Cyclic Tetrapeptides and Metal Complexes Thereof
20240025946 ยท 2024-01-25
Inventors
Cpc classification
International classification
Abstract
Provided herein is a cyclic tetrapeptides including alternating - and 3-amino acids and metal complexes thereof. The cyclic tetrapeptides are useful for coordinating a metal selected from Pb, Cd, Hg and As. Also provided herein is the use of the cyclic tetrapeptides in treating a disease, particularly metal poisoning, and the use in remediation of contaminated water and soil. Also provided herein are methods for detecting said metals in various substrates are provided.
Claims
1. A compound having the structure of formula 1; ##STR00014## (1a), wherein each R independently from any other R is independently selected from CH.sub.3 and H, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-4-alkyl or phenyl, wherein the C.sub.1-4-alkyl or phenyl is substituted by one or more substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, NHC(NH)(NH.sub.2) a 5- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 10 carbon atoms, wherein the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety may optionally be substituted by one or more substituents selected from C.sub.1-4-alkyl, SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, (O), COOH, NH.sub.2, CONH.sub.2, R.sup.B1 and R.sup.B2 are independently from each other; H, or a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a 5- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from OH, (O), SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle, or a linker suitable for binding to a detectable marker or a solid support, a detectable marker, optionally linked by a linker, or a linker bound to a solid support.
2. The compound according to claim 1, wherein the compound has the structure of formula 2, 3, 4, 5, 6 or 7 ##STR00015## ##STR00016##
3. The compound according to claim 1, wherein R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-4-alkyl, substituted by one or more substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, a five- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 6 carbon atoms, wherein the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety may optionally be substituted by one or more substituents selected from C.sub.1-4-alkyl, SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, (O), COOH, NH.sub.2, CONH.sub.2.
4. The compound according to claim 1, wherein the cyclic hydrocarbon moiety at R.sup.A1 and R.sup.A2 is selected from cyclopentyl, cyclohexyl, and phenyl.
5. The compound according to claim 1, wherein R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-3-alkyl, substituted by 1 or 2 substituents independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl, and phenyl, wherein the phenyl may optionally be substituted by one or more substituents selected from SH, and SeH.
6. The compound according to claim 1, wherein R.sup.A1 and R.sup.A2 are independently selected from CH.sub.2SH, (CH.sub.2).sub.2SH, CH.sub.2SCH.sub.3, (CH.sub.2).sub.2SCH.sub.3, CH(SH)(CH.sub.2SH), CH.sub.2CH(SH)(CH.sub.2SH), CH(SH)(COOH), CH(SH)CH.sub.2COOH, CH.sub.2CH(SHXCOOH), -phenyl-SH, CH.sub.2SO.sub.3H, (CH.sub.2).sub.2SO.sub.3H CH.sub.2COOH, (CH.sub.2).sub.2COOH, CH.sub.2NH.sub.2, (CH.sub.2).sub.2NH.sub.2, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2CONH.sub.2, CH.sub.2 imidazolyl, CH.sub.2-mercaptoimidazolyl, and CH.sub.2-phenyl.
7. The compound according to claim 1, wherein R.sup.B1 and R.sup.B2 are independently selected from H, or a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a 5- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from OH, (O), SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle.
8. The compound according to claim 1, wherein R.sup.B1 and R.sup.B2 are independently selected from H, or a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a 5- to 10-membered heterocycle, a cyclopentyl, a cyclohexyl, phenyl or a C.sub.1-4-alkyl, wherein the cyclopentyl, a cyclohexyl, phenyl or the C.sub.1-4-alkyl is optionally substituted by one or more substituents independently selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle.
9. The compound according to claim 1, wherein R.sup.B1 and R.sup.B2 are independently selected from H, C.sub.3-6-alkyl, CH.sub.2-phenyl, SH, (CH.sub.2).sub.mSH, (CH.sub.2).sub.mCOOH and (CH.sub.2).sub.rCONH.sub.2 with m and r being 0, 1, 2 or 3.
10. The compound according to claim 1, wherein the heterocycle at R.sup.A1 and R.sup.A2 and/or at R.sup.BI and R.sup.B2 is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl, indolyl, mercaptopurinyl, and benzothiophenyl.
11. The compound according to claim 1, wherein R.sup.A1 and R.sup.A2 are identical and/or R.sup.B1 and R.sup.B2 are identical.
12. The compound according to claim 1, wherein the detectable marker is selected from a dye, an affinity tag, a magnetic bead and a moiety comprising a radioisotope, and/or the linker is a hydrocarbon moiety comprising up to 50 C atoms, wherein one or more C atoms may optionally be replaced by O, S or N, and/or the solid support is a resin, a bead, a surface of an electrode or the bottom/wall of a reaction vessel.
13. A metal complex consisting of a ligand and a metal, wherein the ligand is a compound according to claim 1.
14. A method of treating metal poisoning, comprising administering an amount effective of compound according to claim 1 to a person in need thereof.
15. A method of removing and/or detecting a metal from/in a substrate, wherein the method comprises applying the compound according to claim 1 to the substrate.
16. The compound according to claim 1, wherein the compound has the structure of Formula 1a: ##STR00017##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0049] Terms and definitions
[0050] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.
[0051] The terms comprising, having, containing, and including, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article comprising components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that comprises and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of consisting essentially of or consisting of.
[0052] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
[0053] Reference to about a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to about X includes description of X.
[0054] As used herein, including in the appended claims, the singular forms a, or, and the include plural referents unless the context clearly dictates otherwise.
[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.
[0056] The term tetrapeptide in the context of the present specification relates to a molecule consisting of 4 amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The tetrapeptide comprises two -amino acids and two -amino acids.
[0057] The term cyclic tetrapeptide relates to the tetrapeptide described above, wherein the amino acids form a head-to-tail cyclic as represented in formula 1.
[0058] Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3.sup.rd ed. p. 21). Lower case letters for amino acid sequence positions or D written before the amino acid name or amino acid code refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, -amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, 1), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). The three letter or single letter code is also used after the Greek letter s for -amino acids that comprise a residue at the -carbon which is identical to the residue of the corresponding -amino acid, e.g. -Ala or Ala indicates the -amino acid -alanine. Homologues of -amino acids or -amino acids that differ by an additional methylene bridge (CH.sub.2) in the side chain are referred to as homo-amino acid, e.g. homocysteine. homo is also abbreviated by h, e.g. hCys depicts for the -amino acid homocysteine and shGlu depicts for -homoglutamic acid.
[0059] In the context of the present invention, the term 5- to 10-membered heterocycle relates to a compound that consists of 5 to 10 carbon atoms, wherein one or more carbon atoms are replaced by a heteroatom N, S or O, particularly N. Similarly, a 5- to 6-membered heterocycle consists of 5 to 6 carbon atoms, wherein one or more carbon atoms are replaced by a heteroatom N, S or O, particularly N. The carbon atoms and one or more heteroatoms are connected by single and/or double bonds to form a ring structure. The ring structure may be monocyclic or bicyclic.
[0060] The term hydrocarbon moiety comprising 3 to 10 carbon atoms relates to a hydrocarbon moiety that comprises carbon-carbon single, double and/or triple bonds, particularly carbon-carbon single bonds and/or carbon-carbon double bonds. The carbon atoms may form a linear, branched or cyclic structure or combinations thereof.
[0061] The term alkyl refers to a linear or branched hydrocarbon moiety. A C.sub.1-4-alkyl in the context of the present specification relates to a saturated linear or branched hydrocarbon having 1, 2, 3 or 4 carbon atoms. Similarly, a C.sub.1-3-alkyl relates to a linear or branched hydrocarbon having up to 3 carbon atoms. Non-limiting examples for a C.sub.1-C.sub.4 alkyl include methyl, ethyl, propyl, n-butyl, 2-methylpropyl, tert-butyl. In certain embodiments, a C.sub.1-4 alkyl refers to methyl (Me), ethyl (Et), propyl (Pr), isopropyl (iPr), n-butyl (Bu) and tertbutyl (tBu).
[0062] The term cyclic hydrocarbon moiety relates to a mono- or polycyclic hydrocarbon moiety that comprises carbon-carbon single, double and/or triple bonds, particularly carbon-carbon single bonds and/or carbon-carbon double bonds. The ring structures of a polycyclic hydrocarbon moiety may be bridged, fused or spirocyclic. Non-limiting examples for cyclic hydrocarbon moieties are aryls, e.g. phenyl, and cycloalkyls, e.g. cyclohexyl.
[0063] The term C.sub.5-6-cycloalkyl in the context of the present specification relates to a saturated hydrocarbon ring having 5 or 6 carbon atoms.
[0064] The term fluorescent dye in the context of the present specification relates to a small molecule capable of fluorescence in the visible or near infrared spectrum.
DESCRIPTION OF THE INVENTION
[0065] A first aspect of the invention relates to a compound of formula 1, particularly of formula 1a,
##STR00002## [0066] wherein [0067] each R independently from any other R is independently selected from CH.sub.3 and H, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-4-alkyl or phenyl, wherein the C.sub.1-4-alkyl or phenyl is substituted by one or more substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, NHC(NH)(NH.sub.2) a five- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 10, particularly 3 to 6, carbon atoms, wherein [0068] the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety may optionally be substituted by one or more substituents selected from C.sub.1-4-alkyl, SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, (O), COOH, NH.sub.2, CONH.sub.2, particularly C.sub.1-4-alkyl, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, [0069] R.sup.B1 and R.sup.B2 are independently form each other [0070] H, or [0071] a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a five- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from OH, (O), SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle, or [0072] a linker suitable for binding to a detectable marker or a solid support, [0073] a detectable marker, optionally linked by a linker, or [0074] a linker bound to a solid support.
[0075] In certain embodiments, at least one R is H and the other ones are CH.sub.3.
[0076] In certain embodiments, at least two R are H and the other ones are CH.sub.3.
[0077] In certain embodiments, at least three R are H and the other R is CH.sub.3.
[0078] In certain embodiments, at least one of the moieties R.sup.A1 and R.sup.A2 comprises a heteroatom S, N or O, particularly S. When the compound of formula 1 is used for binding a metal, the heteroatom at R.sup.A forms a bond to the metal such as Pb, Hg, As and Cd, particularly Pb. Binding of Pb, Hg, As and Cd, particularly Pb, may not be achieved by a hydroxyl moiety such as in the side chain of serine. Thus, -serine is not a suitable amino acid to provide suitable R.sup.A. However, -serine might still be used to provide a moiety R.sup.B that enhances the water solubility of the cyclic tetrapeptide.
[0079] In certain embodiments, the compound is a compound of formula 2, 3, 4, 5, 6 or 7, particularly of formula 2a, 3a, 4a, 5a, 6a or 7a,
##STR00003## ##STR00004##
[0080] The cyclic tetrapeptides may be formed of L- or D-amino acids or a mix thereof. Due to economic reasons, particularly L-amino acids are used since they are usually cheaper than the corresponding D-amino acids.
[0081] For stable metal complex formation, the metal binding moieties R.sup.A1 and R.sup.A2 should orient the same direction, particularly for capturing Pb.sup.2+ in its favored unique hemidirected geometry.
[0082] In certain embodiments, the -amino acids of the cyclic tetrapeptide are both L-amino acids or are both D-amino acids, particularly are both L-amino acids. This means, R.sup.A1 and R.sup.A2 are both bound to the -carbon atom by an up-wedge bond or R.sup.A1 and R.sup.A2 are both bound to the -carbon atom by a down-wedge bond.
[0083] In certain embodiments, the compound is a compound of formula 2, 5, 6 or 7, particularly of formula 2a, 5a, 6a or 7a.
[0084] In certain embodiments, the compound is a compound of formula 2 or 5, particularly of formula 2a or 5a.
[0085] In certain embodiments, the compound is a compound of formula 2, particularly of formula 2a.
[0086] In certain embodiments, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-4-alkyl, particularly a C.sub.1-3-alkyl, more particularly a C.sub.1-2-alkyl, substituted by one or more, particularly 1 or 2, substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, a five- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 6 carbon atoms, wherein the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety may optionally be substituted by one or more, particularly 1, substituents selected from C.sub.1-4-alkyl, SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, (O), COOH, NH.sub.2, CONH.sub.2, particularly C.sub.1-4-alkyl, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2.
[0087] In certain embodiments, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-4-alkyl, particularly a C.sub.1-3-alkyl, more particularly a C.sub.1-2-alkyl, substituted by one or more, particularly 1 or 2, substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, a five- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 6 carbon atoms, wherein the cyclic hydrocarbon moiety may optionally be substituted by one or more, particularly 1, substituents selected from C.sub.1-4-alkyl, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2.
[0088] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl, indolyl, mercaptopurinyl, benzothiophenyl, particularly imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl, more particularly, mercaptoimidazolyl.
[0089] Thiofuran is also referred to as thiophene.
[0090] Benzothiophene is also referred to as benzothiofuran.
[0091] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl.
[0092] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl.
[0093] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from imidazolyl, mercaptoimidazolyl, thiofuranyl.
[0094] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from pyrrolyl, pyrazolyl, imidazolyl.
[0095] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from imidazolyl, indolyl.
[0096] In certain embodiments, the heterocycle at R.sup.A1 and R.sup.A2 is selected from imidazolyl.
[0097] In certain embodiments, the imidazolyl is 1H-imidazol-4-yl. For example, R.sup.A is 1H-imidazol-4-yl when histidine is used as -amino acid.
[0098] In certain embodiments, the indolyl is 1H-indol-3-yl. For example, R.sup.A is 1H-indol-3-yl when tryptophan is used as -amino acid.
[0099] In certain embodiments, the cyclic hydrocarbon moiety at R.sup.A1 and R.sup.A2 is selected from cyclopentyl, cyclohexyl and phenyl.
[0100] In certain embodiments, the cyclic hydrocarbon moiety at R.sup.A1 and R.sup.A2 is phenyl.
[0101] In certain embodiments, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, imidazolyl, indolyl and phenyl, wherein the phenyl may optionally be substituted by one or more, particularly 1, substituents selected from SH, and SeH, particularly SH.
[0102] In certain embodiments, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2.
[0103] In certain embodiments, R.sup.A1 and R.sup.A2 are independently from each other a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, imidazolyl, indolyl and phenyl, wherein the phenyl may optionally be substituted by one or more, particularly 1, substituents selected from SH, and SeH, particularly SH.
[0104] In certain embodiments, R.sup.A1 and R.sup.A2 are independently selected from CH.sub.2SH, (CH.sub.2).sub.2SH, CH.sub.2SCH.sub.3, (CH.sub.2).sub.2-SCH.sub.3, CH(SH)(CH.sub.2SH), CH.sub.2CH(SH)(CH.sub.2SH), CH(SH)(COOH), CH(SH)CH.sub.2COOH, CH.sub.2CH(SH)(COOH), -phenyl-SH, CH.sub.2SO.sub.3H, (CH.sub.2).sub.2-SO.sub.3H CH.sub.2COOH, (CH.sub.2).sub.2COOH, CH.sub.2NH.sub.2, (CH.sub.2).sub.2-NH.sub.2, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2CONH.sub.2, CH.sub.2-imidazolyl, CH.sub.2-mercaptoimidazolyl and CH.sub.2-phenyl.
[0105] In certain embodiments, R.sup.A1 and R.sup.A2 are independently selected from CH.sub.2SH, (CH.sub.2).sub.2SH, CH.sub.2SCH.sub.3, (CH.sub.2).sub.2SCH.sub.3, CH(SH)(CH.sub.2SH), CH.sub.2CH(SH)(CH.sub.2SH), CH(SH)(COOH), CH(SH)CH.sub.2COOH, CH.sub.2CH(SH)(COOH), -phenyl-SH, CH.sub.2SO.sub.3H, (CH.sub.2).sub.2SO.sub.3H CH.sub.2COOH, (CH.sub.2).sub.2COOH, CH.sub.2NH.sub.2, (CH.sub.2).sub.2NH.sub.2, CH.sub.2CONH.sub.2, (CH.sub.2).sub.2CONH.sub.2.
[0106] In certain embodiments, R.sup.A1 and R.sup.A2 are independently selected from CH.sub.2SH, (CH.sub.2).sub.2SH, (CH.sub.2).sub.2SCH.sub.3, CH.sub.2CH(SH)(CH.sub.2SH), CH(SH)(COOH), -phenyl-SH, CH.sub.2SO.sub.3H, CH.sub.2COOH, CH.sub.2NH.sub.2, CH.sub.2CONH.sub.2, CH.sub.2-imidazolyl, and CH.sub.2-phenyl.
[0107] In certain embodiments, R.sup.A1 and R.sup.A2 are independently selected from CH.sub.2SH, (CH.sub.2).sub.2SCH.sub.3, CH.sub.2COOH.
[0108] In certain embodiments, [0109] R.sup.A1 and R.sup.A2 are identical and selected from a C.sub.1-4-alkyl, particularly a C.sub.1-3-alkyl, more particularly a C.sub.10.2-alkyl, substituted by one or more, particularly 1 or 2, substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, a 5- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 6 carbon atoms, wherein [0110] the cyclic hydrocarbon moiety is substituted by one or more substituents, particularly 1 substituent, selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, and wherein [0111] the 5- to 10-membered heterocycle is optionally substituted by one or more substituents, particularly 1 substituent, selected from C.sub.1-4-alkyl, SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, (O), COOH, NH.sub.2, CONH.sub.2, particularly C.sub.1-4-alkyl, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, and/or [0112] R.sup.A1 is selected from a C.sub.1-4-alkyl, particularly a C.sub.1-3-alkyl, more particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl and COOH, particularly SH, SC.sub.1-4-alkyl and COOH, and [0113] R.sup.A2 is selected from a C.sub.1-4-alkyl, particularly a C.sub.1-3-alkyl, more particularly a C.sub.1-2-alkyl, substituted by one or more, particularly 1 or 2, substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, a 5- to 10-membered heterocycle, a cyclic hydrocarbon moiety comprising 3 to 6 carbon atoms, wherein [0114] the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety may optionally be substituted by one or more substituents, particularly 1 substituent, selected from C.sub.1-4-alkyl, SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, (O), COOH, NH.sub.2, CONH.sub.2, particularly C.sub.1-4-alkyl, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, [0115] particularly R.sup.A2 is selected from a C.sub.1-4-alkyl, particularly a C.sub.1-3-alkyl, more particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents, particularly 1 substituent, independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, CONH.sub.2, a five- to 6-membered heterocycle, particularly imidazolyl, mercaptoimidazolyl or thiofuranyl, a phenyl, particularly an unsubstituted phenyl, [0116] wherein the phenyl may optionally be substituted by one or more substituents, particularly 1 substituent, selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, wherein R.sup.A2 is selected in such a way that it differs from R.sup.A.
[0117] In certain embodiments, [0118] R.sup.A1 and R.sup.A2 are identical and selected from a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl and phenyl, wherein the phenyl is substituted by one or more substituents, particularly 1 substituent, selected from SH, and SeH, particularly SH, and/or [0119] R.sup.A1 is selected from a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted 1 or 2 substituents selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl and COOH, particularly SH, SC.sub.1-4-alkyl and COOH, and [0120] R.sup.A2 is selected from a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, SO.sub.3H, COOH, NH.sub.2, CONH.sub.2, imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl and phenyl, [0121] wherein the phenyl may optionally be substituted by one or more substituents, particularly 1 substituent, selected from SH, and SeH, particularly SH, [0122] particularly R.sup.A2 is selected from a C.sub.1-3-alkyl, particularly a C.sub.1-2-alkyl, substituted by 1 or 2 substituents, particularly 1 substituent, independently selected from SH, SCH.sub.3, SeH, SeCH.sub.3, COOH, NH.sub.2, CONH.sub.2, imidazolyl and phenyl, particularly an unsubstituted phenyl or imidazoyl, wherein the phenyl may optionally be substituted by one or more substituents, particularly 1 substituent, selected from SH, and SeH, particularly SH, wherein R.sup.A2 is selected in such a way that it differs from R.sup.A1.
[0123] In certain embodiments, [0124] R.sup.A1 and R.sup.A2 are identical and selected from CH.sub.2SH, (CH.sub.2).sub.2SH, (CH.sub.2).sub.2SCH.sub.3, CH.sub.3CH(SH)(CH.sub.2SH), CH(SH)(COOH), -phenyl-SH, CH.sub.2SO.sub.3H, CH.sub.2COOH and CH.sub.2-imidazolyl, and/or [0125] R.sup.A1 is selected from CH.sub.2SH and CH(SH)(COOH), and R.sup.A2 is selected from CH.sub.2SH, (CH.sub.2).sub.2SH, (CH.sub.2).sub.2SCH.sub.3, CH.sub.2COOH, CH.sub.2NH.sub.2, CH.sub.3CONH.sub.2, CH.sub.2-imidazolyl, and CH.sub.2-phenyl, wherein R.sup.A2 is selected in such a way that it differs from R.sup.A1.
[0126] In certain embodiments, R.sup.A1 and R.sup.A2 are identical.
[0127] In certain embodiments of any aspect of the invention, the alkyl moiety of R.sup.A1 or of R.sup.A2 is not substituted by a 5- to 6-membered heterocycle or a cyclic hydrocarbon moiety.
[0128] In certain embodiments of any aspect of the invention, the alkyl moiety of R.sup.A1 or of R.sup.A2 is not substituted by a cyclic hydrocarbon moiety.
[0129] In certain embodiments, R.sup.B1 and R.sup.B2 are independently form each other [0130] H, or [0131] a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a 5- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from OH, (O), SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle.
[0132] In certain embodiments, R.sup.B1 and R.sup.B2 are independently selected from [0133] H, or [0134] a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a five- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle.
[0135] To enhance the water solubility of the cyclic tetrapeptide according to the invention, one and/or both moieties R.sup.B1 and R.sup.2 may comprise a hydrophilic moiety. In certain embodiments, at least one of R.sup.B1 and R.sup.2 is independently selected from a moiety selected from OH, COOH, NH.sub.2, CONH.sub.2, SO.sub.3H, a five- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from OH, COOH, NH.sub.2, CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle.
[0136] To enhance the metal binding affinity, R.sup.B1 and R.sup.2 may comprise a moiety that provides additional coordination sites and/or a second coordination sphere. In certain embodiments, R.sup.B1 and R.sup.2 are independently form each other a moiety selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a 5- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from (O), SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle. In certain embodiments, at least one of R.sup.B1 and R.sup.2 is independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a five- to 10-membered heterocycle or a hydrocarbon moiety comprising 1 to 12 C atoms, wherein the hydrocarbon moiety is optionally substituted by one or more substituents independently selected from SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle.
[0137] In certain embodiments, R.sup.B1 and R.sup.B2 are independently selected from [0138] H, or [0139] a moiety selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, a 5- to 10-membered heterocycle, a cyclopentyl, a cyclohexyl, phenyl or a C.sub.1-4-alkyl, particularly C.sub.1-4-alkyl, wherein the cyclopentyl, a cyclohexyl, phenyl or the C.sub.1-4-alkyl, particularly C.sub.1-4-alkyl, is optionally substituted by one or more substituents independently selected from OH, SH, SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H, and a five- to 10-membered heterocycle, and wherein the 5- to 10-membered heterocycle is optionally substituted by one or more substituents independently selected from OH, (O), SH, (S), SC.sub.1-4-alkyl, SeH, SeC.sub.1-4-alkyl, COOH, NH.sub.2, NHC.sub.1-4-alkyl, NHC(NH)(NH.sub.2), CONH.sub.2, SO.sub.3H.
[0140] In certain embodiments, the cyclopentyl, a cyclohexyl or phenyl at R.sup.B is unsubstituted.
[0141] In certain embodiments, the heterocycle at R.sup.B1 and R.sup.B2 is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, particularly imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl, indolyl, mercaptopurinyl, benzothiophenyl benzimidazolyl, quinolyl, isoquinolyl, diazanaphthalenyl.
[0142] In certain embodiments, the heterocycle at R.sup.B1 and R.sup.B2 is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, particularly imidazolyl, indolyl.
[0143] In certain embodiments, the heterocycle at R.sup.B1 and/or R.sup.B2 is defined as described for R.sup.A1 and R.sup.A2. Reference is made to the respective embodiments relating to R.sup.A1 and R.sup.A2.
[0144] In certain embodiments, R.sup.B1 and R.sup.B2 are independently selected from H, C.sub.3-6-alkyl, particularly CH.sub.2CH(CH.sub.3).sub.2, CH.sub.2-phenyl, SH, (CH.sub.2).sub.mSH, (CH.sub.2).sub.mCOOH and (CH.sub.2).sub.rCONH.sub.2 with m and r being 0, 1, 2 or 3.
[0145] In certain embodiments, R.sup.B1 and R.sup.B2 are independently selected from H, SH, (CH.sub.2).sub.mSH, (CH.sub.2).sub.mCOOH and (CH.sub.2).sub.rCONH.sub.2 with m and r being 0, 1, 2 or 3.
[0146] In certain embodiments, R.sup.B1 and R.sup.B2 are independently selected from H, (CH.sub.2).sub.mCOOH and (CH.sub.2).sub.rCONH.sub.2 with m and r being 0, 1, 2 or 3.
[0147] In certain embodiments, R.sup.B1 and R.sup.B2 are independently selected from H, (CH.sub.2).sub.mCOOH and CONH.sub.2 with m being 1, 2 or 3.
[0148] In certain embodiments, m is 1, 2 or 3.
[0149] In certain embodiments, r is 0 or 1, particularly 1.
[0150] In certain embodiments, R.sup.B1 and R.sup.B2 are H.
[0151] In certain embodiments, R.sup.B1 and R.sup.B2 are identical.
[0152] To facilitate detection by the cyclic tetrapeptide and/or a metal complex comprising the cyclic tetrapeptide according to the invention, the cyclic tetrapeptide may comprise a detectable marker.
[0153] In certain embodiments, the detectable marker is selected from a dye, an affinity tag, a magnetic bead, and a moiety comprising a radioisotope.
[0154] Suitable dyes are for example fluorescent dyes that are known to someone of skill in the art.
[0155] For detection of the cyclic tetrapeptide via an affinity tag, commonly known tags may be used.
[0156] Non-limiting examples for an affinity tag are strep-tag, glutathione-S-transferase (GST) tag, poly(His) tag.
[0157] In certain embodiments, the linker is a hydrocarbon moiety comprising up to 50 C atoms, particularly up to 20 C atoms, wherein one or more C atoms may optionally be replaced by 0, S or N.
[0158] In certain embodiments, the solid support is a resin, a bead, a surface of an electrode or the bottom/wall of a reaction vessel, particularly a surface of an electrode, a resin or a bead, more particularly a resin or a bead.
[0159] The compound according to the first aspect of the invention might be bound via a linker to a reaction vessel such as a 96-well plate or to a flow through device. This would facilitate the use of the compound in diagnostic/detection methods and the use of the compound in the remediation of contaminated water and soil, respectively.
[0160] In certain embodiments, the compound according to the first aspect of the invention is a compound of formula X1 to X22, particularly of formula X1-11 or X14-22,
##STR00005## ##STR00006## ##STR00007## ##STR00008##
[0161] In certain embodiments of any aspect of the invention described herein, R.sup.A1 and R.sup.A2 are not CH.sub.2-imidazolyl, and R.sup.A1 and R.sup.A2 are not CH.sub.2-phenyl.
[0162] In certain embodiments of any aspect of the invention described herein, R.sup.A1 and R.sup.A2 are not CH.sub.2-imidazoly.
[0163] In certain embodiments of any aspect of the invention described herein, R.sup.A1 and R.sup.A2 are not CH.sub.2-phenyl.
[0164] In certain embodiments of any aspect of the invention described herein, the compound of formula 1 is not a compound of formula D1 or D2,
##STR00009##
[0165] A second aspect of the invention relates to a metal complex consisting of a ligand and a metal, wherein the ligand is a compound according to the first aspect of the invention.
[0166] As described above, the compound according to the first aspect of the invention may bind to a metal via suitable moieties at R.sup.A and R.sup.B.
[0167] In certain embodiments, the binding moiety of the compound according to the first aspect of the invention binds to the metal in its deprotonated from. For example, a thiol and/or carboxylic acid moiety in its deprotonated form may form a complex with Pb.sup.2+ as shown below (see also
##STR00010##
[0168] In certain embodiments, the ligand is an anion.
[0169] Usually the ratio of metal to peptide is 1:1 or 1:2, i.e. the complex is monomeric or dimeric.
[0170] In certain embodiments, the complex is dimeric, particularly homodimeric.
[0171] In certain embodiments, the metal is selected from Pb, As, Cd and Hg, in particular the metal is Pb.
[0172] With regard to the ligand, reference is made to the embodiments of the first aspect of the invention.
[0173] A third aspect of the invention relates to the use of the compound according to the first aspect of the invention in the treatment of a disease.
[0174] In certain embodiments, the compound according to the first aspect of the invention is for use in the treatment of a disease.
[0175] With regard to the compound, reference is made to the embodiments of the first aspect of the invention.
[0176] A fourth aspect of the invention relates to the use of the compound according to the first aspect of the invention in the treatment of metal poisoning.
[0177] In certain embodiments, the compound according to the first aspect of the invention is for use in the treatment of metal poisoning.
[0178] In certain embodiments, the metal poisoning is selected from Pb poisoning, As poisoning, Cd poisoning and Hg poisoning.
[0179] In certain embodiments, the metal poisoning is Pb poisoning.
[0180] In a medical context, the compound according to the first aspect of the invention may be applied by standard methods as described in Sears, M.E. (2013).
[0181] With regard to the compound, reference is made to the embodiments of the first aspect of the invention.
[0182] A fifth aspect of the invention relates to a method of determining whether a patient has, or is at risk of developing metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning comprising [0183] a. determining the level of the metal, particularly of Pb, As, Cd and/or Hg using a compound according to the first aspect of the invention in an ex vivo blood, plasma or serum sample taken from the patient, and [0184] b. establishing the statistical significance of the concentration of the metal.
[0185] Particularly compounds according to the first aspect of the invention that comprise a detectable marker, optionally linked by a linker, at R.sup.B are suitable for the determination of the amount of metal in a sample.
[0186] Statistical significance might be established by determining the ratio of free ligand, i.e. the compound according to the first aspect of the invention, to the metal complex. The signal obtained when detecting the marker, may be compared to a standard.
[0187] With regard to the compound, reference is made to the embodiments of the first aspect of the invention.
[0188] The invention further encompasses the use of a compound according to the first aspect of the invention for use in the manufacture of a kit for the detection of developing metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning.
[0189] Wherever alternatives for single separable features are laid out herein as embodiments, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a detectable label may be combined with any of the alternative embodiments of ligand/compound according to the first aspect of the invention and these combinations may be combined with any medical indication or diagnostic method mentioned herein.
[0190] A sixth aspect of the invention relates to a method of removing a metal, particularly a metal selected from Pb, As, Cd and Hg, more particularly Pb, from a substrate, particularly soil or an aqueous solution or aqueous suspension, wherein the method comprises using a compound according to the first aspect of the invention.
[0191] Particularly compounds according to the first aspect of the invention that comprise a detectable marker, e.g. an affinity tag, or that are bound via a linker to a solid support are suitable for this method.
[0192] With regard to the compound, reference is made to the embodiments of the first aspect of the invention.
[0193] A seventh aspect of the invention relates to a method of detecting a metal, particularly a metal selected from Pb, As, Cd and Hg, more particularly Pb, in a substrate, particularly soil or an aqueous solution or aqueous suspension, wherein the method comprises using a compound according the first aspect of the invention.
[0194] Particularly compounds according to the first aspect of the invention that comprise a detectable marker, optionally linked by a linker, at R.sup.B are suitable for the determination of the amount of metal in a sample.
[0195] With regard to the compound, reference is made to the embodiments of the first aspect of the invention.
[0196] Another aspect of the invention relates to the preparation of the compound according to the first aspect of the invention. The preparation comprises the following steps: [0197] Providing a tetrapeptide consisting of two -amino acids Xaa and two -amino acids Xaa, wherein the tetrapeptide is characterized by the sequence Xaa-Xaa-Xaa-Xaa (SEQ ID NO: 13) or Xaa-Xaa-Xaa-Xaa (SEQ ID NO: 1414) from N- to C-terminus, particularly Xaa-Xaa-Xaa-Xaa (SEQ ID NO: 13), [0198] adding a coupling reagent and a base yielding a reaction mix, [0199] in a diluting step, diluting the reaction mix in an organic solvent, particularly CH.sub.2Cl.sub.2 or DMF, more particularly CH.sub.2Cl.sub.2.
[0200] In certain embodiments, the coupling reagent is selected from PyBOP, HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, CAS No. 148893-10-1), HCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, CAS No. 330645-87-9), HOBt/DIC (benzotriazol-1-ol , CAS No. 2592-95-2) and N,N-di(propan-2-yl)methanediimine, CAS No. 693-13-0), DCC (N,N-dicyclohexylmethanediimine, CAS No. 538-75-0), DPPA (diphenyl phosphorazidate, CAS No. 26386-88-9).
[0201] In certain embodiments, the coupling reagent is PyBOP. The term PyBOP relates to benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (CAS No. 128625-52-5).
[0202] In certain embodiments, 1 to 2 mole equivalents of the coupling reagent in relation to the mole amount of tetrapeptide are used.
[0203] In certain embodiments, 1.5 mole equivalents in relation to the mole amount of tetrapeptide are used.
[0204] In certain embodiments, the base is Hunig's base. The term Hunig's base relates to N-Ethyl-N-(propan-2-yl)propan-2-amine (CAS No. 7087-68-5).
[0205] In certain embodiments, 2 to 6 mole equivalents of base in relation to the mole amount of tetrapeptide are used.
[0206] In certain embodiments, 3 mole equivalents of base in relation to the mole amount of tetrapeptide are used.
[0207] In certain embodiments, the concentration of the tetrapeptide in the diluting step is 0.01 mM to 10 mM, particularly 0.05 mM to 2 mM.
[0208] In certain embodiments, the concentration of the tetrapeptide in the diluting step is 0.1 mM.
[0209] In certain embodiments, the dilution step is performed for 12 h to 72 h, particularly for 16 h to 48 h.
[0210] In certain embodiments, the diluting step is followed by an evaporation step. To allow fast evaporation, a solvent with a low boiling point such as CH.sub.2Cl.sub.2 may be used. When the boiling point of the solvent, e.g. DMF, is higher, evaporation may become tedious.
[0211] In certain embodiments, the method is performed at a temperature ranging from 15 C. to 40 C., particularly ranging from 20 C. to 25 C. The method may be performed at ambient temperature. There is no need to heat or cool down the reaction mixture.
[0212] The tetrapeptide may comprise protecting groups. Suitable protecting groups as well as methods for deprotection are known to one of skill in the art.
[0213] Medical treatment, Dosage Forms and Salts
[0214] Similarly, within the scope of the present invention is a method for treating metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning, in a patient in need thereof, comprising administering to the patient a compound according to the first aspect of the invention.
[0215] Similarly, a dosage form for the prevention or treatment of metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning is provided, comprising a compound according to any of the above aspects or embodiments of the invention.
[0216] The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.
[0217] Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms.
[0218] Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.
[0219] Topical administration is also within the scope of the advantageous uses of the invention. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemplified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2.sup.nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1.sup.st Ed. CRC Press 1989; ISBN-13: 978-0824781835).
[0220] Pharmaceutical Compositions and Administration
[0221] Another aspect of the invention relates to a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
[0222] In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.
[0223] In embodiments of the invention relating to topical uses of the compounds of the invention, the pharmaceutical composition is formulated in a way that is suitable for topical administration such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like, comprising the active ingredient together with one or more of solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives that are known to those skilled in the art.
[0224] The pharmaceutical composition can be formulated for enteral administration, particularly oral administration or rectal administration. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
[0225] The pharmaceutical composition can be formulated for parenteral administration, for example by i.v. infusion, intradermal, subcutaneous or intramuscular administration.
[0226] The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
[0227] In certain embodiments, the pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
[0228] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).
[0229] Method of Manufacture and Method of Treatment according to the invention
[0230] The invention further encompasses, as an additional aspect, the use of a compound according to the first aspect of the invention, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning.
[0231] Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with a disease associated with metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning. This method entails administering to the patient an effective amount of compound according to the first aspect of the invention, or its pharmaceutically acceptable salt, as specified in detail herein.
[0232] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
EXAMPLES
Example 1: Synthesis of Cyclic Tetrapeptides
[0233] For compounds described in this example, a scaffold composed of the sequence cyc-[Xaa-Ala-Xaa-Ala] (SEQ ID NO: 15) (Xaa depicts for any -AA; Scheme 1) was chosen, where in addition to enhancing stability, Ala was expected to facilitate the challenging intramolecular cyclization of the tetrapeptide.
##STR00011##
[0234] Herein the inventors present a family of cyclic tetrapeptides that were designed to detoxify Pb.sup.2+ ions. The peptides were examined for their ability to recover Pb-exposed bacteria and human cells, where one particular peptide (8) excelled to a greater extent than the benchmark chelating agents (CA). Mechanistic studies of the successful peptide shed light on its biological outcome and medicinal potential.
[0235] The inventors started their studies by synthesizing nine sidechain-protected linear peptides (Table 1, 1-9). Typically, head-to-tail cyclization occurs in dimethylformamide (DMF) as a solvent and only rarely of peptides shorter than pentamers (White et al, 2011). The inventors aimed at cyclizing tetrapeptides in the absence of a high boiling-point solvent as DMF. Upon condition screening, the inventors found the following ideal conditions: PyBOP and Hunig's base (1.5 and 3.0 equivalents, respectively) as the coupling reagent and the base, respectively, and ultrahigh dilution of the peptide (0.1 mM) in CH.sub.2Cl.sub.2 for 16-48 h until full conversion was obtained. The cyclic peptides were then side-chain deprotected and purified without the need for HPLC, reaching above 95% purity and with a yield of 62-87% over two steps (cyclization and deprotection). HR-ESI-MS and .sup.1H and .sup.13C NMR indicated exclusively intramolecular cyclization to form the desired tetramers.
TABLE-US-00001 TABLE 1 Peptides and benchmark compounds studied Toxicity.sup.c,e Recovery.sup.c,d Maximal Name Aqueous IC.sub.50.sup.e inhibition.sup.f (SEQ ID NO) Sequence solubility DH5 (%) HT-29 (%) (mM) (%) 1 [Cys-Ala].sub.2 insoluble 879 (164) (SEQ ID NO: 1) 2 Cys-Ala- insoluble 436 (176) (SEQ ID NO: 2) DCys-Ala 3 [Met-Ala].sub.2 insoluble 139 (48) (SEQ ID NO: 3) 4 [His-Ala].sub.2 Soluble 80 (11) 106 (3) 5.8 (0.7) 62 (3) (SEQ ID NO: 4) 5 [Asp-Ala].sub.2 soluble.sup.b 150 (80) 121 (16) n.d..sup.g 44 (8) (SEQ ID NO: 5) 6 Cys-Ala- insoluble 403 (51) (SEQ ID NO: 6) Met-Ala 7 Cys-Ala- insoluble 47 (41) (SEQ ID NO: 7) His-Ala 8 Cys-Ala- soluble.sup.b 279 (21) 334 (42) n.d..sup.g 15 (5) (SEQ ID NO: 8) Asp-Ala 9 Cys-Ala- insoluble 180 (4) (SEQ ID NO: 9) Phe-Ala 1a [Cys-Asp].sub.2 soluble.sup.b 2 (2) 106 (10) n.d..sup.g 0 (1) (SEQ ID NO: 10) 1b [Cys-hGlu].sub.2 soluble.sup.b 30 (29) (SEQ ID NO: 11) Na.sub.2EDTA 107 (72) 41 (6) 0.6 (0.2) 72 (4) Na.sub.2CaEDTA 182 (39) 95 (16) 3.9 (0.4) 63 (11) DMSA 165 (81) 110 (10) 3.7 (0.1) 77 (3) GSH.sup.h 39 (15) 126 (9) 9.6 (1.1) 57 (8) .sup.athree-letter code of AAs, h depicts for homo; .sup.bwith the addition of 2 equiv. NaOH or 1 equiv. of Ca(OH).sub.2; .sup.cvalues are mean SD of >3 repeats each performed in triplicate; .sup.dpresented values are at the highest concentrations; .sup.ethe concentration at 50% viability; .sup.fthe inhibition at the highest concentration; .sup.gnot detected as is it too high; .sup.hglutathione
Example 2: In Vivo and In Vitro Detoxification
[0236] Desalted peptides were then assessed for their ability to detoxify Pb (
[0237] Within the nine peptides, four exhibited outstanding results in detoxifying poisoned E. coli compared to the benchmark compounds (
[0238] Despite being highly active in vivo, peptides 1, 2, and 6 showed low aqueous solubility, reducing their effectiveness as potential CAs. Attempts to solubilize them, including in different pH conditions, formulation with PEG or co-solvent systems with DMSO failed. Therefore, two analogs of 1 where Ala is substituted by PAsp or PhGlu, to form peptides 1a and 1b, respectively, were synthesized. These peptides proved high solubility as Na or Ca salts, but their detoxification ability in bacteria was unsatisfied (
[0239] Nevertheless, the inventors tested 1a and the other soluble peptides in vitro for their ability to recover poisoned human cells (
[0240] The administration of EDTA as a CA has been evolved from its Na salt to Na.sub.2CaEDTA, to decrease undesired depletion of Ca.sup.2+ ions. Therefore, it was tested whether also in the case of 8, the counter cations affect its activity (
[0241] To conclude the effectiveness of 8, the inventors assessed its in vitro toxicity (
[0242] Material and Methods
[0243] The peptides described herein are synthesized according to the reaction shown in Scheme 2. R depicts for the side chain of an - or -amino acid. The side chain may be protected by a suitable protecting group (R). The tetrapeptide is obtained by standard solid phase peptide synthesis (SPPS) using a standard Fmoc-based protocol on a chlorotrityl chloride resin. Cleavage (1% TFA) is achieved by using TFA in CH.sub.2Cl.sub.2 in 5 rounds of 1 min each. Cyclization is obtained by reacting the sidechain-protected peptide with PyBOP (as a coupling reagent) and Hunig's base (DIPEA; as the base) in a ratio of 1.5 equivalents for PyBOP and 3 equivalents for the base, with respect to the peptide. The peptide is highly diluted (0.1 mM) to avoid dimerization and the solvent is solely CH.sub.2Cl.sub.2. The reaction mixture is incubated overnight (16-48 h). The sidechains are deprotected with a TFA cocktail that is adjusted to the respective amino acid composition.
[0244] Typically, a mixture of TFA:TIPS:EDT:H.sub.2O (87.5:2.5:7.5:2.5) is applied for 1 h. Finally, the cyclic tetrapeptide is purified by precipitation in an aqueous solution without the need for HPLC. Purities of 95% and higher and yields in the range of 62% to 87% (after purification) are reached. In a last step, the peptide is reacted with HCl so that Cl.sup. ions replace the TFA anions as TFA is toxic.
[0245] The complete removal of TFA is monitored with .sup.19F NMR.
##STR00012##
[0246] The following cyclic tetrapeptide were synthesized as described above:
TABLE-US-00002 (SEQIDNO:1) Cys-Ala-Cys-Ala
[0247] HRMS (ESI) m/z calculated for C.sub.1-2H.sub.21N.sub.4O.sub.4S.sub.2.sup.+ [M+H].sup.+ 349.09987; found: 349.09946
TABLE-US-00003 (SEQIDNO:6) Cys-Ala-Met-Ala
[0248] HRMS (ESI) m/z calculated for C.sub.14H.sub.25N.sub.4O.sub.4S.sub.2.sup.+ [M+H]+ 377.13117; found: 377.13120
TABLE-US-00004 (SEQIDNO:4) His-Ala-His-Ala
[0249] HRMS (ESI) m/z calculated for C.sub.18H.sub.26N.sub.6O.sub.4.sup.2+ [M+2H].sup.2. 209.10330; found: 209.10341
TABLE-US-00005 (SEQIDNO:7) Cys-Ala-His-Ala
[0250] HRMS (ESI) m/z calculated for C.sub.15H.sub.23N.sub.6O.sub.4.sup.2+ [M+H]+ 383.14960; found: 383.14971
TABLE-US-00006 (SEQIDNO:5) Asp-Ala-Asp-Ala
[0251] HRMS (ESI) m/z calculated for C.sub.14H.sub.19N.sub.4O.sub.8.sup. [MH].sup. 371.12084; found: 371.12065
TABLE-US-00007 (SEQIDNO:8) Cys-Ala-Asp-Ala
[0252] HRMS (ESI) m/z calculated for C.sub.1-3H.sub.21N.sub.4O.sub.6S.sup.+ [M+H].sup.+ 361.11763; found: 361.11771
TABLE-US-00008 (SEQIDNO:2) Cys-Ala-DCys-Ala
[0253] HRMS (ESI) m/z calculated for C.sub.1-2H.sub.21N.sub.4O.sub.4S.sub.2.sup.+ [M+H].sup.+ 349.09987; found: 349.09978
TABLE-US-00009 (SEQIDNO:10) Cys-Asp-Cys-Asp
[0254] HRMS (ESI) m/z calculated for C.sub.1-4H.sub.19N.sub.4O.sub.8S.sub.2.sup. [MH].sup. 435.06498; found: 435.06564
TABLE-US-00010 (SEQIDNO:9) Cys-Ala-Phe-Ala
[0255] HRMS (ESI) m/z calculated for C.sub.18H.sub.25N.sub.4O.sub.4S.sup.+ [M+H]+ 393.15910; found: 393.15888
TABLE-US-00011 (SEQIDNO:3) Met-Ala-Met-Ala
[0256] HRMS (ESI) m/z calculated for C.sub.16H.sub.26N.sub.4O.sub.4SNa.sup.+ [M+Na].sup.+ 427.14442; found: 427.14447 In vivo recovery tests A single colony of DH5a E. coli WT cells was grown overnight at 37 C. and 220 rpm in Tris Minimal Medium (TMM, pH 6.0; 5 mL) without antibiotics. The culture was then diluted to an OD.sub.600 of 0.03 with additional TMM to a total volume of 5 mL, and its OD.sub.600 was monitored. Upon cell density of 0.25 (which was achieved after 3-5 h), 1 mL of the culture was transferred into a cell culture tube and was labeled as the positive control. To additional 3 mL of the culture, 36 L of Pb(NO.sub.3).sub.2 1 M were added (final concentration of 12 mM). Both cultures were shaken at 37 C. and 220 rpm for an additional 5 h.
[0257] Aqueous stock solutions of each CA were plated on freshly prepared agar-LB plates such that the final concentration of each compound is equal to 0.5, 1, 2, 5, and 10 equivalents, compared to the amount of Pb(NO.sub.3).sub.2 in 50 L pre-toxified culture. The stock solutions were prepared such that plating and equally spreading 30 L of each solution will provide the desired amount of CA. To additional two plates, 30 L of H.sub.2O was added.
[0258] 50 L of the pre-toxified culture were homogenously spread to each CA-containing plate, 5 h after adding the metal to the culture. The Pb-containing culture was also plated on one of the two H.sub.2O-containing plates and was labeled as the negative control. Lastly, 50 L of the non-toxified culture were platted on the second H.sub.2O-containing plate and was labeled as positive control. All plates (positive and negative control and five plates for each examined compound) were then incubated at 37 C. overnight. The plates were then imaged, and the colonies were counted (with Vilber Quantum Visualization System). The recovery of each concentration of CA was calculated according to equation 1:
[0259] #CA.sub.xnumber of colonies of pre-toxified culture in a plate containing X mM of CA
[0260] #NEGnumber of colonies of pre-toxified culture in a plate containing no CA
[0261] Each experiment was performed on three independent occasions. Values are meanSD of >3 repeats each performed in triplicate.
[0262] In vitro recovery tests
[0263] HT-29 cells (purchased from ATCC) were grown in 25 mM HEPES RPMI-1640 medium, supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 10% fetal calf serum (FCS) superior (standardized) at 37 C. and 5% CO.sub.2. 96-well plates were prepared such that every well contains 10,000 cells in 100 L medium, and the cells were allowed to adhere overnight. To all wells but the positive control, 10 L of 22 mM Pb(NO.sub.3).sub.2 were added (final concentration 2 mM). 10 L of H.sub.2O was added to the positive control wells. 60 min after addition of metal, 10 L of each solution of the examined CA (2.4, 6, 12, 24, 48, and 120 mM) were added to reach final concentrations of 0.2, 0.5, 1, 2, 4, and 10 mM (which are 0.1, 0.25, 0.5, 1, 2, and 5 equivalents, respectively). To the positive control wells containing no metal and the negative control wells containing metal but no CA, 10 L of H.sub.2O were added. Each condition was performed in triplicates. The plates were incubated at 37 C. and 5% CO.sub.2 for an additional 23 h, after which the medium was removed, washed with fresh medium and 50 L of crystal violet solution (0.5% crystal violet powder in 20 mL MeOH and 80 mL H.sub.2O) to each well and the plates were gently shaken (60 rpm) for 20 min. The plates were then washed with H.sub.2O until no more unbound dye was observed and allowed to dry overnight. 200 L of MeOH were added to each well, and the plates were gently shaken (60 rpm) for 20 min, after which their absorbance at 560 nm was read with a plate reader. The recovery of each concentration of CA was calculated according to equation 2:
[0264] A[CA.sub.x]absorbance of the pre-toxified well in the presence of X mM of CA
[0265] A[blank]absorbance of blank wells (contain nothing)
[0266] A[NEG]absorbance of the pre-toxified well that contains no CA
[0267] Each experiment was performed on three independent occasions. Values are meanSD of >3 repeats each performed in triplicate.
[0268] In Vitro Toxicity Tests
[0269] HT-29 cells (purchased from ATCC) were grown in 25 mM HEPES RPMI-1640 medium, supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 10% fetal calf serum (FCS) superior (standardized) at 37 C. and 5% CO.sub.2. 96-well plates were prepared such that every well contains 10,000 cells in 100 L medium, and the cells were allowed to adhere overnight.
[0270] To all wells but the positive control, 10 L of each solution of the examined CA (2.4, 6, 12, 24, 48, and 120 mM) were added to reach final concentrations of 0.2, 0.5, 1, 2, 4, and 10 mM. To the positive control wells, 10 L of H.sub.2O was added. Each condition was performed in triplicates. The plates were incubated at 37 C. and 5% CO.sub.2 for 24 h, after which the medium was removed, washed with fresh medium and 50 L of crystal violet solution (0.5% crystal violet powder in 20 mL MeOH and 80 mL H.sub.2O) to each well and the plates were gently shaken (60 rpm) for 20 min. The plates were then washed with H.sub.2O until no more unbound dye was observed and allowed to dry overnight. 200 L of MeOH were added to each well, and the plates were gently shaken (60 rpm) for 20 min, after which their absorbance at 560 nm was read with a plate reader. The toxicity of each concentration of CA was calculated according to equation 3:
[0271] A[CA.sub.x]absorbance of the pre-toxified well in the presence of X mM of CA
[0272] A[blank]absorbance of blank wells (contain nothing)
[0273] A[POS]absorbance of well that contains no CA
[0274] Each experiment was performed on three independent occasions. Values are meanSD of >3 repeats each performed in triplicate.
[0275] The set-ups of in vitro and in vivo assays for determination of Pb detoxification ability could be seen in Table 2.
TABLE-US-00012 TABLE 2 Set-ups of in vitro and in vivo assays for determination of Pb detoxification ability In vitro assay In vivo assay Cells HT-29.sup.a DH5 E. coli.sup.b Cell density at t = 0 10,000 cells per well OD.sub.600 0.25 [Pb(NO.sub.3).sub.2] (mM) 2.sup.c 12.sup.c Incubated time with Pb.sup.2+ (h) 1 5 Concentrations of compounds (mM) 0-10.sup.d 0-120.sup.e Incubation with compounds Overnight, 37 C., 5% CO.sub.2 Overnight, 37 C., 220 rpm Further steps Washing with fresh medium Plating on LB agar Positive control Non-poisoned cells Non-poisoned cells Negative control Untreated cells Untreated cells Detection of viability Crystal violet.sup.f Colony counting.sup.g .sup.aHT-29 cells were chosen as model human cells since they show high sensitivity to Pb, and the latter does not precipitate in their medium (RPMI-1640); .sup.bDH5 E. coli strain was chosen due to its low minimal inhibitory concentration of Pb; .sup.cThe lowest concentration that shows a significant effect at the shortest timeframe; .sup.d0-5 equiv.; .sup.e0-10 equiv.; .sup.fDyes based on reduction (such as MTT) cannot be of use due to competing reduction by thiols; .sup.gDue to Pb precipitation over time, colonies counting was found to be more accurate and indicative than optical density.
Example 3: Peptide 8a
[0276] In Vitro and In Vivo Detoxification Results
[0277] The peptide that revealed the best results within all investigated peptides and also outcompeted the standards of care (SOCs) (
##STR00013##
[0278] Peptide 8a was then tested in mice. 40 male mice (C.sub.57BL/6) aged 6-8 weeks were provided with 20 mM Pb(OAc).sub.2 solution as the sole water supply for seven days (days 1-7). This poisoning route mimics chronic exposure in humans. Two days after returning to clean water (day 9), they were randomly divided into five groups of eight mice. They received a single treatment per day for seven days (days 10-16) of either CaNa.sub.2EDTA, DMSA, or 8a at a concentration of 30 mg kg.sup.1 except for group 1 that served as the negative control (Table 3).
[0279] Blood samples (100 L) were collected before dosing the mice on days 10-15 and on day 18, which is two days after the last dosing and the day by which the experiment was terminated. Urine was also collected from 34 animals on day 18 and was kept frozen until analysis.
TABLE-US-00013 TABLE 3 The treatments administered to each group of eight mice and the quantified values Dose BLL Urinary Pb Group Treatment Administration (mg kg.sup.1 day.sup.1) (g dL.sup.1) (ppm) 1 None 33.4 2.2 154 38 2 CaNa.sub.2EDTA IV 30 25.7 1.6 210 30 3 DMSA Oral 23.3 2.3 340 28 4 8a IV 16.0 1.5 452 47 5 Oral 17.7 1.3 435 28
[0280] The blood samples from the last day clearly indicate that 8a is more efficient than the two SOCs, both when administered orally and IV (Table 3,
[0281] The Pb content in the urine of 34 mice (out of 40;
[0282] Water remediation with immobilized peptides Two peptides that are expected to tightly and selectively bind Pb.sup.2+ ions were immobilized to a solid support with a long and flexible linker ((PEG2).sub.2) and a photocleavable moiety (
[0283] In addition, a negative control by which the second PEG2 is acetylated (Of) was synthesized. All three devices were then tested for their ability to capture Pb.sup.2+ ions from a 25 mM Pb(NO.sub.3).sub.2 solution. 1 h after adding the metal solution to the devices, the solutions were filtered, and the Pb concentration in each of them was quantified by ICP-MS. The efficacy was calculated by dividing the concentration of each solution by the concentration detected in the original solution as the 100% Pb content (
[0284] While 0f was incapable of reducing Pb-concentration of the contaminated solution, 1f and 8f reduced Pb concentration by 624% and 367%, respectively (
[0285] The resins were then treated with 100 mM Na.sub.2EDTA solutions for 10 min and quantified their Pb concentration to indicate an effective resin regeneration (
[0286] To detect the metal selectivity of our devices, similar filtration experiments were performed in equimolar mixtures of ZnCl.sub.2+Pb(NO.sub.3).sub.2 and CaCl.sub.2+Pb(NO.sub.3).sub.2 and in human blood serum (HBS) that was spiked with 25 mM of Pb(NO.sub.3).sub.2(
LIST OF REFERENCES
[0287] Sears, Margaret E. Chelation: Harnessing and Enhancing Heavy Metal DetoxificationA Review. 2013. The Scientific World Journal, Volume 2013, Article ID 219840, 13 pages [0288] White, C. J.; Yudin, A. K. Contemporary Strategies for Peptide Macrocyclization. Nat. Chem. 2011, 3 (7), 509-524. https-J/doi.org/10.1038/nchem.1062. [0289] Feoktistova, M.; Geserick, P.; Leverkus, M. Crystal Violet Assay for Determining Viability of Cultured Cells. Cold Spring Harb. Protoc. 2016, 2016 (4), 343-346. httpsJ/doi.org/10.1101/pdb.prot087379.