WO2016207626A1 - Fluorogenic compounds, process of preparation thereof and methods of use - Google Patents

Fluorogenic compounds, process of preparation thereof and methods of use Download PDF

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Publication number
WO2016207626A1
WO2016207626A1 PCT/GB2016/051864 GB2016051864W WO2016207626A1 WO 2016207626 A1 WO2016207626 A1 WO 2016207626A1 GB 2016051864 W GB2016051864 W GB 2016051864W WO 2016207626 A1 WO2016207626 A1 WO 2016207626A1
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protected
group
derivative
tryptamine
tryptophan
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PCT/GB2016/051864
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French (fr)
Inventor
Marc Vendrell Escobar
Ramon SUBIROS FUNOSAS
Lorena MENDIVE TAPIA
Fernando Albericio Palomera
Rodolfo Lavilla Grifols
Nick D. READ
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The University Court Of The University Of Edinburgh
Universitat De Barcelona
The University Of Manchester
Fundació Institut De Recerca Biomèdica (Irb Barcelona)
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Application filed by The University Court Of The University Of Edinburgh, Universitat De Barcelona, The University Of Manchester, Fundació Institut De Recerca Biomèdica (Irb Barcelona) filed Critical The University Court Of The University Of Edinburgh
Publication of WO2016207626A1 publication Critical patent/WO2016207626A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages

Definitions

  • the invention relates to compounds, particularly fluorogenic amino acid derivatives, which can be used as optical probes.
  • the invention further relates to processes for the preparation of such compounds, the use of such compounds as probes and methods of detecting a target using such compounds as probes.
  • IPA Invasive pulmonary aspergillosis
  • IPA is a highly fatal disease in immunocompromised patients. IPA results from the infection with the fungal pathogen Aspergillus fumigatus, and it is a frequent cause of fungal pneumonia with mortality rates up to 40%.
  • Current diagnostic approaches for IPA rely on histological analysis, cultures from bronchoalveolar lavage fluid and sampling peripheral blood. These methods are fraught with problems of upper airway contamination and diagnostic delays, by which time the disease may have progressed or been treated empirically with inappropriate drugs.
  • blood markers are unlikely to provide useful information about events deep in pulmonary tissue, especially in patients with multi-system disease, such as immunosuppressed patients affected by IPA.
  • Fluorescent probes based on antibiotics and antimicrobial peptides are chemical entities with enormous potential for imaging infection sites due to their high selectivity for microbial cell structures over mammalian cells. These probes have been prepared by conjugating peptides of interest to suitable fluorophores via chemical spacers. While such approaches have been useful to functionalise long peptides or proteins, alternative strategies are needed for shorter peptides, where relevant modifications can compromise their specificity. For instance the use of chemical spacers could compromise the molecular recognition of the peptide for its target. Molecular recognition may be affected by a number of factors, such as changes in the polarity or hydrophobicity of the peptide, H-bonding pattern, or
  • Peptide AntiFungal 26 is a synthetic antimicrobial hexapeptide with high affinity for fungal cells and selectivity over bacterial and mammalian cells (Lopez- Garcia, B., Perez-Paya, E. & Marcos, J. F. "Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library", Appl. Environ. Microbiol. 68, 2453-2460 (2002); Munoz, A.
  • PAF26 has a highly conserved sequence with a C-terminal hydrophobic domain (Trp-Phe- Trp) and an /V-terminal cationic domain (Arg-Lys-Lys) that are essential to exert its antifungal action.
  • Fluorogenic amino acids are advantageous in that they provide high signal-to-noise ratios without the need for washing or additional labelling steps.
  • fluorogenic amino acids exhibit inherent limitations as fluorophores (e.g. short emission wavelengths, low extinction coefficients, compromised cell permeability) and may not behave as surrogates of the amino acids they replace, leading to alterations in the molecular recognition properties of the peptide. A need therefore exists to provide fluorogenic amino acids which address these problems.
  • the present invention provides a novel fluorogenic compound based on the 4,4-difluoro-4- bora-3a,4a-diaza-s-indacene (BODIPY) scaffold.
  • BODIPY 4,4-difluoro-4- bora-3a,4a-diaza-s-indacene
  • R 1 , R 2 , R 3 , R 5 , R 6 and R 7 are independently selected from the group comprising H, CMO alkyl, C 2 -io alkenyl, C 2 -io alkynyl, CM 0 alkyl carboxylic acid, Ci_i 0 alkoxy, CM 0 acyl, Ci_i 0 acyloxy, C 6 -io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C;
  • L is a conjugated divalent linker selected from a C 6 -io arylene group; and wherein one or more of said R 1 , R 2 , R 3 , R 5 , R 6 , R 7 and L groups may be optionally
  • A is a group comprising an aromatic group and is selected from tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; wherein the conjugated divalent linker L is covalently bonded to the aromatic group of group
  • the conjugated divalent linker L is free from chemical spacers.
  • a conjugated system comprising the BODIPY aromatic core, the conjugated divalent linker and the aromatic group of the amino acid is provided.
  • Such a conjugated system is advantageous because it is capable of strong fluorogenic behaviour and because it has minimal impact on the molecular properties of the peptide.
  • Direct carbon-carbon coupling of an amino acid residue in a peptide and a fluorogen without a chemical spacer minimizes any change in the polarity of a peptide modified to contain the fluorogen, thereby minimizing any changes in molecular recognition of a target for the peptide. This allows such fluorogen modified peptides to be used as probes while retaining the original selectivity.
  • the amino acids tryptophan and phenylalanine to which the fluorogen is linked are both hydrophobic, and so they are commonly present in the hydrophobic domain of a peptide.
  • BODIPY is a hydrophobic fluorogen such that the introduction of a BODIPY-derived substituent will not alter the hydrophobic character of these amino acids and may even increase the stability of such a domain. Consequently, the addition of the BODIPY-derived fluorogen minimises any modification of the hydrophobicity of the peptide, leading to a retention of the molecular recognition compared to the unmodified peptide.
  • the conjugated divalent linker L is selected from the group comprising a divalent phenyl group and a divalent naphthyl group, which may be unsubstituted or substituted.
  • the conjugated divalent linker L is a group selected from L3 or L4 having the structural formulae:
  • the conjugated divalent linker L may be independently substituted with 1 or more substituents independently selected from the group comprising Ci_ 6 alkyl and Ci_ 6 alkoxy. More preferably, when substituted, the conjugated divalent linker L may be independently substituted with from 1 to 3 substituents selected from methyl and methoxy. In one embodiment, the divalent linker L is independently substituted with 1 or more substituents independently selected from the group comprising C 2 -6 alkyl and C 2 -6 alkoxy. In another embodiment, the divalent linker L is independently substituted with 1 or more substituents independently selected from methyl and methoxy.
  • the conjugated divalent linker L is a group selected from L1 or L2 having the structural formulae:
  • the conjugated divalent linker L may be selected from the group comprising 1 ,3- phenylene (L1) and 1 ,3-naphthdiyl (L2).
  • the conjugated divalent linker L is a 1 ,3- phenylene group, for instance when the group is tryptophan or a derivative thereof or a peptide comprising tryptophan or a derivative thereof.
  • the groups R 1 , R 3 , R 5 and R 7 are preferably methyl. In another embodiment, the groups R 2 and R 6 are preferably H. In a further embodiment, the groups R 1 , R 3 , R 5 and R 7 are preferably methyl and the groups R 2 and R 6 are preferably H.
  • the groups R 1 , R 2 , R 3 , R 5 , R 6 and R 7 are unsubstituted. In another embodiment, one or more of the groups R 1 , R 2 , R 3 , R 5 , R 6 and R 7 are independently substituted with 1 or more substituents independently selected from the group comprising C 2 -6 alkyl and C 2 -6 alkoxy. In another embodiment, one or more of the groups R 1 , R 2 , R 3 , R 5 , R 6 and R 7 are independently substituted with 1 or more substituents independently selected from methyl and methoxy.
  • derivatives of tryptaminyl, tryptophanyl, phenylalaninyl, N- protected-tryptaminyl, N-protected tryptophanyl or N-protected phenylalaninyl include peptides, proteins, peptidomimetics, peptoids, and peptide nucleic acids comprising residues of tryptamine, tryptophan, phenylalanine, N-protected tryptamine, N-protected tryptophan or N-protected phenylalanine.
  • the derivatives are obtainable by condensing tryptophan, phenylalanine, N-protected tryptophan or N-protected phenylalanine with a -COOH or -NH group on an amino acid, amino acid residue, peptidomimetic, peptoid or peptide nucleic acid to form a peptide bond.
  • the derivatives of tryptaminyl or N-protected tryptaminyl are obtainable by condensing the -NH group of tryptamine or N-protected tryptamine with a -COOH group on an amino acid, amino acid residue, peptidomimetic, peptoid or peptide nucleic acid to form a peptide bond
  • the group A is a derivative of tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl or N-protected phenylalaninyl, then these derivatives may be formed by known techniques, such as solid phase synthesis or synthetic biology. Examples of suitable solid phase syntheses are discussed below.
  • an organism such as a bacteria can be altered, for instance by the alteration of its RNA, to accept compounds of formula (I) in which the group A is tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl or N- protected phenylalaninyl as a synthetic amino acid, allowing the synthesis of derivatives of tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl or N-protected phenylalaninyl, for instance peptides or proteins incorporating compounds of formula (I).
  • the group A comprising an aromatic group may be selected from: tryptaminyl; N-protected tryptaminyl; an amino acid comprising an aromatic group selected from tryptophanyl,
  • phenylalaninyl and derivatives thereof an N-protected derivative of an amino acid comprising an aromatic group selected from N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; a peptide obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an
  • a peptoid derivative comprising an amino acid residue comprising an aromatic group, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; or a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a protein derivative
  • the group A is an amino acid comprising an aromatic group selected from tryptophanyl, phenylalaninyl and derivatives thereof; an N-protected derivative of an amino acid comprising an aromatic group selected from N-protected tryptophanyl, N- protected phenylalaninyl and derivatives thereof; or a peptide obtainable from one or more amino acids, particularly one or more types of amino acids, comprising an amino acid comprising an aromatic group selected from tryptophanyl, phenylalaninyl and derivatives thereof.
  • the amino acid comprising an aromatic group, N-protected derivative of an amino acid comprising an aromatic group and peptides obtainable from such amino acids may independently be in the D- or L-configuration.
  • the group A is selected from the group comprising: tryptaminyl; N- protected tryptaminyl; a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine; a peptide nucleic acid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with tryptamine or
  • a peptoid derivative comprising an amino acid residue, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; or a protein derivative obtainable from
  • the proteins may be obtainable from amino acids independently be in the D- or L- configuration.
  • the group A may selected from: a natural amino acid selected from tryptophan, such as L-tryptophan, or an N- protected derivative thereof, including D-tryptophan and N-protected derivatives of D-tryptophan;
  • a synthetic D or L amino acid selected from the group comprising phenylalanine, phenylalanine derivatives, tryptophan derivatives or an N-protected derivative thereof;
  • peptide obtainable from at least one of said natural or synthetic amino acids; a peptidomimetic obtainable from at least one of said natural or synthetic amino acids, or
  • a protein obtainable from at least one of said natural or synthetic amino acids.
  • the nitrogen protecting groups for the N-protected tryptaminyl, N-protected amino acid or N- protected amino acid derivative such as peptides, proteins, peptidomimetics, peptoid derivative and peptide nucleic acid derivative obtainable from such N-protected amino acids may be selected from the group comprising Fmoc (fluorenylmethyloxycarbonyl), Boc (t- butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), Ts
  • tryptamine, tryptophan and phenylalanine contain an aromatic group, indolyl in the case of tryptamine and tryptophan and phenyl in the case of phenylalanine, to which the conjugated divalent linking group L is bound without a chemical spacer, forming a larger conjugated system.
  • the conjugated divalent linker is directly covalently bonded to the aromatic group of A by a single covalent bond i.e. there is no intervening bridging group.
  • the derivative of tryptophanyl, N-protected tryptophanyl, phenylalaninyl or N-protected phenylalaninyl may be the residue of tryptophan, N-protected tryptophan, phenylalanine or N-protected phenylalanine after the formation of at least one peptide bond.
  • a peptide bond may be formed between the -NH or -COOH group of the tryptophan, N-protected tryptophan, phenylalanine or N-protected phenylalanine and a -COOH or -NH 2 group respectively of a further species, such as an amino acid, peptide, protein or peptide nucleic acid.
  • tryptophan, N-protected tryptophan, phenylalanine and N- protected phenylalanine are capable of forming two peptide bonds, such that the amino acid residue of tryptophan, N-protected tryptophan, phenylalanine or N-protected phenylalanine may be incorporated into the terminal position of a polyamide, when a single peptide bond is formed or mid-chain, when two peptide bonds are formed.
  • the derivative of tryptaminyl, N-protected tryptaminyl may be the residue of tryptamine or N- protected tryptamine after the formation of a peptide bond.
  • a peptide bond may be formed between the -NH group of the tryptamine or N-protected tryptamine and a -COOH group of a further species, such as an amino acid, peptide, protein or peptide nucleic acid. It will be apparent that tryptamine and N-protected tryptamine are capable of forming a single peptide bond, such that the residue of tryptamine or N-protected tryptamine is incorporated into the terminal position of a polyamide.
  • the group A comprises tryptophanyl, an N-protected tryptophanyl or a derivative thereof. Still more preferably, the tryptophanyl, N-protected tryptophanyl or a derivative thereof is bonded to the conjugated divalent linker L via the indole C2 position of the tryptophanyl or tryptophanyl residue.
  • the group A is an amino acid, it is tryptophanyl or an N-protected tryptophanyl. Still more preferably, the tryptophan or N-protected tryptophanyl is bonded to the conjugated divalent linker L via the indole C2 position of the tryptophanyl.
  • the compound of formula (I) is compound selected from:
  • Z is an N-protecting group, such as a group selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), tnfluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl), Ts (toluenesulfonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4- biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2- (4-Nitrophenylsulfonyl
  • the group A is a peptide, such as a linear or cyclic peptide, wherein the peptide is obtainable from one or more amino acids, at least one of which is an amino acid comprising an aromatic group, an N-protected amino acid comprising an aromatic group or derivatives thereof as defined above.
  • the amino acid in the peptide may be an N- protected amino acid when it is in the N-terminal position of the peptide.
  • the conjugated linker group L is covalently bonded to the aromatic group of the amino acid comprising an aromatic group, N-protected amino acid comprising an aromatic group or derivatives thereof, without a chemical spacer, such that a larger conjugated system is formed.
  • the peptide is obtainable from one or more kinds of amino acids, at least one of which is tryptophan or an N-protected tryptophan, for instance if it is in the N-terminal position of the peptide.
  • the group A is a protein obtainable from one or more amino acids, at least one of which is an amino acid comprising an aromatic group, an N-protected amino acid comprising an aromatic group or derivatives thereof as defined above.
  • the amino acid in the protein may be an N-protected amino acid when it is in the N-terminal position of the protein.
  • the conjugated linker group L is covalently bonded to the aromatic group of the amino acid, aromatic group of the N-protected amino acid or derivatives thereof, without a chemical spacer, such that a larger conjugated system is formed.
  • the protein is obtainable from one or more kinds of amino acids, at least one of which is tryptophan or an N-protected tryptophan, for instance if it is in the N-terminal position of the protein.
  • the group A may be a peptide, such as a linear or cyclic peptide, obtainable from a plurality of amino acids, or derivatives thereof as defined above or an N- protected amino acid, for instance in the N-terminal position of the peptide, in combination with at least one amino acid or derivative thereof as defined above, which comprise a plurality of fluorophores of formula (i):
  • each conjugated divalent linker L is covalently bonded to an aromatic group of the peptide and the groups R 1 , R 2 , R 3 , R 5 , R 6 , R 7 and L are as defined above.
  • At least one of said plurality of amino acids, N-protected amino acids or derivatives thereof are covalently bonded to conjugated divalent linkers covalently bonded to a BODIPY core as shown in Formula (I).
  • conjugated divalent linkers covalently bonded to a BODIPY core as shown in Formula (I).
  • At least two of said plurality of amino acids, N-protected amino acids or derivatives thereof are covalently bonded to conjugated divalent linkers covalently bonded to a BODIPY core as shown in Formula (I).
  • the group A comprising an aromatic group may be selected from: a peptide obtainable from a plurality of amino acids, at least two of said plurality of amino acids comprising an aromatic group and independently selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptide nucleic acid derivative comprising at least two amino acid residues comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with at least two amino acids comprising an aromatic group, said amino acid comprising an aromatic group independently selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptidomimetic derivative comprising at least two an amino acid residues comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with at least two amino acids comprising an aromatic group, said at least two amino
  • the conjugated divalent linker of the compound of formula (I) may be attached to the peptide (ii) at one or both of positions 1 and 2 shown:
  • a hydrogen atom is present at position 1 or position 2 if this is not covalently attached to the conjugated divalent linker.
  • the group A may be a linear peptide.
  • the group A may be a linear peptide comprising PAF26, namely:
  • H-Arg-Lys-Lys-Trp-Phe-Trp-NH 2 (Seq. I.D. No. 2) or H-Ala-Ala-Ala-Trp-Phe-Trp-NH 2 (Seq. I.D. No. 5) or
  • H-Arg-Lys-Lys-Trp-Ala-Ala-NH 2 (Seq. I.D. No. 6). in which one or both of the tryptophan residues in Seq. I.D. Nos. 1 , 2 and 5 are covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group or in which the tryptophan residue in Seq. I.D. No. 6 is covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group.
  • the amino acids forming these linear peptides may be independently in the D- or L- configuration.
  • the linear peptides may comprise amino acids in both the D- and L- configuration, for instance either as the same amino acid in different configurations, such as D-Lys and L-Lys, or different amino acids in different configurations, such as D-Arg and L- Trp.
  • the compound of formula (I) is selected from:
  • the group A may be a cyclic peptide.
  • the group A may comprise a cyclic peptide analogue of PAF26, namely:
  • Cyclo(-Arg-Lys-Lys-Trp-Phe-Trp-Gly-) (Seq. I.D. No. 3) in which one or both of the tryptophan residues are covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group.
  • the amino acids forming these cyclic peptides may be independently in the D- or L-configuration.
  • the cyclic peptides may comprise amino acids in both the D- and L-configuration, for instance either as the same amino acid in different configurations, such as D-Lys and L-Lys, or different amino acids in different configurations, such as D-Arg and L-Trp. It will be apparent that the cyclic version of PAF26 contains an additional glycine residue. The presence of this residue ensures that the cyclisation does not lead to any epimerisation of the peptide.
  • the group A may comprise a cyclic peptide analogue of lactadherin ('EV- GREEN'), namely: Cyclo(-Trp-Asp-Gly-Gly-Gly-Arg-Gly-Gly-Gln-lle-His-Gly-Phe-) (Seq. I.D. No. 4). in which the tryptophan residue is covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group.
  • the amino acids forming these cyclic peptides may be independently in the D- or L-configuration.
  • the cyclic peptides may comprise amino acids in both the D- and L-configuration, for instance either as the same amino acid in different configurations, such as D-GIn and L-GIn, or different amino acids in different configurations, such as D-Arg and L-Trp.
  • the group A is a cyclic peptide of formula (iii) connected to the conjugated divalent linker L of the compound of formula (I) at position 3:
  • position 3 may be covalently bonded to a fluorogen group of formula (i).
  • the compound of formula (I) is:
  • the compound of formula (I) is 'APOGREEN': the D-amino acid derivative thereof or the compound of formula (14) derived from a combination of L- and D- amino acids.
  • the group A is a cyclic peptide of formula (iv) connected to the conjugated divalent linker L of the compound of formula (I) at position 4:
  • position 4 may be bonded to a fluorogen group of formula (i).
  • the compound of formula (I) ('EV-GREEN') is:
  • R 1 , R 3 , R 5 and R 7 may be methyl and R 2 and R 6 may be hydrogen.
  • R 3 and/or R 5 may be a triazole radical, such as a 1 ,2,3-triazole group or a 1 ,2,4-triazole group.
  • the compound of formula (I) may be radiolabelled.
  • one of both of the F atoms in the compound of formula (I) may be radioisotopes of fluorine, such as 18 F.
  • the phenylene group is covalently bonded to the indolyl group of the N- protected tryptamine, N-protected tryptophan or derivative thereof.
  • the N-protected tryptophan or derivative thereof may be in the D- or L-configuration.
  • the phenylene group is covalently bonded to the indolyl group of the N- protected tryptamine or N-protected tryptophan at the C2-position.
  • the group A is N-protected tryptophan or a derivative thereof.
  • the group A is N-protected tryptamine or a derivative thereof.
  • the Pd catalyst used in the reaction of the second aspect may be a Pd(0) catalyst or a Pd(l l) catalyst.
  • An exemplary palladium (0) compound is tetrakistriphenyl phosphine palladium (0).
  • a preferred Pd(l l) catalyst is a Pd(Ci. 5 acyloxy) 2 catalyst, such as Pd(OAc) 2 .
  • the halide abstraction reagent may be a silver (I) salt, such as silver tetrafluoroborate or silver hexafluorophosphate, with silver tetrafluoroborate being preferred.
  • the carboxylic acid may be nitrobenzoic acid or fluorocarboxylic acid such as trifluoroacetic acid, with trifluoroacetic acid being preferred.
  • the reaction is carried out in an organic liquid, such as dimethyl formamide.
  • the N-protecting group is selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-Nitrophenylsulfonyl) e
  • the compound of formula (VI) is the N-protected tryptophan derivative of formula (3).
  • the N-protected tryptophan may be in the D- or L-configuration.
  • the process may further comprise the step of deprotecting the N- protected group to provide a deprotected derivative. For instance, deprotecting the N- protected amino acid group of tryptophan to provide a deprotected amino acid derivative, such as a compound of formula (13).
  • the process may further comprise the step of condensing the deprotected -NH 2 group of the compound of formula (I) with one or more amino acids to form a peptide or peptide derivative.
  • a process according to the second aspect in which: the N-protected tryptophan or a derivative thereof is replaced with a peptide obtainable from one or more amino acids comprising tryptophan or a derivative thereof; or
  • the N-protected tryptamine or a derivative thereof is preplaced with a peptide derivative, said peptide derivative obtainable from the condensation of one or more amino acids and N-protected tryptamine.
  • a process for the preparation of a compound of formula (I) as described above wherein the amino acid A is selected from N-protected 3- phenylalaninyl, N-protected 4-phenylalaninyl or a derivative thereof comprising at least the step of: - reacting an N-protected 4-phenylalanine derivative of formula (Vila), an N-protected 3-phenylalanine derivative of formula (Vllb) or a derivative thereof, including a N-protected 4- D-phenylalanine derivative, N-protected 4-L-phenylalanine derivative, N-protected 3-D- phenylalanine derivative and
  • the groups R 1 , R 2 , R 3 , R 5 , R 6 and R 7 of the compounds of formulae (I) and (VIII) are as defined for the compound of formula (I) above and the group A is an N- protected 4-phenylalaninyl, N-protected 3-phenylalaninyl or a derivative thereof.
  • the N-protected 4-phenylalaninyl may be N-protected 4-D-phenylalaninyl or N- protected 4-L-phenylalaninyl.
  • the N-protected 3-phenylalaninyl may be N-protected 3-D- phenylalaninyl or N-protected 3-L-phenylalaninyl.
  • the conjugated divalent linker L is a group selected from L1 or L2 having the structural formulae:
  • the group A is selected from groups of structural formulae (10a), (10b) or a derivative thereof:
  • the derivative of the N-protected 4-phenylalanine derivative or N- protected 3-phenylalanine derivative is a peptide obtainable from one or more amino acids comprising the N-protected 4-phenylalanine or N-protected 3-phenylalanine.
  • the N- protected 3- or 4- phenylalanine derivative in such a peptide may be independently in D- or L- configuration.
  • the Pd catalyst used in the reaction of the fourth aspect may be a palladium compound, such as a palladium (0) compound or a palladium (II) compound.
  • a palladium (0) compound is tetrakistriphenyl phosphine palladium (0).
  • exemplary palladium (II) compounds are palladium (II) acetate, palladium (II) chloride or diphenylphosphinoferrocene dichloropalladium (II).
  • the base is an alkali metal alkoxide, such as an alkali metal Ci_ 6 alkoxide.
  • reaction is carried out in an organic liquid, such as toluene or a mixture of toluene and water.
  • the N-protecting group is selected from Fmoc
  • the carboxylic acid protecting group is selected from a Ci_ 5 alkyl group such as methyl, ethyl or tert-butyl, allyl, a benzyl derivative such as a benzyl group, 2- chlorotrityl, 9-fluorenylmethyl, carbamoylmethyl and 2-phenylisopropyl.
  • the process may further comprise the step of deprotecting the N- protected amino acid group to provide a deprotected amino acid group.
  • the boronic acid moiety Q is selected from a boronic acid or a group of formula:
  • the process further comprises the step of providing the compound of formula (Vila or b) by the step of: reacting an N-protected 4-iodo phenylalanine derivative of formula (Xla), an N— protected 3-iodo phenylalanine derivative of formula (Xlb) or a derivative thereof, including a N-protected 4-iodo-D-phenylalanine derivative, N-protected 4-iodo-L- phenylalanine derivative, N-protected 3-iodo-D-phenylalanine derivative and N- p e:
  • the process further comprises the step of providing the compound of formula (VII) by the step of: reacting an N-protected 4-bromo phenylalanine of formula (Xlla), a N-protected 3-bromo phenylalanine derivative of formula (XI I b) or a derivative thereof, including N-protected 4-bromo-D-phenylalanine derivative, N-protected 4-bromo- L-phenylalanine derivative, N-protected 3-bromo-D-phenylalanine derivative and N-protected 3-bromo-L-phenylalanine derivative:
  • the Pd catalyst is a Pd(0) or a Pd(ll) catalyst, such as 1 , 1 '- bis(diphenylphosphino)ferrocene dichloropalladium.
  • reaction is carried out in an organic solvent, such as dimethyl sulfoxide.
  • the diboronate is bis(pinacolato)diboron or 4, 6,4', 6'- tetraphenyl[2,2']bis[(1 ,3,2)dioxaborinanyl.
  • the derivative of the N-protected 4-phenylalanine derivative or N-protected 3-phenylalanine derivative is a peptide obtainable from one or more amino acids comprising the N-protected 4-phenylalanine or N-protected 3-phenylalanine.
  • a compound of formula (I) as described above as a probe, preferably an optical probe.
  • a method of detecting the presence and/or function (i.e. activity, inhibition, blockade) of a target in a cell or a component of a cell comprising at least the steps of:
  • detection of the above is indicative of the presence and/or function of the target in the cell or in the component of the cell.
  • the target zone is a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample. Accordingly, the compound of formula (I) may be used in vivo, ex vivo or in vitro.
  • the cell is a fungus, such as Aspergillus fumigatus.
  • the compound of formula (I) is radiolabeled and the detection of fluorescence is the detection of gamma rays.
  • the cell is a cell with phosphatidylserine molecules such as apoptotic cells, platelets or other cells.
  • the component of a cell is a microvesicle.
  • the group A in the compound of formula (I) is a peptide PAF26 having the sequence:
  • the group A in the compound of formula (I) is a peptide having the sequence:
  • the group A in the compound of formula (I) is a peptide having the sequence:
  • H-Ala-Ala-Ala-Trp-Phe-Trp-NH 2 (Seq. I.D. No. 5) in which the conjugated divalent linker L of the fluorogen group of formula (i) is covalently bonded to the indole group, preferably at the C2 position, of one or both tryptophan residues, such as compound 5a described above; and H-Arg-Lys-Lys-Trp-Ala-Ala-NH 2 (Seq. I.D. No. 6) in which the conjugated divalent linker L is covalently bonded to the indole group of the tryptophan residue.
  • Figure 1 a shows the spectral characterisation of peptides 5, 6 and 7.
  • Spectra represented as means from three independent experiments with n 3.
  • Figure 1 b shows the fluorogenic behaviour of peptides 5, 6 and 7in phospholipid
  • Figure 1 c shows the spectral characterisation of peptide 5 and red fluorescent dye Syto82.
  • Figure 2a shows the fluorogenic behaviour of peptide 8 in the cell membrane of fungal cells.
  • Figure 2b shows the stability of cyclic (8) and linear (5) mono-BODIPY labelled peptides in human bronchoalveolar lavage samples from patients with acute respiratory distress syndrome.
  • Figure 2c shows MALDI analysis of cyclic (8) and linear (5) mono-BODIPY labelled peptides in bronchoalveolar lavage samples from patients with acute respiratory distress syndrome.
  • Figure 3a shows the fluorogenic behaviour of the cyclic peptide 8 in phospholipid membranes.
  • Figure 3b shows the fluorescence emission of mono-BODIPY labelled peptides 5, 6 (linear) and 8 (cyclic) upon incubation with A. fumigatus.
  • Figure 3c shows the spectral characterization of cyclic peptide 8 and red fluorescent dye Syto82.
  • Figure 4b shows direct imaging of A. fumigatus in human pulmonary tissue using multi- photon microscopy.
  • Figure 5 shows stability studies of Trp-BODIPY derivatives.
  • HPLC analysis of Fmoc-Trp(C 2 - BODIPY)-OH (3) in TFA:DCM (1 :99) at r.t. after 10 min (83% purity) is shown in Figure 5A
  • HPLC analysis of Fmoc-Trp(C 2 -BODIPY)-OH (3) in in TFA:DCM (1 :9) at r.t. after 30 min (46% purity) is shown in Figure 5B
  • HPLC analysis of crude (12), a BODIPY-labelled linear precursor of the cyclic peptide (8) directly after cleavage from the resin with TFA:DCM (1 :99) at r.t. is shown in Figure 5C (98% purity).
  • Black arrows in Figures 5A and 5B point at the peak corresponding to the amino acid 3.
  • Figure 5D displays long-term stability of Trp- BODIPY in solid- and solution-state at various temperatures.
  • Figure 6 shows live fungal cell imaging after incubation with cyclic peptide 8.
  • Peptide 8 was used at 2 ⁇ for all fungi except C. albicans for which 10 ⁇ was used.
  • the four images shown as Figure 6A were fluorescence images while the four images shown as Figure 6B are the corresponding bright field images.
  • the images were acquired under a confocal microscope at r.t. Scale bars: C. albicans: 5 ⁇ ; C. neoformans: 7.5 ⁇ ; F. oxysporum and N. crassa: 10 ⁇ .
  • Figure 7 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V- AF647and APOGREEN [cyclic peptide (14)] in buffer containing 2 mM CaCI 2 .
  • APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils to a similar extent than ANNEXIN V.
  • Figure 8A shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and APOGREEN [cyclic peptide (14)] in the absence of Ca 2+ .
  • ANNEXIN V Unlike ANNEXIN V,
  • APOGREEN [cyclic peptide (14)] binding does not depend on Ca 2+ .
  • Figure 8B shows that APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils in the presence of Ca 2+ , and as shown in Figure 8C, even in the presence of 2.5 mM EDTA.
  • Figure 9 shows a comparison of the binding of ANNEXIN V in the presence and absence of Ca 2+ and APOGREEN [cyclic peptide (14)] in the presence and absence of EDTA to monocytes, together with a phosphate buffered saline (PBS) control.
  • PBS phosphate buffered saline
  • APOGREEN [cyclic peptide (14)] does not bind to monocytes, therefore its major target is unlikely to be phosphatidylserine.
  • Figure 10 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and linear peptides 1-3 [corresponding to compounds (5), (6) and (7)] derived from APOGREEN [cyclic peptide (14)].
  • Derived peptides of APOGREEN can bind to apoptotic neutrophils to different extent. The cyclic peptide shows better discrimination between viable and apoptotic neutrophils.
  • Figure 11 shows the results of kinetic studies of apoptotic and viable human BL2 cells with APOGREEN [cyclic peptide (14)] over time.
  • Figure 11 A shows the staining of non-apoptotic and apoptotic BL2 cells with APOGREEN [cyclic peptide (14)] over time
  • Figure 1 1 B shows the fluorescence of the non-apoptotic and apoptotic BL2 cells in the presence of APOGREEN [cyclic peptide (14)] over time. It is apparent that APOGREEN [cyclic peptide (14)] binds rapidly to apoptotic cells and reaches saturation within minutes.
  • Figure 12 shows a comparison of the staining of apoptotic neutrophils of different origin between APOGREEN [cyclic peptide (14)] and ANNEXIN V.
  • APOGREEN [cyclic peptide (14)] binds to human apoptotic cells of different origin, such as human BL2 (B-lymphoma cells) ( Figure 12A) and A549 as human epithelial lung cancer cells ( Figure 12B).
  • Figure 13 shows the phagocytic activity of macrophages as indicated by the staining with the commercial dye pHRODO. As reported, macrophages are more phagocytic upon activation with dexamethasone (B, D). APOGREEN [cyclic peptide (14)] does not impair the phagocytosis levels of macrophages (A vs C, B vs D), which is important to ensure clearance of apoptotic neutrophils at activation sites.
  • Figure 14 shows that APOGREEN [cyclic peptide (14)] can label apoptotic neutrophils in vivo in the lungs of mice after intratracheal instillation (Figure 14C right plot), with no toxicity and comparative results as obtained from ex vivo ANNEXIN V staining ( Figure 14C left plot).
  • Graph A shows the total cell number obtained from lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)].
  • Graph B shows the proportion of live versus dead cells in lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)].
  • Figure 15 shows bright field and fluorescence images of APOGREEN [cyclic peptide (14)] and ANNEXIN V-AF647 co-staining in A549 cells after chemical induction of apoptosis. Only ANNEXIN V-AF647 stained cells are APOGREEN-positive, with no staining in viable cells. APOGREEN shows intracellular staining, potentially correlated to binding to negatively charged phospholipids.
  • Figure 16A shows a plot of fluorescence intensity versus wavelength for EV-GREEN [cyclic peptide (10)] upon incubation with various ratios of phosphatidylserine (PS) to
  • PC phosphatidylcholine
  • Figure 16A shows specific fluorescence increase upon incubation with increasing phosphatidylserine (PS) content.
  • Figure 16B shows quantification of the fluorescence increase with phosphatidylserine (PS) over other lipids (PC:
  • Figure 17 shows a plot of fluorescence for binding of EV-GREEN [cyclic peptide (10)] to phosphatidylserine (PS) in phosphate buffered saline (PBS) in the absence of Ca 2+ (-Ca), in the presence of Ca 2+ (+Ca) and in the presence of EDTA (+EDTA) together with the corresponding blanks of phosphate buffered saline in the absence of Ca 2+ , PBS(-Ca), in the presence of Ca 2+ ,PBS (+Ca) and in the presence of EDTA, PBS (+EDTA).
  • Figure 17 shows binding of EV-GREEN [cyclic peptide (10)] to PS is independent of Ca 2+ , unlike Annexin V. ns: non-statistical difference; *** for
  • Figure 18 shows a comparison of relative fluorescence for phosphate buffered saline (PBS) and EV-GREEN [cyclic peptide (10)] in the presence of either viable cells (PBS) or fixed and permeabilized neutrophils (Fix/Lyse solution). This shows that EV-GREEN [cyclic peptide (10)] does not bind to viable cells. EV-green fluoresces brightly when cells are fixed and permeabilized (i.e. treated with Fix/lyse solution).
  • PBS phosphate buffered saline
  • EV-GREEN cyclic peptide (10)
  • Figure 19 shows a comparison of relative fluorescence for phosphate buffered saline (PBS), ANNEXIN V in the presence of Ca 2+ (+Ca), ANNEXIN V in the absence of Ca 2+ (-Ca) and EV-GREEN [cyclic peptide (10)] in the presence of intact apoptotic cells.
  • EV-GREEN [cyclic peptide (10)] does not bind to intact apoptotic cells (i.e. UV-induced apoptotic BL2 cells).
  • ANNEXIN V is used as a positive control for apoptosis.
  • Figure 20 shows the measurement of the tensioactivity induced by EV-GREEN [cyclic peptide (10)].
  • EV-GREEN [cyclic peptide (10)] shows tensioactive properties; it is able to form a monolayer in the interface air-water, with a saturation pressure ( ⁇ 3 ) of 20.4 mN/m and a saturation concentration (C s ) of 3 ⁇ .
  • Figure 21 shows the interaction of EV-GREEN [cyclic peptide (10)] with lipid monolayers.
  • TT c is observed in phosphatidylserine (DOPS)- containing monolayers for EV-GREEN [cyclic peptide (10)] indicating selective affinity for this lipid (42 mN/m vs 36.5 mN/m).
  • Trp-BODIPY label increases the affinity of the unlabeled peptide for phosphatidylserine (DOPS)-containing monolayers (42 mN/m vs 30 mN/m).
  • Figure 22 shows the interaction of EV-GREEN [cyclic peptide (10)] with negatively-charged lipid monolayers.
  • EV-GREEN [cyclic peptide (10)] can also bind to other negatively-charged lipid monolayers.
  • eggPC egg phosphatidylcholine/egg phosphatidylglycerol
  • DOPS phosphatidylserine
  • EV-Green [cyclic peptide (10)] displayed an almost identical TT c (42 mN/m vs 42.4 mN/m), opening the possibility to detect other negatively-charged lipids.
  • Figure 23 shows fluorescence confocal images of unilamellar vesicles without phosphatidylserine (eggPC) and with phosphatidylserine (eggPC:DOPS 70:30) after staining with EV-GREEN [cyclic peptide (10)] (right column). Liss-Rho-DOPE is used as positive control for vesicle staining (left column).
  • EV-GREEN [cyclic peptide (10)] stains phosphatidylserine-containing vesicles with significantly brighter fluorescence than vesicles without phosphatidylserine.
  • Figure 24 shows flow cytometry experiments on extracellular vesicles from apoptotic BL2 cells, a) shows unstained vesicles, b) shows those incubated with 1 ⁇ EV-GREEN [cyclic peptide (10)] for 20 min at 4°C, c) shows those incubated with 2 ⁇ EV-GREEN for 30 min at 4°C. 5 min prior to measurement ANNEXIN V- Pacific Blue was added.
  • Figure 24 c) is a 2D dot plot showing double staining of apoptotic vesicles with both EV-GREEN (x-axis) and ANNEXIN V- Pacific Blue (y-axis).
  • the EV-GREEN stained vesicles are substantially also stained with ANNEXIN V- Pacific blue (36.3% compared to 14.0%), which correlates to the presence of phosphatidylserine.
  • the present invention provides a class of fluorogenic compounds, particularly fluorogenic amino acids and peptides based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • BODIPY (BODIPY) scaffold.
  • the BODIPY core is incorporated without the use of a chemical spacer such that the conjugated divalent linker connects the aromatic group of the amino acid or peptide with the aromatic core of the BODIPY to form a conjugated system comprising the aromatic group of the amino acid or protein, the conjugated divalent linker and the aromatic core of the BODIPY.
  • This conjugated system forms the fluorogenic centre of the compound.
  • the fluorogenic amino acids described herein can be incorporated into peptides, to provide a fluorogenic peptide.
  • the fluorogenic peptide may be used as an optical probe.
  • the peptide may be a targeting peptide, which preferentially binds to a particular biological substrate e.g. a cell such as an apoptotic cell or fungus; an enzyme substrate or an antibody or a fragment of an antibody.
  • a targeting peptide which preferentially binds to a particular biological substrate e.g. a cell such as an apoptotic cell or fungus; an enzyme substrate or an antibody or a fragment of an antibody.
  • the disclosure herein encompasses both D- and L- configurations.
  • a specific enantiomeric configuration is disclosed in a structural formula, such as an L-configuration, particularly of an amino acid or derivative thereof such as a peptide, the present disclosure encompasses the corresponding alternate configuration, such as the D-configuration.
  • N-protected is intended to encompass the chemical protection of an -NH 2 group in a molecule, such as tryptamine, an amino acid or a derivative thereof with a chemical protecting group, such as those described above.
  • an N-protected -NH 2 group may be represented by -NHZ, in which the N-protecting group Z is selected from the group comprising: Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4- methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), B
  • peptide is intended to encompass shorter chain polymers made by the condensation of two or more amino acids and includes oligopeptides and
  • Peptides may be linear, branched or cyclic. The term also encompasses glycopeptides and lipopeptides. Peptides typically comprise from 2 to 100 amino acid residues.
  • protein is intended to encompass longer chain polymers made by the condensation of amino acids and includes enzymes, antibodies, structural proteins and signalling proteins etc.
  • the protein is composed of amino acid residues which no longer contain all the free carboxylic acid and amino groups of the amino acid, which have been converted into a peptide bond and comprise more amino acid residues than a peptide.
  • Such proteins typically comprise >100 amino acid residues.
  • peptide nucleic acid is intended to encompass artificial polyamide analogues of DNA and RNA made of repeating units of N-(2-aminoethyl)-glycine linked by peptide bonds to which nucleic acid bases are linked to the secondary amide by the removal of the hydrogen atom from the secondary amide via a methylene bridge and carbonyl group.
  • peptidomimetic is intended to encompass protein-like molecules which mimic peptides, such as peptide analogues in which the peptide bonds are replaced with other polar bonds or analogues in which the C a atom is substituted by a N a one.
  • Non- limiting examples of peptidomimetics include foldamers, desipeptides, thiopeptides, selenoxopeptides or azapeptides.
  • the term "peptoid” is intended to represent peptide analogues derived from poly-N-substituted glycines in which the side chains, such as amino acid side chains, are attached to the nitrogen atom of the peptide, rather than the a- carbon as in peptides.
  • the present invention requires that the conjugated divalent linker L is covalently bonded to the aromatic group of group A.
  • tryptophanyl represents a tryptophan molecule from which ⁇ has been removed from a C on the indole ring.
  • N-protected tryptophanyl represents an N-protected tryptophan molecule from which ⁇ has been removed from a C on the indolyl ring.
  • Phenylalaninyl represents phenylalanine from which ⁇ has been removed from a C on the phenyl ring.
  • N-protected phenylalaninyl represents N-protected phenylalanine from which ⁇ has been removed from a C on the phenyl ring.
  • derivative is used to refer to the residue of a chemical compound, such as an amino acid, after it has undergone chemical modification. For instance, N- protection of an amino acid can produced an N-protected amino acid derivative.
  • an amino acid derivative when used in relation to an amino acid or N-protected amino acid encompasses the condensation product of the amino acid or N-protected amino acid.
  • an amino acid derivative may encompass peptides, proteins, peptide nucleic acid derivatives comprising an amino acid reside and peptoid derivatives comprising an amino acid residue.
  • the condensation of an amino acid with further amino acids may form a peptides or proteins as the amino acid derivative.
  • Such condensation reactions produce a polymerised amino acid derivative in the form of a polyamide.
  • the condensation of tryptamine with amino acids may form a peptide derivative or protein derivative.
  • Such condensation reactions produce a polymerised amino acid derivative in the form of a polyamide.
  • the tryptamine does not contain a carboxylic acid group and therefore forms a terminal position on the polyamide chain.
  • the condensation of an amino acid with a peptide nucleic acid may form a peptide nucleic acid derivative as the amino acid derivative.
  • the condensation of tryptamine with a peptide nucleic acid may form a peptide nucleic acid derivative.
  • group A is a peptide nucleic acid derivative
  • the peptide nucleic acid derivative comprises a peptide nucleic acid with tryptamine, tryptophan or phenylalanine residue at the point of connection between the polyamide chain of the peptide nucleic acid and the linker group L.
  • a tryptophan or phenylalanine can be linked to the polyamide chain of the peptide nucleic acid by a peptide bond via the -NH or -COOH group of the tryptophan or phenylalanine.
  • a tryptamine can be linked to the polyamide chain of the peptide nucleic acid by a peptide bond via the -NH of the ethanamine group of the tryptamine.
  • the condensation of tryptamine or an amino acid with a peptidomimetic may form a peptidomimetic derivative.
  • group A is a peptidomimetic derivative
  • the peptidomimetic derivative comprises a tryptamine, tryptophan or phenylalanine residue at the point of connection between the peptidomimetic and the linker group L.
  • phenylalanine can be linked to the peptidomimetic by a peptide bond via the -NH or -COOH group of the tryptophan or phenylalanine.
  • a tryptamine can be linked to the peptidomimetic by a peptide bond via the -NH of the ethanamine group of the tryptamine.
  • the condensation of an amino acid with a peptoid may form a peptoid derivative as the amino acid derivative.
  • the condensation of tryptamine with a peptoid may form a peptoid derivative.
  • group A is a peptoid derivative
  • the peptoid derivative comprises a peptoid with tryptamine, tryptophan or phenylalanine residue at the point of connection between the polyglycine chain of the peptoid and the linker group L.
  • the tryptophan or phenylalanine can be linked to the polyglycine chain of the peptoid by a peptide bond via the -NH or -COOH group of the tryptophan or phenylalanine.
  • a tryptamine can be linked to the polyglycine chain of the peptoid by a peptide bond via the -NH of the ethanamine group of the tryptamine
  • a tryptamine derivative or N-protected tryptamine derivative may comprise a peptide derivative obtained from a plurality of amino acids and tryptamine or N-protected tryptamine (i.e. the peptide comprises tryptamine residue or N-protected tryptamine residue); a protein derivative obtained from a plurality of amino acids and tryptamine or N-protected tryptamine (i.e.
  • the protein derivative comprises tryptamine residue or N-protected tryptamine residue); a peptoid derivative obtained from the condensation of a peptoid and tryptamine or N- protected tryptamine (i.e. the peptoid derivative comprises tryptamine residue or N-protected tryptamine residue); and a peptide nucleic acid derivative obtained from the condensation of a peptide nucleic acid and tryptamine or N-protected tryptamine (i.e. the peptide nucleic acid derivative comprises tryptamine residue or N-protected tryptamine residue).
  • a tryptophan derivative or N-protected tryptophan derivative may comprise a peptide obtained from a plurality of amino acids including tryptophan or N-protected tryptophan (i.e. the peptide comprises tryptophan residue or N-protected tryptophan residue); a protein obtained from a plurality of amino acids including tryptophan or N-protected tryptophan (i.e. the protein comprises tryptophan residue or N-protected tryptophan residue); a peptoid derivative obtained from the condensation of a peptoid and tryptophan or N-protected tryptophan (i.e.
  • the peptoid derivative comprises tryptophan residue or N-protected tryptophan residue
  • a peptide nucleic acid derivative obtained from the condensation of a peptide nucleic acid and tryptophan or N-protected tryptophan i.e. the peptide nucleic acid derivative comprises tryptophan residue or N-protected tryptophan residue.
  • a phenylalanine derivative or N-protected phenylalanine derivative may comprise a peptide obtained from a plurality of amino acids including phenylalanine or N-protected phenylalanine (i.e. the peptide comprises phenylalanine residue or N-protected
  • phenylalanine residue a protein obtained from a plurality of amino acids including phenylalanine or N-protected phenylalanine (i.e. the protein comprises phenylalanine residue or N-protected phenylalanine residue); a peptoid derivative obtained from the condensation of a peptoid and phenylalanine or N-protected phenylalanine (i.e. the peptoid derivative comprises phenylalanine residue or N-protected phenylalanine residue); and a peptide nucleic acid derivative obtained from the condensation of a peptide nucleic acid and phenylalanine or N-protected phenylalanine (i.e. the peptide nucleic acid derivative comprises phenylalanine residue or N-protected phenylalanine residue).
  • Peptidomimetics obtained from the condensation of a peptide nucleic acid and phenylalanine or N-protecte
  • a derivative of tryptophan may include a synthetic amino acid in which one or more of the hydrogen atoms, particularly from 1 to 3 of the hydrogen atoms, especially the hydrogen atoms on the indole ring, more especially the hydrogen atoms on the indole ring with the exception of the hydrogen atom at the C2-position, have been replaced with a group selected from the group comprising methyl and methoxy.
  • a derivative of phenylalanine may include a synthetic amino acid n which one or more of the hydrogen atoms, particularly from 1 to 3 of the hydrogen atoms, especially the hydrogen atoms on the phenyl ring, have been replaced with a group selected from the group comprising Ci_ 6 alkyl and Ci_ 6 alkoxy, preferably methyl and methoxy.
  • conjugated is a system of alternating single and multiple bonds. This can be viewed as an overlapping system of p-orbitals across an intervening o-bonds allowing a derealization of ⁇ -electrons across all the adjacent aligned p-orbitals.
  • aromatic is intended to represent ring structures comprising a delocalised conjugated ⁇ -system of electrons forming an aromatic core, which may typically be an arrangement of alternating single and double bonds.
  • An aromatic group may be an aryl group, particularly an aryl group having only carbon atoms in the aromatic core (i.e.
  • an aromatic group may be a heteroaryl group, such as a heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C.
  • the aromatic group, including the heteroaryl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms bonded to the aromatic core may be substituted with a substituent independently selected from the group comprising methyl and methoxy.
  • arylene is synonymous with arenediyl and represents an organic group derived from an aromatic hydrocarbon in which a hydrogen atom has been removed from two ring carbon atoms.
  • alkyl represents a linear, branched or cyclic alkyl group, particularly a linear or branched C M0 alkyl or a C 4 .i 0 cycloalkyl, more particularly a linear Ci_ 5 alkyl or a branched C 3 . 5 alkyl.
  • the alkyl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms in the alkyl group may be substituted with a substituent independently selected from the group comprising methyl or methoxy.
  • alkenyl represents a linear, branched or cyclic alkenyl group, particularly a linear or branched C 2 -io alkenyl or a C 4 _i 0 cycloalkenyl, more particularly a linear C 2 - 5 alkenyl or a branched C 3 . 5 alkyl.
  • the alkenyl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms in the alkenyl group may be substituted with a substituent independently selected from the group comprising methyl or methoxy.
  • alkynyl represents a linear, branched or cyclic alkynyl group, particularly a linear or branched C2-10 alkynyl or a C7.10 cycloalkynyl, more particularly a linear C2-5 alkynyl or a branched C 3 . 5 alkynyl.
  • the alkynyl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms in the alkynyl group may be substituted with a substituent independently selected from the group comprising methyl or methoxy.
  • alkyloxy represents an alkyi group having an oxy (-0-) linker, such as C-1 -10 alkyl-O- group, preferably a Ci _ 5 alkyl-O- group, in which the alkyi group may be as defined above.
  • halo represents a halogen atom, such as fluorine, chlorine or bromine, preferable fluorine or chlorine, more preferably fluorine.
  • the group A of the compound of formula (I) may be a peptide, such as a biologically active peptide.
  • the peptide may be a targeting peptide, which preferentially binds to a particular biological substrate e.g. a cell such as an apoptotic cell or fungus; an enzyme substrate or an antibody or a fragment of an antibody.
  • PAF26 has a highly conserved sequence with a C-terminal hydrophobic domain (Trp-Phe- Trp) and an /V-terminal cationic domain (Arg-Lys-Lys) that are essential to exert its antifungal action.
  • the use of fluorogenic amino acids in the peptide is advantageous in that they provide high signal-to-noise ratios without the need for washing or additional labelling steps.
  • BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s- indacene
  • Lactadherin also known as Milk Fat Globule-EGF Factor 8 protein (Mfge8) is a cell adhesion protein containing a phosphatidyl serine binding domain. The levels of phosphatidyl serine in apoptotic cells are much higher than in quiescent or normal cells. Lactadherin analogues, such as cyclo(-Trp-Asp-Gly-Gly-Gly-Arg-Gly-Gly-Gln-lle-His-Gly-Phe-) (Seq. I.D. No. 4) contain a tryptophan residue.
  • the compounds of the invention are operable as probes to detect a target in a cell or in a component of a cell.
  • the target zone may be a portion of tissue within a subject, and the method may be carried out in vivo.
  • the portion of tissue may be a portion of the lung, brain, muscle, heart, liver, kidneys, connective tissue, skin, intestine, or joints of a subject.
  • the compounds of the invention may be used as probes in the immune system, the circulatory system, the nervous system, the digestive system or the reproductive system.
  • the target area may be a portion of the lung of a subject.
  • the target may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
  • the compounds of the invention may be used as probes, and in methods using such compounds, in vivo, ex vivo or in vitro.
  • the compounds of the invention used as probes may be delivered to a target zone by any means known in the art.
  • they may be delivered by endoscope, spray, injection, topically, or ingestion.
  • the compounds may be delivered to the target zone using a bronchoscope.
  • Illumination light of a suitable wavelength to excite fluorophore of the compound may be delivered to the target zone by any conventional means known in the art.
  • the light is delivered by means of an optical fibre or similar.
  • the fluorescence from the compound in the target zone may be collected by an optical fibre or similar.
  • the fluorescence from the compounds in the target zone may be collected by the same optical fibre that delivered the illumination light.
  • the collected fluorescence, fluorescence life-time or fluorescence polarization is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar.
  • a recording device such as a charge-coupled device (CCD) or similar.
  • the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar.
  • a recording device such as a CCD or similar.
  • individual instruments may be used to deliver the probe to the target zone, to deliver light to the target zone and to detect fluorescence from the target zone.
  • fluorescence may be detected from the tissue of a target area using fibered confocal fluorescence microscopy (fCFM).
  • fCFM fibered confocal fluorescence microscopy
  • optical properties of these fluorogenic compounds also enables their use in
  • Photoacoustic imaging can provide high resolution images of the target area with very good tissue or in vivo penetration.
  • the fluorogenic compounds would absorb energy from an irradiation source and subsequently generate ultrasonic waves that would be detected by ultrasonic transducers to produce photoacoustic images of the target area.
  • the subject is a human subject.
  • the subject may be a non-human animal such as equine, ovine, bovine, canine, feline, rodent or fish, for example.
  • the term "significant fluorescence” as used herein refers to the fluorescence of a fluorophore that is above the background or, where present, autofluorescence in the target area.
  • the autofluorescence of the indigenous cells or tissue within the target area may have a different fluorescent lifetime than the fluorophore of the compound.
  • the autofluorescence of the indigenous cells or tissue within the target area may reduce over time at a different rate than that of the fluorophore of the compound.
  • autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the BODIPY fluorophore of the compound.
  • fluorescence observed in the target area that decays more slowly over time may be indicative of the probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence.
  • the compounds of the invention display uniform fluorescent lifetimes in the nanosecond range, allowing their identification and differentiation from other fluorophores.
  • the fluorescent lifetime of a fluorogen is the time between excitation, for instance by the absorption of incident light, and the corresponding emission.
  • the fluorescent lifetime of a fluorogen is independent of concentration and can be used to discriminate between emissions of similar wavelengths from different fluorophores. For instance, some of the compounds of the invention have a fluorescent lifetime in the range of from 1 to 5 ns. Such fluorescent lifetimes can be used to differentiate the emissions from those of the compounds of the invention from other fluorophores exhibiting longer or shorter fluorescent lifetimes, which may emit at similar wavelengths.
  • the method may comprise the step of determining the absorbance or
  • fluorescence intensity or fluorescence polarization of the target zone over a period of time determining the rate of decay of fluorescence during the period of time, and determining the fluorescence intensity of that fluorescence with a slower rate of decay, wherein the fluorescence with a slower rate of decay corresponds to the fluorescence of the compound.
  • the target zone may be a portion of tissue within a subject, and the method may be carried out in vivo.
  • the portion of tissue may be a portion of the lung, heart, liver, connective tissue, skin, intestine, brain, muscle, or joints of a subject, for example.
  • the target zone may be a portion of the lung of a subject.
  • the method of the invention may be carried out in the immune system, the circulatory system, the nervous system, the digestive system or the reproductive system.
  • the target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
  • the target zone may be a target area.
  • the target zone may be a target volume.
  • the compound of formula (I) may be radiolabelled.
  • one of both of the F atoms in the compound of formula (I) may be radioisotopes of fluorine, such as 18 F.
  • Such radiolabelled compounds of formula (I) can be used in positron emission tomography. This technique detects pairs of gamma rays emitted by a positron-emitting radionuclide such as 18 F.
  • the group A of an 18 F radiolabelled compound formula (I) is a peptide, it may be a targeting peptide with a specificity for a particular biological substrate. In this way, the radiolabelled compound of formula (I) can allow the imaging and detection of particular biological cells or components of a cell, such as cell apoptotic cells or fungal cells.
  • the compounds of the invention can be prepared via a number of synthetic routes.
  • Disclosed herein is a process for the preparation of fluorogenic tryptophan derivatives utilising a palladium catalysed direct C-2 arylation of the indolyl group to add a BODIPY fluorogen. Also disclosed is a process for the preparation of fluorogenic phenylalanine derivatives using a Suzuki-Miyaura coupling of a borono-phenylalanine derivative and a BODIPY fluorogen. Both preparative reactions utilise an iodo-C 6 -ioaryl BODIPY reagent, such as that of formula (VIII), to introduce the fluorogenic centre into the amino acid.
  • this amino acid is N-protected, for instance as shown in compound (1 1), prior to reaction with the iodo-C 6 -ioaryl BODIPY reagent.
  • N-protecting group Z is selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl and Ts (toluenesulfonyl).
  • this amino acid can be N-protected prior to reaction with the iodo-C 6 -ioaryl BODIPY reagent.
  • the N-protection of the D- or L- tryptophan or a derivative thereof can be carried out by methods known in the art.
  • the coupling of the N-protected tryptophan and the iodo-C 6 -ioaryl BODIPY reagent is carried out in the presence of a Pd catalyst, such as a Pd(ll) acyloxy compound, for instance Pd(OAc) 2 and a halide abstraction agent, such as a silver (I) salt.
  • an N-protected iodo-phenylalanine is first prepared.
  • a 3- or 4- iodo- or bromo-phenylalanine or a derivative thereof may be N-protected, for instance with any of the groups described in the previous paragraph for the N-protection of tryptophan.
  • the N-protected iodo- or bromo-phenylalanine such as a compound of formula (XI) or (XII) can then be converted into the corresponding N-protected borono-phenylalanine, such as a compound of formula (VII), as disclosed in J. Org. Chem. 1998, 63, 8019.
  • This can be carried out by the reaction of the N-protected iodo- or bromo-phenylalanine or its derivative with a diboronate in the presence of a Pd catalyst.
  • the Pd catalyst may be 1 ,1 '- bis(diphenylphosphino)ferrocene dichloropalladium (II).
  • the reaction can be carried out in an organic solvent, such as dimethyl sulfoxide.
  • the diboronate should contain boronates protected by reducible groups, such as pinacolato or 1 ,3-diphenyl-1 ,3-propanedioxy groups.
  • the diboronate can be prepared by the reaction of tetrakis(dimethyamino)diboron with a suitable diol, such as pinacol or 1 ,3-diphenyl-1 ,3- propanediol, which can provide a reducible ligand.
  • the N-protected borono-phenylalanine such as a compound of formula (VII) can then be coupled with an iodo-C 6 -ioaryl BODIPY reagent, such as that of formula (VIII), in a Suzuki- Miyaura reaction, to introduce the fluorogenic centre into the amino acid.
  • the coupling reaction is carried out in the presence of a Pd catalyst and a base.
  • the Pd catalyst may be an aryl palladium(ll) alkoxide complex.
  • the base may be an alkali metal alkoxide, such sodium tert.-butoxide.
  • the palladium catalyst may be tetrakistriphenyl phosphine palladium (0), palladium (II) acetate, palladium (II) chloride or
  • the nitrogen protecting group can be removed to provide the fluorogenic amino acid derivative.
  • the fluorogenic amino acid derivative can then be incorporated into peptides using conventional techniques.
  • Resin loading (only for 2-chlorotrityl polystyrene resin).
  • Fmoc-AA-OH (1 eq.) was attached to the resin (1 eq.) with DIPEA (3 eq.) in DCM at r.t for 10 min and then DIPEA (7.0 eq.) for 40 min.
  • the remaining trityl groups were capped adding 0.8 ⁇ _ MeOH/mg resin for 10 minutes. After that, the resin was filtered and washed with DCM (4 x 1 min), DMF (4 x 1 min).
  • the loading of the resin was determined by titration of the Fmoc group.
  • Peptides were purified by semi-preparative RP-HPLC (XB RIDGETM BEH 130, Ci 8 , 5 ⁇ OBD 19x50 mm column).
  • Mobile phase ACN (0.1 % HCOOH)/H 2 0 (0.1 % HCOOH); flow rate: 20 mL/min. Pure fractions were lyophilized furnishing the corresponding peptides.
  • the protected linear peptide (119 mg, 0.073 mmol) was dissolved in 1.3 ml_ of DMF (0.055 M). DIPEA (2.5 eq., 32 ⁇ _, 0.182 mmol) and HATU (1.0 eq., 28 mg, 0.073 mmol) were added. The solution was stirred at r.t. until the cyclisation was complete (approx. 2 h). The cyclic peptide was precipitated by adding H 2 0 to the solution. The precipitate was washed with H 2 0, decanted and dried, obtaining 109 mg of crude protected cyclic peptide (87% yield).
  • the A. fumigatus strain CEA10 was grown on Vogel's medium at 37 °C for 5 days before the spores were harvested. Peptides 4-8 were incubated at different concentrations with A. fumigatus conidia to reach a final volume of 100 ⁇ _ per well. The final conidia concentration was 5 ⁇ 10 5 cells/mL in 10% Vogel's medium. After 24 h incubation at 37 °C in 96 well-plates, fungal growth was determined by measuring OD 6 ionm in a
  • Human lung A549 epithelial cells were grown using Dulbecco's Modified Eagle Medium (DM EM) supplemented with 10% fetal bovine serum (FBS), antibiotics (100 U ml_ "1 penicillin and 100 mg ml_ “1 streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5% C0 2 . Cells were regularly passaged in T-75 cell culture flasks. For co-cultures, human lung epithelial cells were plated on glass chamber slides Lab-TekTM I I (Nunc) two days before imaging and incubated for 16 h with A. fumigatus conidia reaching 75% to 90% confluence on the day of the experiment.
  • DM EM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • antibiotics 100 U ml_ "1 penicillin and 100 mg ml_ "1 streptomycin
  • 2 mM L-glutamine in a humidified
  • Peptides 5 and 8 (20 ⁇ ) were dissolved in human bronchoalveolar lavage samples (total volume: 100 ⁇ _) and incubated at 37 °C for the indicated times. Samples were injected into an HPLC Agilent 1 100 separations module connected to a UV detector with a Discovery Ci 8 column (5 ⁇ , 4.6 ⁇ 50 mm). MALDI data was recorded on a Bruker Ultraflex mass spectrometer using sinapinic acid as the matrix.
  • Multi-photon and lifetime imaging of A. fumigatus in ex vivo human pulmonary tissue Multi-photon and lifetime imaging of A. fumigatus in ex vivo human pulmonary tissue.
  • Human lung tissue was obtained from the periphery (non-cancerous) region of patients undergoing resection for lung cancer. 1 cm 3 tissue was inflated with optimum cutting temperature (O.C.T.) formulation and stored at -80 °C. Embedded tissue was cryo- sectioned at 10 micron intervals and fixed onto glass slides for imaging. RFP-expressing A. fumigatus conidia were grown overnight at 37 °C the day before the experiments and incubated with human lung tissue sections for 2-3 h hours before imaging.
  • O.C.T. optimum cutting temperature
  • a custom-built multi-photon microscope was used to acquire second harmonic generation (SHG) and two-photon fluorescence images.
  • a picoEmerald (APE) laser provided both a tunable pump laser (720-990 nm, 7 ps, 80 MHz repetition rate) and a spatially overlapped Stokes laser (1 ,064 nm, 5-6 ps, 80 MHz repetition rate).
  • GFP two-photon fluorescence signals were filtered using the following series of filters: FF520-Di02,
  • Fluorescence lifetime images were acquired by connecting the relevant detector to a PicoHarp 300 (Picoquant, Berlin) and configuring the PMT for photon counting mode for TCSPC-FLIM. SHG and GFP images were taken with the laser tuned to 950 nm and RFP images were recorded using a 1 ,064 nm laser. Lifetime images were recorded at 20 mW with a 10 ⁇ pixel dwell using the SymPhoTime software (Picoquant). All images were analysed and processed using ImageJ.
  • Peptides 5-7 exhibited similar spectral properties (Figure 1 a) to 3 with an equally strong fluorogenic behaviour in phospholipid membranes ( Figure 1 b).
  • Double-labelled peptide 7 displayed a weaker fluorescence response than mono-labelled peptides (5,6), partially due to the self-quenching derived from two neighbouring BODIPY fluorophores.
  • Peptide 5 was further used to image live A. fumigatus in co-cultures with human lung epithelial cells.
  • the fluorogenic properties of 5 enabled direct live fungal cell imaging without the need of any washing steps.
  • lung epithelial cells were counterstained with the red fluorescent dye Syto82 (the spectral characterisation of which is shown in Figure 1 c) and plot profile analysis was performed to confirm that 5 specifically labelled A. fumigatus without staining human lung epithelial cells.
  • Table 1 shows the IC 50 in Aspergillus Fumigatus the antimicrobial and haemolytic activity of peptides 5-7 determined as described in the methods section above.
  • cyclic peptide 8 was synthesised as the corresponding BODIPY-labelled cyclic analog. Cyclic peptides do not contain free N- and C-terminal groups, leading to increased resistance to degradation by proteases. Compound 8 was synthesised as described in Example 4 using 2-chlorotrityl polystyrene resin, which enabled the preparation and subsequent cleavage of the protected linear peptide under mild acidic conditions (Scheme 2).
  • NMR analysis of 8 showed no evidence of relevant structural modifications with respect to the parent unlabeled peptide, in agreement with molecular simulations. Importantly, the peptide 8 remained intact after 24 h in human bronchoalveolar lavages from patients with ARDS (Table 2, Figures 2a, 2b and 2c). Peptides were incubated in human bronchoalveolar lavage samples at 37 °C and analysed by HPLC or MALDI at the time points indicated in Figures 2b and 2c respectively.
  • the peptide 8 also displayed stronger fluorogenic response than the linear peptides (5, 6) and remarkable fluorescence emission in phospholipid bilayer membranes in PBS (A exc .: 450 nm), with quantum yields reaching 30% ( Figures 3a and 3b). Quantum yields were determined using fluorescein in basic ethanol as reference according to P. G. Seybold, M. Gouterman, J. Callis. Photochem. Photobiol., 1969, 9, 229-242.
  • peptide 8 was employed for direct imaging of A. fumigatus in human pulmonary tissue using multi-photon microscopy.
  • a transgenic strain of A. fumigatus was employed expressing red fluorescent protein (RFP) in the cytoplasm.
  • RFP red fluorescent protein
  • multi-photon excitation enabled the acquisition of second harmonic generation (cyan) from the collagen structures of the fibrillar network of human pulmonary tissue (Fig 4b C and D).
  • fluorescence lifetime imaging revealed that 8-stained A. fumigatus and tissue structures could be readily distinguished by their fluorescence lifetimes (Fig 4). This feature is important to distinguish the signal from autofluorescence signals of the tissue could potentially overlap with the emission of BODIPY fluorogens.
  • fluorogenic BODIPY-labelled cyclic peptide 8 validate fluorogenic BODIPY-labelled cyclic peptide 8 as a highly stable imaging agent for direct and straightforward visualisation of A. fumigatus in human pulmonary tissue.
  • Trp-BODIPY derivative (3) was developed with high extinction coefficients and significant fluorescence enhancement in hydrophobic microenvironments.
  • Compound 3 is fully compatible with SPPS and was used to prepare fluorogenic analogs of PAF26, a short antifungal hexapeptide, as imaging agents for the fungal pathogen A.
  • the spacer-free C-C linkage of the BODIPY fluorogen to the exposition of Trp did not impair the affinity and selectivity properties of PAF26 derivatives while providing a suitable imaging reporter.
  • amino acid 3 can be readily incorporated and has general applicability to both linear and cyclic peptides, we envisage that the site-specific introduction of this spacer-free BODIPY fluorogen at strategic positions in relevant peptides will enable the development of novel imaging peptide tools with high sensitivity and specificity.
  • Neurospora crassa (strain 74-OR23-1V A, source: FGSC 2489) was grown on standard Vogel's agar at 25°C under constant artificial light for 5 days. Conidia were collected using sterile dH 2 0 and then diluted in 20% Vogel's liquid medium for imaging. Fusarium oxysporum (strain 4287, source: FGSC 9935) was grown in liquid potato dextrose broth (PDB) at 28°C with shaking. Conidia were resuspended in 20% Vogel's liquid medium and imaged after incubation for 12 h at 30 °C.
  • Candida albicans (strain SC5314, source: ATCC MYA-2876) was grown on yeast peptone dextrose (YPD) liquid medium at 30 °C for 12 h with shaking and then diluted using minimal medium (0.7% yeast nitrogen base plus 2% glucose) before imaging.
  • Cryptococcus neoformans (strain H99, source: FGSC 9487) was grown on YPD agar at 30 °C for 3 days. To collect the cells for imaging, a single colony 1 -2 mm in diameter was resuspended in PBS and washed once with fresh PBS before imaging. Compound 8 was used at 2 ⁇ for all fungi except C. albicans for which 10 ⁇ was used.
  • Fluorescence and bright field images were acquired under a confocal microscope at r.t. and are shown in Figure 6.
  • the four images shown as Figure 6A were fluorescence images while the four images shown as Figure 6B are the corresponding bright field images.
  • the images were acquired under a confocal microscope at r.t. Scale bars: C. albicans: 5 ⁇ ; C. neoformans: 7.5 ⁇ ; F. oxysporum and N. crassa: 10 ⁇ .
  • Table 4 a) shows the chemical structures of fluorogenic linear (5a, 5b) and cyclic peptides (8, 9).
  • Apoptotic human neutrophils or monocytes obtained from human peripheral blood were plated in a 96-well round bottom plate on ice (300.000 cells/well). Cells were pelleted down by spinning at 4° C for 3 min, the supernatant was flicked out and the plate was vortexed to resuspend the cells in residual liquid. Different probes were dissolved in PBS (for Ca2+-free conditions) or in HEPES with CaCI 2 (for Ca2+ containing media at a final concentration of 2 rtiM), added to their respective wells and incubated at 4° C for 20 min. EDTA (final concentration 2.5 mM) was added to chelate any residual Ca 2+ when appropriate.
  • Figure 7 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V- AF647and APOGREEN [cyclic peptide (14)] in buffer containing 2 mM CaCI 2 .
  • APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils to a similar extent than ANNEXIN V.
  • Figure 8A shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and APOGREEN [cyclic peptide (14)] in the absence of Ca 2+ . Unlike ANNEXIN V, APOGREEN [cyclic peptide (14)] binding does not depend on Ca 2+ .
  • Figure 8B shows that APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils in the presence of Ca 2+ , and as shown in Figure 8C, even in the presence of 2.5 mM EDTA.
  • Figure 9 shows a comparison of the binding of ANNEXIN V in the presence and absence of Ca 2+ and APOGREEN [cyclic peptide (14)] in the presence and absence of EDTA to monocytes, together with a phosphate buffered saline (PBS) control.
  • APOGREEN cyclic peptide (14)
  • PBS phosphate buffered saline
  • Figure 10 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and linear peptides 1-3 [corresponding to compounds (5), (6) and (7)] derived from APOGREEN [cyclic peptide (14)].
  • Derived peptides of APOGREEN can bind to apoptotic neutrophils to different extent.
  • the cyclic peptide shows better discrimination between viable and apoptotic neutrophils.
  • Figure 17 shows a plot of fluorescence for binding of EV-GREEN [cyclic peptide (10)] to phosphatidylserine (PS) in phosphate buffered saline (PBS) in the absence of Ca 2+ (-Ca), in the presence of Ca 2+ (+Ca) and in the presence of EDTA (+EDTA) together with the corresponding blanks of phosphate buffered saline in the absence of Ca 2+ , PBS(-Ca), in the presence of Ca 2+ ,PBS (+Ca) and in the presence of EDTA, PBS (+EDTA).
  • Figure 17 shows binding of EV-GREEN [cyclic peptide (10)] to PS is independent of Ca 2+ , unlike ANNEXIN V. ns: non-statistical difference; *** for p values ⁇ 0.001.
  • Figure 19 shows a comparison of relative fluorescence for phosphate buffered saline (PBS), ANNEXIN V in the presence of Ca 2+ (+Ca), ANNEXIN V in the absence of Ca 2+ (-Ca) and EV-GREEN [cyclic peptide (10)] in the presence of intact apoptotic cells.
  • EV-GREEN [cyclic peptide (10)] does not bind to intact apoptotic cells (i.e. UV-induced apoptotic BL2 cells).
  • ANNEXIN V is used as a positive control for apoptosis.
  • BL2 Human Burkitt's lymphoma (BL2) cells were induced into apoptosis by UV dose radiation (300 mJ/ cm 2 ) and subsequent culture for a few hours at 37 0 C. Compounds were dissolved in PBS (for Ca 2+ -free conditions) or in HEPES with CaCI 2 (for Ca 2+ containing media at a final concentration of 2 rtiM), added to their respective wells and incubated at 4 °C for 20 min. After incubation, cells were resuspended again by spinning, flicking out the supernatant and finally diluting in 300 ⁇ _ the final cell suspensions in separate tubes. Data acquisition was carried out using FACSCalibur or BD LSRFortessa flow cytometers (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
  • Figure 11 shows the results of kinetic studies of apoptotic and viable human BL2 cells with APOGREEN [cyclic peptide (14)] over time.
  • Figure 1 1 A shows the staining of non-apoptotic and apoptotic BL2 cells with APOGREEN [cyclic peptide (14)] over time
  • Figure 11 B shows the fluorescence of the non-apoptotic and apoptotic BL2 cells in the presence of APOGREEN [cyclic peptide (14)] over time. It is apparent that APOGREEN [cyclic peptide (14)] binds rapidly to apoptotic cells and reaches saturation within minutes.
  • Figure 12A shows the staining of apoptotic neutrophils, specifically human BL2 (B-lymphoma cells) by APOGREEN [cyclic peptide (14)] and ANNEXIN V.
  • APOGREEN cyclic peptide (14)
  • Human lung epithelial (A549) cells were grown using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), antibiotics (100 U ml_ "1 penicillin and 100 mg ml_ “1 streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5% C0 2 .
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS foetal bovine serum
  • antibiotics 100 U ml_ "1 penicillin and 100 mg ml_ "1 streptomycin
  • 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5% C0 2 .
  • Cells were regularly passaged in T-75 cell culture flasks, and plated on glass chamber slides Lab-TekTM II (Nunc) the day before imaging, reaching 60% to 90% confluence on the day of the experiment.
  • A549 cells were treated with hTNF-a (10 ng/mL) and flavopiridol (500 nM) in MEM with 0.5% FBS for 6 h.
  • Cells were incubated at 37 °C with fluorescent probes for 20 min and imaged under an epifluorescence EVOS microscope or a confocal Zeiss LSM 510 Meta fluorescence microscope equipped with a live cell imaging stage.
  • Cell images were acquired using 40X (EVOS) or 63X oil (ZEISS) objectives. Fluorescent probes were excited with suitable lasers: 488 nm (compound 14) and 633 nm (ANNEXIN V-AF647). Images were acquired with the corresponding microscope software and processed and analyzed with ImageJ.
  • Figure 12B shows a comparison of the staining of apoptotic neutrophils, specifically A549 human epithelial lung cancer cells, by APOGREEN [cyclic peptide (14)] and ANNEXIN V.
  • Figure 15 shows bright field and fluorescence images of APOGREEN [cyclic peptide (14)] and ANNEXIN V-AF647 co-staining in A549 cells after chemical induction of apoptosis. Only ANNEXIN V-AF647 stained cells are APOGREEN-positive, with no staining in viable cells. APOGREEN shows intracellular staining, potentially correlated to binding to negatively charged phospholipids.
  • Macrophages were treated with dexamethasone in order to induce phagocytosis, and incubated or not with compound 14 for 20 min. Afterwards, macrophages were treated with the commercial dye pHRODO for staining of the phagocytic population. Data acquisition was carried out using FACSCalibur or BD LSRFortessa flow cytometers (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
  • Figure 13 shows the phagocytic activity of macrophages as indicated by the staining with the commercial dye pHRODO. As reported, macrophages are more phagocytic upon activation with dexamethasone (B, D). APOGREEN [cyclic peptide (14)] does not impair the phagocytosis levels of macrophages (A vs C, B vs D), which is important to ensure clearance of apoptotic neutrophils at activation sites.
  • APOGREEN cyclic peptide (14)
  • mice were nebulised in a chamber with LPS (1 mg/mL) and then 24 hours later instilled with compound 14 (2.5 ⁇ in PBS) or vehicle (equal amount of DMSO in PBS) by intratracheal instillation. After 30 min, mice were sacrificed and cells were lavaged from the lungs. A fraction of the cells was cytospined and stained for visual inspection under microscopy to determine cell numbers and the ratio of dead/live cells. Lavaged cells were also counterstained with fluorescent ANNEXIN V and/or TOPRO dyes and analysed by flow cytometry. Data acquisition was carried out using FACSCalibur or BD LSRFortessa flow cytometers (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
  • Figure 14 shows that APOGREEN [cyclic peptide (14)] can label apoptotic neutrophils in vivo in the lungs of mice after intratracheal instillation (Figure 14C right plot), with no toxicity and comparative results as obtained from ex vivo ANNEXIN V staining ( Figure 14C left plot).
  • Graph A shows the total cell number obtained from lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)].
  • Graph B shows the proportion of live versus dead cells in lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)].
  • Figure 16A shows a plot of fluorescence intensity versus wavelength for EV-GREEN [cyclic peptide (10)] upon incubation with various ratios of phosphatidylserine (PS) to phosphatidylcholine (PC).
  • Figure 16A shows specific fluorescence increase upon incubation with increasing phosphatidylserine (PS) content.
  • Figure 16B shows quantification of the fluorescence increase with phosphatidylserine (PS) over other lipids (PC: phosphatidylcholine).
  • Flow cytometry experiments were conducted with EV-Green [cyclic peptide (10)] and apoptotic human neutrophils using the protocol described for Fig. 17 in Example 11. The procedure also included a treatment with fix-lyse solution to induce cell membrane perforation and subsequent necrosis. Neutrophils were incubated at various EV-Green [cyclic peptide (10)] concentrations ranging from 0.16 to 5.0 ⁇ in PBS at 4 C for 20 min.
  • Figure 18 shows a comparison of relative fluorescence for phosphate buffered saline (PBS) and EV-GREEN [cyclic peptide (10)] in the presence of either viable cells (PBS) or fixed and permeabilized neutrophils (Fix/Lyse solution). This shows that EV-GREEN [cyclic peptide (10)] does not bind to viable cells. EV-green fluoresces brightly when cells are fixed and permeabilized (i.e. treated with Fix/lyse solution). Fig. 18 shows that EV-Green [cyclic peptide (10)] stains permeabilised necrotic cells.
  • Tensioactivity was analyzed by measuring the surface pressure of the resulting lipid- aqueous mixtures, using a DeltaPi-4 (Kibron, Helsinki, Finland) at 22°C, with constant stirring.
  • EV-Green cyclic peptide (10)] displays remarkable tensioactivity, and able to form monolayers in the air-water interphase with a saturation pressure ( ⁇ 3 ) of 20.4 mN m "1 and a saturation concentration of 3 ⁇ .
  • EV-Green [cyclic peptide (10)] displayed stronger tensioactivity, which is a measure of the area occupied by the probe in the interphase, indicating the optimal positioning of the BODIPY fluorogen within the hydrophobic domain of the peptide.
  • Figure 20 shows the measurement of the tensioactivity induced by EV-GREEN [cyclic peptide (10)].
  • EV-GREEN [cyclic peptide (10)] shows tensioactive properties; it is able to form a monolayer in the interphase air-water, with a saturation pressure ( ⁇ 3 ) of 20.4 rtiN/m and a saturation concentration (C s ) of 3 ⁇ .
  • Lipids dissolved in chloroform:methanol (2: 1), egg phosphatidylcholine (eggPC), 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and egg phosphatidylglycerol (eggPG) were spread gently over the surface until the desired initial surface pressure was attained.
  • the peptides, either the unlabeled peptide (Fig. 21a) or EV-GREEN [cyclic peptide (10)] (Fig. 21 b) were injected with a micropipette through a hole connected to the subphase. The increment in surface pressure was recorded until a stable signal was obtained. Values of critical pressure (TTC) were determined by linear regression using the values over the saturation pressure (TTS).
  • initial pressure ( ⁇ ,) is plotted against variation of pressure ( ⁇ ), either in eggPC or eggPC:DOPS (70:30) monolayers.
  • Higher TT c is observed in phosphatidylserine (DOPS)-containing monolayers for EV-GREEN [cyclic peptide (10)] indicating selective affinity for this lipid (42 mN/m vs 36.5 rtiN/m) (Fig. 21 b).
  • Trp- BODIPY label dramatically increases the affinity of the unlabeled peptide for phosphatidylserine (DOPS)-containing monolayers (42 mN/m vs 30 mN/m) (Fig. 21a vs. Fig.
  • EV-GREEN [cyclic peptide (10)] was extended to lipid monolayers containing other negatively-charged phospholipids, such as cardiolipin or eggPC: eggPG (70:30) (Fig. 22).
  • Fig. 22 shows a comparison of the initial pressure ( ⁇ ,) versus variation of pressure ( ⁇ ) of eggPC: eggPG and eggPC:DOPS (70:30) when incubated with EV-Green [cyclic peptide (10)].
  • EV-Green [cyclic peptide (10)] displayed an almost identical TT c (42 mN/m vs 42.4 mN/m) (Fig. 22).
  • EV-Green [cyclic peptide (10)] as a fluorescent probe for staining phosphatidylserine (DOPS)-containing vesicles
  • GMVs giant unilamellar vesicles
  • Fig. 23 Giant unilamellar vesicles (GUV) were prepared by electroformation on a pair of platinum (Pt) wires. Lipid stock solutions were prepared in 2: 1 (v/v) chloroform/methanol at 0.2 mg/ml, and appropriate volumes of each one were mixed.
  • Labelling was carried out by pre-mixing the desired fluorescent probes with the lipids in organic solvent.
  • GUVs containing phosphatidylcholine (eggPC)-only (Fig. 23a and 23b) or eggPC:DOPS (7:3) (Fig. 23c and 23d) were prepared, mimicking the phosphatidylserine content of apoptotic EVs.
  • N- (Lissamine Rhodamine B sulfonyl) dioleoylphosphatidylethanolamine (Liss-Rho-DOPE) was used as marker for lipid membrane (Fig. 23a and 23c).
  • the average concentration of individual fluorescent probes in each sample was 0.2 mol%.
  • AC field was applied in three steps, all of them performed at 37°C: 1), frequency 500 Hz, amplitude 220 mV (35 V/m) for 5 min; 2), frequency 500 Hz, amplitude 1900 mV (313 V/m) for 20 min; 3), frequency 500 Hz, amplitude 5.3 V (870 V/m) for 90 min.
  • the temperatures used for GUV formation correspond to those at which the different membranes display a single fluid phase.
  • GUVs were imaged with confocal microscopy using a Nikon D-eclipse C1 confocal system (Nikon corporation, Tokyo, Japan).
  • the excitation wavelengths used for excitation were 488 nm for EV-Green [cyclic peptide (10)] and 561 nm for Liss-Rho-DOPE. Fluorescence emission was retrieved at 500-530 for EV-Green [cyclic peptide (10)] and at 573-613 for Liss-Rho-PE.
  • EV-Green [cyclic peptide (10)] at 3,3 ⁇ g/ml, were added to study its effect on the GUVs. All these experiments were performed at 22° C. Image treatment was performed using the software EZ-C1 3.20 (Nikon Inc., Melville, N.Y.).
  • EV-Green [cyclic peptide (10)] showed significantly higher staining in phosphatidylserine-containing GUVs (Fig. 23d vs 23b), in agreement with our in vitro characterization data. Altogether, these results confirmed the potential of EV-Green [cyclic peptide (10)] as a probe for the fluorescence labelling of apoptotic vesicles.
  • ANNEXIN V-Pacific Blue vesicles were prepared by mixing 100 ⁇ of vesicle solution with 0.5 ⁇ of ANNEXIN V and 11 ⁇ of concentrated Ca 2+ -rich binding buffer. The mixture was incubated 10 minutes at 4°C and diluted to 1 ml_ with diluted ANNEXIN V binding buffer, prior to measurement.
  • a single EV-Green [cyclic peptide (10)]- stained sample was carried out by preparing a 2 ⁇ solution of the probe with EV (100 ⁇ total volume), followed by 40 min incubation at 4°C.
  • Fig. 24a and Fig. 24b show fluorescence histogram of unlabeled vesicles and 2 ⁇ EV-Green [cyclic peptide (10)] stained vesicles respectively.
  • Fig. 24c shows a 2D-plot displaying ANNEXIN V-Pacific Blue staining on y-axis and 2 ⁇ EV-Green [cyclic peptide (10)] on x-axis.
  • ANNEXIN V and EV-Green [cyclic peptide (10)] are observed (36.3%) indicating a correlation with PS binding.
  • Data acquisition was carried out using an Altune acoustic focusing cytometer (Thermo Fisher) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).

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Abstract

The invention relates to compounds, particularly fluorogenic amino acid derivatives, which can be used as optical probes. The invention further relates to processes for the preparation of such compounds, the use of such compounds as probes and methods of detecting a target using such compounds as probes.

Description

FLUOROGENIC COMPOUNDS PROCESS OF PREPARATION THEREOF AND
METHODS OF USE
Field of the Invention The invention relates to compounds, particularly fluorogenic amino acid derivatives, which can be used as optical probes. The invention further relates to processes for the preparation of such compounds, the use of such compounds as probes and methods of detecting a target using such compounds as probes.
Background of the Invention
Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunocompromised patients. IPA results from the infection with the fungal pathogen Aspergillus fumigatus, and it is a frequent cause of fungal pneumonia with mortality rates up to 40%. Current diagnostic approaches for IPA rely on histological analysis, cultures from bronchoalveolar lavage fluid and sampling peripheral blood. These methods are fraught with problems of upper airway contamination and diagnostic delays, by which time the disease may have progressed or been treated empirically with inappropriate drugs. Moreover, blood markers are unlikely to provide useful information about events deep in pulmonary tissue, especially in patients with multi-system disease, such as immunosuppressed patients affected by IPA. These limitations of current diagnostic tools have prompted the development of imaging probes that can provide in situ and real-time information on the progression of infection.
Fluorescent probes based on antibiotics and antimicrobial peptides are chemical entities with enormous potential for imaging infection sites due to their high selectivity for microbial cell structures over mammalian cells. These probes have been prepared by conjugating peptides of interest to suitable fluorophores via chemical spacers. While such approaches have been useful to functionalise long peptides or proteins, alternative strategies are needed for shorter peptides, where relevant modifications can compromise their specificity. For instance the use of chemical spacers could compromise the molecular recognition of the peptide for its target. Molecular recognition may be affected by a number of factors, such as changes in the polarity or hydrophobicity of the peptide, H-bonding pattern, or
conformational structure resulting from the incorporation of substituent groups. It is therefore essential to ensure that the labelling step has no or marginal impact on the structural and conformational features of the original peptide. l For instance, Peptide AntiFungal 26 (PAF26) is a synthetic antimicrobial hexapeptide with high affinity for fungal cells and selectivity over bacterial and mammalian cells (Lopez- Garcia, B., Perez-Paya, E. & Marcos, J. F. "Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library", Appl. Environ. Microbiol. 68, 2453-2460 (2002); Munoz, A. et al "Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells", PloS One 8, e54813 (2013)). However, the incorporation of fluorophores in short antimicrobial peptides is challenging as chemical modifications are likely to alter the distribution of positive charges as well as their amphipathic character.
PAF26 has a highly conserved sequence with a C-terminal hydrophobic domain (Trp-Phe- Trp) and an /V-terminal cationic domain (Arg-Lys-Lys) that are essential to exert its antifungal action.
One option to prepare such peptides is to utilize fluorogenic amino acids to integrate a fluorogenic label within the peptide sequence. Fluorogenic amino acids are advantageous in that they provide high signal-to-noise ratios without the need for washing or additional labelling steps. Known fluorogenic amino acids exhibit inherent limitations as fluorophores (e.g. short emission wavelengths, low extinction coefficients, compromised cell permeability) and may not behave as surrogates of the amino acids they replace, leading to alterations in the molecular recognition properties of the peptide. A need therefore exists to provide fluorogenic amino acids which address these problems.
Statements of the Invention
The present invention provides a novel fluorogenic compound based on the 4,4-difluoro-4- bora-3a,4a-diaza-s-indacene (BODIPY) scaffold. The incorporation of the spacer-free BODIPY- fluorogen into a probe, such as a peptide probe, for instance the incorporation of BODIPY-TRP into the hydrophobic domain of PAF26, was found to maintain the recognition features of the peptide while providing an excellent reporter of the interaction with a target microbe, such as fungal cells. The resulting BODIPY-labelled antimicrobial peptides are highly stable and are the first fluorogenic probes to image A. fumigatus in ex vivo human tissue.
In a first aspect, there is provided a compound of formula (I):
Figure imgf000005_0001
wherein R1 , R2, R3, R5, R6 and R7 are independently selected from the group comprising H, CMO alkyl, C2-io alkenyl, C2-io alkynyl, CM0 alkyl carboxylic acid, Ci_i0 alkoxy, CM0 acyl, Ci_i0 acyloxy, C6-io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C;
L is a conjugated divalent linker selected from a C6-io arylene group; and wherein one or more of said R1 , R2, R3, R5, R6, R7 and L groups may be optionally
independently substituted with 1 or more substituents independently selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy; and
A is a group comprising an aromatic group and is selected from tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; wherein the conjugated divalent linker L is covalently bonded to the aromatic group of group
The conjugated divalent linker L is free from chemical spacers. In this way, a conjugated system comprising the BODIPY aromatic core, the conjugated divalent linker and the aromatic group of the amino acid is provided. Such a conjugated system is advantageous because it is capable of strong fluorogenic behaviour and because it has minimal impact on the molecular properties of the peptide.
Direct carbon-carbon coupling of an amino acid residue in a peptide and a fluorogen without a chemical spacer minimizes any change in the polarity of a peptide modified to contain the fluorogen, thereby minimizing any changes in molecular recognition of a target for the peptide. This allows such fluorogen modified peptides to be used as probes while retaining the original selectivity.
Furthermore, the amino acids tryptophan and phenylalanine to which the fluorogen is linked are both hydrophobic, and so they are commonly present in the hydrophobic domain of a peptide. BODIPY is a hydrophobic fluorogen such that the introduction of a BODIPY-derived substituent will not alter the hydrophobic character of these amino acids and may even increase the stability of such a domain. Consequently, the addition of the BODIPY-derived fluorogen minimises any modification of the hydrophobicity of the peptide, leading to a retention of the molecular recognition compared to the unmodified peptide. In one embodiment, the conjugated divalent linker L is selected from the group comprising a divalent phenyl group and a divalent naphthyl group, which may be unsubstituted or substituted. Preferably, the conjugated divalent linker L is a group selected from L3 or L4 having the structural formulae:
Figure imgf000006_0001
14 in which the group R is selected from H, Ci_6 alkyl and Ci_6 alkoxy. When substituted, the conjugated divalent linker L may be independently substituted with 1 or more substituents independently selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy. More preferably, when substituted, the conjugated divalent linker L may be independently substituted with from 1 to 3 substituents selected from methyl and methoxy. In one embodiment, the divalent linker L is independently substituted with 1 or more substituents independently selected from the group comprising C2-6 alkyl and C2-6 alkoxy. In another embodiment, the divalent linker L is independently substituted with 1 or more substituents independently selected from methyl and methoxy.
Most preferably, the conjugated divalent linker L is a group selected from L1 or L2 having the structural formulae:
Figure imgf000007_0001
Li 12
Thus, the conjugated divalent linker L may be selected from the group comprising 1 ,3- phenylene (L1) and 1 ,3-naphthdiyl (L2). Preferably, the conjugated divalent linker L is a 1 ,3- phenylene group, for instance when the group is tryptophan or a derivative thereof or a peptide comprising tryptophan or a derivative thereof.
In one embodiment, the groups R1 , R3, R5 and R7 are preferably methyl. In another embodiment, the groups R2 and R6 are preferably H. In a further embodiment, the groups R1 , R3, R5 and R7 are preferably methyl and the groups R2 and R6 are preferably H.
In one embodiment, the groups R1 , R2, R3, R5, R6 and R7 are unsubstituted. In another embodiment, one or more of the groups R1 , R2, R3, R5, R6 and R7 are independently substituted with 1 or more substituents independently selected from the group comprising C2-6 alkyl and C2-6 alkoxy. In another embodiment, one or more of the groups R1 , R2, R3, R5, R6 and R7 are independently substituted with 1 or more substituents independently selected from methyl and methoxy. With regard to the group A, derivatives of tryptaminyl, tryptophanyl, phenylalaninyl, N- protected-tryptaminyl, N-protected tryptophanyl or N-protected phenylalaninyl include peptides, proteins, peptidomimetics, peptoids, and peptide nucleic acids comprising residues of tryptamine, tryptophan, phenylalanine, N-protected tryptamine, N-protected tryptophan or N-protected phenylalanine. The derivatives are obtainable by condensing tryptophan, phenylalanine, N-protected tryptophan or N-protected phenylalanine with a -COOH or -NH group on an amino acid, amino acid residue, peptidomimetic, peptoid or peptide nucleic acid to form a peptide bond. Similarly, the derivatives of tryptaminyl or N-protected tryptaminyl are obtainable by condensing the -NH group of tryptamine or N-protected tryptamine with a -COOH group on an amino acid, amino acid residue, peptidomimetic, peptoid or peptide nucleic acid to form a peptide bond
When the group A is a derivative of tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl or N-protected phenylalaninyl, then these derivatives may be formed by known techniques, such as solid phase synthesis or synthetic biology. Examples of suitable solid phase syntheses are discussed below. With regard to synthetic biological methods, an organism such as a bacteria can be altered, for instance by the alteration of its RNA, to accept compounds of formula (I) in which the group A is tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl or N- protected phenylalaninyl as a synthetic amino acid, allowing the synthesis of derivatives of tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl or N-protected phenylalaninyl, for instance peptides or proteins incorporating compounds of formula (I).
In one embodiment, the group A comprising an aromatic group may be selected from: tryptaminyl; N-protected tryptaminyl; an amino acid comprising an aromatic group selected from tryptophanyl,
phenylalaninyl and derivatives thereof; an N-protected derivative of an amino acid comprising an aromatic group selected from N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; a peptide obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N- protected phenylalanine or a peptide nucleic acid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with tryptamine or N-protected tryptamine; a peptidomimetic derivative comprising an amino acid residue comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptidomimetic derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptidomimetic derivative obtainable by condensing a
peptidomimetic with tryptamine or N-protected tryptamine; a peptoid derivative comprising an amino acid residue comprising an aromatic group, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; or a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a protein derivative obtainable from a plurality of amino acids and comprising further comprising a tryptamine residue or N- protected tryptamine residue, said protein derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine.
In another embodiment, the group A is an amino acid comprising an aromatic group selected from tryptophanyl, phenylalaninyl and derivatives thereof; an N-protected derivative of an amino acid comprising an aromatic group selected from N-protected tryptophanyl, N- protected phenylalaninyl and derivatives thereof; or a peptide obtainable from one or more amino acids, particularly one or more types of amino acids, comprising an amino acid comprising an aromatic group selected from tryptophanyl, phenylalaninyl and derivatives thereof. The amino acid comprising an aromatic group, N-protected derivative of an amino acid comprising an aromatic group and peptides obtainable from such amino acids may independently be in the D- or L-configuration.
In another embodiment, the group A is selected from the group comprising: tryptaminyl; N- protected tryptaminyl; a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine; a peptide nucleic acid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with tryptamine or N-protected tryptamine; a peptidomimetic derivative comprising an amino acid residue comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptidomimetic derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptidomimetic derivative obtainable by condensing a
peptidomimetic with tryptamine or N-protected tryptamine; a peptoid derivative comprising an amino acid residue, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; or a protein derivative obtainable from a plurality of amino acids and comprising further comprising a tryptamine residue or N-protected tryptamine residue, said protein derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine.
The proteins may be obtainable from amino acids independently be in the D- or L- configuration.
In another embodiment, the group A may selected from: a natural amino acid selected from tryptophan, such as L-tryptophan, or an N- protected derivative thereof, including D-tryptophan and N-protected derivatives of D-tryptophan;
a synthetic D or L amino acid selected from the group comprising phenylalanine, phenylalanine derivatives, tryptophan derivatives or an N-protected derivative thereof;
- a peptide obtainable from at least one of said natural or synthetic amino acids; a peptidomimetic obtainable from at least one of said natural or synthetic amino acids, or
a protein obtainable from at least one of said natural or synthetic amino acids.
The nitrogen protecting groups for the N-protected tryptaminyl, N-protected amino acid or N- protected amino acid derivative such as peptides, proteins, peptidomimetics, peptoid derivative and peptide nucleic acid derivative obtainable from such N-protected amino acids may be selected from the group comprising Fmoc (fluorenylmethyloxycarbonyl), Boc (t- butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), Ts
(toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5- dimethoxybenzyloxycarbony), Nsc [2-(4-Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4- Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1- ylidene)-3-methylbutyl], Pms [2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4- Dinitrobenzenesulfonyl), Troc (2,2,2-Trichloroethyloxycarbonyl), pNZ (p- Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4-Bromophenyl)-9-fluorenyl], HFA
(Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2-Dimethylindole-3-sulfonyl).
It will be apparent that tryptamine, tryptophan and phenylalanine contain an aromatic group, indolyl in the case of tryptamine and tryptophan and phenyl in the case of phenylalanine, to which the conjugated divalent linking group L is bound without a chemical spacer, forming a larger conjugated system.
Thus, the conjugated divalent linker is directly covalently bonded to the aromatic group of A by a single covalent bond i.e. there is no intervening bridging group.
The derivative of tryptophanyl, N-protected tryptophanyl, phenylalaninyl or N-protected phenylalaninyl may be the residue of tryptophan, N-protected tryptophan, phenylalanine or N-protected phenylalanine after the formation of at least one peptide bond. A peptide bond may be formed between the -NH or -COOH group of the tryptophan, N-protected tryptophan, phenylalanine or N-protected phenylalanine and a -COOH or -NH2 group respectively of a further species, such as an amino acid, peptide, protein or peptide nucleic acid. It will be apparent that tryptophan, N-protected tryptophan, phenylalanine and N- protected phenylalanine are capable of forming two peptide bonds, such that the amino acid residue of tryptophan, N-protected tryptophan, phenylalanine or N-protected phenylalanine may be incorporated into the terminal position of a polyamide, when a single peptide bond is formed or mid-chain, when two peptide bonds are formed.
The derivative of tryptaminyl, N-protected tryptaminyl may be the residue of tryptamine or N- protected tryptamine after the formation of a peptide bond. A peptide bond may be formed between the -NH group of the tryptamine or N-protected tryptamine and a -COOH group of a further species, such as an amino acid, peptide, protein or peptide nucleic acid. It will be apparent that tryptamine and N-protected tryptamine are capable of forming a single peptide bond, such that the residue of tryptamine or N-protected tryptamine is incorporated into the terminal position of a polyamide. Preferably, the group A comprises tryptophanyl, an N-protected tryptophanyl or a derivative thereof. Still more preferably, the tryptophanyl, N-protected tryptophanyl or a derivative thereof is bonded to the conjugated divalent linker L via the indole C2 position of the tryptophanyl or tryptophanyl residue. Preferably, when the group A is an amino acid, it is tryptophanyl or an N-protected tryptophanyl. Still more preferably, the tryptophan or N-protected tryptophanyl is bonded to the conjugated divalent linker L via the indole C2 position of the tryptophanyl.
In another embodiment, the compound of formula (I) is compound selected from:
Figure imgf000012_0001
(13), (3), and the corresponding D-tryptophan derivatives, wherein Z is an N-protecting group, such as a group selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), tnfluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl), Ts (toluenesulfonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4- biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2- (4-Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)- 3-ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl), MIS (1 ,2- Dimethylindole-3-sulfonyl).
In another embodiment, the group A is a peptide, such as a linear or cyclic peptide, wherein the peptide is obtainable from one or more amino acids, at least one of which is an amino acid comprising an aromatic group, an N-protected amino acid comprising an aromatic group or derivatives thereof as defined above. The amino acid in the peptide may be an N- protected amino acid when it is in the N-terminal position of the peptide. The conjugated linker group L is covalently bonded to the aromatic group of the amino acid comprising an aromatic group, N-protected amino acid comprising an aromatic group or derivatives thereof, without a chemical spacer, such that a larger conjugated system is formed. Preferably, the peptide is obtainable from one or more kinds of amino acids, at least one of which is tryptophan or an N-protected tryptophan, for instance if it is in the N-terminal position of the peptide.
In another embodiment, the group A is a protein obtainable from one or more amino acids, at least one of which is an amino acid comprising an aromatic group, an N-protected amino acid comprising an aromatic group or derivatives thereof as defined above. The amino acid in the protein may be an N-protected amino acid when it is in the N-terminal position of the protein. The conjugated linker group L is covalently bonded to the aromatic group of the amino acid, aromatic group of the N-protected amino acid or derivatives thereof, without a chemical spacer, such that a larger conjugated system is formed. Preferably, the protein is obtainable from one or more kinds of amino acids, at least one of which is tryptophan or an N-protected tryptophan, for instance if it is in the N-terminal position of the protein.
In another embodiment, the group A may be a peptide, such as a linear or cyclic peptide, obtainable from a plurality of amino acids, or derivatives thereof as defined above or an N- protected amino acid, for instance in the N-terminal position of the peptide, in combination with at least one amino acid or derivative thereof as defined above, which comprise a plurality of fluorophores of formula (i):
Figure imgf000014_0001
(i)
in which each conjugated divalent linker L is covalently bonded to an aromatic group of the peptide and the groups R1 , R2, R3, R5, R6, R7 and L are as defined above.
Typically, at least one of said plurality of amino acids, N-protected amino acids or derivatives thereof are covalently bonded to conjugated divalent linkers covalently bonded to a BODIPY core as shown in Formula (I). In this way, the fluorogenic properties of the peptide may be enhanced.
In another embodiment, at least two of said plurality of amino acids, N-protected amino acids or derivatives thereof are covalently bonded to conjugated divalent linkers covalently bonded to a BODIPY core as shown in Formula (I).
In one embodiment, the group A comprising an aromatic group may be selected from: a peptide obtainable from a plurality of amino acids, at least two of said plurality of amino acids comprising an aromatic group and independently selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptide nucleic acid derivative comprising at least two amino acid residues comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with at least two amino acids comprising an aromatic group, said amino acid comprising an aromatic group independently selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; a peptidomimetic derivative comprising at least two an amino acid residues comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with at least two amino acids comprising an aromatic group, said at least two amino acids comprising an aromatic group selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine; a peptoid derivative comprising at least two amino acid residues comprising an aromatic group, said peptoid derivative obtainable by condensing a peptoid with at least two amino acids comprising an aromatic group, said at least two amino acids comprising an aromatic group independently selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine; or a protein obtainable from a plurality of amino acids, at least two of said plurality of amino acids comprising an aromatic group and independently selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine, wherein at least two of the aromatic groups of the at least two amino acids comprising an aromatic group are covalently bonded to a conjugated divalent linker L of formula (i).
For instance, when the group A is peptide PAF26 shown as group (ii) below, the conjugated divalent linker of the compound of formula (I) may be attached to the peptide (ii) at one or both of positions 1 and 2 shown:
Figure imgf000015_0001
A hydrogen atom is present at position 1 or position 2 if this is not covalently attached to the conjugated divalent linker.
In another embodiment, the group A may be a linear peptide. For instance, the group A may be a linear peptide comprising PAF26, namely:
H-Arg- Lys- Lys-Trp- Phe-Trp-0 H (Seq. I.D. No. 1) or
H-Arg-Lys-Lys-Trp-Phe-Trp-NH2 (Seq. I.D. No. 2) or H-Ala-Ala-Ala-Trp-Phe-Trp-NH2 (Seq. I.D. No. 5) or
H-Arg-Lys-Lys-Trp-Ala-Ala-NH2 (Seq. I.D. No. 6). in which one or both of the tryptophan residues in Seq. I.D. Nos. 1 , 2 and 5 are covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group or in which the tryptophan residue in Seq. I.D. No. 6 is covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group. The amino acids forming these linear peptides may be independently in the D- or L- configuration. The linear peptides may comprise amino acids in both the D- and L- configuration, for instance either as the same amino acid in different configurations, such as D-Lys and L-Lys, or different amino acids in different configurations, such as D-Arg and L- Trp.
In another embodiment, the compound of formula (I) is selected from:
Figure imgf000016_0001
Figure imgf000016_0002
Figure imgf000017_0001
the corresponding D-amino acid derivatives thereof or the compounds of formulae (5a), (5b), (5), (6), (7) or (12) derived from a combination of L- and D- amino acids, wherein Z is as defined above and is preferably Cbz (benzyloxycarbonyl).
In another embodiment, the group A may be a cyclic peptide. For instance, the group A may comprise a cyclic peptide analogue of PAF26, namely:
Cyclo(-Arg-Lys-Lys-Trp-Phe-Trp-Gly-) (Seq. I.D. No. 3) in which one or both of the tryptophan residues are covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group. The amino acids forming these cyclic peptides may be independently in the D- or L-configuration. The cyclic peptides may comprise amino acids in both the D- and L-configuration, for instance either as the same amino acid in different configurations, such as D-Lys and L-Lys, or different amino acids in different configurations, such as D-Arg and L-Trp. It will be apparent that the cyclic version of PAF26 contains an additional glycine residue. The presence of this residue ensures that the cyclisation does not lead to any epimerisation of the peptide.
Alternatively, the group A may comprise a cyclic peptide analogue of lactadherin ('EV- GREEN'), namely: Cyclo(-Trp-Asp-Gly-Gly-Gly-Arg-Gly-Gly-Gln-lle-His-Gly-Phe-) (Seq. I.D. No. 4). in which the tryptophan residue is covalently bonded to the group of formula (i) via the indolyl group, particularly at the C2-position of the indolyl group. The amino acids forming these cyclic peptides may be independently in the D- or L-configuration. The cyclic peptides may comprise amino acids in both the D- and L-configuration, for instance either as the same amino acid in different configurations, such as D-GIn and L-GIn, or different amino acids in different configurations, such as D-Arg and L-Trp.
Thus, in one embodiment, the group A is a cyclic peptide of formula (iii) connected to the conjugated divalent linker L of the compound of formula (I) at position 3:
Figure imgf000018_0001
Thus, position 3 may be covalently bonded to a fluorogen group of formula (i). In another embodiment, the compound of formula (I) is:
Figure imgf000018_0002
the D-amino acid derivative thereof or the compound of formula (8) derived from a combination of L- and D- amino acids.
In another embodiment, the compound of formula (I) is 'APOGREEN':
Figure imgf000019_0001
the D-amino acid derivative thereof or the compound of formula (14) derived from a combination of L- and D- amino acids.
In another embodiment, the group A is a cyclic peptide of formula (iv) connected to the conjugated divalent linker L of the compound of formula (I) at position 4:
Figure imgf000019_0002
(iv).
Thus, position 4 may be bonded to a fluorogen group of formula (i). In another embodiment, the compound of formula (I) ('EV-GREEN') is:
Figure imgf000020_0001
the D-amino acid derivative thereof or the compound of formula (10) derived from a combination of L- and D- amino acids.
In another embodiment, in the compound of formula (I), R1 , R3, R5 and R7 may be methyl and R2 and R6 may be hydrogen.
In another embodiment, in the compound of formula (I), R3 and/or R5 may be a triazole radical, such as a 1 ,2,3-triazole group or a 1 ,2,4-triazole group.
In another embodiment, the compound of formula (I) may be radiolabelled. Typically, one of both of the F atoms in the compound of formula (I) may be radioisotopes of fluorine, such as 18F.
In a second aspect, there is provided a process for the preparation of a compound of formula (I) as described above wherein the conjugated divalent linker L is a 1 ,3-phenylene group and the group A comprising an aromatic group is selected from N-protected tryptaminyl, N- protected tryptophanyl or a derivative thereof, comprising at least the step of: - reacting an N-protected tryptamine, N-protected tryptophan or a derivative thereof with a compound of formula (V):
Figure imgf000020_0002
(V) in the presence of a Pd catalyst, a halide abstraction reagent and a carboxylic acid to provide a compound of formula (VI):
Figure imgf000021_0001
(VI)
wherein the groups R1 , R2, R3, R5, R6 and R7 of the compounds of formulae (V) and (VI) are as defined for the compound of formula (I) above and the group A is an N-protected amino acid comprising an aromatic group selected from N-protected tryptophan or a derivative thereof, and
wherein the phenylene group is covalently bonded to the indolyl group of the N- protected tryptamine, N-protected tryptophan or derivative thereof. The N-protected tryptophan or derivative thereof may be in the D- or L-configuration.
In one embodiment, the phenylene group is covalently bonded to the indolyl group of the N- protected tryptamine or N-protected tryptophan at the C2-position.
In another embodiment, the group A is N-protected tryptophan or a derivative thereof.
In another embodiment, the group A is N-protected tryptamine or a derivative thereof. In another embodiment, the Pd catalyst used in the reaction of the second aspect may be a Pd(0) catalyst or a Pd(l l) catalyst. An exemplary palladium (0) compound is tetrakistriphenyl phosphine palladium (0). A preferred Pd(l l) catalyst is a Pd(Ci.5acyloxy)2 catalyst, such as Pd(OAc)2.
In another embodiment, the halide abstraction reagent may be a silver (I) salt, such as silver tetrafluoroborate or silver hexafluorophosphate, with silver tetrafluoroborate being preferred.
In another embodiment, the carboxylic acid may be nitrobenzoic acid or fluorocarboxylic acid such as trifluoroacetic acid, with trifluoroacetic acid being preferred.
In another embodiment, the reaction is carried out in an organic liquid, such as dimethyl formamide. In another embodiment, the N-protecting group is selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-Nitrophenylsulfonyl) ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl], ivDde [1-(4,4- Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2-[Phenyl(methyl)sulfonio] ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p- nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
In another embodiment, the compound of formula (VI) is the N-protected tryptophan derivative of formula (3). The N-protected tryptophan may be in the D- or L-configuration.
In one embodiment, the process may further comprise the step of deprotecting the N- protected group to provide a deprotected derivative. For instance, deprotecting the N- protected amino acid group of tryptophan to provide a deprotected amino acid derivative, such as a compound of formula (13).
In another embodiment, the process may further comprise the step of condensing the deprotected -NH2 group of the compound of formula (I) with one or more amino acids to form a peptide or peptide derivative.
In a third aspect, there is provided a process according to the second aspect in which: the N-protected tryptophan or a derivative thereof is replaced with a peptide obtainable from one or more amino acids comprising tryptophan or a derivative thereof; or
the N-protected tryptamine or a derivative thereof is preplaced with a peptide derivative, said peptide derivative obtainable from the condensation of one or more amino acids and N-protected tryptamine.
In a fourth aspect, there is provided a process for the preparation of a compound of formula (I) as described above wherein the amino acid A is selected from N-protected 3- phenylalaninyl, N-protected 4-phenylalaninyl or a derivative thereof, comprising at least the step of: - reacting an N-protected 4-phenylalanine derivative of formula (Vila), an N-protected 3-phenylalanine derivative of formula (Vllb) or a derivative thereof, including a N-protected 4- D-phenylalanine derivative, N-protected 4-L-phenylalanine derivative, N-protected 3-D- phenylalanine derivative and
Figure imgf000023_0001
in which Q is a boronic acid moiety, R' is H or a carboxylic acid protecting group and R is a N-protecting group, with a compound of formula (VIII):
Figure imgf000023_0002
(VIII) in which L is a conjugated divalent linker selected from a C6-io arylene group and I is an iodine atom, in the presence of a Pd catalyst and a base to provide a compound of formula (I):
Figure imgf000023_0003
(I) wherein the groups R1 , R2, R3, R5, R6 and R7 of the compounds of formulae (I) and (VIII) are as defined for the compound of formula (I) above and the group A is an N- protected 4-phenylalaninyl, N-protected 3-phenylalaninyl or a derivative thereof. The N-protected 4-phenylalaninyl may be N-protected 4-D-phenylalaninyl or N- protected 4-L-phenylalaninyl. The N-protected 3-phenylalaninyl may be N-protected 3-D- phenylalaninyl or N-protected 3-L-phenylalaninyl.
It will be apparent that reacting an N-protected 4-phenylalanine derivative of formula (Vila) provides a compound of formula (I) in which A is N-protected 4-phenylalaninyl and reacting an N-protected 3-phenylalanine derivative of formula (VI lb) provides a compound of formula (I) in which A is N-protected 3-phenylalaninyl.
In one embodiment, the conjugated divalent linker L is a group selected from L1 or L2 having the structural formulae:
Figure imgf000024_0001
11
In another embodiment, the group A is selected from groups of structural formulae (10a), (10b) or a derivative thereof:
Figure imgf000024_0002
In another embodiment, the derivative of the N-protected 4-phenylalanine derivative or N- protected 3-phenylalanine derivative is a peptide obtainable from one or more amino acids comprising the N-protected 4-phenylalanine or N-protected 3-phenylalanine. The N- protected 3- or 4- phenylalanine derivative in such a peptide may be independently in D- or L- configuration.
In another embodiment, the Pd catalyst used in the reaction of the fourth aspect may be a palladium compound, such as a palladium (0) compound or a palladium (II) compound. An exemplary palladium (0) compound is tetrakistriphenyl phosphine palladium (0). Exemplary palladium (II) compounds are palladium (II) acetate, palladium (II) chloride or diphenylphosphinoferrocene dichloropalladium (II).
In another embodiment, the base is an alkali metal alkoxide, such as an alkali metal Ci_6 alkoxide.
In another embodiment, the reaction is carried out in an organic liquid, such as toluene or a mixture of toluene and water.
In another embodiment, the N-protecting group is selected from Fmoc
(fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-
Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3- ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
In another embodiment, the carboxylic acid protecting group is selected from a Ci_5 alkyl group such as methyl, ethyl or tert-butyl, allyl, a benzyl derivative such as a benzyl group, 2- chlorotrityl, 9-fluorenylmethyl, carbamoylmethyl and 2-phenylisopropyl.
In one embodiment, the process may further comprise the step of deprotecting the N- protected amino acid group to provide a deprotected amino acid group.
In another embodiment, the boronic acid moiety Q is selected from a boronic acid or a group of formula:
Figure imgf000026_0001
In another embodiment, the process further comprises the step of providing the compound of formula (Vila or b) by the step of: reacting an N-protected 4-iodo phenylalanine derivative of formula (Xla), an N— protected 3-iodo phenylalanine derivative of formula (Xlb) or a derivative thereof, including a N-protected 4-iodo-D-phenylalanine derivative, N-protected 4-iodo-L- phenylalanine derivative, N-protected 3-iodo-D-phenylalanine derivative and N- p e:
Xla),
Figure imgf000026_0002
with a diboronate in the presence of a Pd catalyst.
In another embodiment, the process further comprises the step of providing the compound of formula (VII) by the step of: reacting an N-protected 4-bromo phenylalanine of formula (Xlla), a N-protected 3-bromo phenylalanine derivative of formula (XI I b) or a derivative thereof, including N-protected 4-bromo-D-phenylalanine derivative, N-protected 4-bromo- L-phenylalanine derivative, N-protected 3-bromo-D-phenylalanine derivative and N-protected 3-bromo-L-phenylalanine derivative:
Figure imgf000027_0001
with a diboronate in the presence of a Pd catalyst.
In one embodiment, the Pd catalyst is a Pd(0) or a Pd(ll) catalyst, such as 1 , 1 '- bis(diphenylphosphino)ferrocene dichloropalladium.
In another embodiment, the reaction is carried out in an organic solvent, such as dimethyl sulfoxide.
In another embodiment, the diboronate is bis(pinacolato)diboron or 4, 6,4', 6'- tetraphenyl[2,2']bis[(1 ,3,2)dioxaborinanyl. In a fifth aspect, there is provided a process according to the fourth aspect in which the derivative of the N-protected 4-phenylalanine derivative or N-protected 3-phenylalanine derivative is a peptide obtainable from one or more amino acids comprising the N-protected 4-phenylalanine or N-protected 3-phenylalanine.
In a sixth aspect, there is provided the use of a compound of formula (I) as described above as a probe, preferably an optical probe.
In a seventh aspect, there is provided a method of detecting the presence and/or function (i.e. activity, inhibition, blockade) of a target in a cell or a component of a cell, comprising at least the steps of:
- providing a compound of formula (I) as described above to a target zone;
- illuminating the target zone with a wavelength of light suitable to excite the compound of formula (I);
- detecting emission of fluorescence, fluorescence life-time, fluorescence polarization, ultrasonic waves or gamma rays;
wherein detection of the above is indicative of the presence and/or function of the target in the cell or in the component of the cell.
In one embodiment, the target zone is a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample. Accordingly, the compound of formula (I) may be used in vivo, ex vivo or in vitro.
In another embodiment the cell is a fungus, such as Aspergillus fumigatus.
In another embodiment, the compound of formula (I) is radiolabeled and the detection of fluorescence is the detection of gamma rays.
In another embodiment the cell is a cell with phosphatidylserine molecules such as apoptotic cells, platelets or other cells.
In another embodiment, the component of a cell is a microvesicle.
In another embodiment of the method of the seventh aspect, the group A in the compound of formula (I) is a peptide PAF26 having the sequence:
H-Arg-Lys-Lys-Trp-Phe-Trp-NH2 (Seq. I.D. No. 2) in which the conjugated divalent linker L of the fluorogen group of formula (i) is covalently bonded to the indole group, preferably at the C2 position, of one or both tryptophan residues, such as compounds 5, 6 or 7 described above or
H-Arg-Lys-Lys-Trp-Phe-Trp-OH (Seq. I.D. No. 1) in which the conjugated divalent linker L of the fluorogen group of formula (i) is covalently bonded to the indole group, preferably at the C2 position,
cyclo (-Arg-Lys-Lys-Trp-Phe-Trp-Gly-) (Seq. I.D. No. 3) in which the conjugated divalent linker L of the fluorogen group of formula (i) is covalently bonded to the indole group, preferably at the C2 position, of one or both tryptophan residues, such as compound 8 as described above.
In another embodiment of the method of the seventh aspect, the group A in the compound of formula (I) is a peptide having the sequence:
- cyclo(-Trp-Asp-Gly-Gly-Gly-Arg-Gly-Gly-Gln-lle-His-Gly-Phe-) (Seq. I.D. No. 4) in which the conjugated divalent linker L of the fluorogen group of formula (i) is covalently bonded to the indole group, preferably at the C2 position, of the tryptophan residue, such as compound 10 described above.
In another embodiment of the method of the seventh aspect, the group A in the compound of formula (I) is a peptide having the sequence:
H-Ala-Ala-Ala-Trp-Phe-Trp-NH2 (Seq. I.D. No. 5) in which the conjugated divalent linker L of the fluorogen group of formula (i) is covalently bonded to the indole group, preferably at the C2 position, of one or both tryptophan residues, such as compound 5a described above; and H-Arg-Lys-Lys-Trp-Ala-Ala-NH2 (Seq. I.D. No. 6) in which the conjugated divalent linker L is covalently bonded to the indole group of the tryptophan residue.
Brief Description of the Figures Figure 1 a shows the spectral characterisation of peptides 5, 6 and 7. Absorbance (solid lines) and emission (dashed lines) spectra (Aexc. : 450 nm) of peptides 5-7 in PBS (phosphate buffer saline). Spectra represented as means from three independent experiments with n=3.
Figure 1 b shows the fluorogenic behaviour of peptides 5, 6 and 7in phospholipid
membranes. Figure 1 c shows the spectral characterisation of peptide 5 and red fluorescent dye Syto82.
Figure 2a shows the fluorogenic behaviour of peptide 8 in the cell membrane of fungal cells.
Figure 2b shows the stability of cyclic (8) and linear (5) mono-BODIPY labelled peptides in human bronchoalveolar lavage samples from patients with acute respiratory distress syndrome. Figure 2c shows MALDI analysis of cyclic (8) and linear (5) mono-BODIPY labelled peptides in bronchoalveolar lavage samples from patients with acute respiratory distress syndrome.
Figure 3a shows the fluorogenic behaviour of the cyclic peptide 8 in phospholipid membranes. Emission spectra of (8) in suspensions of phospholipid bilayer membranes in PBS (Aexc. : 450 nm). Spectra represented as means from three independent experiments with n=3. Quantum yields were determined using fluorescein in basic ethanol as reference (QY: 0.97).
Figure 3b shows the fluorescence emission of mono-BODIPY labelled peptides 5, 6 (linear) and 8 (cyclic) upon incubation with A. fumigatus. Peptides (5), (6), and (8) were incubated in PBS alone or in suspensions of A. fumigatus in PBS (Aexc. : 485 nm; Aem- : 515 nm). Values are represented as means ± SD from two independent experiments with n=3. ** for p < 0.01 and *** for p < 0.005 were determined as statistically significant differences between the fluorescence emission values in PBS and in suspensions of A. fumigatus in PBS.
Figure 3c shows the spectral characterization of cyclic peptide 8 and red fluorescent dye Syto82. Figure 4a shows cell proliferation assays. Values are represented as means ± SD from two independent experiments with n=4. No significant differences (p > 0.05) were determined between the control and any of the treatments. Figure 4b shows direct imaging of A. fumigatus in human pulmonary tissue using multi- photon microscopy.
Figure 5 shows stability studies of Trp-BODIPY derivatives. HPLC analysis of Fmoc-Trp(C2- BODIPY)-OH (3) in TFA:DCM (1 :99) at r.t. after 10 min (83% purity) is shown in Figure 5A, HPLC analysis of Fmoc-Trp(C2-BODIPY)-OH (3) in in TFA:DCM (1 :9) at r.t. after 30 min (46% purity) is shown in Figure 5B, and HPLC analysis of crude (12), a BODIPY-labelled linear precursor of the cyclic peptide (8) directly after cleavage from the resin with TFA:DCM (1 :99) at r.t. is shown in Figure 5C (98% purity). Black arrows in Figures 5A and 5B point at the peak corresponding to the amino acid 3. Figure 5D displays long-term stability of Trp- BODIPY in solid- and solution-state at various temperatures.
Figure 6 shows live fungal cell imaging after incubation with cyclic peptide 8. Peptide 8 was used at 2 μΜ for all fungi except C. albicans for which 10 μΜ was used. The four images shown as Figure 6A were fluorescence images while the four images shown as Figure 6B are the corresponding bright field images. The images were acquired under a confocal microscope at r.t. Scale bars: C. albicans: 5 μηι; C. neoformans: 7.5 μηι; F. oxysporum and N. crassa: 10 μηι.
Figure 7 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V- AF647and APOGREEN [cyclic peptide (14)] in buffer containing 2 mM CaCI2. APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils to a similar extent than ANNEXIN V. Figure 8A shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and APOGREEN [cyclic peptide (14)] in the absence of Ca2+. Unlike ANNEXIN V,
APOGREEN [cyclic peptide (14)] binding does not depend on Ca2+. Figure 8B shows that APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils in the presence of Ca2+, and as shown in Figure 8C, even in the presence of 2.5 mM EDTA. Figure 9 shows a comparison of the binding of ANNEXIN V in the presence and absence of Ca2+ and APOGREEN [cyclic peptide (14)] in the presence and absence of EDTA to monocytes, together with a phosphate buffered saline (PBS) control. Unlike ANNEXIN V, APOGREEN [cyclic peptide (14)] does not bind to monocytes, therefore its major target is unlikely to be phosphatidylserine. Figure 10 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and linear peptides 1-3 [corresponding to compounds (5), (6) and (7)] derived from APOGREEN [cyclic peptide (14)]. Derived peptides of APOGREEN can bind to apoptotic neutrophils to different extent. The cyclic peptide shows better discrimination between viable and apoptotic neutrophils. Figure 11 shows the results of kinetic studies of apoptotic and viable human BL2 cells with APOGREEN [cyclic peptide (14)] over time. Figure 11 A shows the staining of non-apoptotic and apoptotic BL2 cells with APOGREEN [cyclic peptide (14)] over time, while Figure 1 1 B shows the fluorescence of the non-apoptotic and apoptotic BL2 cells in the presence of APOGREEN [cyclic peptide (14)] over time. It is apparent that APOGREEN [cyclic peptide (14)] binds rapidly to apoptotic cells and reaches saturation within minutes.
Figure 12 shows a comparison of the staining of apoptotic neutrophils of different origin between APOGREEN [cyclic peptide (14)] and ANNEXIN V. APOGREEN [cyclic peptide (14)] binds to human apoptotic cells of different origin, such as human BL2 (B-lymphoma cells) (Figure 12A) and A549 as human epithelial lung cancer cells (Figure 12B).
Figure 13 shows the phagocytic activity of macrophages as indicated by the staining with the commercial dye pHRODO. As reported, macrophages are more phagocytic upon activation with dexamethasone (B, D). APOGREEN [cyclic peptide (14)] does not impair the phagocytosis levels of macrophages (A vs C, B vs D), which is important to ensure clearance of apoptotic neutrophils at activation sites.
Figure 14 shows that APOGREEN [cyclic peptide (14)] can label apoptotic neutrophils in vivo in the lungs of mice after intratracheal instillation (Figure 14C right plot), with no toxicity and comparative results as obtained from ex vivo ANNEXIN V staining (Figure 14C left plot). Graph A shows the total cell number obtained from lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)]. Graph B shows the proportion of live versus dead cells in lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)].
Figure 15 shows bright field and fluorescence images of APOGREEN [cyclic peptide (14)] and ANNEXIN V-AF647 co-staining in A549 cells after chemical induction of apoptosis. Only ANNEXIN V-AF647 stained cells are APOGREEN-positive, with no staining in viable cells. APOGREEN shows intracellular staining, potentially correlated to binding to negatively charged phospholipids.
Figure 16A shows a plot of fluorescence intensity versus wavelength for EV-GREEN [cyclic peptide (10)] upon incubation with various ratios of phosphatidylserine (PS) to
phosphatidylcholine (PC). Figure 16A shows specific fluorescence increase upon incubation with increasing phosphatidylserine (PS) content. Figure 16B shows quantification of the fluorescence increase with phosphatidylserine (PS) over other lipids (PC:
phosphatidylcholine). Fluorescence quantum yields (100% PS): 0.46 vs in blank (phosphate buffer saline, PBS): 0.08. *** for p values O.001. Figure 17 shows a plot of fluorescence for binding of EV-GREEN [cyclic peptide (10)] to phosphatidylserine (PS) in phosphate buffered saline (PBS) in the absence of Ca2+ (-Ca), in the presence of Ca2+ (+Ca) and in the presence of EDTA (+EDTA) together with the corresponding blanks of phosphate buffered saline in the absence of Ca2+, PBS(-Ca), in the presence of Ca2+,PBS (+Ca) and in the presence of EDTA, PBS (+EDTA). Figure 17 shows binding of EV-GREEN [cyclic peptide (10)] to PS is independent of Ca2+, unlike Annexin V. ns: non-statistical difference; *** for p values <0.001.
Figure 18 shows a comparison of relative fluorescence for phosphate buffered saline (PBS) and EV-GREEN [cyclic peptide (10)] in the presence of either viable cells (PBS) or fixed and permeabilized neutrophils (Fix/Lyse solution). This shows that EV-GREEN [cyclic peptide (10)] does not bind to viable cells. EV-green fluoresces brightly when cells are fixed and permeabilized (i.e. treated with Fix/lyse solution).
Figure 19 shows a comparison of relative fluorescence for phosphate buffered saline (PBS), ANNEXIN V in the presence of Ca2+ (+Ca), ANNEXIN V in the absence of Ca2+ (-Ca) and EV-GREEN [cyclic peptide (10)] in the presence of intact apoptotic cells. EV-GREEN [cyclic peptide (10)] does not bind to intact apoptotic cells (i.e. UV-induced apoptotic BL2 cells). ANNEXIN V is used as a positive control for apoptosis.
Figure 20 shows the measurement of the tensioactivity induced by EV-GREEN [cyclic peptide (10)]. EV-GREEN [cyclic peptide (10)] shows tensioactive properties; it is able to form a monolayer in the interface air-water, with a saturation pressure (π3) of 20.4 mN/m and a saturation concentration (Cs) of 3 μΜ.
Figure 21 shows the interaction of EV-GREEN [cyclic peptide (10)] with lipid monolayers. Measurement of initial pressure (ττ,) versus variation of pressure (Δττ) of egg phosphatidylcholine (eggPC) and egg PC: phosphatidylserine (DOPS) (70:30) when incubated with a) unlabeled peptide and b) EV-GREEN [cyclic peptide (10)]. The critical surface pressure (TTc) can be calculated from the linear regression curve of every set of data, being TTc the expected ττ, when Δπ=0. Higher TTc is observed in phosphatidylserine (DOPS)- containing monolayers for EV-GREEN [cyclic peptide (10)] indicating selective affinity for this lipid (42 mN/m vs 36.5 mN/m). Remarkably, the presence of Trp-BODIPY label increases the affinity of the unlabeled peptide for phosphatidylserine (DOPS)-containing monolayers (42 mN/m vs 30 mN/m).
Figure 22 shows the interaction of EV-GREEN [cyclic peptide (10)] with negatively-charged lipid monolayers. EV-GREEN [cyclic peptide (10)] can also bind to other negatively-charged lipid monolayers. Measurement of initial pressure (ττ,) versus variation of pressure (Δττ) of egg phosphatidylcholine/egg phosphatidylglycerol (eggPC: egg PG) (a negatively-charged lipid) and egg PC: phosphatidylserine (DOPS) (70:30) when incubated with EV-Green [cyclic peptide (10)]. The critical surface pressure (TTc) can be calculated from the linear regression curve of every set of data, being TTc the expected ττ, when Δπ=0. EV-Green [cyclic peptide (10)] displayed an almost identical TTc (42 mN/m vs 42.4 mN/m), opening the possibility to detect other negatively-charged lipids.
Figure 23 shows fluorescence confocal images of unilamellar vesicles without phosphatidylserine (eggPC) and with phosphatidylserine (eggPC:DOPS 70:30) after staining with EV-GREEN [cyclic peptide (10)] (right column). Liss-Rho-DOPE is used as positive control for vesicle staining (left column). EV-GREEN [cyclic peptide (10)] stains phosphatidylserine-containing vesicles with significantly brighter fluorescence than vesicles without phosphatidylserine.
Figure 24 shows flow cytometry experiments on extracellular vesicles from apoptotic BL2 cells, a) shows unstained vesicles, b) shows those incubated with 1 μΜ EV-GREEN [cyclic peptide (10)] for 20 min at 4°C, c) shows those incubated with 2 μΜ EV-GREEN for 30 min at 4°C. 5 min prior to measurement ANNEXIN V- Pacific Blue was added. Figure 24 c) is a 2D dot plot showing double staining of apoptotic vesicles with both EV-GREEN (x-axis) and ANNEXIN V- Pacific Blue (y-axis). The EV-GREEN stained vesicles are substantially also stained with ANNEXIN V- Pacific blue (36.3% compared to 14.0%), which correlates to the presence of phosphatidylserine.
Specific Description of Embodiments of the Invention
The present invention provides a class of fluorogenic compounds, particularly fluorogenic amino acids and peptides based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(BODIPY) scaffold. The BODIPY core is incorporated without the use of a chemical spacer such that the conjugated divalent linker connects the aromatic group of the amino acid or peptide with the aromatic core of the BODIPY to form a conjugated system comprising the aromatic group of the amino acid or protein, the conjugated divalent linker and the aromatic core of the BODIPY. This conjugated system forms the fluorogenic centre of the compound.
The fluorogenic amino acids described herein can be incorporated into peptides, to provide a fluorogenic peptide. When the peptide is biologically active, the fluorogenic peptide may be used as an optical probe. For instance, the peptide may be a targeting peptide, which preferentially binds to a particular biological substrate e.g. a cell such as an apoptotic cell or fungus; an enzyme substrate or an antibody or a fragment of an antibody. For the avoidance of doubt, where the enantiomeric configuration of a compound, such as an amino acid, is undefined, the disclosure herein encompasses both D- and L- configurations. Where a specific enantiomeric configuration is disclosed in a structural formula, such as an L-configuration, particularly of an amino acid or derivative thereof such as a peptide, the present disclosure encompasses the corresponding alternate configuration, such as the D-configuration.
As used herein, the term "N-protected" is intended to encompass the chemical protection of an -NH2 group in a molecule, such as tryptamine, an amino acid or a derivative thereof with a chemical protecting group, such as those described above. For instance, an N-protected -NH2 group may be represented by -NHZ, in which the N-protecting group Z is selected from the group comprising: Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4- methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4- biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2- (4-Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)- 3-ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
As used herein, the term "peptide" is intended to encompass shorter chain polymers made by the condensation of two or more amino acids and includes oligopeptides and
polypeptides. Peptides may be linear, branched or cyclic. The term also encompasses glycopeptides and lipopeptides. Peptides typically comprise from 2 to 100 amino acid residues.
As used herein, the term "protein" is intended to encompass longer chain polymers made by the condensation of amino acids and includes enzymes, antibodies, structural proteins and signalling proteins etc. The protein is composed of amino acid residues which no longer contain all the free carboxylic acid and amino groups of the amino acid, which have been converted into a peptide bond and comprise more amino acid residues than a peptide. Such proteins typically comprise >100 amino acid residues.
As used herein, the term "peptide nucleic acid" is intended to encompass artificial polyamide analogues of DNA and RNA made of repeating units of N-(2-aminoethyl)-glycine linked by peptide bonds to which nucleic acid bases are linked to the secondary amide by the removal of the hydrogen atom from the secondary amide via a methylene bridge and carbonyl group.
As used herein, the term "peptidomimetic" is intended to encompass protein-like molecules which mimic peptides, such as peptide analogues in which the peptide bonds are replaced with other polar bonds or analogues in which the Ca atom is substituted by a Na one. Non- limiting examples of peptidomimetics include foldamers, desipeptides, thiopeptides, selenoxopeptides or azapeptides.
As used herein, the term "peptoid" is intended to represent peptide analogues derived from poly-N-substituted glycines in which the side chains, such as amino acid side chains, are attached to the nitrogen atom of the peptide, rather than the a- carbon as in peptides. The present invention requires that the conjugated divalent linker L is covalently bonded to the aromatic group of group A. Thus, within the meaning of the present invention
tryptophanyl represents a tryptophan molecule from which Η· has been removed from a C on the indole ring. N-protected tryptophanyl represents an N-protected tryptophan molecule from which Η· has been removed from a C on the indolyl ring. Phenylalaninyl represents phenylalanine from which Η· has been removed from a C on the phenyl ring. N-protected phenylalaninyl represents N-protected phenylalanine from which Η· has been removed from a C on the phenyl ring.
As used herein, the term "derivative" is used to refer to the residue of a chemical compound, such as an amino acid, after it has undergone chemical modification. For instance, N- protection of an amino acid can produced an N-protected amino acid derivative.
Similarly, the term "derivative" when used in relation to an amino acid or N-protected amino acid encompasses the condensation product of the amino acid or N-protected amino acid. For instance, an amino acid derivative may encompass peptides, proteins, peptide nucleic acid derivatives comprising an amino acid reside and peptoid derivatives comprising an amino acid residue.
The condensation of an amino acid with further amino acids may form a peptides or proteins as the amino acid derivative. Such condensation reactions produce a polymerised amino acid derivative in the form of a polyamide. The polyamide is composed of amino acid residues which no longer contain all the free carboxylic acid and amino groups of the amino acid, which have been converted into a peptide bond (-C(=0)-NH-) by the condensation reaction.
Similarly, the condensation of tryptamine with amino acids may form a peptide derivative or protein derivative. Such condensation reactions produce a polymerised amino acid derivative in the form of a polyamide. The polyamide is composed of tryptamine residue and amino acid residues which no longer contain all the free carboxylic acid and amino groups of the amino acid, which have been converted into a peptide bond (-C(=0)-NH-) by the condensation reaction. The tryptamine does not contain a carboxylic acid group and therefore forms a terminal position on the polyamide chain. Furthermore, the condensation of an amino acid with a peptide nucleic acid may form a peptide nucleic acid derivative as the amino acid derivative. The condensation of tryptamine with a peptide nucleic acid may form a peptide nucleic acid derivative. When group A is a peptide nucleic acid derivative, the peptide nucleic acid derivative comprises a peptide nucleic acid with tryptamine, tryptophan or phenylalanine residue at the point of connection between the polyamide chain of the peptide nucleic acid and the linker group L. A tryptophan or phenylalanine can be linked to the polyamide chain of the peptide nucleic acid by a peptide bond via the -NH or -COOH group of the tryptophan or phenylalanine. A tryptamine can be linked to the polyamide chain of the peptide nucleic acid by a peptide bond via the -NH of the ethanamine group of the tryptamine. The condensation of tryptamine or an amino acid with a peptidomimetic may form a peptidomimetic derivative. When group A is a peptidomimetic derivative, the peptidomimetic derivative comprises a tryptamine, tryptophan or phenylalanine residue at the point of connection between the peptidomimetic and the linker group L. A tryptophan or
phenylalanine can be linked to the peptidomimetic by a peptide bond via the -NH or -COOH group of the tryptophan or phenylalanine. A tryptamine can be linked to the peptidomimetic by a peptide bond via the -NH of the ethanamine group of the tryptamine.
The condensation of an amino acid with a peptoid may form a peptoid derivative as the amino acid derivative. The condensation of tryptamine with a peptoid may form a peptoid derivative. When group A is a peptoid derivative, the peptoid derivative comprises a peptoid with tryptamine, tryptophan or phenylalanine residue at the point of connection between the polyglycine chain of the peptoid and the linker group L. The tryptophan or phenylalanine can be linked to the polyglycine chain of the peptoid by a peptide bond via the -NH or -COOH group of the tryptophan or phenylalanine. A tryptamine can be linked to the polyglycine chain of the peptoid by a peptide bond via the -NH of the ethanamine group of the tryptamine Thus, a tryptamine derivative or N-protected tryptamine derivative may comprise a peptide derivative obtained from a plurality of amino acids and tryptamine or N-protected tryptamine (i.e. the peptide comprises tryptamine residue or N-protected tryptamine residue); a protein derivative obtained from a plurality of amino acids and tryptamine or N-protected tryptamine (i.e. the protein derivative comprises tryptamine residue or N-protected tryptamine residue); a peptoid derivative obtained from the condensation of a peptoid and tryptamine or N- protected tryptamine (i.e. the peptoid derivative comprises tryptamine residue or N-protected tryptamine residue); and a peptide nucleic acid derivative obtained from the condensation of a peptide nucleic acid and tryptamine or N-protected tryptamine (i.e. the peptide nucleic acid derivative comprises tryptamine residue or N-protected tryptamine residue).
Thus, a tryptophan derivative or N-protected tryptophan derivative may comprise a peptide obtained from a plurality of amino acids including tryptophan or N-protected tryptophan (i.e. the peptide comprises tryptophan residue or N-protected tryptophan residue); a protein obtained from a plurality of amino acids including tryptophan or N-protected tryptophan (i.e. the protein comprises tryptophan residue or N-protected tryptophan residue); a peptoid derivative obtained from the condensation of a peptoid and tryptophan or N-protected tryptophan (i.e. the peptoid derivative comprises tryptophan residue or N-protected tryptophan residue); and a peptide nucleic acid derivative obtained from the condensation of a peptide nucleic acid and tryptophan or N-protected tryptophan (i.e. the peptide nucleic acid derivative comprises tryptophan residue or N-protected tryptophan residue).
Thus, a phenylalanine derivative or N-protected phenylalanine derivative may comprise a peptide obtained from a plurality of amino acids including phenylalanine or N-protected phenylalanine (i.e. the peptide comprises phenylalanine residue or N-protected
phenylalanine residue); a protein obtained from a plurality of amino acids including phenylalanine or N-protected phenylalanine (i.e. the protein comprises phenylalanine residue or N-protected phenylalanine residue); a peptoid derivative obtained from the condensation of a peptoid and phenylalanine or N-protected phenylalanine (i.e. the peptoid derivative comprises phenylalanine residue or N-protected phenylalanine residue); and a peptide nucleic acid derivative obtained from the condensation of a peptide nucleic acid and phenylalanine or N-protected phenylalanine (i.e. the peptide nucleic acid derivative comprises phenylalanine residue or N-protected phenylalanine residue). Peptidomimetics
Additionally, a derivative of tryptophan may include a synthetic amino acid in which one or more of the hydrogen atoms, particularly from 1 to 3 of the hydrogen atoms, especially the hydrogen atoms on the indole ring, more especially the hydrogen atoms on the indole ring with the exception of the hydrogen atom at the C2-position, have been replaced with a group selected from the group comprising methyl and methoxy.
A derivative of phenylalanine may include a synthetic amino acid n which one or more of the hydrogen atoms, particularly from 1 to 3 of the hydrogen atoms, especially the hydrogen atoms on the phenyl ring, have been replaced with a group selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy, preferably methyl and methoxy.
As used herein, the term "conjugated" is a system of alternating single and multiple bonds. This can be viewed as an overlapping system of p-orbitals across an intervening o-bonds allowing a derealization of ττ-electrons across all the adjacent aligned p-orbitals. As used herein, the term "aromatic" is intended to represent ring structures comprising a delocalised conjugated ττ-system of electrons forming an aromatic core, which may typically be an arrangement of alternating single and double bonds. An aromatic group may be an aryl group, particularly an aryl group having only carbon atoms in the aromatic core (i.e. no heteroatoms such as O, S and N), such as a C6-io aryl group, more preferably a C6 aryl group. Alternatively, an aromatic group may be a heteroaryl group, such as a heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C. The aromatic group, including the heteroaryl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms bonded to the aromatic core may be substituted with a substituent independently selected from the group comprising methyl and methoxy.
As used herein, the term "arylene" is synonymous with arenediyl and represents an organic group derived from an aromatic hydrocarbon in which a hydrogen atom has been removed from two ring carbon atoms. As used herein, the term "alkyl" represents a linear, branched or cyclic alkyl group, particularly a linear or branched CM0 alkyl or a C4.i0 cycloalkyl, more particularly a linear Ci_5 alkyl or a branched C3.5 alkyl. The alkyl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms in the alkyl group may be substituted with a substituent independently selected from the group comprising methyl or methoxy.
As used herein, the term "alkenyl" represents a linear, branched or cyclic alkenyl group, particularly a linear or branched C2-io alkenyl or a C4_i0 cycloalkenyl, more particularly a linear C2-5 alkenyl or a branched C3.5 alkyl. The alkenyl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms in the alkenyl group may be substituted with a substituent independently selected from the group comprising methyl or methoxy.
As used herein, the term "alkynyl" represents a linear, branched or cyclic alkynyl group, particularly a linear or branched C2-10 alkynyl or a C7.10 cycloalkynyl, more particularly a linear C2-5 alkynyl or a branched C3.5 alkynyl. The alkynyl group may be unsubstituted or substituted. When substituted, 1 or more, preferably from 1 to 10, more preferably from 1 to 5 of the hydrogen atoms in the alkynyl group may be substituted with a substituent independently selected from the group comprising methyl or methoxy.
As used herein, the term "alkyi carboxylic acid" represents an alkyi group having a carboxylic acid (-C(=0)OH) substituent, such as d.10 alkyl-C(=0)OH, preferably a C1.5 alkyl-C(=0)OH group, in which the alkyi group may be as defined above.
As used herein, the term "alkyloxy" represents an alkyi group having an oxy (-0-) linker, such as C-1 -10 alkyl-O- group, preferably a Ci _5 alkyl-O- group, in which the alkyi group may be as defined above. As used herein, the term "acyl" represents an alkyi group having a carbonyl (-C(=0)-) linker, such as C-1 -10 alkyl-C(=0)-, preferably a Ci _5 alkyl-C(=0)- group, in which the alkyi group may be as defined above.
As used herein, the term "acyloxy" represents an alkyi group having a -C(=0)0- linker, such as C1 -10 alkyl-C(=0)0-, preferably a Ci _5 alkyl-C(=0)0- group, in which the alkyi group may be as defined above.
As used herein, the term "halo" represents a halogen atom, such as fluorine, chlorine or bromine, preferable fluorine or chlorine, more preferably fluorine.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc., Library of Congress, USA); Molecular Cloning: A
Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984);
Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Targeting peptide
The group A of the compound of formula (I) may be a peptide, such as a biologically active peptide. For instance, the peptide may be a targeting peptide, which preferentially binds to a particular biological substrate e.g. a cell such as an apoptotic cell or fungus; an enzyme substrate or an antibody or a fragment of an antibody.
PAF26 has a highly conserved sequence with a C-terminal hydrophobic domain (Trp-Phe- Trp) and an /V-terminal cationic domain (Arg-Lys-Lys) that are essential to exert its antifungal action. The use of fluorogenic amino acids in the peptide is advantageous in that they provide high signal-to-noise ratios without the need for washing or additional labelling steps.
Disclosed herein is a fluorogenic amino acid based on the 4,4-difluoro-4-bora-3a,4a-diaza-s- indacene (BODIPY) scaffold conjugated with tryptophan. The incorporation of the spacer- free BODIPY-Trp fluorogen in the hydrophobic domain of PAF26 maintains the recognition features of the peptide while providing an excellent reporter of the interaction with fungal cells. This approach has rendered fluorogenic BODIPY-labelled antimicrobial peptides as highly stable probes to image A. fumigatus in ex vivo human tissue.
Lactadherin, also known as Milk Fat Globule-EGF Factor 8 protein (Mfge8) is a cell adhesion protein containing a phosphatidyl serine binding domain. The levels of phosphatidyl serine in apoptotic cells are much higher than in quiescent or normal cells. Lactadherin analogues, such as cyclo(-Trp-Asp-Gly-Gly-Gly-Arg-Gly-Gly-Gln-lle-His-Gly-Phe-) (Seq. I.D. No. 4) contain a tryptophan residue. The incorporation of the spacer-free BODIPY fluorogen into this lactadherin analogue via the tryptophan residue retains the recognition features of the peptide for apoptotic cells while providing an excellent reporter of the interaction with phosphatidyl serine.
Typically, the compounds of the invention are operable as probes to detect a target in a cell or in a component of a cell. The target zone may be a portion of tissue within a subject, and the method may be carried out in vivo. The portion of tissue may be a portion of the lung, brain, muscle, heart, liver, kidneys, connective tissue, skin, intestine, or joints of a subject. The compounds of the invention may be used as probes in the immune system, the circulatory system, the nervous system, the digestive system or the reproductive system. For example, the target area may be a portion of the lung of a subject.
The target may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
Accordingly, the compounds of the invention may be used as probes, and in methods using such compounds, in vivo, ex vivo or in vitro.
The compounds of the invention used as probes may be delivered to a target zone by any means known in the art. For example, they may be delivered by endoscope, spray, injection, topically, or ingestion. For example, where they are to be delivered to a portion of the lung, the compounds may be delivered to the target zone using a bronchoscope.
Illumination light of a suitable wavelength to excite fluorophore of the compound may be delivered to the target zone by any conventional means known in the art. Typically, in embodiments where the compound is to be used within the body of a subject, the light is delivered by means of an optical fibre or similar. The fluorescence from the compound in the target zone may be collected by an optical fibre or similar. The fluorescence from the compounds in the target zone may be collected by the same optical fibre that delivered the illumination light.
The collected fluorescence, fluorescence life-time or fluorescence polarization is typically delivered to a recording device, such as a charge-coupled device (CCD) or similar.
Alternatively, the fluorescence from the probes in the target zone may be directly collected by a recording device, such as a CCD or similar. For example to both deliver the compound to the target zone, to deliver light to the target zone, and to detect fluorescence from the target zone. Alternatively, individual instruments may be used to deliver the probe to the target zone, to deliver light to the target zone and to detect fluorescence from the target zone.
For example, fluorescence may be detected from the tissue of a target area using fibered confocal fluorescence microscopy (fCFM).
The optical properties of these fluorogenic compounds also enables their use in
photoacoustic imaging. Photoacoustic imaging can provide high resolution images of the target area with very good tissue or in vivo penetration. In this case, the fluorogenic compounds would absorb energy from an irradiation source and subsequently generate ultrasonic waves that would be detected by ultrasonic transducers to produce photoacoustic images of the target area. Preferably, the subject is a human subject. However, the subject may be a non-human animal such as equine, ovine, bovine, canine, feline, rodent or fish, for example.
The term "significant fluorescence" as used herein refers to the fluorescence of a fluorophore that is above the background or, where present, autofluorescence in the target area. The autofluorescence of the indigenous cells or tissue within the target area may have a different fluorescent lifetime than the fluorophore of the compound. The autofluorescence of the indigenous cells or tissue within the target area may reduce over time at a different rate than that of the fluorophore of the compound.
For instance, autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the BODIPY fluorophore of the compound.
Consequently, fluorescence observed in the target area that decays more slowly over time may be indicative of the probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence.
The compounds of the invention display uniform fluorescent lifetimes in the nanosecond range, allowing their identification and differentiation from other fluorophores. The fluorescent lifetime of a fluorogen is the time between excitation, for instance by the absorption of incident light, and the corresponding emission. The fluorescent lifetime of a fluorogen is independent of concentration and can be used to discriminate between emissions of similar wavelengths from different fluorophores. For instance, some of the compounds of the invention have a fluorescent lifetime in the range of from 1 to 5 ns. Such fluorescent lifetimes can be used to differentiate the emissions from those of the compounds of the invention from other fluorophores exhibiting longer or shorter fluorescent lifetimes, which may emit at similar wavelengths.
Therefore, the method may comprise the step of determining the absorbance or
fluorescence intensity or fluorescence polarization of the target zone over a period of time, determining the rate of decay of fluorescence during the period of time, and determining the fluorescence intensity of that fluorescence with a slower rate of decay, wherein the fluorescence with a slower rate of decay corresponds to the fluorescence of the compound.
The target zone may be a portion of tissue within a subject, and the method may be carried out in vivo. The portion of tissue may be a portion of the lung, heart, liver, connective tissue, skin, intestine, brain, muscle, or joints of a subject, for example. For example, the target zone may be a portion of the lung of a subject. In addition, the method of the invention may be carried out in the immune system, the circulatory system, the nervous system, the digestive system or the reproductive system. The target zone may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample.
The target zone may be a target area. The target zone may be a target volume.
In another embodiment, the compound of formula (I) may be radiolabelled. Typically, one of both of the F atoms in the compound of formula (I) may be radioisotopes of fluorine, such as 18F. Such radiolabelled compounds of formula (I) can be used in positron emission tomography. This technique detects pairs of gamma rays emitted by a positron-emitting radionuclide such as 18F. When the group A of an 18F radiolabelled compound formula (I) is a peptide, it may be a targeting peptide with a specificity for a particular biological substrate. In this way, the radiolabelled compound of formula (I) can allow the imaging and detection of particular biological cells or components of a cell, such as cell apoptotic cells or fungal cells.
Syntheses
The compounds of the invention can be prepared via a number of synthetic routes.
Disclosed herein is a process for the preparation of fluorogenic tryptophan derivatives utilising a palladium catalysed direct C-2 arylation of the indolyl group to add a BODIPY fluorogen. Also disclosed is a process for the preparation of fluorogenic phenylalanine derivatives using a Suzuki-Miyaura coupling of a borono-phenylalanine derivative and a BODIPY fluorogen. Both preparative reactions utilise an iodo-C6-ioaryl BODIPY reagent, such as that of formula (VIII), to introduce the fluorogenic centre into the amino acid.
In the case of L-tryptophan or its derivatives, this amino acid is N-protected, for instance as shown in compound (1 1), prior to reaction with the iodo-C6-ioaryl BODIPY reagent.
Figure imgf000043_0001
(1 1) in which the N-protecting group Z is selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl and Ts (toluenesulfonyl).
Similarly, in the case of D-tryptophan or its derivatives, this amino acid can be N-protected prior to reaction with the iodo-C6-ioaryl BODIPY reagent. The N-protection of the D- or L- tryptophan or a derivative thereof can be carried out by methods known in the art. The coupling of the N-protected tryptophan and the iodo-C6-ioaryl BODIPY reagent is carried out in the presence of a Pd catalyst, such as a Pd(ll) acyloxy compound, for instance Pd(OAc)2 and a halide abstraction agent, such as a silver (I) salt.
In the case of the phenylalanine or its derivatives, an N-protected iodo-phenylalanine is first prepared. For instance, a 3- or 4- iodo- or bromo-phenylalanine or a derivative thereof may be N-protected, for instance with any of the groups described in the previous paragraph for the N-protection of tryptophan.
The N-protected iodo- or bromo-phenylalanine, such as a compound of formula (XI) or (XII) can then be converted into the corresponding N-protected borono-phenylalanine, such as a compound of formula (VII), as disclosed in J. Org. Chem. 1998, 63, 8019. This can be carried out by the reaction of the N-protected iodo- or bromo-phenylalanine or its derivative with a diboronate in the presence of a Pd catalyst. The Pd catalyst may be 1 ,1 '- bis(diphenylphosphino)ferrocene dichloropalladium (II). The reaction can be carried out in an organic solvent, such as dimethyl sulfoxide. The diboronate should contain boronates protected by reducible groups, such as pinacolato or 1 ,3-diphenyl-1 ,3-propanedioxy groups. The diboronate can be prepared by the reaction of tetrakis(dimethyamino)diboron with a suitable diol, such as pinacol or 1 ,3-diphenyl-1 ,3- propanediol, which can provide a reducible ligand.
The N-protected borono-phenylalanine, such as a compound of formula (VII), can then be coupled with an iodo-C6-ioaryl BODIPY reagent, such as that of formula (VIII), in a Suzuki- Miyaura reaction, to introduce the fluorogenic centre into the amino acid. The coupling reaction is carried out in the presence of a Pd catalyst and a base. The Pd catalyst may be an aryl palladium(ll) alkoxide complex. The base may be an alkali metal alkoxide, such sodium tert.-butoxide. Alternatively, the palladium catalyst may be tetrakistriphenyl phosphine palladium (0), palladium (II) acetate, palladium (II) chloride or
diphenylphosphinoferrocene dichloropalladium (II).
After the coupling step to form the N-protected fluorogenic amino acid derivative, the nitrogen protecting group can be removed to provide the fluorogenic amino acid derivative. The fluorogenic amino acid derivative can then be incorporated into peptides using conventional techniques.
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of". It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such these are within the scope of the present invention.
All documents and sequence database entries mentioned in this specification are
incorporated herein by reference in their entirety for all purposes. The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above and the following tables. Peptide Synthesis
General procedures for SPPS. All peptides were manually synthesized in polystyrene syringes fitted with a polyethylene porous disc using Fmoc-based SPPS. Solvents and soluble reagents were removed by suction. The Fmoc group was removed with piperidine- DMF (1 :4) (1 x 1 min, 2 x 5 min). Peptide synthesis transformations and washings were performed at r.t.
Resin loading (only for 2-chlorotrityl polystyrene resin). Fmoc-AA-OH (1 eq.) was attached to the resin (1 eq.) with DIPEA (3 eq.) in DCM at r.t for 10 min and then DIPEA (7.0 eq.) for 40 min. The remaining trityl groups were capped adding 0.8 μΙ_ MeOH/mg resin for 10 minutes. After that, the resin was filtered and washed with DCM (4 x 1 min), DMF (4 x 1 min). The loading of the resin was determined by titration of the Fmoc group.
Peptide elongation. After the Fmoc group was removed, the resin was washed with DMF (4 x 1 min), DCM (3 x 1 min), DMF (4 x 1 min). Unless otherwise noted, standard coupling procedures used DIC (3 eq.) and OxymaPure (3 eq.) in DMF for 1 h and 5-min of pre- activation. The completion of the coupling was monitored by the Kaiser test as described in E. Kaiser, R.L. Colescott, CD. Bossinger, P.I. Cook. Anal. Biochem., 1970, 34, 595-598. Then, the resin was filtered and washed with DCM (4 x 1 min) and DMF (4 x 1 min).
Final cleavage (for Sieber amide and for 2-chlorotrityl polystyrene resins). The resin bound peptide was treated for 5 times with 1 % TFA in DCM (1 min in each treatment) and washed with DCM. The combined filtered mixtures were poured over DCM and evaporated under vacuum. Then, the residue was dissolved in ACN:H20 and lyophilised.
Reference Example 1
4,4-Difluoro-8-(4-iodophenyl)-1 ,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (1). 4-iodobenzaldehyde (500 mg, 2.2 mmol) was dissolved in anhydride DCM (50 ml_) under N2. Then, 2,4-dimethylpyrrole (492 μΙ_, 4.8 mmol) and 2 drops of TFA were added and the reaction was stirred overnight at r.t in N2 atmosphere or until the consumption of the aldehyde was complete (TLC). DDQ (490 mg, 2.2 mmol) dissolved in DCM (20 ml_) was added dropwise (10-15 min) to the reaction mixture and the reaction was stirred for 15 min at r.t. Finally, TEA (4 ml_, 45 mmol) and BF3OEt2 (4 ml_, 30 mmol) were added and the mixture stirred for 3 h. Workup was done by diluting with DCM (50 ml_) and washing with H20 (4 x 100 ml_). The organic layers were combined, dried over sodium sulfate, filtered and concentrated under vacuum. The crude was purified via flash column chromatography using and DCM/hexane gradient on silica gel. The expected compound was isolated as a red amorphous solid (290 mg, 29%).
Characterisation data: 1 H NMR (500 MHz, CDCI3): δ 7.87 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 6.02 (s, 2H), 2.58 (s, 6H), 1.45 (s, 6H); 13C NMR (125 MHz, CDCI3): δ 155.9, 142.9, 138.3, 134.6, 131.1 , 130.0, 121 .5, 94.7, 14.7, 14.6; HRMS (m/z): [M+H]+ calcd. for Ci9H18BF2IN2, 451.0654; found, 451 .0651 .
Reference Example 2
Unsuccessful coupling of 4,4-Difluoro-8-(4-iodophenyl)-1 ,3,5,7-tetramethyl-4-bora- 3a,4a-diaza-s-indacene (1 ) with Fmoc-Trp-OH. The interaction of Fmoc-Trp-OH with the p-iodophenyl-BODI PY 1 was explored using 5 mol% Pd(OAc)2, 1 (4.0 eq.), AgBF4 (1 .0 eq.) and o-nitrobenzoic acid (1 .5 eq.) in DM F under microwave irradiation at 150 °C for 5 min. Under these conditions, no reaction occurred.
Example 1 4,4-Difluoro-8-(3-iodophenyl)-1 ,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (2).
3-iodobenzaldehyde (500 mg, 2.2 mmol) was dissolved in anhydride DCM (50 mL) under N2. Then, 2,4-dimethylpyrrole (492 μΙ_, 4.8 mmol) and three drops of TFA were added and the reaction was stirred overnight at r.t in N2 atmosphere or until the consumption of the aldehyde was complete (TLC). DDQ (490 mg, 2.2 mmol) dissolved in DCM (20 mL) was added dropwise (10-15 min) to the reaction mixture and the reaction was stirred for 15 min at r.t. Finally, TEA (4 mL, 45 mmol) and BF3OEt2 (4 mL, 30 mmol) were added and the mixture stirred for 3 h. Workup was done by diluting with DCM (50 mL) and washing with H20 (4 x 100 mL). The organic layers were combined, dried over sodium sulfate, filtered and concentrated under vacuum. The crude was purified via flash column chromatography using and DCM/hexane gradient on silica gel. The expected compound was isolated as a red amorphous solid (401 mg, 41 %).
Characterisation data: 1 H NMR (400 MHz, CDCI3): δ 7.76 (dt, J = 7.7, 1.5 Hz, 1 H), 7.62 (t, J = 1.6 Hz, 1 H), 7.24 - 7.20 (m, 1 H), 7.19 - 7.14 (m, 1 H), 5.92 (s, 2H), 2.48 (d, J = 1.3 Hz, 6H), 1 .36 (s, 6H); 13C NMR (100 MHz, CDCI3): δ 155.9, 142.9, 139.3, 138.0, 137.1 , 136.8, 130.7, 127.3, 121.5, 94.3, 14.7, 14.6 (one quaternary carbon signal not seen); HRMS (m/z): [M+H]+ calcd. for Ci9H18BF2IN2, 451.0654; found: 451 .0651 . Example 2
Fmoc-Trp(C2-BODIPY)-OH (3).
Initial arylation: Fmoc-Trp-OH, 5 mol% Pd(OAc)2, 2 (4.0 eq.), AgBF4 (1.0 eq.) and o- nitrobenzoic acid (1.5 eq.) in DMF were placed in a microwave reactor vessel. The mixture was heated under microwave irradiation at 150 °C for 5 min. The formation of expected product 3 was observed.
Optimised arylation: Fmoc-Trp-OH (100 mg, 0.234 mmol), 2 (1.5 eq., 158 mg, 0.352 mmol), AgBF4 (1.0 eq., 46 mg, 0.234 mmol), TFA (1.0 eq., 18 μΙ_, 0.234 mmol) and Pd(OAc)2 (0.05 eq., 2.6 mg, 0.01 17 mmol) were placed in a microwave reactor vessel in 1.8 ml_ DMF. The mixture was heated under microwave irradiation (250 W) at 80°C for 20 min. AcOEt was added and the resulting suspension was filtered through Celite and concentrated under vacuum. The resulting crude was purified by flash column chromatography using and EtOAc/hexane gradient on silica gel. The expected adduct was isolated as a red solid (130 mg, 74%). Characterisation data: 1 H N MR (400 MHz, CDCI3): 5 8.12 (s, 1 H), 7.69 - 7.56 (m, 4H), 7.48 (t, J = 7.7 Hz, 1 H), 7.41 (t, J = 1.7 Hz, 1 H), 7.37 (d, J = 4.9 Hz, 2H), 7.30 (t, J = 8.0 Hz, 3H), 7.25 - 7.21 (m, 1 H), 7.20 - 7.13 (m, 3H), 7.07 (ddd, J = 8.0, 7.0, 1.1 Hz, 1 H), 5.90 (s, 1 H), 5.87 (s, 1 H), 5.09 (d, J = 8.0 Hz, 1 H), 4.55 (d, J = 7.5 Hz, 1 H), 4.17 (q, J = 10.3, 9.4 Hz, 2H), 4.01 (s, 1 H), 3.44 - 3.37 (m, 1 H), 3.37 - 3.28 (m, 1 H), 2.47 (s, 3H), 2.46 (s, 3H), 1.38 (s, 3H), 1.35 (s, 3H); 13C NMR (100 MHz, CDCI3): δ 174.8, 163.1 , 156.0, 155.9, 143.9, 143.2, 141.4, 140.7, 136.1 , 136.0, 135.1 , 133.9, 131.5, 130.1 , 129.1 , 128.8, 127.9, 127.8, 127.2, 125.2, 123.2, 121.6, 120.5, 120.1 , 119.3, 11 1.2, 108.2, 67.2, 47.2, 36.9, 28.0, 14.8, 14.7; HRMS (m/z): [M+Na]+ calcd. for C45H39BF2N404, 771.2930; found: 771.2925.
Comparing Example 2, initial arylation with Reference Example 2, the p-iodophenyl-BODIPY 1 failed to couple with the Fmoc-Trp-OH, while the m-iodophenyl-BODIPY 2 under the same reaction conditions formed the product 3. These findings are summarized in Scheme 1 below.
Figure imgf000048_0001
(not formed) 74 Scheme 1. C-H activation processes with Fmoc-Trp-OH and BODI PY derivatives 1 and 2.
Reference Example 3
Procedure for the preparation of linear reference peptide 4 H-Arg-Lys-Lys-Trp-Phe-Trp-NH2 (4). Reference peptide, synthesized as described in Morisse, H. et al, "In vivo molecular microimaging of pulmonary aspergillosis", Med. Mycol. 51 , 352-360 (2013).
White powder (5.0 mg).
Characterisation data: HPLC: tR: 3.37 min (95% purity); HRMS (m/z): [M+H]+ calcd. for C49H67N1307, 950.5365; found: 950.5364.
Example 3
General procedures for the preparation of linear peptides 5-7 (structures shown in Table 1 below) Syntheses were performed on Sieber amide resin (0.69 mmol/g). Amino acid 3 (1 .5 eq.) was incorporated with a 5-min pre-activation using DIC (1.5 eq.) and OxymaPure (1 .5 eq.) in DM F for 1 h.
For Fmoc-Arg-OH (3 eq.), several treatments (up to 9) with DIC (3 eq.) and HOBt (3 eq.) in DMF for 15 min without pre-activation were performed. The rest of amino acids (Fmoc-AA-OH, 3 eq.) were incorporated with a 5-min pre-activation with DIC (3 eq.) and OxymaPure (3 eq.) in DMF for 1 h.
Peptides were purified by semi-preparative RP-HPLC (XB RIDGE™ BEH 130, Ci8, 5 μΜ OBD 19x50 mm column). Mobile phase: ACN (0.1 % HCOOH)/H20 (0.1 % HCOOH); flow rate: 20 mL/min. Pure fractions were lyophilized furnishing the corresponding peptides. H-Arg-Lys-Lys-Trp(C2-BODIPY)-Phe-Trp-NH2 (5).
Red powder (3.6 mg).
Characterisation data: HPLC: tR: 3.28 min (90% purity); HRMS (m/z): [M+H]+ calcd. for C68H85BF2N1606, 1271.6977; found: 1271 .6947.
H-Arg-Lys-Lys-Trp-Phe-Trp(C2-BODIPY)-NH2 (6). Red powder (1.8 mg).
Characterisation data: HPLC: tR: 3.95 min (90% purity); HRMS (m/z): [M+H]+ calcd. for C68H85BF2N1606, 1271.6977; found: 1271.6937.
H-Arg-Lys-Lys-Trp(C2-BODIPY)-Phe-Trp(C2-BODIPY)-NH2 (7). Red powder (16.8 mg).
Characterisation data: HPLC: tR: 6.24 min (91 % purity); HRMS (m/z): [M+H]+ calcd. for C87Hio3B2F4N1806, 1593.8449; found: 1593.8532.
Example 4 Procedure of the preparation of cyclic peptide 8 (structure shown in Table 2 below)
Cyclo(Arg-Lys-Lys-Trp(C2-BODIPY)-Phe-Trp-Gly) (8). The synthesis was performed on 91 mg of 2-chlorotrityl polystyrene resin (0.94 mmol/g). Amino acid 3 (1.5 eq.) was incorporated with PyBOP (1.5 eq.), HOBt (1.5 eq.) and DIPEA (2.0 eq.) in DMF for 1 h. Other Fmoc amino acids (3 eq.) were incorporated with a 5 min pre-activation with DIC (3 eq.) and OxymaPure (3 eq.) in DMF for 1 h. After cleavage as described above, the protected linear peptide (119 mg, 0.073 mmol) was dissolved in 1.3 ml_ of DMF (0.055 M). DIPEA (2.5 eq., 32 μΙ_, 0.182 mmol) and HATU (1.0 eq., 28 mg, 0.073 mmol) were added. The solution was stirred at r.t. until the cyclisation was complete (approx. 2 h). The cyclic peptide was precipitated by adding H20 to the solution. The precipitate was washed with H20, decanted and dried, obtaining 109 mg of crude protected cyclic peptide (87% yield). The crude protected macrocycle (108 mg, 0.067 mmol) and 20% Pd(OH)2-C (54 mg) were dissolved in 5% HCOOH/MeOH (10.8 ml_), previously purged with Ar. Then, the reaction flask was flushed again with Ar, evacuated and filled with H2. The reaction mixture was stirred under H2 for 72 h (H2 balloons were refilled periodically during the reaction along with re-addition of Pd(OH)2-C (4 times). The catalyst was removed by filtration and the filtrate was evaporated to afford 46 mg of the crude deprotected peptide. The final peptide was purified by PoraPak Rxn RP 60 cc reverse phase column. Mobile phase: ACN (0.1 % HCOOH)/H20 (0.1 % HCOOH). Pure fractions were lyophilized furnishing the corresponding peptide. Red powder (30 mg).
Characterisation data: 1 H NMR (600 MHz, CD3OD): δ 8.55 (s, 3H), 7.89 - 7.84 (m, 1 H), 7.71 (t, J = 7.7 Hz, 1 H), 7.65 (d, J = 7.8 Hz, 1 H), 7.60 - 7.55 (m, 2H), 7.42 (dt, J = 8.1 , 0.9 Hz, 1 H), 7.40 - 7.36 (m, 2H), 7.18 - 7.12 (m, 5H), 7.1 1 (s, 1 H), 7.06 (m, 3H), 6.94 (m, 1 H), 6.10 (s, 1 H), 6.06 (s, 1 H), 4.45 (t, J = 7.4 Hz, 1 H), 4.32 (d, J = 10.5 Hz, 1 H), 4.25 (m, 1 H), 4.17 - 4.07 (m, 2H), 4.04 (d, J = 16.5 Hz, 1 H), 4.00 (m, 1 H), 3.63 (dd, J = 14.9, 9.5 Hz, 1 H), 3.51 - 3.45 (m, 1 H), 3.38 (m, 1 H), 3.30 (1 H), 3.27 - 3.23 (m, 2H), 3.15 (dd, J = 14.2, 7.5 Hz, 1 H), 3.10 (dd, J = 14.0, 5.9 Hz, 1 H), 2.90 (td, J = 8.6, 4.2 Hz, 2H), 2.72 (t, J = 7.7 Hz, 2H), 2.63 (dd, J = 14.2, 8.5 Hz, 1 H), 2.51 (s, 3H), 2.50 (s, 3H), 2.09 - 1.88 (m, 4H), 1.76 (m, 1 H), 1.67 (m, 3H), 1.52 (m, 7H), 1.48 - 1.41 (m, 4H), 1.37 (m, 2H), 1.26 - 1.14 (m, 2H);
HPLC: tR: 4.45 min (98% purity); HRMS (m/z): [M+H]+ calcd. for C7oH86BF2N1607, 1311.6926; found: 1311.6864.
Methods
Synthetic procedures and chemical characterisation of all the probes are described below.
In vitro characterisation: spectral measurements, IC50 determination, antimicrobial and haemolytic activity. Spectral measurements. Spectroscopic and quantum yield data were recorded on a Synergy HT spectrophotometer (Biotek). Compounds were dissolved at the indicated concentrations and spectra were recorded at r.t. Spectra are represented as means from at least two independent experiments with n=3. Quantum yields were calculated by measuring the integrated emission area of the fluorescence spectra and comparing it to the area measured for fluorescein basic ethanol as reference (QY: 0.97).
IC50 determination. The A. fumigatus strain CEA10 was grown on Vogel's medium at 37 °C for 5 days before the spores were harvested. Peptides 4-8 were incubated at different concentrations with A. fumigatus conidia to reach a final volume of 100 μΙ_ per well. The final conidia concentration was 5 χ 105 cells/mL in 10% Vogel's medium. After 24 h incubation at 37 °C in 96 well-plates, fungal growth was determined by measuring OD6ionm in a
spectrophotometer. IC50 values were determined using four parameter logistic regression. Data is represented as means ± SEM from two independent experiments with n=3.
Antimicrobial activity. Bacteria were grown on Lysogeny Broth (LB) agar plates and stored at 4 °C. For assays, a single colony of bacteria was taken into 10 mL liquid broth and incubated at 37 °C for 16 h. Cultures were centrifuged at 4,000 rpm for 5 min and the pellet was re-suspended in 1 mL of fresh phosphate buffered saline (PBS) and washed three times. Cultures were reconstituted to 1.0 OD595nm, then diluted 1 : 1000 and incubated with compounds 4-8 at the indicated concentrations. Cell viability was monitored over 18 h by measuring OD595nm in a spectrophotometer. Data is represented as % of cell viability as means from two independent experiments with n=3.
Haemolytic activity. Erythrocytes were isolated from freshly drawn, anticoagulated human blood and re-suspended to 20 vol. % in PBS. 100 μΙ_ of erythrocyte suspension was added to 100 μΙ_ of compounds 4-8 at the indicated concentrations. 0.2% Triton X-100 was used as positive control and PBS as negative control. The plate was incubated at 37 °C for 1 h, each well was diluted with 150 μΙ_ of PBS and the plate was centrifuged at 1 ,200 g for 15 min. 100 μΙ_ of the supernatant from each well was transferred to a fresh plate, and the absorbance at 350 nm was measured in a microplate reader. Data is represented as % of cell viability as means from three independent experiments with n=3.
Live cell confocal microscopy in co-cultures of A. fumigatus and human epithelial cells.
Human lung A549 epithelial cells were grown using Dulbecco's Modified Eagle Medium (DM EM) supplemented with 10% fetal bovine serum (FBS), antibiotics (100 U ml_"1 penicillin and 100 mg ml_"1 streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5% C02. Cells were regularly passaged in T-75 cell culture flasks. For co-cultures, human lung epithelial cells were plated on glass chamber slides Lab-Tek™ I I (Nunc) two days before imaging and incubated for 16 h with A. fumigatus conidia reaching 75% to 90% confluence on the day of the experiment. For imaging experiments, cells were incubated at 37 °C with compounds 5-8 for 10 min unless otherwise stated, and imaged without washing in phenol red-free DMEM under a Zeiss LSM 510 META fluorescence confocal microscope equipped with a live cell imaging stage. Fluorescence and bright field images were acquired using 40X or 63X oil objectives. Fluorescent probes were excited with 488 nm (compounds 5-8) or 543 nm (Syto82) lasers. All images were analysed and processed with ImageJ. Chemical stability assays in human bronchoalveolar lavage.
Peptides 5 and 8 (20 μΜ) were dissolved in human bronchoalveolar lavage samples (total volume: 100 μΙ_) and incubated at 37 °C for the indicated times. Samples were injected into an HPLC Agilent 1 100 separations module connected to a UV detector with a Discovery Ci8 column (5 μηι, 4.6 χ 50 mm). MALDI data was recorded on a Bruker Ultraflex mass spectrometer using sinapinic acid as the matrix.
Multi-photon and lifetime imaging of A. fumigatus in ex vivo human pulmonary tissue.
Ex vivo human lung tissue experiments were approved by the N HS Lothian Tissue
Governance Committee and Regional Ethics Committee (REC reference: 13/ES/0126). Human lung tissue was obtained from the periphery (non-cancerous) region of patients undergoing resection for lung cancer. 1 cm3 tissue was inflated with optimum cutting temperature (O.C.T.) formulation and stored at -80 °C. Embedded tissue was cryo- sectioned at 10 micron intervals and fixed onto glass slides for imaging. RFP-expressing A. fumigatus conidia were grown overnight at 37 °C the day before the experiments and incubated with human lung tissue sections for 2-3 h hours before imaging.
A custom-built multi-photon microscope was used to acquire second harmonic generation (SHG) and two-photon fluorescence images. Briefly, a picoEmerald (APE) laser provided both a tunable pump laser (720-990 nm, 7 ps, 80 MHz repetition rate) and a spatially overlapped Stokes laser (1 ,064 nm, 5-6 ps, 80 MHz repetition rate). GFP two-photon fluorescence signals were filtered using the following series of filters: FF520-Di02,
FF483/639-Di01 and ET500/40m. RFP two-photon fluorescence signals were filtered using FF520-Di02, FF757-Di01 and FF01-609/181 , and SHG signals were filtered using FF520- Di02, FF483/639-Di01 and FF01 -466/40.
Fluorescence lifetime images were acquired by connecting the relevant detector to a PicoHarp 300 (Picoquant, Berlin) and configuring the PMT for photon counting mode for TCSPC-FLIM. SHG and GFP images were taken with the laser tuned to 950 nm and RFP images were recorded using a 1 ,064 nm laser. Lifetime images were recorded at 20 mW with a 10 με pixel dwell using the SymPhoTime software (Picoquant). All images were analysed and processed using ImageJ.
Example 5
In vitro imaging of A. fumigatus in co-cultures with human epithelial cells.
Peptides 5-7, the structures of which are shown below, exhibited similar spectral properties (Figure 1 a) to 3 with an equally strong fluorogenic behaviour in phospholipid membranes (Figure 1 b). Double-labelled peptide 7 displayed a weaker fluorescence response than mono-labelled peptides (5,6), partially due to the self-quenching derived from two neighbouring BODIPY fluorophores.
In view of the excellent properties of 5-7 as fungi-targeting fluorogenic peptides, their potential as live cell imaging agents of A. Fumigatus were evaluated. Peptides 5 and 6 brightly stained fungal cells, whereas 7 showed significantly weaker fluorescence, in accordance with its lower fluorogenicity (Figure 1 b).
Peptide 5 was further used to image live A. fumigatus in co-cultures with human lung epithelial cells. The fluorogenic properties of 5 enabled direct live fungal cell imaging without the need of any washing steps. Furthermore, lung epithelial cells were counterstained with the red fluorescent dye Syto82 (the spectral characterisation of which is shown in Figure 1 c) and plot profile analysis was performed to confirm that 5 specifically labelled A. fumigatus without staining human lung epithelial cells.
Table 1 shows the IC50 in Aspergillus Fumigatus the antimicrobial and haemolytic activity of peptides 5-7 determined as described in the methods section above.
Table 1. Affinity for fungal, bacteria and human cells ia PseyifcKSftas Human
Figure imgf000054_0001
;-: iimi ftiSic s- wsis hydrophobic domain
R,: H, ¾: BOOIPV (5)
Rl.' 8G0iPY, R;.;-. H (6)
SODiPV (7)
Example 6
Optimisation of a fluorogenic probe for direct ex vivo imaging of fungal infection in human tissue.
Direct tissue imaging of infection sites is often hampered by the high concentration of proteolytic enzymes, which can compromise the integrity of imaging agents. Hence, we decided to examine the chemical stability of peptide 5 in human bronchoalveolar lavages from patients with acute respiratory distress syndrome (ARDS) to assess the potential for ex vivo human tissue imaging. The linear peptide 5 was rapidly degraded in human lavages with a half-life shorter than 60 min (Figures 2a, 2b and 2c). In Figure 2b, peptides were incubated in human bronchoalveolar lavage samples at 37 °C and analysed by HPLC at the time points indicated. Arrows point at the peaks corresponding to the intact linear peptide. In order to enhance the stability required for direct ex vivo imaging in human pulmonary tissue, cyclic peptide 8 was synthesised as the corresponding BODIPY-labelled cyclic analog. Cyclic peptides do not contain free N- and C-terminal groups, leading to increased resistance to degradation by proteases. Compound 8 was synthesised as described in Example 4 using 2-chlorotrityl polystyrene resin, which enabled the preparation and subsequent cleavage of the protected linear peptide under mild acidic conditions (Scheme 2).
CTC-PS resin
(0.94 rnrnoi/g)
J SPPS
Figure imgf000055_0001
H-Arg(N02)-Lys(Z)-Lys(Z)-Tr (C2-BODIPy)-Phe-Tr -Giy-OH i) cyclisation:
HATU {1.0 eq), DiEA (2.5 eq)
DMF, r.t ii) removal protecting groups:
H2, Pd(OH)2-C (20%)
!f 5% HC02H/MeO r.t, 72 h
cyclo{-Arg~Lys-Lys-Trp(C2-BOD!PY}~Phe-Trp-Gly-) Scheme 2. Schematic synthesis of cyclic peptide 8
Head-to-tail cyclisation was performed in solution with 87% yield using HATU as the coupling reagent. We optimised the reaction conditions to remove all the protecting groups without affecting the BODIPY scaffold. Reduction of the protected peptide in H2 atmosphere with Pd(OH)2-C using mild acidic conditions led to the desired product with yields around 60% and purities over 90%. The peptide 8 showed around 2-fold enhanced affinity for fungal cells compared to peptide 5, and maintained very high selectivity over bacteria and human cells (Table 1). This observation is in line with the fact that peptide cyclisation can restrict conformational flexibility, which often leads to enhanced affinity and activity. NMR analysis of 8 showed no evidence of relevant structural modifications with respect to the parent unlabeled peptide, in agreement with molecular simulations. Importantly, the peptide 8 remained intact after 24 h in human bronchoalveolar lavages from patients with ARDS (Table 2, Figures 2a, 2b and 2c). Peptides were incubated in human bronchoalveolar lavage samples at 37 °C and analysed by HPLC or MALDI at the time points indicated in Figures 2b and 2c respectively. The peptide 8 also displayed stronger fluorogenic response than the linear peptides (5, 6) and remarkable fluorescence emission in phospholipid bilayer membranes in PBS (Aexc.: 450 nm), with quantum yields reaching 30% (Figures 3a and 3b). Quantum yields were determined using fluorescein in basic ethanol as reference according to P. G. Seybold, M. Gouterman, J. Callis. Photochem. Photobiol., 1969, 9, 229-242.
It was also confirmed that the peptide 8 brightly selectively stained A. fumigatus in co- cultures with human lung epithelial cells (Figure 3c). Peptides 5, 6, and 8 were incubated in PBS alone or in suspensions of A. fumigatus in PBS (Aexc.: 485 nm; Aem-: 515 nm). Values are represented as means ± SD from two independent experiments with n=3. ** for p < 0.01 and *** for p < 0.005 were determined as statistically significant differences between the fluorescence emission values in PBS and in suspensions of A. fumigatus in PBS.
These results validate 8 as a fluorogenic peptide with high stability in samples from patients with multi-system respiratory disease and potential for direct ex vivo imaging of A. fumigatus in human pulmonary tissue.
Table 2. Proportion of peptide remaining in human bronchoalveolar lavages from patients with ARDS over time
Figure imgf000056_0002
Figure imgf000056_0001
Example 7
Ex vivo direct imaging of A. fumigatus in human pulmonary tissue. Peptide 8 was also employed for high-resolution imaging of A. fumigatus. Time-lapse imaging showed the fluorogenic response of 8 upon interaction with the fungal cell membrane and after being internalised and accumulated in lipid-rich intracellular compartments (Fig 3a). The kinetic analysis shows that the peptide 8 labelled fungal cells very rapidly, within few minutes after addition of the probe and without requiring any washing steps. Moreover, the peptide 8 showed no cytotoxicity in lung epithelial cells, even at high concentrations (Figure 4a). The viability of human lung A549 epithelial cells was determined after incubation with different concentrations of the peptide 8. Values are represented as means ± SD from two independent experiments with n=4. No significant differences (p > 0.05) were determined between the control and any of the treatments.
In view of these properties, peptide 8 was employed for direct imaging of A. fumigatus in human pulmonary tissue using multi-photon microscopy. In order to confirm the specific staining of 8, a transgenic strain of A. fumigatus was employed expressing red fluorescent protein (RFP) in the cytoplasm. As shown in Fig 4b, the peptide 8 (green) clearly stained RFP-expressing A. fumigatus (red), which confirmed the selectivity of the probe.
Furthermore, multi-photon excitation enabled the acquisition of second harmonic generation (cyan) from the collagen structures of the fibrillar network of human pulmonary tissue (Fig 4b C and D). Further examination of the tissue samples by fluorescence lifetime imaging revealed that 8-stained A. fumigatus and tissue structures could be readily distinguished by their fluorescence lifetimes (Fig 4). This feature is important to distinguish the signal from autofluorescence signals of the tissue could potentially overlap with the emission of BODIPY fluorogens. Altogether, these results validate fluorogenic BODIPY-labelled cyclic peptide 8 as a highly stable imaging agent for direct and straightforward visualisation of A. fumigatus in human pulmonary tissue.
In conclusion, fluorogenic Trp-BODIPY derivative (3) was developed with high extinction coefficients and significant fluorescence enhancement in hydrophobic microenvironments. Compound 3 is fully compatible with SPPS and was used to prepare fluorogenic analogs of PAF26, a short antifungal hexapeptide, as imaging agents for the fungal pathogen A.
fumigatus. Notably, the spacer-free C-C linkage of the BODIPY fluorogen to the exposition of Trp did not impair the affinity and selectivity properties of PAF26 derivatives while providing a suitable imaging reporter. Further optimisation yielded the peptide 8 as a highly fluorogenic cyclic structure with bright fluorescence emission in fungal cells and high chemical stability in human bronchoalveolar lavages from patients with ARDS. Multi-photon imaging with the peptide 8 has enabled direct visualisation of A. fumigatus in ex vivo human tissue. In view that the amino acid 3 can be readily incorporated and has general applicability to both linear and cyclic peptides, we envisage that the site-specific introduction of this spacer-free BODIPY fluorogen at strategic positions in relevant peptides will enable the development of novel imaging peptide tools with high sensitivity and specificity.
Example 8
Stability studies of Trp-BODIPY derivatives
Fmoc-Trp(C2-BODIPY)-OH (3) or the BODIPY-labelled linear precursor (12) of the cyclic peptide (8) in which the N-protecting group Z is Cbz (benzyloxycarbonyl) were dissolved in different solutions of trifluoroacetic acid (TFA) and the resulting solutions analysed by HPLC (UV detection) at the time points indicated. The results are shown in Figure 5 in which the conditions are as follows: a) TFA:DCM (1 :99), 10 min, r.t. b) TFA:DCM (1 :9), 30 min, r.t. c) crude (12), a BODIPY-labelled linear precursor of the cyclic peptide (8) directly after cleavage with TFA:DCM (1 :99), r.t.
The long-term stability of the fluorogenic probe was tested both in the solid and solution state at various temperatures. Prolonged stability would be advantageous for a facile and safe shipping, as well as ensuring chemical integrity of Fmoc-Trp(C2-BODIPY)-OH (3) during long-term storage. Solution-phase chemical stability was assessed in solvents typically used in peptide synthesis such as dichloromethane (DCM), A/,A/-dimethylformamide (DMF) and methanol (MeOH). Three temperature values were considered in the study: room temperature (r.t.), 4°C and -20°C, relevant for both shipping and storage (shelf, fridge and freezer) purposes.
For solid-state studies, 5 mg of the amino acid were weighed in a glass vial covered with aluminium foil and left at the corresponding temperature. For monthly HPLC analysis, an aliquot was taken, dissolved in MeOH (50 μΙ) and 30 μΙ_ were injected in HPLC-MS using a 0-100% gradient of CH3CN/0.1 % formic acid over H2O/0.1 % formic acid in 8 min at flow 1.0 ml/min, with detection and integration at 500 nm. Regarding solution-phase studies, 30 mL of a 100 μΜ solution of the fluorogenic amino acid in the corresponding solvent was prepared (3 μηιοΙ, 2.2 mg). This solution was distributed in 3 separate 15-mL Falcon tubes and stored at various temperatures detailed above, wrapped in aluminium foil. Solutions were analyzed monthly by HPLC-MS analysis, injecting 20 μΙ of the given solution in the abovementioned HPLC conditions. Percentages of remaining Fmoc-Trp(C2-BODIPY)-OH (3) were detected by integration at 500 nm as shown in Table 3. Whereas the fluorogenic amino acid was shelf stable as a solid and as DCM and MeOH solution, progressive loss of Fmoc (and subsequent formylation sometimes) was observed in DMF. After 2 months, only 55% of the protected amino acid remained at room temperature in DMF, although at 4°C Fmoc removal took place in a much slower rate and was negligible at -20°C.
Table 3. STABILITY DATA FOR THE AMINO ACID FMOC-TRP-BODIPY-OH
Solid DCM DMF MeOH rt j 4°C -20°C rt 4°C j-20°C rt j 4°C |-20°C rt 4°C j -20°C t=0 97% 97% 97% 98% 98% 98% 98% 98% 98% 98% 98% 98% t= 1 96% 96% 96% 96%a 98% 98% 87%B 97%¾ 98% 98% 98% 98% month
t= 2 96% 96% 96% 98% 98% 55%B 95% 98% 97% 98% 98% months 3 Solvent evaporated after 1 week leaving a pure solid.
b Partial loss of Fmoc was observed as the main side-product.
Example 9
Live fungal cell imaging
Neurospora crassa (strain 74-OR23-1V A, source: FGSC 2489) was grown on standard Vogel's agar at 25°C under constant artificial light for 5 days. Conidia were collected using sterile dH20 and then diluted in 20% Vogel's liquid medium for imaging. Fusarium oxysporum (strain 4287, source: FGSC 9935) was grown in liquid potato dextrose broth (PDB) at 28°C with shaking. Conidia were resuspended in 20% Vogel's liquid medium and imaged after incubation for 12 h at 30 °C. Candida albicans (strain SC5314, source: ATCC MYA-2876) was grown on yeast peptone dextrose (YPD) liquid medium at 30 °C for 12 h with shaking and then diluted using minimal medium (0.7% yeast nitrogen base plus 2% glucose) before imaging. Cryptococcus neoformans (strain H99, source: FGSC 9487) was grown on YPD agar at 30 °C for 3 days. To collect the cells for imaging, a single colony 1 -2 mm in diameter was resuspended in PBS and washed once with fresh PBS before imaging. Compound 8 was used at 2 μΜ for all fungi except C. albicans for which 10 μΜ was used. Fluorescence and bright field images were acquired under a confocal microscope at r.t. and are shown in Figure 6. The four images shown as Figure 6A were fluorescence images while the four images shown as Figure 6B are the corresponding bright field images. The images were acquired under a confocal microscope at r.t. Scale bars: C. albicans: 5 μηι; C. neoformans: 7.5 μηι; F. oxysporum and N. crassa: 10 μηι.
Example 10
Characterisation of the amino acid 3 and peptides 5a, 5b and 9 for live cell imaging of A. fumigatus
Table 4 a) shows the chemical structures of fluorogenic linear (5a, 5b) and cyclic peptides (8, 9). Table 4 b) shows the activity in A. fumigatus, several bacterial strains and in human red blood cells (RBCs).[1] IC50 (μΜ) values as means ± s.e.m. (n=3), [2] cell viability upon 16 h incubation with 3, 5a, 5b or 9 at their respective IC50 concentrations or 20 μΜ for compounds 3 and 5a (n=3), [3] cell viability upon 1 h incubation with 3, 5a, 5b or 9 at their respective IC50 concentrations or 20 μΜ for compounds 3 and 5a (n=3). Table 4 Chemical structures and activit in A. fumigatus
Figure imgf000060_0001
Aspergillus Klebsiella Escherichia Pseudomonas Human
fumigatusm pneumoniae^} coH[2] aemgi'nosa[2j RBCs[3]
3 > 50 94% gg% >98% 99%
5a > S0 99% 87% 99%
5b 4.6 ± 0.4 93% >99% 34% 99%
9 7.7 ± 0,7 97% >99% 84% 99%
Example 11
Imaging of apoptotic neutrophils
Apoptotic human neutrophils or monocytes obtained from human peripheral blood were plated in a 96-well round bottom plate on ice (300.000 cells/well). Cells were pelleted down by spinning at 4° C for 3 min, the supernatant was flicked out and the plate was vortexed to resuspend the cells in residual liquid. Different probes were dissolved in PBS (for Ca2+-free conditions) or in HEPES with CaCI2 (for Ca2+ containing media at a final concentration of 2 rtiM), added to their respective wells and incubated at 4° C for 20 min. EDTA (final concentration 2.5 mM) was added to chelate any residual Ca2+ when appropriate. After incubation, cells were resuspended again by spinning, flicking out the supernatant, vortexing the plate and finally diluting in 300 μΙ_ the final cell suspension of every well in a separate tube. Data acquisition was carried out using a FACSCalibur flow cytometer (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
Figure 7 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V- AF647and APOGREEN [cyclic peptide (14)] in buffer containing 2 mM CaCI2. APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils to a similar extent than ANNEXIN V.
Figure 8A shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and APOGREEN [cyclic peptide (14)] in the absence of Ca2+. Unlike ANNEXIN V, APOGREEN [cyclic peptide (14)] binding does not depend on Ca2+. Figure 8B shows that APOGREEN [cyclic peptide (14)] stains apoptotic neutrophils in the presence of Ca2+, and as shown in Figure 8C, even in the presence of 2.5 mM EDTA.
Figure 9 shows a comparison of the binding of ANNEXIN V in the presence and absence of Ca2+ and APOGREEN [cyclic peptide (14)] in the presence and absence of EDTA to monocytes, together with a phosphate buffered saline (PBS) control. Unlike ANNEXIN V, APOGREEN [cyclic peptide (14)] does not bind to monocytes, therefore its major target is unlikely to be phosphatidylserine.
Figure 10 shows a comparison of the staining of apoptotic neutrophils between ANNEXIN V and linear peptides 1-3 [corresponding to compounds (5), (6) and (7)] derived from APOGREEN [cyclic peptide (14)]. Derived peptides of APOGREEN can bind to apoptotic neutrophils to different extent. The cyclic peptide shows better discrimination between viable and apoptotic neutrophils.
Figure 17 shows a plot of fluorescence for binding of EV-GREEN [cyclic peptide (10)] to phosphatidylserine (PS) in phosphate buffered saline (PBS) in the absence of Ca2+ (-Ca), in the presence of Ca2+ (+Ca) and in the presence of EDTA (+EDTA) together with the corresponding blanks of phosphate buffered saline in the absence of Ca2+, PBS(-Ca), in the presence of Ca2+,PBS (+Ca) and in the presence of EDTA, PBS (+EDTA). Figure 17 shows binding of EV-GREEN [cyclic peptide (10)] to PS is independent of Ca2+, unlike ANNEXIN V. ns: non-statistical difference; *** for p values <0.001.
Figure 19 shows a comparison of relative fluorescence for phosphate buffered saline (PBS), ANNEXIN V in the presence of Ca2+ (+Ca), ANNEXIN V in the absence of Ca2+ (-Ca) and EV-GREEN [cyclic peptide (10)] in the presence of intact apoptotic cells. EV-GREEN [cyclic peptide (10)] does not bind to intact apoptotic cells (i.e. UV-induced apoptotic BL2 cells). ANNEXIN V is used as a positive control for apoptosis. Example 12
Kinetic studies and staining of apoptotic and viable human BL2 cells
Human Burkitt's lymphoma (BL2) cells were induced into apoptosis by UV dose radiation (300 mJ/ cm2) and subsequent culture for a few hours at 37 0 C. Compounds were dissolved in PBS (for Ca2+-free conditions) or in HEPES with CaCI2 (for Ca2+ containing media at a final concentration of 2 rtiM), added to their respective wells and incubated at 4 °C for 20 min. After incubation, cells were resuspended again by spinning, flicking out the supernatant and finally diluting in 300 μΙ_ the final cell suspensions in separate tubes. Data acquisition was carried out using FACSCalibur or BD LSRFortessa flow cytometers (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
Figure 11 shows the results of kinetic studies of apoptotic and viable human BL2 cells with APOGREEN [cyclic peptide (14)] over time. Figure 1 1 A shows the staining of non-apoptotic and apoptotic BL2 cells with APOGREEN [cyclic peptide (14)] over time, while Figure 11 B shows the fluorescence of the non-apoptotic and apoptotic BL2 cells in the presence of APOGREEN [cyclic peptide (14)] over time. It is apparent that APOGREEN [cyclic peptide (14)] binds rapidly to apoptotic cells and reaches saturation within minutes.
Figure 12A shows the staining of apoptotic neutrophils, specifically human BL2 (B-lymphoma cells) by APOGREEN [cyclic peptide (14)] and ANNEXIN V. Example 13
Imaging of A549 human epithelial lung cancer cells
Human lung epithelial (A549) cells were grown using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), antibiotics (100 U ml_"1 penicillin and 100 mg ml_"1 streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5% C02. Cells were regularly passaged in T-75 cell culture flasks, and plated on glass chamber slides Lab-Tek™ II (Nunc) the day before imaging, reaching 60% to 90% confluence on the day of the experiment. For induction of apoptosis, A549 cells were treated with hTNF-a (10 ng/mL) and flavopiridol (500 nM) in MEM with 0.5% FBS for 6 h. Cells were incubated at 37 °C with fluorescent probes for 20 min and imaged under an epifluorescence EVOS microscope or a confocal Zeiss LSM 510 Meta fluorescence microscope equipped with a live cell imaging stage. Cell images were acquired using 40X (EVOS) or 63X oil (ZEISS) objectives. Fluorescent probes were excited with suitable lasers: 488 nm (compound 14) and 633 nm (ANNEXIN V-AF647). Images were acquired with the corresponding microscope software and processed and analyzed with ImageJ.
Figure 12B shows a comparison of the staining of apoptotic neutrophils, specifically A549 human epithelial lung cancer cells, by APOGREEN [cyclic peptide (14)] and ANNEXIN V. Figure 15 shows bright field and fluorescence images of APOGREEN [cyclic peptide (14)] and ANNEXIN V-AF647 co-staining in A549 cells after chemical induction of apoptosis. Only ANNEXIN V-AF647 stained cells are APOGREEN-positive, with no staining in viable cells. APOGREEN shows intracellular staining, potentially correlated to binding to negatively charged phospholipids.
Example 14
Imaging of macrophages
Macrophages were treated with dexamethasone in order to induce phagocytosis, and incubated or not with compound 14 for 20 min. Afterwards, macrophages were treated with the commercial dye pHRODO for staining of the phagocytic population. Data acquisition was carried out using FACSCalibur or BD LSRFortessa flow cytometers (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
Figure 13 shows the phagocytic activity of macrophages as indicated by the staining with the commercial dye pHRODO. As reported, macrophages are more phagocytic upon activation with dexamethasone (B, D). APOGREEN [cyclic peptide (14)] does not impair the phagocytosis levels of macrophages (A vs C, B vs D), which is important to ensure clearance of apoptotic neutrophils at activation sites. Example 15
Imaging of apoptotic neutrophils
Mice were nebulised in a chamber with LPS (1 mg/mL) and then 24 hours later instilled with compound 14 (2.5 μΜ in PBS) or vehicle (equal amount of DMSO in PBS) by intratracheal instillation. After 30 min, mice were sacrificed and cells were lavaged from the lungs. A fraction of the cells was cytospined and stained for visual inspection under microscopy to determine cell numbers and the ratio of dead/live cells. Lavaged cells were also counterstained with fluorescent ANNEXIN V and/or TOPRO dyes and analysed by flow cytometry. Data acquisition was carried out using FACSCalibur or BD LSRFortessa flow cytometers (BD Biosciences) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).
Figure 14 shows that APOGREEN [cyclic peptide (14)] can label apoptotic neutrophils in vivo in the lungs of mice after intratracheal instillation (Figure 14C right plot), with no toxicity and comparative results as obtained from ex vivo ANNEXIN V staining (Figure 14C left plot). Graph A shows the total cell number obtained from lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)]. Graph B shows the proportion of live versus dead cells in lavages of mice that have been treated with the vehicle only or with APOGREEN [cyclic peptide (14)]. Example 16
Imaging of phosphatidylserine and phosphatidylcholine
A 5 μΜ solution of EV-Green [cyclic peptide (10)] in PBS was incubated with lipid films bearing different phosphatidylcholine (PC): phosphatidylserine (PS) ratios, from PC-only (100:0) to PS-only (0:100) in a 96-well plate. Samples were irradiated at 450 nm and fluorescence spectra were recorded in a spectrophotometer using PBS as a negative control. EV-Green [cyclic peptide (10)] displayed dramatic fluorescence enhancement at 514 nm with lipid films containing increasing PS-content, proving the enhanced affinity of the probe for PS (Fig. 16a and Fig. 16b). Quantum yields were determined using fluorescein in basic ethanol as the reference (QY: 0.97). Quantum yields for experiments with PS-only films and PBS as blank were 0.46 and 0.08 respectively.
Figure 16A shows a plot of fluorescence intensity versus wavelength for EV-GREEN [cyclic peptide (10)] upon incubation with various ratios of phosphatidylserine (PS) to phosphatidylcholine (PC). Figure 16A shows specific fluorescence increase upon incubation with increasing phosphatidylserine (PS) content. Figure 16B shows quantification of the fluorescence increase with phosphatidylserine (PS) over other lipids (PC: phosphatidylcholine). Fluorescence quantum yields (100% PS): 0.46 vs in blank (phosphate buffer saline, PBS): 0.08. *** for p values O.001.
Example 17
Imaging of permeabilised necrotic cells
Flow cytometry experiments were conducted with EV-Green [cyclic peptide (10)] and apoptotic human neutrophils using the protocol described for Fig. 17 in Example 11. The procedure also included a treatment with fix-lyse solution to induce cell membrane perforation and subsequent necrosis. Neutrophils were incubated at various EV-Green [cyclic peptide (10)] concentrations ranging from 0.16 to 5.0 μΜ in PBS at 4 C for 20 min.
Figure 18 shows a comparison of relative fluorescence for phosphate buffered saline (PBS) and EV-GREEN [cyclic peptide (10)] in the presence of either viable cells (PBS) or fixed and permeabilized neutrophils (Fix/Lyse solution). This shows that EV-GREEN [cyclic peptide (10)] does not bind to viable cells. EV-green fluoresces brightly when cells are fixed and permeabilized (i.e. treated with Fix/lyse solution). Fig. 18 shows that EV-Green [cyclic peptide (10)] stains permeabilised necrotic cells. Example 18
Affinity of EV-GREEN [cyclic peptide (10)] for lipid monolayers
In order to gain further insight into the biomolecular interactions between EV-Green [cyclic peptide (10)] and different phospholipids, a physicochemical analysis of the affinity of EV- Green [cyclic peptide (10)] for various lipid monolayers was performed. First, the tensioactive potential of EV-Green [cyclic peptide (10)] was determined, which can give insight into the affinity of the probe for phospholipids. Peptides were incubated at various concentrations in an aqueous subphase consisting of 1.25 ml of 25 mM HEPES, 150 mM NaCI (pH 7.4). Tensioactivity was analyzed by measuring the surface pressure of the resulting lipid- aqueous mixtures, using a DeltaPi-4 (Kibron, Helsinki, Finland) at 22°C, with constant stirring. In these assays, we observed that EV-Green [cyclic peptide (10)] displays remarkable tensioactivity, and able to form monolayers in the air-water interphase with a saturation pressure (π3) of 20.4 mN m"1 and a saturation concentration of 3 μΜ. Furthermore, in a comparison of the behaviour of EV-Green [cyclic peptide (10)] with the corresponding non-labelled analogue at the same saturation concentration, EV-Green [cyclic peptide (10)] displayed stronger tensioactivity, which is a measure of the area occupied by the probe in the interphase, indicating the optimal positioning of the BODIPY fluorogen within the hydrophobic domain of the peptide. Figure 20 shows the measurement of the tensioactivity induced by EV-GREEN [cyclic peptide (10)]. EV-GREEN [cyclic peptide (10)] shows tensioactive properties; it is able to form a monolayer in the interphase air-water, with a saturation pressure (π3) of 20.4 rtiN/m and a saturation concentration (Cs) of 3 μΜ.
Example 19
Binding affinity of EV-GREEN [cyclic peptide (10)] for lipid monolayers containing phosphatidylserine
In order to quantify the binding affinity of EV-GREEN [cyclic peptide (10)] for monolayers containing phosphatidylserine, the critical pressure of EV-GREEN [cyclic peptide (10)] in lipid monolayers with different composition was determined. Langmuir lipid monolayers at the air- water interface were used as a model membrane system to study lipid-peptide interaction. Surface pressure experiments were carried out with a DeltaPi-4 (Kibron, Helsinki, Finland) at 22°C, with constant stirring. The aqueous phase consisted of 1.25 ml of 25 mM HEPES, 150 mM NaCI (pH 7.4). Lipids dissolved in chloroform:methanol (2: 1), egg phosphatidylcholine (eggPC), 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and egg phosphatidylglycerol (eggPG) were spread gently over the surface until the desired initial surface pressure was attained. The peptides, either the unlabeled peptide (Fig. 21a) or EV-GREEN [cyclic peptide (10)] (Fig. 21 b) were injected with a micropipette through a hole connected to the subphase. The increment in surface pressure was recorded until a stable signal was obtained. Values of critical pressure (TTC) were determined by linear regression using the values over the saturation pressure (TTS).
In all experiments, initial pressure (ττ,) is plotted against variation of pressure (Δττ), either in eggPC or eggPC:DOPS (70:30) monolayers. Higher TTc is observed in phosphatidylserine (DOPS)-containing monolayers for EV-GREEN [cyclic peptide (10)] indicating selective affinity for this lipid (42 mN/m vs 36.5 rtiN/m) (Fig. 21 b). Remarkably, the presence of Trp- BODIPY label dramatically increases the affinity of the unlabeled peptide for phosphatidylserine (DOPS)-containing monolayers (42 mN/m vs 30 mN/m) (Fig. 21a vs. Fig. 21 b). The analysis of EV-GREEN [cyclic peptide (10)] was extended to lipid monolayers containing other negatively-charged phospholipids, such as cardiolipin or eggPC: eggPG (70:30) (Fig. 22). Fig. 22 shows a comparison of the initial pressure (ττ,) versus variation of pressure (Δττ) of eggPC: eggPG and eggPC:DOPS (70:30) when incubated with EV-Green [cyclic peptide (10)]. EV-Green [cyclic peptide (10)] displayed an almost identical TTc (42 mN/m vs 42.4 mN/m) (Fig. 22). These experiments confirmed the strong affinity of EV- GREEN [cyclic peptide (10)] for monolayers containing negatively-charged phospholipids, which are present in high levels in apoptotic microvesicles.
Example 20 Staining of phosphatidylserine-containing vesicles by EV-GREEN [cyclic peptide (10)]
In order to assess the capabilities of EV-Green [cyclic peptide (10)] as a fluorescent probe for staining phosphatidylserine (DOPS)-containing vesicles, giant unilamellar vesicles (GUVs) were prepared and stained with EV-Green [cyclic peptide (10)] to compare the degree of fluorescence labelling by confocal microscopy (Fig. 23). Giant unilamellar vesicles (GUV) were prepared by electroformation on a pair of platinum (Pt) wires. Lipid stock solutions were prepared in 2: 1 (v/v) chloroform/methanol at 0.2 mg/ml, and appropriate volumes of each one were mixed. Labelling was carried out by pre-mixing the desired fluorescent probes with the lipids in organic solvent. GUVs containing phosphatidylcholine (eggPC)-only (Fig. 23a and 23b) or eggPC:DOPS (7:3) (Fig. 23c and 23d) were prepared, mimicking the phosphatidylserine content of apoptotic EVs. N- (Lissamine Rhodamine B sulfonyl) dioleoylphosphatidylethanolamine (Liss-Rho-DOPE) was used as marker for lipid membrane (Fig. 23a and 23c). The average concentration of individual fluorescent probes in each sample was 0.2 mol%. 2.5 μΙ lipid mixtures containing the fluorescent probes were deposited on Pt wires. The Pt wires were placed under vacuum for 2 h to completely remove the organic solvent. The sample was covered to avoid light exposure and allowed to precipitate onto the Pt wires for 5 min. One side of the chamber was then sealed with a coverslip. 500 μΙ assay buffer, prepared with high-purity water (Millipore SuperQ) heated at 37°C was added to the chamber until it covered the Pt wires and connected to a TG330 function generator (Thurlby Thandar Instruments, Huntingdon, UK). AC field was applied in three steps, all of them performed at 37°C: 1), frequency 500 Hz, amplitude 220 mV (35 V/m) for 5 min; 2), frequency 500 Hz, amplitude 1900 mV (313 V/m) for 20 min; 3), frequency 500 Hz, amplitude 5.3 V (870 V/m) for 90 min. The temperatures used for GUV formation correspond to those at which the different membranes display a single fluid phase.
GUVs were imaged with confocal microscopy using a Nikon D-eclipse C1 confocal system (Nikon corporation, Tokyo, Japan). The excitation wavelengths used for excitation were 488 nm for EV-Green [cyclic peptide (10)] and 561 nm for Liss-Rho-DOPE. Fluorescence emission was retrieved at 500-530 for EV-Green [cyclic peptide (10)] and at 573-613 for Liss-Rho-PE. When required EV-Green [cyclic peptide (10)], at 3,3 μg/ml, were added to study its effect on the GUVs. All these experiments were performed at 22° C. Image treatment was performed using the software EZ-C1 3.20 (Nikon Inc., Melville, N.Y.).
As shown in Fig. 23, EV-Green [cyclic peptide (10)] showed significantly higher staining in phosphatidylserine-containing GUVs (Fig. 23d vs 23b), in agreement with our in vitro characterization data. Altogether, these results confirmed the potential of EV-Green [cyclic peptide (10)] as a probe for the fluorescence labelling of apoptotic vesicles.
Example 21
Staining of extracellular vesicles by EV-GREEN [cyclic peptide (10)] The ability of EV-Green [cyclic peptide (10)] to stain extracellular vesicles was assessed using apoptotic vesicles from BL2 cells. In order to verify that EV-Green labels apoptotic EV via PS-binding interaction, double staining experiments with ANNEXIN V-Pacific Blue as positive control at different EV-Green [cyclic peptide (10)] concentrations were performed. Human BL2 cells were grown in serum-free cell media and induced to undergo apoptosis under UV dose radiation (300 mJ/ cm2) and re-cultured 3-6 hours post-UV. Samples were centrifuged for 10 minutes, and the supernatant (containing mainly EV and exosomes) was collected. Single stained ANNEXIN V-Pacific Blue vesicles were prepared by mixing 100 μΙ of vesicle solution with 0.5 μΙ of ANNEXIN V and 11 μΙ of concentrated Ca2+-rich binding buffer. The mixture was incubated 10 minutes at 4°C and diluted to 1 ml_ with diluted ANNEXIN V binding buffer, prior to measurement. A single EV-Green [cyclic peptide (10)]- stained sample was carried out by preparing a 2 μΜ solution of the probe with EV (100 μΙ total volume), followed by 40 min incubation at 4°C. After that time, Ca2+-binding buffer was added and diluted to 1 ml_ with diluted binding buffer (Fig. 24b). A sample containing unlabeled EV with the abovementioned dilution in Ca2+-ANNEXIN V-binding buffer was also prepared as a negative control (Fig. 24a).
For double-staining experiments, 0.5, 1 , and 2 μΜ EV-Green [cyclic peptide (10)] in pre- mixed samples with EVs were incubated at 4 °C for 40 min as abovementioned. After that time, ANNEXIN V-Pacific Blue was added (0.5 μΙ), together with its binding buffer, incubated 10 min at 4°C, and diluted to 1 ml_ with binding buffer prior to measurement (Fig. 24c). Fig. 24a and Fig. 24b show fluorescence histogram of unlabeled vesicles and 2 μΜ EV-Green [cyclic peptide (10)] stained vesicles respectively. Subpopulation of bigger EVs (above 500 nm) were selected due to its enhanced and clear shift in staining at 530 nm. Fig. 24c shows a 2D-plot displaying ANNEXIN V-Pacific Blue staining on y-axis and 2 μΜ EV-Green [cyclic peptide (10)] on x-axis. Considerable amount of particles simultaneously stained with ANNEXIN V and EV-Green [cyclic peptide (10)] are observed (36.3%) indicating a correlation with PS binding. Data acquisition was carried out using an Altune acoustic focusing cytometer (Thermo Fisher) with post-acquisition data analysis with Flowjo software (Flowjo, Ashland, OR, USA).

Claims

A compound of formula (I):
Figure imgf000069_0001
(I)
wherein R1 , R2, R3, R5, R6 and R7 are independently selected from the group comprising H, CMO alkyl, C2-io alkenyl, C2-io alkynyl, CM0 alkyl carboxylic acid, Ci_i0 alkoxy, CM0 acyl, Ci_i0 acyloxy, C6-io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C,
L is a conjugated divalent linker selected from a C6-io arylene group; and wherein one or more of said R1 , R2, R3, R5, R6, R7 and L groups may be optionally
independently substituted with 1 or more substituents independently selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy; and
A is a group comprising an aromatic group and is selected from tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; wherein the conjugated divalent linker L is covalently bonded to the aromatic group of group A.
2. The compound of claim 1 wherein the group A comprising an aromatic group may be selected from: tryptaminyl;
N-protected tryptaminyl; an amino acid comprising an aromatic group selected from tryptophanyl, phenylalaninyl and derivatives thereof; an N-protected derivative of an amino acid comprising an aromatic group selected from N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; a peptide obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N- protected phenylalanine or a peptide nucleic acid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with tryptamine or N-protected tryptamine; a peptidomimetic derivative comprising an amino acid residue comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptidomimetic derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptidomimetic derivative obtainable by condensing a
peptidomimetic with tryptamine or N-protected tryptamine; a peptoid derivative comprising an amino acid residue comprising an aromatic group, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; or a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a protein derivative obtainable from a plurality of amino acids and comprising further comprising a tryptamine residue or N- protected tryptamine residue, said protein derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine.
3. The compound of claim 1 or claim 2 wherein the group A may selected from:
(i) a natural amino acid selected from tryptophan, such as L-tryptophan, or an N- protected derivative thereof, including D-tryptophan and N-protected derivatives of D-tryptophan;
(ii) a synthetic D or L amino acid selected from the group comprising
phenylalanine, phenylalanine derivatives, tryptophan derivatives or an N- protected derivative thereof;
(iii) a peptide obtainable from at least one of said natural amino acid (i) or
synthetic amino acid (ii);
(iv) a peptidomimetic obtainable from at least one of said natural or synthetic amino acids (ii) or
(v) a protein obtainable from at least one of said natural amino acid (i) or
synthetic amino acid (ii).
4. The compound of claim 2 or claim 3 wherein when the group A comprises N- protected tryptamine, N-protected tryptophan or N-protected phenylalanine, wherein the N- protective group is independently selected from the group comprising Fmoc
(fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl), Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-
Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3- ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
5. The compound of any of the preceding claims wherein the group A comprises tryptophanyl, N-protected tryptophanyl or a derivative thereof, preferably wherein the tryptophanyl, N-protected tryptophanyl or a derivative thereof is bonded to the conjugated divalent linker L via the indole C2 position of the tryptophanyl or N-protected tryptophanyl residue.
The compound of any of the preceding claims wherein the compound of formula (I) i pound selected from:
Figure imgf000072_0001
(13), (3),
and the corresponding D-tryptophan derivatives, wherein Z is an N-protecting group, such as a group selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4- methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4- biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2- (4-Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)- 3-ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
7. The compound of any of claims 1-5, wherein the group A comprising an aromatic group is a derivative of tryptophan, phenylalanine, N-protected tryptophan or N-protected phenylalanine, said derivative comprising at least two amino acid residues comprising an aromatic group independently selected from tryptophan residue, phenylalanine residue, N- protected tryptophan residue, and N-protected phenylalanine residue, wherein at least two aromatic groups of the at least two amino acid residues comprising an aromatic group are covalently bonded to the conjugated divalent linker L of a fluorophore of formula (i):
Figure imgf000073_0001
(i)
in which the groups R1 , R2, R3, R5, R6, R7 and L are as defined in claim 1.
8. The compound of claim 7 wherein:
the group A is obtainable by the condensation of a plurality of amino acids, at least two of which are amino acids comprising an aromatic group independently selected from tryptophan residue, phenylalanine residue, N-protected tryptophan residue, and N-protected phenylalanine residue, for instance such that the group A is a peptide or a protein; or
the group A is obtainable by the condensation of a peptide nucleic acid and at least two amino acids comprising an aromatic group independently selected from tryptophan, phenylalanine, N-protected tryptophan, and N-protected phenylalanine, for instance such that the group A is a peptide nucleic acid derivative;
the group A is obtainable by the condensation of a peptidomimetic and at least two amino acids comprising an aromatic group independently selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine, for instance such that the group A is a peptidomimetic derivative; or the group A is obtainable by the condensation of a peptoid and at least two amino acids comprising an aromatic group independently selected from tryptophan, phenylalanine, N-protected tryptophan, and N-protected phenylalanine, for instance such that the group A is a peptoid derivative.
9. The compound of claim 7 or claim 8 wherein the group A is a peptide of formula (ii),
Figure imgf000074_0001
in which the conjugated divalent linker L of the compound of formula (I) may be attached to the peptide (ii) at one or both of positions 1 and 2 with the proviso that a hydrogen atom is present at position 1 or position 2 if this is not covalently attached to the conjugated divalent linker.
10. A compound of claim 1 wherein the compound of formula (I) is selected from the group comprising:
Figure imgf000074_0002
Figure imgf000075_0001
the D-amino acid derivatives of the compounds of formulae (5a), (5b), (5), (6), (7) and (12) and the compounds of formulae (5a), (5b), (5), (6), (7) and (12) derived from a combination of L- and D- amino acids, wherein Z is independently selected from the group comprising Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl), Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-
Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3- ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
1 1. A compound of claim 1 wherein the compound of formula (I) is selected from the group comprising:
Figure imgf000076_0001
the D-amino acid derivatives of the compounds of formulae (8), (10) and (14) and the compounds of formulae (8), (10) and (14) derived from a combination of L- and D- amino acids.
12. A process for the preparation of a compound of formula (VI):
Figure imgf000077_0001
(VI) wherein R1 , R2, R3, R5, R6 and R7 are independently selected from the group comprising H, CMO alkyl, C2-io alkenyl, C2-io alkynyl, CM0 alkyl carboxylic acid, Ci_i0 alkoxy, Ci_i0 acyl, CM0 acyloxy, C6-io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C, wherein one or more of said R1 , R2, R3, R5, R6, R7 groups may be optionally independently substituted with 1 or more substituents independently selected from the group comprising Ci-6 alkyl and Ci_6 alkoxy; and the group A comprising an aromatic group is selected from N-protected tryptaminyl, N- protected tryptophanyl or a derivative thereof, said process comprising at least the step of:
- reacting an N-protected amino acid comprising an aromatic group selected from N- protected tryptamine, N-protected tryptophan or a derivative thereof with a compound of formula (V):
Figure imgf000077_0002
(V)
wherein R1 , R2, R3, R5, R6 and R7 are as defined above, in the presence of a Pd catalyst, a halide abstraction reagent and a carboxylic acid to provide a compound of formula (VI),
wherein the phenylene group is covalently bonded to the indolyl group of the N- protected tryptamine, N-protected tryptophan or derivative thereof.
13. The process of claim 12 wherein, the phenylene group is covalently bonded to the indolyl group of the N-protected tryptamine or N-protected tryptophan at the C2-position.
14. The process of claim 12 or claim 13 wherein the Pd catalyst is a Pd(0) catalyst or a Pd(ll) catalyst such as tetrakistriphenyl phosphine palladium (0) or Pd(OAc)2.
15. The process of any of claims 12 to 14 wherein the halide abstraction reagent is a silver (I) salt, such as silver tetrafluoroborate or silver hexafluorophosphate.
16. The process of any of claims 12 to 15 wherein the carboxylic acid is nitrobenzoic acid or a fluorocarboxylic acid such as trifluoroacetic acid.
17. The process of any of claims 12 to 16 wherein the N-protecting group is selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-
Nitrophenylsulfonyl)ethoxycarbony], Dde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3- ethyl], ivDde [1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
18. The process of any of claims 12 to 17 further comprising the step of deprotecting the N-protected group to provide a deprotected derivative.
19. The process of claim 18 further comprising the step of condensing the deprotected NH2 group of the compound of formula (VI) with one or more further amino acids to form a peptide or peptide derivative.
20. A process for the preparation of a compound of formula (I):
Figure imgf000079_0001
(I)
wherein R1 , R2, R3, R5, R6 and R7 are independently selected from the group comprising H, CMO alkyl, C2-io alkenyl, C2-io alkynyl, CM0 alkyl carboxylic acid, Ci_i0 alkoxy, Ci_i0 acyl, CM0 acyloxy, C6-io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C;
L is a conjugated divalent linker selected from a C6-io arylene group; wherein one or more of said R1 , R2, R3, R5, R6, R7 and L groups may be optionally
independently substituted with 1 or more substituents independently selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy; and the group A comprising an aromatic group is selected from N-protected 4- phenylalaninyl, N-protected 3-phenylalaninyl or a derivative thereof, said process comprising at least the step of:
- reacting an N-protected 4-phenylalanine derivative of formula (Vila), an N-protected 3-phenylalanine derivative of formula (VI lb) or a derivative thereof:
Figure imgf000080_0001
in which Q is a boronic acid moiety, R' is H or a carboxylic acid protecting group and R is a N-protecting group, with a compound of formula (VIII):
Figure imgf000080_0002
(VIII), in which L is a conjugated divalent linker selected from a C6-io arylene group, the groups R1 , R2, R3, R5, R6 and R7 are as defined for the compound of formula (I) and I is an iodine atom, in the presence of a Pd catalyst and a base to provide a compound of formula (I).
21. The process of claim 20 wherein the conjugated divalent linker L is a group selected from L1 or L2 having the structural formulae:
Figure imgf000080_0003
It
22. The process of claim 20 or claim 21 in which the derivative of the N-protected 4- phenylalanine derivative or the derivative of the N-protected 3-phenylalanine derivative is a peptide obtainable from one or more amino acids comprising the N-protected 4- phenylalanine or N-protected 3-phenylalanine respectively.
23. The process of any of claims 20 to 22 wherein the Pd catalyst is a palladium (0) compound or a palladium (I I) compound such as tetrakistriphenyl phosphine palladium (0), palladium (II) acetate, palladium (I I) chloride or diphenylphosphinoferrocene
dichloropalladium (II).
24. The process of any of claims 20 to 23 wherein the base is an alkali metal alkoxide.
25. The process of any of claims 20 to 24 wherein the N-protecting group is selected from Fmoc (fluorenylmethyloxycarbonyl), Boc (t-butoxycarbonyl), Cbz (benzyloxycarbonyl), Ac (acetyl), trifluoromethylcarbonyl, Bn (benzyl), Tr (triphenylmethyl), Pbf (2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl), Ts (toluenesulfonyl), Mtt (4-methyltrytil), Alloc (allyloxycarbonyl), Nps (2-nitrophenylsulfenyl), Bpoc [2-(4-biphenyl)isopropoxycarbonyl], Ddz (a,a-Dimethyl-3,5-dimethoxybenzyloxycarbony), Nsc [2-(4-
Nitrophenylsulfonyl)ethoxycarbony], Dde [1 -(4,4-Dimethyl-2,6-dioxocyclohex-1 -ylidene)-3- ethyl], ivDde [1 -(4,4-Dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl], Pms [2- [Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate], oNBS (o-Nitrobenzenesulfonyl), pNBS (p-nitrobenzenesulfonyl), dNBS (2,4-Dinitrobenzenesulfonyl), Troc (2,2,2- Trichloroethyloxycarbonyl), pNZ (p-Nitrobenzyloxycarbonyl), Poc (Propargyloxycarbonyl), oNZ (o-Nitrobenzyloxycarbonyl), NVOC (6-Nitroveratryloxycarbonyl), BrPhF [9-(4- Bromophenyl)-9-fluorenyl], HFA (Hexafluoroacetone), CIZ (2-chlorobenzyloxycarbonyl), Mmt (monomethoxytrityl), Tfa (Trifluoroacetyl), Pmc (2,2,5,7,8-Pentamethylchroman-6-sulfonyl), Mts (Mesityl-2-sulfonyl), Mtr (4-Methoxy-2,3,6-trimethylphenylsulfonyl) and MIS (1 ,2- Dimethylindole-3-sulfonyl).
26. The process of any of claims 20 to 25 wherein the carboxylic acid protecting group is selected from a Ci_5 alkyl group, allyl or a benzyl derivative.
27. The process of any of claims 20 to 26 further comprising the step of deprotecting the N-protected amino acid group to provide a deprotected amino acid group.
28. The process of any of claims 20 to 27 wherein the boronic acid moiety Q is selected from a boronic acid or a group of formulae:
Figure imgf000082_0001
29. Use of a compound of formula (I) as a probe, preferably an optical probe:
Figure imgf000082_0002
wherein R1 , R2, R3, R5, R6 and R7 are independently selected from the group comprising H, CMO alkyl, C2-io alkenyl, C2-io alkynyl, CM0 alkyl carboxylic acid, Ci_i0 alkoxy, Ci_i0 acyl, CM0 acyloxy, C6-io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C,
L is a conjugated divalent linker selected from a C6-io arylene group; and wherein one or more of said R1 , R2, R3, R5, R6, R7 and L groups may be optionally independently substituted with 1 or more substituents independently selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy; and
A is a group comprising an aromatic group and is selected from tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl, N-protected phenylalaninyl and derivatives thereof; wherein the conjugated divalent linker L is covalently bonded to the aromatic group of group
30. The use of claim 29 wherein the group A comprising an aromatic group may be selected from: tryptaminyl; N-protected tryptaminyl; a peptide obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N- protected phenylalanine or a peptide nucleic acid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with tryptamine or N-protected tryptamine; a peptidomimetic derivative comprising an amino acid residue comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptidomimetic derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptidomimetic derivative obtainable by condensing a
peptidomimetic with tryptamine or N-protected tryptamine; a peptoid derivative comprising an amino acid residue comprising an aromatic group, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; or a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanineor a protein derivative obtainable from a plurality of amino acids and comprising further comprising a tryptamine residue or N- protected tryptamine residue, said protein derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine.
31. A method of detecting the presence and/or function of a target in a cell or a component of a cell, comprising at least the steps of:
- providing a compound of formula (I) to a target zone:
Figure imgf000084_0001
wherein R1 , R2, R3, R5, R6 and R7 are independently selected from the group comprising H, CM0 alkyl, C2-io alkenyl, C2-io alkynyl, CM0 alkyl carboxylic acid, CM0 alkoxy, Ci_i0 acyl, Ci_i0 acyloxy, C6-io aryl, heteroaryl group having from 5 to 10 atoms in the aromatic core in which from 1 to 3 atoms are independently selected from O, S and N, with the remainder being C,
L is a conjugated divalent linker selected from a C6-io arylene group; and wherein one or more of said R1 , R2, R3, R5, R6, R7 and L groups may be optionally independently substituted with 1 or more substituents independently selected from the group comprising Ci_6 alkyl and Ci_6 alkoxy; and
A is a group comprising an aromatic group and is selected from tryptaminyl, tryptophanyl, phenylalaninyl, N-protected tryptaminyl, N-protected tryptophanyl, N- protected phenylalaninyl and derivatives thereof;
wherein the conjugated divalent linker L is covalently bonded to the aromatic group of group A;
- illuminating the target zone with a wavelength of light suitable to excite the compound of formula (I);
- detecting emission of fluorescence, fluorescence life-time, fluorescence polarization, ultrasonic waves or gamma rays; wherein detection of the above is indicative of the presence and/or function of the target in the cell or in the component of the cell.
33. The method of claim 32 wherein the group A comprising an aromatic group may be selected from: tryptaminyl;
N-protected tryptaminyl; a peptide obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a peptide derivative obtainable from a plurality of amino acids and tryptamine or N-protected tryptamine, said peptide derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine; a peptide nucleic acid derivative comprising an amino acid residue comprising an aromatic group, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N- protected phenylalanine or a peptide nucleic acid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptide nucleic acid derivative obtainable by condensing a peptide nucleic acid with tryptamine or N-protected tryptamine; a peptidomimetic derivative comprising an amino acid residue comprising an aromatic group, said peptidomimetic derivative obtainable by condensing a peptidomimetic with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptidomimetic derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptidomimetic derivative obtainable by condensing a
peptidomimetic with tryptamine or N-protected tryptamine; a peptoid derivative comprising an amino acid residue comprising an aromatic group, said peptoid derivative obtainable by condensing a peptoid with an amino acid comprising an aromatic group, said amino acid comprising an aromatic group selected from a tryptophan, phenylalanine, N-protected tryptophan and N-protected phenylalanine or a peptoid derivative comprising a tryptamine residue or N-protected tryptamine residue, said peptoid derivative obtainable by condensing a peptoid with tryptamine or N-protected tryptamine; or a protein obtainable from a plurality of amino acids, at least one of said plurality of amino acids comprising an aromatic group and selected from tryptophan, phenylalanine, N- protected tryptophan and N-protected phenylalanine or a protein derivative obtainable from a plurality of amino acids and comprising further comprising a tryptamine residue or N- protected tryptamine residue, said protein derivative obtainable by condensing plurality of amino acids and tryptamine or N-protected tryptamine.
34. The method of claim 32 or claim 33 wherein the target zone is a portion of a cell culture, a tissue sample or a liquid sample such as a bodily fluid sample.
35. The method of any of claims 32 to 34 wherein the step of providing a compound of formula (I) to a target zone occurs in vivo, ex vivo or in vitro.
36. The method of any one of claims 32 to 35 wherein the group A in the compound of formula (I) is a peptide having the sequence selected from:
H-Arg-Lys-Lys-Trp-Phe-Trp-NH2 (Seq. I.D. No. 2) in which the conjugated divalent linker L is covalently bonded to the indole group of one tryptophan residue;
H-Arg-Lys-Lys-Trp-Phe-Trp-OH (Seq. I.D. No. 1) in which the conjugated divalent linker L is covalently bonded to the indole group of one tryptophan residue;
cyclo (-Arg-Lys-Lys-Trp-Phe-Trp-Gly-) (Seq. I.D. No. 3) in which the conjugated divalent linker L is covalently bonded to the indole group of one tryptophan residue;
- cyclo(-Trp-Asp-Gly-Gly-Gly-Arg-Gly-Gly-Gln-lle-His-Gly-Phe-) (Seq. I.D. No. 4) in which the conjugated divalent linker L is covalently bonded to the indole group of the tryptophan residue;
H-Ala-Ala-Ala-Trp-Phe-Trp-NH2 (Seq. I.D. No. 5) in which the conjugated divalent linker L is covalently bonded to the indole group of one tryptophan residue; and H-Arg-Lys-Lys-Trp-Ala-Ala-NH2 (Seq. I.D. No. 6) in which the conjugated divalent linker L is covalently bonded to the indole group of the tryptophan residue.
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