform for evolving a carbon–silicon bond-forming enzyme.

      The wild-type protein chosen must exhibit some degree of activity for the desired reaction. When compared to hemin, hemin with bovine serum, a range of cytochrome P450 and myoglobin variants, it was found that Rma cyt c showed 97% ee. This was chosen as the enzyme that would form the starting point for directed evolution.

    13. wild-type Rma cyt c

      Natural variant of cytochrome c protein from the natural variant Rhodothermus marinus.

    14. Carbon–silicon bond formation catalyzed by heme and purified heme proteins.

      Heme proteins that were readily available were screened to identify the one that gave the highest enantioselectivity. This served as a starting point for directed evolution. Purified heme protein, silane, diazo ester, thiosulfate, methyl cyanide and M9-N buffer as the medium for microbial growth were stirred at room temperature in anaerobic conditions. Reactions were performed in triplicate. Unreacted starting material was obtained in all cases and no further purification was carried out.

    15. genetically encoded

      The sequence of nucleotides that is translated into proteins

    16. isostere

      Elements that have the same number of electrons in the outermost shell (also known as valence shell) and have similar electronic properties. For example, carbon and silicon are isosteres as they both have four valence electrons.

    17. turnover

      The turnover number of an enzyme, is the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate.

    18. directed evolution

      IA method of engineering proteins towards a defined property. Process of directed evolution: Directed evolution mimics "real" evolution and is accelerated in the laboratory by focusing on individual genes expressed in fast‐growing microorganisms such as E. coli. Enzyme chosen (known as wild-type) must show at least a minimal desired reactivity. Mutations are randomly or site specifically introduced to the gene of the wild type protein. Then, the library of protein variants is screened for the ones with enhanced reactivity. The improved enzymes are used as parents for the next round of mutation and screening. Additional beneficial mutations are introduced if needed. This can continue for several cycles until a desired and new property of the enzyme is attained.

    19. carbene insertion

      Carbene is a neutral reactive intermediate; a carbene insertion reaction is the insertion of a carbene into a chemical bond.

    20. physiological

      conditions that occur in the natural host organism in contrast to laboratory conditions

  2. Jul 2021
    1. B. D. Levin, K. A. Walsh, K. K. Sullivan, K. L. Bren, S. J. Elliott, Inorg. Chem. 54, 38–46

      The study shows the loss of axial methionine from cyt c. The same phenomenon was observed over a range of cyt orthologs. In Professor Arnold's work, the labile nature of methionine in cyt c is believed to be responsible for the improved efficacy of the C-Si bond forming biocatalyst.

    2. V. Tyagi, R. B. Bonn, R. Fasan, Chem. Sci. 6, 2488–2494 (2015).

      This paper discusses the directed evolution via mutation of the amino acids of myoglobin that lead to 49% enantiomeric excess. Engineered myoglobin catalysts are used to synthesize thioethers via a carbene S-H insertion reaction. Conversions as high as 99% and turnover numbers as high as 5400 were observed.

    3. P. J. O’Brien, D. Herschlag, Chem. Biol. 6, R91–R105 (1999)

      O'Brien and Herschlag discuss the evolution of a superfamily of enzymes and the diverse reactions they catalyze. Single point mutations can lead to the evolution of new enzymatic activities. Professor Arnold's work also involves mutations of the WT to alter the reactivity of the enzyme.

    4. bioorthogonal chemistry

      A new approach to conducting chemical reactions in which reactants must react rapidly and selectively with each other under physiological conditions. Two key and relevant features of bioorthogonal reactions are high selectivity and compatibility with naturally occurring functional groups.

    5. metabolic engineering

      Metabolic engineering is the production of specific target chemicals in high yield and stereoselectivity by altering the metabolic pathways. Metabolic pathways are changed via recombinant DNA technology.

    6. streamlined alternative to transition-metal catalysis

      Although transition metal catalysis offers several advantages, the metals typically used are toxic. Removal of these metals after the synthetic process is time consuming and expensive. The process described in the research ensures sustainability and reduces cost.

    7. functional-group protection and/or manipulation

      Functional group protection and deprotection increases the number of steps and auxiliary agents during synthesis and is not considered green. The methodology described in this research obeys one of the principles of green chemistry: "Unnecessary derivatization (use of blocking groups, protection/deprotection, temporary modification of physical/chemical processes) should be minimized or avoided if possible, because such steps require additional reagents and can generate waste."

    8. Fig. 2 Scope of Rma cyt c V75T M100D M103E-catalyzed carbon–silicon bond formation.

      Rma cyt c V75T M100D M103E shows excellent enantioselectivity and turnover over a wide range of substrates. Silicon substrates with weakly electron-donating or -activating methyl substituents (4), strongly electron donating -OMe (5), weakly deactivating -Cl (6), strongly deactivating -CF3 (7), and moderately deactivating COOMe and CONMe (9 and 10 respectively) show moderate to excellent turnover and high selectivity. No direct relationship exists between turnover number and substituent effects from this study. Enantioselectivity is excellent in all substrates. All products were identified using GC-MS, and no traditional organic chemistry techniques were used.

    9. In addition, diazo compounds other than Me-EDA could be used for carbon–silicon bond formation

      Additional diazo compounds that were successful were R3 = -CH3, -CH2CH3, -Ph.

    10. heteroarenes

      aromatic compounds in which one or more ring carbon atoms are replaced by a heteroatom such as nitrogen, sulfur or oxygen.

    11. amides

      organic compounds that contain a -CONH2 structural feature

    12. esters

      organic compounds that contain a -COOR functional group

    13. alkyl halides

      organic compounds that contain a halogen connected to an alkyl group such as methyl, ethyl, etc.

    14. ethers

      organic compounds that contain a C-O-C structural feature

    15. silicon and diazo reagents

      General method for the preparation of phenyl dimethyl silanes: In a 100 mL round bottom flask, chlorodimethylsilane in THF was cooled to zero degrees. A solution of the appropriate Grignard reagent was added dropwise over ~15 minutes. The reaction was allowed to reach room temperature and stirred for ~8 hours. The product mixture was quenched with ammonium chloride solution. The product was extracted into ether and then isolated. Purification was done by column chromatography.

    16. Relative to the wild-type protein, the evolved triple mutant catalyzes the reaction more than seven times faster, with turnover frequency (TOF) of 46 min–1 (Fig. 1E).

      Via site-saturation mutagenesis, V75 and M103 positions along the protein sequence were identified as likely beneficial mutations and were randomized, i.e., the amino acids at these positions are replaced by random ones. A large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. The evolved triple mutant fits the bill.

    17. a 12-fold improvement over the wild-type protein (Fig. 1D).

      Recombinant protein is a protein encoded by a gene that has been cloned in a system that supports expression of the gene (in this case, it is M100). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. In this study, M100D mutation is more highly activating than the wild-type protein.

    18. anaerobic

      oxygen-free conditions

    19. recombinantly expressed in Escherichia coli.

      At the theoretical level, the steps needed for obtaining a recombinant protein are straightforward. Take your gene of interest, clone it, transform it into the host of choice (here it is E. coli), induce, and then the protein is ready for purification and characterization.

    20. distal

      located at a farther distance

    21. eukaryotic

      cells with membrane-bound organelles

    22. Carbon–silicon bond forming rates over four generations of Rma cyt c.

      Turnover frequency for each variant relative to wild-type protein: WT: 1 M100D 2.8 +/- 0.2 V75T M100D 4.6 +/- 0.3 V75T M100D M103E 7.1 +/- 0.4

      From this experimental data, it is clear that directed evolution has resulted from changing the enzyme from unselective wild-type into a highly enantioselective variant.

    23. using lysates of E. coli expressing Rma cyt c

      Experiments were conducted with purified heme protein, silane, diazo ester, sodium dithionate, MeCN, and M9-N buffer at room temperature in anaerobic conditions for 1.5 h. Three trials were conducted. Turnover number reported is the average of the three trials. Unreacted reactants were not isolated.

    24. “Active site” structure of wild-type Rma cyt c showing a covalently bound heme cofactor ligated by axial ligands H49 and M100. Amino acid residues M100, V75, and M103 residing close to the heme iron were subjected to site-saturation mutagenesis.

      Axial methionine in cyt c is known to be labile. The proposed model for the binding for the iron-silane complex is one where the complex forms such that the silane molecule takes the place of the axial methionine. The silane may approach from the more exposed side in the protein. This further explains the observed stereochemistry of the organosilicon product. The V75T, M100D, and M103E mutations are thought to improve reactivity by providing better access of the substrate to the iron center.

    25. 22

      In this study, Professor Arnold's group used protein-engineered variants of cytochrome P450 BM3 to bring about highly diastereoselective and enantioselective cyclopropanation reaction of styrenes from diazoester. Variant BM3-CIS was identified as a competent cyclopropanation catalyst. It exhibits a strong preference for the cis product and forms both diastereomers over 90% ee and is as stable as the wild-type enzyme. P450 BM3 works on a wide range of substrates with both electron-donating and electron-withdrawing substituents in styrene.

    26. molecular biology

      branch of biology that deals with the structure and function of nucleic acids and proteins

    27. selectivity

      The preference shown by an enzyme when exposed to a competitive attack on two or more substrates or two or more positions in the same substrate.

    28. specificity

      The ability of a protein's binding site to bind to only specific ligands. The fewer ligands a protein can bind to, the greater its specificity.

    29. halogenated solvents

      Solvents that contain halogens such as fluorine, chlorine, bromine and iodine. For example, methylene chloride, CH2Cl2, is a halogenated solvent.

    30. Synthetic methodologies such as carbene insertion into silanes can be rendered enantioselective using chiral transition metal complexes based on rhodium (11, 12), iridium (13), and copper (14, 15).

      Certain iridium catalytic systems show 97% ee, while copper catalysts show 35% ee and rhodium has been shown to exhibit 77% ee.

    31. Rhodothermus marinus

      gram-negative, rod-shaped bacterium

    32. cytochrome

      Cytochromes are proteins that contain heme as the prosthetic group.

    33. heme proteins

      A type of metalloprotein that contains a heme group, which is required for the functionality of the protein

  3. Jun 2021
    1. silicon can also be used to optimize and repurpose the pharmaceutical properties of bioactive molecules

      AP Essential Knowledge Enduring Understanding 1.C

    2. A. M. Tondreau et al., Science 335, 567–570 (2012).

      Iron compounds are found abundantly in nature and are cheap. The paper discusses the use of iron complexes as catalysts to add Si-H (hydrosilylation) across alkene double bonds. The catalysis proceeds with high regioselectivity, thereby eliminating the need for product purification.

    3. J. G. Kleingardner, K. L. Bren, Acc. Chem. Res. 48, 1845–1852 (2015).

      The article presents a wealth of information about heme c and cytochrome c. This knowledge is essential for engineering enzymes to catalyze novel reactions.

    4. E. Scharrer, M. Brookhart, J. Organomet. Chem. 497, 61–71 (1995).

      Iron carbene complexes react with organosilanes leading to insertion of carbene in the Si-H bond. This is another example of the use of organometallic catalyst to carry out the insertion reaction.

    5. M. Stelter et al., Biochemistry 47, 11953–11963 (2008)

      This paper presents the crystal structure of cyt c. X-ray crystallography shows that cytochrome c consists of alpha helices wrapped around the compact heme core. In addition, there is a singular alpha helix that wraps around the back of the molecule.

    6. The crystal structure of wild-type Rma cyt c

      AP Chemistry Science Practice 1: The student can use representations and models to communicate scientific phenomena and solve scientific problems.

    7. Reactions performed in triplicate.

      AP Chemistry Science Practice 4: The student can plan and implement data collection strategies in relation to a particular scientific question.

    8. carbene insertion

      Common Core ELA - Literacy RST 11.12.4 Determine the meaning of symbols, key terms, and other domain-specific words and phrases as they are used in a specific scientific or technical context relevant to grades

    9. enzymatic carbon–silicon bond formation

      Common Core ELA Literacy RST 11.12.10 By the end of grade 12, read and comprehend science/technical texts in the grades 11-CCR text complexity band independently and proficiently.

    10. accelerate chemical transformations

      AP Chemistry Essential Knowledge 4D: Reaction rates may be increased by the presence of a catalyst.

    11. G. A. Showell, J. S. Mills, Drug Discov. Today 8, 551–556 (2003).

      This paper describes how silicon isosteres can be critical to drug discovery success. Professor Arnold's paper uses an approach whose benefits can be applied to drug design.

    12. A. A. Toutov et al., Nature 518, 80–84 (2015).

      This paper describes how potassium tert-butoxide can catalyze the silylation of C-H bonds in aromatic heterocycles. The reaction is one step, occurs under mild conditions and is scalable to ~100 g. This methodology replaces the expensive route of using Rh or Ir complexes.

    13. racemic

      equal amounts of enantiomers (mirror images) of a chiral (asymmetric) compound

    14. These in vitro and in vivo examples of carbon–silicon bond formation using an enzyme and Earth-abundant iron affirm the notion that nature’s protein repertoire is highly evolvable and poised for adaptation:

      Rma cyt c V75T M100D M103E variant is not the most evolved protein. This is considered only as a starting point to future enzymes that will show greater selectivity.

    15. 98% ee

      Enantiomeric excess was determined using chiral SFC.

    16. Product distribution was quantified after 2 hours of reaction time

      Products were analyzed using gas chromatography.

    17. Chemoselectivity and in vivo activity of evolved Rma cyt c.

      With each mutation, the chemoselectivity of the enzyme is greatly enhanced. V75T M100D M103E favors the carbon-silicon bond 29 times more than the wild type. This is attributed to the improved binding and orientation of the silicon donor in the enzyme's active site.

    18. coordinatively labile

      Electrochemical analysis revealed the loss of methionine from a range of cytochrome proteins.

    19. cytochrome P450 and myoglobin

      types of heme proteins

    20. enantiomeric excess (ee)

      excess of one enantiomer over the other in a mixture of enantiomers

    21. No product formation was observed in the absence of heme,

      Heme proteins are required for the reaction to occur.

    22. M9-N minimal medium

      a microbial growth medium

    23. heme proteins

      A very large class of proteins that contain heme as the prosthetic group. Examples of heme proteins are hemoglobin, myoglobin and cytochrome c.

  4. Jul 2019
    1. directed evolution
    2. downstream processing

      Refers to the process of separating desired products from biosynthetic pathways

    3. chiral reagents

      Any reagent that exhibits chirality (or asymmetry) in its molecular structure

    4. Evolution, natural or in the laboratory, can use these promiscuous functions to generate catalytic novelty
    5. Such enzymes would expand the catalytic repertoire of biology
    6. the triple mutant catalyzed the formation of 20 silicon-containing products,

      A generally applicable protocol, as a result of directed evolution, over a variety of substrates illustrates the synthetic utility of the mutant.

    7. naphthalenes

      compounds that contain two fused benzene rings; also referred to as polycyclic aromatic hydrocarbon

    8. aryl halides

      organic compounds that contain a halogen connected to a benzene ring

    9. adventitious “active site”

      an active site created by chance rather than by design

    10. Met

      The amnio acid, methionine

    11. proximal

      located at a closer distance

    12. hydrophobic

      repels or has no affinity towards water

    13. functionally conserved

      relatively unchanged when one goes back in genealogical time

    14. thermohalophilic

      An organism that thrives in extreme high temperature and high salt concentrations

    15. enantioinduction

      Enantioinduction is also popularly known as asymmetric induction. This process is the preferential formation of one enantiomer over the other as a result of the influence of a chiral feature present in reactants or the catalyst.

    16. biocompatible

      not harmful to living cells

    17. organosilicon

      Compounds that contain carbon-silicon bonds

    18. enantiopure

      A compound available in one enantiomeric form

    19. chemo- and enantioselectivity

      Chemoselectivity is the preferential reaction of a reagent with a specific functional group over others. Enantioselectivity is the extent to which one enantiomer is formed over the other in a chemical reaction.

    20. catalyze

      speed up a reaction with the use of external agent, typically a chemical compound