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WO2011038259A1 - Glycosyle hydrolase synthétique à base de nano-armures d'adn - Google Patents

Glycosyle hydrolase synthétique à base de nano-armures d'adn Download PDF

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Publication number
WO2011038259A1
WO2011038259A1 PCT/US2010/050246 US2010050246W WO2011038259A1 WO 2011038259 A1 WO2011038259 A1 WO 2011038259A1 US 2010050246 W US2010050246 W US 2010050246W WO 2011038259 A1 WO2011038259 A1 WO 2011038259A1
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Prior art keywords
artificial
glycosyl hydrolase
hydrolase enzyme
acid
dna
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PCT/US2010/050246
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English (en)
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Peter Mikochik
Aviad Cahana
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Incitor Incorporated
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Publication of WO2011038259A1 publication Critical patent/WO2011038259A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01074—Glucan 1,4-beta-glucosidase (3.2.1.74)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)

Definitions

  • the present invention concerns DNA-based nanostructures that display hydrolytic activity against oligosaccharide polymers.
  • All complex carbohydrates are comprised of repeating saccharide units; the nature of these monomers, as well as their relative connectivity, determines the overall chemical stability and function of the polysaccharide or oligosaccharide.
  • Cellulose for example, consists exclusively of D- Glucose monomers bearing ⁇ -1,4 linkages, which render it extremely stable to environmental stresses due to the uniform hydrogen bonding network (see Fig. 1).
  • hemicellulose is heterogeneously comprised of glucose, mannose, and galactose, and the relative linkages between the monomer units vary considerably. This randomness renders the oligomer susceptible to hydrolysis under mild conditions.
  • Other saccharide constructs such as pectin and glycogen vary in their monomer constitutions, connectivities, and biological functions.
  • the amino acids are 5.5A apart, and it is the conjugate base which intercepts the oxocarbenium ion.
  • This glycosyl acetate adduct is highly susceptible to hydrolysis, and does so under a S n 2-like mechanism to afford the ⁇ -product.
  • the glycosidic bond is also susceptible to hydrolysis by a number of organic and mineral acids, however many of these acids lead to substrate dehydration, rearrangement or degradation, and furthermore present issues regarding recyclability.
  • the thiourea group is related to these acids in terms of hydrogen bonding ability, and is also able to cleave glycosidic bonds in a manner similar to that of acid catalysts (Fig. 3).
  • 1,2:5, 6-di-isopropylidene glucose can be deketalized in a 0.8M aqueous thiourea sol ution (Fig. 4).
  • Enzymes based on oligonucleotides include catalytic NA or ribozymes, which are essential for the production of proteins, and catalytic DNA, which regulates the hydrolysis of RNA.
  • the present invention can be constructed on a matrix comprising a multitude of biopolymeric helical bundles. These bundles are comprised of com puter designed, custom synthesized single stranded DNA molecules, which will anneal in solution according to Watson-Crick base pairing.
  • the phenomenon of helical cross linking is well known to those in the art of DNA weaving or DNA origami, and originated with the discovery of the Holliday Junction quadruplex (See Holliday, Genet Res 5, 282 (2005)). For instance, if four single strands (one half of a double helix) of DNA with the following sequences were placed in solution, they would naturally form a cross-link between two helices: Strand 1: CCACCTTTTCAGCTCGCGCCCCAAAT
  • the present invention concerns the synthesis of an artificial glycosyl hydrolase enzyme, comprising an active site capable of bifunctional catalysis, which has been covalently bonded to a rigid organic matrix through the use of a tethering construct.
  • the active site of this enzyme can comprise natural amino acids, unnatural amino acids, which incl ude enantiomers, disastereomers, or homologues of a natural amino acid, or peptidomimetic amino acids that contain a catalytic moiety typically found on an amino acid, including carboxylic acid, hydroxyl, thiol, amine, amide, guanidinyl, imidazoyl, selenyl, or aryl.
  • the rigid organic matrix of this artificial enzyme comprises a singularity or plurality of biopolymeric matrices, including deoxyribonucleic acid, ribonucleic acid, locked nucleic acid, morpholino nucleic acid, or peptide nucleic acid helices or helical bundles.
  • the unique chemical function can comprise glycosidic bond hydrolysis using a bifunctional catalytic mechanism.
  • Fig. 1 shows exemplary amino acids typically found in the active sites of all glycosyl hydrolases, along with typical polysaccharide bonding motifs.
  • Fig. 2 shows the "inversion” and “retention” mechanisms of hydrolysis, along with requisite intermolecular distances.
  • Fig. 3 shows exemplary thiourea catalysts, including a chiral and resin bound variants.
  • Fig. 4 shows exemplary synthetic mechanisms and methods for glycosyl bond modifications using thiourea catalysis.
  • Fig. 5a illustrates the interaction of four semi-complementary single strands of DNA to create a "Holliday Junction" (See Holliday, Genet Res 5, 282 (2005)).
  • Fig. 5b illustrates a ball-and-stick model of a Holliday Junction.
  • Fig. 6 shows an exemplary catalytic system attached to a DNA helix using an acridine intercalator. This system was effective in the cycloaddition reaction between cyclopentadiene and chalcone (See Roelfes and Feringa, Angew Chem Int Ed 44, 3230 (2005)).
  • Fig. 7 shows an exemplary catalytic system wherein DNA cleavage could be affected by a chemically modified DNA strand containing a lysine and histidine peptidomimetic (See May et al., J Am Chem Soc 126, 4145, (2004)).
  • Fig. 8 shows an exemplary tetra-helical DNA construct, containing 6 aspartic acid pairings which are covalently attached to helices 1 and 4.
  • Fig. 9 shows a space-filling model of an artificial enzyme for use in the hydrolysis of glycosidic bonds.
  • Fig. 10 shows an exemplary synthetic representation of the attachment of a multiplicity of thiourea groups onto a single modified nucleotide base using a dendrimer-like approach.
  • Fig. 11 shows an exemplary multihelical thiourea-based artificial enzyme.
  • Fig. 12 shows an exemplary synthetic "click" reaction between an alkyne-containing thymidine derivative, with an azido-acid molecule to generate a peptidomimetic aspartic acid residue for use in hydrolysis.
  • Fig. 13 shows the synthetic route for the production of the preferred phosphoramidite base for use in solid phase DNA synthesis to produce the synthetic enzyme.
  • Fig. 14 shows an analytic gel electrophoresis image showing successive additions of single strands to the growing structure.
  • the second lane from the right corresponds to the addition of all of the DNA strands, to produce a single band, with no indication of unannealed strands below it.
  • Fig. 15 is a schematic illustration of enzymatic hydrolysis of glycosidic bonds.
  • Fig. 16 is a schematic illustration of an inversion mechanism and a retention mechanism.
  • Fig. 17 is a schematic view of an example bionanolattice according to the present invention.
  • Fig. 18 is a schematic illustration of an example application of a target molecule according to the present invention.
  • Fig. 19 is a schematic illustration of properties of thiourea. Illustrated in Fig. 19: 1: unfunctionalized thiourea; 2: a highly-reactivity thiourea with two 3,5-bis-trifluoromethylphenyl groups; 3: a bi- functional thiourea catalyst capable of generating high levels of enantioselectivity in the Michael reaction ; 4: a thiourea catalyst bound to a polystyrene resin.
  • Fig. 20 is a schematic illustration of bonding properties of thiourea.
  • Fig. 21 is a schematic illustration of the front view of a bionanolattice according to the present invention.
  • Fig. 22 is a schematic illustration of the top view of a bionanolattice according to the present invention.
  • Fig. 23 is a schematic illustration of the use of attached thiourea moieties to serve as a binding site for glycosidic polymers containing uronic acid derivatives.
  • Fig. 24 is a schematic illustration of the chemical synthesis of a target compound.
  • Fig. 25 is a depiction of a multi-helical protein that spans the cellular membrane.
  • Fig. 26 is a depiction of an example six-helical DNA synthetic ion channel selective for positive ions.
  • the molecules in blue represent compounds capable of hydrophobic interactions, of which a few examples are shown.
  • the central red crown ether represents the ionophilic interior to facilitate ion transport.
  • Fig. 27 is a depiction of an example six-helical DNA synthetic ion channel selective for negative ions.
  • Fig. 28 is a depiction of an example of a dipole-based ion gated channel, beginning from crown ether (neutral) to a carboxylate (mono-anionic) to a phosphate (di-anionic).
  • Fig. 29 is a depiction of an example dipole-based ion gated channel, representing an increase in local concentrations of carboxylate residues.
  • Fig. 30 is a depiction of a representative monomer unit of the creation of a functional DNA bionanolattice synthetic ion pore.
  • an artificial glycosyl hydrolase can meet the requirement of having two carboxylic acid residues, either glutamic acid, aspartic acid, or mimic of such, at specified intermolecular distances.
  • a distance of about 10A is required for the "inversion mechanism” of hydrolysis, allowing for both a water molecule and the substrate to be contained in the active site.
  • An effective distance of 5.5A between these residues allows for the "retention mechanism” of hydrolysis, which is seen in other members of this enzymatic family.
  • the appropriate intermolecular distance between the active catalytic residues can be between 2-20A, with 5.5A the preferred distance in some embodiments.
  • An example of an artificial glycosyl hydrolase that is capable of this requirement comprises the utilization of a singularity or plurality of individual DNA single strands, the sequences of which have been engineered such that annealing in solution will generate a rigid construct based on Watson-Crick base pairing tenets.
  • An embodiment of this invention includes hairpin loops of DNA that will generate pockets into which catalytic residues can be inserted, such as in the following sequence:
  • Another embodiment of the present invention can also comprise the utilization of several semi-complementary single DNA strands, such that when annealed in solution will generate a complex three dimensional shape, drawing on techniques concerning DNA weaving or DNA origami.
  • Cross links between DNA helices can be designed pre-synthesis through computer aided design programs, such as Tiamat.
  • the development of three dimensional shapes can arise from helical torque phenomena, using the assumption that a full DNA helical turn requires approximately 10.5 base pairs. In this way, it is possible to design a trough-like or barrel-like design through minimal helical domains.
  • An example of an artificial glycosyl hydrolase that is capable of this requirement can also comprise the use of /V-phenyl thiourea moieties, which have been covalently attached onto a multiplicity of DNA helices.
  • /V-phenyl thiourea moieties which have been covalently attached onto a multiplicity of DNA helices.
  • These thioureas can also decorate the exterior of a multi-helical barrel construct, or the periphery of a single DNA duplex (Fig. 11).
  • RNA ribonucleic acids
  • LNA locked nucleic acids
  • PNA peptide nucleic acids
  • morpholino- base nucleic acids although for this embodiment the deoxyribonucleic acid bases (A, C, T, G) are preferred.
  • the modified "X" base refers to any of the above nucleobases, which have been modified using methods known in the art of organic chemistry.
  • the aspartic acid - like mimetic molecule can be covalently attached to any point on these nucleobases, including the phosphate, the ribose, the deoxyribose, the locked ribose bicycle, the morpholino ring positions, or the nucleobase heterocycle or other pendant groups, using etherification, esterification, amidation, amination, alkylation, cycloaddition, cross-coupling, or other appropriate reactions.
  • An example embodiment can include the halogenation of thymidine at the 5' position, followed by azide displacement and reduction to afford aminomethyl thymidine.
  • the amine group can serve as a handle for subsequent manipulations.
  • halogen sources include bromine, chlorine, iodine, N-bromosuccinimide, N-chlorosuccinimide, or N-iodosuccinimide, or other electrophilic halogen sources, with N-bromosuccinimide being the preferred method in some embodiments.
  • halogenation of uracil is followed by Sonagashira cross- coupling with a copper acetylide to form an alkynyl uracil derivative.
  • halogen sources include bromine, chlorine, iodine, N-bromosuccinimide, N-chlorosuccinimide, or N-iodosuccinimide, or other electrophilic halogen sources, with iodine being the preferred method in some embodiments.
  • the method includes the hydroxymethylenation of uracil with a hydroxymethylene precursor, such as formaldehyde, paraformaldehyde, or 1,3,5-trioxane, with paraformaldehyde as the preferred method in some embodiments.
  • a hydroxymethylene precursor such as formaldehyde, paraformaldehyde, or 1,3,5-trioxane
  • the present invention would include the halogenation of adenosine, cytosine, or guanosine at positions known to be nucleophilic, by electrophilic halogen sources such as bromine, chlorine, iodine, N-bromosuccinimide, N-chlorosuccinimide, or N- iodosuccinimide, with N-bromosuccinimide being the preferred method.
  • electrophilic halogen sources such as bromine, chlorine, iodine, N-bromosuccinimide, N-chlorosuccinimide, or N- iodosuccinimide, with N-bromosuccinimide being the preferred method.
  • the method includes attachment of the linker to the 5'- phosphate at the conclusion of solid phase DNA synthesis.
  • the tethering construct can comprise any organic chain of known length that serves to covalently attach the peptidomimetic group to the biopolymeric matrix.
  • This chain can be designed to project the catalytic group into a specific location, and can be comprised of carbon, oxygen, sulfur or nitrogen, in combinations typically found in bioconjugate constructs, such as (poly)ethylene glycol (PEG), ketal, triazole, dithiol, phthalimide, maleimide or alkyl.
  • the preferred tethering constructs can be designed to project carboxylic acid residues such that the relative spacing between two acid residues is approximately 5.5A. These distances can also be varied to achieve distances between 2 - 20A.
  • the present invention can make use of a maleimide-containing molecule that is combined with a thiolized fragment through a conjugate addition reaction, to generate the polymer-tether- catalytic residue construct.
  • the present invention can also include a copper-catalyzed [3+2] dipolar cycloaddition between a terminal alkyne and an azide fragment to form a triazole attachment point (Fig. 12).
  • the exemplary reactions known to those in the art as "bioconjugate reactions" can be performed before or after the DNA synthesis or DNA weaving procedures, as these are known to involve minimal byproducts and very mild reaction conditions.
  • the copper-catalyzed [3+2] dipolar cycloaddition is the preferred method of attachment of the carboxylic acid group; wherein the copper catalyst can include copper (II) sulfate, copper (I) iodide, copper (I) bromide, copper (I) chloride, copper (I) acetate or copper metal, with the preferred method being copper (I) bromide; wherein the preferred stabilizing ligand is TBTA, Tris[(l-benzyl-lH-l,2,3-triazol-4-yl)methyl]amine; wherein the stoichiometric oxidizing agent is ascorbic acid or any salt thereof, or atmospheric oxygen, with the preferred method being sodium ascorbate.
  • the copper catalyst can include copper (II) sulfate, copper (I) iodide, copper (I) bromide, copper (I) chloride, copper (I) acetate or copper metal, with the preferred method being copper (I) bromide; wherein
  • the phosphoramidite was incorporated into twelve positions labeled with an X, as shown above using an Expedite Nucleic Acid Synthesis System.
  • the resin was heated to 65 Q C in concentrated NH 4 OH for 15h, followed by removal of volatile components and purified by gel electrophoresis.
  • DNA solution concentrations were quantified by absorbance analysis by a Nanodrop spectrophotomer. Equimolar DNA solutions were annealed by variable temperature step gradients, and gel electrophoresis showed the production of a single band in the appropriate lane ( Figure 14).
  • Example Embodiments relate to synthesis of enzymes, and the use of enzymes, in various applications. Several representative applications are described; those skilled in the art will appreciate variations within the scope of the described invention(s), and other applications that are also within the scope of the described inventions.
  • PCE Perchloroethylene
  • Trichloroethylene TCE
  • PCE and TCE pose such a prevalent problem are both migrate quickly through the unsaturated soil in the form of dense non-aqueous phase liquid (DNAPL) and pool on top of a confining layer.
  • DNAPL dense non-aqueous phase liquid
  • PCE and TCE serve as a long-term (hundreds of years) source for the contamination of drinking water. Due to the extremely high volatility of PCE, pooled PCE in soil will partition into the soil vapor and migrate upwards into residential facilities, degrading air quality and posing serious health hazards in large areas around where leaks had occurred.
  • PCE and TCE can be divided into three types: physical, chemical and biological.
  • Physical approaches such as pump-and-treat and soil-vapor-extraction have failed in capturing the majority of the contaminated plume.
  • In-situ chemical remediation Ozonation, permeable reactive barrier, etc.
  • Ozonation, permeable reactive barrier, etc. are usually very expensive and fail to achieve cost-effective results within a reasonable timeframe.
  • One promising technology is biological. Biodegradation mechanisms of PCE have been studied and defined for a variety of organisms. Bacteria use specific enzymes to degrade both PCE and TCE (TCE is manufactured artificially and is also a daughter product of PCE).
  • bacteria that can degrade PCE and TCE are highly susceptible to environmental conditions such as O P, DO, pH, electron donors and acceptors, nutrients, bacteria scavengers, etc.
  • the large size of the bacteria prevents it from accessing many of the small pores that are saturated with PCE in the soil, hence compromising the activity of this technology.
  • Another disadvantage of the biological process is that vinyl chloride (VC), a potent toxic molecule, is produced as part of the degradation pathway. If for any reason the biological degradation pathway is not complete, water and air quality will be reduced even further.
  • VC vinyl chloride
  • the present example embodiments can provide a novel, cost-effective nanotechnology for treating both PCE and TCE in situ. Rapid degradation of PCE and TCE to Ethylene, a non-toxic organic compound, can be achieved by immobilizing two enzymes (tetrachloroethene reductive dehydrogenase and trichloroethene reductive dehydrogenase) onto a well-defined nanostructure. Once nanostructure concept has been established, a completely synthetic version of it can be built, utilizing only the active sites mechanisms from both enzymes. This synthetic enzymatic catalytic pathway can degrade PCE and TCE efficiently, while avoiding all of the problems that are associated with bacteria, including the production of VC.
  • bi-functional chemical linkers can be attached to the two enzymes, and the enzymes linked to the nanostructure.
  • the degradation rates of PCE and TCE as the starting substrate can be determined, and compared to the literature.
  • the enzymatic active sites can be replaced with computer modeled synthetic counterparts, either both at once, or one at a time. Displaying high structural rigidity, this complex structure can show enhanced durability as compared to the multi-enzyme counterpart.
  • This example embodiment involves a metal-based synthetic enzyme for the generation of substituted phenols from lignin.
  • Lignin comprises approximately 20% of the world's biomass, though its practical application towards the fuel or specialty chemicals industry has been limited due to its unusual chemical bonding motifs. Depolymerization of this material using enzymatic methods has proven difficult, though 48-60% of the cross-linking bonds are benzylic ethers. Irreversible cleavage of this bond can be affected through transfer hydrogenation with formic acid, however this requires exceptionally high temperatures. State of the art organic methods typically utilize palladium, either on charcoal or substituted with ligands, as a catalyst for the cleavage of this bond such that it can be run at ambient temperatures. This example embodiment generates a synthetic enzyme in which palladium has been immobilized by surface displayed thiourea ligands.
  • this enzyme will be facile, and leaching of palladium into solution can be minimal.
  • this synthetic enzyme can be robust concerning pH, salinity, and temperature in comparison to known lignases.
  • palladium, an appropriate hydrogen source pressurized hydrogen gas, formic acid, cyclohexa-l,4-diene
  • crude lignin, in solution are reacted in a polar solvent.
  • This reaction can be monitored by GC-MS to observe the formation of phenol products.
  • the reaction rates at ambient temperatures in this catalytic system can be compared to the non-catalytic literature reaction.
  • a synthetic enzyme can be constructed such that thiourea ligands are displayed on the surface in a square formation. This structure can be incubated with palladium to introduce the metal cofactor onto the solid support. In analogy to the first example embodiment, the same reaction can be run with the synthetic enzyme to determine catalytic efficiency.
  • An effect of this example is the immobilization of this array of enzymes onto a well-defined nanostructure, to create a supramolecular multi-enzyme. Covalently linking these enzymes to a matrix will aid in their recovery from fermentation mixtures, and allow for their reuse.
  • This multi- enzyme can be applicable to a wide range of cellulosic sources, and its efficiency of hydrogen production can be compared to those found in literature sources.
  • bi-functional chemical linkers are attached to the necessary enzymes, and these are linked to the nanostructure.
  • the yield of evolved hydrogen based on the digestion of cellobiose as the starting substrate can be determined, and yield and rate compared to the literature source.
  • the enzymatic active sites can be replaced with computer modeled synthetic counterparts, either both at once, or one at a time. Displaying high structural rigidity, this complex structure can provide enhanced durability as compared to the multi-enzyme counterpart.
  • Example Embodiments The enzymatic hydrolysis of glycosidic bonds follows one of two operative mechanisms, with the amino acid residues actively involved in this process being highly conserved across all families. Situated directly above and below the anomeric carbon center, two carboxylic acid bearing amino acids, glutamic acid (Glu) or aspartic acid (Asp, Fig. 15), have been proven to operate in concert by a general acid - nucleophile/base mechanism. Within this mechanism, either “inversion” or “retention” can be in operation, and these sub-mechanisms can be determined based on the relative stereochemistry of the resultant saccharide, or else based on the relative distance between the two active amino acids in the active site.
  • Glu glutamic acid
  • Asp aspartic acid
  • the amino acids are 5.5A apart, and it is the conjugate base which intercepts the oxocarbenium ion.
  • This glycosyl acetate adduct is highly susceptible to hydrolysis, and does so under a S n 2-like mechanism to afford the ⁇ -product.
  • the present examples contemplate mimicking the function of glycosyl hydrolase enzymes by incorporating peptidomimetic constructs onto multi-helical DNA bionanolattices.
  • These glutamic acid / aspartic acid surrogates can be positioned to a high degree of precision in space through linkages to modified thymidine derivatives comprising the DNA helical backbone of the lattice.
  • These DNA helices can be ⁇ 63 base pairs in length, and assuming 10.5 base pairs per turn, up to 6 active sites can be displayed per tetrameric bionanolattice (Fig. 17).
  • the target molecule in Fig. 18 contains the necessary covalent linker to position two carboxylic acid units approximately 5.5A apart, when placed in the opposed 1 and 4 helices. These calculations are based on the fact that both the effective trough distance and the width of a DNA helix will be 30A. By placing these surrogates at this distance, the present examples can provide the ability to mimic the function of the retention mechanism. The present examples also contemplate a molecule containing spaces to separate two carboxylic acid groups at 10A, thus simulating the inversion mechanism. Disproportionation into a carboxylic acid - conjugate base pair on the lattice can be accomplished by adjusting the solution pH to approximately 5.5.
  • This monomer can be divided into three subsections: the nucleobase (green), the linker (red), and the amino acid surrogate (blue).
  • the chemical synthesis of this compound can take advantage of disconnections between the amide bonds.
  • the green and red retrons shown in Fig. 18 can be covalently linked via standard peptide coupling conditions (EDCI, HOBt), and the red and blue retrons can be condensed under ambient conditions owing to the reactivity of succinic anhydride (blue).
  • EDCI, HOBt standard peptide coupling conditions
  • HOBt succinic anhydride
  • Using a PEG (poly-ethylene glycol) linker (red) was chosen due to ease of synthesis and ubiquity in the literature, though other covalent linkers containing other atoms would be amenable.
  • Example Embodiments Every naturally occurring cellulase operates via a general acid / nucleophilic mechanism.
  • the exocyclic glycosidic oxygen atom is protonated by a glutamic or aspartic acid, thus weakening the C-0 bond and facilitating cleavage.
  • the two major mechanisms termed “inversion” and “retention,” diverge after this step, but both involve interception of the oxocarbenium intermediate by a proximal aspartate or glutamate residue and subsequent hydrolysis to produce a shortened cellulose polymer.
  • the present examples provide a non-peptidomimetic approach to cellulose hydrolysis using thiourea catalysis.
  • a second example is put forth by Kotke and Shreiner, who were able to show the converse; namely the ketalization of free alcohols with dihydropyran (DHP) under anhydrous conditions.
  • DHP dihydropyran
  • ketalization is an equilibrium process which is greatly affected by the presence of exogenous water, it can be assumed that the former reaction would not proceed unless water was vigorously excluded, and indeed their experimental shows the use of oven dried glassware in which the reaction was performed. It can also be inferred that the inclusion of water after the ketalization step would most likely affect the reverse reaction.
  • the present examples can provide the ability to mimic the function of traditional aspartate- based glycosyl hydrolase enzymes by incorporating thiourea catalysts onto DNA bionanolattices (Fig. 21 and Fig. 22).
  • the manipulation of DNA into various two and three dimensional shapes has been pioneered by the work of Seeman and Shih, though the application of these constructs towards practical purposes has yet to be disclosed.
  • the present examples can use multi-helical bundles which are capable of creating clefts or pockets in which catalytic activity could take place, in analogy to enzymatic active sites. These helices can be interlocked using shorter "staple strands" of DNA, in analogy to the "DNA Origami” approach by otheman.
  • the present examples can use a chemically modified thymidine derivative for this purpose, although the other four DNA bases (cytosine, adenine, guanine, and uracil) can be used for this purpose.
  • the reaction requires a highly concentrated 0.8 M solution of thiourea (61 g/L) to affect the desired transformation.
  • the present examples provide at least two advantages. First, by attaching the catalyst to a solid support, removal from solution can be facile. This particular solid support, a DNA bionanolattice, is also expected to display a high degree of thermal stability due to its massive hydrogen bonding network. Therefore it is expected that a nanostructure according to the present invention will continue to function at high temperatures, in the case of recalcitrant ketals / glycosides seen in cellulose polymers.
  • the present examples can emulate local concentrations around the lattice close to 0.8 M, though the overall average solution molarity of thiourea would be substantially less. This can further be enhanced through the use of dendrimer-type branching, where several catalytic residues would share a single anchor point onto the lattice (Fig. 22).
  • thiourea While not peptidomimetic, thiourea shares some similarity to arginine in its effectiveness for complexing carboxylic acid salts (carboxylates).
  • the polygalactonurase family of hydrolases makes use of an arginine residue in their active site, which is believed to function as a molecular recognition factor for binding with the free carboxylate of galactonuric acid.
  • the present examples can use the attached thiourea moieties to serve as a binding site for glycosidic polymers containing uronic acid derivatives (Fig. 23).
  • a derivatized nucleobase in this case will be thymidine, although this can be extended to the other four DNA bases (guanine, cytosine, adenine, uracil, Fig. 24).
  • the linking chain can be altered to accommodate any physical length, and can be comprised of (poly)ether, amide, ester, sulfide, or any related connection of atoms generally found in organic molecules.
  • the nature of the R group on the thiourea can include H, but preferably any electron-deficient aromatic group, where said electron-deficient groups include trifluoromethyl, carbonyl (amide, ester, ketone), nitro or nitrile located on the o, m, or p positions of the ring.
  • the chemical synthesis of the target compound in Fig. 24 can take advantage of the shown disconnections, involving an amide coupling between the green and blue fragments, a reduction of the terminal azide, and a condensation between the resultant amine and the red aryl thioisocyanate derivative.
  • Example Embodiments Proteins that span cellular membranes and serve as conduits for ion transport between the cytosol and the environment have been a source of intense study. While the function of such channels is well understood, their isolation and crystallization in non- membrane environments has met with great difficulty. In order to gain a better understanding of their natural conformations, synthetic variants of these structures have been synthesized and assayed, employing two key structural elements: an ionophoric interior to facilitate ion transport, and a hydrophobic exterior to allow for incorporation into the cellular membrane.
  • Cystic Fibrosis which is an aberration of the chloride transport protein.
  • Bacteria have capitalized on this phenomenon in the production of the natural antibiotics Gramicidin and Amphotericin, which will self-assemble into amphiphilic synthetic pores and rapidly depolarize the cellular ion gradient of target organisms. Though a good starting point for synthetic design, it is the end goal of this field to create a true "gated" channel which will not simply act as a non-selective pore, but can rather respond to external stimuli or gradient concentrations.
  • the present examples apply DNA bionanolattice technology to the creation of synthetic ion channels, through the covalent attachment of both hydrophobic and hydrophilic residues to the exterior and interior of the lattice, respectively.
  • the manipulation of DNA into various two and three dimensional shapes has been pioneered by the work of Seeman and Shih, though the application of these constructs towards practical purposes has yet to be disclosed.
  • the present examples can use multi-helical bundles, which are capable of creating clefts or pockets in which catalytic activity could take place, in analogy to enzymatic active sites, or in this case a centralized pore which has been lined with hydrophilic moieties.
  • helices can be interlocked using shorter "staple strands" of DNA, in analogy to the "DNA Origami” approach by otheman.
  • the present examples can use a chemically modified thymidine derivative for this purpose; the other four DNA bases (cytosine, adenine, guanine, and uracil) can also be used for this purpose.
  • the present examples can connect hydrophobic compounds to the perimeter of the lattice through covalent linkages to modified thymidine derivatives. These compounds include saturated or unsaturated aliphatic chains, phenyl, naphthyl, indoyl, or other aromatic groups, or any steroidal compound (Fig. 26).
  • the present examples can attach different functional groups, depending on the nature of the ion to be transported.
  • cyclic polyethers (crown ethers) can be attached to the DNA weave as amine or catechol derivatives, though the present examples can also utilize carboxylate and hydroxyalkyl moieties for the same function.
  • the present examples can also use tetraalkyl ammonium derivatives for negatively charged ions.
  • the second method is based on a report by Fyles, and makes use only of carboxylate residues; however, the relative charge per m3 is varied along the length of the pore.
  • carboxylate mono-anionic
  • succinate two carboxylates, di-anionic
  • a gradient is developed for the shuttling of cationic atoms.
  • the present examples can create a charge gradient by increasing the concentration of carboxylate residues along the interior of the DNA bionanolattice to create a gradual gradient from one end to the other (Fig. 29).
  • the present examples can provide the advantage in the ability to place these charged groups at all positions in the interior of the pore. Accordingly, the present examples can facilitate ion transfer through the remainder of the pore, as the two previous examples contained charge-neutral groups along the interior.
  • the monomers needed to create both the ionophilic and hydrophobic residues will be based on a thymidine - linker - functional group motif, with covalent linkages being created either before or after DNA solid phase synthesis.
  • R' represent a covalent linkage between the thymidine base and the linking molecule, or the linking molecule and the functional group, respectively, and can include triazole, amide, ether, sulfide, disulfide or alkene linkage; R" represents the active functional group.
  • this can encompass any of the "R" groups shown in blue displayed in Fig. 26 and Fig. 27.
  • any carboxylate, phosphate, succinate, hydroxyalkyl, tetraalkyl-ammonium or (aza)-crown ether can be incorporated.
  • NTP National Toxicology Program

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Abstract

La présente invention concerne la synthèse d'une enzyme glycosyle hydrolase artificielle, comprenant un site actif capable de catalyse bifonctionnelle, qui a été lié de façon covalente à une matrice organique rigide par l'utilisation d'un produit de recombinaison fixant. Le site actif de cette enzyme peut comprendre des acides aminés naturels, des acides aminés non naturels, notamment des énantiomères, des diastéréomères, ou des homologues d'un acide aminé naturel, ou des acides aminés peptidomimétiques qui contiennent un fragment catalytique que l'on trouve classiquement sur un acide aminé, notamment l'acide carboxylique, un hydroxyle, un thiol, une amine, un amide, un guanidinyle, un imidazoyl, un sélényle, ou un aryle. D'autres groupes non biotiques connus pour participer à la coupure de la liaison glycosidique comprennent l'urée et la thio-urée. La matrice organique rigide de cette enzyme artificielle comprend une ou plusieurs matrice(s) biopolymère(s), notamment des hélices ou des faisceaux d'hélices d'acide désoxyribonucléique, d'acide ribonucléique, d'acide nucléique bloqué, d'acide nucléique morpholino, ou d'acide nucléique peptidique. La fonction chimique unique peut comprendre une hydrolyse de la liaison glycosidique à l'aide d'un mécanisme catalytique bifonctionnel.
PCT/US2010/050246 2009-09-25 2010-09-24 Glycosyle hydrolase synthétique à base de nano-armures d'adn WO2011038259A1 (fr)

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JP2013151468A (ja) * 2011-11-30 2013-08-08 Agilent Technologies Inc オリゴマーの合成及び精製の新規方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013151468A (ja) * 2011-11-30 2013-08-08 Agilent Technologies Inc オリゴマーの合成及び精製の新規方法

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