# US 11,897,917 B2 · Bioconjugation of polypeptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,897,917 B2 - Document ID: US11897917B2 - Status: Granted - Kind: B2 - Application number: 16/650,357 - Filed: 2017-09-27 - Published: 2020-10-08 - Granted: 2024-02-13 - Earliest claimed: 2017-09-27 - Assignees: The University of York - Applicants: The University of York - Inventors: Martin A. Fascione; Richard J. Spears; Robin Brabham; Darshita Budhadev; Tessa Keenan - Web page: https://patentgrep.com/patent/US11897917B2 ## CPC - A61K 47/542: {Carboxylic acids, e.g. a fatty acid or an amino acid} - C07K 1/1075: {by covalent attachment of amino acids or peptide residues} - B01J 27/24: Nitrogen compounds - C07D 205/04: having no double bonds between ring members or between ring members and non-ring members - C07D 207/06: with radicals, containing only hydrogen and carbon atoms, attached to ring carbon atoms - C07D 207/16: Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals - C07D 211/06: having no double bonds between ring members or between ring members and non-ring members - C07D 295/027: containing only one hetero ring - C07D 403/04: directly linked by a ring-member-to-ring-member bond - C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids} - C07K 1/13: Labelling of peptides - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) ## Abstract Certain embodiments of the present invention relate to methods of forming and manipulating bioconjugates. Particularly, but not exclusively certain embodiments relate to methods of reversible carbon-carbon bond bioconjugation using aldol based chemical reactions at physiological conditions. ## Claims ### Claim 1 A method of modifying a polypeptide, the method comprising: a. contacting a first polypeptide comprising a first aldehyde moiety with: i. an aldehyde donor molecule comprising a second aldehyde moiety; and ii. a catalyst molecule, under conditions sufficient for an aldol reaction to occur between the first aldehyde group and the second aldehyde group, such that a second polypeptide is formed; wherein the second polypeptide comprises a third aldehyde moiety which is a beta-hydroxy aldehyde moiety and further wherein the catalyst molecule comprises a secondary amine moiety or an acid addition salt thereof. ### Claim 2 (depends on 1) The method of claim 1, wherein the polypeptide comprises at least one accessible aldehyde moiety. ### Claim 3 (depends on 2) The method according to claim 2, wherein the catalyst molecule comprises a cyclic secondary amine molecule. ### Claim 4 (depends on 3) The method according to claim 3 wherein the catalyst molecule comprises a secondary amine moiety of the formula HNR1R2 wherein R1 and R2 together with the nitrogen atom to which they are attached form a 3 to 7 membered ring. ### Claim 5 (depends on 4) The method according to claim 4, wherein the catalyst molecule is selected from the group consisting of: ### Claim 6 (depends on 4) The method according to claim 4, wherein the catalyst molecule comprises a substituted cyclic secondary amine molecule, wherein optionally the substituted cyclic secondary amine molecule is substituted pyrrolidine. ### Claim 7 (depends on 6) The method according to claim 6, wherein the catalyst molecule comprises a tetrazole-substituted cyclic secondary amine molecule. ### Claim 8 (depends on 7) The method according to claim 7, wherein optionally the tetrazole-substituted cyclic molecule is pyrrolidine substituted with tetrazole. ### Claim 9 (depends on 8) The method according to claim 8, wherein the tetrazole-substituted cyclic molecule is 2S-tetrazolylpyrrolidine. ### Claim 10 (depends on 1) The method according to claim 1, which comprises contacting the first polypeptide, the aldehyde donor molecule and the catalyst molecule in a buffer solution having a pH of between about 7 and 8 and/or at a temperature of between about 35° C. and about 38° C. ### Claim 11 (depends on 1) The method according to claim 1, wherein the first polypeptide comprises the first aldehyde moiety at an internal sequence of the first polypeptide; at a terminal loop, a C-terminus, or an N-terminus of the first polypeptide; on a solvent-accessible region of the first polypeptide when folded; and/or at a site of post-translational modification of the first polypeptide that is native or non-native to the amino acid sequence of the first polypeptide. ### Claim 12 (depends on 1) The method according to claim 1, wherein the second polypeptide comprises the third aldehyde moiety which is a beta hydroxy aldehyde moiety at an internal sequence of the second polypeptide; at a terminal loop, a C-terminus, or an N-terminus of the second polypeptide; on a solvent-accessible region of the second polypeptide when folded; and/or at a site of post-translational modification of the second polypeptide that is native or non-native to the amino acid sequence of the first polypeptide. ### Claim 13 (depends on 1) The method according to claim 1, wherein the aldehyde donor moiety further comprises a functional moiety and the method comprises incorporating the functional moiety into the second polypeptide, wherein the functional moiety is selected from the group consisting of a fluorescent label, an affinity tag, a conjugation moiety, a water soluble polymer and a targeting moiety and wherein the method comprises, if the functional moiety is a conjugation moiety, conjugating a further functional moiety to the second polypeptide via the conjugation moiety. ### Claim 14 (depends on 13) The method according to claim 13, which comprises incorporating the functional moiety into the second polypeptide at the alpha carbon position of the third aldehyde moiety. ### Claim 15 (depends on 1) The method according to claim 1, which comprises, prior to step (a), contacting a polypeptide with an oxidising reagent to generate the first aldehyde moiety on the polypeptide and form the first polypeptide, wherein: (i) the oxidising reagent is periodate and wherein the method comprises oxidising a N-terminal serine residue or a N-terminal threonine residue to form an alpha-oxy aldehyde moiety, or (ii) the oxidising reagent is pyridoxal-5-phosphate (PLP) and the method comprises oxidising a N-terminal lysine residue to form an alpha-oxy aldehyde moiety. ### Claim 16 (depends on 1) The method according to claim 1, which comprises, prior to step (a), a step of modifying a polypeptide to incorporate an aldehyde moiety at a pre-determined location to form the first polypeptide. ### Claim 17 (depends on 1) The method according to claim 1, further comprising a step of: a). contacting the second polypeptide with: i. a substituted or unsubstituted aniline catalyst molecule; and ii. a substituted hydroxylamine molecule, wherein the substituent of the hydroxylamine molecule comprises a further functional moiety, under conditions sufficient for an oxime reaction to occur between the substituted hydroxylamine molecule and the beta-hydroxy aldehyde moiety to form a third polypeptide comprising the further function moiety. ### Claim 18 (depends on 17) A method according to claim 17, wherein the substituted hydroxylamine molecule has a structure of a formula selected from the group consisting of: wherein one or more, but not all of Rx, Ry and Rz is H. ### Claim 19 (depends on 17) A method according to claim 17, wherein the aniline catalyst molecule is selected from the group consisting of: ### Claim 20 (depends on 17) A method according to claim 17, which comprises contacting the polypeptide, the aniline catalyst in a buffer solution, wherein the buffer solution has a concentration of between at or below 100 mM and/or wherein the method comprises contacting the second polypeptide, the substituted or unsubstituted aniline catalyst molecule and the substituted hydroxylamine molecule in a buffer solution having a pH of between about 6.5 to about 8. ### Claim 21 (depends on 20) A method according to claim 20, which comprises contacting the second polypeptide, the substituted or unsubstituted aniline catalyst molecule and the substituted hydroxylamine molecule in a buffer solution having a pH of between about 7.3 to about 7.6.