WO2019219002A1 - Procédé de fonctionnalisation n-terminale de polypeptides et produits associés - Google Patents

Procédé de fonctionnalisation n-terminale de polypeptides et produits associés Download PDF

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WO2019219002A1
WO2019219002A1 PCT/CN2019/086825 CN2019086825W WO2019219002A1 WO 2019219002 A1 WO2019219002 A1 WO 2019219002A1 CN 2019086825 W CN2019086825 W CN 2019086825W WO 2019219002 A1 WO2019219002 A1 WO 2019219002A1
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polypeptide
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Sui-Yi Kwok
Norman Fung-Man WAI
Man-Kin Wong
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Vision Global Holdings Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids

Definitions

  • the present disclosure relates to methods for functionalization of polypeptides, such as proteins and antibodies, and products thereof.
  • Covalent conjugation of a biomolecule to a therapeutic agent opens the door to a new generation biotherapeutic agents with improved pharmaceutical properties.
  • conjugation of a therapeutic agent to biomolecules as done with antibody-drug conjugates, polymer conjugates, immobilized biomaterials and controlled release of bioconjugates are excellent demonstrations of the potential of protein-based conjugates to improve efficacy, pharmacokinetics, biodistribution and safety of bioconjugates.
  • (Gemtuzumab ozogamicin) the first antibody-drug conjugate approved by the FDA in 2000, includes a non-specific linkage of the cytotoxic agent at one or more free amines present on the antibody.
  • Mylotarg was withdrawn from the market in 2010, and then a post-marketing study for insufficient clinical benefit and adverse side-effects associated with its heterogeneity was conducted. It was found that Mylotarg’s drug-antibody ratio strongly impacted Mylotarg's therapeutic efficacy with a large portion of the unlabeled antibody ( ⁇ 50%) serving as a competitive inhibitor to the antibody-drug conjugate for cellular uptake, which is of importance for its activity.
  • the statistical conjugation of the cytotoxic drug can affect the therapeutic window and safety of the antibody-drug conjugate.
  • Additional, multiple drug-loaded forms of antibody-drug conjugates (usually with drug-antibody ratio from 0 to 8) can have distinct pharmacokinetics in vivo and differ in clinical performance, immunogenicity, clearance and stability.
  • Purifying antibody-drug conjugates can be particularly challenging in instances where there are multiple sites on the antibody capable of forming bonds with the drug.
  • the side chains of 70 out of 88 lysines in (Trastuzumab emtansine) are conjugated, leading to the generation of a complex mixture of antibody-drug conjugate species with significant differences in clinical performance and pharmacokinetic properties.
  • the N-terminal ⁇ -amino group of polypeptide is a unique reactive site, which can render site-specific selectivity in polypeptide bioconjugation possible. Compared with the side chain amine of lysine with pKa of 10, pKa of N-terminal ⁇ -amino group ranges from 6 to 8. Additionally, the N-terminal residues are usually exposed on the surface of proteins over an estimated 80%of cases.
  • N-terminal modification of a polypeptide with a therapeutic agent e.g., chemotherapeutic/radiotherapeutic agent
  • a therapeutic agent e.g., chemotherapeutic/radiotherapeutic agent
  • a highly N-terminal selective chemical linker is therefore a powerful approach to polypeptide modification as most proteins contain a unique N-terminus in their amino acid sequences and no extra bioorthogonal handles, such as ketones, aldehydes, azides and alkynes, are required to be genetically and/or chemically incorporated into the protein before coupling with a therapeutic agent (e.g., chemotherapeutic/radiotherapeutic agent) .
  • chemotherapeutic/radiotherapeutic agent e.g., chemotherapeutic/radiotherapeutic agent
  • compositions and methods useful for N-terminal selective functionalization of polypeptides and intermediates and products thereof are highly N-terminal selective and are capable of yielding N-terminal functionalized polypeptides with site selectivity of 99%or greater.
  • Most protein-based therapeutic agents are heterogeneous mixtures. The method described herein provides a reliable tool to reduce the overall product heterogeneity, and hence deliver more consistent pharmaceutical and/or therapeutic properties.
  • a method for preparing a functionalized polypeptide comprising the step of: contacting a polypeptide comprising an N-terminal amino acid represented by the structure shown below:
  • R 1 is the side chain of the N-terminal amino acid of the polypeptide and the N-terminal amino acid is a natural amino acid or a non-natural amino acid; with a compound of Formula 1:
  • n 1, 2, or 3;
  • R 3 is –A–R 4 , wherein A is a linker or absent;
  • R 4 is selected from the group consisting of hydroxyl, thiol, aldehyde, ketone, maleimide, N-hydroxysuccinimide ester, imidoester, haloacetyl, pyridyl disulfide, alkyne, azide, nitrone, nitrile oxide, norbornene, oxanorbornadiene, tetrazine, cyclooctene, isocyanide, quadricyclane, and bis (dithiobenzil) nickel (II) ; and
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl;
  • N* is the N-terminal nitrogen of the functionalized polypeptide.
  • m is 1 and R 2 is hydrogen, -OR 5 , or halide.
  • n is a whole number selected from 1-10 and p is a whole number selected from 1-4.
  • R 4 is maleimide, azide, alkyne, hydroxyl, norbornene, or cyclooctene.
  • n is a whole number selected from 1-4;
  • R 2 is hydrogen, alkyl, or –OR 5 ;
  • R 4 is maleimide or alkyne
  • R 5 is alkyl
  • the functionalized polypeptide comprises at least one lysine residue and less than 5%of the side chains of the at least one lysine residue undergo a reaction with the compound of Formula 3.
  • polypeptide is a protein, enzyme, antibody, glycoprotein, or lipoprotein.
  • the method of the seventh embodiment of the first aspect wherein the protein is a hemoglobin protein.
  • hemoglobin protein is a hemoglobin tetramer, hemoglobin dimer, or hemoglobin monomer.
  • hemoglobin tetramer is a cross-linked hemoglobin tetramer.
  • the method of the first aspect further comprising the step of contacting the functionalized polypeptide with an active agent comprising a mercaptan or conjugate salt thereof.
  • the active agent is a chemotherapeutic agent, a radiotherapeutic agent, a fluorescent dye, or affinity tag.
  • a method for preparing a functionalized polypeptide comprising the step of: contacting a polypeptide comprising an N-terminal amino acid represented by the structure shown below:
  • R 1 is the side chain of the N-terminal amino acid of the polypeptide and the N-terminal amino acid is a natural amino acid or a non-natural amino acid; with a compound of Formula 3:
  • n 1, 2, or 3;
  • A is a linker or is absent
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl;
  • R 6 is an active agent
  • R 7 is a spacer or absent
  • R 8 is selected from the group consisting of:
  • ⁇ indicates the position of a covalent bond with A and ⁇ indicates the position of a covalent bond with R 7 ;
  • N* is the N-terminal nitrogen of the functionalized polypeptide.
  • the spacer is selected from the group consisting of
  • R 6 indicates the position of a covalent bond with R 8 ; each instance of n is independently a whole number selected from 1-20; p is an integer selected from 0-20; and R for each instance is independently selected from hydrogen, alkyl, cycloalkyl, and aryl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • ⁇ indicates the position of a covalent bond with R 8 ; each instance of n is independently a whole number selected from 1-20; p is an integer selected from 0-20; and R for each instance is independently selected from hydrogen, alkyl, cycloalkyl, and aryl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • n is a whole number selected from 1-4.
  • the active agent is a chemotherapeutic agent, a radiotherapeutic agent, a fluorescent dye, or an affinity tag.
  • a functionalized polypeptide comprising an N-terminal amino acid represented by the structure of Formula 2:
  • n 1, 2, or 3;
  • N* is the N-terminal nitrogen of the functionalized polypeptide
  • R 1 is the side chain of the N-terminal amino acid of the functionalized polypeptide and the N-terminal amino acid is a natural amino acid or a non-natural amino acid;
  • R 3 is selected from the group consisting of –R 4 or –A–R 4 , wherein A is a linker;
  • R 4 is selected from the group consisting of hydroxyl, thiol, aldehyde, ketone, maleimide, N-hydroxysuccinimide ester, imidoester, haloacetyl, pyridyl disulfide, alkyne, azide, nitrone, nitrile oxide, norbornene, oxanorbornadiene, tetrazine, cyclooctene, isocyanide, quadricyclane, and bis (dithiobenzil) nickel (II) ; and
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl.
  • the functionalized peptide of the third aspect wherein the functionalized polypeptide is a protein, enzyme, antibody, glycoprotein, or lipoprotein.
  • the functionalized peptide of the third aspect wherein the functionalized polypeptide is hemoglobin.
  • a fifth embodiment of the third aspect provided herein is the functionalized peptide of the fourth embodiment of the third aspect, wherein the linker is ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , or ** – (OCR 2 CR 2 ) n – *** .
  • n is a whole number selected from 1-10 and p is a whole number selected from 1-4.
  • a seventh embodiment of the third aspect provided herein is the functionalized peptide of the third aspect, wherein R 4 is maleimide, azide, alkyne, hydroxyl, norbornene, or cyclooctene.
  • n is a whole number selected from 1-4;
  • R 2 is hydrogen, alkyl, or –OR 5 ;
  • R 4 is maleimide or alkyne
  • R 5 is alkyl
  • Figure 1 depicts the design approach for construction of an exemplary N-terminal selective hemoglobin-based therapeutic agent.
  • One or more active agents e.g., therapeutic drugs
  • Figure 2 depicts the structure of the exemplary N-terminal selective bifunctional linkers comprising an N-terminal selective functional group and a functional group for active agent (e.g., therapeutic) attachment, such as maleimide, thiol, aldehyde, ketone, N-hydroxysuccinimide ester, imidoester, haloacetyl, pyridyl disulfide, alkyne, azide, nitrone, nitrile oxide, norbornene, oxanorbornadiene, tetrazine, cyclooctene, isocyanide, quadricyclane and bis (dithiobenzil) nickel (II) ) .
  • active agent e.g., therapeutic
  • Figure 3A depicts an exemplary synthetic sequences for two-step N-terminal modification of hemoglobin followed by reaction with a thiol containing active agent and one-step N-terminal modification with a reagent that includes the linked active agent.
  • Figure 3B depicts exemplary functionalized polypeptides comprising (1) alkyl, (2) aryl, (3) polyethylene glycol (PEG) , (4) ether, (5) thioether, (6) amide, (7) ester, (8) N-carbamate, (9) carbonate, (10) acylamino, (11) acyloxy, and (12) O-carbamate linkages.
  • Figure 4 depicts the N-terminal modification of hemoglobin with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde thereby forming a functionalized hemoglobin for subsequent azide-alkyne [3+2] cycloaddition reaction with a fluorescent dye-, affinity-tag-, therapeutic drug-or active agent-linked azide.
  • Figure 5 depicts an exemplary synthetic scheme for the synthesis of polyethylene (PEG) linked maleimide containing 2-ethynylbenzaldehyde according to certain embodiments of the present disclosure.
  • Figure 6 depicts an exemplary synthetic scheme for an alkyl linked maleimide containing 2-ethynylbenzaldehyde according to certain embodiments of the present disclosure.
  • Figure 7 depicts an exemplary synthetic sequence for the preparation of an exemplary active agent conjugate by Michael reaction of an active agent comprising a thiol or conjugate base thereof with a maleimide linked functionalized polypeptide according to certain embodiments of the present disclosure.
  • Figure 8 depicts exemplary 2-ethynylbenzaldehydes analogs that can be used in connection with the methods described herein: (A) maleimide linked 2-ethynylbenzaldehyde (e.g., PEG and/or alkyl linked) , (B) benzylmercaptan linked 2-ethynylbenzaldehyde and (C) N-Boc-L-cysteine methyl ester linked 2-ethynylbenzaldehyde, (D) alkyne linked 2-ethynylbenzaldehyde, and (E) fluorescein linked 2-ethynylbenzldehyde.
  • A maleimide linked 2-ethynylbenzaldehyde (e.g., PEG and/or alkyl linked)
  • B benzylmercaptan linked 2-ethynylbenzaldehyde
  • C N-Boc-L-cysteine
  • Figure 9A depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of hemoglobin with an N-terminal amine depicted.
  • Figure 9B depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of hemoglobin N-terminal functionalized with 2-ethynylbenzaldehyde OCH 2 CH 2 CH 2 linked maleimide according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 9C depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of hemoglobin N-terminal functionalized with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 9D depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of hemoglobin N-terminal functionalized with 2-ethynylbenzldehyde OCH 2 CH 2 CH 2 O conjugated fluorescein according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 9E depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of the product of the [3+2] cycloaddition reaction of hemoglobin N-terminal functionalized with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde and a modified rhodamine comprising a linked azide according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 9F depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of hemoglobin N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 9G depicts an expanded view of the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of hemoglobin N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 10A depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of lysozyme with the N-terminal amine depicted.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 10B depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of lysozyme N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 10C depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of lysozyme N-terminal functionalized with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 10D depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of lysozyme N-terminal functionalized with 2-ethynylbenzldehyde OCH 2 CH 2 CH 2 O conjugated fluorescein according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 11A depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of ribonuclease A (RNase A) with the N-terminal amine depicted.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 11B depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of RNase A N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 11C depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of RNase A N-terminal functionalized with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 11D depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of RNase A N-terminal functionalized with 2-ethynylbenzldehyde OCH 2 CH 2 CH 2 O conjugated fluorescein according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 12A depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of tryptic lysozyme N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 12B depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of tryptic lysozyme N-terminal functionalized with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 12C depicts liquid chromatography–mass spectrometry-mass spectrometry LC-MS-MS results for tryptic lysozyme N-terminal functionalized with 2-ethynylbenzldehyde OCH 2 CH 2 CH 2 O conjugated fluorescein.
  • Figure 12D depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of RNase A N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 12E depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of tryptic RNase A N-terminal functionalized with 2-ethynyl-5- (prop-2-yn-1-yloxy) benzaldehyde according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 12F depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of tryptic RNase A N-terminal functionalized with 2-ethynylbenzldehyde OCH 2 CH 2 CH 2 O conjugated fluorescein according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 13A depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of YTSSSKNVVR (SEQ ID NO: 1) .
  • Figure 13B depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of YTSSSKNVVR (SEQ ID NO: 1) N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 13C depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of YTSSSKNVVR (SEQ ID NO: 1) N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with N-Boc-L-cysteine methyl ester according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 13D depicts the liquid chromatography–mass spectrometry (LC-MS) mass chromatogram of YTSSSKNVVR (SEQ ID NO: 1) N-terminal functionalized with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • Figure 14A depicts the liquid chromatography (LC) results for the selectivity of the N-terminal functionalization of YTSSSKNVVR (SEQ ID NO: 1) with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide according to certain embodiments described herein.
  • Figure 14B depicts the liquid chromatography (LC) results for the selectivity of the N-terminal functionalization of YTSSSKNVVR (SEQ ID NO: 1) with 2-ethynylbenzaldehyde (OCH 2 CH 2 ) 2 linked maleimide conjugated with benzylmercaptan according to certain embodiments described herein.
  • LC-MS liquid chromatography–mass spectrometry
  • Figure 16 depicts the LC-MS results of stability tests of hemoglobin N-terminal functionalized with 2, 5-diethynylbenzaldehyde (2n) stored for 1-8 weeks at 4 °C.
  • antibody as used herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
  • antibodies are polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, Fv, Fab fragments, F (ab’ ) fragments, F(ab’ ) 2 fragments, anti-idiotypic (anti-Id) antibodies, CDR's , and epitope-binding fragments of any of the above which immunospecifically bind to an antigen, such as cancer cell antigens, viral antigens, or microbial antigens.
  • an antigen such as cancer cell antigens, viral antigens, or microbial antigens.
  • amino acid refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • non-natural amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine; other terms that may be used synonymously with the term “non-natural amino acid” is “non-naturally encoded amino acid, " “unnatural amino acid, " “non-naturally-occurring amino acid, “ and the like.
  • the term “non-natural amino acid” includes, but is not limited to, amino acids that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, as well as amino acids that amino acids in which the amino group is attached at the ⁇ or ⁇ carbon.
  • hemoglobin refers generally to the protein contained within red blood cells that transports oxygen.
  • Each molecule of hemoglobin has 4 subunits, 2 ⁇ chains and 2 ⁇ chains, which are arranged in a tetrameric structure.
  • Each subunit also contains one heme group, which is the iron-containing center that binds oxygen.
  • each hemoglobin molecule can bind 4 oxygen molecules.
  • modified hemoglobin includes, but is not limited to, hemoglobin altered by a chemical reaction such as intra-and inter-molecular cross-linking, genetic manipulation and polymerization.
  • hemoglobin by itself refers both to native, unmodified, hemoglobin, as well as modified hemoglobin.
  • protein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof.
  • polypeptide or “peptide” includes amino acid polymers of any length including full length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called an oligopeptide.
  • amino acid refers to any of the twenty naturally occurring amino acids.
  • heteroatom is art-recognized and refers to an atom of any element other than carbon or hydrogen.
  • Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
  • alkyl is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C 1 -C 30 for straight chain, C 3 -C 30 for branched chain) , and alternatively, about 20 or fewer.
  • cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.
  • lower alkyl refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure.
  • lower alkenyl and “lower alkynyl” have similar chain lengths.
  • aralkyl is art-recognized and refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group) .
  • alkenyl and alkynyl are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • aryl is art-recognized and refers to 5-, 6-and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl heterocycles or "heteroaromatics.
  • the aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF 3 , -CN, or the like.
  • substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings” ) wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • ortho, meta and para are art-recognized and refer to 1, 2-, 1, 3-and 1, 4-disubstituted benzenes, respectively.
  • the names 1, 2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
  • heterocyclyl refers to 3-to about 10-membered ring structures, alternatively 3-to about 7-membered rings, whose ring structures include one to four heteroatoms.
  • Heterocycles may also be polycycles.
  • Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, o
  • the heterocyclic ring may be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxy
  • optionally substituted refers to a chemical group, such as alkyl, cycloalkyl aryl, and the like, wherein one or more hydrogen may be replaced with a with a substituent as described herein, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF 3 , -CN, or the like
  • polycyclyl or “polycyclic group” are art-recognized and refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings” . Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle may be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, si
  • carrier is art-recognized and refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
  • nitro is art-recognized and refers to -NO 2 ;
  • halogen is art-recognized and refers to -F, -Cl, -Br or -I;
  • sulfhydryl is art-recognized and refers to -SH;
  • hydroxyl means -OH;
  • sulfonyl and “sulfone” is art-recognized and refers to -SO 2 -.
  • Halide designates the corresponding anion of the halogens.
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action.
  • Classes of chemotherapeutic agents include, but are not limited to: alkyating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors.
  • Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy.
  • fluorescent dye refers interchangeably to molecules, groups or radicals which are fluorescent.
  • fluorescent as applied to a molecule or compound is used to refer to the property of the compound of absorbing energy (such as UV, visible or IR radiation) and re-emitting at least a fraction of that energy as light over time.
  • Fluorescent dyes include, but are not limited to small molecules, proteins and macromolecular complexes.
  • the methods described herein are capable of N-terminal selective functionalization, under mild conditions (e.g., pH 6.3 at 37 °C) , of the N-terminal amine by reaction of a polypeptide with 2-ethynylbenzaldehyde or an analog thereof thereby forming an N-terminal functionalized isoquinolinium with selectivity up to 99%or greater for the N-terminal amine as shown in the general reaction sequence below.
  • the methods described herein can afford the N-terminal functionalized polypeptide with yields of 86%or greater and moderate to excellent N-terminal selectivity (12: 1 to >99: 1) .
  • the resulting isoquinolonium is relatively stable and can be stored for extended periods of time without substantial degradation (See Figure 17, which shows LCMS data for N-terminal functionalized hemoglobin stored at 4 °C for 1-8 weeks storage) .
  • the methods described herein can thus be used as a means of selectively functionalizing a polypeptide and controlling the extent of functionalization (e.g., at least partially limiting the functionalization reaction to e.g., one per polypeptide) .
  • the methods described herein can be used as a means for selectively conjugating an active agent, such as chemotherapeutic agent, a radiotherapeutic agent, a fluorescent dye, or affinity tag, by covalent attachment either directly or indirectly (e.g., via a linker or linker and spacer) to the 2-ethynylbenzaldehyde, to the N-terminal of a polypeptide in a selective manner.
  • an active agent such as chemotherapeutic agent, a radiotherapeutic agent, a fluorescent dye, or affinity tag
  • N-terminal selective conjugation of an active agent to a polypeptide using the methods described herein can be accomplished using a one-step or two-step method.
  • the polypeptide can be a protein, enzyme, antibody, glycoprotein, or lipoprotein.
  • the polypeptide is hemoglobin.
  • the hemoglobin is derived from substantially any mammalian source. Exemplary sources of hemoglobin include common livestock animals, e.g., cows, pigs, sheep and the like and humans. In certain embodiments, the hemoglobin is recombinant hemoglobin.
  • the polypeptide is a hemoglobin tetramer, dimer, or subunit thereof.
  • the antibody is an intact antibody, single chain antibody, Fv, Fab fragment, F (ab’ ) fragment, or F (ab’ ) 2 fragment.
  • Chemotherapeutic agents suitable for use in connection with the methods described herein include any chemotherapeutic agent known to those skilled in the art including, but not limited to erlotinib ( Genentech/OSI Pharm. ) , docetaxel ( Sanofi-Aventis) , 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8) , gemcitabine ( Lilly) , PD-0325901 (CAS No. 391210-10-9, Pfizer) , cisplatin (cis-diamine, dichloroplatinum (II) , CAS No. 15663-27-1) , carboplatin (CAS No.
  • paclitaxel Bristol-Myers Squibb Oncology, Princeton, N.J.
  • trastuzumab Genentech
  • temozolomide (4-methyl-5-oxo-2, 3, 4, 6, 8-pentazabicyclo [4.3.0] nona-2, 7, 9-triene-9-carboxamide, CAS No. 85622-93-1, Schering Plough)
  • tamoxifen (Z) -2- [4- (1, 2-diphenylbut-1-enyl) phenoxy] -N, N-dimethyl-ethanamine, )
  • doxorubicin Akti-1/2, HPPD, and rapamycin doxorubicin Akti-1/2, HPPD, and rapamycin.
  • chemotherapeutic agents include: oxaliplatin ( Sanofi) , bortezomib ( Millennium Pharm. ) , sutent ( SU1 1248, Pfizer) , letrozole ( Novartis) , imatinib mesylate ( Novartis) , XL-518 (Mek inhibitor, Exelixis, WO 2007/044515) , ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca) , SF-1 126 (PI3K inhibitor, Semafore Pharmaceuticals) , BEZ-235 (PI3K inhibitor, Novartis) , XL-147 (PI3K inhibitor, Exelixis) , PTK787/ZK 222584 (Novartis) , fulvestrant ( AstraZeneca) , leucovorin (folinic acid) , rapamycin (sirolimus, Wyeth)
  • dynemicin dynemicin A
  • bisphosphonates such as clodronate
  • an esperamicin as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores
  • aclacinomysins actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , epirubicin, esorubicin, i
  • Fluorescent dyes are well known in the art, and include, but are not limited to xanthene dyes, e.g., fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC) , 6-carboxyfluorescein, 6-carboxy-2', 4', 7', 4, 7-hexachlorofluorescein (HEX) , 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein, N, N, N', N'-tetramethyl6-carboxyrhodamine, 6-carboxy-xrhodmnine, 5-carboxyrhodamine-6G, 6-carboxyrhodamine-6G, and rhodmnine 110; cyanine dyes, e.g.
  • Cy3, Cy5 and Cy7 dyes include coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes.
  • Affinity tags suitable for use in connection with the methods described herein include any affinity tag known to those skilled in the art including, but not limited to Biotin-tag, epitope-tag, FLAG-tag, poly (His) tag, streptag II, HA-tag, softag1, softag 3, c-myc, T7-tag, S-tag, elastin-like peptides, chitin-binding domain, thioredoxin, xylanase 10A, glutathione S-transferase, maltose binding protein, NusA, and the like.
  • affinity tag known to those skilled in the art including, but not limited to Biotin-tag, epitope-tag, FLAG-tag, poly (His) tag, streptag II, HA-tag, softag1, softag 3, c-myc, T7-tag, S-tag, elastin-like peptides, chitin-binding domain, thior
  • Active agents suitable for use in connection with the methods described herein can also include crosslinking agents, which can be used to covalently bond with other chemical or biological moieties (N-hydroxysuccinimide (NHS) and sulfo-NHS, maleimide, iodoacetyl groups or pyridyl disulfides, primary amines, carbonyls, hydrazines and alkoxyamines.
  • crosslinking agents can be used to covalently bond with other chemical or biological moieties (N-hydroxysuccinimide (NHS) and sulfo-NHS, maleimide, iodoacetyl groups or pyridyl disulfides, primary amines, carbonyls, hydrazines and alkoxyamines.
  • chemical modifications may be required to introduce the necessary functionality in the chemotherapeutic agent, a radiotherapeutic agent, a fluorescent dye, or affinity tag in order to provide a functional group capable of conjugating the chemotherapeutic agent, a radiotherapeutic agent, a fluorescent dye, or affinity tag to the 2-ethynylbenzaldehyde analogs described herein (e.g., the compound of Formula 1 and the compound of Formula 3) .
  • the choice of such functional group, the site of incorporation and the methods for incorporating the functional group in the molecule is well within the skill of a person of ordinary skill in the art.
  • reactive functionality can be incorporated into the 2-ethynylbenzaldehyde analog.
  • Such reactive functionality can have orthogonal reactivity with and/or a slower reaction rate than the 2-ethynylbenzaldehyde analog with the N-terminal amine.
  • the 2-ethynylbenzaldehyde analog can be represented by the compound of Formula 1:
  • n 1, 2, or 3;
  • R 3 is selected from the group consisting of –R 4 or –A–R 4 , wherein A is a linker;
  • R 4 is selected from the group consisting of hydroxyl, thiol, aldehyde, ketone, maleimide, N-hydroxysuccinimide ester, imidoester, haloacetyl, pyridyl disulfide, alkyne, azide, nitrone, nitrile oxide, norbornene, oxanorbornadiene, tetrazine, cyclooctene, isocyanide, quadricyclane, and bis (dithiobenzil) nickel (II) ; and
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl.
  • the moiety R 3 comprises reactive functional groups that can be further reacted with an active agent comprising at least one complimentary reactive functional group.
  • m is 1.
  • R 2 independently selected from is halide, cyano, nitro, hydrogen, or alkyl.
  • m is 1 and R 2 is halide, cyano, nitro, hydrogen, or alkyl.
  • R 2 is hydrogen.
  • R 4 can be covalently attached directly to the 2-ethynylbenzaldehyde analog or can be covalently attached via a linker.
  • the linker can be any linker known to those of skill in the art. The selection of which is well within the skill of a person of skill in the art. In the broadest sense, a linker is a divalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches two groups (e.g., R 4 and the 2-ethynylbenzaldehyde analog) .
  • the linker A comprises 2 or 3.
  • the linker can be represented by –A 1 –A 2 –A 3 –, wherein each of A 1 , A 2 , and A 3 is independently selected from the linkers recited in the definition of A herein; or A 3 is absent.
  • ** indicates the position of a covalent bond with the moiety:
  • n is a whole number selected from the group consisting of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, and 1-3.
  • p is an integer selected from the group consisting of 0-10, 0-9, 0-8, 0-7, 0-6, 1-5, 1-4, and 1-3.
  • n is a whole number selected from the group consisting of 1-5, 1-4, and 1-3; and p is an integer selected from the group consisting of 0-6, 1-5, 1-4, and 1-3.
  • R for each instance is independently selected from hydrogen and alkyl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • R for each instance is independently selected from hydrogen and C 1 -C 6 alkyl.
  • R is hydrogen.
  • the linker is at least one moiety selected from the group consisting of ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , ** –O (CR 2 ) n O– *** , and ** – (OCR 2 CR 2 ) n – *** .
  • R is hydrogen or C 1 -C 6 alkyl. In certain embodiments of the compound of Formula 1, R is hydrogen.
  • R 4 is maleimide, alkyne, azide, norbornene, or cyclooctene. In certain embodiments of the compound of Formula 1, R 4 is maleimide, alkyne, or azide.
  • m is 1;
  • R 2 is halide, cyano, nitro, hydrogen, or alkyl; the linker is absent, ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , ** –O (CR 2 ) n O– *** , or ** – (OCR 2 CR 2 ) n – *** , wherein R is hydrogen and n is a whole number selected from 1-4; and R 4 is maleimide, alkyne, azide, norbornene, or cyclooctene.
  • m is 1; R 2 is hydrogen; the linker is absent, ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , ** –O (CR 2 ) n O– *** , or ** – (OCR 2 CR 2 ) n – *** ; R 4 is maleimide, alkyne, or azide; R is hydrogen; and n for each instance is independently selected from 1, 2, 3, or 4.
  • the compound of Formula 1 is selected from the group consisting of:
  • n is a whole number selected from 1-4;
  • R 2 is hydrogen, alkyl, or –OR 5 ;
  • R 4 is maleimide or alkyne
  • R 5 is alkyl
  • the compound of Formula 1 is selected from the group consisting of:
  • the compound of Formula 1 can be brought into contact with a polypeptide comprising an N-terminal amino acid under reaction conditions as described herein thereby forming an N-terminal functionalized polypeptide comprising an N-terminal amino acid represented by the structure of Formula 2:
  • n 1, 2, or 3;
  • N* is the N-terminal nitrogen of the functionalized polypeptide
  • R 1 is the side chain of the N-terminal amino acid of the functionalized polypeptide and the N-terminal amino acid is a natural amino acid or a non-natural amino acid;
  • R 3 is selected from the group consisting of –R 4 or –A–R 4 , wherein A is a linker;
  • R 4 is selected from the group consisting of hydroxyl, thiol, aldehyde, ketone, maleimide, N-hydroxysuccinimide ester, imidoester, haloacetyl, pyridyl disulfide, alkyne, azide, nitrone, nitrile oxide, norbornene, oxanorbornadiene, tetrazine, cyclooctene, isocyanide, quadricyclane, and bis (dithiobenzil) nickel (II) ; and
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl.
  • m is 1.
  • R 2 independently selected from is halide, cyano, nitro, hydrogen, or alkyl.
  • m is 1 and R 2 is halide, cyano, nitro, hydrogen, or alkyl.
  • R 2 is hydrogen.
  • R 4 can be covalently attached directly to the 2-ethynylbenzaldehyde analog or can be covalently attached via a linker.
  • the linker can be any linker known to those of skill in the art. The selection of which is well within the skill of a person of skill in the art. In the broadest sense, a linker is a divalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches two groups (e.g., R 4 and the 2-ethynylbenzaldehyde analog) .
  • the linker A comprises 2 or 3.
  • the linker can be represented by –A 1 –A 2 –A 3 –, wherein each of A 1 , A 2 , and A 3 is independently selected from the linkers recited in the definition of A herein; or A 3 is absent.
  • ** indicates the position of a covalent bond with the moiety:
  • the linker is at least one moiety selected from the group consisting of ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , ** –O (CR 2 ) n O– *** , and ** – (OCR 2 CR 2 ) n – *** .
  • R is hydrogen or C 1 -C 6 alkyl. In certain embodiments of the compound of Formula 2, R is hydrogen.
  • R 4 is maleimide, alkyne, azide, norbornene, or cyclooctene. In certain embodiments of the compound of Formula 2, R 4 is maleimide, alkyne, or azide.
  • m is 1;
  • R 2 is halide, cyano, nitro, hydrogen, or alkyl; the linker is absent, ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , ** –O (CR 2 ) n O– *** , or ** – (OCR 2 CR 2 ) n – *** , wherein R is hydrogen and n is a whole number selected from 1-4; and R 4 is maleimide, alkyne, azide, norbornene, or cyclooctene.
  • m is 1; R 2 is hydrogen; the linker is absent, ** – (CR 2 ) n – *** , ** –O (CR 2 ) n – *** , ** –O (CR 2 ) n O– *** , or ** – (OCR 2 CR 2 ) n – *** ; R 4 is maleimide, alkyne, or azide; R is hydrogen; and n for each instance is independently selected from 1, 2, 3, or 4.
  • the compound of Formula 1 is selected from the group consisting of:
  • n is a whole number selected from 1-4;
  • R 2 is hydrogen, alkyl, or –OR 5 ;
  • R 4 is maleimide or alkyne
  • R 5 is alkyl
  • the compound of Formula 2 is selected from the group consisting of:
  • the compound of Formula 2 can be brought into contact with an active agent comprising a complimentary reactive functional group capable of reacting with functionality present in R 4 thereby forming an active agent conjugate represented by the structure of Formula 4:
  • n 1, 2, or 3;
  • N* is the N-terminal nitrogen of a polypeptide
  • R 1 is the side chain of the N-terminal amino acid of the polypeptide and the N-terminal amino acid is a natural amino acid or a non-natural amino acid;
  • A is a linker or is absent
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl;
  • R 6 is an active agent
  • R 7 is a spacer or absent
  • R 8 is selected from the group consisting of:
  • ⁇ indicates the position of a covalent bond with A and ⁇ indicates the position of a covalent bond with R 7 .
  • R 2 independently selected from is halide, cyano, nitro, hydrogen, or alkyl.
  • m is 1 and R 2 is halide, cyano, nitro, hydrogen, or alkyl.
  • R 2 is hydrogen.
  • the spacer is at least one moiety selected from the group consisting of a natural amino acid, a non-natural amino acid, piperazine, 4-amino piperidine, 4- (aminomethyl) piperidine, indicates the position of a covalent bond with R 6 indicates the position of a covalent bond with R 8 ; each instance of n is independently a whole number selected from 1-20; p is an integer selected from 0-20; and R for each instance is independently selected from hydrogen, alkyl, cycloalkyl, and aryl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • ⁇ indicates the position of a covalent bond with R 8 ; each instance of n is independently a whole number selected from 1-20; each instance of p is independently an integer selected from 0-20; and R for each instance is independently selected from hydrogen, alkyl, cycloalkyl, and aryl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • the linker A comprises 2 or 3.
  • the linker can be represented by –A 1 –A 2 –A 3 –, wherein each of A 1 , A 2 , and A 3 is independently selected from the linkers recited in the definition of A herein; or A 3 is absent.
  • A comprises 2 or 3 linkers
  • ** indicates the position of a covalent bond with the moiety:
  • the linker is at least one moiety selected from the group consisting of ⁇ – (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n O– ⁇ , and ⁇ – (OCR 2 CR 2 ) n – ⁇ .
  • R 8 is selected from the group consisting of:
  • R 8 is selected from the group consisting of:
  • R is hydrogen or C 1 -C 6 alkyl. In certain embodiments of the compound of Formula 4, R is hydrogen.
  • the spacer is at least one of piperazine
  • R 8 is selected from the group consisting of: the linker is at least one moiety selected from the group consisting of ⁇ – (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n O– ⁇ , and ⁇ – (OCR 2 CR 2 ) n – ⁇ ;
  • R is hydrogen; and n for each instance is independently selected from 1, 2, 3, or 4.
  • the compound is selected from the group consisting of:
  • the selection of the appropriate complimentary reactive functional group can depend on the chemical reactivity of R 4 .
  • the complimentary reactive functional group when R 4 is a maleimide, can be a thiol or conjugate salt thereof; when R 4 is an alkyne, the complimentary reactive functional group can be an azide, nitrile oxide, or nitrone; and when R 4 is an azide, the complimentary reactive functional group can be an alkyne.
  • the selection of the appropriate complimentary reactive functional group in the active agent and reaction conditions for the reaction of the active agent comprising the complimentary reactive functional group is well within the skill of a person of ordinary skill in the art.
  • the 2-ethynylbenzaldehyde analog can comprise the active agent.
  • the 2-ethynylbenzaldehyde analog can be represented by the compound of Formula 3:
  • n 1, 2, or 3;
  • A is a linker or is absent
  • R 5 for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocycloalkyl, and heteraryl; or two instances of R 5 taken together with the atoms to which they are attached form a 5-6 membered heterocycloalkyl;
  • R 6 is an active agent
  • R 7 is a spacer or absent
  • R 8 is selected from the group consisting of:
  • ⁇ indicates the position of a covalent bond with A and ⁇ indicates the position of a covalent bond with R 7 .
  • R 2 independently selected from is halide, cyano, nitro, hydrogen, or alkyl. In certain embodiments of the compound of Formula 3, m is 1 and R 2 is halide, cyano, nitro, hydrogen, or alkyl. In certain embodiments of the compound of Formula 3, R 2 is hydrogen.
  • the spacer is at least one moiety selected from the group consisting of a natural amino acid, a non-natural amino acid, piperazine, 4-amino piperidine, 4- (aminomethyl) piperidine, indicates the position of a covalent bond with R 6 indicates the position of a covalent bond with R 8 ; each instance of n is independently a whole number selected from 1-20; p is an integer selected from 0-20; and R for each instance is independently selected from hydrogen, alkyl, cycloalkyl, and aryl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • ⁇ indicates the position of a covalent bond with R 8 ; each instance of n is independently a whole number selected from 1-20; each instance of p is independently an integer selected from 0-20; and R for each instance is independently selected from hydrogen, alkyl, cycloalkyl, and aryl; or two instances of R taken together with the carbons to which they are attached form a 3-6 membered carbocyclic ring; or two instances of R taken together with the atoms to which they are attached form a 5-6 membered heterocyclic ring.
  • the linker A comprises 2 or 3.
  • the linker can be represented by –A 1 –A 2 –A 3 –, wherein each of A 1 , A 2 , and A 3 is independently selected from the linkers recited in the definition of A herein; or A 3 is absent.
  • ** indicates the position of a covalent bond with the moiety:
  • the linker is at least one moiety selected from the group consisting of ⁇ – (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n O– ⁇ , and ⁇ – (OCR 2 CR 2 ) n – ⁇ .
  • R 8 is selected from the group consisting of:
  • R 8 is selected from the group consisting of:
  • R is hydrogen or C 1 -C 6 alkyl. In certain embodiments of the compound of Formula 3, R is hydrogen.
  • the spacer is at least one of 4-amino piperidine, 4- (aminomethyl) piperidine, or the spacer is absent;
  • R 8 is selected from the group consisting of: the linker is at least one moiety selected from the group consisting of ⁇ – (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n – ⁇ , ⁇ –O (CR 2 ) n O– ⁇ , and ⁇ – (OCR 2 CR 2 ) n – ⁇ ;
  • R is hydrogen; and n for each instance is independently selected from 1, 2, 3, or 4.
  • the compound is selected from the group consisting of:
  • Conjugate salts include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from nontoxic organic or inorganic acids.
  • such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
  • the compounds described herein may contain one or more acidic functional groups and, thus, are capable of forming conjugate salts with pharmaceutically-acceptable bases.
  • conjugate salts in these instances refer to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • the compounds described herein can comprise a cleavable linker and/or a cleavable spacer.
  • a cleavable linkers or spacer is a linker or spacer that contains one or more covalent bonds and/or functional groups that are susceptible to being cleaved under physiological conditions, e.g., under the action of an enzyme, water, acidic conditions, basic conditions, oxygen, reducing agents, oxidizing agents, or the like.
  • cleavable functional groups include, but are not limited to carbinoamine, disulfide, carbamide, aminal, acetal, ester, carbonate, carbamate, phosphate, imine, oxime, and hydrazone groups.
  • the linker and/or spacer comprises or further comprises at least one cleavable functional group selected from the group consisting of carbinoamine, disulfide, carbamide, aminal, N, O aminal, acetal, ester, carbonate, carbamate, phosphate, imine, oxime, and hydrazone. In certain embodiments, the linker and/or spacer comprises or further comprises at least one cleavable functional group selected from the group consisting of carbinoamine, disulfide, aminal, N, O aminal, and acetal.
  • the methods provided herein can be conducted under mild conditions under which the risk of polypeptide denaturing, degradation, and/or isomerization is minimized.
  • the step of contacting a polypeptide comprising a N-terminal amine and a compound of Formula 1 or 3 can be conducted at a temperature of about 10 to about 60 °C. In certain embodiments, the step of contacting a polypeptide comprising a N-terminal amine and a compound of Formula 1 or 3 can be conducted at a temperature of about 10 to about 60, 20 to about 60, 20 to about 40, or 20 to about 30 °C. In certain embodiments, the step of contacting a polypeptide comprising an N-terminal amine and a compound of Formula 1 or 3 can be conducted at about 37 °C.
  • the step of contacting a polypeptide comprising an N-terminal amine and a compound of Formula 1 or 3 can be conducted at a pH of about 5 to about 10. In certain embodiments, the step of contacting a polypeptide comprising an N-terminal amine and a compound of Formula 1 or 3 can be conducted at a pH of about 5 to about 9; about 5 to about 8; about 5 to about 7; about 6 to about 7; or about 6 to about 6.5. In certain embodiments, the step of contacting a polypeptide comprising an N-terminal amine and a compound of Formula 1 or 3 can be conducted at a pH of about 6.3.
  • the step of contacting a polypeptide comprising an N-terminal amine and a compound of Formula 1 or 3 can be conducted in dimethylsulfoxide (DMSO) , N, N-dimethylformamide (DMF) , N, N-dimethylacetamide (DMA) , methanol, ethanol, ethylene glycol, isopropanol, tetrahydrofuran, tetrahydropyran, water, and combinations thereof.
  • the step of contacting a polypeptide comprising an N-terminal amine and a compound of Formula 1 or 3 can be conducted in a mixture of DMSO and water.
  • the ratio of DMSO to water can be about 1: 1 to about 12: 1. In certain embodiments, the ratio of DMSO to water is about 9: 1.
  • the compound of Formula 1 or 3 can present in any amount relative to the polypeptide. However, to ensure higher yields of the desired product and/or higher rates of reaction, an excess of the compound of Formula 1 or 3 can be used. 1-500 molar equivalents of the compound of Formula 1 or 3 can be used to relative to the polypeptide. In certain embodiments, 1-5, 5-10, 5-15, 5-20, or 5-25 equivalents of the compound of Formula 1 or 3 can be used to relative to the polypeptide. In certain embodiments, about 10 equivalents of the compound of Formula 1 or 3 can be used to relative to the polypeptide.
  • the methods for N-terminal amine functionalization are capable of selectively functionalizing the N-terminal amine of a polypeptide with a selectivity in favor of the N-terminal amine over amines present in side chains of amino acids in the polypeptide (such as lysine) of about 10: 1, about 15: 1, about 20: 1, about 25: 1, about 30: 1, about 35: 1, about 40: 1, about 45: 1, about 50: 1, about 55: 1, about 60: 1, about 65: 1, about 70: 1, about 75: 1, about 80: 1, about 85: 1, about 90: 1, about 95: 1, about 99: 1 or greater.
  • the methods for N-terminal amine functionalization are capable of selectively functionalizing the N-terminal amine of a polypeptide with a selectivity in favor of the N-terminal amine over amines present in side chains of the polypeptide of between about 10: 1 and 99: 1, 20: 1 and 99: 1, 30: 1 and 99: 1, 40: 1 and 99: 1, 50: 1 and 99: 1, 50: 1 and 90: 1, and 50: 1 and 80: 1.
  • the N-terminal selectivity results are shown in Table 1.
  • Suitable reaction conditions for the reaction of the active agent comprising a functional group capable of reacting with the functionalized polypeptide of Formula 2 depends on the chemical reactivity of the R 4 and the selection of which is well within the skill of a person of ordinary skill in the art.
  • reaction intermediate was reacted with potassium carbonate (0.50 g, 3.64 mmol) in methanol (30 mL) for 30 min at room temperature, and the solvent was removed under the reduced pressure.
  • the residue was extracted with dichloromethane and washed with saturated sodium carbonate, and dried over magnesium sulfate.
  • the filtrate was concentrated under reduced pressure and purified by column chromatography over silica gel with n-hexane/ethyl acetate (50: 1) to give 2-ethynylbenzaldehydes 2a-2p.
  • the reaction mixture is kept at 20-37 °C for 0-24 h.
  • the conversion of the peptide is subjected to LC/MS and LC/MS-MS analysis to analyze the product distribution.

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Abstract

L'invention concerne des réactifs et des procédés utiles pour la fonctionnalisation N-terminale de polypeptides et d'intermédiaires et des produits associés.
PCT/CN2019/086825 2018-05-14 2019-05-14 Procédé de fonctionnalisation n-terminale de polypeptides et produits associés Ceased WO2019219002A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10913770B2 (en) 2019-05-14 2021-02-09 Vision Global Holdings Limited Mertansine polypeptide conjugates

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008106104A2 (fr) * 2007-02-26 2008-09-04 Massachusetts Institute Of Technology Fluorophores sensibles à l'environnement
CN102477066A (zh) * 2010-11-30 2012-05-30 首都医科大学 N-(s-1,2,3,4-四氢-6,7-二羟基异喹啉-3-甲酰)氨基酸及其合成方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008106104A2 (fr) * 2007-02-26 2008-09-04 Massachusetts Institute Of Technology Fluorophores sensibles à l'environnement
CN102477066A (zh) * 2010-11-30 2012-05-30 首都医科大学 N-(s-1,2,3,4-四氢-6,7-二羟基异喹啉-3-甲酰)氨基酸及其合成方法和应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10913770B2 (en) 2019-05-14 2021-02-09 Vision Global Holdings Limited Mertansine polypeptide conjugates

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