+

WO2012034043A1 - Amorceur génétiquement codé pour la croissance de polymères à partir de protéines - Google Patents

Amorceur génétiquement codé pour la croissance de polymères à partir de protéines Download PDF

Info

Publication number
WO2012034043A1
WO2012034043A1 PCT/US2011/051043 US2011051043W WO2012034043A1 WO 2012034043 A1 WO2012034043 A1 WO 2012034043A1 US 2011051043 W US2011051043 W US 2011051043W WO 2012034043 A1 WO2012034043 A1 WO 2012034043A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
formula
initiator
protein
unnatural amino
Prior art date
Application number
PCT/US2011/051043
Other languages
English (en)
Inventor
Ryan A. Mehl
Krzysztof Matyjaszewski
Saadyah Averick
Original Assignee
Franklin And Marshall College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Franklin And Marshall College filed Critical Franklin And Marshall College
Publication of WO2012034043A1 publication Critical patent/WO2012034043A1/fr
Priority to US13/788,710 priority Critical patent/US8816001B2/en
Priority to US13/837,590 priority patent/US9790305B2/en
Priority to US14/452,060 priority patent/US9243274B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C229/36—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/53—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/54—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of a saturated carbon skeleton

Definitions

  • This invention pertains to the bioconjugation of polymers with proteins at specific sites on the protein.
  • Protein-polymer bioconjugates have already shown an impressive range of altered or improved properties (see Borner et al., J.
  • Protein-polymer bioconjugates also have shown efficient pharmacokinetics and therapeutic potency (see Gao et al.; Krishna et al.; Lutz et al.; Nicolas et al.; Lele et al., Biomacromolecules 2005, 6, 3380).
  • Protein polymers bioconjugates have been prepared in two general ways: either by graft-to methods where a preformed functionalized polymer is attached to an amino acid, cofactor or end group, or by the graft-from method where a location on the purified protein is functionalized with an initiator and then the polymer is grown from that site (see Krishna et al.; Liu et al., Ang. Chem., Int. Ed. 2007, 46, 3099; Zeng et al., Chem. Comm. 2007, 1453; Heredia etal., J. Am. Chem. Soc. 2005, 127, 16955.
  • Previous protein-polymers have been prepared using an atom transfer radical polymerization (ATRP) technique (see Wang et al., Am. Chem. Soc. 1995, 117, 5614; Matyjaszewski & Xia, Chem. Rev. 2001, 101, 2921; Matyjaszewski &Tsarevsky, Nature Chem. 2009, 1, 276) wherein an ATRP initiator attached to the protein provides a linkage between the protein and growing polymer chain.
  • ATRP atom transfer radical polymerization
  • Atom-transfer radical polymerization and o.ther controlled/living radical polymerization (CRP) methodologies including nitroxide mediated polymerization (NMP) and reversible addition fragmentation transfer (RAFT) systems allow unprecedented control over polymer dimensions (molecular weight), uniformity (polydispersity), topology (geometry), composition and functionality.
  • NMP nitroxide mediated polymerization
  • RAFT reversible addition fragmentation transfer
  • ATRP is a controlled radical polymerization (CRP) technique; therefore, monomers and cross-linkers may be incorporated in a predictable, controlled, and programmed manner to yield polymer chains of essentially equal length, as defined by the ratio of consumed monomer to the added initiator.
  • the functionality present on the introduced initiator is preserved and forms both the ⁇ - and ⁇ -chain end functionality on the formed polymer segment.
  • the polymers synthesized using ATRP show tolerance to many functional groups, such as hydroxy, amino, amido, esters, carboxylic acid, that can be incorporated into a copolymer then used for post-polymerization modifications including covalent linking of biomolecules and drug delivery. As disclosed below, this enables formation of bioconjugates between synthetic polymers and biomolecules.
  • the delivery system synthesized using ATRP offer customizable and tunable structure for precise targeted delivery of biologically active molecules.
  • This invention provides a general method for the quantitative, site-specific incorporation of a polymer initiator initially exemplified by an unnatural amino acid of formula 6, and further exemplified by the modified amino acid of formula 1, into a recombinant protein. This method overcomes the technical challenges of attaching an initiator to the protein of interest prior to polymerization and
  • An embodiment of this invention is a general method for the quantitative, site-specific incorporation into a recombinant protein of an unnatural amino acid further comprising a functionality that directly acts as a polymer initiator function as exemplified by the initiator of formula 6.
  • the unnatural amino acid may alternatively comprise a functionality that can be converted into the desired initiating functionality.
  • the utility of this approach is further exemplified by growing polymers of oligo(ethylene oxide) monomethyl ether methacrylate from a green fluorescent protein with initiator 1 site-specifically incorporated on its surface and showing that the attached polymer does not affect the general structure or solubility of the green fluorescent protein.
  • the resulting amide linkage between the protein and polymer should be stable to drug delivery and material science applications. While we have shown that this initiator on GFP functions well for generating protein-polymers in aqueous conditions by standard ATRP chemistry it should also function with other controlled radical polymerization agents as well.
  • An embodiment of the invention is the unnatural amino acid of formula 6 and salts thereof, designed to be incorporated in a protein site-specifically and function as an initiator for atom-transfer radical polymerization.
  • Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
  • X is F, CI, Br, I, N3, alkoxyamine, or a thiocarbonyl thio moiety;
  • A is O, S, or NR wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3.
  • a preferred embodiment of the invention is a unnatural amino acid having the structure of formula 6 and salts thereof, wherein Rl and R2 are
  • the unnatural , amino acid is comprised of compounds wherein Rl and R2 are independently H, methyl, or phenyl; X is F, CI, Br, or I; A is O; and n is 1 and are represented by the generic structure 7 and salts thereof.
  • the unnatural amino acid 7 has the structure 7b and salts thereof .
  • An embodiment of the invention is the unnatural amino acid of formula 6 and salts thereof, wherein Rl and R2 are independently H, methyl, or phenyl; X is F, CI, Br, or I; A is O, S, or NR, wherein R is H, methyl, or phenyl; and n is 0.
  • a more preferred embodiment is the unnatural amino acid is comprised of compounds of formula 6 wherein Rl and R2 are independently H, methyl, or phenyl; X is F, CI, Br, or I; A is O; and n is 0 and is represented by the generic structure of formula 8 and salts thereof.
  • the unnatural amino acid has the structure of formula 8b and salts thereof.
  • a more preferred embodiment of the invention is the unnatural amino acid of formula 6 and salts thereof, wherein Rl and R2 are independently H, methyl, or phenyl; X is F, CI, Br, or I; A is NR, wherein R is H, methyl, or phenyl; and n is 0, and is represented by the generic structure of formula 9 and salts thereof.
  • the unnatural amino acid has the structure of formula 1 and salts thereof.
  • Another embodiment of the invention is a process of preparing the unnatural amino acid of formula 6, said process comprising the steps of
  • Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl,
  • heterocycloalkyl aryl, or heteroaryl
  • X is F, CI, Br, I, N3, alkoxyamine, or a thiocarbonyl thio moiety
  • A is O, S, or NR, wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3.
  • Rl and R2 are independently H, methyl, or phenyl; X is F, CI, Br, or I; Y is F, CI, Br, I, or trifluoroacetate; A is O, S, or NR, wherein R is H, methyl, or phenyl; and n is 1.
  • Rl and R2 are H, methyl, or phenyl; X is Br; Y is CI or Br; A is O or NH.
  • An embodiment of the invention is a protein-based initiator comprising an unnatural amino acid or its salt thereof, wherein said unnatural amino acid comprises an initiator for a controlled polymerization process and wherein the unnatural amino acid is incorporated site -specifically within the protein via translation using an orthogonal aminoacyl-tRNA synthetase/ orthogonal tRNA pair and selector codon.
  • the protein-based initiator comprises an unnatural amino acid of formula 6 or a salt thereof, wherein Rl and R2 of said amino acid initiator are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
  • X is F, CI, Br, I, N3, alkoxyamine, or a thiocarbonyl thio moiety;
  • A is O, S, or NR, wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3.
  • Another embodiment of the invention is a process of preparing a protein- based initiator containing a site-specifically incorporated unnatural amino acid of formula 6 comprising an initiator functionality, where the process comprises the steps,
  • nucleic acid (a) providing a nucleic acid, wherein the nucleic acid further includes a selector codon;
  • Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
  • X is F, CI, Br, I, N3, alkoxyamine, or a thiocarbonyl thio moiety;
  • A is O, S, or NR, wherein R is H, Cl- C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3.
  • the process uses an unnatural amino acid initiator of formula 7. It is also preferred that the process uses and unnatural amino acid initiator of formula 8. It is more preferred that the process uses an unnatural amino acid initiator of formula 9. It is most preferred that the process uses an unnatural amino acid initiator of formula 1.
  • An embodiment of the invention is a process of preparing a site-specific protein-polymer bioconjugate from a protein-based initiator incorporating an unnatural amino acid herein exemplified by an initiator of formula 6 or salt thereof, comprising the step of,
  • Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;
  • X is F, CI, Br, I, N3, alkoxyamine, or a thiocarbonyl thio moiety;
  • A is O, S, or NR, wherein R is H, Cl- C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3.
  • the protein-based initiator incorporates an initiator amino acid of formula 7. It is more preferred that the protein-based initiator incorporates an unnatural initiator amino acid of formula 8. It also is more preferred that the protein-based initiator incorporates an unnatural initiator amino acid of formula 9. It is most preferred that the protein-based initiator incorporates an unnatural initiator amino acid of formula 1.
  • втори ⁇ codon refers to a codon recognized by the O-tRNA in the translation process and not typically recognized by an endogenous tRNA.
  • the O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates its amino acid, e.g., an initiator amino acid, at this site in the polypeptide.
  • Selector codons can include, e.g., nonsense codons, such as stop codons ⁇ e.g., amber, ochre, and opal codons), four or more base codons, rare codons, codons derived from natural or unnatural base pairs, or the like.
  • translation system refers to the components that incorporate an amino acid into a growing polypeptide chain (protein).
  • Components of a translation system can include, e.g., ribosomes, tRNAs, synthetases, mRNA, and the like.
  • Typical translation systems include cells, such as bacterial cells ⁇ e.g., Escherichia coli), archeaebacterial cells, eukaryotic cells ⁇ e.g., yeast cells, mammalian cells, plant cells, insect cells), or the like.
  • the translation system comprises an in vitro translation system, e.g., a translation extract including a cellular extract.
  • the O-tRNA or the O-RSs of the invention can be added to or be part of an in vitro or in vivo translation system, e.g., in an eukaryotic cell, e.g., a bacterium (such as & coli), or in a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, an insect cell, or the like.
  • the translation system can also be a cell-free system, e.g., any of a variety of commercially available in vitro transcription/translation systems in combination with an O-tRNA O-RS pair and an initiator amino acid as described herein.
  • the translation system may optionally include multiple O-tRNA/O-RS pairs, which allow incorporation of more than one unnatural amino acid, e.g., an initiator amino acid and another unnatural amino acid.
  • the cell can further include an additional different O-tRNA/O-RS pair and a second unnatural amino acid, where this additional O-tRNA recognizes a second selector codon and this additional O-RS preferentially aminoacylates the O-tRNA with the second unnatural amino acid.
  • a cell that includes an O- tRNA/O-RS pair can further comprise a second orthogonal pair, where the second O-tRNA recognizes a different selector codon ⁇ e.g., an opal codon, four-base codon, or the like).
  • the different orthogonal pairs are derived from different sources, which can facilitate recognition of different selector codons.
  • Matyjaszewski and coworkers disclosed the fundamental four component Atom Transfer Radical Polymerization (ATRP) process comprising the addition, or in situ formation, of an initiator, in this case a molecule with a transferable atom or group that is completely incorporated into the final product, a transition metal and a ligand that form, a partially soluble transition metal complex that participates in a reversible redox reaction with the added initiator or a dormant polymer to form the active species to copolymerize radically polymerizable monomers, and a number of improvements to the basic ATRP process, in a number of patents and patent applications: U.S. Pat. Nos. 5,763,546; 5,807,937; 5,789,487; 5,945,491; 6, 111,022; 6,121,371; 6,124,411; 6, 162,882; 6,624,262;
  • amino acid initiator is, in this case a molecule containing a primary amine functionality and carboxylic acid functionality that can be incorporated into a protein primary sequence with a transferable atom or group that is completely incorporated into the final product, a transition metal and a ligand that form, a partially soluble transition metal complex that participates in a reversible redox reaction with the added initiator or a dormant polymer to form the active species to copolymerize radically polymerizable monomers.
  • a "protein-based initiator” is where an amino acid initiator has been incorporated into the primary sequence of a protein producing a protein with a transferable atom or group that is completely incorporated into the final product, a transition metal and a ligand that form, a partially soluble transition metal complex that participates in a reversible redox reaction with the added initiator or a dormant polymer to form the active species to copolymerize radically polymerizable monomers.
  • Figure 1 shows the genetic incorporation of ATRP initiator into proteins.
  • the evolved A//RS/tRNAcuA pair in pDule-BIBAF allows for site-specific incorporation of 1 in response to an amber codon.
  • Lane 2 shows expression levels of GFP-wt from pBad-GFP-Hise. Production of GFP-1 from pBad-GFP-134TAG- His6 is dependent on 1 in the growth media, lane 3 without 1 present, lane 4 with 1 mM 1 present. Protein was purified by Co +2 affinity chromatography, separated by SDS-PAGE and stained with Coomassie.
  • Figure 2 shows the fluorescence measurements of 92 synthetases with GFP clones.
  • the lighter lines represent colonies induced in media containing 1 mM 1 while darker black lines represent colonies induced in the absence of UAA.
  • Figure 3 shows the fluorescence measurements of 20 highest-expressing synthetases with GFP clones.
  • the striped bar lines represent colonies induced in media containing 1 mM 1 while the solid lines represent colonies induced in the absence of UAA.
  • Expressions of 3 mL were grown for 40 hours before dilution of suspended cells directly from culture 100-fold with phosphate buffer saline (PBS). Fluorescence measurements were collected using a HORIBA Jobin Yvon FluoroMax®-4.
  • Figures 4a and 4b are the respective ESI-MS of GFP-wt and GFP-1 proteins and demonstrate the efficient high fidelity incorporation of a single 1 in response to an amber stop codon.
  • Fig. 4a is an ESI-MS-Tof analysis of sfGFP showing a single major peak at 27827.0 Da ⁇ 1 Da.
  • Fig. 4b is an ESI-MS-Tof analysis of GFP-1 showing a single major peak at 28024.0 Da ⁇ 1 Da.
  • These spectra show the expected molecular weigh difference of 197 Da from native indicating a single efficient incorporation of 1 at the expected site.
  • Figures 5a and 5b are isotopic abundance patterns indicating the presence of bromine.
  • Fig. 5a is the experimentally observed isotopic pattern for [M+2H] 2+ at 589-592 Da.
  • Fig. 5b is the predicted isotopic pattern for [C5oH77Ni40i Br + 2H] Z+ as derived from various on-line isotopic pattern generators.
  • Figure 6 is the E(BIBAF)GNILGHK MS/MS spectrum of 589 Da.
  • the signal at 580 Da retains the characteristic isotopic pattern associated with the presence of bromine in a 2+ charge state, and is consistent with the doubly charged species resulting from the loss of water, [M-18 + 2H] 2+ .
  • Loss of water is a recognized low-energy fragmentation pathway for N-terminal glutamic acid peptides
  • the isotopic pattern for the peak at 540 Da indicates a +2 charge absent bromine and is consistent with the loss of HBr from the side chain 4-(2'- bromoisobutyramido)phenylalanine.
  • Figure 7a is a characterization of ATRP grafting from GFP-wt and GFP-1 with OEO 30 0MA monomer in PBS at 24°C. SDS-PAGE of crude time points (5 ⁇ g of protein was loaded on each lane of a 4-12% gel). The reaction produced no size change for GFP-wt (Lane 2 and 3), while the majority of GFP-1 showed significant size increases with increasing ATRP reaction time (Lane 4-7).
  • Figure 7b is a SEC of GFP-wt. SEC of 0.1 mg of desalted reaction time- points on Superdex 200 at flow rate of 0.8 mL/min of PBS buffer monitored at 230 nm. GFP-wt eluted at the expected volume of 17.3 mL (black line) and was unaltered by the ATRP reaction (white line).
  • Figure 7c is a SEC of GFP-1 ATRP.
  • Figure 8 is a SDS PAGE analysis of SEC fractionated ATRP reactions.
  • ATRP reactions were separated by SEC, (Fig. 7c), and the individual fractions were concentrated and separated on a 4-12% gel and stained with Coomassie.
  • the full 3 hr reaction mixture is in lane 6 and fractions 1-5 are in lanes 1-5 respectively.
  • the GFP -polymer hybrid's change in size is due to polymer growth, as indicated by SEC and SDS-PAGE separation. Characterization of ATRP grafting from GFP-wt and GFP-1 with OEO300MA monomer in PBS at 24°C.
  • A SDS-PAGE of crude time points (5 ⁇ of protein was loaded on each lane of a 4- 12% gel). The reaction produced no size change for GFP-wt (Lane 2 and 3), while the majority of GFP-1 showed significant size increases with increasing ATRP reaction time (Lane 4-7).
  • GFP Green Fluorescent Protein
  • MPEG oligo(ethylene oxide) monomethyl ether methacrylate
  • the unnatural amino acid containing an ATRP initiator functionality, 1, was prepared as outlined in Scheme 2.
  • Initiator 1 may be used directly in the subsequent reactions or its hydrochloride salt la may be used and neutralized under the reaction conditions to afford GFP-1. It is important to synthesize 1 in large quantities since relatively large quantities of initiator-containing protein are needed for polymerization experiments.
  • the synthetic route need not be asymmetric since the MJRS only utilizes the L form.
  • the initiator la was synthesized in two steps in 63% yield from commercially available material.
  • the salt was neutralized with base, for example one equivalent of NaOH, to afford initiator 1.
  • Initiator 1 is an exemplification of a wider class of functional unnatural amino acids that the inventors have discovered are useful for the site-specific incorporation of the initiator functionality into proteins, thereby allowing site specific incorporation of grafting of polymers into proteins.
  • acyl derivatives of 4-aminophenylalanine as initiators acyl derivatives of tyrosine and other initiators having at least one methylene spacer between the amide or ester group and the benzene ring of the amino acid have been found to be useful for the invention.
  • One class of these unnatural amino acids, with incorporated initiator functionality is represented by the generalized structure 6.
  • Initiator 6 may be prepared by the synthesis route shown in Scheme 3, wherein Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; X is F, CI, Br, or I; Y is F, CI, Br, I, or trifluoroacetate; A is O, S, or NR, wherein R is H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and n is 0, 1, 2, or 3.
  • substituents provides an unnatural amino acid with an initiating group suitable for ATRP the utility of this exemplary unnatural amino acid can be modified to provide functionality for other CRP procedures.
  • X may also comprise an alkoxyamine for nitroxide mediated polymerization or a thiocarbonyl thio moiety suitable for a reversible addition fragmentation transfer polymerization.
  • the N-boc protected intermediate 4 can be purchased commercially or readily prepared by methods well known in the art. Intermediate 4 is reacted with an D-haloacylhalide optionally in the presence of a base to afford protected compound 5. Reactions may be done at ambient temperature in a non-protic solvent. Suitable solvents include the ether solvents as tetrahydrofuran, ether, dioxane, and glyme, aromatic solvents as benzene and toluene, halogenated solvents such as methylene chloride, chloroform, carbontetrachloride, and chlorobutane. Generally, no additional bases are needed when A is nitrogen.
  • A is oxygen or sulfur it is useful to have a tertiary amine base, such as ⁇ , ⁇ -diisopropylethylamine, present in the reaction mixture.
  • the N-boc protecting group may be removed by reacting 5 with an acid halide in dioxane/methylene chloride or other suitable deprotection agent, such as trifluoroacetic acid, to afford the salt 6a.
  • the initiator 6a is readily neutralized in the presence of base to afford initiator 6.
  • 6a can be neutralized by one equivalent of a base, such as one equivalent of sodium hydroxide to afford 6 immediately prior to use in subsequent initiator reactions.
  • 6a may be used and neutralized under reaction conditions to afford the protein-based initiator comprising 6. Both initiators 6a and 6 may be used as reactants for evolving the orthogonal pair and incorporating the desired initiating functionality into specific sites within the protein of interest.
  • Preferred initiators include 7a and 7 having a methylene spacer between the amino acid phenyl group and the ester group. These compounds are analogous to compounds of general structures 6a and 6 wherein n is 1 and A is O, and wherein Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl,
  • heterocycloalkyl aryl, or heteroaryl
  • X is F, CI, Br, or I
  • Y is F, CI, Br, I, or trifluoroacetate.
  • More preferred initiators include the ester derivatives 8a and 8. These compounds are analogous to compounds of general structures 6a and 6 wherein n is 0 and A is O, and wherein Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; X is F, CI, Br, or I; Y is F, CI, Br, I, or trifluoroacetate. '
  • amide derivatives 9a and 9 are analogous to compounds of general structures 6a and 6 wherein n is 1 and A is NR, wherein R is H, methyl, or phenyl; and wherein Rl and R2 are independently H, C1-C8 alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; X is F, CI, Br, or I; Y is F, CI, Br, I, or trifluoroacetate.
  • Table X is a partial listing of some of the unnatural initiator amino that may be prepared by the methods described above. Data for prepared compounds is given.
  • Scheme 1 (above) is an exemplification of the overall process of preparing a recombinant protein-polymer.
  • an orthogonal tRNA pair capable of incorporating 1 is evolved and a library of synthetase gene is randomized for codons specific to the desired site residue.
  • unnatural amino acid comprising an initiator functionality, 1, is site-specifically incorporated into the GFP to produce the protein-based initiator GFP-1.
  • GFP-1 is then reacted with a monomer MPEG under ATRP reaction conditions to afford the recombinant protein-polymer, polyMPEG-GFP.
  • the most active RS was cloned into a pDuIe vector that contains one copy of ⁇ /> ' tRNAcuA to create pDule-BIBAF.
  • Expression of GFP gene interrupted by an amber codon at site 134 in the presence of pDule- BIBAF was efficient and dependent on the presence of 1 (Fig. 1).
  • 0.42 g of GFP-1 was purified per liter of media, while GFP-wt yielded 1.27 g/L under similar conditions (no GFP is produced in the absence of 1).
  • Methods of producing a protein in a cell ⁇ e.g., a non-eukaryotic cell, such as an K coli cell or the like, or a eukaryotic cell) with an initiator amino acid at a specified position are a feature of the invention.
  • Proteins or polypeptides of interest having at least one initiator amino acid are a feature of the invention.
  • a protein of the invention may include a post-translational modification.
  • the protein comprises an amino acid sequence that is at least 75% identical to that of a known protein, e.g., a therapeutic protein, a diagnostic protein, an industrial enzyme, or portion thereof.
  • any protein (or portion thereof) that includes an amino acid further comprising an initiator for a CRP, or that encodes multiple different unnatural amino acids (and any corresponding coding nucleic acid, e.g., which includes one or more selector codons) can be produced using the compositions and methods herein. No attempt is made to identify the hundreds of thousands of known proteins, any of which may be modified to include one or more unnatural amino acid, e.g., by tailoring any available mutation methods to include one or more appropriate selector codon in a relevant translation system. Common sequence repositories for known proteins include GenBank, EMBL, DDBJ, and the NCBI, among others.
  • the proteins are, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to any available protein ⁇ e.g., a therapeutic protein, a diagnostic protein, an industrial enzyme, or portion thereof, and the like), and they comprise one or more unnatural amino acid.
  • any protein of interest can be modified to include an initiator comprising an unnatural amino acid.
  • Enzymes ⁇ e.g., industrial enzymes) or portions thereof with at least one initiator amino acid are also provided by the invention.
  • enzymes include, but are not limited to, e.g., amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases ⁇ e.g., p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases.
  • Host cells are genetically engineered (e.g., transformed, transduced, or transfected) with one or more vectors that express the orthogonal tRNA, the orthogonal tRNA synthetase, and a vector that encodes the protein to be derivatized.
  • Each of these components can be on the same vector, or each can be on a separate vector, or two components can be on one vector and the third component on a second vector.
  • the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
  • compositions of the invention and compositions made by the methods of the invention are optionally made in a cell.
  • the O-tRNA/O-RS pairs or individual components of the invention may then be used in a host system's translation machinery, which results in an initiator amino acid being
  • an O-tRNA/O-RS pair when introduced into a host, e.g., Escherichia coli, the pair leads to the in vivo incorporation of an initiator amino acid, which can be exogenously added to the growth medium, into a protein, e.g., any protein where a polymer attachment is of interest, in response to a selector codon, e.g., an amber nonsense codon.
  • a selector codon e.g., an amber nonsense codon.
  • compositions of the invention can be in an in vitro translation system, or in an in vivo system(s) with the initiator amino acid and may be used to facilitate production of a protein polymer hybrid.
  • Kits are also a feature of the invention.
  • a kit for producing a protein with an initator amino acid at a specified position is provided, where the kit includes a cell comprising an orthogonal tRNA that functions in the cell and recognizes a selector codon and an orthogonal aminoacyl-tRNA synthetase, packaged in one or more containers.
  • the kit further includes an initiator amino acid.
  • the kit further comprises instructional materials for producing the protein, an appropriate cell growth medium, reagents for introducing a target nucleic acid encoding the protein of interest and including the selector codon into the cell, or the like.
  • Any composition, system or device of the invention can also be associated with appropriate packaging materials ⁇ e.g., containers, etc.) for production in kit form.
  • a kit may also include a plasmid and instructions for practicing a method of the invention.
  • the simple power of this method allows one to (a) form proteins that require no further modification, such as random attachment of initiating groups; (b) control the location of the initiating group so the polymer can be grown from one or more sites selected so that they can either be positioned to be totally free from the active site or are situated in ways to regulate active site activity; and (c) grow an exact number of well defined polymer chains with targeted molecular weight and site specific functionality from the protein.
  • This method overcomes nearly all limitations of other previous methods to produce protein polymer hybrids.
  • an initiation functionality for any controlled radical polymerization process can be introduced into nearly any protein thereby providing the ability to advance the field of protein polymer hybrids from the current class of nonfunctional proteins, e.g. bovine serum albumin, towards enzymes or
  • this invention allows one to assay the efficacy of the system and properly study the effects of polymer placement using commercially available enzyme assays.
  • N-Boc 4-aminophenylalanine (3.62 g, 0.01447 mol) was dissolved in 50 mL of dry THF.
  • 2-bromoisobutyryl bromide (1.757 mL,
  • N- Boc-4-(2'-bromoisobutyramido)phenylalanine was obtained in 65% yield (3.63 g).
  • Example 2 4-(2'-bromoisobutyramido)phenylalanine
  • N-Boc-4-(2'-bromoisobutyramido)-phenylalanine (4.8 g, 0.0112 mol) was dissolved in 50 mL ethyl acetate under argon and dry 4 M HC1 in dioxane (50 mL) was subsequently added to the solution while stirring at room temperature overnight. The reaction mixture was then evaporated under reduced pressure to a final volume of 5-10 mL. Pentane was then added to the solution, and the precipitate was filtered using an M type filter crucible and dried under reduced pressure. 4-(2'-bromoisobutyramido)phenylalanine hydrochloride was obtained in 97% yield (3.93 g).
  • Example 3 Selection of an aminoacyl-tRNA synthetase specific for 4-(2'- bromoisob uty r amido)phenylalanine .
  • the library of aminoacyl-tRNA synthetases was encoded on a kanamycin (Kn) resistant plasmid (pBK, 3000 bp) under control of the constitutive
  • Escherichia coli GlnRS promoter and terminator The aminoacyl synthetase library (3D-Lib) was randomized as follows: Leu65, His70, Glnl55, and He 159 were randomized to all 20 natural amino acids; Tyr32 was randomized to 15 natural amino acids (less Trp, Phe, Tyr, Cys, and He); Asp 158 was restricted to Gly, Ser, or Val; Leu 162 was restricted to Lys, Ser, Leu, His, and Glu; and
  • Phe 108 and Gin 109 were restricted to the pairs Trp-Met, Ala -Asp, Ser-Lys, Arg- Glu, Arg-Pro, Ser-His, or Phe-Gln.
  • the library plasmid, pBK-3D-Lib was moved between cells containing a positive selection plasmid (pCG) and cells containing a negative selection plasmid (pNEG).
  • Mj Methanococc s jannaschii
  • T7 RNA polymerase an amber codon-disrupted T7 RNA polymerase that drives the production of green fluorescent protein
  • Tet tetracycline
  • the negative selection plasmid, pNEG (7000 bp), encodes the mutant tyrosyl-tRNAcuA, an amber codon-disrupted barnase gene under control of an arabinose promoter and rrnC terminator, and the ampicillin (Amp) resistance marker.
  • pCG electrocompetent cells and pNEG electrocompetent cells were made from DH10B cells carrying the respective plasmids and stored in 100 DL aliquots at Q80 °C for future rounds of selection.
  • the synthetase library in pBK-3D-Lib was transformed by electroporation into DH10B cells containing the positive selection plasmid, pCG.
  • the resulting pCG/pBK-3D-Lib-containing cells were amplified in 1 L of 2xYT with 50 Dg/mL Kn and 25 Dg/mL Tet with shaking at 37 °C.
  • the cells were grown to saturation, then pelleted at 5525 rcf, resuspended in 30 mL of 2xYT and 7.5 mL of 80% glycerol, and stored at D80 °C in 1 mL aliquots for use in the first round of selections.
  • pCG/pBK-3D-Lib cells were thawed on ice before addition to 1.2 L of room temperature 2xYT media containing 50 Dg/mL Kn and 25 Dg/mL Tet. After incubation (11 h, 250 rpm, 37 °C), a 200 DL aliquot. of these cells was plated on eleven 15 cm GMML-agar plates containing 50 Dg/mL Kn, 25 Dg/mL Tet, and 60 Dg/mL chloramphenicol (Cm). The positive selection agar medium also contained 1 mM 4-(2'- bromoisobutyramido)phenylalanine hydrochloride.
  • plasmid purification steps a Qiagen miniprep kit was used to purify the plasmid DNA. .
  • the smaller pBK-3D-Lib plasmid was separated from the larger pCG plasmid by agarose gel electrophoresis and extracted from the gel using the Qiagen gel extraction kit.
  • the purified pBK-3D-Lib was then transformed into pNEG-containing
  • DH10B cells A 100 DL sample of pNEG electrocompetent cells was transformed with 50 ng of purified pBK-3D-Lib DNA. Cells were rescued in 1 mL of SOC for 1 h (37 °C, 250 rpm) and the entire 1 mL of rescue solution was plated on three 15 cm LB plates containing 100 Dg/mL Amp, 50 Og/mL Kn, and 0.2% L-arabinose. Cells were collected from plates and pBK-3D-Lib plasmid DNA was isolated in the same manner as described above for positive selections.
  • one plate was spread with 250 DL of rescued cells, and two plates were spread with 50 DL of rescued cells and then incubated (12D 16 h, 37 °C).
  • the cells were plated on LB agar containing 100 Og/mL Amp, 50 Dg/mL Kn, and 0.04% L-arabinose.
  • the synthetases resulting from the selection rounds were tested with the pALS plasmid.
  • This plasmid contains the sfGFP reporter with a TAG codon at residue 150 as well as tyrosyl-tRNAcuA.
  • a pBK plasmid with a functional synthetase is transformed with the pALS plasmid and the cells are grown in the presence of the appropriate amino acid on autoinduction agar, sfGFP is expressed and the colonies are visibly green.
  • Table 1 column A with an autoclaved solution of 40 g of agarose in 400 mL water. Sterile water was added to a final volume of 500 mL. Antibiotics were added to a final concentration of 25 ⁇ g/mL Tet and 25 ⁇ g/mL Kan. Nine plates were poured with 1 mM 4-(2'-bromoisobutyramido)phenylalanine hydrochloride, and nine plates were maintained as controls without UAA.
  • a total of 92 visually green colonies were selected from the two 1 mM 4-(2'- bromoisobutyramido)phenylalanine hydrochloride plates and used to inoculate a 96-well plate containing 0.5 mL per well non-inducing minimal media (Table 2, column B, with sterile water added to a final volume of 500 mL) with 25 Dg/mL Kn, 25 Dg/mL Tet. After 24 hours of growth (37 °C, 250 rpm), 5 DL of these non- inducing samples were used to inoculate 96-well plates with 0.5 mL
  • Fluorescence measurements of 92 synthetases with GFP clones were conducted. Expressions of 500 DL were grown for 40 hours before dilution of suspended cells directly from culture 100-fold with phosphate buffer saline (PBS). Fluorescence measurements were collected using a HORIBA Jobin Yvon FluoroMax®-4. The emission from 500 to 520 nm (1 nm bandwidth) was summed with excitation at 488 nm (1 nm bandwidth). See Figure 2.
  • Non-inducing cultures (3 mL) with 25 Dg/mL Kn and 25 Dg/mL Tet were grown to saturation (37 °C with shaking at 250 rpm) from the 20 highest- fluorescing colonies.
  • Autoinduction cultures (3 mL) with 25 Dg/mL Kn and 25 ⁇ g/mL Tet were inoculated with 30 DL of non-inducing cultures and grown with and without 1 mM 1 at 37 °C with shaking at 250 rpm. After approximately 40 hours, fluorescence was assessed (see Fig. 3). The top eight performing clones were sequence revealing five unique members (Table 3).
  • the top performing clone (G2) was moved from the pBK-G2 plasmid to the pDule plasmid (pDuh- BIBAJF).
  • pDule plasmid was generated by amplifying the MjYRS gene from the pBKplasmid isolated from the library using primers RSmovef (5'- CGCGCGCCATGGACGAATTTGAAATG-3') and RSmover (5'- GACTCAGTCTAGGTACCCGTTTGAAACTGCAGTTATA-3'). The amplified DNA fragments were cloned in to the respective sites on the pDule plasmids using the incorporated Ncol and Kpnl sites.
  • DH10B E. coli cells co-transformed with the pBad-sfGFP-134TAG vector and the machinery plasmid pDule-BIBAF were used to inoculate 5 mL of non- inducing medium containing 100 Dg/mL Amp and 25 Dg/mL Tet.
  • the non- inducing medium culture was grown to saturation with shaking at 37 °C, and 5.0 mL was used to inoculate 0.5 L autoinduction medium with 100 Dg/mL Amp, 25 Dg/mL Tet, and 1 mM 4-(2'-bromoisobutyramido)phenylalanine hydrochloride (0.5 L of media grown in 2 L plastic baffled flasks). After 40 hours of shaking at 37 °C, cells were collected by centrifugation.
  • the protein was purified using BD-TALON cobalt ion-exchange chromatography.
  • the cell pellet was resuspended in wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, pH 7) containing 1 mg/mL chicken egg white lysozyme, and sonicated 3 1 min while cooled on ice.
  • wash buffer 50 mM sodium phosphate, 300 mM sodium chloride, pH 7
  • the lysate was clarified by centrifugation, applied to 6-9 mL bed- volume resin, and bound for 30 min. Bound resin was washed with >50 volumes wash buffer.
  • the protein was eluted from the bound resin with 2.5 mL aliquots of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole pH 7) until the resin turned pink and the color of the eluent the column was no longer green.
  • elution buffer 50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole pH 7
  • the elusions concentrations were check with a Bradford protein assay.
  • the protein was desalted into PBS using PD10 columns and concentrated with 3000 MWCO centrifuge filters.
  • Trypsin digestion of GFP and GFP-1 were performed using Trypsin In-Gel Digest Kit from Sigma Aldrich (PP0100). Mass spectral analyses were performed on an Agilent 1100 series LC MSD SL ion trap mass spectrometer x with electrospray ionization and MS/MS capabilities. Ten microliters of the protein digests were injected onto a Zorbax 300SB-C8 column (narrow-bore 2.1x150mm 5-micron) for separation using a gradient of 5-95% CH3CN (with 0.1% formic acid) in water (with 0.1% formic acid) over 75 min (5-25% 0-40 minutes, 25-95% 40-60 minutes, 95-5% 60-75 minutes).
  • the flow rate was set to 0.25 mL/min.
  • the SL Trap MS was operated in the SPS mode under the normal scan setting.
  • the dry temperature was 325°C, with dry gas flow of 10.0 L/min and a nebulizer pressure of 40 psi.
  • the instrument was operated in the auto MS/MS mode selecting two precursor ions with preference given to doubly charged ions while singly charged ions were excluded.
  • An extracted ion chromatograph of mass 589 showed a single peak eluting at 36.3 minutes containing an isotopic cluster with a unique pattern consistent with that expected for the bromine containing peptide.
  • the observed and predicted isotopic abundance patterns compare favorably (see Figure 5a and 5b).
  • MS/MS analysis of the peptide at 589 Da further confirmed incorporation of the bromine into the peptide (see Figure 6).
  • Example 7 ATRP reactions grafting from GFP-wt and GFP-1.
  • Initiator stock solution Bpy (16.70 mg, 1.07*10-3 m mol) and Cu(II)Br 2 (6 mg, 2.68*10 mmol) were dissolved in 10 mL of H2O; the solution was deoxygenated with nitrogen. Cu(I)Br (3.8 mg, 2.68*10 mmol) was added to the mixture.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne des procédés de production de protéines recombinantes homogènes qui contiennent des amorceurs de polymères à des sites définis. L'acide aminé non naturel, 4-(2'-bromoisobutyramido)phényl- alanine de formule 1, a été conçu et synthétisé sous la forme d'une molécule contenant un groupe fonctionnel comprenant, en outre, un amorceur pour une polymérisation radicalaire par transfert d'atomes ( » ATRP ») qui créera, en plus, une liaison stable entre la protéine et le polymère en cours de croissance. Une paire tyrosyl-ARNt synthétase/ARNt-cuA de Methanococcus jannaschii (Mj) a été élaborée pour coder génétiquement cet acide aminé non naturel en réponse à un codon ambre. Pour démontrer l'utilité de cet acide aminé fonctionnel, une protéine fluorescente verte a été produite avec l'amorceur de l'acide aminé non naturel de formule 1 incorporé de manière spécifique de site sur sa surface (GFP-1). La GFP-1 purifiée a ensuite été utilisée sous forme d'amorceur dans des conditions d'ATRP standards avec un méthacrylate d'éther monoéthylique d'oligo(oxyde d'éthylène), et a produit de manière efficace un bioconjugué polymère-GFP dans lequel le polymère est lié à un site spécifiquement choisi sur la GFP.
PCT/US2011/051043 2010-09-10 2011-09-09 Amorceur génétiquement codé pour la croissance de polymères à partir de protéines WO2012034043A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US13/788,710 US8816001B2 (en) 2010-09-10 2013-03-07 Genetically encoded initiator for polymer growth from proteins
US13/837,590 US9790305B2 (en) 2011-09-09 2013-03-15 Site specifically incorporated initiator for growth of polymers from proteins
US14/452,060 US9243274B2 (en) 2010-09-10 2014-08-05 Genetically encoded initiator for polymer growth from proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38175710P 2010-09-10 2010-09-10
US61/381,757 2010-09-10

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/788,710 Continuation US8816001B2 (en) 2010-09-10 2013-03-07 Genetically encoded initiator for polymer growth from proteins

Publications (1)

Publication Number Publication Date
WO2012034043A1 true WO2012034043A1 (fr) 2012-03-15

Family

ID=45810984

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/051043 WO2012034043A1 (fr) 2010-09-10 2011-09-09 Amorceur génétiquement codé pour la croissance de polymères à partir de protéines

Country Status (1)

Country Link
WO (1) WO2012034043A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8816001B2 (en) 2010-09-10 2014-08-26 Franklin And Marshall College Genetically encoded initiator for polymer growth from proteins
US9533297B2 (en) 2012-02-23 2017-01-03 Carnegie Mellon University Ligands designed to provide highly active catalyst complexes
EP3003342A4 (fr) * 2013-05-30 2017-03-08 Duke University Synthèse catalysée par une enzyme de conjugués de biomolécule-polymère spécifiques d'un site et st chiométriques
US9644042B2 (en) 2010-12-17 2017-05-09 Carnegie Mellon University Electrochemically mediated atom transfer radical polymerization
US9790305B2 (en) 2011-09-09 2017-10-17 Franklin And Marshall College Site specifically incorporated initiator for growth of polymers from proteins
US9982070B2 (en) 2015-01-12 2018-05-29 Carnegie Mellon University Aqueous ATRP in the presence of an activator regenerator
US10072042B2 (en) 2011-08-22 2018-09-11 Carnegie Mellon University Atom transfer radical polymerization under biologically compatible conditions
US11174325B2 (en) 2017-01-12 2021-11-16 Carnegie Mellon University Surfactant assisted formation of a catalyst complex for emulsion atom transfer radical polymerization processes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4033998A (en) * 1976-02-20 1977-07-05 Morton-Norwich Products, Inc. Vinylbenzyl ester of an N-BOC amino acid
US6197794B1 (en) * 1998-01-08 2001-03-06 Celltech Therapeutics Limited Phenylalanine derivatives
US6492421B1 (en) * 1997-07-31 2002-12-10 Athena Neurosciences, Inc. Substituted phenylalanine type compounds which inhibit leukocyte adhesion mediated by VLA-4
US20040110753A1 (en) * 1999-09-24 2004-06-10 Genentech, Inc. Tyrosine derivatives
US20050283021A1 (en) * 2004-06-17 2005-12-22 Ajinomoto, Co., Inc. Production method of O-substituted tyrosine compound

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4033998A (en) * 1976-02-20 1977-07-05 Morton-Norwich Products, Inc. Vinylbenzyl ester of an N-BOC amino acid
US6492421B1 (en) * 1997-07-31 2002-12-10 Athena Neurosciences, Inc. Substituted phenylalanine type compounds which inhibit leukocyte adhesion mediated by VLA-4
US6197794B1 (en) * 1998-01-08 2001-03-06 Celltech Therapeutics Limited Phenylalanine derivatives
US20040110753A1 (en) * 1999-09-24 2004-06-10 Genentech, Inc. Tyrosine derivatives
US20040158076A1 (en) * 1999-09-24 2004-08-12 Genentech, Inc. Tyrosine derivatives
US20050283021A1 (en) * 2004-06-17 2005-12-22 Ajinomoto, Co., Inc. Production method of O-substituted tyrosine compound

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8816001B2 (en) 2010-09-10 2014-08-26 Franklin And Marshall College Genetically encoded initiator for polymer growth from proteins
US9243274B2 (en) 2010-09-10 2016-01-26 Franklin And Marshall College Genetically encoded initiator for polymer growth from proteins
US9644042B2 (en) 2010-12-17 2017-05-09 Carnegie Mellon University Electrochemically mediated atom transfer radical polymerization
US10072042B2 (en) 2011-08-22 2018-09-11 Carnegie Mellon University Atom transfer radical polymerization under biologically compatible conditions
US9790305B2 (en) 2011-09-09 2017-10-17 Franklin And Marshall College Site specifically incorporated initiator for growth of polymers from proteins
US9533297B2 (en) 2012-02-23 2017-01-03 Carnegie Mellon University Ligands designed to provide highly active catalyst complexes
EP3003342A4 (fr) * 2013-05-30 2017-03-08 Duke University Synthèse catalysée par une enzyme de conjugués de biomolécule-polymère spécifiques d'un site et st chiométriques
US9982070B2 (en) 2015-01-12 2018-05-29 Carnegie Mellon University Aqueous ATRP in the presence of an activator regenerator
US11174325B2 (en) 2017-01-12 2021-11-16 Carnegie Mellon University Surfactant assisted formation of a catalyst complex for emulsion atom transfer radical polymerization processes

Similar Documents

Publication Publication Date Title
WO2012034043A1 (fr) Amorceur génétiquement codé pour la croissance de polymères à partir de protéines
US9243274B2 (en) Genetically encoded initiator for polymer growth from proteins
EP2380982B1 (fr) Composants de translation orthogonale pour une incorporation in vivo d'acides aminés non naturels
EP2665744B1 (fr) Composés macrocycliques ayant un squelette hybride peptidique/non-peptidique et procédés pour leur préparation
EP3030545B1 (fr) Dérivé d'acides aminés
US20110076718A1 (en) In vivo incorporation of an unnatural amino acid comprising a 1,2-aminothiol group
Zeng et al. Genetic introduction of a diketone-containing amino acid into proteins
CN118420494B (zh) 一种羟基自由基反应性非天然氨基酸及其制备方法、应用
US20230313180A1 (en) Multimeric antibody platform and methods of making and using multimeric antibody platform
US20230295613A1 (en) Long chain carbon and cyclic amino acids substrates for genetic code reprogramming
WO2017213158A1 (fr) Complexe d'affichage de ribosome et procédé de production associé
CN113651756A (zh) 一种遗传编码的光交联非天然氨基酸盐及其制备方法与应用
US9790305B2 (en) Site specifically incorporated initiator for growth of polymers from proteins
CN103571804B (zh) 3-吡唑基酪氨酸翻译系统及其应用
CN117730076A (zh) 含有四嗪部分的氨基酸
Campbell et al. Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers
Duca et al. Synthesis of bisaminoacylated pdCpAs and tandemly activated transfer RNAs
JP2011239686A (ja) ペプチド及びペプチド誘導体の製造方法
Wang et al. Ring-opening Polymerization of Unprotected Hydroxyproline N-Carboxyanhydride for the Facile Synthesis of Linear and Branched Poly-L-hydroxyproline
Radtke Unnatural Amino Acids for Protein Modification by Oxime Ligation
Lee Expanding the Genetic Code for Synthesis of Proteins with Native Biological Modifications and Novel Chemical, Biophysical Probes
Huang Genetic Incorporation of Noncanonical Amino Acids into Proteins for Protein Function Investigation
Wu Synthesis of Proteins with Homogenous Chemical and Posttranslational Modifications
Talukder Small Molecules as Probes of Biological Systems
Averick Preparation of Precise Bioconjugates Using Atom Transfer Radical Polymerization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11824191

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11824191

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载