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WO2006105199A2 - Compositions et methodes de synthese d'un peptide et d'un conjugue apparente - Google Patents

Compositions et methodes de synthese d'un peptide et d'un conjugue apparente Download PDF

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Publication number
WO2006105199A2
WO2006105199A2 PCT/US2006/011470 US2006011470W WO2006105199A2 WO 2006105199 A2 WO2006105199 A2 WO 2006105199A2 US 2006011470 W US2006011470 W US 2006011470W WO 2006105199 A2 WO2006105199 A2 WO 2006105199A2
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WIPO (PCT)
Prior art keywords
seq
amino acid
peptide fragment
acid sequence
peptide
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Application number
PCT/US2006/011470
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English (en)
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WO2006105199A3 (fr
Inventor
Stephen Schneider
Catherine J. Mader
Brian Bray
Nicolai Tvermoes
Huyi Zhang
Original Assignee
Trimeris, Inc.
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Publication of WO2006105199A2 publication Critical patent/WO2006105199A2/fr
Publication of WO2006105199A3 publication Critical patent/WO2006105199A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to novel methods of synthesis of a synthetic peptide having the amino acid sequence of SEQ ID NO:4, and of a conjugate comprised of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid.
  • the present invention relates to a process by which smaller peptides, each comprising a separate portion of the synthetic peptide desired to be chemically synthesized (and therefore the smaller peptides are referred to as "peptide fragments" with respect to the synthetic peptide or conjugate which is produced by combining a set of peptide fragments), are covalently coupled in assembling the desired synthetic peptide having the amino acid sequence of SEQ ID NO:4, or a conjugate comprised thereof.
  • HR1 peptides derived from the native sequence of either the HR1 region (having an amino acid sequence of SEQ ID NO:1 ) ("HR1 peptides") or HR2 region (having and amino acid sequence of SEQ ID NO:2) ("HR2 peptides") of HIV gp41 inhibit transmission of HIV to host cells both in in vitro assays and in in vivo clinical studies.
  • HR2 peptides as exemplified by DP178 (also known as T20, enfuvirtide, and Fuzeon®; SEQ ID NO:3) blocked infection of target cells with a potency of 0.5 ng/ml (EC50 against HIV-1 L AI)- Efforts have been made to improve the biological activity of HIV gp41 -derived peptides, such as by trying to stabilize the helical structure of the HR2 peptide. Additionally, various efforts have also been made to improve the pharmacological properties of HIV gp41 -derived peptides.
  • conjugates each conjugate comprising a fatty acid operatively linked to synthetic peptide, wherein the synthetic peptide is derived from the HR2 region of HIV gp41 (as disclosed in a co-pending application by Applicant).
  • synthetic peptide alone e.g., not part of a conjugate
  • conjugates demonstrate one or more improved pharmaceutical activities (e.g., one or more of increased antiviral activity against HIV-1 , increased antiviral potency against HIV strains resistant to current HIV fusion inhibitor peptides, a reduction in clearance, and a marked prolongation of terminal half-life).
  • a preferred conjugate comprises fatty acid operatively linked to a synthetic peptide having the amino acid sequence of SEQ ID NO:4 (see Table 3 herein).
  • this preferred conjugate comes to be used more extensively, for example as a therapeutic and/or as a medicament, the need arises for an ability to synthesize the conjugate, and its component parts such as the synthetic peptide having the amino acid sequence of SEQ ID NO:4 (containing 36 amino acids), in large scale quantities.
  • methods for synthesis of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 utilize either solid phase and/or liquid phase synthesis procedures to synthesize specific peptide fragments, that may comprise a set of peptide fragments, and then combine the specific peptide fragments in a set of peptide fragments to yield the synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • the methods of the present invention involve synthesizing specific (with respect to amino acid sequence), side chain protected (using chemical protecting groups), peptide fragments which serve as intermediates, which are then chemically coupled in a process of assembly in a manner to result in the desired synthetic peptide having an amino acid sequence of SEQ ID NO:4 having side chain protection.
  • the method may further comprise deprotection of the side chains to yield a synthetic peptide having an amino acid sequence of SEQ ID NO:4, which may then be used to produce a conjugate comprised of the synthetic peptide operatively linked to fatty acid.
  • each peptide fragment serving as an intermediate that may be covalently coupled with the one or more other peptide fragments of the at least 2 peptide fragments, to yield the synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • the peptide fragments within a set of peptide fragments according to the present invention are coupled in a solution phase process in a manner to result in the desired synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • At least one peptide fragment, used in the assembly of the synthetic peptide having an amino acid sequence of SEQ ID NO:4, may be operatively linked to fatty acid directly or through a linker, so that when the peptide fragments are combined, assembled is a conjugate comprised of fatty acid operatively linked to a synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • a novel modified amino acid comprising an amino acid, and more preferably a lysine, having covalently coupled thereto a linker-fatty acid combination, and more preferably a linker-fatty acid combination comprising (PEG-3)-C18.
  • the modified amino acid is incorporated into one of the peptide fragments (preferably during solid phase synthesis) resulting in a peptide fragment comprising a peptide fragment-linker- fatty acid combination.
  • the peptide fragment-linker-fatty acid combination is then combined with one or more other peptide fragments to yield a conjugate comprising the synthetic peptide having an amino acid sequence of SEQ ID NO:4, operatively linked to fatty acid through a linker.
  • the peptide fragments (including a peptide fragment operatively linked to fatty acid), within a set of peptide fragments according to the present invention, are covalently coupled in a solution phase process in a manner to result in the desired conjugate comprised of a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to a fatty acid (directly or through a linker).
  • the present invention also provides the use of a set of peptide fragments according to the present invention, in the manufacture of a medicament for use within therapy of HIV infection (e.g., used in a method of inhibiting transmission of HIV, a method of inhibiting HIV fusion, or a method of treating HIV infection), as described herein.
  • the medicament is preferably in the form of a conjugate produced according to the compositions and methods of the present invention together with a pharmaceutically acceptable carrier.
  • the present invention further extends to methods of making a conjugate comprising the steps of: (a) synthesizing at least two peptide fragments, wherein at least one of the peptide fragments comprises a peptide fragment operatively linked to fatty acid (directly or through a linker), and wherein when assembled together the at least two peptide fragments form a synthetic peptide having the amino acid sequence of SEQ ID NO:4 which is operatively linked to fatty acid; (b) chemically coupling the 2 or more peptide fragments in a process of assembly to produce the conjugate comprised of a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to a fatty acid.
  • FIG. 1 shows a comparison of the sequences contained within the HR2 region of HIV-1 gp41 for purposes of illustration, and not limitation, as determined from various laboratory strains and clinical isolates, wherein illustrated are some of the variations in amino acid sequence (e.g., polymorphisms), as indicated by the single letter amino acid code.
  • amino acid sequence e.g., polymorphisms
  • FIG. 2 is a schematic showing synthesis of a synthetic peptide, SEQ ID NO:4, using 3 peptide fragments (SEQ ID NOs:5, 6, & 7) assembled by chemically coupling in a fragment condensation approach.
  • FIG. 3 is a schematic showing synthesis of a synthetic peptide, SEQ ID NO:4, using 2 peptide fragments (SEQ ID NOs:9 & 10 or SEQ ID NOs:9 & 11 ) assembled by chemically coupling in a fragment condensation approach.
  • FIG. 4 is a schematic showing synthesis of a conjugate comprised of a synthetic peptide (SEQ ID NO:4), a linker, and a fatty acid, using 2 peptide fragments (e.g., SEQ ID NOs:13 & 20, or SEQ ID NOs:13 & 21 ) assembled by chemically coupling in a fragment condensation approach.
  • SEQ ID NO:4 synthetic peptide
  • linker linker
  • a fatty acid e.g., 2 peptide fragments (e.g., SEQ ID NOs:13 & 20, or SEQ ID NOs:13 & 21 ) assembled by chemically coupling in a fragment condensation approach.
  • 2 peptide fragments e.g., SEQ ID NOs:13 & 20, or SEQ ID NOs:13 & 21
  • amino acid is meant, for purposes of the specification and claims and in reference to the synthetic peptide or peptide fragment used in the present invention, to refer to a molecule that has at least one free amine group and at least one free carboxyl group.
  • the amino acid may have more than one free amine group, or more than one free carboxyl group, or may further comprise one or more free chemical reactive groups other than an amine or a carboxyl group (e.g., a hydroxyl, a sulfhydryl, etc.).
  • the amino acid may be a naturally occurring amino acid (e.g., L-amino acid), a non-naturally occurring amino acid (e.g., D-amino acid), a synthetic amino acid, a modified amino acid, an amino acid derivative, an amino acid precursor, and a conservative substitution.
  • L-amino acid a naturally occurring amino acid
  • D-amino acid a non-naturally occurring amino acid
  • synthetic amino acid a modified amino acid
  • an amino acid derivative e.g., an amino acid precursor
  • conservative substitution e.g., amino acid derivative of amino acid
  • One skilled in the art would know that the choice of amino acids incorporated into a peptide will depend, in part, on the specific physical, chemical or biological characteristics required of the antiviral peptide. Such characteristics are determined, in part, by determination of structure and function (e.g., antiviral activity; as described herein in more detail).
  • amino acids in a synthetic peptide may be comprised of one or more of naturally occurring (L)-amino acid and non-naturally occurring (D)-amino acid.
  • a preferred amino acid may be used to the exclusion of amino acids other than the preferred amino acid.
  • the term "internal”, when used to refer to an amino acid means that the amino acid is other than the N-terminal amino acid and the C-terminal amino acid in an amino acid sequence (e.g., of a peptide fragment or synthetic peptide or conjugate).
  • an internal amino acid is located in the amino acid sequence in a position in-between the N-terminal amino acid and the C-terminal amino acid.
  • a "conservative substitution”, in relation to amino acid sequence of a synthetic peptide used in the present invention, is a term used hereinafter for the purposes of the specification and claims to mean one or more amino acids substitution in the sequence of the synthetic peptide such that its biological activity is substantially unchanged (e.g., if the peptide inhibits HIV gp41-mediated fusion at a concentration in the nanomolar range before the substitution, after the substitution inhibition of HIV gp41 -mediated fusion is still observed in the nanomolar range).
  • substitutions are defined by aforementioned function, and includes substitutions of amino acids having substantially the same charge, size, hydrophilicity, and/or aromaticity as the amino acid replaced.
  • substitutions are known to those of ordinary skill in the art to include, but are not limited to, glycine-alanine-valine; isoleucine-leucine; methionine-leucine; tryptophan-tyrosine; aspartic acid-glutamic acid; arginine-lysine; asparagine-glutamine; and serine-threonine.
  • a conserved substitution is known in the art to also include substituting lysine with ornithine, in providing a free amine group (e.g., epsilon amine).
  • a free amine group e.g., epsilon amine
  • substitutions may also comprise polymorphisms at the various amino acid positions along the relevant HR region (HR2) of gp41 found in any one or more of various clades, laboratory strains, or clinical isolates of HIV, which are readily available from public databases and are well known in the art (see also, FIG. 1 as illustrative example).
  • fatty acid when used herein for purposes of the specification and claims, means a fatty acid comprising no less than 5, and no more than 30, carbon atoms; and is also used to mean a fatty acid analog produced by operatively linking fatty acid to either a linker or an amino acid.
  • operatively linking a fatty acid to a C-terminal amino acid results in an alkylamide (also referred to herein as "fatty acid”).
  • a fatty acid” or “fatty acid” when used in reference to operatively linking to a synthetic peptide or peptide fragment, means one or more fatty acids, unless otherwise specifically indicated herein.
  • the more preferred fatty acids useful in this invention are the fatty acids of 12 to 20 carbon atoms.
  • the fatty acid may comprise a monoacid or a diacid.
  • the fatty acid may be saturated (Table 1 , "S") or unsaturated (Table 1 , "U”).
  • Examples of fatty acids that may be useful in producing a conjugate according to the present invention include, but are not limited to the following illustrated in Table 1. Table 1
  • a preferred fatty acid may be applied in the present invention to the exclusion of a fatty acid other than the preferred fatty acid.
  • synthetic peptide is used herein, and for the purposes of the specification and claims, to mean a peptide comprising an amino acid sequence, wherein the amino acid sequence has the amino acid sequence of SEQ ID NO:4.
  • isolated when used in reference to a synthetic peptide means that it is substantially free (e.g., at least 90% pure, and more preferably at least 95% pure) of components which have not become part of the integral structure of the peptide itself; e.g., such as substantially free of chemical precursors or other chemicals when chemically synthesized, produced, or modified using biological, biochemical, or chemical processes.
  • peptide fragment and “intermediate” are used synonymously herein, in relation to a synthetic peptide having an amino acid sequence of SEQ ID NO:4, and for the purposes of the specification and claims, to mean a peptide comprising an amino acid sequence of no less than about 4 amino acids and no more than about 32 amino acid residues in length (and more preferably, no less than 5 amino acids, and no more than 30 amino acids), and comprises at least a portion (as contiguous amino acids) of the amino acid sequence of SEQ ID NO:4 (see Tables 2 and 4 for illustrative examples).
  • peptide fragment is also used herein to refer to a peptide fragment having operatively linked thereto fatty acid (either directly or through a linker).
  • isolated when used in reference to a peptide fragment means that it is substantially free of components which have not become part of the integral structure of the peptide fragment itself; e.g., such as substantially free of chemical precursors or other chemicals when chemically synthesized, produced, or modified using biological, biochemical, or chemical processes.
  • the peptide fragment may comprise, in its amino acid sequence, one or more conservative substitutions and/or one or more polymorphisms found in the corresponding amino acid sequence of HIV gp41.
  • a lysine in the amino acid sequence of a peptide may be substituted with another amino acid (naturally occurring or not naturally occurring) having a side chain with a free amino group (e.g., epsilon amine).
  • Ornithine is an illustrative example of such amino acid that may be used to substitute a lysine.
  • peptide fragments are synthesized such that peptidic bonds are formed between the amino acid residues
  • non-peptidic bonds may be formed using reactions known to those skilled in the art (e.g., imino, ester, hydrazide, azo, semicarbazide, and the like).
  • the present invention also encompasses particular sets of peptide fragments which act as intermediates in a method of synthesis of either (a) a synthetic peptide having the amino acid sequence of SEQ ID NO:4, or (b) a conjugate comprised of a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid.
  • the peptide fragments, in a set of peptide fragments are covalently coupled ("combined” or "assembled") in a method of synthesizing a synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • peptide fragments in a set of peptide fragments are combined in a method of synthesizing a conjugate comprising a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid.
  • the peptide fragments of the invention may include, but are not limited to, those having the amino acid sequences depicted in the following Table 2.
  • no less than 2 peptide fragments, and no more than 5 peptide fragments are combined to form a synthetic having an amino acid sequence of SEQ ID NO:4, or a conjugate comprised of the synthetic peptide.
  • Preferred peptide fragment(s) and sets(s) of peptide fragments may be used in the present invention to the exclusion of peptide fragment(s) and sets(s) of peptide fragments other than the preferred peptide fragment(s) and sets(s) of peptide fragments.
  • the corresponding amino acids in SEQ ID NO:4 of each peptide fragment are also indicated.
  • each peptide fragment of the at least 2 peptide fragments is selected from a group consisting of a peptide fragment having an amino acid sequence of any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:22.
  • each peptide fragment is made up of a number of contiguous amino acids of the amino acid sequence of SEQ ID NO:4.
  • the sets of peptide fragments according to the invention include Sets 1-30, as designated in Table 2 (the numbering of a set is for ease of description only).
  • some peptide fragments (see e.g., Sets 9-16; SEQ ID NOs:16- 21 ) comprise a peptide fragment operatively linked to fatty acid through a linker (a "peptide fragment-linker-fatty acid combination").
  • Such peptide fragment-linker-fatty acid combinations are illustrated (for purposes of ease of description only) by a lysine residue ("K") with a vertical line (“
  • some peptide fragments comprise a peptide fragment operatively linked to fatty acid directly or through a linker at a terminal (C-terminal or N-terminal) amino acid of the peptide fragment (a "peptide fragment-fatty acid combination").
  • a "peptide fragment-fatty acid combination” Such peptide fragment-fatty acid combinations are illustrated (for purposes of ease of description only) by an "X * ", wherein X * is a fatty acid operatively linked either directly or through a linker to a terminal (C-, N-) amino acid of a peptide fragment.
  • reactive functionality when used herein for purposes of the specification and claims, means a chemical group or chemical moiety that is capable of forming a bond with another chemical group or chemical moiety.
  • a reactive functionality of a molecule is reacted with a reactive functionality of another molecule in chemically coupling (or otherwise, operatively linking) the two molecules.
  • a first reactive functionality of a linker is reacted with a reactive functionality of an amino acid being incorporated into the amino acid sequence of the peptide fragment, in chemically coupling the linker to the amino acid to be incorporated into the amino acid sequence of the peptide fragment.
  • a second reactive functionality of a linker is, or has been, reacted with a reactive functionality of a fatty acid in chemically coupling the linker to the fatty acid.
  • a reactive functionality is known to those skilled in the art to comprise a group that includes, but is not limited to, maleimide, thiol, carboxylic acid, hydrogen, phosphoryl, acyl, hydroxyl, acetyl, hydrophobic, amine, amido, dansyl, sulfo, a succinimide, a thiol- reactive, an amine-reactive, a carboxyl-reactive, and the like.
  • a preferred reactive functionality may be used, in application to the present invention, to the exclusion of a reactive functionality other than the preferred reactive functionality.
  • operatively linked means that the two or more molecules are physically associated by a linking means that does not interfere with the ability of either of the linked molecules to function as described herein.
  • a synthetic peptide or peptide fragment may be operatively linked to fatty acid.
  • Operatively linked to fatty acid means that the synthetic peptide or peptide fragment is either operatively linked directly to fatty acid, or is operatively linked to fatty acid through a linker.
  • the synthetic peptide or peptide fragment via its reactive functionality, may be operatively linked to a reactive functionality of a fatty acid using standard chemical techniques.
  • an amino acid as an amino acid already part of the amino acid sequence of a synthetic peptide or peptide fragment or as an amino acid to be incorporated into the amino acid sequence of a synthetic peptide or peptide fragment, via its reactive functionality may be operatively linked to a linker via a reactive functionality of the linker.
  • the linker may already be operatively linked to a fatty acid (a linker-fatty acid combination), or is subsequently operatively linked to a fatty acid.
  • a bond formed by operatively linking two molecules may comprise, but is not limited to, one or more of: covalent, ionic, hydrogen, van der Waals and the like.
  • Coupled when used herein for purposes of the specification and claims and in relation to assembling two or more peptide fragments, means that there is a chemical bonding, and more preferably a covalent chemical bonding, between a peptide fragment with another peptide fragment in the process of assembling the peptide fragments to form a synthetic peptide having an amino acid sequence of SEQ ID NO:4, or a conjugate comprised of the synthetic peptide.
  • linker when used herein for purposes of the specification and claims, means a compound or moiety that acts as a molecular bridge to operatively link two different molecules.
  • a first reactive functionality of a linker is covalently coupled to a reactive functionality of a fatty acid
  • a second reactive functionality of the linker is covalently coupled to a reactive functionality of an amino acid, wherein the amino acid is then incorporated into the amino acid sequence of a peptide fragment during its synthesis, or the amino acid is already part of the amino acid sequence of the peptide fragment (in forming a "peptide fragment-linker- fatty acid combination").
  • the peptide fragment may then be assembled with other peptide fragments to form the synthetic peptide, in forming the conjugate according to the methods of the present invention.
  • linker there is no particular size or content limitations for the linker so long as it can fulfill its purpose as a molecular bridge.
  • Linkers are known to those skilled in the art to include, but are not limited to, chemical chains, chemical compounds (e.g., reagents), amino acids, and the like.
  • the linkers may include, but are not limited to, homobifunctional linkers, heterobifunctional linkers, biostable linkers, and biodegradable linkers, as well known in the art.
  • a linker when used, it is a non-planar (e.g., so that the synthetic peptide in the conjugate is not rigidly fixed to the fatty acid in the conjugate but remains flexible in the linkage).
  • Heterobifunctional linkers well known to those skilled in the art, contain one end having a first reactive functionality to specifically link a first molecule, and an opposite end having a second reactive functionality to specifically link to a second molecule. It will be evident to those skilled in the art that a variety of monofunctional, difunctional, and polyfunctional reagents (such as those described in the catalog of the Pierce Chemical Co., Rockford, III.) may be employed as a linker with respect to the present invention.
  • the linker may vary in length and composition for optimizing such properties as preservation of biological function stability, resistance to certain chemical and/or temperature parameters, and of sufficient stereo-selectivity or size.
  • the linker should not significantly interfere with the ability of the synthetic peptide (to which it is linked) to function as an inhibitor of either or both of HIV fusion and HIV transmission to a target cell.
  • Preferred linkers comprise from no less than 2 and no more than 100 units of ethylene glycol; as may be represented by the formula: R 1 - (CH 2 CH 2 O) n -R 2 ; wherein R is a reactive functionality, and n is from 2 to 100, and more preferably, n is from 2 to 30.
  • Preferred linkers include PEG3; discrete units comprised of PEG3 such as (PEG 3) 2 , (PEG 3) 4 , (PEG 3) 5 , (PEG 3) 9 ; PEG13; (PEG 13) 2 ; PEG25; PEG29; a combination of preferred linkers (e.g., a linker made by combining two or more PEG linkers together; for example, a linker comprised of a PEG13 operatively linked to a PEG3); and amino acid linkers (e.g., comprising combinations of GIy, Ser, and like amino acids; ranging from no less than 3 amino acids to no more than 15 amino acids).
  • a preferred linker may be used, in application to the present invention, to the exclusion of a linker other than the preferred linker.
  • chemical protecting group when used herein for purposes of the specification and claims, means a chemical moiety that is used to block a reactive functionality from chemically reacting with another reactive functionality.
  • Chemical protecting groups are well known by those in the art of peptide synthesis to include, but are not limited to, tBu (t-butyl), trt (triphenylmethyl(trityl)), OtBu (tert-butoxy), Boc or t-Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Aoc (t-amyloxy-carbonyl), TEOC ( ⁇ -trimethylethyloxycarbonyl), CLIMOC (2-chloro-1-indanyl methoxyl carbonyl), BIMOC (benz-[f]-indene-3-methoxylcarbonyl), PBF (2,2,4,6,7-pentamethyldihydro- benzofuan-5-sulfonyl), 2-
  • deprotection when used herein for purposes of the specification and claims, is known in the art to mean a process by which chemical protecting group(s) is removed from a molecule containing one or more chemical protecting groups, wherein the molecule comprises an amino acid, peptide fragment, synthetic peptide, or conjugate comprised of a synthetic peptide.
  • the deprotection process involves reacting the molecule protected by one or more chemical protecting groups with a chemical agent that removes the chemical protecting group.
  • a chemical agent that removes the chemical protecting group.
  • an N-terminal alpha amino group which is protected by a chemical protecting group, may be reacted with a base to remove base labile chemical protecting groups (e.g., Fmoc, and the like).
  • Chemical , protecting groups e.g., Boc, TEOC, Aoc, Adoc, Bopc, Ddz, Cbz, and the like
  • Other chemical protecting groups particularly those derived from carboxylic acids, may be removed by acid or a base.
  • the terms “first”, “second”, “third” and the like, may be used herein to: (a) indicate an order; or (b) to distinguish between molecules or chemical groups of a molecule (e.g., for ease of description); or (c) a combination of (a) and (b).
  • the terms “first”, “second”, “third” and the like are not otherwise to be construed as limiting the invention.
  • the present invention relates to methods and compositions (e.g., peptide fragments, Sets of peptide fragments, and a modified amino acid) for synthesizing a synthetic peptide, or conjugate comprising a synthetic peptide, having the amino acid sequence of SEQ ID NO:4.
  • a conjugate comprised of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid may be represented by the following Formula 1 , with B being operatively linked to the N-terminus, Z being operatively linked to the C-terminus, and J being operatively linked to an internal amino acid, lysine: Formula 1
  • B, Z, and J may each be a member selected from the group consisting of a fatty acid, a linker-fatty acid combination, and a chemical group.
  • no more than 2 of B, J, or Z comprise a fatty acid or linker-fatty acid combination.
  • a fatty acid may comprise one or more fatty acid molecules.
  • a linker-fatty acid combination may comprise one or more linkers and one or more fatty acid molecules.
  • a chemical group may comprise, but is not limited to, one or more of: a reactive functionality, or a chemical protecting group.
  • B and Z each be comprise a chemical group
  • J is a linker-fatty acid combination comprising PEG3-C18 which is operatively linked (via a reactive functionality of the PEG3 linker) to a synthetic peptide having the amino acid sequence of SEQ ID NO:4 via the lysine at amino acid position 30 of SEQ ID NO:4 (See also, Table 3, Ref# 4-2).
  • this preferred conjugate When the antiviral activity of this preferred conjugate was compared with the antiviral activity of synthetic peptide alone (SEQ ID NO:4) against a panel of several clinical isolates of HIV-1, this preferred conjugate consistently demonstrated an increased antiviral potency, typically ranging from 2 fold more potent (e.g., 2 fold decrease in IC50 value) to 50 fold more potent. Additionally, this preferred conjugate was compared to synthetic peptide alone (SEQ ID NO:4; not part of a conjugate) for activity against a representative HIV-2 strain (HIV2, NIIHZ).
  • This preferred conjugate showed significant antiviral activity against HIV-2 (Category C) as compared to the synthetic peptide alone (Category D), and further demonstrated about a 10 fold increase in antiviral potency against HIV-2.
  • B is a fatty acid or linker-fatty acid combination operatively linked to the N-terminal amino acid of SEQ ID NO:4 ( See, Table 3, Ref# 4-7 and Ref# 4-8).
  • Z is a fatty acid or linker-fatty acid combination operatively linked to the C-terminal amino acid of SEQ ID NO:4 ( See, Table 3, Ref# 4-9 and Ref# 4-10).
  • more than one of B, J, and Z is a fatty acid or linker-fatty acid combination operatively linked to the amino acid sequence of SEQ ID NO:4 ( See, Table 3, Ref# 4-11).
  • Table 3 shows a comparison of various conjugates and synthetic peptide alone, and that the conjugates demonstrate one or more improved pharmaceutical activities as compared to the synthetic peptide alone.
  • IC50 antiviral activity in the absence of HSA
  • IC50 + HSA FrV antiviral activity in the presence of HSA with fatty acids
  • CL terminal elimination half-life
  • Category A is used herein to designate compositions with a calculated IC50 value less than or equal to 0.005 ⁇ g/ml.
  • Category C is used herein to designate compositions with a calculated IC50 value less than or equal to 0.10 ⁇ g/ml, but greater than 0.005 ⁇ g/ml.
  • Category D is used herein to designate compositions with a calculated IC50 value less than or equal toi .O ⁇ g/ml, but greater than 0.10 ⁇ g/ml.
  • This invention is based, in part, on the unexpected discovery that certain combinations of peptide fragments may be utilized in a synthetic process, such as a combining (through chemically coupling) a set of peptide fragments (a synthetic process that may also be referred to as "combinatorial synthesis") , to allow for the manufacture of a synthetic peptide having the amino acid sequence of SEQ ID NO:4, or a conjugate comprised of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid directly or through a linker, on a large scale with high throughput and high yield (as compared to conventional linear solid phase synthesis only).
  • Such peptide fragments and methods allow for manufacture of the synthetic peptide or a conjugate to be synthesized on a scale of kilogram amounts. Further, using peptide fragments and methods according to the present invention, the efficiency of synthesis of the synthetic peptide or conjugate may be increased, as compared to a standard automated solid phase synthesis on a peptide synthesizer (e.g., using approximately 3 to 5 fold less raw material and reagents). Further, the peptide fragments and methods according to the present invention reduce impurities, and therefore allow for synthetic peptides and conjugates to be synthesized in high purity, as compared to conventional techniques for producing such synthetic peptide or conjugate (e.g., solely conventional solid phase synthesis).
  • the individual peptide fragments themselves, and a synthetic peptide having the amino acid sequence of SEQ ID NO:4 produced from combining peptide fragments of a set of peptide fragments according to the present invention (or a conjugate comprised of the synthetic peptide), may each be made using techniques known to those skilled in the art for synthesizing peptide sequences.
  • a conjugate comprised of synthetic peptide operatively linked to fatty acid may be produced solely using solid phase synthesis.
  • the peptide fragments may be synthesized in solid phase, and then combined in solution phase in a process of assembly to produce the synthetic peptide or conjugate.
  • solution phase synthesis may be used to produce the peptide fragments, which then are combined in solid phase in a process of assembly to produce the synthetic peptide or conjugate.
  • each peptide fragment may be synthesized using solid phase synthesis, and then combined in solid phase in a process of assembly to produce the synthetic peptide or conjugate.
  • each peptide fragment is produced using solid phase synthesis known to those skilled in the art.
  • a synthetic peptide having the amino acid sequence of SEQ ID NO:4 produced using an assembly process that combines solid phase and solution phase techniques using a set of peptide fragments according to the present invention.
  • a conjugate comprised of a synthetic peptide having the amino acid sequence of SEQ ID NO:4, a linker, and a fatty acid is produced using an assembly process that combines solid phase and solution phase techniques using a set of peptide fragments according to the present invention.
  • Protected peptide fragments according to the present invention include, but are not limited to, the peptide fragments listed in Table 4.
  • K - represents the epsilon amine of lysine protected by CPG CPG
  • CPG is chemical protecting group (e.g., Fmoc or other N-terminal chemical protecting group, as described in more detail in the "Definitions” section herein).
  • any one or more of the side- chains of the amino acids in the amino acid sequence may be protected with a chemical protecting group.
  • t-Bu comprises the chemical protecting group for side chains of amino acid residues tyrosine (Y), threonine (T), and serine (S).
  • OtBu comprises the chemical protecting group for side chains of amino acid residues glutamic acid (E) and aspartic acid (D).
  • trt comprises the chemical protecting group for side chains of amino acid residues glutamine (Q), histidine (H), and asparagine (N).
  • Boc comprises the chemical protecting group for side chains of amino acid residues lysine (K), tryptophan (W), histidine (H), or arginine (R; using (Boc)2).
  • PBF comprises the chemical protecting group for side chains of amino acid residue arginine (R).
  • a process of assembly in which peptide fragments of a set of peptide fragments are combined to produce a synthetic peptide having the amino acid sequence of SEQ ID NO:4, or a conjugate comprised of such synthetic peptide operatively linked to a fatty acid, is also referred to as a fragment condensation approach, or a fragment assembly approach.
  • each peptide fragment, of a set of peptide fragments is preferably synthesized using solid phase peptide synthesis techniques. However, some peptide fragments may be preferred to be produced using solution phase synthesis, as will be described in more detail herein.
  • fragment condensation approaches specifically described herein are preferred embodiments, wherein one approach involves assembly of 3 fragments (e.g., as illustrated in Example 2 herein, and FIG. 2), and another approach involves assembly of 2 fragments (e.g., as illustrated in Examples 3, 4, & 5 herein, and FIGs. 3 & 4).
  • one approach involves assembly of 3 fragments (e.g., as illustrated in Example 2 herein, and FIG. 2), and another approach involves assembly of 2 fragments (e.g., as illustrated in Examples 3, 4, & 5 herein, and FIGs. 3 & 4).
  • FIG. 2 illustrated is a method for synthesis of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 using 3 specific peptide fragments (e.g., SEQ ID NOs:5-7 + Leu; or SEQ ID NOs:5, 6, and 8), and using a fragment condensation approach involving combining the 3 peptide fragments to produce the synthetic peptide.
  • 3 specific peptide fragments e.g., SEQ ID NOs:5-7 + Leu; or SEQ ID NOs:5, 6, and 8
  • each of these peptide fragments demonstrated physical properties and solubility characteristics that make them preferred peptide fragments to be used in a method for synthesis of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 in high yield and high purity.
  • a peptide fragment having the amino acid sequence of SEQ ID NO:5, and comprising the first 12 amino acids of SEQ ID NO:4 was synthesized by standard solid phase synthesis (using a super acid sensitive resin; e.g., 4-hydroxy-methyl-3-methoxyphenoxy-butyric acid resin, or 2- chlorotrityl chloride resin- "CTC", FIG.2 ), with acetylation of ("Ac", as a chemical group) the N-terminus, while having a hydroxyl group (-OH) at the C-terminus (see, FIG. 2, "Ac- AA(1-12)-OH").
  • a super acid sensitive resin e.g., 4-hydroxy-methyl-3-methoxyphenoxy-butyric acid resin, or 2- chlorotrityl chloride resin- "CTC", FIG.2
  • a peptide fragment having the amino acid sequence of SEQ ID NO:6, and comprising amino acids 13-28 of SEQ ID NO:4 was synthesized by standard solid phase synthesis with Fmoc at the N-terminus (as a chemical protecting group), and -OH at the C-terminus (see, FIG. 2, "Fmoc-AA(13-28)-OH”).
  • a peptide fragment having the amino acid sequence of SEQ ID NO:7, and comprising amino acids 29-35 of SEQ ID NO:4 was synthesized by standard solid phase synthesis with Fmoc at the N-terminus (as a chemical protecting group), and -OH at the C-terminus (see, FIG. 2, "Fmoc-AA(29-35)- OH").
  • Each peptide fragment was cleaved from the resin used for its solid phase synthesis by cleavage reagents, solvents, and techniques well known to those skilled in the art. Each peptide fragment was then isolated by removing the majority of above mentioned solvents by distillation and precipitating the peptide fragment by the addition of water with or without an alcohol containing-cosolvent. The resulting solid was isolated by filtration, washed, reslurried in water or alcohol/water, refiltered, and dried in a vacuum oven
  • a peptide fragment was produced by solution phase synthesis, when the peptide fragment having the amino acid sequence of SEQ ID NO:7 (see, FIG. 2, "Fmoc-AA(29-35)-OH”) was chemically coupled to Leu, amino acid 36 of SEQ ID NO:4 which has been amidated, in solution phase to result in a peptide fragment having the amino acid sequence of SEQ ID NO:8 (comprising amino acids 29-36 of SEQ ID NO:4) with amidation of the C-terminus (as a chemical group) (see, FIG. 2, "Fmoc- AA(29-36)-NH 2 ").
  • reaction is run in a polar, aprotic solvent such as DMF (dimethyl formamide) or NMP (N-methyl pyrrolidinone) at O 0 C to 30 0 C.
  • a polar, aprotic solvent such as DMF (dimethyl formamide) or NMP (N-methyl pyrrolidinone)
  • alcohol or a water miscible solvent and/or water are added to precipitate the peptide fragment having the amino acid sequence of SEQ ID NO:8 with amidation of the C-terminus (Fmoc-AA(29-36)-NH 2 ).
  • Fmoc-AA(29-36)-NH 2 As schematically illustrated in FIG. 2, to produce peptide fragment Fmoc-AA(29-
  • the Fmoc chemical protecting group of the peptide fragment Fmoc-AA(29-36)- NH 2 was removed using a base such as piperidine or potassium carbonate in organic solvents such as DMF 1 NMP, or mixtures thereof.
  • a base such as piperidine or potassium carbonate in organic solvents such as DMF 1 NMP, or mixtures thereof.
  • piperidine 1.5 ml, 15.2 mmol, 2.4 eq
  • the reaction was stirred overnight.
  • the reaction mixture was then poured into 400 ml of water, and the solids formed were collected via filtration and dried.
  • the dry solid was suspended in 400 ml 3:1 MTBE:hexanes and stirred for 4 hours, then collected and dried.
  • the solids were then suspended in 400 mL 1 :1 ethanol:water and stirred for 1 hour.
  • the solids were collected and dried.
  • FIG. 2 see, e.g., Table 4, SEQ ID NO:8), the result was a substantially pure preparation of
  • a solution phase reaction was then performed in which peptide fragment H-AA(29-36)-NH 2 (SEQ ID NO:8) is combined with peptide fragment Fmoc-AA(13-28)-OH (SEQ ID NO:6) to yield a peptide fragment Fmoc-AA(13-36)-NH 2 (Table 4, SEQ ID NO:22 with chemical groups at each of the N-terminus and C-terminus).
  • Peptide fragment H-AA(29-36)-NH 2 (8.74 g, 4.7 mmol, 1 eq), peptide fragment Fmoc- AA(13-28)-OH (16.32 g, 4.7 mmol, 1 eq), and HOAT (0.96 g, 7.1 mmol, 1 .5 eq) were dissolved in DMF (100 ml, 20 vol), cooled with an ice bath, and treated with DIEA (4.0 ml, 23.5 mmol, 5 eq). Added to the reaction was TBTU (1.81 g, 5.6 mmol, 1.2 eq), and the reaction was stirred for about 5 minutes at O 0 C, then 2 hours at room temperature.
  • peptide fragment H-AA(13-36)-N H 2 (SEQ ID NO:22) was then assembled in a solution phase reaction with peptide fragment Ac-(I -12)-OH (SEQ ID NO:5) to yield a synthetic peptide having the amino acid sequence of SEQ ID NO:4 (see, e.g., FIG. 2, Ac-(I -36)-NH 2 ).
  • Peptide fragment H-AA(13-36)-NH 2 (1 1.18 g, 2.20 mmol, 1 eq), peptide fragment Ac-AA(I -12)-OH (6.69 g, 2.20 mmol, 1 eq), and HOAT (0.450 g, 3.31 mmol, 1.5 eq) were dissolved in DMF (120 ml, 10 vol), cooled with an ice bath, and treated with DIEA (2.0 ml, 11.5 mmol, 5 eq). Added to the reaction was TBTU (0.847 g, 2.64 mmol, 1.2 eq). After stirring 5 minutes at O 0 C, the reaction was stirred at room temperature for 2 hours, after which HPLC showed the reaction was complete.
  • the side chain chemical protecting groups of synthetic peptide Ac-AA(I -36)-N H 2 may be removed by acidolysis or any other method known to those skilled in the art for deprotecting a peptide by removing side chain chemical protecting groups.
  • synthetic peptide Ac-AA(I -36J-NH 2 (16.98 g, 2.09 mmol) was treated with TFA (trifluoracetic acid):DTT(dithiothreitol):water (95:5:5; 200 ml) and stirred at room temperature for 4 hours.
  • MTBE approximately 500 ml was added, and a precipitate was collected by filtration.
  • the dried powder was then dissolved in 400 ml of 3:1% water/acetonitrile containing 1% HOAc (acetic acid), and was reacted for 20 hours to allow for substantially pure preparation (deprotected and decarboxylated) of synthetic peptide having an amino acid sequence of SEQ ID NO:4, as determined by HPLC analysis for purity.
  • HOAc acetic acid
  • EXAMPLE 3 In referring to Table 2 (Set 3 and Set 4) and FIG. 3, illustrated is a method for synthesis of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 using 2 specific peptide fragments (e.g., SEQ ID NOs:9-10 + Leu; or SEQ ID NOs:9 and 11), and using a fragment assembly approach involving combining the 2 peptide fragments by chemically coupling them to produce the synthetic peptide.
  • each of these peptide fragments demonstrated physical properties and solubility characteristics that make them preferred peptide fragments to be used in a method for synthesis of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 in high yield and greater purity.
  • leucine and/or isoleucine residues at the point of juncture between the two fragments being assembled together e.g., the C-terminal amino acid of a peptide fragment having the amino acid sequence of SEQ ID NO:9, and the N-terminal amino acid of a peptide fragment having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:11
  • leucine and/or isoleucine residues at the point of juncture between the two fragments being assembled together e.g., the C-terminal amino acid of a peptide fragment having the amino acid sequence of SEQ ID NO:9, and the N-terminal amino acid of a peptide fragment having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:11
  • a peptide fragment having the amino acid sequence of SEQ ID NO:9, and comprising the first 20 amino acids of SEQ ID NO:4 was synthesized by standard solid phase synthesis, with acetylation of ("Ac", as a chemical group) the N-terminus, while having a hydroxyl group (-OH) at the C-terminus (see, FIG. 3; also referred to herein as "Ac-AA(I -2O)-OH”).
  • a peptide fragment having the amino acid sequence of SEQ ID NO:10, and comprising amino acids 21-35 of SEQ ID NO:4 was synthesized by standard solid phase synthesis with Fmoc at the N-terminus (as a chemical protecting group), and -OH at the C-terminus (see, FIG.
  • Fmoc-AA(21-35)-OH a peptide fragment was produced by solution phase synthesis, when the peptide fragment Fmoc-AA(21-35)-OH was chemically coupled to Leu, amino acid 36 of SEQ ID NO:4 which has been amidated, in solution phase to result in a peptide fragment having the amino acid sequence of SEQ ID NO:11 (comprising amino acids 21- 36 of SEQ ID NO:4) with amidation of the C-terminus (as a chemical group) ("Fmoc- AA(21-36)-NH 2 ").
  • the Fmoc chemical protecting group of the peptide fragment Fmoc-AA(21-36)- NH 2 (Table 4, SEQ ID NO:11) was then removed prior to isolation of the fragment H- AA(21-36)-NH 2 by adding piperidine (27.5 mL, 278 mmol, 5.5 eq), and the solution was stirred for 1 hour at 25 ⁇ 5 0 C or until analysis by HPLC showed that substantially all the Fmoc was removed from the peptide fragment.
  • the reactor was cooled, and water (2000 ml, 10 vol) was added, and the free-flowing slurry was stirred 30 minutes at ⁇ 10°C, and then isolated by filtration.
  • the collected solid was washed with 1 :1 EtOH/water and dried in a vacuum oven at 35 ⁇ 5 0 C.
  • the peptide fragment was then reslurried in MTBE (2000 mL, 10 vol) at 30 ⁇ 5 0 C for 4 hours and then isolated by filtration and redried.
  • the MTBE reslurry may be repeated if necessary to remove additional piperidine.
  • the result is a substantially pure preparation of peptide fragment H-AA(21-36)-NH 2 (see FIG.3; and Table 4, SEQ ID NO:11).
  • a solution phase reaction was then performed in which peptide fragment H-AA(21-36)-NH 2 (SEQ ID NO:11) was combined with peptide fragment Ac-AA(I -2O)-OH (SEQ ID NO:9; Table 4) to yield a synthetic peptide having the amino acid sequence of SEQ ID NO:4 (see, e.g., FIG. 3, Ac-(I -36)-NH 2 ).
  • the reaction was stirred for 15 minutes at 0 ⁇ 5 0 C, and at 25 ⁇ 5 0 C for 30 minutes or until the reaction was shown to be complete by HPLC.
  • the reactor was cooled, and water (4200 ml, 20 vol) was slowly added. A slurry was formed and stirred at ⁇ 10°C for at least 30 minutes.
  • the solid was isolated by filtration and washed with additional water. The collected solid dried in a vacuum oven at 35 ⁇ 5°C.
  • the result was a substantially pure preparation of fully protected synthetic peptide Ac-AA(I -36)-N H 2 (SEQ ID NO:4), as determined by HPLC analysis for purity.
  • the synthetic peptide Ac-AA(I -36)-N H 2 was then deprotected (by removing the side chain chemical protecting groups) and decarboxylated (at the tryptophan residues) by using the methods described herein in Example 2, or any other method known to those skilled in the art, for deprotection and decarboxylation.
  • the result was a substantially pure preparation (deprotected and decarboxylated) of synthetic peptide having an amino acid sequence of SEQ ID NO:4, as determined by HPLC analysis for purity.
  • the peptide was then treated with TFA/water/DTT to remove the acid labile side chain protecting groups, and then purified by HPLC to yield a substantially pure preparation of synthetic peptide having the amino acid sequence of SEQ ID NO:4 (Ac-AA(I -36)-NH 2 ).
  • operatively linking fatty acid to a terminal (C- or N-) amino acid of a peptide fragment and use of that peptide fragment in a fragment condensation approach to produce a conjugate comprised of synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid.
  • the first example illustrates operatively linking fatty acid to the C-terminus of the synthetic peptide by operatively linking the fatty acid to a peptide fragment used in the synthesis of synthetic peptide.
  • the fatty acid was covalently coupled to the amino acid to be incorporated into the peptide fragment as the terminal amino acid.
  • CPG-Leu-C18 to be incorporated as the C-terminal amino acid of a peptide fragment comprising amino acids 21-36 of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 ("AA(21-36)").
  • CPG-Leu-OH (1.05 g, 3.96 mmol, 1.0 eq) was dissolved in DMF (10 mL, 10 vol) and DCM (20 mL, 20 vol), and HOAT (1.076 g, 7.91 mmol, 2 eq) and DIEA (2.75 mL, 15.8 mmol, 4 eq) were added.
  • the resulting yellow solution was cooled to 0 ⁇ 5 0 C, and then TBTU (1.525 g, 4.75 mmol, 1.2 eq) and octadecylamine (C18 fatty acid; 1.274 g, 4.73 mmol, 1.2 eq) were added, and the reaction was stirred at 0 ⁇ 5 0 C for 5 minutes.
  • the reaction had thickened, so it was allowed to heat to 25 ⁇ 5 0 C and additional DCM (10 mL, 10 vol) was added.
  • the reaction was stirred at 25 ⁇ 5 0 C for 2 hours, after which HPLC showed no more starting material present.
  • the dichloromethane was taken off under reduced pressure, and the resulting oily solid was suspended in 100 mL water.
  • peptide fragment Fmoc-AA(21-36)-NHC18 (see Tables 2 & 4, SEQ ID NO:29) using a peptide fragment Fmoc-AA(21-35)-OH combined with H-Leu-C18 in a solution phase process
  • the peptide fragment Fmoc-AA(21-37)-OH (2.01 g, 0.508 mmol, 1.0 eq)
  • H-Leu-C18 0.234 g, 0.611 mmol, 1.2 eq
  • HOAT (1.04 g, 0.764 mmol, 1.5 eq
  • DIEA 0.354 ml, 2.03 mmol, 4 eq
  • TBTU (0.197 g, 0.613 mmol, 1.2 eq) was added, stirred for 5 minutes at 0 ⁇ 5 0 C, and then allowed to react at 25 ⁇ 5 0 C for 2 hours or until the reaction was shown complete by HPLC.
  • the Fmoc chemical protecting group of the peptide fragment Fmoc-AA(21-36)-NHC18 was then removed prior to isolation of the fragment H-AA(21-36)-NHC18.
  • piperidine (0.301 mL, 3.04 mmol, 6 eq) was added, and the solution was stirred for 1 hour at 25 ⁇ 5 0 C or until analysis by HPLC showed that substantially all the Fmoc was removed from the peptide fragment.
  • Peptide fragment H-AA(21-36)-NHC18 (SEQ ID NO:29; 1.501 g, 0.366 mmol, 1 eq), peptide fragment comprising amino acids 1-20 of SEQ ID NO:33 ("Ac-AA(I -2O)-OH"; SEQ ID NO:9; 1.628 g, 0.368 mmol, 1.0 eq), and HOAT (0.075 g, 0.551 mmol, 1.5 eq) and DIEA (0.255 ml, 0.1.46 mmol, 4 eq) were dissolved in DMF (15 ml, 10 vol), and the reaction was cooled to 0 ⁇ 5 0 C.
  • the conjugate Ac-AA(I -36)-NHC18 was then deprotected (by removing the side chain chemical protecting groups) and decarboxylated (at the tryptophan residues) by using the methods described herein, or any other method known to those skilled in the art, for deprotection and decarboxylation, with subsequent purification.
  • the result was a substantially pure preparation (deprotected and decarboxylated) of a conjugate comprising a synthetic peptide operatively linked to a fatty acid, wherein the fatty acid is operatively linked to the C-terminus of the synthetic peptide.
  • peptide fragments in Set 20 (SEQ ID NO:25 & SEQ ID NO:10 + Leu36) or Set 21 (SEQ ID NO:25 & SEQ ID NO:11 ), produced by a fragment condensation approach is a conjugate comprising a synthetic peptide operatively linked to a fatty acid, wherein the fatty acid is operatively linked to the N-terminus of the synthetic peptide.
  • the fragment approach is essentially the same as that described in this example for the conjugate comprising a synthetic peptide operatively linked to a fatty acid, wherein the fatty acid is operatively linked to the C-terminus of the synthetic peptide.
  • SEQ ID NO:29 for a peptide fragment comprising amino acids 21- 36 of SEQ ID NO:4, used is SEQ ID NO:11.
  • SEQ ID NO:9 for amino acids 1-20
  • SEQ ID NO:25 having a fatty acid operatively linked to the N-terminal amino acid of the peptide fragment.
  • Fatty acid can be operatively linked to the N-terminal amino acid of the peptide fragment by standard coupling methods known in the art. For example, H-AA(I -2O)-CPG (SEQ ID NO:9) and fatty acid (C18-OPFP), and DIEA are dissolved in DMF, and stirred for 2 hours at room temperature.
  • a fragment condensation approach to synthesizing a conjugate wherein the conjugate comprises synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid at two different locations of the synthetic peptide (see, e.g., Table 3, Ref# 4-11).
  • the conjugate comprises synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid at two different locations of the synthetic peptide (see, e.g., Table 3, Ref# 4-11).
  • more than one of the peptide fragments used in the fragment condensation approach is operatively linked to fatty acid.
  • This example also illustrates use of peptide fragments from multiple sets as listed in Table 2. For example, illustrated is a peptide fragment having the amino acid sequence of SEQ ID NO:25 (Table 2, Set 20) combined with a peptide fragment having the amino acid sequence of SEQ ID NO.19 (Table 2, Set 12) to form the conjugate.
  • a synthetic peptide having an amino acid sequence of SEQ ID NO:4 was synthesized by a fragment condensation approach using a peptide fragment having an amino acid sequence of SEQ ID NO:25 having operatively linked at the N-terminus a linker-fatty acid combination ("C18-PEG3"), and a peptide fragment having an amino acid sequence of SEQ ID NO:19 having a linker- fatty acid combination operatively linked to an internal lysine ("K30”) ("AA(21-30(PEG3- C18)-36)").
  • Fmoc-AA(1-20)-O-2-CTC resin was put into peptide reactor, which was temperature-controlled at 3O 0 C with circulating bath. 10vol of DCM was charged into the reactor, and the reaction was agitated with nitrogen for 15 minutes, and then drained. Fmoc protection group was removed by the treatment of 20% piperidine in NMP (N- methyl pyrrolidinone; 10vol) for 20 minutes, twice. NMP washed the resin until a negative chloranil test was obtained.
  • NMP N- methyl pyrrolidinone
  • the reactor was charged with pre-activated C18-PEG3-OH/6- CI-HOBt/TBTU/DIEA (2/2/2/2.3eq) in DMF/DCM(1 :1 , 8 vol) and the mixture was agitated by nitrogen until a negative ninhydrine test was obtained.
  • the resin was washed by NMP (10 vol x 4) and DCM (10 vol x 4).
  • the reactor was charged with 1%TFA in DCM (10 vol) and the suspension was agitated by nitrogen for 2 minutes at O 0 C, then DCM solution was drained into a flask with pyridine (1.26 vol relative to TFA). After this treatment was repeated ten times, the resin was washed by DCM twice and IPA twice.
  • the resulting slurry was stirred at room temperature for 30 minutes before vacuum filtration. After being washed with MTBE, the solid was reslurried in acetonitrile with pH adjust to 4 by AcOH and DIEA overnight. The suspension was diluted by 5OmM NH 4 OAc and the solution was filtered and directly loaded onto HPLC for purification.
  • the result is a substantially homogeneous conjugate comprised of fatty acid operatively linked (through a linker) to the N-terminus, and fatty acid operatively linked (through a linker) to an internal amino acid (lysine in amino acid position 30), of a synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • PEG3 linker comprising (PEG3) n (where n > 1 )
  • starting material known as 8-amino-3,6 dioxaoctanoic acid, and what is referenced herein as "PEG3", generally represented by the structure:
  • R 1 and R 2 are each a reactive functionality, and more preferably a reactive functionality selected from the group consisting of a hydrogen, oxygen, a hydroxyl, an amine-reactive group, a carboxyl-reactive group, and a chemical protecting group
  • CPG used in peptide synthesis to protect a reactive functionality from further chemical reactivity.
  • Various discrete length linkers having PEG3 as a subunit, were produced by covalently coupling two or more PEG3 molecules together to get the desired discrete length of the linker.
  • PEG3 poly(ethylene glycol)
  • the specific number of ethylene glycol units (i.e., - OCH 2 CH 2 ) in the chemical backbone of the linker ranges from 2 to 30 ethylene glycol units, depending on the value of n, one or two of which ethylene glycol units may be broken or substituted for in forming a reactive functionality or for linking a molecule of PEG3 to a another molecule of PEG3 in forming (PEG3) n .
  • a reactive functionality of a PEG linker e.g., PEG13, PEG25, PEG29, etc.
  • a reactive functionality of a molecule are essentially the same as those described herein for the (PEG3) 2 linker.
  • a (PEG3) 2 linker is produced by solid phase synthesis.
  • 2-CTC resin (15g, 1.61 mmol/g, 24.15 mmol) was swelled in 150 ml of CH 2 CI 2 twice for 5 minutes each time.
  • PEG3 Fmoc-PEG3-OH; 3.32g, 9 mmol
  • DIEA N,N-diisopropylethylamine; 2.90 ml_, 18 mmol.
  • the solution containing PEG3 was added to the swelled resin, and the resin was agitated for 2 hours. The resin was then washed three times with CH 2 CI 2 .
  • a linker may be produced which comprises a combination of any of PEG13, PEG3, and PEG25. Also illustrated in this embodiment is a method of making a linker-fatty acid combination.
  • An illustrative and preferred fatty acid combination comprises C18-PEG3-R, where R is a reactive functionality.
  • C18-PEG3-OH may be represented by the formula:
  • Example 7 A method of making C18-PEG3-OH is illustrated in Example 7.
  • other reactive functionalities have been used besides a hydroxyl.
  • a linker-fatty acid combination comprising C18-PEG3-OPFP was produced as follows.
  • C18-PEG3-OH (25.08g, 1eq) and pentafluorophenol (PFP, 10.74g, 1eq) were mixed in DCM (500ml).
  • the resulted suspension was cooled to O 0 C, then 1 ,3-diisopropylcarbodiimide (DIC, 9.14ml, 1eq) was added into it.
  • EXAMPLE 7 The present invention also provides a novel composition comprising a modified amino acid comprising a lysine which is operatively linked to fatty acid through a linker (i.e., an amino acid-linker-fatty acid combination) which is incorporated into the amino acid sequence of a peptide fragment or synthetic peptide during synthesis.
  • a linker i.e., an amino acid-linker-fatty acid combination
  • the modified amino acid may be represented by the formula: CPG-LyS(PEG) n -FA)-R 1 wherein n is from 2 to 100, and more preferably, n is from 3 to 30; wherein FA is a fatty acid (see, e.g., Table 1 ; a preferred fatty acid is in the range of C-12 to C-20); wherein R 1 is a reactive functionality, and more preferably a reactive functionality selected from the group consisting of a hydrogen, oxygen, a hydroxyl, an amine-reactive group (including, but not limited to an activated ester), a carboxyl-reactive group; and wherein CPG comprises a chemical protecting group, preferably a chemical protecting group useful in peptide synthesis.
  • Preferred chemical protecting groups include, but are not limited to, Fmoc, Boc (Fmoc and Boc are preferred amine protection groups), benzyl (Bz), trityl, and t-butyl.
  • CPG is at the amino terminus of the lysine
  • the PEG linker (with attached fatty acid) is covalently coupled to the lysine epsilon amine
  • Ri is at the carboxy terminus of the lysine.
  • Lys-PEG3-C18 (wherein the epsilon amine of lysine is covalently coupled to one reactive functionality of the linker PEG3, and the fatty acid C18 is covalently coupled to another reactive functionality of the linker PEG3) may be represented as CPG-Lys(PEG3-C18)-Ri.
  • This modified lysine may be incorporated into a peptide fragment, or a synthetic peptide, of the present invention during its synthesis, at a position in the amino acid sequence position corresponding to the lysine in an amino acid sequence of any one of SEQ ID NOs: 16-21 , and 23, as further illustrated in Tables 2 & 4.
  • Assembly of this peptide fragment with one or more peptide fragments in a set of peptide fragments can result in production of a conjugate comprised of a synthetic peptide having the amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid.
  • CPG-Lys(PEG3-C18)-Ri as exemplified by Fmoc-Lys(PEG3-C18)-OH, was synthesized by a process which included utilizing a solid phase coupling.
  • Fmoc-PEG3-OH was coupled to a solid phase resin by dissolving Fmoc- PEG 3 -OH (30.3 g, 218.4 mmol, 0.36 eq) and DIEA (76 ml, 218.4 mmol, 2 eq) in DCM (700 ml, 5 vol) and adding the solution to 2-CTC resin (140 g, 218.4 mmol Cl, 1 eq) which had been pre-swelled in DCM. The coupling was allowed to proceed with agitation for 40 minutes at 2O 0 C to 25 0 C.
  • the resin was drained and unreacted sites were capped by adding a solution of 9:1 methanol: DIEA (840 ml, 6 vol). After 45 minutes at 25 ⁇ 5 0 C, the resin was drained, and washed with methanol. The Fmoc group was removed from the resin-bound PEG3 by reswelling the resin in DCM and then treating it twice with 10% piperidine in NMP (700 ml, 5 vol) for 20 to 30 minutes. The resin was then washed with NMP to a negative chloranil test.
  • Stearic acid (C18) was activated by dissolving stearic acid (20.5 g, 72.2 mmol, 1.1 eq) in 1 :1 NMP/DCM (840 ml, 6 vol) by the addition of DIEA (6.3 ml, 85.3 mmol, 1.3 eq).
  • TBTU (23.2 g, 72.2 mmol, 1.1 eq) was added and the solution was stirred.
  • resin bound PEG3 was added the activated C18 solution and the mixture was allowed to react for 1 to 3 hours or until the reaction was complete by the Kaiser test.
  • the resin was washed twice with 1 :1 DCM/NMP, 3 times with DCM, and then the resultant compound comprising a linker-fatty acid combination (C-18-PEG3) was cleaved from the resin by treatment with 2% TFA in DCM for 45 to 50 minutes.
  • the resin was drained and washed repeatedly with DCM.
  • the DCM was removed by distillation and replaced by MTBE which caused the product to precipitate.
  • the product was isolated by filtration, reslurried in MTBE, re-isolated, and dried.
  • the resultant product was a substantially pure preparation of C18-PEG3-OH.
  • CPG-LyS(PEGS-CIS)-R 1 Alternate methods may be used to produce CPG-LyS(PEGS-CIS)-R 1 .
  • CPG-Lys-OH 50 mg, 0.134 mmol, 1.0 eq
  • ethyl acetate 1 ml_
  • DIEA 57 ⁇ L, 0.339 mmol, 2.5 eq
  • C18-PEG3-OH 80.8 mg, 0.134 mmol, 1.0 eq
  • the product was purified by flash chromatography using DCM/MeOH as the mobile phase.
  • the resultant product was a preparation of isolated CPG-Lys(PEG3-C18)OH.
  • Fmoc-Lys-OH 11.65 g. 1 eq
  • DIEA 5 ml, 1 eq
  • C18-PEG3-OPFP was added into the solution, and the reaction mixture was stirred at room temperature for 3 hours or until analysis of a sample showed that the reaction was complete.
  • the reaction mixture was cooled to O 0 C to 5 0 C.
  • HCI (0.01 N, 342 ml, 20 vol) was added to the reaction mixture.
  • a fragment condensation approach for synthesizing a conjugate comprised of fatty acid operatively linked to a synthetic peptide having an amino acid sequence of SEQ ID NO:4.
  • the modified amino acid according to the present invention (as described in Example 7) is used as an amino acid in the synthesis of a peptide fragment according to the present invention.
  • a method of making a conjugate comprises the steps of: (a) synthesizing two or more peptide fragments, wherein one of the peptide fragments is synthesized so as to comprise a peptide fragment operatively linked to fatty acid through a linker; (b) combining the peptide fragments by chemically coupling to produce the conjugate comprising a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid through a linker.
  • SEQ ID NO:13 As an illustration of a method for producing such conjugate, refer to FIG. 4 and Tables 2 & 4 (SEQ ID NO:13, and either SEQ ID NO:20 or SEQ ID NO:21 ).
  • a peptide fragment having an amino acid sequence of SEQ ID NO:20 (“Fmoc-AA(17- Lys 30 (PEG3-C18)-35)-OH”) was built on H-Leu-CTC resin (0.59 mmol/g) using standard SPPS conditions and recovered as white solid.
  • a peptide fragment having an amino acid sequence of SEQ ID NO:21 was synthesized. Fmoc-AA(17- Lys 30 (PEG3-C18)-35)-OH (1.50 g, 1 eq), H-LeU-NH 2 (46.8 mg, 1.2 eq) and 6 CI-HOBt (61 mg, 1.2 eq) were dissolved in DMF (15 mL).
  • a conjugate comprised of a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid was produced by a fragment condensation approach when a peptide fragment having the amino acid sequence of SEQ ID NO:21 (H-AA(17-Lys 30 (PEG3-C18)-36-NH 2 ) was operatively linked (covalently coupled) to a peptide fragment having the amino acid sequence of SEQ ID NO:13 ("Ac-AA(I -16)-OH").
  • the reaction mixture was stirred at room temperature for 4.5 hours, and then cooled to O 0 C to 5 0 C.
  • the pre-cooled MTBE 50ml was added into the reactor slowly to maintain the temperature below 1O 0 C.
  • the resulted slurry was stirred for 30 minutes with ice bath, and 4 hours at room temperature.
  • the solid was collected by vacuum filtration, washed by MTBE, and then returned to the flask for decarboxylation.
  • Acetonitrile (10 mL) was added into the flask, then 150 ⁇ l of DIEA and 100 ⁇ l of HOAc.
  • the suspension with pH at 3.5 to 4 was stirred at room temperature overnight.
  • the crude conjugate was collected as an off-white solid by vacuum filtration. Purification was achieved by HPLC. The result is a substantially pure preparation
  • a conjugate comprised of synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to fatty acid through a linker.
  • EXAMPLE 9 illustrated is a method of producing a conjugate comprised of a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to a fatty acid through a linker, wherein the method is by linear synthesis (linear fashion by standard solid phase synthesis), and the modified amino acid according to the present invention (as described in Example 7) is incorporated into the amino acid sequence at position 30 of SEQ ID NO:4.
  • amide resins are examples of peptide synthesis resins often referred to as "amide" resins, because synthesis of a peptide on these resins, followed by cleavage of the synthesized peptide, can provide a peptide with a C-terminal amide group, and in some cases, a peptide with a preferred chemical group, other than an amide, at the C- terminal amino acid.
  • the C-terminal amino acid of the synthetic peptide has a chemical group comprising an amide.
  • a method of synthesizing a conjugate comprises: linearly synthesizing a synthetic peptide having an amino acid sequence of SEQ ID NO:4 using an amide resin and solid phase peptide synthesis; incorporating a modified amino acid comprising a lysine operatively linked to fatty acid as an amino acid in the amino acid sequence of SEQ ID NO:4 (e.g., at position 30 of SEQ ID NO:4) during synthesis of the synthetic peptide to produce the conjugate; and cleaving the resultant conjugate from the resin.
  • the conjugate may be made with higher throughput (e.g., fewer isolation steps); and fewer impurities (such as those that may be generated in solution phase reactions or additional coupling reactions).
  • the conjugate, synthesized on the resin was the cleaved from the resin, deprotected, and then purified.
  • the resin was treated with 2% TFA in DCM (10 vol) at 0°C for 2 minutes. This treatment was repeated for 10 times.
  • the resin was then washed with DCM (10vol) and IPA (10vol X 2).
  • the solvent was removed on the rotavap under reduced pressure. The residue was re-dissolved in IPA (10 vol) and precipitated by water. After being re-slurried in 10% EtOH (30 vol) for 2 hours, the solid was collected by vacuum filtration and dried in vacuum oven overnight. 1.44g of white solid was collected after drying.
  • the solution was collected in a flask with pre-cooled MTBE.
  • the solid was collected by vacuum filtration. It was decarboxylated in acetonitrile with pH adjustment by AcOH and DIEA overnight.
  • the resulting solid was collected as a crude preparation of conjugate.
  • This conjugate was purified on HPLC or using other chromatographic methods or purification methods known in the art of peptide synthesis, and the resulting white fluffy solid is a substantially pure preparation of conjugate comprised of a synthetic peptide having an amino acid sequence of SEQ ID NO:4 operatively linked to a fatty acid through a linker (92.6% purity).

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Abstract

L'invention concerne des fragments de peptide pouvant être utilisés dans une méthode de production d'un peptide synthétique ou d'un conjugué constitué du peptide synthétique lié de manière fonctionnelle à un acide gras. La méthode consiste à lier chimiquement ensemble deux ou plusieurs fragments de peptide, dans un processus d'assemblage, aux fins de produire le peptide synthétique ou le conjugué.
PCT/US2006/011470 2005-03-30 2006-03-28 Compositions et methodes de synthese d'un peptide et d'un conjugue apparente WO2006105199A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009155064A1 (fr) * 2008-05-28 2009-12-23 New York Blood Center Molécules bifonctionnelles destinées à inhiber la pénétration du vih
US8828932B2 (en) 2010-05-03 2014-09-09 New York Blood Center, Inc. Bifunctional molecules for inactivating HIV and blocking HIV entry
US8936789B2 (en) 2008-10-16 2015-01-20 New York Bood Center, Inc. Immunoenhancer-linked oligomeric HIV envelope peptides
US9115186B2 (en) 2009-02-09 2015-08-25 New York Blood Center Stabilized trimeric HIV-1 gp41 fusion polypeptides comprising the N-terminal heptad repeat (NHR) and foldon (FD) trimerization motif

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6750008B1 (en) * 1999-07-09 2004-06-15 Trimeris, Inc. Methods and compositions for inhibition of membrane fusion-associated events, including HIV transmission
US20040115774A1 (en) * 2000-09-08 2004-06-17 Kochendoerfer Gerd G Polymer-modified synthetic proteins
US6767993B2 (en) * 1999-07-07 2004-07-27 Brian Bray Methods and compositions for peptide synthesis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6767993B2 (en) * 1999-07-07 2004-07-27 Brian Bray Methods and compositions for peptide synthesis
US6750008B1 (en) * 1999-07-09 2004-06-15 Trimeris, Inc. Methods and compositions for inhibition of membrane fusion-associated events, including HIV transmission
US20040115774A1 (en) * 2000-09-08 2004-06-17 Kochendoerfer Gerd G Polymer-modified synthetic proteins

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009155064A1 (fr) * 2008-05-28 2009-12-23 New York Blood Center Molécules bifonctionnelles destinées à inhiber la pénétration du vih
JP2011521648A (ja) * 2008-05-28 2011-07-28 ニューヨーク ブラッド センター， インコーポレイテッド Ｈｉｖの侵入を阻害するための二機能性分子
US8828931B2 (en) 2008-05-28 2014-09-09 New York Blood Center, Inc. Bifunctional molecules for inhibiting HIV entry
US8936789B2 (en) 2008-10-16 2015-01-20 New York Bood Center, Inc. Immunoenhancer-linked oligomeric HIV envelope peptides
US9115186B2 (en) 2009-02-09 2015-08-25 New York Blood Center Stabilized trimeric HIV-1 gp41 fusion polypeptides comprising the N-terminal heptad repeat (NHR) and foldon (FD) trimerization motif
US9724383B2 (en) 2009-02-09 2017-08-08 New York Blood Center, Inc. Stabilized trimeric HIV-1 GP41 fusion inhibitor polypeptides comprising the N-terminal heptad repeat (NHR) and foldon (FD) trimerization motif
US8828932B2 (en) 2010-05-03 2014-09-09 New York Blood Center, Inc. Bifunctional molecules for inactivating HIV and blocking HIV entry

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