EP4637801A1 - Polypeptides - Google Patents

Polypeptides

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Publication number
EP4637801A1
EP4637801A1 EP23840678.9A EP23840678A EP4637801A1 EP 4637801 A1 EP4637801 A1 EP 4637801A1 EP 23840678 A EP23840678 A EP 23840678A EP 4637801 A1 EP4637801 A1 EP 4637801A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
linker
amino acid
group
independently
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23840678.9A
Other languages
German (de)
French (fr)
Inventor
Andrew George JAMIESON
Danielle MORGAN
Laura MCDOUGALL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Glasgow
Original Assignee
University of Glasgow
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Glasgow filed Critical University of Glasgow
Publication of EP4637801A1 publication Critical patent/EP4637801A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • POLYPEPTIDES Related Application This present case is related to, and claims the benefit of, GB 2219576.2 filed on 22 December 2022 (22.12.2022), the contents of which are hereby incorporated by reference in their entirety.
  • This invention provides polypeptides, in particular stapled polypeptides. Also provided are methods for preparing stapled polypeptides, as well as amino acids suitable for preparing stapled polypeptides.
  • Background Stapled peptides are an important class of ⁇ -helical peptides which are conformationally constrained. They are used in the regulation of Protein-Protein Interactions (PPIs), utilising their ability to bind to shallow PPI interfaces.
  • PPIs Protein-Protein Interactions
  • Conformationally constraining wild type peptides using staples could pay the entropic penalty of folding, resulting in favourable binding affinities and improved selectivities.
  • a frequently used stapling method makes use of two commercially available unnatural alkenyl amino acids (R8 and S5) and a ring closing metathesis (RCM) reaction using Grubbs’ catalyst. Cyclisation on resin forms an all-hydrocarbon alkene bridge using relatively mild conditions (Kim et al., 2011). The first biological example of alkene stapling was applied to peptides derived from the BCL-2 domain (Walensky et al., 2004).
  • the specific section of the protein that was being mimicked was the BH3 domain from the BID protein (EDIIRNIARHLAQVGDSMDRSIW).
  • Stapling increased the helicity of the peptides, proteolytic stability and binding affinity for the target receptor. Additionally, the peptide had a long in vivo half-life in a mouse model of T cell leukemia.
  • the alkene staple type includes the production of cis/trans isomers during synthesis. The yield of the purified bioactive product is lowered as one isomer is purified from the other. There is a need for new approaches for the stapling of polypeptides.
  • the present inventors have developed new stapled polypeptides and amino acids for the synthesis of the polypeptides.
  • 008528580 Summary of the Invention relates to a polypeptide having a diyne linkage.
  • the invention also relates to polypeptides and amino acids for the preparation of the diyne linked polypeptides, and methods for the preparation of the diyne linked polypeptides.
  • the secondary structure of polypeptides is important for their three-dimensional shape. ⁇ -helical peptides are a prevalent type of secondary structure that play an important functional role in protein-protein interactions.
  • a diyne linkage according to the present invention provides a stable covalent attachment between two amino acid side chains, which conformationally constrains the polypeptide.
  • the stapled polypeptide has improved helicity, as well as improved protease stability compared to the corresponding native peptide that lacks the diyne linkage.
  • the stapled polypeptide is suitable for therapeutic applications.
  • the invention provides a polypeptide wherein two amino acid residues are bridged at their respective ⁇ -carbon positions by a diynylene group, and typically a 1,3-diyne group.
  • the formation of the staple uses two amino acids each comprising a terminal alkynyl group which are linked to form a 1,3-diyne linkage.
  • the connection formed between the ⁇ -carbon positions is a hydrocarbon chain, without the presence of heteroatoms, such as without oxygen, within that chain.
  • a polypeptide comprising a group of Formula (I) wherein -R 1 and -R 2 are each independently selected from optionally substituted C 1-4 alkyl and -H; -R 3 and -R 4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue, such as an ⁇ -amino acid residue; -L 1 - and -L 2 - are each independently an optionally substituted C 2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof.
  • the polypeptide has a substantially ⁇ -helical structure secondary structure, such as in aqueous solution.
  • the 1,3-diyne staple increases the stability of the ⁇ -helical structure.
  • the diyne linkage may be readily formed from a corresponding non-stapled polypeptide possessing two amino acid residues each comprising a terminal alkynyl side chain. The linkage is formed under mild conditions that are compatible with other functionalities that may be present within the polypeptide.
  • the polypeptide may have a percentage helicity of 50% or more, such as 60% or more, such as 70% or more. Helicity may be measured by circular dichroism such as by a method described herein.
  • Each of -L 1 - and -L 2 - may independently be an optionally substituted C 2-7 alkylene, such as optionally substituted C4-7 alkylene, such as optionally substituted C4-6 alkylene, such as optionally substituted C 5 or C 6 alkylene. Varying the size of the macrocycle within the stapled polypeptide in this way helps to further alter, such as improve, helicity. These polypeptides can also be obtained in high yield by cyclisation of a corresponding linear polypeptide by a method as described herein. In alternative embodiments, -L 1 - and -L 2 - may independently be an optionally substituted C5-10 alkylene, such as optionally substituted C5-7 alkylene.
  • -L 1 - and -L 2 - are independently C 5-10 alkylene, such as C 5-7 alkylene. These polypeptides have excellent helicity, and they can be obtained in high yield by cyclisation of a corresponding linear polypeptide by a method, such as described herein.
  • -L 1 - and -L 2 - are each independently C4-10 alkylene, such as C4-7 alkylene, such as C5 alkylene or C6 alkylene.
  • -L 1 - and -L 2 - may be the same, such as where -L 1 - and -L 2 - are both C 5 alkylene or both C 6 alkylene, or -L 1 - and -L 2 - may be different.
  • Polypeptides having a 14 or 16 carbon atom bridge between the ⁇ -carbons of the stapled amino acids are particularly preferred, such as where -L 1 - and -L 2 - are both C 5 alkylene or both C 6 alkylene.
  • the total number of carbon atoms in -L 1 -(C ⁇ C-C ⁇ C)-L 2 - in Formula (I) is in the range of 14 to 16, such as 14 or 16, more preferably 14.
  • n may be 6 to provide an i, i+7 diyne linkage.
  • Each of -L 1 - and -L 2 - may independently be unsubstituted, or may be substituted, such as substituted with one or more halo groups, such as one or more fluoro groups.
  • An amino acid residue may be a natural or a non-natural amino acid residue.
  • An amino acid residue may comprise a protecting group, and thus an amino acid residue as described herein encompasses a protected natural or non-natural amino acid residue.
  • the 008528580 protecting group wherein present is a side-chain protecting group.
  • an amino acid reside is located at a C or N terminal of a polypeptide
  • the terminal carbonyl or carboxyl group, or terminal amino group, of the polypeptide may be protected.
  • an amino acid residue is selected from a natural amino acid or a non-natural amino acid, such as an ⁇ -amino acid, including a proteinogenic amino acid.
  • An amino acid residue may have L- or D- stereochemistry.
  • amino acid residue such as -A-
  • -A- may be selected from: Alanine [Ala, A], Aminobenzoic acid [PABA], Aminobutyric acid [Abu], Aminohexanoic acid [Ahx], Aminoisobutyric acid [Aib], Arginine [Arg, R], Asparagine [Asn, N], Aspartic acid [Asp, D], Butylglycine, Citrulline [Cit], Cyclohexylalanine [Cha], Cysteine [Cys], Diaminobutanoic acid [Dab], Diaminopropionic acid [Dpr or Dap], Dihydroxyphenylalanine [DOPA], Glutamic acid [Glu, E], Glutamine [Gln, Q], Glycine [Gly, G], Histidine [His, H], Homoserine [Hse], Hydroxyproline [Hyp], Isoleucine [Ile, I], Isonipecotic
  • n is an integer from 2 to 8, such as from 3 to 8, such as from 3 to 6, such as 3 or 6.
  • n may be an integer selected from 2, 3, 6 and 10, such as an integer selected from 3, 6 and 10.
  • Polypeptides with this number of amino acids residues between two stapled amino acid residues are readily prepared by cyclisation of a corresponding linear polypeptide under mild conditions.
  • these polypeptides can also be obtained in high yield such as by a coupling reaction as described herein.
  • Each of -R 1 and -R 2 may independently be selected from methyl, ethyl and -H.
  • -R 1 and -R 2 are independently methyl or -H, and most preferably -R 1 and -R 2 are methyl.
  • Methyl is particularly preferred as this facilitates the coupling of terminal alkynyl groups during synthesis of the stapled polypeptide.
  • Each of -R 3 and -R 4 may independently be -H or methyl.
  • -R 3 and -R 4 are both -H.
  • the ⁇ -carbon atoms that are bonded to -R 1 and -L 1 -, and to -R 2 and -L 2 -, respectively, are chiral. These ⁇ -carbon atoms may independently be in a R or S configuration.
  • the polypeptide comprises a group of Formula (IRS), (ISR) and/or (ISS).
  • the polypeptide comprises a group of Formula (I SS ).
  • the polypeptide is of Formula (Ia) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue, such as an ⁇ -amino acid residue or an N-methylated ⁇ -amino acid residue; -R A is selected from -H, a protecting group, acyl, C 1-4 alkyl, a protected linker, and a tag optionally with a linker; -R B is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200.
  • a tag may comprise a chromophore; a fluorescent or a phosphorescent label; or a radiolabel.
  • a tag may comprise a group for targeting the polypeptide to a cell membrane, such where the tag comprises a lipid group, such as farnesyl or geranyl.
  • a solid phase may be connected to the C terminal end of the polypeptide, such as at an amino acid residue -A'-, by a linker.
  • a tag may be connected to the N terminal end or the C terminal of the amino acid, such as to an amino acid residue -A'-, by a linker.
  • a tag is connected to the N terminal end.
  • the moieties represented by -[A’] m - and -[A’] m' - independently comprise up to one N-methylated amino acid residue.
  • the N-methylated amino acid residue is a terminal residue.
  • -R 3 and -R 4 may be -H.
  • each -A’- is independently an amino acid residue, such as wherein -A’- is not an N-methylated amino acid residue.
  • -A’- may be represented by -A- as described herein.
  • an acyl group is C1-12 acyl, such as C1-6 acyl, such as acetyl.
  • m and m’ are each independently an integer from 1 to 200. More preferably, m and m’ are each independently an integer from 2 to 50.
  • the polypeptide is of Formula (Ib) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -A’-, -L 1 -, -L 2 -, -R A , -R B , n, m and m’ are as described herein, and z is an integer from 1 to 4.
  • z is 1 or 2, such as 1.
  • a polypeptide comprising a group of Formula (II) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein, and the salts, solvates and protected forms thereof.
  • the groups -L 1 - and -L 2 - are preferably each independently C4-10 alkylene, such as wherein -L 1 - and -L 2 - are both C5 alkylene or both C6 alkylene.
  • -L 1 - and -L 2 - are each independently C 5-10 alkylene, such as C 5-7 alkylene.
  • n may is an integer from 2 to 8, such as from 3 to 8, such as an integer selected from 2, 3 and 6. In some embodiments n is an integer selected from 2, 3, 6 and 10. In some preferred embodiments, n is 6.
  • Each of -R 1 and -R 2 is preferably independently selected from methyl and -H.
  • Each of -R 3 and -R 4 is preferably -H.
  • the polypeptide may comprise a group selected from Formula (IIRS), (IISR) or (IISS). Alternatively, the polypeptide comprises a group of Formula (II SS ).
  • the polypeptide may have the Formula (IIa) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue;
  • -R A is selected from -H, a protecting group, acyl, C 1-4 alkyl, a protected linker, and a tag optionally with a linker;
  • -R B is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and
  • m and m’ are each independently an integer from 0 to 200.
  • m and m’ are each independently an integer from 1 to 200. More preferably, m and m’ are each independently an integer from 2 to 50.
  • each -A’- is an independently an amino acid residue.
  • -A’- is represented by -A- as described herein.
  • the polypeptide may have the Formula (IIb) 008528580 wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -A-‘, -L 1 -, -L 2 -, -R A , -R B , n, m and m’ are as described herein, and z is an integer from 1 to 4.
  • z is 1 or 2.
  • a method of preparing a diyne stapled polypeptide such as a polypeptide of the first aspect, the method comprising steps of: (i) providing a polypeptide according to the second aspect; and (ii) coupling the terminal alkynyl groups of the polypeptide to provide a 1,3-diyne linkage.
  • Step (ii) may be performed in the presence of a metal salt.
  • a metal salt may be a transition metal salt, such as a copper salt.
  • a metal salt may be selected from CuCl, CuBr, CuI, and CuOAc.
  • Step (ii) may be performed in the presence of a ligand selected from 4,4’-bis(hydroxymethyl)-2,2’-bipyridine, tetramethylethylenediamine, and 2,2’-bipyridine, such as in addition to the metal salt.
  • a ligand selected from 4,4’-bis(hydroxymethyl)-2,2’-bipyridine, tetramethylethylenediamine, and 2,2’-bipyridine, such as in addition to the metal salt.
  • the reaction mixture may be heated.
  • microwave radiation may be applied to the reaction mixture.
  • a pharmaceutical composition comprising a polypeptide comprising a group of Formula (I) and a pharmaceutically acceptable carrier. Preferences for the first aspect apply equally to the fourth aspect.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or excipient.
  • a polypeptide comprising a group of Formula (I), or the pharmaceutical composition described herein, for use in a method of treatment or prophylaxis.
  • the polypeptide or pharmaceutical composition may be for the treatment of cancer.
  • the polypeptide or pharmaceutical composition may be for use in a method of treating cancer wherein the polypeptide inhibits dimerization of Sam68, or wherein the polypeptide inhibits interaction between p53 and MDM2.
  • the polypeptide or pharmaceutical composition may be for use in a method of treating a disease with altered Sam68 activity, or a disease with altered p53 and/or MDM2 activity.
  • the polypeptide or pharmaceutical composition may be for use in a method of treatment wherein the polypeptide inhibits Sam68 activity, or inhibits MDM2 activity.
  • Preferences for the first aspect apply equally to the fifth aspect.
  • a compound of Formula (III) wherein -L X - is optionally substituted C 5-10 alkylene; -R 1 is selected from optionally substituted C1-4 alkyl and -H; -R 3 is selected from -H and methyl; -R N is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; and -R C is selected from -OH and a protecting group, or -R C together with the carbonyl group to which it is attached forms an activated acid, and the salts and solvates thereof.
  • 008528580 -L X - is optionally substituted C 5-10 alkylene and preferably is optionally substituted C6-10 alkylene, such as C6-10 alkylene.
  • -R 3 is hydrogen.
  • a tag may be a lipid group, such as farnesyl, geranyl; or may comprise a chromophore, a fluorescent or a phosphorescent label, or a radiolabel.
  • An acyl group may be a C1-12 acyl group. In some embodiments an acyl group is acetyl.
  • -L X - is optionally substituted C2-10 alkylene in the compounds of formula (III).
  • the compound may be of Formula (IIIa) wherein -L X -, -R 1 , -R 3 and -R N are as defined herein.
  • the compounds of Formula (III) are useful in the preparation of polypeptides, in particular stapled polypeptides in high yield that have high helicity.
  • Preferences for -R 1 and -L X - are as defined herein for -R 1 and -L 1 -, respectively.
  • -L X - is optionally substituted C6-10 alkylene, such as optionally substituted C6-8 alkylene.
  • -R 1 is selected from methyl and -H, such as methyl.
  • the group -R N may be selected from -H, a protecting group, farnesyl, geranyl and a tag optionally with a linker.
  • -R N may be a protecting group wherein -R N together with the nitrogen group it is bonded to forms a carbamate.
  • Figure 1 shows (A) CD analysis for T-STAR-1, T-STAR-HCS-2-cis and T-STAR-HCS-2-trans; and (B) calculated helicity at 222 nm for peptides according to an embodiment of the invention (from top to bottom at 240 nm: T-STAR-HCS-2-trans, T-STAR-HCS-2-cis).
  • Figure 4 shows (A) CD spectra of C 16 diyne linked peptides according to an embodiment of the invention (from top to bottom at start of x-axis: S 8 R 8 , R 8 S 8 , S 8 S 8 , R 8 R 8 ); (B) calculated helicity of C16 bridges; (C) CD spectra of C14 diyne bridges (from top to bottom at start of x-axis: S 7 S 7 , R 7 S 7 , R 7 R 7 , S 7 R 7 ); and (D) calculated helicity of C 16 bridges.
  • Figure 5 shows (A) CD spectra of C 14, C 12 and C 10 diyne linked peptides according to an embodiment of the invention (from top to bottom at x-axis: S7S7, S6S6, S5S5); (B) calculated helicity of C 14, C 12 and C 10 bridges; (C) CD spectra of C 14 and C 14 (monosubstituted) diyne bridges (from top to bottom at x-axis: S7S7, S7HS7H); and (D) calculated helicity of C14 and C14 (monosubstituted) bridges.
  • Figure 6 shows the percentage helicity calculated at 222 nm of diyne peptides according to an embodiment of the invention.
  • FIGs 2, 3, 5-7, 9, 11, 14 are > 60% (green); columns 10, 12 and 13 are > 50% (yellow); and columns 4 and 8 are ⁇ 40% (red).
  • Figure 8 shows CD spectra of the linear and stapled peptides according to an embodiment of the invention at 50 ⁇ M concentration (from top to bottom at 220 nm: 145a, 153a’, 153a, 160a).
  • Figure 9 shows CD spectra of the linear and stapled peptides according to an embodiment of the invention at 25 ⁇ M concentration (from top to bottom at 220 nm: 145a, 153a’, 153a, 160a).
  • Figure 10 shows fluorescence imaging of A) T-STAR-S 7 S 7 stapled peptide, B and C) alkene stapled peptides T-STAR-HCS-2-cis, T-STAR-HCS-2-trans, and D) T-STAR-S7HS7H internalised in cells; E) The native T-STAR-1 peptide showing no internalisation; and F) DMSO negative control. Microscopy images were acquired with a custom-built multi-modal microscope setup.
  • FIG. 11 shows solid-state Raman spectra of A) diyne stapled T-STAR-S7S7 showing that the diyne functionality gives a peak at ⁇ 2,255 cm -1 in the cell-silent region; and B) the acetylated native T-STAR-1 showing the absence of the diyne functionality peak.
  • the present invention relates to a polypeptide having a diyne linkage.
  • the invention also provides polypeptides and amino acids for the preparation of the diyne linked polypeptides, as well as methods for the preparation of the diyne linked polypeptides.
  • the diyne linked polypeptides are stapled polypeptides which can be readily prepared from a polypeptide having two ⁇ -amino acid residues, each having an alkynyl side chain connected to the ⁇ -carbon. These side chains readily undergo cyclisation under mild conditions at high conversion and high yield.
  • Polypeptides with a diyne staple as described herein have improved helicity compared to a corresponding linear (unstapled) polypeptide.
  • the stapled polypeptide also has increased protease stability.
  • alkene staples in a polypeptide are relatively flexible, apart from the two sp 2 hybridised carbons.
  • a 1,3-diyne (diacetylene) functionality can be introduced into the bridge instead of an alkene to provide enhanced rigidity.
  • the 1,3-diyne can be readily accessed synthetically using a Glaser oxidative coupling between two terminal alkyne amino acids within the peptide sequence. This eliminates the use of expensive Grubbs’ catalysts for producing the cyclic peptide.
  • the diyne-stapled peptide can be obtained as a single isomer, improving overall yield and simplifying purification compared to an alkene staple.
  • Cistrone et al. describe a diyne stapled peptide derived from cyclisation of two propargyl serine residues.
  • the cyclisation involves an intramolecular Glaser reaction between i and i + 4 to i + 7 spaced amino acids, which is carried out on-resin.
  • the authors report that high conversion can be achieved between i and i + 4 spaced residues within 72 hours, whilst conversion is substantially reduced under the same conditions for i and i + 5; i and i + 6; and i and i + 7 spaced residues.
  • the percentage helicity of the stapled peptides is reported to be between 6% to 42%.
  • Verlinden et al. also describe the use of a 1,3-diyne linker for helix ⁇ -stabilisation.
  • Peptides containing two propargyl serine residues are described, which are spaced apart at positions i and i +7. Cyclisation via Glaser stapling is carried out in solution, and full conversion is reported within after 24 hours. A stapled peptide based on linked D-Ser and L-Ser residues was reported to display a full ⁇ -helical structure, whilst the L-Ser, L-Ser peptide displayed helical structure within the stapled macrocycle but the exocyclic segment was not part of the helix.
  • WO 2017/040990 describes peptidomimetic macrocycles and their use. The peptides have an i, i+7 staple, and hydrocarbon staples are described.
  • the diyne linked polypeptides of the present invention have improved helicity.
  • a polypeptide where -L 1 - and -L 2 - are C 5 alkylene has good helicity (76% as shown for the polypeptide of Figure 5A and 5B).
  • the polypeptides of the present invention are also beneficial in that conversion from the linear precursor peptide to the stapled peptide is high (at about 100% as shown for the polypeptides of Figure 3A and 3B), and therefore the polypeptides are also conveniently prepared.
  • the diyne-stapled peptides of the present invention can be prepared from a linear peptide having two terminal alkynyl amino acids, preferably hydrocarbon alkynyl amino acids.
  • the rate of conversion of these amino acids as described herein via a Glaser reaction is higher than for a corresponding reaction using alkyne-functionalised serine residues, where the staple comprises a chain with oxygen chain atoms.
  • the rate of cyclisation is further improved when the length of the alkylene-alkyne side chain is increased from C 3 alkylene, which corresponds to the side chain in propargyl serine (-CH2OCH2-), to C4 alkylene, and in particular the rate of cyclisation is high when the alkyne is connected to the ⁇ -carbon by a C5 or C6 alkylene group.
  • polypeptide Comprising a Group of Formula (I)
  • the invention provides a polypeptide wherein two amino acid residues are bridged at their respective ⁇ -carbon positions by a diynylene group, and typically a 1,3-diyne group.
  • the formation of the staple uses two amino acids each comprising a terminal alkynyl group side chain, which are linked to form a 1,3-diyne linkage.
  • a polypeptide comprising a group of Formula (I) wherein -R 1 and -R 2 are each independently selected from optionally substituted C 1-4 alkyl and -H; -R 3 and -R 4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue; -L 1 - and -L 2 - are each independently an optionally substituted C2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof.
  • Formula (I) wherein -R 1 and -R 2 are each independently selected from optionally substituted C 1-4 alkyl and -H; -R 3 and -R 4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue; -L 1 - and -L 2 - are each independently an optionally substituted C2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof.
  • a polypeptide end group attached to a carbonyl group may be selected from: -H; C1-4 alkyl, such as methyl, ethyl, iso-propyl, n-propyl and tert-butyl, such as methyl and ethyl; hydroxyl (-OH); amino (-NH 2 ); alkoxy, such as C1-4 alkoxy (-O-C1-4 alkyl), such as methoxy and ethoxy; aminoalkyl, such C1-4 aminoalkyl (-N(H)C1-4 alkyl), such as -N(H)Me; a tag optionally with a linker, where the linker may link the tag to carbonyl group; a solid phase optionally with a linker, where the linker may link the tag to the carbonyl group; a protecting group; and a protected linker.
  • C1-4 alkyl such as methyl, ethyl, iso-propyl, n-propyl and tert-
  • a polypeptide end group attached to a nitrogen atom may be selected from -H; a protecting group, such as Fmoc or Boc; acyl, such as acetyl or propionyl, such as acetyl; C1-4 alkyl, such as methyl, ethyl, iso-propyl, n-propyl and tert-butyl, such as methyl and ethyl; 008528580 a protected linker; and a tag optionally with a linker where the linker may link the tag to the nitrogen atom.
  • a protecting group such as Fmoc or Boc
  • acyl such as acetyl or propionyl, such as acetyl
  • C1-4 alkyl such as methyl, ethyl, iso-propyl, n-propyl and tert-butyl, such as methyl and ethyl
  • 008528580 a protected linker
  • a tag
  • a polypeptide end group attached to a nitrogen atom may be selected from -H, a protecting group, acyl and C 1-4 alkyl, such as -H, acetyl, methyl and ethyl; such as -H, acetyl and methyl.
  • a polypeptide end group attached to a carbonyl group is preferably selected from -OH, -NH2, -O-C1-4 alkyl, -N(H)-C1-4 alkyl, C1-4 alkyl and a solid phase optionally with a linker; such as -OH, -NH 2 , and a solid phase optionally with a linker.
  • a tag may comprise a chromophore; a fluorescent or a phosphorescent label; a radiolabel; or a group for targeting the polypeptide to a cell membrane, such as a lipid group, such as farnesyl or geranyl.
  • a tag is connected to a peptide terminal, such as the N terminal, via a linker.
  • a solid phase may comprise a solid support material or resin, and may be ad such as a polystyrene bead.
  • a solid phase may be connected to a peptide terminal, such as the C terminal, via a linker.
  • a linker is a divalent moiety that links together two moieties together, for example an amino acid residue or peptide and a tag, a solid phase or a protecting group.
  • a linker may comprise one or more of: a covalent bond; one or more C 2-4 oxyalkylene groups, such where the linker is a PEG linker; and one or more groups selected from C1-20 alkylene, ether, carbonyl, amide and ester.
  • linkers examples include beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and PEG linkers such as PEG2, PEG3, or PEG4.
  • GABA 4-aminobutyric acid
  • AEA (2-aminoethoxy) acetic acid
  • Ava 5-aminovaleric acid
  • PEG linkers such as PEG2, PEG3, or PEG4.
  • a linker, where present, may be between the terminal nitrogen of a polypeptide of Formula (Ia) and a chromophore, a fluorescent label, a phosphorescent label, a radiolabel or a protecting group.
  • a linker may be between the C terminal of a polypeptide and a solid phase, a chromophore, a fluorescent label, a phosphorescent label, a radiolabel or a protecting group.
  • a linker may be protected with a protecting group.
  • a protecting group may be bonded directly to an end of the group of Formula (I), or a protecting group may be bonded to a linker.
  • the protecting group may be an amino protecting group that is attached to a nitrogen, or a carbonyl protecting group that is attached to a carbonyl group.
  • the protecting group When the protecting group is attached to a linker, it may be a protecting group for a moiety on the linker, such as a nitrogen or oxygen atom or a carbonyl group.
  • a polypeptide end group attached to a nitrogen atom may comprise a fluorescent label and a linker, such as fluorescent label that is fluorescein-5-isothiocyanate (FITC) and linker that is 6-aminohexanoic acid (Ahx) or beta-alanine.
  • FITC fluorescein-5-isothiocyanate
  • a protecting group may be selected from 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), -oMe and -oBn.
  • a protecting group attached to a nitrogen atom may be Fmoc, Boc or Cbz.
  • a protecting group attached to a carbonyl group may be -oMe or -oBn.
  • a polypeptide comprising a group of Formula (I) comprises an end group at the N terminal that is selected from -H, a protected linker, acyl, and a tag optionally with a linker, such as -H, acetyl, a fluorescent tag with a linker and a lipid group, such as -H, acetyl, and -Ahx-FITC, -beta-alanine-FITC, farnesyl and geranyl.
  • a polypeptide comprising a group of Formula (I) comprises an end group at the C terminal that is selected from -OH, -NH 2 , and a solid phase optionally with a linker; such as -NH2 and a solid phase optionally with a linker.
  • a polypeptide comprising a group of Formula (I) may be referred to as a stapled polypeptide.
  • a polypeptide comprising a group of Formula (I) may be bonded to an amino acid residue or a peptide segment.
  • a polypeptide may comprise a group of Formula (I’) where * and ** each independently represent the point of attachment to an amino acid residue or a peptide segment 008528580 wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein.
  • the polypeptide comprises Formula (I’) where * and/or ** indicates the point of attachment to a polypeptide end group.
  • the polypeptide may comprise Formula (I’) where * indicates the point of attachment to a group selected from -H, acyl such as acetyl, an amino protecting group, a protected linker, a tag optionally with a linker, or * indicates the point of attachment to a moiety that together with the nitrogen atom it is bonded to forms an N-terminal modification.
  • N-terminal modifications include N-acetyl, N-formyl, pyroglutamyl, urea, fatty acids, carbamate, sulfonamide and alkylamine.
  • the polypeptide may comprise Formula (I’) where ** indicates the point of attachment to a group selected from -OH, -NH2, a protecting group, a protected linker, or a solid phase optionally with a linker, and a tag optionally with a linker, or * indicates the point of attachment to a moiety that together with the carbonyl group it is bonded to forms a C-terminal modification.
  • C-terminal modifications include amide, N-alkyl amide, aldehyde and esters.
  • a polypeptide comprising a group of Formula (I) may comprise one or more peptide segments according to Formula (I), such as two or more segments of Formula (I).
  • Each of -L 1 - and -L 2 - is independently optionally substituted C 2-10 alkylene.
  • An alkylene group may be substituted or unsubstituted, and preferably, -L 1 - and -L 2 - are both unsubstituted.
  • Each alkylene group may be linear or branched, and is preferably linear.
  • Each alkylene group maybe unsubstituted or substituted.
  • An alkylene group may be substituted, for example, with one or more halo groups, such as one or more of fluoro, chloro or bromo, and preferably one or more fluoro.
  • the alkylene group is 008528580 substituted with 1 to 4 fluoro, such as 1, 2, 3 or 4 fluoro.
  • the alkylene group may be per-halogenated, such as per-fluorinated.
  • one or both of -L 1 - and -L 2 - is independently optionally substituted C2-10 alkylene, more preferably optionally substituted C 3-10 alkylene, more preferably optionally substituted C4-10 alkylene, more preferably optionally substituted C4-7 alkylene, such as optionally substituted C5 alkylene or optionally substituted C6 alkylene.
  • One or both of -L 1 - and -L 2 - may independently be optionally substituted C2-7 alkylene, such as optionally substituted C 3-7 alkylene, such as optionally substituted C 3-6 alkylene, such as optionally substituted C4-6 alkylene, such as optionally substituted C5-6 alkylene, and most preferably C 5 alkylene.
  • -L 1 - and -L 2 - may be unsubstituted.
  • one or both of -L 1 - and -L 2 - is independently optionally substituted C5-10 alkylene, preferably optionally substituted C5-8 alkylene, more preferably optionally substituted C 5-7 alkylene, most preferably optionally substituted C 5 alkylene or optionally substituted C 6 alkylene.
  • one or both of -L 1 - and -L 2 - is independently optionally substituted C6-10 alkylene, more preferably optionally substituted C 6-8 alkylene, such as optionally substituted C 6 alkylene or C 7 alkylene.
  • -L 1 - and -L 2 - may be unsubstituted.
  • the groups -L 1 - and -L 2 - may be the same or they may be different. In some preferred embodiments -L 1 - and -L 2 - are the same, such as where both are selected from C 4-10 alkylene, such as C4-7 alkylene, such as C5 or C6 alkylene. Additionally or alternatively, L 1 - and -L 2 - are the same and are both are selected from C5-10 alkylene, such as C5-8 alkylene, such as C5-7 alkylene; or C6-10 alkylene, such as C6-8 alkylene, such as C 6-7 alkylene.
  • Each -A- in Formula (I) is an amino acid residue, which may be a natural amino acid residue or a non-natural amino acid residue.
  • Each -A- may be an ⁇ -amino acid residue or a ⁇ -amino acid residue.
  • -A- is an ⁇ -amino acid residue.
  • Non-natural amino acids which may also be referred to as modified amino acids, include enantiomers of natural amino acids and amino acid analogues having a modified side chain, as well as enantiomers of amino acid analogues. These may be ⁇ -amino acid residues or ⁇ -amino acid residues, such as ⁇ -amino acid residues.
  • non-natural amino acid residues include natural amino acid residues having a D-configuration (D-amino acid residues).
  • non-natural amino acid residues include: norleucine (Nle), cyclobutylalanine (Cba), citrulline (Cit), hydroxyproline (Hyp), 3-nitrotyrosine, nitroarginine and ornithine (Orn), which each may be in the L- or D-configuration.
  • An amino acid residue as described herein may comprise a protecting group.
  • An amino acid residue thus encompasses: a natural or non-natural amino acid residue; and a protected natural or non-natural amino acid residue. Where an amino acid residue is protected, the protection may be provided on the side chain.
  • protection may be additionally or alternatively provided at the N terminal or the C terminal.
  • Suitable protecting groups for use are well known to those skill in the art.
  • -A- is not an N-alkylated amino acid residue.
  • Typical protecting groups are those suitable for use in solid phase peptide synthesis, and may include protecting groups that are removable under acidic conditions, for example with TFA.
  • -A- is selected from: Alanine [Ala, A], Aminobenzoic acid [PABA], Aminobutyric acid [Abu], Aminohexanoic acid [Ahx], Aminoisobutyric acid [Aib], Arginine [Arg, R], Asparagine [Asn, N], Aspartic acid [Asp, D], Butylglycine, Citrulline [Cit], Cyclohexylalanine [Cha], Cysteine [Cys], Diaminobutanoic acid [Dab], Diaminopropionic acid [Dpr or Dap], Dihydroxyphenylalanine [DOPA], Glutamic acid [Glu, E], Glutamine [Gln, Q], Glycine [Gly, G], Histidine [His, H], Homoserine [Hse], Hydroxyproline [Hyp], Isoleucine [Ile, I], Isonipecotic acid [Isn], Leucine
  • one or more amino acid residues may be crosslinked to another amino acid residue, in addition to the diyne linkage between the ⁇ -carbon atoms that are bonded to -R 1 and -R 2 , respectively.
  • An additional crosslink may be a further diyne linkage as described herein, or may be a different amino acid crosslink.
  • Suitable crosslinks 008528580 include alkene bridges, amide bridges, ester bridges, disulfide bridges and lanthionine bridges.
  • the polypeptide may comprise 10 or more, such as 12 or more, such as 15 or more, such as 18 or more, such as 20 or more, such as 25 or more, contiguous amino acid residues, such as ⁇ -amino acid residues, including [A]n together with the two stapled amino acid residues comprising -R 1 and -R 2 , respectively.
  • the amino acid residue -A- may be represented by Formula (AA) where -R 1 is as defined herein, and -R AA is an amino acid side chain, such as that of a natural amino acid, such as a proteinogenic amino acid.
  • -R 1 is -H or methyl, such as -H.
  • the amino acid residue of Formula (AA) is an ⁇ -amino acid residue.
  • Integer n n is an integer from 2 to 10, and preferably is from 3 to 6. More preferably n is 3 or 6. n may be an integer selected from 2, 3, 6 and 10, and preferably an integer selected from 3, 6 and 10. In some embodiments n is 6. In these embodiments the diyne may be referred to as an i, i + 7 stapled polypeptide.
  • -L 1 - and -L 2 - are C4 alkylene or higher, such as C5 alkylene or C6 alkylene.
  • n is 6 and -L 1 - and -L 2 - are both C5 or C 6 alkylene, such as C 6 alkylene.
  • -R 1 and -R 2 Each of -R 1 and -R 2 is independently selected from C1-4 alkyl and -H.
  • a C1-4 alkyl group may be methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl or sec-butyl.
  • -R 1 and -R 2 are independently selected from methyl, ethyl, and -H, such as where -R 1 and -R 2 are independently selected from methyl and -H.
  • -R 1 and -R 2 may be the same, or -R 1 and -R 2 may be different.
  • -R 1 and -R 2 are the same, such as where -R 1 and -R 2 are both methyl or both -H.
  • -R 1 and -R 2 may both be methyl.
  • the Thorpe-Ingold effect aids in the success of the Glaser reaction which improves the conversion rate during peptide stapling.
  • increased peptide helicity can be induced by the incorporation of a quaternary ⁇ -carbon into ⁇ -amino acids.
  • -R 3 and -R 4 Each of -R 3 and -R 4 is independently selected from -H and methyl. -R 3 and -R 4 may be the same or they may be different. In some preferred embodiments -R 3 and -R 4 are the same and are both -H. In these embodiments the polypeptide may comprise a group of Formula (Ic) wherein -R 1 , -R 2 , -A-, -L 1 -, -L 2 - and n are as described herein.
  • -R 3 and -R 4 are both -H, and -R 1 and -R 2 are each independently selected from -H and methyl, such where -R 1 and -R 2 are both methyl.
  • -L 1 - and -L 2 - may independently be optionally substituted C 5-10 alkylene, such as optionally substituted C 5-8 alkylene, such as optionally substituted C 5 alkylene or optionally substituted C6 alkylene.
  • -L 1 - and - L 2 - are unsubstituted.
  • Stereoisomers The ⁇ -carbons to which -R 1 and -L 1 -, or -R 2 - and -L 2 -, are bonded, respectively, are chiral. These carbon atoms may independently be in an R or S configuration.
  • a polypeptide of the invention comprises a group of Formula (I XS ), where the ⁇ -carbon that is bonded to -R 2 and -L 2 - is in a S configuration, and the ⁇ -carbon that is bonded to -R 1 - and -L 1 - may be in a R or S configuration, or a mixture thereof, wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as defined herein.
  • the polypeptide may be a diastereomer according to any one of Formula (IRR), (IRS), (ISR) and (ISS) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein.
  • the polypeptide comprises Formula (IRS), (ISR) and/or (ISS), more preferably Formula (I RS ) and/or (I SS ), and most preferably Formula (I SS ).
  • n 6 and/or when -L 1 - and -L 2 - are C4-6 alkylene, such as C5 or C 6 alkylene, such as such as C 5 alkylene.
  • These polypeptides are associated with high percentage ⁇ -helicity, such as 50% or more. Any remaining amino acid residues present in the polypeptide, such as each -A-, may independently be in a D-configuration or an L-configuration, and preferably are in the L-configuration.
  • Helicity A polypeptide comprising a group of Formula (I) may have a substantially ⁇ -helical structure secondary structure, such as in aqueous solution.
  • the polypeptide may have a helicity, which is a percentage helicity, of 40% or more, 50% or more, such as 60% or more, such as 65% or more, such as 70% or more, such as 75% or more.
  • the polypeptide may have a helicity of 90% or less, such as 85% or less, such as 80% or less, such as 75% or less.
  • the polypeptide may have a helicity in a range with upper and lower limits as described above, such as 40% to 90%, including 50 to 80%, such as 60% to 80%. Additionally or alternatively, the polypeptide has a helicity of 50% or more, such as 55% or more, such as 50% to 90%, including 50% to 80%, such as 55% to 80%.
  • the polypeptide may have a helicity that is higher by 5% or more than a corresponding linear (non-stapled) polypeptide, such as 10% or more, such as 15% or more, such as 20% or more.
  • a linear (non-stapled) polypeptide may be according to Formula (II) as described herein.
  • Helicity may be measured by circular dichroism as described herein. Scanning may be carried out in a range of about 190-260 nm, such as with a scanning speed of about 50 nm/min, optionally with a 1 nm data pitch, further optionally with a 1 nm bandwidth, further optionally with an 8 s response time.
  • the polypeptide may be measured at a concentration in the range of about 10 ⁇ M to 100 ⁇ M, such as about 20 ⁇ M to 75 ⁇ M, such as about 25 ⁇ M to 50 ⁇ M, such as about 25 ⁇ M or 50 ⁇ M. Measurements may be carried out at a pH in the range of about pH 7 to about pH 8, such as about pH 7.4. Circular dichroism data may be processed according to Equation B and/or Equation C as described herein. Helicity may be measured at a temperature of about 4 °C to about 50 °C, such as about 20 °C to about 40 °C, such as about 25 °C or about 37 °C.
  • a group of Formula (I) may be functionalised at one or both terminals, and optionally may be attached to one or more amino acid residues at either end.
  • the invention also provides a polypeptide of Formula (Ia) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue;
  • -R A is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker;
  • -R B is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and
  • m and m’ are each independently an integer from
  • N-methylated amino acid residue is an amino acid residue as described herein, having a monomethylated amino group.
  • methylated amino group refers to the amino group within a connecting amide bond or at the terminal, such as in the peptide backbone, and not an amino group within the side chain of the residue.
  • each -A’- is independently an ⁇ -amino acid residue or an N-methylated ⁇ -amino acid residue.
  • Each of -R 3 and -R 3 may be -H. In embodiments where m is an integer of 1 or more, preferably -R 3 is -H. In embodiments where m’ is an integer of 1 or more, preferably -R 4 is -H.
  • the moieties represented by -[A’] m - and -[A’] m' - may independently each comprise zero or one N-methylated amino acid residues, and the remaining -A’- groups are independently an amino acid residue.
  • the moieties represented by -[A’] m - and -[A’]m'- independently may each comprise one N-methylated amino acid residue and the remaining -A’- groups are independently an amino acid residue.
  • the moiety represented by -[A’]m- or -[A’]m'- comprises an N-methylated amino acid residue
  • the N-methylated amino acid residue is a terminal residue.
  • each -A’- is an independently an amino acid residue and -R 3 and -R 4 are each independently -H.
  • -A’- may be represented by -A- and the polypeptide may be represented by Formula (Ia’).
  • -R 1 , -R 2 , -A-, -L 1 -, -L 2 -, n, m, m’, R A and R B are as described herein.
  • An alkyl group may be a C1-4 alkyl group, such as methyl, ethyl, iso-propyl or tert-butyl, such as methyl or ethyl.
  • a protecting group attached to an amino group may be a carbamate protecting group, such as Fmoc (9-fluorenylmethoxycarbonyl), Boc (tert- butyloxycarbonyl) or (Cbz (carboxybenzyl).
  • a protected amino group may be a carbamate protected amino group, such as -NHFmoc, -NHBoc or -NHCbz.
  • An acyl group may be formyl, acetyl or propionyl, such as acetyl.
  • a linker may comprise one or more C 2-4 oxyalkylene groups, such where the linker is a PEG linker.
  • a linker may be one or more groups selected from C1-20 alkylene, ether, carbonyl, amide and ester.
  • a linker is selected from beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and PEG linkers such as PEG2, PEG3, or PEG4.
  • GABA 4-aminobutyric acid
  • AEA (2-aminoethoxy) acetic acid
  • Ava 5-aminovaleric acid
  • 6aminohexanoic acid Ahx
  • a linker may be protected.
  • a linker is protected such as at a nitrogen atom, such as with a protecting group selected from Fmoc and Boc.
  • the linker which is attached to the polypeptide may be capable of being deprotected and can undergo a reaction to link the polypeptide to a moiety.
  • -R A is a protected linker, such as an Fmoc protected 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), or 6-aminohexanoic acid (Ahx) linker.
  • GABA 2-aminoethoxy
  • AEA (2-aminoethoxy) acetic acid
  • Ava 5-aminovaleric acid
  • 6aminohexanoic acid (Ahx) linker 6-aminohexanoic acid
  • -R A is an Fmoc protected 6-aminohexanoic acid Ahx linker (-Ahx-Fmoc).
  • a linker is unprotected, and may be a linker between an end group and a tag or a solid phase.
  • Each of -R A and -R B may independently be a tag.
  • a tag may be a chromophore, a fluorescent label, a phosphorescent label or a radiolabel.
  • a tag may comprise a group for targeting the polypeptide to a cell membrane, such as where the tag is a lipid group, such as farnesyl or geranyl.
  • a tag may be connected, such as to an amino acid residue -A- or a peptide terminal, by a linker.
  • a linker may be 6-aminohexanoic acid or a PEG linker.
  • a linker is preferably between the terminal nitrogen of a polypeptide of Formula (Ia) and a chromophore, a fluorescent label, a phosphorescent label or a radiolabel, or the linker may be between the C terminal of Formula (Ia) and a chromophore, a fluorescent label, a phosphorescent label or a radiolabel.
  • -R A is selected from -H, C 1-4 alkyl, -Fmoc, -L-Fmoc, acetyl, and -L-X T , where -X T is a tag selected from chromophore; a fluorescent or a phosphorescent label and a radiolabel; and a lipid group such as farnesyl and geranyl, and where -L- is a linker.
  • a linker is selected from a covalent bond, beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and a PEG linker such as PEG2, PEG3, or PEG4.
  • GABA 4-aminobutyric acid
  • AEA (2-aminoethoxy) acetic acid
  • Ava 5-aminovaleric acid
  • Ahx 6-aminohexanoic acid
  • PEG linker such as PEG2, PEG3, or PEG4.
  • the group -R A may be selected from -H, C 1-4 alkyl, acetyl, -Fmoc, -Ahx-Fmoc, -beta-alanine-Fmoc, -Ahx-FITC, and -beta-alanine-FITC; such as acetyl, -Ahx-Fmoc, -beta-alanine-Fmoc, -Ahx-FITC, and -beta-alanine-FITC.
  • -R A is -H, the polypeptide has an unmodified N terminal.
  • -R B may be selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a tag optionally with a linker, and a solid phase optionally with a linker.
  • -R B is selected from hydroxyl and amino, and a solid phase optionally with a linker.
  • -R B is hydroxyl the polypeptide has an unmodified C terminal. This terminal may also be in carboxylate form.
  • R B is amino, the polypeptide has a C-terminal amide modification.
  • -R B may be a solid phase with a linker present between the solid phase and the carbonyl to which -R B is attached to.
  • a linker may be, or may comprise, a group selected from -O-, -N(H)-, or -N(Me)-.
  • R B is selected from -O-L-P SP and -N(H)-L-P SP , where -L- is a linker and -P SP is a solid phase.
  • -R B is selected from hydroxyl, amino, -O-L-P SP and -N(H)-L-P SP , where -L- is a linker and -P SP is a solid phase.
  • the group -R B may be selected from hydroxyl and amino.
  • An alkoxy group may be C1-4 alkoxy, such as methoxy or ethoxy.
  • each of m and m’ is independently an integer from 0 to 100, such as 1 to 100, such as 2 to 100, such as 2 to 75, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 10, such as 2 to 8.
  • m and m’ are each greater than 1.
  • Preference for amino acid stereochemistry is as described herein, such as according to one or more of Formula (I ), (I ), (I ) and (I ).
  • the ⁇ -carbon bonded to -R and -L 2 - is in a S configuration, and the ⁇ -carbon bonded to -R 1 and -L 1 - is in a S or R configuration, more preferably S.
  • the polypeptide is of Formula (Ib) or Formula (Ib’) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -A’-, -L 1 -, -L 2 -, -R A , -R B , n, m and m’ are as described herein, and z is an integer from 1 to 4.
  • z is an integer from 1 to 3, more preferably 1 or 2, most preferably 1.
  • m’ is not 0, and preferably m’ is 2 or more.
  • z is greater than 1, preferably m’ in a moiety that is positioned between two diyne staples is an integer of 5 or more, such as 6 or more, such as 7 or more.
  • the polypeptide comprises Formula (IRS’), (ISS’) or a mixture thereof wherein -R 1 and -R 2 are independently selected from methyl and -H, such as methyl; each -A- is independently an amino acid residue; -L 1 - and -L 2 - are independently C 4-6 alkylene; and n is an integer from 3 to 6, such as 3 or 6, such as 6.
  • polypeptide is according to Formula (IaRS’), (IaSS’) or a mixture thereof 008528580 wherein -R A and -R B are as defined herein, and -R 1 and -R 2 are independently selected from methyl and -H, such as methyl; each -A- is independently a natural or non-natural amino acid residue; -L 1 - and -L 2 - are independently C4-6 alkylene; and n is an integer from 3 to 6, such as 3 or 6, such as 6.
  • polypeptides include a polypeptide of Formula (Id), (IdRS) or (IdSS), wherein -R 1 , -R 2 , -L 1 - and -L 2 - are as defined herein, and: (i) -R A is selected from -H, an amino protecting group, acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a fluorescent tag optionally with a linker; -[A]m- is -Leu-Thr-Phe-; -[A]n- is -Glu-Tyr-Trp-Ala-Gln-Cba-; -[A]m’- is -Ser-Ala-Ala-; and -R B is selected from hydroxy, amino, protected amino, -O-L-P SP and -N
  • polypeptide Comprising a Group of Formula (II) In an aspect the present invention provides a polypeptide comprising a group of Formula (II) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein, and the salts and solvates and protected forms thereof.
  • a polypeptide comprising a group of Formula (II) may be referred to as a linear polypeptide.
  • a linear polypeptide may be a precursor to a stapled polypeptide, and the terminal alkynyl resides are available for cyclisation to form the corresponding stapled polypeptide.
  • Preferences for -L 1 -, -L 2 -, -R 1 , -R 2 , -A-, n, stereochemistry at the ⁇ -carbons, and linkages at the N-terminal and C-terminal of the segment shown in Formula (II) are as described herein for polypeptides comprising a group of Formula (I).
  • the groups -L 1 - and -L 2 - are preferably independently C4-10 alkylene, such as C5 or C6 alkylene.
  • the groups -L 1 - and -L 2 - are independently C5-10 alkylene, such as C5-8 alkylene.
  • N is preferably an integer from 2 to 8, such as from 3 to 8, such as from 3 to 6, and more preferably n is 3 or 6, such as 6.
  • Each of -R 1 and -R 2 is preferably independently selected from methyl and -H. More preferably, -R 1 and -R 2 are methyl.
  • the polypeptide may comprise a group of Formula (IIXS), where the ⁇ -carbon that is bonded to -R 2 and -L 2 - is in a S configuration, and the ⁇ -carbon that is bonded to -R 1 and -L 1 - may be in a R or S configuration, or a mixture thereof.
  • -R 1 , -R 2 , -R 3 , -R 4 , -L 1 -, -L 2 -, -A- and n are as defined herein.
  • the polypeptide may be a diastereomer according to any one of Formula (IIRR), (IIRS), (IISR) and (I SS ) 008528580 where -R 1 , -R 2 , -R 3 , -R 4 , -L 1 -, -L 2 -, -A- and n are as defined herein.
  • the polypeptide comprises Formula (II RS ), (II SR ) and/or (II SS ), more preferably Formula (IIRS) and/or (IISS), and most preferably Formula (IISS).
  • the remaining amino acid residues in the polypeptide may be in a D-configuration or an L-configuration, and preferably are in the L-configuration.
  • a peptide segment of Formula (II) may be functionalised at either end. One or both ends of the peptide segment may be bonded to one or more amino acid residues.
  • the invention also provides a polypeptide of Formula (IIa), (IIa’), (IIb) or (IIb’) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein, and each -A’- is independently an amino acid residue or an N-methylated amino acid reside;
  • -R A is selected from -H, a protecting group, acyl, C 1-4 alkyl, a protected linker, and a tag optionally with a linker;
  • -R B is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and
  • m and m’ are each independently an integer from 0 to 200; 008528580 wherein -R 1 , -R 2
  • the group -R A is selected from -H, C1-4 alkyl, -Fmoc, -L-Fmoc, acetyl, and -L-X T , where -X T is a tag selected from chromophore; a fluorescent or a phosphorescent label and a radiolabel; and a lipid group such as farnesyl and geranyl, and where -L- is a linker.
  • the group -R B may be selected from hydroxyl, amino, -O-L-P SP and -N(H)-L-P SP , where -L- is a linker as described herein and -P SP is a solid phase.
  • a linker is selected from a covalent bond, beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and a PEG linker such as PEG2, PEG3, or PEG4.
  • GABA 4-aminobutyric acid
  • AEA (2-aminoethoxy) acetic acid
  • Ava 5-aminovaleric acid
  • 6aminohexanoic acid Ahx
  • PEG linker such as PEG2, PEG3, or PEG4.
  • Each of m and m’ may be independently an integer from 0 to 100, such as 1 to 100, such as 2 to 100, such as 2 to 75, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 10, such as 2 to 8.
  • Z may be an integer from 1 to 3, more preferably 1 or 2, most
  • polypeptide is according to Formula (IIc) where R 1 , -R 2 , -A-, -L 1 -, -L 2 - and n are as described herein.
  • polypeptide is according to Formula (IiaRS’), (IiaSS’) or a mixture thereof wherein -R A and -R B are as defined herein, and -R 1 and -R 2 are independently selected from methyl and -H, such as methyl; each -A- is independently a natural or non-natural amino acid residue; 008528580 -L 1 - and -L 2 - are independently C 4-6 alkylene; and n is an integer from 3 to 6, such as 3 or 6, such as 6.
  • polypeptides include a polypeptide of Formula (IId), (IIdRS) or (IIdSS), (i) -R A is selected from -H, an amino protecting group, acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a fluorescent tag optionally with a linker; -[A] m - is -Leu-Thr-Phe-; -[A]n- is -Glu-Tyr-Trp-Ala-Gln-Cba-; -[A]m’- is -Ser-Ala-Ala-; and -R B is selected from hydroxy, amino, protected amino, -O-L-P SP and -N(H)-L-P SP , where -L- is a linker, such as a covalent bond, and
  • -R 1 and -R 2 are independently methyl and/or -L 1 - and -L 2 - are independently selected from C 5-6 alkylene.
  • Amino Acid Disclosed herein is a compound of Formula (A) wherein -L Y - is optionally substituted C 2-10 alkylene; -R 1 is selected from optionally substituted C 1-4 alkyl and -H; -R 3 is selected from -H and methyl; -R N is selected from -H, a protecting group, acyl, C 1-4 alkyl, a protected linker, and a tag optionally with a linker; such as -H, Fmoc, Boc, methyl, ethyl, iso-propyl, acetyl, farnesyl and geranyl; and -R C is selected from -OH and a protecting group, or -R C together with the carbonyl group -R C is attached to forms an activated acid, and the salts
  • -L Y - may be an optionally substituted C4-10 alkylene, such as C5 alkylene or C6 alkylene.
  • R 1 may be methyl or -H, such as methyl.
  • R 3 may be -H. 008528580
  • the invention provides a compound of Formula (III) wherein -L X - is optionally substituted C5-10 alkylene; -R 1 is selected from optionally substituted C 1-4 alkyl and -H; -R 3 is selected from -H and methyl; -R N is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker, such as -H, Fmoc, Boc, methyl, ethyl, iso-propyl, acetyl, farnesyl and geranyl; and -R C is selected from -OH and a protecting group, or -R C together with the carbon
  • a compound of Formula (A) or Formula (III) is a non-natural amino acid or protected non-natural amino acid, and is suitable for use in the synthesis of linear polypeptides, and in turn, stapled polypeptides as described herein.
  • Preference for -R 1 , -R3, -LX and stereochemistry may be as described herein for a polypeptide comprising a group of Formula (I) or (II).
  • Preference for -R N may be as described herein for -R A for a polypeptide comprising a group of Formula (I) or (II).
  • -L X - may be C 5-9 alkylene, such as C 5-8 alkylene, such as C 5 or C 6 alkylene.
  • -L X - is C 6-10 alkylene, more preferably C 6-9 alkylene, more preferably C 6-8 alkylene, such as C6 or C7 alkylene. Most preferably, -L X - is C6 alkylene.
  • the group -R 1 may be selected from -H, methyl, ethyl, n-propyl, i-propyl, or n-butyl, s-butyl or tert-butyl.
  • -R 1 is selected from -H, methyl and ethyl, more preferably from -H and methyl, and most preferably -R 1 is methyl.
  • the group -R N may be -H or a protecting group.
  • -R N is selected from -H, Fmoc and Boc.
  • the group -R N may be an acyl group such as a C 1-12 acyl group, such as formyl, acetyl or propionyl.
  • -R N is acetyl.
  • -R N is -H.
  • -R N may be a tag that is optionally with a linker.
  • a tag may be selected from: a chromophore; a fluorescent label; a phosphorescent label; a radiolabel; or a group for targeting the compound to a cell membrane, such as a lipid group, such as farnesyl or geranyl.
  • a linker where present, links the tag and the nitrogen atom to which -R N is attached.
  • -R N comprises a fluorescent label and a linker, such as fluorescein-5-isothiocyanate (FITC) and a linker which may be 6-aminohexanoic acid (Ahx) or beta-alanine.
  • FITC fluorescein-5-isothiocyanate
  • Ahx 6-aminohexanoic acid
  • beta-alanine alanine
  • -R N may be FITC-Ahx- or FITC-beta-alanine-.
  • -R C may be selected from -OH, -NH 2 and a protecting group.
  • protecting groups include -Ome, -O t Bu and benzyloxy (Obn).
  • the group -R C together with the carbonyl group to which it is attached may form an activated acid.
  • activated acids include acyl chloride, 1-hydroxybenzotriazole activated ester, and carbodiimide activated ester.
  • -L X is C5-10 alkylene, such as C5 alkylene; or C6-10 alkylene, such as C6 alkylene; -R 1 is selected from methyl or -H, such as methyl; -R 3 is -H; -R N is selected from Fmoc and Boc, farnesyl and geranyl, such as Fmoc and Boc; and -R C is selected from -OH and a protecting group, or -R C together with the carbonyl group to which it is attached forms an activated acid.
  • a compound of Formula (A) or Formula (III) may be compound A or compound B. In some embodiments, the compound is not compound A or compound B.
  • a compound of Formula (A) or Formula (III) may be in a R configuration or an S configuration.
  • the invention provides a compound of Formula (IIIR) and a compound of Formula (IIIS). (IIIR) (III S ) where -R N , -R 1 , -R 3 , and -L X - and -R C are as described herein.
  • the compound is of Formula (IIIa), (IIIa R ) or (IIIa S ). where -R N , -R 1 , -R 3 , and -L X - are as described herein.
  • Compounds of Formula (III) may be prepared by a method as described herein, such as by reaction of an Ni(II) Schiff base complex derived from an amino acid, such as alanine, glycine, homoalanine and valine, with an iodo-alkyne.
  • an amino acid such as alanine, glycine, homoalanine and valine
  • Polypeptides and compounds of the invention may be provided in salt form, solvate from or protected form.
  • Compounds having a carboxyl group, such as polypeptides comprising a group of Formula (I) or (II) or compounds of Formula (III) may be provided as salts, for example base addition salts of strong mineral bases and addition salts of strong organic bases.
  • Compounds having an amine group such as the polypeptides comprising a group of Formula (I) or (II) or compounds of Formula (III) may be provided as salts, for example acid 008528580 addition salts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt.
  • Further examples of salts include sulfates and acetates such as trifluoroacetate or trichloroacetate.
  • a reference to a compound or polypeptide described herein is also a reference to a solvate of that compound. Examples of solvates include hydrates.
  • a protected form of a compound or polypeptide may comprise an amino protecting group, such as benzyloxycarbonyl (Cbz), Fmoc or Boc; a carboxyl protecting group, such as alkoxy, such as -Ome or -Obn; and/or a side chain protecting group, such as benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), 9-xanthenyl (Xan), tosyl (Tos), benzyloxymethyl (Bom), formyl, tert-butyldimethylsilyl (TBDMS), allyl, o-nitrobenzyl (ONB), p-methylbenzyl (Meb) and acetamidomethyl (Acm).
  • an amino protecting group such as benzyloxycarbonyl (Cbz), Fm
  • a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof.
  • Methods for the preparation e.g., asymmetric synthesis
  • separation e.g., fractional crystallisation and chromatographic means
  • the present invention provides an enantiomer or diastereomer of the polypeptides or compounds described herein.
  • An enantiomer as described herein may be substantially pure, and may have an enantiomeric excess of 70% or more, such as 80% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 98% or more, such as 99% or more.
  • Methods of Synthesis In one aspect, there is provided a method of preparing a diyne stapled polypeptide, such as a stapled peptide of Formula (I), the method comprising steps of (i) providing a polypeptide of Formula (II); and (ii) coupling the terminal alkynyl groups of the polypeptide to provide a 1,3-diyne linkage.
  • the method is for preparing a diyne of a stapled peptide comprising a group of Formula (I).
  • Step (ii) may be carried out in the presence of a metal salt, such as a transition metal salt, such as: a copper salt, such as a Cu(I) salt; a cobalt salt, such as a Co(II) salt; a silver salt, such as Ag(I) salt; and a palladium salt, such as a Pd(II) salt, such as described in Akhtar et al.
  • a metal salt such as a transition metal salt, such as: a copper salt, such as a Cu(I) salt; a cobalt salt, such as a Co(II) salt; a silver salt, such as Ag(I) salt; and a palladium salt, such as a Pd(II) salt, such as described in Akhtar et al.
  • a copper salt may be a Cu(I) salt, examples of which include CuCl, CuBr, CuI, CuOAc, Cu 2 (ophen) 2 , Cu 4 (ophen) 4 (tp).
  • Step (ii) may be carried out in the presence of a source of Cu(I).
  • a source of Cu(I) include Cu(II) salts, such as CuSO4, Cu(OAc)2 and CuCl2, optionally together with a reducing agent.
  • a metal salt, where present in step (ii) may be provided in a stoichiometric amount relative to the polypeptide, or the metal salt may be present at an amount that is less than a stoichiometric amount. The metal salt may be regenerated during the radical reaction.
  • the metal salt may be present at an amount that is greater than a stoichiometric amount. This may help to increase the yield of the stapled polypeptide.
  • Step (ii) may be carried out in the presence of a ligand, and preferably is so when a metal salt such as a copper salt is present.
  • a ligand where present, may be 4,4’-bis(hydroxymethyl)-2,2’-bipyridine, 2,2’-bipyridine, tetramethylethylenediamine (TMEDA) or pyridine.
  • the ligand is 4,4’-bis(hydroxymethyl)-2,2’-bipyridine or tetramethylethylenediamine (TMEDA).
  • step (ii) is carried out in the presence of an oxidant, such as oxygen.
  • Step (ii) may be carried out in the presence of air, or in an oxygen atmosphere.
  • Step (ii) may be carried out in the presence of a base, typically an organic base, such as a tertiary amine, such as N,N-diisopropylethylamine (DIPEA), triethylamine and piperidine.
  • Step (ii) may be performed at a temperature of 20 °C or more, such as 30 °C or more, such as 40 °C or more, such as 50 °C or more. The temperature may be 80 °C or less, such as 70 °C or less.
  • the temperature may be in a range with upper and lower values described above, such as from 20 °C to 80 °C, including 50 °C to 70 °C.
  • the temperature during step (ii) is about 60 °C.
  • the reaction mixture may be heated, such as by microwave heating.
  • Step (ii) may be carried out in the presence of microwave radiation.
  • the reaction may be subjected to microwave radiation during step (ii).
  • the reaction time during step (ii) may be 5 minutes or more, such as 10 minutes or more, such as 30 minutes or more, such as 1 hour or more, such as 2 hours or more, such as 3 hours or more.
  • step (ii) two terminal alkynes in the polypeptide, in particular in side chains of the polypeptide, are coupled together and a 1,3-diyne is formed.
  • the coupling reaction provides a stapled peptide.
  • more than two terminal alkyne groups are present in the polypeptide, such as where the polypeptide provided in step (i) is of Formula Iib where z is an integer of 2 or more, typically any two alkynyl groups that are spaced by up to i and i+10 and/or at least i and i+3 may be coupled to form a 1,3-diyne linkage.
  • a polypeptide provided in step (i) may be prepared by peptide synthesis, such as by solid phase peptide synthesis using one or more amino acids of Formula (A) or Formula (III) as described herein.
  • a polypeptide provided in step (i) may be provided attached to a solid phase, or the polypeptide may be provided with free N terminal and a free C terminal, such as wherein the polypeptide has been cleaved from a solid phase following solid phase synthesis.
  • the polypeptide provided in step (i) is provided attached to a solid phase, such as at the C terminal.
  • step (i) comprises synthesising a polypeptide comprising a group of Formula (II), such as a polypeptide of Formula (Iia) or (Iib), by peptide synthesis as described herein using an amino acid of Formula (III) and/or Formula (A).
  • Step (i) may further comprise cleaving the polypeptide from a solid phase.
  • Step (ii) may be carried out on-resin, such as wherein the polypeptide of Formula (II) is attached to a solid phase at the C terminal of the polypeptide, or step (ii) may be carried out off-resin, such as wherein the linear polypeptide has been cleaved from a resin used during solid phase synthesis.
  • step (ii) is carried out on-resin, such as wherein the polypeptide of Formula (II) is attached to a solid phase, and the stapled peptide formed during step (ii) may be cleaved from the solid phase.
  • the N terminal, C terminal, and one or more side chains in the polypeptide may independently be protected during the coupling in step (ii).
  • the N terminal is protected, and the C terminal and one or more side chains are optionally also protected.
  • the method may further comprise a step of deprotecting the polypeptide following step (ii).
  • Coupling in step (ii) may be carried out on a polypeptide having a N terminal protected polypeptide, such as a Fmoc protected polypeptide.
  • a method of preparing a diyne stapled peptide comprising steps of (i) providing a polypeptide comprising a group of Formula (II) wherein -R 1 , -R 2 , -R 3 , -R 4 , -A-, -L 1 -, -L 2 - and n are as described herein; and (ii) contacting the polypeptide with a metal salt, optionally in the presence of a ligand.
  • An amino acid may be prepared by a method as described herein, such as by reaction of an Ni(II) Schiff base complex derived from an amino acid with an iodo-alkyne.
  • Methods of Treatment The polypeptides comprising a group of Formula (I) and pharmaceutical compositions described herein are suitable for use in methods of treatment and prophylaxis.
  • the polypeptides or pharmaceutical compositions may be administered to a subject in need thereof.
  • the polypeptides comprising a group of Formula (I) or pharmaceutical compositions are for use in a method of treatment of the human or animal body by therapy.
  • a polypeptide comprising a group of Formula (I) or pharmaceutical composition may be administered to a mammalian subject, such as a human, in order to treat a proliferative disease, such as cancer.
  • a polypeptide comprising a group of Formula (I) such as a polypeptide of Formula (Id), (Id RS ) or (Id SS ), is for use in a method of treating cancer, wherein the polypeptide inhibits dimerization of Sam68.
  • the polypeptide or pharmaceutical composition comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS), is for use in a method of treatment cancer wherein the polypeptide inhibits interaction between p53 and MDM2, such as where the polypeptide inhibits p53/MDM2 binding.
  • a polypeptide comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS) is for use in a method of treating a disease with altered Sam68 activity or altered p53 and/or MDM2 activity.
  • the disease may be cancer.
  • a polypeptide comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS), is for use in a method of treatment wherein the polypeptide inhibits Sam68 activity or inhibits MDM2 activity.
  • Another aspect of the present invention pertains to use of a polypeptide comprising a group of Formula (I) in the manufacture of a medicament for use in treatment.
  • the medicament comprises a polypeptide comprising a group of Formula (I).
  • the polypeptides of the present case may be useful for the treatment of a proliferative disease, such as cancer.
  • treatment pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis
  • prophylaxis is also included.
  • treatment For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term “treatment.”
  • the term “therapeutically-effective amount,” as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • treatment includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • Formulations The present invention also provides a pharmaceutical composition comprising a polypeptide comprising a group of Formula (I) together with a pharmaceutically acceptable carrier.
  • the polypeptide comprising a group of Formula (I) While it is possible for the polypeptide comprising a group of Formula (I) to be administered alone or together with the second agent, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising a polypeptide comprising a group of Formula (I) as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but 008528580 not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
  • a pharmaceutical formulation e.g., composition, preparation, medicament
  • pharmaceutically acceptable carriers diluents, excipients, adjuvants, fillers, buffers, preserv
  • the formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
  • the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing a polypeptide comprising a group of Formula (I) as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.
  • the composition optionally further comprises the second active agent in a predetermined amount.
  • pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, etc.
  • the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the polypeptide comprising a group of Formula (I) with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
  • carriers e.g., liquid carriers, finely divided solid carrier, etc.
  • Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other 008528580 pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers.
  • Formulations may also suitably be provided in the form of a depot or reservoir. The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients.
  • Formulations suitable for oral administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
  • Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavoured basis, usually sucrose and acacia or tragacanth.
  • Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia.
  • Mouthwashes typically comprise the compound in a suitable liquid carrier.
  • Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
  • Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in- water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
  • Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.
  • Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
  • Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat.
  • Formulations suitable for intranasal administration include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.
  • a dry powder delivery may be used as an alternative to nebulised aerosols.
  • Formulations suitable for intranasal administration, where the carrier is a solid include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Formulations suitable for pulmonary administration include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro- tetrafluoroethane, carbon dioxide, or other suitable gases.
  • a formulaton for pulmonary administration may be formulated for administration from a nebuliser or a dry powder inhaler.
  • the formulation may be provided with carriers or liposomes to provide a suitable particle size to reach the appropriate parts of the lung, to aid delivery of an appropriate does to enhance retention in the lung tissue.
  • Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.
  • Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
  • sterile liquids e.g., solutions, suspensions
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection.
  • the concentration of the compound in the liquid is from about 1 ng/mL to about 100 ⁇ g/mL, for example from about 10 ng/mL to about 10 ⁇ g/mL, for example from about 10 ng/mL to about 1 ⁇ g/mL.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • Routes of Administration A polypeptide comprising a group of Formula (I), a second agent, or a pharmaceutical composition comprising the polypeptide comprising a group of Formula (I), may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
  • Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular
  • the subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine 008528580 (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, bab
  • the subject/patient may be any of its forms of development, for example, a foetus.
  • the subject/patient is a human.
  • the invention may be practised on a non-human animal having a microbial infection.
  • a non-human mammal may be a rodent.
  • Rodents include rats, mice, guinea pigs, chinchillas and other similarly-sized small rodents used in laboratory research. Definitions
  • An alkyl group is monovalent alicyclic or cyclic saturated hydrocarbon group.
  • An alkyl group may be a C 1-20 alkyl group, for example a C 1-15 , C 1-12 , C 1-10 , C 1-8 , C 1-6 , C 1-4 , C 1-3 or a C 1-2 alkyl group.
  • the prefix e.g. C1-6
  • An alkyl group may be linear, branched or cyclic. Examples of C1-6 linear alkyl groups include methyl (-Me), ethyl (-Et), n-propyl (-nPr), n-butyl (-nBu), n-pentyl (-Amyl) and n-hexyl.
  • C 1-6 branched alkyl groups include iso- propyl (-iPr), iso-butyl (-iBu), sec-butyl (-sBu), tert-butyl (-tBu), iso-pentyl, sec-pentyl, tert- pentyl, neo-pentyl, iso-hexyl, sec-hexyl, tert-hexyl and neo-hexyl.
  • Examples of C 1-6 cyclic alkyl groups include cyclo-pentyl and cyclo-hexyl.
  • An alkylene group is a divalent saturated hydrocarbon group in which the two free valencies independently form part of a single bond to separate adjacent atom.
  • An alkylene group is a divalent group that may be derived from an alkyl group as defined above, such as by removal of a hydrogen atom.
  • Examples of linear alkylene groups include methanediyl (methylene bridge), ethane-1,2-diyl (ethylene bridge), propane-1,3-diyl, butan-1,4-diyl, pentan-1,5-diyl and hexan-1,6-diyl.
  • Examples of branched alkylene groups include ethane- 1,1-diyl and propane-1,2-diyl.
  • An alkynyl group is a monovalent unsaturated hydrocarbon group containing one or more carbon-carbon triple bonds.
  • An alkynyl group may be a C 2-20 alkynyl group, for example a C2-10, C2-6 or a C2-4 alkynyl group.
  • the prefix e.g. C1-6
  • An alkynyl group may be linear or branched, or the alkenyl group may be incorporated into a ring system. Examples of linear alkynyl groups include ethynyl and 2-propynyl (propargyl).
  • alkynyl groups incorporated into a ring system include cyclooctyne (OCT).
  • a hydroxyl group is -OH or the hydroxide form of this group.
  • Examples of acyl groups include formyl, acetyl (-Ac), propionyl, tert-butyryl and benzoyl (-Bz).
  • An oxyalkylene group is an alkylene group in which one or more carbon atoms is replaced with a oxygen.
  • the oxyalkylene group may be a C 1-6 oxyalkylene group, for example, a C 1-4 or a C1-3 oxyalkylene group.
  • the prefix e.g. C1-6
  • the oxyalkylene group may be linear or branched. Examples of linear oxyalkylene groups include those derived from oxymethylene (e.g. polyoxymethylene, POM), ethylene glycol (e.g. polyethylene glycol, PEG), and tetramethylene glycol (e.g.
  • An ether group comprises the divalent moiety -O-.
  • An ether group is -OR E , where R E is selected from hydrogen, C1-6 alkyl and C5-20 aryl.
  • a protecting group may be selected from 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), 9-xanthenyl (Xan), tosyl (Tos), benzyloxymethyl (Bom), formyl, tert-butyldimethylsilyl (TBDMS), allyl, o-nitrobenzyl (ONB), p-methylbenzyl (Meb) or acetamidomethyl (Acm).
  • a nitrogen protecting group such as an N-terminal protecting group, may be selected from 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
  • a side chain protecting group may 008528580 be selected from allyloxycarbonyl (Alloc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5- sulfonyl (Pbf), 9-xanthenyl (Xan), tosyl (Tos), benzyloxymethyl (Bom), formyl, tert- butyldimethylsilyl (TBDMS), allyl, o-nitrobenzyl (ONB), p-methylbenzyl (Meb) or acetamidomethyl (Acm).
  • a C terminal protecting group may be an ester and may be selected from methyl ester and benzyl ester.
  • Trifluoroacetic acid (TFA), N,N’-diisopropylethylamine (DIPEA), N,N’-diisopropylcarbodiimide (DIC), ethyl (hydroxyamino)cyanoacetate (Oxyma Pure), fluorescein-5-isothiocyanate (FITC), Fmoc-Lys(Alloc)-OH, Fmoc-6-Ahx-OH, palladium (O)tetrakis(triphenyphosphine) (Pd(PPh3)4) and phenylsilane were purchased from Fluorochem. Morpholine was purchased from Alfa Aesar. Dichloromethane (DCM) was purchased from VWR.
  • DIPEA N,N’-diisopropylethylamine
  • DIPEA N,N’-diisopropylcarbodiimide
  • ethyl (hydroxyamino)cyanoacetate Oxy
  • Acetonitrile was purchased from Fisher Scientific.
  • TentaGel S RAM resin was purchased from Rapp 008528580 Polymere.
  • Chemmatrix Rink Amide resin was purchased from Biotage. All other reagents were purchased from Sigma Aldrich.
  • S-RBD was purchased from Genscript (S-RBD, P330-F541, Product no. Z03479) and AcroBiosystems (S-RBD, R319-F541, Product no. SPD-C52H3). Dry solvents were purified using a PureSolv 500 MD solvent purification system.
  • a linear gradient of 5-95% B over 20 min with a flow rate of 1 mL/min was used with a Reprosil-Gold column (3 mm C18, 150 ⁇ 4 mm).
  • IR Infrared spectroscopy
  • FTIR-8400S Shimadzu Fourier Transform Infrared Spectrophotometer
  • 008528580 Microwave reactions were completed in a CEM Explorer 12 Hybrid Microwave.
  • Optical rotation was determined using an Autopol V polarimeter.
  • Peptide content was analysed on a Nanodrop 2000c using UV absorption of peptides at 280 nm or 214 nm. Samples were centrifuged at 4,500 RPM in a Heraeus Megafuge 8 centrifuge purchased from Thermo Fisher Scientific. Fluorescence polarisation was conducted on a CLARIOstar plate reader purchased from BMG Labtech.
  • CD spectra were obtained at room temperature using a JASCO J-810 CD spectrometer. A range of 190 – 260 nm was scanned at a speed of 50 nm/min, with a 1 nm data pitch, a 1 nm bandwidth and an 8 s response time. Samples were prepared in phosphate buffered saline (PBS; pH 7.4), and CD spectra measured in a 0.2 mm quartz cuvette. Raw data (mdeg) were converted to mean residue ellipticity (MRE; deg cm 2 dmol -1 res -1 ) by normalizing for path length, peptide concentration, and number of amide bonds.
  • PBS phosphate buffered saline
  • Amino Acid Coupling of Fmoc-protected amino acids (5 equiv., 0.2 M in DMF) and unnatural/orthogonally-protected amino acids (2 equiv., 0.1 M in DMF) was achieved by treatment with DIC (5 equiv., 0.5 M in DMF) and Oxyma Pure (5 equiv., 0.5 M in DMF) at 90 °C for 2 min. His residues were coupled at 50 °C for 10 min. Amino acids following unnatural amino acids were double coupled. Arg residues were coupled at room temperature for 45 min, followed by 90 °C for 5 min, then double coupled at 90 °C for 10 min.
  • Fmoc Deprotection was achieved by treatment with morpholine (20% in DMF with 5% formic acid, 4 mL) at 90 °C for 1 min. The resin was washed with DMF between deprotection and coupling (4 ⁇ 4 mL), and after coupling (2 ⁇ 4 mL).
  • 008528580 N-Terminal Acetylation (capping) Peptides requiring N-terminal acetylation were treated on-resin with acetic anhydride (3 equiv.), DIPEA (4.5 equiv.) and DMF (7 mL for 0.1 mmol of resin) for 20 min with agitation.
  • Peptide on resin (1 equiv., 0.1 mmol) was added to a microwave vial followed by a solution of CuCl (5 equiv.) and 4,4’-bis(hydroxymethyl)-2,2’-bipyridine (7.5 equiv.) in DMF (10 mL) and DIPEA (20 equiv.). The reaction was heated to 60 °C for 3 h in a microwave reactor and excess reagents filtered off.
  • Peptides containing cysteines were cleaved with a cocktail of TFA (94 v%), TIPS (1 v%), EDT (2.5 v%) and H2O (2.5 v%).
  • Peptides containing the amino acid Asu were cleaved with a cocktail of TFA (94 v%), TIPS (2.5 v%), EDT (2.5 v%) and dry DCM (1 v%).
  • the resin was subsequently filtered and the 008528580 TFA evaporated using a stream of N 2 , the peptide was precipitated with cold Et 2 O and centrifuged (4,500 rpm for 5 min).
  • Peptides were dissolved in a mixture of H2O and MeCN with 0.1% TFA and lyophilized on a Christ Alpha 2-4 LO plus freeze dryer.
  • Peptide Full Cleavage and Global Deprotection Peptides were cleaved from the resin using a cocktail (10 mL) of TFA (95 v%), TIPS (2.5 v%) and H2O (2.5 v%) for 3-4 h as stated with agitation.
  • Peptides containing cysteines were cleaved with a cocktail of TFA (94 v%), TIPS (1 v%), EDT (2.5 v%) and H2O (2.5 v%).
  • Peptides containing the amino acid Asu were cleaved with a cocktail of TFA (94 v%), TIPS (2.5 v%), EDT (2.5 v%) and dry DCM (1% v). The resin was subsequently filtered and the TFA evaporated using a stream of N 2 , the peptide was precipitated with cold Et 2 O and centrifuged (4,500 rpm for 5 min). Peptides were dissolved in a mixture of H2O and MeCN with 0.1% TFA and lyophilized on a Christ Alpha 2-4 LO plus freeze dryer.
  • LC-MS was performed on a Thermo Scientific LCQ Fleet Ion Trap Mass Spectrometer using positive mode electrospray ionisation (ESI+).
  • General Method C Fluorescence Polarisation (FP) FP was carried out to assess the direct binding kinetics between the peptides and MDM2. The assays were carried out in black, round bottomed, non-binding surface (NBS) 384 well plates. Kd values were calculated using Equation A.
  • Peptides 145-170 Protein (MDM21-138) in 150 mM NaCl, 25 mM Tris, pH 7.6, 1 mM DTT was added and serially diluted across the plate from 10 ⁇ M to 9 pM. Peptide (10 nM in Tris buffer pH 7.6) was then added to every well and mixed using a pipette. Plates were placed in a PerkinElmer Victor X5 plate reader with excitation measured at 531 nm and emission measured at 595 nm. 008528580 Data Analysis. Raw data were normalized using baseline correction, where the baseline was defined as the fluorescence polarisation measured in the absence of peptide.
  • Equation A Y is the fluorescent polarisation signal measured at each datapoint.
  • Rt is the total concentration of (FITC-labelled peptide).
  • X is the total concentration of protein and Kd is the dissociation constant (the inflection point of the curve).
  • General Method D Determination of Binding Affinity using a Fluorescence Polarisation (FP) Assay The binding properties of the peptides were assessed using a biophysical FP assay. Specifically, a direct binding assay was conducted to calculate the K d values of the fluorescent peptide ligands binding to the target proteins.
  • a range of 190-260 nm was scanned at a speed of 50 nm/min, with a 1 nm data pitch, a 1 nm bandwidth and an 8 s response time.
  • Samples were prepared (50 ⁇ M and 25 ⁇ M) in phosphate buffered saline (PBS; pH 7.4), and CD spectra measured in a 1 mm or 0.2 mm quartz cuvette.
  • Raw data (mdeg) were converted to mean residue ellipticity (MRE; deg cm 2 dmol -1 res -1 ) by normalizing for path length, peptide concentration, and number of amide bonds. Percentage helicities can be calculated for ⁇ -helical peptides.
  • Equation B MRE calculation.
  • ⁇ machine units in degrees
  • MRW mean residue weight
  • l path length (cm)
  • c peptide concentration in mg/mL.
  • Equation C (1) % Helicity equation.
  • ⁇ c random coil ellipticity calculation proposed by Luo and Baldwin.
  • ⁇ 222 ⁇ -helix ellipticity calculation determined by Luo and Baldwin reading observed at 222 nm.
  • a picoEmerald S (APE, Berlin, Germany) laser provided both a tunable pump laser (700–990 nm, 2 ps, 80 MHz repetition rate) and a spatially and temporally overlapped Stokes laser (1032nm, 2 ps, 80 MHz repetition rate).
  • the output beams were inserted into the scanning unit of an Olympus FV1000MPE microscope using a series of dielectric mirrors and a 3 ⁇ lens-based beam-expanding module.
  • the resulting 3.6 mm beams were expanded by a further 3.6 ⁇ lens within the microscope and directed into an Olympus XLPL25XWMP N.A.1.05 objective lens using a short-pass 690 nm dichroic mirror (Olympus).
  • the Stokes beam was intensity modulated with a 20 MHz EoM built into the picoEmerald S. Forward scattered light was collected by a further 25 ⁇ Olympus 008528580 XLPL25XWMP N.A.1.05 objective lens and Stokes light was removed by filtering with an ET890/220m filter (Chroma).
  • a telescope focused the light onto an APE silicon photodiode connected to an APE lock in amplifier with the time constant set to 20 ⁇ s.
  • the lock in amplifier signal was fed into an Olympus FV10-Analog unit. Laser powers after the objective were measured up to 20–50 mW for the pump laser and up to 70 mW for the Stokes laser.
  • Triethylamine (Et 3 N) (2.48 mL, 17.8 mmol, 2 equiv.) was added and left to stir for 10 mins at room temperature. The solution was then cooled to 0 °C and methanesulfonyl chloride (MsCl) (1.01 mL, 13.4 mmol, 1.5 equiv.) was added dropwise. The solution was then brought up to room temperature and left to stir under nitrogen overnight. Volatiles were evaporated under reduced pressure. The residue was dissolved in water (H2O) and ethyl acetate (EtOAc) and extracted with EtOAc (3 ⁇ 10 mL).
  • EtOAc ethyl acetate
  • the flask was cooled to 0 °C and dry DMF added (30 mL) before dropwise addition of electrophile (1.52 g, 7.83 mmol, 1.2 equiv.). After 5 mins the ice bath was removed and the flask warmed to rt and stirred for 1 h. The reaction mixture was quenched by pouring into cold 5% acetic acid (50 mL) and concentrated in vacuo. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and the layers separated.
  • the aqueous layer was extracted with DCM (50 mL) and the combined organic layers washed with 5% w/v LiCl solution (3 ⁇ 50 mL), brine (3 ⁇ 50 mL), dried with MgSO 4 and concentrated in vacuo. Purification was carried out using normal phase flash column chromatography with a gradient of 50-100% EtOAc/Pet Ether to give the pure alkylated complex as a red solid (2.80 g, 72%).
  • the aqueous layer which contained amino acid was fully evaporated. The residue was treated with 6% Na 2 CO 3 until the solution reached pH 9-10. EDTA.2Na (171 mg, 0.46 mmol, 1 equiv.) was added and the reaction was left to stir for 10 mins. The solution was cooled to -10 °C in an EtOH-ice bath and FmocOSu (140 mg, 0.420 mmol, 0.9 equiv.) in equal amount of acetonitrile was added dropwisely over 30 mins. The reaction was warmed to room temperature and left to stir for 16 h. The acetonitrile was removed under reduced pressure. The aqueous layer was acidified to pH 1-2 with 1 M HCl.
  • the aqueous solution was extracted with EtOAc (3 ⁇ 10 mL), the organic layer was then washed with brine (3 ⁇ 10 mL), dried over sodium sulfate. The solvent was removed under reduced pressure to give the target compound as a white solid (151 mg, 77%).
  • Boc-L-Serine (755 mg, 3.68 mmol, 1 equiv.) was dissolved in DMF (20 mL) under N 2 and the flask cooled to 0 °C.
  • NaH (60% in oil, 353 mg, 8.84 mmol, 2.4 equiv.) was added portion wise and the reaction stirred at 0 °C for 2 h.5-Iodo-pent-1-yne (1.00 g, 5.15 mmol, 1.4 equiv.) was added dropwise over 10 mins. The ice bath was removed and the reaction left to stir for 16 h at room temperature.
  • reaction mixture was quenched with ethanol (10 mL), H 2 O (30 mL) added and subsequently washed with Et 2 O (4 ⁇ 50 mL). After acidification using 3 M HCl (5 mL), the aqueous layer was extracted with EtOAc (4 ⁇ 75 mL). The organic layer was washed with H 2 O (4 ⁇ 75 mL), dried using MgSO 4, filtered and then concentrated in vacuo to yield as a yellow sticky solid (450 mg, 45%).
  • the aqueous layer was extracted with DCM (100 mL) and the combined organic layers washed with 5% w/v LiCl solution (3 ⁇ 100 mL), brine (3 ⁇ 100 mL), dried with MgSO 4 and concentrated in vacuo. Purification was carried out using normal phase flash column chromatography with a gradient of 50-100% EtOAc/Pet Ether to give the alkylated complex as a red solid (2.89 g, 79%).
  • All proteins in this family contain an extended KH RNA binding domain of around two hundred amino acids, unlike other proteins which contain multiple KH domains.
  • the KH domain is situated between an N-terminal QUA1 domain that is responsible for dimerisation and a C-terminal QUA2 domain which has been proposed to participate in RNA binding (Artzt et al., 2010).
  • the first STAR protein determined to be involved in alternative splicing was Sam68 (Hartmann et al., 1999), and will be the focus in this Example.
  • Sam68 (the Src-Associated substrate in Mitosis of 68 kDa) belongs to the STAR family of RNA binding proteins. Unlike other hnRNP K homology proteins, the STAR proteins contain a single RNA binding KH domain.
  • T-STAR is a tissue specific analogue of Sam68, mainly expressed in neurons. T-STAR differs by the absence of a one hundred amino acid N-terminal region in comparison with Sam68, however it retains 77% sequence homology at the KH domain.
  • Sam68 has been reported to be involved in the alternative splicing of oncogenes, typically favouring the most oncogenic form, with Sam68 levels found to be upregulated in a variety of cancers.
  • the protein has been associated with poor prognosis when linked to cancer due to its implications in tumour progression (Frisone et al., 2015; Busà et al., 2007).
  • the exact mechanism of the protein within the splicing machinery however, remains poorly understood and whether or not RNA binding is the specific cause of the oncogenic characteristics of Sam68 is not known. Another important question is if there is a correlation between the RNA binding ability of Sam68 and its role in signal transduction. A peptide inhibitor strategy was used to probe these interactions.
  • the peptide was synthesised up to and including the Ahx linker and then a test cleavage of the linear peptides was performed.
  • Analytical RP-HPLC and LC-MS revealed a deletion corresponding to Tyr making up around 20% of the crude material.
  • An on-resin RCM 008528580 reaction was performed using Grubbs 1 st generation catalyst in DCE for 2 ⁇ 2 h. To determine conversion, a test cleavage was performed with complete conversion to the stapled product observed with a 50/50 mixture of cis/trans alkene isomers. The N-terminal Fmoc was removed manually and FITC coupled using standard protocols.
  • each isomer was isolated. After a further purification, each isomer was obtained in >95% purity by analytical RP-HPLC in an overall yield of 5%. Both isomers were isolated to compare their activity, since differences in activity have previously been reported for isomers of i, i + 7 staples (Yuen et al., 2019). NMR experiments were performed which revealed the earlier eluting isomer by RP-HPLC as a cis isomer, and the later eluting isomer was assumed to be the trans isomer.
  • NMR spectroscopy is a useful technique to distinguish between the two through a decoupling experiment.
  • Decoupling can be performed by irradiating the signal of adjacent methylene protons coupling to the alkene. Through this process, the multiplet corresponding to the alkene protons can be simplified allowing an accurate J value for the isomer to be calculated.
  • the coupling constants By deconvoluting the coupling constants to only show the 3 JHH coupling constant, the size of the coupling constant can be correlated to each isomer.
  • Coli with resistance to chloramphenicol and kanamycin were transformed with the Sam68 plasmid. Selection for the desired bacteria was then achieved by plating the resulting cells onto agar containing kanamycin and chloramphenicol antibiotics. Colonies were picked and transferred to 20 mL of 2 ⁇ TY medium containing kanamycin and chloramphenicol. These starter cultures were then transferred to 1.6 L of 2 ⁇ Y medium for protein expression. Protein expression is controlled in BL21 DE3 cells by the presence of a T7 promotor next to the gene that is to be expressed. This promotor itself is under the control of the lac operon that is restricted by the presence of lac binding proteins.
  • the cells are prevented from generating T7 polymerase which therefore prevents transcription of the DNA of the gene of interest to RNA and thus inhibits protein expression.
  • a change in the conformation of the lac promotor can be caused by addition of isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) as it binds to the lac repressor protein.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • This process causes release of the T7 promotor and allows for T7 polymerase and subsequent protein expression to occur.
  • protein expression was induced once the cell cultures had reached an optical density of 0.6, within the exponential growth phase.
  • purification of the desired protein was carried out.
  • the desired gene had been engineered to contain a 6-His tag at the C-terminus of the sequence.
  • a 6-His tag provides a facile method of purification as His has a high affinity for nickel.
  • Cells were lysed by sonication; cell debris was removed by centrifugation and the resulting protein purified by affinity chromatography.
  • the protein mixture was passed through a column packed with Ni-NTA agarose resin, with 6-His tag containing proteins binding to the nickel. Non-specifically bound proteins were then removed by a gradient of imidazole (25-250 mM) which competes with His to bind the nickel. Once a sufficient concentration of imidazole was present in the eluent, the 6-His tagged protein is eluted. After this, dialysis of the soluble protein was carried out to remove the high concentrations of imidazole used in the purification.
  • NKKH-Sam68 was confirmed by taking samples at various stages during the expression process. A sample was taken after cell lysis and the soluble protein content was measured by centrifugation of a cell lysis sample followed by analysis of the supernatant. Some target protein was observed in the wash fractions of the His-tag purification due to over-loading of the resin but only a minimal amount was observed on the gel. A band was observed at the correct mass in each of the fractions from the His-trap purification. From 1.6 L of culture, 150 mg of protein was obtained (7 ⁇ M) as determined by a Bradford Assay. 008528580 Under the denaturing conditions of an SDS-PAGE, the protein mass corresponds to that of monomeric NKKH-Sam68.
  • T-STAR-HCS-2-cis and T-STAR-HCS-2-trans show cellular uptake in the cells from visualisation of the FITC channel and overlay with the nuclear dye. No aggregation was observed in this experiment and thus the mutation of His to Arg was included in the design of further analogues.
  • Diyne Staple The alkene stapled T-STAR peptides synthesised have been shown to have increased binding for Sam68 compared to the native, unconstrained peptide and are found to be cell penetrant. This peptide sequence was therefore taken on as a model system for optimisation of the diyne staple.
  • NaI, acetone, Iodo-alkynes may also be synthesised from the appropriate chloro-compound, with reaction conditions as described herein for the synthesis of Compound 166. Yields were generally excellent with a 96% yield for both C7 and C8 iodo-alkynes (Table 3). A slight decrease was observed for C6 and C5 analogues. This decrease in yield is a result of increased volatility for the smaller compounds and thus, some material was lost during evaporation of solvent after the reaction. 008528580 Table 3. Yields for Finkelstein reaction of carbon chain lengths between five and eight.
  • Fmoc protected amino acids 99a-d and 100a-d were obtained in yields ranging from 60-77% for R- and S- configured amino acids incorporating alkynyl side-chains ranging from five to eight carbons (Scheme 3). Purity of all amino acids was calculated using analytical RP-HPLC and deemed to be >95% for all cases. 008528580 Scheme 3: Decomplexation and Fmoc protection forming the terminal alkyne S- and R- Fmoc protected amino acids 99a-d and 100a-d.
  • Circular Dichroism CD analysis was undertaken for each diyne peptide at a concentration of 50 ⁇ M, scanning from 185 to 260 nm as described in General Method E.
  • combinations SS, SR and RS also showed very similar results in terms of helicity ( Figure 4).
  • the RR configured analogue was much lower in helicity.
  • similar helicity was observed for SS (77%) and RS (70%) combination ( Figure 5).
  • the SR bridge had a lower helicity of 54% and the RR had the lowest helicity of 40%.
  • Chymotrypsin was chosen for the experiment as it typically cleaves at the C-terminus of aromatic residues. Since there is a single Tyr located within the centre of the peptide sequence this would provide a good model for resistance due to increased secondary structure.
  • Cleavage site of chymotrypsin (red) in T-STAR sequence After 4 h, T-STAR-1 was completely degraded by the enzyme as confirmed by analytical RP-HPLC and LC-MS ( Figure 7). Of the stapled analogues tested, both alkene and diyne based constraints were extremely stable with >92% of all peptides remaining after the last time point.
  • HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high glucose with GlutaMAX, Gibco) supplemented with 10 % (v/v) foetal bovine serum (Gibco) and 1 % (v/v) penicillin/streptomycin (10,000 units/mL penicillin, 10,000 ⁇ g/mL streptomycin, Gibco). Cultured cells were maintained in a humidified incubator at 37°C, 5 % CO2 and passaged twice weekly in T-25 flasks (Corning).
  • DMEM Modified Eagle Medium
  • Gibco high glucose with GlutaMAX
  • penicillin/streptomycin 10,000 units/mL penicillin, 10,000 ⁇ g/mL streptomycin, Gibco
  • the hole width was 200 ⁇ M with a diffraction grating of 600 g/mm using an Olympus x50LWD objective lens.
  • the recorded spectral range was 600–4000 cm -1 and data acquisition was performed during 5 seconds with 3 repeats and collected with the Synapse OCD detector. 100% power was used for T-STAR-1 peptide and 50 % power was used for the stapled peptide T-STAR-S7S7. Data was analysed using the Labspec 5 software.
  • T-STAR-S7S7 unlabelled analogues of native T-STAR-1 and a preferred stapled analogue, T-STAR-S7S7 were prepared for analysis using Raman spectroscopy.
  • Raman imaging of the solid diyne stapled T-STAR-1 peptide was conducted using Raman spectroscopy with a laser at 532.02 nm.
  • Figure 11(A) shows the spectra from the diyne stapled peptide.
  • a 008528580 significant peak can be observed in the cell-silent region at ⁇ 2,255 cm -1 .
  • Raman was also performed on the native T-STAR-1 peptide to confirm the absence of this peak at ⁇ 2,255 cm -1 ( Figure 11B).
  • T-STAR-1 T-STAR native sequence
  • T-STAR-HCS-2 all-hydrocarbon i, i + 7 stapled peptide
  • T-STAR-HCS-2-cis and T-STAR-HCS-2-trans were significantly more helical than the native sequence, T-STAR-1.
  • the NKKH domain of Sam68 was expressed and purified for FP analysis of the peptides.
  • Results showed that T-STAR-1 did not bind to Sam68, whereas both T-STAR-HCS-2-cis and T-STAR-HCS-2-trans retained binding to Sam68.
  • An in vitro splicing assay was performed to determine whether the peptides synthesised had an impact on the splicing of the Nrxn3 minigene which is a common splicing target for Sam68.
  • Example 2 Application of the Diyne Staple to p53 The p53/MDM2 interaction is a very well researched target, especially for ⁇ -helical peptides. Therefore, this application was selected to trial the diyne staple type on, and compare the conformational properties and biological activity to the alkene stapled peptide (ATSP-7041).
  • ATSP-7041 is the precursor peptide of ALRN-6924 and was chosen for this study.
  • Microwave heating was used for all coupling reactions in the synthesis of the peptide as couplings are more efficient at elevated temperature with shorter reaction times required.
  • Coupling reactions were achieved using Fmoc protected amino acids (5 equiv.), Oxyma Pure and DIC in DMF with heating to 90 °C for 2 mins followed by washing the resin using DMF. Deprotection of N-terminal Fmoc groups was achieved using 20% morpholine/DMF for 1 min at 90 °C. Unlabelled (145a) and fluorescently labelled (145b) peptides were synthesised for the selected assays.
  • the N-terminus was acetyl capped using acetic anhydride in DMF (5 M) for 10 mins at rt (Scheme 13).
  • the fluorescently labelled peptides were synthesised with an Ahx linker between the last N-terminal amino acid and the FITC, to avoid the formation of a fluorescein thiohydantoin. 008528580 .
  • the linear peptide 152 containing R 8 and S 5 was cyclised by RCM using Grubbs catalyst, excluding light (Scheme 14) using conditions as described in General Method B (see Ring Closing Metathesis section).
  • a test cleave was performed and HPLC and LC-MS analysis was used to determine the success of the RCM reaction.
  • removal of two carbon atoms and four hydrogens results in a mass difference of 28, which was observed using LC-MS.
  • a shift in retention time was observed between the linear and closed peptide product using HPLC analysis.
  • the product peptide resulted in two separate peaks with the same mass by LC- MS, which is indicative of the isomers produced from the alkene staple.
  • Diastereoselective alkylation of the S-Ni(II) Schiff base complex 154 was achieved using sodium hydroxide as base and iodo-alkyne as the electrophile. After formation of a planar enolate intermediate, attack is preferred at the Si face of the complex as the 2-fluorobenzyl moiety sterically blocks attack from the Re face facilitating formation of the S-alkylated complex.
  • the diastereomeric ratio (dr) of the crude mixture 156 was determined using 19 F NMR spectroscopy with a ratio of 96:4.
  • n i n wohs ( 0 h eu i d se r eh P gn i t c a r e t n i eh T .a46 1 ed i t pe p d l e p a t s en i y d l o r t no c ev i t age n e h tf o i s se h t ny s ehT:0 2 e meh .
  • the alkene stapled peptide 153 has a lower yield due to the production of cis and trans isomers during the RCM reaction (Yields; Diyne: 10%, Alkene cis: 4%, Alkene trans: 2%).
  • the crude purity of the alkene vs diyne stapled peptide was compared and quantified using RP-HPLC upon cleavage of the crude peptides before purification. The results show that the cis isomer in 51% and the trans isomer in 31%.
  • the crude purity of the diyne 160a is significantly improved at 79%. Conformational Analysis The effectiveness of the diyne staple as an ⁇ -helical constraint was assessed using CD.
  • the output of CD is typically converted from machine units (millidegrees) to MRE with units of deg ⁇ cm 2 ⁇ dmol -1 . This unit is useful as it takes into account the concentration and length of the peptide which normalises the output for comparison with other systems (Sreerama et al., 2004).
  • the raw CD data was converted to MRE and the value at 222 nm was used to calculate the % helicities using Equations B and C (see General Method E).
  • the diyne stapled peptide 160a was the most helical with a 59% helicity, in comparison to the cis (153a) and trans (153a’) alkene stapled peptides with 48% helicity (Table 4). As such, the diyne conformational constraint in this example peptide clearly has an enhanced effect on inducing the helical bioactive conformation.
  • Table 4 The stapled peptide % helicities calculated using Equations B and C.
  • 008528580 FP analysis using MDM2 (1-138) Protein Fluorescently labelled peptides were first tested for binding with MDM2 sequence 1-138 according to General Method D. Chang et al. reported the binding of the linear peptide (ATSP-3848) and ATSP-7041 to MDM2 (1-138) with low nM binding affinity, using SPR and FP. A comparable FP assay was set up to test the binding affinity of the diyne stapled peptide 160b in comparison to the alkene stapled 153b/b’ and linear peptide. A 10 ⁇ M stock concentration of MDM2 (1-138) was prepared in Tris buffer.
  • HCT-116 cells were treated with either 20 ⁇ M fluorescein diyne stapled peptide 160b, fluorescein ATSP-7041153b/b’ or fluorescein native peptide 145b stained with Hoechst 33342 nuclei stain and imaged 4.5 hours post-treatment. 008528580 Fluorescein ATSP-7041153b/b’ and fluorescein diyne stapled peptide 160b showed a diffused intracellular localization, confirming efficient cellular penetration. The native peptide 145b showed no cellular internalisation.

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Abstract

The invention relates to polypeptides, in particular polypeptides comprising a diyne linkage. The invention provides a polypeptide of Formula (I), a polypeptide and amino acids for the preparation of the diyne linked polypeptides, and methods for the preparation of the diyne linked polypeptides. The invention also provides medical uses of the polypeptides.

Description

POLYPEPTIDES Related Application This present case is related to, and claims the benefit of, GB 2219576.2 filed on 22 December 2022 (22.12.2022), the contents of which are hereby incorporated by reference in their entirety. Technical Field This invention provides polypeptides, in particular stapled polypeptides. Also provided are methods for preparing stapled polypeptides, as well as amino acids suitable for preparing stapled polypeptides. Background Stapled peptides are an important class of α-helical peptides which are conformationally constrained. They are used in the regulation of Protein-Protein Interactions (PPIs), utilising their ability to bind to shallow PPI interfaces. Conformationally constraining wild type peptides using staples could pay the entropic penalty of folding, resulting in favourable binding affinities and improved selectivities. A frequently used stapling method makes use of two commercially available unnatural alkenyl amino acids (R8 and S5) and a ring closing metathesis (RCM) reaction using Grubbs’ catalyst. Cyclisation on resin forms an all-hydrocarbon alkene bridge using relatively mild conditions (Kim et al., 2011). The first biological example of alkene stapling was applied to peptides derived from the BCL-2 domain (Walensky et al., 2004). The specific section of the protein that was being mimicked was the BH3 domain from the BID protein (EDIIRNIARHLAQVGDSMDRSIW). Stapling increased the helicity of the peptides, proteolytic stability and binding affinity for the target receptor. Additionally, the peptide had a long in vivo half-life in a mouse model of T cell leukemia. However, there are several disadvantages associated with the alkene staple type, including the production of cis/trans isomers during synthesis. The yield of the purified bioactive product is lowered as one isomer is purified from the other. There is a need for new approaches for the stapling of polypeptides. Accordingly, the present inventors have developed new stapled polypeptides and amino acids for the synthesis of the polypeptides. 008528580 Summary of the Invention At its most general, the present invention relates to a polypeptide having a diyne linkage. The invention also relates to polypeptides and amino acids for the preparation of the diyne linked polypeptides, and methods for the preparation of the diyne linked polypeptides. The secondary structure of polypeptides is important for their three-dimensional shape. Α-helical peptides are a prevalent type of secondary structure that play an important functional role in protein-protein interactions. A diyne linkage according to the present invention provides a stable covalent attachment between two amino acid side chains, which conformationally constrains the polypeptide. The stapled polypeptide has improved helicity, as well as improved protease stability compared to the corresponding native peptide that lacks the diyne linkage. The stapled polypeptide is suitable for therapeutic applications. In a general aspect, the invention provides a polypeptide wherein two amino acid residues are bridged at their respective α-carbon positions by a diynylene group, and typically a 1,3-diyne group. The formation of the staple uses two amino acids each comprising a terminal alkynyl group which are linked to form a 1,3-diyne linkage. The connection formed between the α-carbon positions is a hydrocarbon chain, without the presence of heteroatoms, such as without oxygen, within that chain. In a first aspect of the invention there is provided a polypeptide comprising a group of Formula (I) wherein -R1 and -R2 are each independently selected from optionally substituted C1-4 alkyl and -H; -R3 and -R4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue, such as an α-amino acid residue; -L1- and -L2- are each independently an optionally substituted C2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof. 008528580 Typically, the polypeptide has a substantially α-helical structure secondary structure, such as in aqueous solution. The 1,3-diyne staple increases the stability of the α-helical structure. The diyne linkage may be readily formed from a corresponding non-stapled polypeptide possessing two amino acid residues each comprising a terminal alkynyl side chain. The linkage is formed under mild conditions that are compatible with other functionalities that may be present within the polypeptide. The polypeptide may have a percentage helicity of 50% or more, such as 60% or more, such as 70% or more. Helicity may be measured by circular dichroism such as by a method described herein. Each of -L1- and -L2- may independently be an optionally substituted C2-7 alkylene, such as optionally substituted C4-7 alkylene, such as optionally substituted C4-6 alkylene, such as optionally substituted C5 or C6 alkylene. Varying the size of the macrocycle within the stapled polypeptide in this way helps to further alter, such as improve, helicity. These polypeptides can also be obtained in high yield by cyclisation of a corresponding linear polypeptide by a method as described herein. In alternative embodiments, -L1- and -L2- may independently be an optionally substituted C5-10 alkylene, such as optionally substituted C5-7 alkylene. Preferably, -L1- and -L2- are independently C5-10 alkylene, such as C5-7 alkylene. These polypeptides have excellent helicity, and they can be obtained in high yield by cyclisation of a corresponding linear polypeptide by a method, such as described herein. Preferably, -L1- and -L2- are each independently C4-10 alkylene, such as C4-7 alkylene, such as C5 alkylene or C6 alkylene. -L1- and -L2- may be the same, such as where -L1- and -L2- are both C5 alkylene or both C6 alkylene, or -L1- and -L2- may be different. Polypeptides having a 14 or 16 carbon atom bridge between the α-carbons of the stapled amino acids are particularly preferred, such as where -L1- and -L2- are both C5 alkylene or both C6 alkylene. Preferably, the total number of carbon atoms in -L1-(C≡C-C≡C)-L2- in Formula (I) is in the range of 14 to 16, such as 14 or 16, more preferably 14. In these embodiments, n may be 6 to provide an i, i+7 diyne linkage. Each of -L1- and -L2- may independently be unsubstituted, or may be substituted, such as substituted with one or more halo groups, such as one or more fluoro groups. An amino acid residue may be a natural or a non-natural amino acid residue. An amino acid residue may comprise a protecting group, and thus an amino acid residue as described herein encompasses a protected natural or non-natural amino acid residue. Typically, the 008528580 protecting group wherein present is a side-chain protecting group. Where an amino acid reside is located at a C or N terminal of a polypeptide, the terminal carbonyl or carboxyl group, or terminal amino group, of the polypeptide may be protected. Preferably, an amino acid residue is selected from a natural amino acid or a non-natural amino acid, such as an α-amino acid, including a proteinogenic amino acid. An amino acid residue may have L- or D- stereochemistry. An amino acid residue, such as -A-, may be selected from: Alanine [Ala, A], Aminobenzoic acid [PABA], Aminobutyric acid [Abu], Aminohexanoic acid [Ahx], Aminoisobutyric acid [Aib], Arginine [Arg, R], Asparagine [Asn, N], Aspartic acid [Asp, D], Butylglycine, Citrulline [Cit], Cyclohexylalanine [Cha], Cysteine [Cys], Diaminobutanoic acid [Dab], Diaminopropionic acid [Dpr or Dap], Dihydroxyphenylalanine [DOPA], Glutamic acid [Glu, E], Glutamine [Gln, Q], Glycine [Gly, G], Histidine [His, H], Homoserine [Hse], Hydroxyproline [Hyp], Isoleucine [Ile, I], Isonipecotic acid [Isn], Leucine [Leu, L], Lysine [Lys, K], Methionine [Met, M], Norleucine [Nle], Norvaline [Nva], Ornithine [Orn], Phenylalanine [Phe, F], Phenylglycine [Phg], Proline [Pro, P], Sarcosine [Sar], Serine [Ser, S], Statine and derivatives thereof [Sta], Tetrahydroisoquinoline-3-carboxylic acids [Tic], Thienylalanine [Thi], Threonine [Thr, T], Tryptophan [Trp, W], Tyrosine [Tyr, Y], and Valine [Val, V]. Preferably, n is an integer from 2 to 8, such as from 3 to 8, such as from 3 to 6, such as 3 or 6. n may be an integer selected from 2, 3, 6 and 10, such as an integer selected from 3, 6 and 10. Polypeptides with this number of amino acids residues between two stapled amino acid residues are readily prepared by cyclisation of a corresponding linear polypeptide under mild conditions. Advantageously, these polypeptides can also be obtained in high yield such as by a coupling reaction as described herein. Each of -R1 and -R2 may independently be selected from methyl, ethyl and -H. Preferably, -R1 and -R2 are independently methyl or -H, and most preferably -R1 and -R2 are methyl. Methyl is particularly preferred as this facilitates the coupling of terminal alkynyl groups during synthesis of the stapled polypeptide. Each of -R3 and -R4 may independently be -H or methyl. Preferably, -R3 and -R4 are both -H. The α-carbon atoms that are bonded to -R1 and -L1-, and to -R2 and -L2-, respectively, are chiral. These α-carbon atoms may independently be in a R or S configuration. Preferably, the polypeptide comprises a group of Formula (IRS), (ISR) and/or (ISS). Alternatively, the polypeptide comprises a group of Formula (ISS). These stapled polypeptides display good helicity. 008528580 wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as defined herein. In some embodiments, the polypeptide is of Formula (Ia) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue, such as an α-amino acid residue or an N-methylated α-amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200. 008528580 A tag may comprise a chromophore; a fluorescent or a phosphorescent label; or a radiolabel. A tag may comprise a group for targeting the polypeptide to a cell membrane, such where the tag comprises a lipid group, such as farnesyl or geranyl. A solid phase may be connected to the C terminal end of the polypeptide, such as at an amino acid residue -A'-, by a linker. A tag may be connected to the N terminal end or the C terminal of the amino acid, such as to an amino acid residue -A'-, by a linker. Preferably, a tag is connected to the N terminal end. In some embodiments the moieties represented by -[A’]m- and -[A’]m'- independently comprise up to one N-methylated amino acid residue. Preferably, where the moiety represented by -[A’]m- or -[A’]m'- comprises an N-methylated amino acid residue, the N-methylated amino acid residue is a terminal residue. In these embodiments, -R3 and -R4 may be -H. Preferably, each -A’- is independently an amino acid residue, such as wherein -A’- is not an N-methylated amino acid residue. In these embodiments, -A’- may be represented by -A- as described herein. Preferably, an acyl group is C1-12 acyl, such as C1-6 acyl, such as acetyl. Preferably, m and m’ are each independently an integer from 1 to 200. More preferably, m and m’ are each independently an integer from 2 to 50. In some embodiments, the polypeptide is of Formula (Ib) wherein -R1, -R2, -R3, -R4, -A-, -A’-, -L1-, -L2-, -RA, -RB, n, m and m’ are as described herein, and z is an integer from 1 to 4. Preferably, z is 1 or 2, such as 1. 008528580 In a second aspect, there is provided a polypeptide comprising a group of Formula (II) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein, and the salts, solvates and protected forms thereof. The groups -L1- and -L2- are preferably each independently C4-10 alkylene, such as wherein -L1- and -L2- are both C5 alkylene or both C6 alkylene. Alternatively, -L1- and -L2- are each independently C5-10 alkylene, such as C5-7 alkylene. In some embodiments n may is an integer from 2 to 8, such as from 3 to 8, such as an integer selected from 2, 3 and 6. In some embodiments n is an integer selected from 2, 3, 6 and 10. In some preferred embodiments, n is 6. Each of -R1 and -R2 is preferably independently selected from methyl and -H. Each of -R3 and -R4 is preferably -H. The polypeptide may comprise a group selected from Formula (IIRS), (IISR) or (IISS). Alternatively, the polypeptide comprises a group of Formula (IISS). 008528580 The polypeptide may have the Formula (IIa) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200. Preferably, m and m’ are each independently an integer from 1 to 200. More preferably, m and m’ are each independently an integer from 2 to 50. Preferably, each -A’- is an independently an amino acid residue. In some embodiments -A’- is represented by -A- as described herein. The polypeptide may have the Formula (IIb) 008528580 wherein -R1, -R2, -R3, -R4, -A-, -A-‘, -L1-, -L2-, -RA, -RB, n, m and m’ are as described herein, and z is an integer from 1 to 4. Preferably, z is 1 or 2. In a third aspect, there is provided a method of preparing a diyne stapled polypeptide, such as a polypeptide of the first aspect, the method comprising steps of: (i) providing a polypeptide according to the second aspect; and (ii) coupling the terminal alkynyl groups of the polypeptide to provide a 1,3-diyne linkage. Preferences for the first and second aspects apply equally to the third aspect. Step (ii) may be performed in the presence of a metal salt. A metal salt may be a transition metal salt, such as a copper salt. A metal salt may be selected from CuCl, CuBr, CuI, and CuOAc. Step (ii) may be performed in the presence of a ligand selected from 4,4’-bis(hydroxymethyl)-2,2’-bipyridine, tetramethylethylenediamine, and 2,2’-bipyridine, such as in addition to the metal salt. In step (ii) the reaction mixture may be heated. In step (ii) microwave radiation may be applied to the reaction mixture. In a fourth aspect, there is provided a pharmaceutical composition comprising a polypeptide comprising a group of Formula (I) and a pharmaceutically acceptable carrier. Preferences for the first aspect apply equally to the fourth aspect. The pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or excipient. 008528580 In a fifth aspect, there is provided a polypeptide comprising a group of Formula (I), or the pharmaceutical composition described herein, for use in a method of treatment or prophylaxis. The polypeptide or pharmaceutical composition may be for the treatment of cancer. The polypeptide or pharmaceutical composition may be for use in a method of treating cancer wherein the polypeptide inhibits dimerization of Sam68, or wherein the polypeptide inhibits interaction between p53 and MDM2. The polypeptide or pharmaceutical composition may be for use in a method of treating a disease with altered Sam68 activity, or a disease with altered p53 and/or MDM2 activity. The polypeptide or pharmaceutical composition may be for use in a method of treatment wherein the polypeptide inhibits Sam68 activity, or inhibits MDM2 activity. Preferences for the first aspect apply equally to the fifth aspect. In a sixth aspect, there is a provided a compound of Formula (III) wherein -LX- is optionally substituted C5-10 alkylene; -R1 is selected from optionally substituted C1-4 alkyl and -H; -R3 is selected from -H and methyl; -RN is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; and -RC is selected from -OH and a protecting group, or -RC together with the carbonyl group to which it is attached forms an activated acid, and the salts and solvates thereof. The group -RC may be selected from -OH, -OMe, -OBn, -Cl, -O-benzotriazole, and -O-C(=NR’)NR’’, where R’ and R’’ are independently selected from cyclohexyl, isopropyl, ethyl, and dimethylaminopropyl. 008528580 -LX- is optionally substituted C5-10 alkylene and preferably is optionally substituted C6-10 alkylene, such as C6-10 alkylene. Preferably, -R3 is hydrogen. A tag may be a lipid group, such as farnesyl, geranyl; or may comprise a chromophore, a fluorescent or a phosphorescent label, or a radiolabel. An acyl group may be a C1-12 acyl group. In some embodiments an acyl group is acetyl. Alternatively, -LX- is optionally substituted C2-10 alkylene in the compounds of formula (III). The compound may be of Formula (IIIa) wherein -LX-, -R1, -R3 and -RN are as defined herein. The compounds of Formula (III) are useful in the preparation of polypeptides, in particular stapled polypeptides in high yield that have high helicity. Preferences for -R1 and -LX- are as defined herein for -R1 and -L1-, respectively. In some embodiments, -LX- is optionally substituted C6-10 alkylene, such as optionally substituted C6-8 alkylene. In some embodiments, -R1 is selected from methyl and -H, such as methyl. The group -RN may be selected from -H, a protecting group, farnesyl, geranyl and a tag optionally with a linker. -RN may be a protecting group wherein -RN together with the nitrogen group it is bonded to forms a carbamate. These and other aspects and embodiments of the invention are described in further detail below. Summary of the Figures The present invention is described with reference to the figures listed below. 008528580 Figure 1 shows (A) CD analysis for T-STAR-1, T-STAR-HCS-2-cis and T-STAR-HCS-2-trans; and (B) calculated helicity at 222 nm for peptides according to an embodiment of the invention (from top to bottom at 240 nm: T-STAR-HCS-2-trans, T-STAR-HCS-2-cis). Figure 2 shows (A) the conversion observed after Glaser reaction (3 h) of C14 and C16 bridges, green bar = >70% conversion (columns 1, 3 and 5-7), yellow bar = >60% conversion (columns 2 and 8) and red bar = <60% conversion (column 4); and (B) conversions calculated from analytical RP-HPLC after Glaser reaction. Figure 3 shows (A) the conversion observed after Glaser reaction (3 h) of C10, C12, C14, and C16 SS configured bridges, green bar = >70% conversion (columns 3 and 4), yellow bar = >60% conversion (column 2) and red bar = <60% conversion (column 1); and (B) conversions calculated by analytical RP-HPLC after Glaser reaction. Figure 4 shows (A) CD spectra of C16 diyne linked peptides according to an embodiment of the invention (from top to bottom at start of x-axis: S8R8, R8S8, S8S8, R8R8); (B) calculated helicity of C16 bridges; (C) CD spectra of C14 diyne bridges (from top to bottom at start of x-axis: S7S7, R7S7, R7R7, S7R7); and (D) calculated helicity of C16 bridges. Figure 5 shows (A) CD spectra of C14, C12 and C10 diyne linked peptides according to an embodiment of the invention (from top to bottom at x-axis: S7S7, S6S6, S5S5); (B) calculated helicity of C14, C12 and C10 bridges; (C) CD spectra of C14 and C14 (monosubstituted) diyne bridges (from top to bottom at x-axis: S7S7, S7HS7H); and (D) calculated helicity of C14 and C14 (monosubstituted) bridges. Figure 6 shows the percentage helicity calculated at 222 nm of diyne peptides according to an embodiment of the invention. Columns 2, 3, 5-7, 9, 11, 14 are > 60% (green); columns 10, 12 and 13 are > 50% (yellow); and columns 4 and 8 are < 40% (red). Figure 7 shows a plot of percentage degradation of T-STAR-1, T-STAR-HCS-2-cis, T-STAR-HCS-2-trans, T-STAR-S7S7 and T-STAR-S7HS7H from t = 0 to t = 4 h. The percentage remaining for T-STAR-1 is substantially reduced at 50 minutes and more. Figure 8 shows CD spectra of the linear and stapled peptides according to an embodiment of the invention at 50 µM concentration (from top to bottom at 220 nm: 145a, 153a’, 153a, 160a). Figure 9 shows CD spectra of the linear and stapled peptides according to an embodiment of the invention at 25 µM concentration (from top to bottom at 220 nm: 145a, 153a’, 153a, 160a). 008528580 Figure 10 shows fluorescence imaging of A) T-STAR-S7S7 stapled peptide, B and C) alkene stapled peptides T-STAR-HCS-2-cis, T-STAR-HCS-2-trans, and D) T-STAR-S7HS7H internalised in cells; E) The native T-STAR-1 peptide showing no internalisation; and F) DMSO negative control. Microscopy images were acquired with a custom-built multi-modal microscope setup. Fluorescein excitation was conducted at 495 nm. Scale bars= 40 μm. Images were processed using MetaMorph software. Figure 11 shows solid-state Raman spectra of A) diyne stapled T-STAR-S7S7 showing that the diyne functionality gives a peak at ~2,255 cm-1 in the cell-silent region; and B) the acetylated native T-STAR-1 showing the absence of the diyne functionality peak. Detailed Description of the Invention At its most general, the present invention relates to a polypeptide having a diyne linkage. The invention also provides polypeptides and amino acids for the preparation of the diyne linked polypeptides, as well as methods for the preparation of the diyne linked polypeptides. The diyne linked polypeptides are stapled polypeptides which can be readily prepared from a polypeptide having two α-amino acid residues, each having an alkynyl side chain connected to the α-carbon. These side chains readily undergo cyclisation under mild conditions at high conversion and high yield. Polypeptides with a diyne staple as described herein have improved helicity compared to a corresponding linear (unstapled) polypeptide. The stapled polypeptide also has increased protease stability. The present inventors have found that alkene staples in a polypeptide are relatively flexible, apart from the two sp2 hybridised carbons. A 1,3-diyne (diacetylene) functionality can be introduced into the bridge instead of an alkene to provide enhanced rigidity. The 1,3-diyne can be readily accessed synthetically using a Glaser oxidative coupling between two terminal alkyne amino acids within the peptide sequence. This eliminates the use of expensive Grubbs’ catalysts for producing the cyclic peptide. The diyne-stapled peptide can be obtained as a single isomer, improving overall yield and simplifying purification compared to an alkene staple. Cistrone et al. describe a diyne stapled peptide derived from cyclisation of two propargyl serine residues. The cyclisation involves an intramolecular Glaser reaction between i and i + 4 to i + 7 spaced amino acids, which is carried out on-resin. The authors report that high conversion can be achieved between i and i + 4 spaced residues within 72 hours, whilst conversion is substantially reduced under the same conditions for i and i + 5; i and i + 6; and i and i + 7 spaced residues. The percentage helicity of the stapled peptides is reported to be between 6% to 42%. 008528580 Verlinden et al. also describe the use of a 1,3-diyne linker for helix α-stabilisation. Peptides containing two propargyl serine residues are described, which are spaced apart at positions i and i +7. Cyclisation via Glaser stapling is carried out in solution, and full conversion is reported within after 24 hours. A stapled peptide based on linked D-Ser and L-Ser residues was reported to display a full α-helical structure, whilst the L-Ser, L-Ser peptide displayed helical structure within the stapled macrocycle but the exocyclic segment was not part of the helix. WO 2017/040990 describes peptidomimetic macrocycles and their use. The peptides have an i, i+7 staple, and hydrocarbon staples are described. The diyne linked polypeptides of the present invention have improved helicity. For example, a polypeptide where -L1- and -L2- are C5 alkylene has good helicity (76% as shown for the polypeptide of Figure 5A and 5B). The polypeptides of the present invention are also beneficial in that conversion from the linear precursor peptide to the stapled peptide is high (at about 100% as shown for the polypeptides of Figure 3A and 3B), and therefore the polypeptides are also conveniently prepared. The diyne-stapled peptides of the present invention can be prepared from a linear peptide having two terminal alkynyl amino acids, preferably hydrocarbon alkynyl amino acids. The rate of conversion of these amino acids as described herein via a Glaser reaction is higher than for a corresponding reaction using alkyne-functionalised serine residues, where the staple comprises a chain with oxygen chain atoms. The rate of cyclisation is further improved when the length of the alkylene-alkyne side chain is increased from C3 alkylene, which corresponds to the side chain in propargyl serine (-CH2OCH2-), to C4 alkylene, and in particular the rate of cyclisation is high when the alkyne is connected to the α-carbon by a C5 or C6 alkylene group. Polypeptide Comprising a Group of Formula (I) In a general aspect, the invention provides a polypeptide wherein two amino acid residues are bridged at their respective α-carbon positions by a diynylene group, and typically a 1,3-diyne group. The formation of the staple uses two amino acids each comprising a terminal alkynyl group side chain, which are linked to form a 1,3-diyne linkage. 008528580 In one aspect there is provided a polypeptide comprising a group of Formula (I) wherein -R1 and -R2 are each independently selected from optionally substituted C1-4 alkyl and -H; -R3 and -R4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue; -L1- and -L2- are each independently an optionally substituted C2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof. In the formulas described herein, represents a point of attachment to a moiety, such as an amino acid residue, a peptide segment or a polypeptide end group. A polypeptide end group attached to a carbonyl group (C terminal) may be selected from: -H; C1-4 alkyl, such as methyl, ethyl, iso-propyl, n-propyl and tert-butyl, such as methyl and ethyl; hydroxyl (-OH); amino (-NH2); alkoxy, such as C1-4 alkoxy (-O-C1-4 alkyl), such as methoxy and ethoxy; aminoalkyl, such C1-4 aminoalkyl (-N(H)C1-4 alkyl), such as -N(H)Me; a tag optionally with a linker, where the linker may link the tag to carbonyl group; a solid phase optionally with a linker, where the linker may link the tag to the carbonyl group; a protecting group; and a protected linker. A polypeptide end group attached to a nitrogen atom (N terminal) may be selected from -H; a protecting group, such as Fmoc or Boc; acyl, such as acetyl or propionyl, such as acetyl; C1-4 alkyl, such as methyl, ethyl, iso-propyl, n-propyl and tert-butyl, such as methyl and ethyl; 008528580 a protected linker; and a tag optionally with a linker where the linker may link the tag to the nitrogen atom. A polypeptide end group attached to a nitrogen atom may be selected from -H, a protecting group, acyl and C1-4 alkyl, such as -H, acetyl, methyl and ethyl; such as -H, acetyl and methyl. A polypeptide end group attached to a carbonyl group is preferably selected from -OH, -NH2, -O-C1-4 alkyl, -N(H)-C1-4 alkyl, C1-4 alkyl and a solid phase optionally with a linker; such as -OH, -NH2, and a solid phase optionally with a linker. A tag may comprise a chromophore; a fluorescent or a phosphorescent label; a radiolabel; or a group for targeting the polypeptide to a cell membrane, such as a lipid group, such as farnesyl or geranyl. Preferably, a tag is connected to a peptide terminal, such as the N terminal, via a linker. A solid phase may comprise a solid support material or resin, and may be a bead such as a polystyrene bead. A solid phase may be connected to a peptide terminal, such as the C terminal, via a linker. A linker is a divalent moiety that links together two moieties together, for example an amino acid residue or peptide and a tag, a solid phase or a protecting group. A linker may comprise one or more of: a covalent bond; one or more C2-4 oxyalkylene groups, such where the linker is a PEG linker; and one or more groups selected from C1-20 alkylene, ether, carbonyl, amide and ester. Examples of linkers include beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and PEG linkers such as PEG2, PEG3, or PEG4. A linker, where present, may be between the terminal nitrogen of a polypeptide of Formula (Ia) and a chromophore, a fluorescent label, a phosphorescent label, a radiolabel or a protecting group. 008528580 A linker, where present, may be between the C terminal of a polypeptide and a solid phase, a chromophore, a fluorescent label, a phosphorescent label, a radiolabel or a protecting group. A linker may be protected with a protecting group. A protecting group may be bonded directly to an end of the group of Formula (I), or a protecting group may be bonded to a linker. When the protecting group is bonded directly to an end of the group of Formula (I), the protecting group may be an amino protecting group that is attached to a nitrogen, or a carbonyl protecting group that is attached to a carbonyl group. When the protecting group is attached to a linker, it may be a protecting group for a moiety on the linker, such as a nitrogen or oxygen atom or a carbonyl group. A polypeptide end group attached to a nitrogen atom may comprise a fluorescent label and a linker, such as fluorescent label that is fluorescein-5-isothiocyanate (FITC) and linker that is 6-aminohexanoic acid (Ahx) or beta-alanine. A protecting group may be selected from 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), -oMe and -oBn. A protecting group attached to a nitrogen atom may be Fmoc, Boc or Cbz. A protecting group attached to a carbonyl group may be -oMe or -oBn. In some embodiments a polypeptide comprising a group of Formula (I) comprises an end group at the N terminal that is selected from -H, a protected linker, acyl, and a tag optionally with a linker, such as -H, acetyl, a fluorescent tag with a linker and a lipid group, such as -H, acetyl, and -Ahx-FITC, -beta-alanine-FITC, farnesyl and geranyl. In some embodiments a polypeptide comprising a group of Formula (I) comprises an end group at the C terminal that is selected from -OH, -NH2, and a solid phase optionally with a linker; such as -NH2 and a solid phase optionally with a linker. A polypeptide comprising a group of Formula (I) may be referred to as a stapled polypeptide. The diyne linkage, together with -L1- and -L2-, the α-carbon atoms to which -L1- and -L2- are bonded, and the peptide backbone between these α-carbon atoms, represented by -C(O)-[A]n-N(H)-, form a macrocycle which may be capable of conformationally constraining the polypeptide. In this way the degree of α-helical secondary structure in the polypeptide is increased. A polypeptide comprising a group of Formula (I) may be bonded to an amino acid residue or a peptide segment. In some embodiments, a polypeptide may comprise a group of Formula (I’) where * and ** each independently represent the point of attachment to an amino acid residue or a peptide segment 008528580 wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein. In some embodiments the polypeptide comprises Formula (I’) where * and/or ** indicates the point of attachment to a polypeptide end group. For example, the polypeptide may comprise Formula (I’) where * indicates the point of attachment to a group selected from -H, acyl such as acetyl, an amino protecting group, a protected linker, a tag optionally with a linker, or * indicates the point of attachment to a moiety that together with the nitrogen atom it is bonded to forms an N-terminal modification. N-terminal modifications include N-acetyl, N-formyl, pyroglutamyl, urea, fatty acids, carbamate, sulfonamide and alkylamine. The polypeptide may comprise Formula (I’) where ** indicates the point of attachment to a group selected from -OH, -NH2, a protecting group, a protected linker, or a solid phase optionally with a linker, and a tag optionally with a linker, or * indicates the point of attachment to a moiety that together with the carbonyl group it is bonded to forms a C-terminal modification. C-terminal modifications include amide, N-alkyl amide, aldehyde and esters. A polypeptide comprising a group of Formula (I) may comprise one or more peptide segments according to Formula (I), such as two or more segments of Formula (I). -L1- and -L2- Each of -L1- and -L2- is independently optionally substituted C2-10 alkylene. An alkylene group may be substituted or unsubstituted, and preferably, -L1- and -L2- are both unsubstituted. Each alkylene group may be linear or branched, and is preferably linear. Each alkylene group maybe unsubstituted or substituted. An alkylene group may be substituted, for example, with one or more halo groups, such as one or more of fluoro, chloro or bromo, and preferably one or more fluoro. In some embodiments, the alkylene group is 008528580 substituted with 1 to 4 fluoro, such as 1, 2, 3 or 4 fluoro. The alkylene group may be per-halogenated, such as per-fluorinated. Preferably, one or both of -L1- and -L2- is independently optionally substituted C2-10 alkylene, more preferably optionally substituted C3-10 alkylene, more preferably optionally substituted C4-10 alkylene, more preferably optionally substituted C4-7 alkylene, such as optionally substituted C5 alkylene or optionally substituted C6 alkylene. One or both of -L1- and -L2- may independently be optionally substituted C2-7 alkylene, such as optionally substituted C3-7 alkylene, such as optionally substituted C3-6 alkylene, such as optionally substituted C4-6 alkylene, such as optionally substituted C5-6 alkylene, and most preferably C5 alkylene. In these embodiments, -L1- and -L2- may be unsubstituted. Additionally or alternatively, one or both of -L1- and -L2- is independently optionally substituted C5-10 alkylene, preferably optionally substituted C5-8 alkylene, more preferably optionally substituted C5-7 alkylene, most preferably optionally substituted C5 alkylene or optionally substituted C6 alkylene. In some embodiments, one or both of -L1- and -L2- is independently optionally substituted C6-10 alkylene, more preferably optionally substituted C6-8 alkylene, such as optionally substituted C6 alkylene or C7 alkylene. In these embodiments, -L1- and -L2- may be unsubstituted. The groups -L1- and -L2- may be the same or they may be different. In some preferred embodiments -L1- and -L2- are the same, such as where both are selected from C4-10 alkylene, such as C4-7 alkylene, such as C5 or C6 alkylene. Additionally or alternatively, L1- and -L2- are the same and are both are selected from C5-10 alkylene, such as C5-8 alkylene, such as C5-7 alkylene; or C6-10 alkylene, such as C6-8 alkylene, such as C6-7 alkylene. Amino Acid Residue -A- Each -A- in Formula (I) is an amino acid residue, which may be a natural amino acid residue or a non-natural amino acid residue. Each -A- may be an α-amino acid residue or a β-amino acid residue. Preferably, -A- is an α-amino acid residue. Non-natural amino acids, which may also be referred to as modified amino acids, include enantiomers of natural amino acids and amino acid analogues having a modified side chain, as well as enantiomers of amino acid analogues. These may be α-amino acid residues or β-amino acid residues, such as α-amino acid residues. 008528580 Examples of non-natural amino acid residues include natural amino acid residues having a D-configuration (D-amino acid residues). Examples of non-natural amino acid residues include: norleucine (Nle), cyclobutylalanine (Cba), citrulline (Cit), hydroxyproline (Hyp), 3-nitrotyrosine, nitroarginine and ornithine (Orn), which each may be in the L- or D-configuration. An amino acid residue as described herein may comprise a protecting group. An amino acid residue thus encompasses: a natural or non-natural amino acid residue; and a protected natural or non-natural amino acid residue. Where an amino acid residue is protected, the protection may be provided on the side chain. Where an amino acid residue is provided at a terminal, for example within Formula (Ia), protection may be additionally or alternatively provided at the N terminal or the C terminal. Suitable protecting groups for use are well known to those skill in the art. Typically -A- is not an N-alkylated amino acid residue. Typical protecting groups are those suitable for use in solid phase peptide synthesis, and may include protecting groups that are removable under acidic conditions, for example with TFA. Preferably, -A- is selected from: Alanine [Ala, A], Aminobenzoic acid [PABA], Aminobutyric acid [Abu], Aminohexanoic acid [Ahx], Aminoisobutyric acid [Aib], Arginine [Arg, R], Asparagine [Asn, N], Aspartic acid [Asp, D], Butylglycine, Citrulline [Cit], Cyclohexylalanine [Cha], Cysteine [Cys], Diaminobutanoic acid [Dab], Diaminopropionic acid [Dpr or Dap], Dihydroxyphenylalanine [DOPA], Glutamic acid [Glu, E], Glutamine [Gln, Q], Glycine [Gly, G], Histidine [His, H], Homoserine [Hse], Hydroxyproline [Hyp], Isoleucine [Ile, I], Isonipecotic acid [Isn], Leucine [Leu, L], Lysine [Lys, K], Methionine [Met, M], Norleucine [Nle], Norvaline [Nva], Ornithine [Orn], Phenylalanine [Phe, F], Phenylglycine [Phg], Proline [Pro, P], Sarcosine [Sar], Serine [Ser, S], Statine and derivatives thereof [Sta], Tetrahydroisoquinoline-3-carboxylic acids [Tic], Thienylalanine [Thi], Threonine [Thr, T], Tryptophan [Trp, W], Tyrosine [Tyr, Y], and Valine [Val, V]. In the polypeptides of Formula (I), one or more amino acid residues may be crosslinked to another amino acid residue, in addition to the diyne linkage between the α-carbon atoms that are bonded to -R1 and -R2, respectively. An additional crosslink may be a further diyne linkage as described herein, or may be a different amino acid crosslink. Suitable crosslinks 008528580 include alkene bridges, amide bridges, ester bridges, disulfide bridges and lanthionine bridges. In some embodiments, the polypeptide may comprise 10 or more, such as 12 or more, such as 15 or more, such as 18 or more, such as 20 or more, such as 25 or more, contiguous amino acid residues, such as α-amino acid residues, including [A]n together with the two stapled amino acid residues comprising -R1 and -R2, respectively. The amino acid residue -A- may be represented by Formula (AA) where -R1 is as defined herein, and -RAA is an amino acid side chain, such as that of a natural amino acid, such as a proteinogenic amino acid. Preferably, -R1 is -H or methyl, such as -H. The amino acid residue of Formula (AA) is an α-amino acid residue. Integer n n is an integer from 2 to 10, and preferably is from 3 to 6. More preferably n is 3 or 6. n may be an integer selected from 2, 3, 6 and 10, and preferably an integer selected from 3, 6 and 10. In some embodiments n is 6. In these embodiments the diyne may be referred to as an i, i + 7 stapled polypeptide. Preferably, when n is 3 or more, such as 6 or more, -L1- and -L2- are C4 alkylene or higher, such as C5 alkylene or C6 alkylene. More preferably, n is 6 and -L1- and -L2- are both C5 or C6 alkylene, such as C6 alkylene. -R1 and -R2 Each of -R1 and -R2 is independently selected from C1-4 alkyl and -H. A C1-4 alkyl group may be methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl or sec-butyl. 008528580 Preferably, -R1 and -R2 are independently selected from methyl, ethyl, and -H, such as where -R1 and -R2 are independently selected from methyl and -H. -R1 and -R2 may be the same, or -R1 and -R2 may be different. Preferably, -R1 and -R2 are the same, such as where -R1 and -R2 are both methyl or both -H. -R1 and -R2 may both be methyl. Without wishing to be bound by theory, it is believed that when -R1 and -R2 are alkyl, such as methyl, the Thorpe-Ingold effect aids in the success of the Glaser reaction which improves the conversion rate during peptide stapling. It is also proposed that increased peptide helicity can be induced by the incorporation of a quaternary α-carbon into α-amino acids. -R3 and -R4 Each of -R3 and -R4 is independently selected from -H and methyl. -R3 and -R4 may be the same or they may be different. In some preferred embodiments -R3 and -R4 are the same and are both -H. In these embodiments the polypeptide may comprise a group of Formula (Ic) wherein -R1, -R2, -A-, -L1-, -L2- and n are as described herein. Additionally or alternatively, in some embodiments -R3 and -R4 are both -H, and -R1 and -R2 are each independently selected from -H and methyl, such where -R1 and -R2 are both methyl. In these embodiments, -L1- and -L2- may independently be optionally substituted C5-10 alkylene, such as optionally substituted C5-8 alkylene, such as optionally substituted C5 alkylene or optionally substituted C6 alkylene. Preferably, in these embodiments -L1- and - L2- are unsubstituted. Stereoisomers The α-carbons to which -R1 and -L1-, or -R2- and -L2-, are bonded, respectively, are chiral. These carbon atoms may independently be in an R or S configuration. 008528580 Preferably, a polypeptide of the invention comprises a group of Formula (IXS), where the α-carbon that is bonded to -R2 and -L2- is in a S configuration, and the α-carbon that is bonded to -R1- and -L1- may be in a R or S configuration, or a mixture thereof, wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as defined herein. The polypeptide may be a diastereomer according to any one of Formula (IRR), (IRS), (ISR) and (ISS) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein. 008528580 Preferably, the polypeptide comprises Formula (IRS), (ISR) and/or (ISS), more preferably Formula (IRS) and/or (ISS), and most preferably Formula (ISS). These configurations are particularly preferred when n is 6 and/or when -L1- and -L2- are C4-6 alkylene, such as C5 or C6 alkylene, such as such as C5 alkylene. These polypeptides are associated with high percentage α-helicity, such as 50% or more. Any remaining amino acid residues present in the polypeptide, such as each -A-, may independently be in a D-configuration or an L-configuration, and preferably are in the L-configuration. Helicity A polypeptide comprising a group of Formula (I) may have a substantially α-helical structure secondary structure, such as in aqueous solution. The polypeptide may have a helicity, which is a percentage helicity, of 40% or more, 50% or more, such as 60% or more, such as 65% or more, such as 70% or more, such as 75% or more. The polypeptide may have a helicity of 90% or less, such as 85% or less, such as 80% or less, such as 75% or less. The polypeptide may have a helicity in a range with upper and lower limits as described above, such as 40% to 90%, including 50 to 80%, such as 60% to 80%. Additionally or alternatively, the polypeptide has a helicity of 50% or more, such as 55% or more, such as 50% to 90%, including 50% to 80%, such as 55% to 80%. The polypeptide may have a helicity that is higher by 5% or more than a corresponding linear (non-stapled) polypeptide, such as 10% or more, such as 15% or more, such as 20% or more. A linear (non-stapled) polypeptide may be according to Formula (II) as described herein. Helicity may be measured by circular dichroism as described herein. Scanning may be carried out in a range of about 190-260 nm, such as with a scanning speed of about 50 nm/min, optionally with a 1 nm data pitch, further optionally with a 1 nm bandwidth, further optionally with an 8 s response time. The polypeptide may be measured at a concentration in the range of about 10 µM to 100 µM, such as about 20 µM to 75 µM, such as about 25 µM to 50 µM, such as about 25 µM or 50 µM. Measurements may be carried out at a pH in the range of about pH 7 to about pH 8, such as about pH 7.4. Circular dichroism data may be processed according to Equation B and/or Equation C as described herein. Helicity may be measured at a temperature of about 4 °C to about 50 °C, such as about 20 °C to about 40 °C, such as about 25 °C or about 37 °C. 008528580 Further Preferences A group of Formula (I) may be functionalised at one or both terminals, and optionally may be attached to one or more amino acid residues at either end. Thus, the invention also provides a polypeptide of Formula (Ia) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200. An N-methylated amino acid residue is an amino acid residue as described herein, having a monomethylated amino group. Here, methylated amino group refers to the amino group within a connecting amide bond or at the terminal, such as in the peptide backbone, and not an amino group within the side chain of the residue. Preferably, each -A’- is independently an α-amino acid residue or an N-methylated α-amino acid residue. Each of -R3 and -R3 may be -H. In embodiments where m is an integer of 1 or more, preferably -R3 is -H. In embodiments where m’ is an integer of 1 or more, preferably -R4 is -H. In some embodiments the moieties represented by -[A’]m- and -[A’]m'- may independently each comprise zero or one N-methylated amino acid residues, and the remaining -A’- groups are independently an amino acid residue. For example, the moieties represented by -[A’]m- and -[A’]m'- independently may each comprise one N-methylated amino acid residue and the remaining -A’- groups are independently an amino acid residue. Preferably, where the moiety represented by -[A’]m- or -[A’]m'- comprises an N-methylated amino acid residue, the N-methylated amino acid residue is a terminal residue. 008528580 Preferably, each -A’- is an independently an amino acid residue and -R3 and -R4 are each independently -H. In these embodiments, -A’- may be represented by -A- and the polypeptide may be represented by Formula (Ia’). where -R1, -R2, -A-, -L1-, -L2-, n, m, m’, RA and RB are as described herein. An alkyl group may be a C1-4 alkyl group, such as methyl, ethyl, iso-propyl or tert-butyl, such as methyl or ethyl. A protecting group attached to an amino group (amino protecting group) may be a carbamate protecting group, such as Fmoc (9-fluorenylmethoxycarbonyl), Boc (tert- butyloxycarbonyl) or (Cbz (carboxybenzyl). A protected amino group may be a carbamate protected amino group, such as -NHFmoc, -NHBoc or -NHCbz. An acyl group may be formyl, acetyl or propionyl, such as acetyl. A linker may comprise one or more C2-4 oxyalkylene groups, such where the linker is a PEG linker. A linker may be one or more groups selected from C1-20 alkylene, ether, carbonyl, amide and ester. Preferably, a linker is selected from beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and PEG linkers such as PEG2, PEG3, or PEG4. A linker may be protected. In some embodiments a linker is protected such as at a nitrogen atom, such as with a protecting group selected from Fmoc and Boc. In these embodiments the linker which is attached to the polypeptide may be capable of being deprotected and can undergo a reaction to link the polypeptide to a moiety. In some embodiments, -RA is a protected linker, such as an Fmoc protected 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), or 6-aminohexanoic acid (Ahx) linker. In some preferred embodiments, -RA is an Fmoc protected 6-aminohexanoic acid Ahx linker (-Ahx-Fmoc). In some embodiments a linker is unprotected, and may be a linker between an end group and a tag or a solid phase. 008528580 Each of -RA and -RB may independently be a tag. A tag may be a chromophore, a fluorescent label, a phosphorescent label or a radiolabel. A tag may comprise a group for targeting the polypeptide to a cell membrane, such as where the tag is a lipid group, such as farnesyl or geranyl. A tag may be connected, such as to an amino acid residue -A- or a peptide terminal, by a linker. A linker may be 6-aminohexanoic acid or a PEG linker. A linker, where present, is preferably between the terminal nitrogen of a polypeptide of Formula (Ia) and a chromophore, a fluorescent label, a phosphorescent label or a radiolabel, or the linker may be between the C terminal of Formula (Ia) and a chromophore, a fluorescent label, a phosphorescent label or a radiolabel. Preferably, -RA is selected from -H, C1-4 alkyl, -Fmoc, -L-Fmoc, acetyl, and -L-XT, where -XT is a tag selected from chromophore; a fluorescent or a phosphorescent label and a radiolabel; and a lipid group such as farnesyl and geranyl, and where -L- is a linker. Preferably, a linker is selected from a covalent bond, beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and a PEG linker such as PEG2, PEG3, or PEG4. The group -RA may be selected from -H, C1-4 alkyl, acetyl, -Fmoc, -Ahx-Fmoc, -beta-alanine-Fmoc, -Ahx-FITC, and -beta-alanine-FITC; such as acetyl, -Ahx-Fmoc, -beta-alanine-Fmoc, -Ahx-FITC, and -beta-alanine-FITC. When -RA is -H, the polypeptide has an unmodified N terminal. In some embodiments -RB may be selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a tag optionally with a linker, and a solid phase optionally with a linker. Preferably, -RB is selected from hydroxyl and amino, and a solid phase optionally with a linker. When -RB is hydroxyl the polypeptide has an unmodified C terminal. This terminal may also be in carboxylate form. When RB is amino, the polypeptide has a C-terminal amide modification. In embodiments where -RB comprises a solid phase, -RB may be a solid phase with a linker present between the solid phase and the carbonyl to which -RB is attached to. A linker may be, or may comprise, a group selected from -O-, -N(H)-, or -N(Me)-. In some embodiments RB is selected from -O-L-PSP and -N(H)-L-PSP, where -L- is a linker and -PSP is a solid phase. Preferably, -RB is selected from hydroxyl, amino, -O-L-PSP and -N(H)-L-PSP, where -L- is a linker and -PSP is a solid phase. The group -RB may be selected from hydroxyl and amino. An alkoxy group may be C1-4 alkoxy, such as methoxy or ethoxy. 008528580 An amino group is -NH2. Preferably, each of m and m’ is independently an integer from 0 to 100, such as 1 to 100, such as 2 to 100, such as 2 to 75, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 10, such as 2 to 8. Preferably, m and m’ are each greater than 1. Preference for amino acid stereochemistry is as described herein, such as according to one or more of Formula (I ), (I ), (I ) and (I ). Prefe 2 RR RS SR SS rably, the α-carbon bonded to -R and -L2- is in a S configuration, and the α-carbon bonded to -R1 and -L1- is in a S or R configuration, more preferably S. In some embodiments the polypeptide is of Formula (Ib) or Formula (Ib’) wherein -R1, -R2, -R3, -R4, -A-, -A’-, -L1-, -L2-, -RA, -RB, n, m and m’ are as described herein, and z is an integer from 1 to 4. Preferably, z is an integer from 1 to 3, more preferably 1 or 2, most preferably 1. Typically, when z is greater than 1, m’ is not 0, and preferably m’ is 2 or more. When z is greater than 1, preferably m’ in a moiety that is positioned between two diyne staples is an integer of 5 or more, such as 6 or more, such as 7 or more. 008528580 In some preferred embodiments the polypeptide comprises Formula (IRS’), (ISS’) or a mixture thereof wherein -R1 and -R2 are independently selected from methyl and -H, such as methyl; each -A- is independently an amino acid residue; -L1- and -L2- are independently C4-6 alkylene; and n is an integer from 3 to 6, such as 3 or 6, such as 6. In some embodiments the polypeptide is according to Formula (IaRS’), (IaSS’) or a mixture thereof 008528580 wherein -RA and -RB are as defined herein, and -R1 and -R2 are independently selected from methyl and -H, such as methyl; each -A- is independently a natural or non-natural amino acid residue; -L1- and -L2- are independently C4-6 alkylene; and n is an integer from 3 to 6, such as 3 or 6, such as 6. Examples of preferred polypeptides include a polypeptide of Formula (Id), (IdRS) or (IdSS), wherein -R1, -R2, -L1- and -L2- are as defined herein, and: (i) -RA is selected from -H, an amino protecting group, acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a fluorescent tag optionally with a linker; -[A]m- is -Leu-Thr-Phe-; -[A]n- is -Glu-Tyr-Trp-Ala-Gln-Cba-; -[A]m’- is -Ser-Ala-Ala-; and -RB is selected from hydroxy, amino, protected amino, -O-L-PSP and -N(H)-L-PSP, where -L- is a linker, such as a covalent bond, and -PSP is a solid phase; such as hydroxy, amino, -O-L-PSP and -N(H)-L-PSP; such as amino and -N(H)-L-PSP; or 008528580 (ii) -RA is selected from -H, an amino protecting group, acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a fluorescent tag optionally with a linker; -[A]m- is -Ala-Lys-; -[A]n- is -Tyr-Ala-Arg-Nle-Gly-His-; -[A]m’- is -Leu-Lys-Glu-Ile-Ala-Lys-; and -RB is selected from hydroxy, amino, protected amino, -O-L-PSP and -N(H)-L-PSP, where -L- is a linker, such as a covalent bond, and -PSP is a solid phase; such as hydroxy, amino, -O-L-PSP and -N(H)-L-PSP; such as amino and -N(H)-L-PSP, where Cba is cyclobutylalanine and Nle is norleucine. Preferably, in Formula (Id), (IdRS) or (IdSS), -R1 and -R2 are independently methyl and -L1- and -L2- are independently selected from C5-6 alkylene. Polypeptide Comprising a Group of Formula (II) In an aspect the present invention provides a polypeptide comprising a group of Formula (II) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein, and the salts and solvates and protected forms thereof. A polypeptide comprising a group of Formula (II) may be referred to as a linear polypeptide. A linear polypeptide may be a precursor to a stapled polypeptide, and the terminal alkynyl resides are available for cyclisation to form the corresponding stapled polypeptide. Preferences for -L1-, -L2-, -R1, -R2, -A-, n, stereochemistry at the α-carbons, and linkages at the N-terminal and C-terminal of the segment shown in Formula (II) are as described herein for polypeptides comprising a group of Formula (I). The groups -L1- and -L2- are preferably independently C4-10 alkylene, such as C5 or C6 alkylene. Alternatively, in some embodiments, the groups -L1- and -L2- are independently C5-10 alkylene, such as C5-8 alkylene. 008528580 N is preferably an integer from 2 to 8, such as from 3 to 8, such as from 3 to 6, and more preferably n is 3 or 6, such as 6. Each of -R1 and -R2 is preferably independently selected from methyl and -H. More preferably, -R1 and -R2 are methyl. The polypeptide may comprise a group of Formula (IIXS), where the α-carbon that is bonded to -R2 and -L2- is in a S configuration, and the α-carbon that is bonded to -R1 and -L1- may be in a R or S configuration, or a mixture thereof. where -R1, -R2, -R3, -R4, -L1-, -L2-, -A- and n are as defined herein. The polypeptide may be a diastereomer according to any one of Formula (IIRR), (IIRS), (IISR) and (ISS) 008528580 where -R1, -R2, -R3, -R4, -L1-, -L2-, -A- and n are as defined herein. Preferably, the polypeptide comprises Formula (IIRS), (IISR) and/or (IISS), more preferably Formula (IIRS) and/or (IISS), and most preferably Formula (IISS). The remaining amino acid residues in the polypeptide, such as each -A-, may be in a D-configuration or an L-configuration, and preferably are in the L-configuration. A peptide segment of Formula (II) may be functionalised at either end. One or both ends of the peptide segment may be bonded to one or more amino acid residues. Thus, the invention also provides a polypeptide of Formula (IIa), (IIa’), (IIb) or (IIb’) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein, and each -A’- is independently an amino acid residue or an N-methylated amino acid reside; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200; 008528580 wherein -R1, -R2, -A-, -L1-, -L2-, -RA, -RB, n, m and m’ are as described herein; wherein -R1, -R2, -R3, -R4, -A-, -A’-, -L1-, -L2-, -RA, -RB, n, m and m’ are as described herein, and z is an integer from 1 to 4; wherein -R1, -R2, -A-, -L1-, -L2-, -RA, -RB, n, m, m’ and z are as described herein. The group -RA is selected from -H, C1-4 alkyl, -Fmoc, -L-Fmoc, acetyl, and -L-XT, where -XT is a tag selected from chromophore; a fluorescent or a phosphorescent label and a radiolabel; and a lipid group such as farnesyl and geranyl, and where -L- is a linker. The group -RB may be selected from hydroxyl, amino, -O-L-PSP and -N(H)-L-PSP, where -L- is a linker as described herein and -PSP is a solid phase. 008528580 Preferably, a linker is selected from a covalent bond, beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), and a PEG linker such as PEG2, PEG3, or PEG4. Each of m and m’ may be independently an integer from 0 to 100, such as 1 to 100, such as 2 to 100, such as 2 to 75, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 10, such as 2 to 8. Z may be an integer from 1 to 3, more preferably 1 or 2, most preferably 1. Typically, when z is greater than 1, m’ is not 0, and preferably m’ is 2 or more. In some embodiments the polypeptide is according to Formula (IIc) where R1, -R2, -A-, -L1-, -L2- and n are as described herein. In some embodiments the polypeptide is according to Formula (IiaRS’), (IiaSS’) or a mixture thereof wherein -RA and -RB are as defined herein, and -R1 and -R2 are independently selected from methyl and -H, such as methyl; each -A- is independently a natural or non-natural amino acid residue; 008528580 -L1- and -L2- are independently C4-6 alkylene; and n is an integer from 3 to 6, such as 3 or 6, such as 6. Examples of preferred polypeptides include a polypeptide of Formula (IId), (IIdRS) or (IIdSS), (i) -RA is selected from -H, an amino protecting group, acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a fluorescent tag optionally with a linker; -[A]m- is -Leu-Thr-Phe-; -[A]n- is -Glu-Tyr-Trp-Ala-Gln-Cba-; -[A]m’- is -Ser-Ala-Ala-; and -RB is selected from hydroxy, amino, protected amino, -O-L-PSP and -N(H)-L-PSP, where -L- is a linker, such as a covalent bond, and -PSP is a solid phase; such as hydroxy, amino, -O-L-PSP and -N(H)-L-PSP; such as amino and -N(H)-L-PSP; or (ii) -RA is selected from -H, an amino protecting group, acyl, a protected linker, and a tag optionally with a linker; such as acyl, a protected linker, and a tag 008528580 optionally with a linker; such as acyl, a protected linker, and a fluorescent tag optionally with a linker; -[A]m- is -Ala-Lys-; -[A]n- is -Tyr-Ala-Arg-Nle-Gly-His-; -[A]m’- is -Leu-Lys-Glu-Ile-Ala-Lys-; and -RB is selected from hydroxy, amino, protected amino, -O-L-PSP and -N(H)-L-PSP, where -L- is a linker, such as a covalent bond, and -PSP is a solid phase; such as hydroxy, amino, -O-L-PSP and -N(H)-L-PSP; such as amino and -N(H)-L-PSP, where Cba is cyclobutylalanine and Nle is norleucine. Preferably, -R1 and -R2 are independently methyl and/or -L1- and -L2- are independently selected from C5-6 alkylene. Amino Acid Disclosed herein is a compound of Formula (A) wherein -LY- is optionally substituted C2-10 alkylene; -R1 is selected from optionally substituted C1-4 alkyl and -H; -R3 is selected from -H and methyl; -RN is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; such as -H, Fmoc, Boc, methyl, ethyl, iso-propyl, acetyl, farnesyl and geranyl; and -RC is selected from -OH and a protecting group, or -RC together with the carbonyl group -RC is attached to forms an activated acid, and the salts and solvates thereof. -LY- may be an optionally substituted C4-10 alkylene, such as C5 alkylene or C6 alkylene. R1 may be methyl or -H, such as methyl. R3 may be -H. 008528580 In an aspect, the invention provides a compound of Formula (III) wherein -LX- is optionally substituted C5-10 alkylene; -R1 is selected from optionally substituted C1-4 alkyl and -H; -R3 is selected from -H and methyl; -RN is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker, such as -H, Fmoc, Boc, methyl, ethyl, iso-propyl, acetyl, farnesyl and geranyl; and -RC is selected from -OH and a protecting group, or -RC together with the carbonyl group to which it is attached forms an activated acid, and the salts and solvates thereof. A compound of Formula (A) or Formula (III) is a non-natural amino acid or protected non-natural amino acid, and is suitable for use in the synthesis of linear polypeptides, and in turn, stapled polypeptides as described herein. Preference for -R1, -R3, -LX and stereochemistry may be as described herein for a polypeptide comprising a group of Formula (I) or (II). Preference for -RN may be as described herein for -RA for a polypeptide comprising a group of Formula (I) or (II). -LX- may be C5-9 alkylene, such as C5-8 alkylene, such as C5 or C6 alkylene. Preferably, -LX- is C6-10 alkylene, more preferably C6-9 alkylene, more preferably C6-8 alkylene, such as C6 or C7 alkylene. Most preferably, -LX- is C6 alkylene. The group -R1 may be selected from -H, methyl, ethyl, n-propyl, i-propyl, or n-butyl, s-butyl or tert-butyl. Preferably, -R1 is selected from -H, methyl and ethyl, more preferably from -H and methyl, and most preferably -R1 is methyl. The group -RN may be -H or a protecting group. Examples of preferred protecting groups include carbamate, such as Fmoc (9-fluorenylmethoxycarbonyl) and Boc (tert-butyloxycarbonyl) and benzyloxycarbonyl (Cbz). In some embodiments, -RN is selected from -H, Fmoc and Boc. 008528580 The group -RN may be an acyl group such as a C1-12 acyl group, such as formyl, acetyl or propionyl. In some embodiments -RN is acetyl. In some embodiments -RN is -H. -RN may be a tag that is optionally with a linker. A tag may be selected from: a chromophore; a fluorescent label; a phosphorescent label; a radiolabel; or a group for targeting the compound to a cell membrane, such as a lipid group, such as farnesyl or geranyl. A linker, where present, links the tag and the nitrogen atom to which -RN is attached. In some embodiments -RN comprises a fluorescent label and a linker, such as fluorescein-5-isothiocyanate (FITC) and a linker which may be 6-aminohexanoic acid (Ahx) or beta-alanine. For example, -RN may be FITC-Ahx- or FITC-beta-alanine-. -RC may be selected from -OH, -NH2 and a protecting group. Examples of protecting groups include -Ome, -OtBu and benzyloxy (Obn). The group -RC together with the carbonyl group to which it is attached may form an activated acid. Examples of activated acids include acyl chloride, 1-hydroxybenzotriazole activated ester, and carbodiimide activated ester. -RC may be selected from -Cl, -O-benzotriazole, and -O-C(=NR’)NR’’, where -R’ and -R’’ are independently selected from cyclohexyl, isopropyl, ethyl, and dimethylaminopropyl. For example, -C(=NR’)NR’’ may be selected from dicyclohexylcarbodiimidyl, diisopropylcarbodiimidyl, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidyl. In some preferred embodiments -LX is C5-10 alkylene, such as C5 alkylene; or C6-10 alkylene, such as C6 alkylene; -R1 is selected from methyl or -H, such as methyl; -R3 is -H; -RN is selected from Fmoc and Boc, farnesyl and geranyl, such as Fmoc and Boc; and -RC is selected from -OH and a protecting group, or -RC together with the carbonyl group to which it is attached forms an activated acid. A compound of Formula (A) or Formula (III) may be compound A or compound B. In some embodiments, the compound is not compound A or compound B. 008528580 A compound of Formula (A) or Formula (III) may be in a R configuration or an S configuration. The invention provides a compound of Formula (IIIR) and a compound of Formula (IIIS). (IIIR) (IIIS) where -RN, -R1, -R3, and -LX- and -RC are as described herein. 008528580 Preferably, the compound is of Formula (IIIa), (IIIaR) or (IIIaS). where -RN, -R1, -R3, and -LX- are as described herein. Compounds of Formula (III) may be prepared by a method as described herein, such as by reaction of an Ni(II) Schiff base complex derived from an amino acid, such as alanine, glycine, homoalanine and valine, with an iodo-alkyne. Other Forms Polypeptides and compounds of the invention may be provided in salt form, solvate from or protected form. Compounds having a carboxyl group, such as polypeptides comprising a group of Formula (I) or (II) or compounds of Formula (III) may be provided as salts, for example base addition salts of strong mineral bases and addition salts of strong organic bases. Compounds having an amine group, such as the polypeptides comprising a group of Formula (I) or (II) or compounds of Formula (III) may be provided as salts, for example acid 008528580 addition salts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulfates and acetates such as trifluoroacetate or trichloroacetate. A reference to a compound or polypeptide described herein is also a reference to a solvate of that compound. Examples of solvates include hydrates. A protected form of a compound or polypeptide may comprise an amino protecting group, such as benzyloxycarbonyl (Cbz), Fmoc or Boc; a carboxyl protecting group, such as alkoxy, such as -Ome or -Obn; and/or a side chain protecting group, such as benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), 9-xanthenyl (Xan), tosyl (Tos), benzyloxymethyl (Bom), formyl, tert-butyldimethylsilyl (TBDMS), allyl, o-nitrobenzyl (ONB), p-methylbenzyl (Meb) and acetamidomethyl (Acm). Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner. In some embodiments the present invention provides an enantiomer or diastereomer of the polypeptides or compounds described herein. An enantiomer as described herein may be substantially pure, and may have an enantiomeric excess of 70% or more, such as 80% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 98% or more, such as 99% or more. Methods of Synthesis In one aspect, there is provided a method of preparing a diyne stapled polypeptide, such as a stapled peptide of Formula (I), the method comprising steps of (i) providing a polypeptide of Formula (II); and (ii) coupling the terminal alkynyl groups of the polypeptide to provide a 1,3-diyne linkage. In some embodiments, the method is for preparing a diyne of a stapled peptide comprising a group of Formula (I). 008528580 Step (ii) may be carried out in the presence of a metal salt, such as a transition metal salt, such as: a copper salt, such as a Cu(I) salt; a cobalt salt, such as a Co(II) salt; a silver salt, such as Ag(I) salt; and a palladium salt, such as a Pd(II) salt, such as described in Akhtar et al. A copper salt may be a Cu(I) salt, examples of which include CuCl, CuBr, CuI, CuOAc, Cu2(ophen)2, Cu4(ophen)4(tp). Step (ii) may be carried out in the presence of a source of Cu(I). Examples of a source of Cu(I) include Cu(II) salts, such as CuSO4, Cu(OAc)2 and CuCl2, optionally together with a reducing agent. A metal salt, where present in step (ii), may be provided in a stoichiometric amount relative to the polypeptide, or the metal salt may be present at an amount that is less than a stoichiometric amount. The metal salt may be regenerated during the radical reaction. Alternatively, the metal salt may be present at an amount that is greater than a stoichiometric amount. This may help to increase the yield of the stapled polypeptide. Step (ii) may be carried out in the presence of a ligand, and preferably is so when a metal salt such as a copper salt is present. A ligand, where present, may be 4,4’-bis(hydroxymethyl)-2,2’-bipyridine, 2,2’-bipyridine, tetramethylethylenediamine (TMEDA) or pyridine. Preferably, the ligand is 4,4’-bis(hydroxymethyl)-2,2’-bipyridine or tetramethylethylenediamine (TMEDA). Typically, step (ii) is carried out in the presence of an oxidant, such as oxygen. Step (ii) may be carried out in the presence of air, or in an oxygen atmosphere. Step (ii) may be carried out in the presence of a base, typically an organic base, such as a tertiary amine, such as N,N-diisopropylethylamine (DIPEA), triethylamine and piperidine. Step (ii) may be performed at a temperature of 20 °C or more, such as 30 °C or more, such as 40 °C or more, such as 50 °C or more. The temperature may be 80 °C or less, such as 70 °C or less. The temperature may be in a range with upper and lower values described above, such as from 20 °C to 80 °C, including 50 °C to 70 °C. Preferably, the temperature during step (ii) is about 60 °C. The reaction mixture may be heated, such as by microwave heating. 008528580 Step (ii) may be carried out in the presence of microwave radiation. The reaction may be subjected to microwave radiation during step (ii). The reaction time during step (ii) may be 5 minutes or more, such as 10 minutes or more, such as 30 minutes or more, such as 1 hour or more, such as 2 hours or more, such as 3 hours or more. In step (ii) two terminal alkynes in the polypeptide, in particular in side chains of the polypeptide, are coupled together and a 1,3-diyne is formed. The coupling reaction provides a stapled peptide. Where more than two terminal alkyne groups are present in the polypeptide, such as where the polypeptide provided in step (i) is of Formula Iib where z is an integer of 2 or more, typically any two alkynyl groups that are spaced by up to i and i+10 and/or at least i and i+3 may be coupled to form a 1,3-diyne linkage. A polypeptide provided in step (i) may be prepared by peptide synthesis, such as by solid phase peptide synthesis using one or more amino acids of Formula (A) or Formula (III) as described herein. A polypeptide provided in step (i) may be provided attached to a solid phase, or the polypeptide may be provided with free N terminal and a free C terminal, such as wherein the polypeptide has been cleaved from a solid phase following solid phase synthesis. Preferably, the polypeptide provided in step (i) is provided attached to a solid phase, such as at the C terminal. In some embodiments, step (i) comprises synthesising a polypeptide comprising a group of Formula (II), such as a polypeptide of Formula (Iia) or (Iib), by peptide synthesis as described herein using an amino acid of Formula (III) and/or Formula (A). Step (i) may further comprise cleaving the polypeptide from a solid phase. Step (ii) may be carried out on-resin, such as wherein the polypeptide of Formula (II) is attached to a solid phase at the C terminal of the polypeptide, or step (ii) may be carried out off-resin, such as wherein the linear polypeptide has been cleaved from a resin used during solid phase synthesis. Preferably, step (ii) is carried out on-resin, such as wherein the polypeptide of Formula (II) is attached to a solid phase, and the stapled peptide formed during step (ii) may be cleaved from the solid phase. The N terminal, C terminal, and one or more side chains in the polypeptide may independently be protected during the coupling in step (ii). Preferably, the N terminal is protected, and the C terminal and one or more side chains are optionally also protected. In these embodiments, the method may further comprise a step of deprotecting the polypeptide following step (ii). Coupling in step (ii) may be carried out on a polypeptide having a N terminal protected polypeptide, such as a Fmoc protected polypeptide. 008528580 In some embodiments, there is provided a method of preparing a diyne stapled peptide, such as a peptide comprising a group of Formula (I), comprising steps of (i) providing a polypeptide comprising a group of Formula (II) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described herein; and (ii) contacting the polypeptide with a metal salt, optionally in the presence of a ligand. Preference for steps (i) and (ii) are as described herein. An amino acid may be prepared by a method as described herein, such as by reaction of an Ni(II) Schiff base complex derived from an amino acid with an iodo-alkyne. Methods of Treatment The polypeptides comprising a group of Formula (I) and pharmaceutical compositions described herein are suitable for use in methods of treatment and prophylaxis. The polypeptides or pharmaceutical compositions may be administered to a subject in need thereof. The polypeptides comprising a group of Formula (I) or pharmaceutical compositions are for use in a method of treatment of the human or animal body by therapy. In some aspects of the invention, a polypeptide comprising a group of Formula (I) or pharmaceutical composition may be administered to a mammalian subject, such as a human, in order to treat a proliferative disease, such as cancer. In some embodiments a polypeptide comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS), is for use in a method of treating cancer, wherein the polypeptide inhibits dimerization of Sam68. In some embodiments the polypeptide or pharmaceutical composition comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS), is for use in a method of treatment cancer wherein the polypeptide inhibits interaction between p53 and MDM2, such as where the polypeptide inhibits p53/MDM2 binding. 008528580 In some embodiments a polypeptide comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS), is for use in a method of treating a disease with altered Sam68 activity or altered p53 and/or MDM2 activity. The disease may be cancer. In some embodiments a polypeptide comprising a group of Formula (I), such as a polypeptide of Formula (Id), (IdRS) or (IdSS), is for use in a method of treatment wherein the polypeptide inhibits Sam68 activity or inhibits MDM2 activity. Another aspect of the present invention pertains to use of a polypeptide comprising a group of Formula (I) in the manufacture of a medicament for use in treatment. In one embodiment, the medicament comprises a polypeptide comprising a group of Formula (I). The polypeptides of the present case may be useful for the treatment of a proliferative disease, such as cancer. The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term “treatment.” The term “therapeutically-effective amount,” as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. The term “treatment” includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. Formulations The present invention also provides a pharmaceutical composition comprising a polypeptide comprising a group of Formula (I) together with a pharmaceutically acceptable carrier. While it is possible for the polypeptide comprising a group of Formula (I) to be administered alone or together with the second agent, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising a polypeptide comprising a group of Formula (I) as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but 008528580 not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents. Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing a polypeptide comprising a group of Formula (I) as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound. The composition optionally further comprises the second active agent in a predetermined amount. The term “pharmaceutically acceptable,” as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005. The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the polypeptide comprising a group of Formula (I) with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary. Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols. Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other 008528580 pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir. The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses. Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavoured basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier. Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills. Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in- water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs. Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs. Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base. 008528580 Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound. As an alternative method of administration, a dry powder delivery may be used as an alternative to nebulised aerosols. Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro- tetrafluoroethane, carbon dioxide, or other suitable gases. Additionally or alternatively, a formulaton for pulmonary administration may be formulated for administration from a nebuliser or a dry powder inhaler. For example, the formulation may be provided with carriers or liposomes to provide a suitable particle size to reach the appropriate parts of the lung, to aid delivery of an appropriate does to enhance retention in the lung tissue. Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound. Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate. 008528580 Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection. Typically, the concentration of the compound in the liquid is from about 1 ng/mL to about 100 μg/mL, for example from about 10 ng/mL to about 10 μg/mL, for example from about 10 ng/mL to about 1 μg/mL. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Routes of Administration A polypeptide comprising a group of Formula (I), a second agent, or a pharmaceutical composition comprising the polypeptide comprising a group of Formula (I), may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action). Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. The Subject/Patient The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine 008528580 (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orang-utan, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus. In one preferred embodiment, the subject/patient is a human. It is also envisaged that the invention may be practised on a non-human animal having a microbial infection. A non-human mammal may be a rodent. Rodents include rats, mice, guinea pigs, chinchillas and other similarly-sized small rodents used in laboratory research. Definitions An alkyl group is monovalent alicyclic or cyclic saturated hydrocarbon group. An alkyl group may be a C1-20 alkyl group, for example a C1-15, C1-12, C1-10, C1-8, C1-6, C1-4, C1-3 or a C1-2 alkyl group. In this context, the prefix (e.g. C1-6) denotes the number of carbon atoms in the hydrocarbon backbone. An alkyl group may be linear, branched or cyclic. Examples of C1-6 linear alkyl groups include methyl (-Me), ethyl (-Et), n-propyl (-nPr), n-butyl (-nBu), n-pentyl (-Amyl) and n-hexyl. Examples of C1-6 branched alkyl groups include iso- propyl (-iPr), iso-butyl (-iBu), sec-butyl (-sBu), tert-butyl (-tBu), iso-pentyl, sec-pentyl, tert- pentyl, neo-pentyl, iso-hexyl, sec-hexyl, tert-hexyl and neo-hexyl. Examples of C1-6 cyclic alkyl groups include cyclo-pentyl and cyclo-hexyl. An alkylene group is a divalent saturated hydrocarbon group in which the two free valencies independently form part of a single bond to separate adjacent atom. An alkylene group is a divalent group that may be derived from an alkyl group as defined above, such as by removal of a hydrogen atom. Examples of linear alkylene groups include methanediyl (methylene bridge), ethane-1,2-diyl (ethylene bridge), propane-1,3-diyl, butan-1,4-diyl, pentan-1,5-diyl and hexan-1,6-diyl. Examples of branched alkylene groups include ethane- 1,1-diyl and propane-1,2-diyl. An alkynyl group is a monovalent unsaturated hydrocarbon group containing one or more carbon-carbon triple bonds. An alkynyl group may be a C2-20 alkynyl group, for example a C2-10, C2-6 or a C2-4 alkynyl group. In this context, the prefix (e.g. C1-6) denotes the number of carbon atoms in the hydrocarbon backbone. An alkynyl group may be linear or branched, or the alkenyl group may be incorporated into a ring system. Examples of linear alkynyl groups include ethynyl and 2-propynyl (propargyl). Examples of alkynyl groups incorporated into a ring system include cyclooctyne (OCT). A hydroxyl group is -OH or the hydroxide form of this group. 008528580 An acyl group is -C(=O)H or -C(=O)R, where -R is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. Examples of acyl groups include formyl, acetyl (-Ac), propionyl, tert-butyryl and benzoyl (-Bz). An ester group comprises a divalent moiety -C(=O)O- or -OC(=O)-. A ester group is -C(=O)OR or -OC(O)R, where -R is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. Examples of ester groups include acetoxy (-OAc). An amide group comprises a divalent moiety -C(=O)N(RNA)(RNB)- or -N(RNC)C(=O)-. An amide group is -C(=O)NRNARNB or -N(RNC)C(=O)-, where -RNA, -RNB and -RNC are independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. Examples of amide groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHEt, -C(=O)nEt2, (-NHC(=O)Me), -NHC(=O)Et, and -NHC(=O)Ph, as well as amido groups in which RNA and RNB, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl, succinimidyl, maleimidyl, and phthalimidyl. An oxyalkylene group is an alkylene group in which one or more carbon atoms is replaced with a oxygen. The oxyalkylene group may be a C1-6 oxyalkylene group, for example, a C1-4 or a C1-3 oxyalkylene group. In this context, the prefix (e.g. C1-6) denotes the number of atoms in the oxyalkylene backbone, whether carbon or oxygen atoms. The oxyalkylene group may be linear or branched. Examples of linear oxyalkylene groups include those derived from oxymethylene (e.g. polyoxymethylene, POM), ethylene glycol (e.g. polyethylene glycol, PEG), and tetramethylene glycol (e.g. polytetramethylene glycol, PTMEG; polytetrahydrofuran). Examples of branched oxyalkylene groups include those derived from propylene glycol (e.g. polypropylene glycol PPG). A carbonyl group comprises the divalent moiety -C(=O)-. A carbonyl group is -C(=O)RCA where RCA is selected from hydrogen, C1-6 alkyl and C5-20 aryl. An ether group comprises the divalent moiety -O-. An ether group is -ORE, where RE is selected from hydrogen, C1-6 alkyl and C5-20 aryl. A protecting group may be selected from 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), 9-xanthenyl (Xan), tosyl (Tos), benzyloxymethyl (Bom), formyl, tert-butyldimethylsilyl (TBDMS), allyl, o-nitrobenzyl (ONB), p-methylbenzyl (Meb) or acetamidomethyl (Acm). A nitrogen protecting group, such as an N-terminal protecting group, may be selected from 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz). A side chain protecting group may 008528580 be selected from allyloxycarbonyl (Alloc), 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5- sulfonyl (Pbf), 9-xanthenyl (Xan), tosyl (Tos), benzyloxymethyl (Bom), formyl, tert- butyldimethylsilyl (TBDMS), allyl, o-nitrobenzyl (ONB), p-methylbenzyl (Meb) or acetamidomethyl (Acm). A C terminal protecting group may be an ester and may be selected from methyl ester and benzyl ester. Other Embodiments Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. “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. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above. Results and Discussion Materials All reagents were purchased from commercial sources and used without further purification unless otherwise stated. Fmoc-protected amino acids were purchased from CEM Corporation, and Pepceuticals. N,N-Dimethylformamide (DMF) and diethyl ether (Et2O) were purchased from Rathburn. (R)-N-Fmoc-α-(7-Octenyl)alanine, (S)-N-Fmoc-α-(4- pentenyl)alanine, triisopropylsilane (TIPS), 1,2-dichloroethane (DCE) and Grubbs 1st Catalyst were purchased from Sigma Aldrich. Trifluoroacetic acid (TFA), N,N’-diisopropylethylamine (DIPEA), N,N’-diisopropylcarbodiimide (DIC), ethyl (hydroxyamino)cyanoacetate (Oxyma Pure), fluorescein-5-isothiocyanate (FITC), Fmoc-Lys(Alloc)-OH, Fmoc-6-Ahx-OH, palladium (O)tetrakis(triphenyphosphine) (Pd(PPh3)4) and phenylsilane were purchased from Fluorochem. Morpholine was purchased from Alfa Aesar. Dichloromethane (DCM) was purchased from VWR. Acetonitrile (MeCN) was purchased from Fisher Scientific. TentaGel S RAM resin was purchased from Rapp 008528580 Polymere. Chemmatrix Rink Amide resin was purchased from Biotage. All other reagents were purchased from Sigma Aldrich. S-RBD was purchased from Genscript (S-RBD, P330-F541, Product no. Z03479) and AcroBiosystems (S-RBD, R319-F541, Product no. SPD-C52H3). Dry solvents were purified using a PureSolv 500 MD solvent purification system. Thin-layer chromatography was performed on aluminium backed plates (0.25 μm) with silica gel 60 coated F254 and visualised by staining with potassium permanganate (KMnO4). General Methods General Method A: Analytical Methods Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was performed on a Shimadzu RP-HPLC system with Shimadzu LC-20AT pumps, a Shimadzu SIL20A autosampler and a Shimadzu SPD-20A UV-vis detector using a Phenomenex Aeris column (5 mm C18, 100 Å, 150 × 10 mm). Compounds were eluted with linear gradients at column-dependent flow rates (1 mL/min for the Aeris, 10 mL/min for the Gemini), where buffer A = 0.1% TFA in H2O and buffer B = 0.1% TFA in MeCN. Data is reported as column retention time (tR) in minutes (min). Liquid chromatography-mass spectrometry (LC-MS) was performed on a Thermo Scientific LCQ Fleet Ion Trap Mass Spectrometer using positive mode electrospray ionisation (ESI+). Where buffer A = 0.1 % TFA in 95% H2O/5% MeCN and buffer B = 0.1% TFA in 95% MeCN/5% H2O, a linear gradient of 5-95% B over 20 min with a flow rate of 1 mL/min was used with a Reprosil-Gold column (3 mm C18, 150 × 4 mm). Proton (1H), carbon (13C) and fluorine (19F) NMR spectra were obtained using a Bruker Avance 400 MHz spectrometer and chemical shifts δ given in parts per million (ppm) relative to TMS (δ = 0 ppm). Proton and carbon chemical shifts were assigned using proton, carbon, Correlation Spectroscopy (COSY), Distortionless enhancement by polarization transfer (DEPT) and Heteronuclear Single Quantum Coherence (HSQC) experiments. High-resolution ESI mass spectrometry was performed on a Bruker micrOTOF- Q II or an Agilent 6546 LC/Q-TOF in positive mode. HRMS data are reported as mass to charge ratio (m/z) = observed/MW. Normal phase column chromatography was performed on a Biotage Isolera One purification system using prepacked silica Biotage SNAP KP-SIL cartridges or Biotage Sfär Silica HC cartridges. Infrared spectroscopy (IR) was performed on a Shimadzu Fourier Transform Infrared Spectrophotometer (FTIR-8400S). 008528580 Microwave reactions were completed in a CEM Explorer 12 Hybrid Microwave. Optical rotation was determined using an Autopol V polarimeter. Peptide content was analysed on a Nanodrop 2000c using UV absorption of peptides at 280 nm or 214 nm. Samples were centrifuged at 4,500 RPM in a Heraeus Megafuge 8 centrifuge purchased from Thermo Fisher Scientific. Fluorescence polarisation was conducted on a CLARIOstar plate reader purchased from BMG Labtech. CD spectra were obtained at room temperature using a JASCO J-810 CD spectrometer. A range of 190 – 260 nm was scanned at a speed of 50 nm/min, with a 1 nm data pitch, a 1 nm bandwidth and an 8 s response time. Samples were prepared in phosphate buffered saline (PBS; pH 7.4), and CD spectra measured in a 0.2 mm quartz cuvette. Raw data (mdeg) were converted to mean residue ellipticity (MRE; deg cm2 dmol-1 res-1) by normalizing for path length, peptide concentration, and number of amide bonds. General Method B: Peptide synthesis Solid Phase Peptide Synthesis (SPPS) and Peptide Modifications Peptides were synthesised on a 0.1 mmol scale using either a Biotage Initiator+ Alstra (Biotage) or CEM Liberty Blue microwave assisted peptide synthesizer. TentaGel S RAM resin (0.24 mmol/g) or Chemmatrix resin (0.45 mmol/g) was used for peptides with C-terminal amides. Resin Swelling Resin was swollen in DMF at 70 °C for 20 min prior to use. Amino Acid Coupling Coupling of Fmoc-protected amino acids (5 equiv., 0.2 M in DMF) and unnatural/orthogonally-protected amino acids (2 equiv., 0.1 M in DMF) was achieved by treatment with DIC (5 equiv., 0.5 M in DMF) and Oxyma Pure (5 equiv., 0.5 M in DMF) at 90 °C for 2 min. His residues were coupled at 50 °C for 10 min. Amino acids following unnatural amino acids were double coupled. Arg residues were coupled at room temperature for 45 min, followed by 90 °C for 5 min, then double coupled at 90 °C for 10 min. Fmoc Deprotection Deprotection was achieved by treatment with morpholine (20% in DMF with 5% formic acid, 4 mL) at 90 °C for 1 min. The resin was washed with DMF between deprotection and coupling (4 × 4 mL), and after coupling (2 × 4 mL). 008528580 N-Terminal Acetylation (capping) Peptides requiring N-terminal acetylation were treated on-resin with acetic anhydride (3 equiv.), DIPEA (4.5 equiv.) and DMF (7 mL for 0.1 mmol of resin) for 20 min with agitation. The resin was then washed with DMF (3 × 5 mL) and DCM (3 × 5 mL) prior to peptide cleave and global deprotection. Ring Closing Metathesis (RCM) Peptides containing (R)-N-Fmoc-α-(7-Octenyl)alanine and (S)-N-Fmoc-α-(4-pentenyl)alanine were stapled by on-resin ring-closing metathesis (RCM). The resin was suspended in dry DCE before adding Grubbs’ 1st Catalyst (20 mol%) in DCE (4 mL for 0.1 mmol of resin), leaving for 2 h with agitation and excluding light. The resin was washed with DCE (3 mL) before repeating the RCM. The resin was then washed with DCM (2 × 5 mL) prior to peptide cleave and global deprotection. Glaser Reaction Peptides containing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylnon-8-ynoic acid or (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-O-(pent-4-yn-yl)-L-serine were cyclised on resin. Peptide on resin (1 equiv., 0.1 mmol) was added to a microwave vial followed by a solution of CuCl (5 equiv.) and 4,4’-bis(hydroxymethyl)-2,2’-bipyridine (7.5 equiv.) in DMF (10 mL) and DIPEA (20 equiv.). The reaction was heated to 60 °C for 3 h in a microwave reactor and excess reagents filtered off. The resin was washed with 0.5% sodium diethyldithiocarbamate in 0.5% DIPEA/DMF (5 × 10 mL), DMF (3 × 10 mL), methanol (MeOH) (3 × 10 mL), DMF (3 × 10 mL) and DCM (3 × 10 mL). N-Terminal Fluorescein Labelling Peptides requiring an N-terminal fluorescent label were treated on-resin with FITC (2 equiv.), DIPEA (8 equiv. or as described, such as 4 equiv.) and DMF (4 mL for 0.1 mmol of resin) at room temperature and excluding light for 12-16 h as described following coupling of Fmoc-6- Ahx-OH as a spacer. The resin was then washed with DMF (2 × 5 mL) and DCM (2 × 5 mL) prior to peptide cleave and global deprotection. Peptide Test Cleavages Test cleavages were performed on ~5 mg of resin using a cocktail (1 mL) of TFA (95 v%), TIPS (2.5 v%) and H2O (2.5 v%) for 1 h with agitation. Peptides containing cysteines were cleaved with a cocktail of TFA (94 v%), TIPS (1 v%), EDT (2.5 v%) and H2O (2.5 v%). Peptides containing the amino acid Asu were cleaved with a cocktail of TFA (94 v%), TIPS (2.5 v%), EDT (2.5 v%) and dry DCM (1 v%). The resin was subsequently filtered and the 008528580 TFA evaporated using a stream of N2, the peptide was precipitated with cold Et2O and centrifuged (4,500 rpm for 5 min). Peptides were dissolved in a mixture of H2O and MeCN with 0.1% TFA and lyophilized on a Christ Alpha 2-4 LO plus freeze dryer. Peptide Full Cleavage and Global Deprotection Peptides were cleaved from the resin using a cocktail (10 mL) of TFA (95 v%), TIPS (2.5 v%) and H2O (2.5 v%) for 3-4 h as stated with agitation. Peptides containing cysteines were cleaved with a cocktail of TFA (94 v%), TIPS (1 v%), EDT (2.5 v%) and H2O (2.5 v%). Peptides containing the amino acid Asu were cleaved with a cocktail of TFA (94 v%), TIPS (2.5 v%), EDT (2.5 v%) and dry DCM (1% v). The resin was subsequently filtered and the TFA evaporated using a stream of N2, the peptide was precipitated with cold Et2O and centrifuged (4,500 rpm for 5 min). Peptides were dissolved in a mixture of H2O and MeCN with 0.1% TFA and lyophilized on a Christ Alpha 2-4 LO plus freeze dryer. Peptide Purification Crude peptides were purified by RP-HPLC using either an Agilent Technologies 1260 Infinity RP-HPLC system or a Dionex RP-HPLC system with Dionex P680 pumps and a Dionex UVD170U UV-vis detector (monitoring at 214 nm and 280 nm), each with a Phenomenex Gemini column (5 mm C18, 250 × 21.2 mm). Purified peptides were analysed on a Shimadzu RP-HPLC system with Shimadzu LC-20AT pumps, a Shimadzu SIL20A autosampler and a Shimadzu SPD-20A UV-vis detector using a Phenomenex Aeris column (5 mm C18, 100 Å, 150 × 10 mm). Peptides were eluted with linear gradients at column- dependent flow rates (1 mL/min for the Aeris, 10 mL/min for the Gemini), where buffer A = 0.1% TFA in H2O and buffer B = 0.1% TFA in MeCN. LC-MS was performed on a Thermo Scientific LCQ Fleet Ion Trap Mass Spectrometer using positive mode electrospray ionisation (ESI+). Where buffer A = 0.1% TFA in H2O and buffer B = 0.1% TFA in MeCN, a linear gradient of 0-100 % B over 20 min with a flow rate of 1 mL/min was used with a Reprosil-Gold column (3 mm C18, 150 × 4 mm). General Method C: Fluorescence Polarisation (FP) FP was carried out to assess the direct binding kinetics between the peptides and MDM2. The assays were carried out in black, round bottomed, non-binding surface (NBS) 384 well plates. Kd values were calculated using Equation A. Peptides 145-170. Protein (MDM21-138) in 150 mM NaCl, 25 mM Tris, pH 7.6, 1 mM DTT was added and serially diluted across the plate from 10 μM to 9 pM. Peptide (10 nM in Tris buffer pH 7.6) was then added to every well and mixed using a pipette. Plates were placed in a PerkinElmer Victor X5 plate reader with excitation measured at 531 nm and emission measured at 595 nm. 008528580 Data Analysis. Raw data were normalized using baseline correction, where the baseline was defined as the fluorescence polarisation measured in the absence of peptide. Normalized data were then plotted in Prism9.0 and fitted by using the quadratic ligand depletion model (Equation A). Equation A: Y is the fluorescent polarisation signal measured at each datapoint. Rt is the total concentration of (FITC-labelled peptide). X is the total concentration of protein and Kd is the dissociation constant (the inflection point of the curve). General Method D: Determination of Binding Affinity using a Fluorescence Polarisation (FP) Assay The binding properties of the peptides were assessed using a biophysical FP assay. Specifically, a direct binding assay was conducted to calculate the Kd values of the fluorescent peptide ligands binding to the target proteins. Fluorescently labelled peptides were tested for binding to MDM2 (1-138) as described. The proteins were obtained from the Beatson Cancer Institute in Glasgow. All FP measurements were performed on a microplate reader (CLARIOstar) in black 384-well microplates with 20 μL of sample solution per well. The raw data was normalized using baseline correction, where the baseline was defined as the fluorescence polarisation measured in the absence of peptide. Normalized data were then plotted and fitted by using the quadratic ligand depletion model (Equation A). General Method E: Circular Dichroism (CD) Spectroscopy CD spectra were obtained at room temperature using a JASCO J-810 CD spectrometer. A range of 190-260 nm was scanned at a speed of 50 nm/min, with a 1 nm data pitch, a 1 nm bandwidth and an 8 s response time. Samples were prepared (50 µM and 25 µM) in phosphate buffered saline (PBS; pH 7.4), and CD spectra measured in a 1 mm or 0.2 mm quartz cuvette. Raw data (mdeg) were converted to mean residue ellipticity (MRE; deg cm2 dmol-1 res-1) by normalizing for path length, peptide concentration, and number of amide bonds. Percentage helicities can be calculated for ^-helical peptides. The raw CD data is converted to mean residue ellipticity (MRE) using Equation B and the value at 222 nm can be used to calculate the % helicity of the peptides using Equation C. 008528580 Equation B: MRE calculation. Where θ = machine units in degrees, MRW (mean residue weight) = molecular mass of peptide/number of residues, l = path length (cm) and c = peptide concentration in mg/mL. Equation C: (1) % Helicity equation. (2) θc = random coil ellipticity calculation proposed by Luo and Baldwin. (3) θ222 = α-helix ellipticity calculation determined by Luo and Baldwin reading observed at 222 nm. T is the temperature in degrees Celsius, Np is the number of amide bonds and k = the peptide length correction factor, 3. General Method F: Fluorescence (two-photon) Imaging Images were acquired on an Olympus FV3000 laser scanning confocal microscope equipped with a 40× or 60× objective. Image analysis and processing was performed using ImageJ 1.53c. General Method G: Fluorescence + SRS (multiphoton) Imaging Images were acquired using a custom-built multi-modal microscope setup. A picoEmerald S (APE, Berlin, Germany) laser provided both a tunable pump laser (700–990 nm, 2 ps, 80 MHz repetition rate) and a spatially and temporally overlapped Stokes laser (1032nm, 2 ps, 80 MHz repetition rate). The output beams were inserted into the scanning unit of an Olympus FV1000MPE microscope using a series of dielectric mirrors and a 3× lens-based beam-expanding module. The resulting 3.6 mm beams were expanded by a further 3.6 × lens within the microscope and directed into an Olympus XLPL25XWMP N.A.1.05 objective lens using a short-pass 690 nm dichroic mirror (Olympus). For SRS measurements the Stokes beam was intensity modulated with a 20 MHz EoM built into the picoEmerald S. Forward scattered light was collected by a further 25× Olympus 008528580 XLPL25XWMP N.A.1.05 objective lens and Stokes light was removed by filtering with an ET890/220m filter (Chroma). A telescope focused the light onto an APE silicon photodiode connected to an APE lock in amplifier with the time constant set to 20 μs. The lock in amplifier signal was fed into an Olympus FV10-Analog unit. Laser powers after the objective were measured up to 20–50 mW for the pump laser and up to 70 mW for the Stokes laser. All images were recorded at 512 × 512 or 1024 × 1024 pixels with a pixel dwell time between 2 and 20 μs, using FluoView FV10-ASW scanning software (Olympus). Image analysis and processing was performed using ImageJ 1.53c. Hyperspectral images (obtained from a drop of concentrated sample in DMSO) were recorded using the inbuilt 'sweep' function of the picoEmerald S software that adjusted the pump laser ~0.3 nm for each new image recorded. Hyperspectral images were analysed using the ‘Measure Stack’ feature on ImageJ 1.53c to quantify the change in signal intensity. 008528580 5 1 9 6 7 1 3 B 1 9 4 0 3 2 3 , . 8 . 2 0 . 7 2 . 5 0 3 . 3 . d e % 2 3 4 4 4 4 t 0 e 4 a l 5 y mi t – t e 0 n 5 7 0 9 3 6 c a = oi 8 8 7 2 0 3 a t 7 6 5 4 n . 4 . 5 . 1 . 8 2 . 1 1 . = c . e e 1 1 2 2 2 2 2 A - i n t e , e 9- r i d n e R e s - 6 3 1 m r o L- ) . 3 . 2 3 5 4 . 1 . 2 . 2 . 0 al l u l a F y - - i n n H y m - = = = = r 9 (( 4- 1 = 2 t + 1 + 1 + 2 + + et 2 - ((- n 5 ] 8 2] 8 2] 7 2] 1 2] 3 H 2 H 6 H 6 2 2 C 2 e 9 -) p ( 7 3 2 9 . 8 2 9 H .2 2 9 . 2 2 0 . H 0 3 2 . 3 = 2 S - ( + H O = - 9 + 7 + 7 + 0 )o M [ 9 M [ 8 M [ 8 M [ 1 + 0 1 M [ 1 1 N , 7 S n i z / i d & c R m VZ a m a )l = = = = 7 =i o X N y n + 8 . 2] 2 +2] 9 +2 7 c Z 1 4 1 ] 1 9 +2] 3 +2] 3 H 3 H 6 H 6 H 2 2 n a N o b 4 2 9 2 9 2 9 0 . H 0 . x X $ e e r 9 . 8 . 2 . 2 2 3 2 3 Z a 7 3 + 9 + 7 + 7 + 0 + 0 h o R ` c ) y M [ 9 M [ 8 M [ 8 M [ 1 1 M [ 1 1 i n ZR x o m \ ' h . t A e = # ' x m ' m y )l t h P y- % i r 9 u 9 9 9 9 9 7 9 9 9 7 9 A [ P , Y 9-e t ; n e a ' r o d n B l a ' $ l e 3 2 4 2 y u % 4 2 F F i Y 2 2 c # - o i h 5 H t 5 9 o S ( ( i ( s & (- 2 2-5 R V 2- H H- Z ) 2 2n i Z N e X N S ( 2 H H H - N- A A c s NX $ = 2 N- N- N- A A S S e r e O H A A 5 5 Z 7 S o N-u R l ` S T A A L S5 S5 S ) S ) f P , d S Q S a b a b = C ' i c e T L A ) S a ) b a C ( C (C 1 a # c Q W b Q QT ' i I c n m e A Y C ( C ( A A F ' o P n u q W H E Q Q W W : y Y A A Y Ys [ -n Y 8- e S H F E Eo E T W W i t ; ' n o F L 8 8 - Y Y R Ri a B x Ev e ' 8 E8 F F $ l n T y L- h R R T T r b E # h t c A - F F L- L- 5 e A C T T L T x x - L h h b A 8 m- I F c -c A- A-. R 2- A A C T C s , e ) o I T I d e n i n F F i t i p n m e a l a p ) f l a l o y t y n . ) ) ) l s u o i b b s ) ) ) ra n i D I 8 4 8 4 1 4 1 1 1 ’ 4 4 ’ 4 a t l o e c y c ) m y 0 e di a 5 8 3 - b P 5 8 3 - a P 3 0 7 - a 0 3 7 - b 3 0 7 - b 0 3 7 - D C x 2 t 4 1 4 5 P 5 P 5 P 5 P : - , p 1 e L o h t S 1 S 1 S 1 S 1 S 1 S t n e T T T T T T l e P b = e i A ( A ( A ( A ( A ( A ( a a b m ) d a T C l y r g 5 6 7 0 8 2 5 2 8 7 . 3 3 9 A / A / 1 . 3 . 8 . 0 N N e 4 4 3 4 mit n 3 1 0 0 oi 4 3 7 3 t 3 . 6 . 5 . 5 A / A / ne 1 1 9 . 0 N N t 2 2 1 2 e R 4. 2 . 1 . 4 . 2 0 0 A / A / - - 0 - N N + = = = 2] 9 6 +2 0 ] 4 2 + = 2] 3 3 +2 6 ] 7 H 6 H 5 0 2 9 . 2 0 . H H 2 9 . 2 0 . A / A / + 0 1 9 2 2 3 8 N N M [ 8 + M [ 1 + 1 5 + M [ 8 M [ 0 1 z/ m + = = = = 2 1 + 7 + 0 + 3 ] 9 2] 3 2 6 2 6 H 6 H 2 ] ] H 4 0 2 9 . 2 0 . H 0 A 1 2 9 . 2 . / A / + 0 + 2 1 2 8 N N 9 M [ 8 M [ 1 + 1 5 M [ 8 + M [ 0 1 y t % i r 7 u 9 7 9 9 9 8 9 A / A / P N N d % l e A i 0 1 9 1 1 9 / A / Y N N 2 2 2 H H H 2 2 N 2 N- N- H - A H H A A N- A N- A N- A A S A S A 7 A S7 A O 7 S7 S ) A S ) S S ) S) a S b 7 S) a b O 7 S) a b ec a C ( a b C ( a b C ( n b Q Q Q e C ( A C ( A C ( A uq Q W Q W Q W e A Y A Y A Y S W E Y 7 W E Y 7 W E S Y O 7 E S 7 F S T E7 A T E O S 7 F F L- S A L- S T T x T x F L- L- h L h c A- -c A- T L x A - h C A T A C c - I T A F I F C T I F e v e i t i v D I a ) t a ) e ) ) l l di a t 0 e n b e n a g e o r b g e o r a b p 6 1 i y 0 61 i y 4 61 n t e n 4 n t o 6 1 e n 0 0 o 7 1 7 1 e D ( D ( n c n c P y i i y D ( D ( Amino Acid Synthesis Compound 155 - 7-Iodohept-1-yne I 6-Heptyn-1-ol (1.00 g, 8.90 mmol, 1 equiv.) was dissolved in dry DCM (10 mL) in a round bottom flask under a nitrogen atmosphere. Triethylamine (Et3N) (2.48 mL, 17.8 mmol, 2 equiv.) was added and left to stir for 10 mins at room temperature. The solution was then cooled to 0 °C and methanesulfonyl chloride (MsCl) (1.01 mL, 13.4 mmol, 1.5 equiv.) was added dropwise. The solution was then brought up to room temperature and left to stir under nitrogen overnight. Volatiles were evaporated under reduced pressure. The residue was dissolved in water (H2O) and ethyl acetate (EtOAc) and extracted with EtOAc (3 × 10 mL). The organic layer was washed with brine (3 × 10 mL), dried over magnesium sulfate (MgSO4), filtered and concentrated under reduced pressure to give compound as a yellow oil (1.08 g, 64%). The crude (785 mg, 4.13 mmol, 1 equiv.) was dissolved in acetone (15 mL) and sodium iodide (NaI) (3.10 g, 20.7 mmol, 5 equiv.) was added. The reaction was heated to 60 °C and left to stir and reflux overnight. The reaction was left to cool to room temperature and volatiles were evaporated under reduced pressure. The residue was dissolved in H2O and EtOAc and extracted with EtOAc (3 × 10 mL). The organic layer was washed with sodium thiosulfate (Na2S2O3) (3 × 10 mL), dried over magnesium sulfate (MgSO4), filtered and concentrated under reduced pressure to give compound as a yellow oil (485 mg, 56 %). 1H NMR: (400 MHz, Chloroform-d) δ 3.15 (t, J = 7.0 Hz, 2H, H-7), 2.16 (td, J = 6.7, 2.8 Hz, 2H, H-3), 1.92 (t, J = 2.7 Hz, 1H, H-1), 1.80 (quin, J = 7.1 Hz, 2H, H-6), 1.55 – 1.42 (m, 4H, H-4 + H-5). 13C NMR: (101 MHz, Chloroform-d) δ 84.0 (C), 68.6 (CH), 33.0 (CH2), 29.6 -1 (CH2), 27.3 (CH2), 18.3 (CH2), 6.7 (CH2). IR (υmax/cm , neat) 3293 (C≡C-H), 2935 (C-H), 2859 (C-H), 2359 (C-H). 5-Chloro-pent-1-yne (775 μL, 7.31 mmol, 1 equiv.) was dissolved in acetone (15 mL) and NaI (5.49 g, 36.6 mmol, 5 equiv.) was added and left to stir for 16 h at 60 °C. After cooling and concentrating in vacuo, the residue was taken up in H2O (50 mL) and EtOAc (50 mL). The aqueous layer was extracted with EtOAc (3 × 100 mL) and the organic layer was 008528580 washed with Na2S2O3 (2 × 100 mL), dried over MgSO4 and concentrated yielding as a yellow oil (1.05 g, 74%). 1H NMR: (400 MHz, Chloroform-d) δ 3.30 (t, J = 6.7 Hz, 2H, H-5), 2.33 (td, J = 6.7, 2.6 Hz, 2H, H-3), 2.05 – 1.94 (m, 3H, H-1 + H-4). 13C NMR: (101 MHz, Chloroform-d) δ 82.3 (C), -1 69.6 (CH), 31.9 (CH2), 19.5 (CH2), 5.3 (CH2). IR (υmax/cm , neat) 3293 (C≡C-H), 2905 (C-H), 2359 (C-H). Compound 156 - (S)-({2-[1-(2-Fluorobenzyl)pyrrolidine-2- carboxamide]phenyl}phenylmethylene)-(S)- heptynylalaninato-N,N’,N’’,O}nickel (II) Sodium t-butoxide (0.750 g, 7.83 mmol, 1.2 equiv.) and L-Ala-Ni-(S)-FBPB complex (3.45 g, 6.52 mmol, 1 equiv.) were added to a dry flask under N2. The flask was cooled to 0 °C and dry DMF added (30 mL) before dropwise addition of electrophile (1.52 g, 7.83 mmol, 1.2 equiv.). After 5 mins the ice bath was removed and the flask warmed to rt and stirred for 1 h. The reaction mixture was quenched by pouring into cold 5% acetic acid (50 mL) and concentrated in vacuo. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and the layers separated. The aqueous layer was extracted with DCM (50 mL) and the combined organic layers washed with 5% w/v LiCl solution (3 × 50 mL), brine (3 × 50 mL), dried with MgSO4 and concentrated in vacuo. Purification was carried out using normal phase flash column chromatography with a gradient of 50-100% EtOAc/Pet Ether to give the pure alkylated complex as a red solid (2.80 g, 72%). 1H NMR: (400 MHz, Chloroform-d) δ 8.28 (td, J = 7.5, 1.8 Hz, 1H, H-9), 8.01 (d, J = 8.5 Hz, 1H, H-Ar), 7.53 – 7.42 (m, 2H, H-2’Ar), 7.41 – 7.27 (m, 3H, H-8 + H-2’Ar), 7.20 (td, J = 7.5, 1.3 Hz, 1H, H-10), 7.17 – 7.06 (m, 2H, H-11 + H-Ar), 7.02 – 6.92 (m, 1H, H-Ar), 6.71 – 6.55 (m, 2H, H-2’Ar), 4.51 (d, J = 13.5 Hz, 1H, H-6), 3.94 (d, J = 13.0 Hz, 1H, H-6’), 3.65 – 3.49 (m, 1H, H-5), 3.41 (dd, J = 10.7, 6.3 Hz, 1H, H-2), 3.33 – 3.18 (m, 1H, H-4), 2.81 – 2.67 (m, 1H, H-3), 2.61 – 2.45 (m, 1H, H-3’), 2.40 – 2.27 (m, 1H, H-30), 2.24 (td, J = 7.0, 2.5 Hz, 2H, H-33), 2.14 – 1.96 (m, 3H, H-4’, H-5’ + H-30’), 1.95 (t, J = 2.6 Hz, 1H, H-35), 1.72 – 1.58 (m, 4H, H-29 + H-32), 1.49 – 1.35 (m, 2H, H-31), 1.24 (s, 3H, H-28). 13C NMR: (101 MHz, Chloroform-d) δ 182.6 (C=O), 180.3 (C=O), 172.6 (C=N), 161.9 (d, J = 248.0 Hz, Ar-C), 141.6 (Ar-C), 136.7 (Ar-C), 134.3 (d, J = 3.3 Hz, Ar-CH), 133.5 (Ar-CH), 131.7 (Ar-CH), 131.4 (d, J = 8.5 Hz, Ar-CH), 130.3 (Ar-CH), 129.6 (Ar-CH), 128.9 (Ar-C), 008528580 128.1 (Ar-CH), 127.4 (Ar-CH), 127.1 (Ar-CH), 124.7 (d, J = 3.6 Hz, Ar-CH), 124.1 (Ar-CH), 120.9 (Ar-CH), 120.6 (d, J = 14.5 Hz, Ar-C), 116.0 (d, J = 22.4 Hz Ar-CH), 84.4 (C), 78.3 (C), 70.3 (CH), 68.7 (CH), 56.8 (CH2), 56.0 (CH2), 40.1 (CH2), 30.7 (CH2), 29.8 (CH3), 28.9 (CH2), 28.4 (CH2), 25.6 (CH2), 23.4 (CH2), 18.6 (CH2). 19F NMR: (377 MHz, Chloroform-d) δ -113.7. Mp 128-130 °C. HRMS-EI (m/z) [M+H]+ calcd for C35H36FN3NaNiO3 [M+Na]+ 646.1986, found 646.1978 (Δ = 1.3 ppm). [α]D 25 = +3474 -1 (c 0.05 in MeOH). IR (υmax/cm , neat) 3287 (C≡C-H), 2932 (C-H), 1667 (C=N). Compound 157 - (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-methylnon-8- ynoic acid Alkylated complex (290 mg, 0.46 mmol, 1 equiv.) was dissolved in MeOH (3 mL) and added dropwisely to 3 mol.L-1 aqueous HCl (1.5 mL) and refluxed at 70 °C over 5 mins. The reaction was monitored by TLC (5% MeOH in DCM). Upon completion, the reaction was cooled to room temperature and MeOH was removed under reduced pressure. The aqueous solution was washed with DCM (5 × 10 mL). The aqueous layer which contained amino acid was fully evaporated. The residue was treated with 6% Na2CO3 until the solution reached pH 9-10. EDTA.2Na (171 mg, 0.46 mmol, 1 equiv.) was added and the reaction was left to stir for 10 mins. The solution was cooled to -10 °C in an EtOH-ice bath and FmocOSu (140 mg, 0.420 mmol, 0.9 equiv.) in equal amount of acetonitrile was added dropwisely over 30 mins. The reaction was warmed to room temperature and left to stir for 16 h. The acetonitrile was removed under reduced pressure. The aqueous layer was acidified to pH 1-2 with 1 M HCl. The aqueous solution was extracted with EtOAc (3 × 10 mL), the organic layer was then washed with brine (3 × 10 mL), dried over sodium sulfate. The solvent was removed under reduced pressure to give the target compound as a white solid (151 mg, 77%). 1H NMR: (400 MHz, Methanol-d4) δ 7.72 (d, J = 7.5 Hz, 2H, H-18 + H-18’), 7.59 (d, J = 7.6 Hz, 2H, H-15 + H-15’), 7.32 (t, J = 7.4 Hz, 2H, H-17 + H-17’), 7.24 (td, J = 7.5, 1.2 Hz, 2H, H- 16 + H-16’), 4.33 – 4.19 (m, 2H, H-12), 4.14 (t, J = 6.5 Hz, 1H, H-13), 2.14 – 2.03 (m, 3H, H- 7 + H-9), 1.88 – 1.67 (m, 2H, H-3), 1.50 – 1.06 (m, 9H, H-4, H-5, H-6 + H-10). 008528580 13C NMR: (101 MHz, Methanol-d4) δ 177.7 (C=O), 157.1 (C=O), 145.3 (Ar-C), 145.3 (Ar-C), 142.6 (2 × Ar-C), 128.7 (2 × Ar-CH), 128.1 (2 × Ar-CH), 126.2 (2 × Ar-CH), 120.9 (2 × Ar-CH), 85.0 (C), 69.5 (CH), 67.4 (CH2), 60.4 (C), 48.4 (CH), 37.8 (CH2), 29.8 (CH2), 29.5 (CH2), 24.4 (CH2), 23.3 (CH3), 18.9 (CH2). -1 IR (υmax/cm , neat) 3295 (C≡C-H), 2940 (C-H), 2682 (C-H), 1701 (C=O). [α]D 25 = +3.1 (c 0.1 + in MeOH). HRMS-Ei (m/z) calcd for C25H27NNaO4 [M+Na] 428.1832, found 428.1849 (Δ = 3.8 ppm). RP-HPLC (20 min gradient) tR = 20.9, (50 min gradient) tR = 40.6, 98 % purity. Boc-L-Serine (755 mg, 3.68 mmol, 1 equiv.) was dissolved in DMF (20 mL) under N2 and the flask cooled to 0 °C. To the flask, NaH (60% in oil, 353 mg, 8.84 mmol, 2.4 equiv.) was added portion wise and the reaction stirred at 0 °C for 2 h.5-Iodo-pent-1-yne (1.00 g, 5.15 mmol, 1.4 equiv.) was added dropwise over 10 mins. The ice bath was removed and the reaction left to stir for 16 h at room temperature. The reaction mixture was quenched with ethanol (10 mL), H2O (30 mL) added and subsequently washed with Et2O (4 × 50 mL). After acidification using 3 M HCl (5 mL), the aqueous layer was extracted with EtOAc (4 × 75 mL). The organic layer was washed with H2O (4 × 75 mL), dried using MgSO4, filtered and then concentrated in vacuo to yield as a yellow sticky solid (450 mg, 45%). 1H NMR: (400 MHz, Methanol-d4) δ 10.82 (br-s, 1H, H-12), 5.43 (d, J = 8.7 Hz, 1H, H-N), 4.48 – 4.38 (m, 1H, H-2), 3.92 - 3.84 (m, 1H, H-3), 3.66 (dd, J = 9.5, 3.5 Hz, 1H, H-3’), 3.54 (t, J = 6.1 Hz, 2H, H-4), 2.26 – 2.20 (m, 2H, H-6), 1.93 (t, J = 2.7 Hz, 1H, H-8), 1.73 (quin, J = 6.6 Hz, 2H, H-5), 1.43 (s, 9H, H-11). 13C NMR: (101 MHz, Methanol-d4) δ 175.5 (C=O), 155.8 (C=O), 83.8 (C), 80.4 (C), 70.5 (CH2), 70.0 (CH2), 68.8 (C), 53.9 (CH), 28.4 (3’CH3), 28.3 (CH2), 15.20 (CH2). -1 IR (υmax/cm , CHCl3) 3310 (C≡C-H), 3021 (C-H), 1713 (C=O). [α]D 25 = +4.2 (c 0.1 in MeOH). + + HRMS-EI (m/z) [M+H] calcd for C13H21NNaO5 [M+Na] 294.1312, found 294.1310 (Δ = 0.7 ppm). 008528580 Compound 168 - (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-O-(pent-4-yn- yl)-L-serine (N)-2-(tert-butoxycarbonyl)-O-(pent-4-yn-yl)-L-serine (220 mg, 0.81 mmol, 1 equiv.) was dissolved in DCM (1.5 mL), TFA was added (1.2 mL) and the reaction left to stir at room temperature for 6 h. Saturated NaHCO3 (aq) (2.4 mL) was added to the oil and then solid NaHCO3 was added until gas evolution stopped. The solution was poured into a solution of Fmoc-OSu (0.246 g, 0.730 mmol, 0.9 equiv.) in dioxane (22 mL) and left to stir for 16 h. After concentrating the reaction mixture in vacuo, H2O (10 mL) was added and washed with Et2O (4 × 10 mL). The aqueous layer was acidified with 3 M HCl, extracted with EtOAc (4 × 10 mL), dried with MgSO4, filtered and concentrated in vacuo. Purification was carried out using normal phase flash column chromatography with a gradient of 0-10% DCM/10% MeOH in DCM yielding as a pale yellow solid (127 mg, 40%). 1H NMR: (400 MHz, Methanol-d4) δ 7.77 (d, J = 7.5 Hz, 2H, H-16 + 1H-16’), 7.65 (t, J = 7.0 Hz, 2H, H-13 + H-13’), 7.36 (t, J = 7.4 Hz, 2H, H-15 + H-15’), 7.29 (t, J = 7.4 Hz, 2H, H-14 + H-14’), 4.41 – 4.28 (m, 3H, 2-H + 10-H), 4.21 (t, J = 7.0 Hz, 1H, 11-H), 3.78 (dd, J = 9.8, 5.3 Hz, 1H, 3-H), 3.68 (dd, J = 9.8, 3.8 Hz, 1H, 3’-H), 3.60 – 3.45 (m, 2H, 4-H), 2.22 (td, J = 7.1, 2.7 Hz, 2H, 6-H), 2.16 (t, J = 2.7 Hz, 1H, 8-H), 1.71 (h, J = 7.0 Hz, 2H, 5-H). 13C NMR: (101 MHz, Methanol-d4) δ 173.7 (C=O), 158.5 (C=O), 145.3 (2 × Ar-C), 145.2 (Ar-C), 142.6 (Ar-C), 128.8 (2 × Ar-CH), 128.2 (2 × Ar- CH), 126.3 (2 × Ar-CH), 120.9 (2 × Ar-CH), 84.5 (C), 71.4 (CH), 70.7 (CH2), 69.7 (C), 68.1 (CH), 55.8 (CH2), 48.4 (CH2), 29.7 (CH2), 15.8 (CH2). -1 IR (υmax/cm , CHCl3) 3021 (C≡C-H) 2938 (C-H), 1720 (C=O). [α]D 25 = +13.4 (c 0.1 in + + MeOH). HRMS-EI (m/z) [M+H] calcd for C23H23NO5 [M + H] 393.1580, found 394.1657 (Δ = 2.1 ppm). RP-HPLC (20 min gradient) tR = 19.078, (50 min gradient) tR = 36.469, 96% purity. 008528580 Compound 117 - ((S)-((2-[1-(2-Fluorobenzyl)pyrrolidine-2- carboxamide]phenyl)phenylmethylene)-(S)-heptynylglycinato-N,N’,N’’,O)nickel(II)) To ground NaOH (1.20 g, 30.0 mmol, 5.0 equiv.) was added dry DMF (15 mL) under N2 and the flask cooled to 0 °C. A solution of L-Gly-Ni-(S)-FBPB complex (3.10 g, 6.00 mmol, 1 equiv.) in dry DMF (10 mL) was added and the flask removed from the ice bath.8-Iodooct-1- yne electrophile (1.47 g, 6.60 mmol, 1.1 equiv.) was added dropwise and the solution left to stir for 30 mins at rt. The reaction mixture was quenched by pouring into H2O (50 mL) and concentrated in vacuo. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and the layers separated. The aqueous layer was extracted with DCM (100 mL) and the combined organic layers washed with 5% w/v LiCl solution (3 × 100 mL), brine (3 × 100 mL), dried with MgSO4 and concentrated in vacuo. Purification was carried out using normal phase flash column chromatography with a gradient of 50-100% EtOAc/Pet Ether to give the alkylated complex as a red solid (2.89 g, 79%). 1H NMR: (400 MHz, CDCl3) ^ 8.28 (td, J = 7.4, 1.8 Hz, 1H, 9-H), 8.16 – 8.14 (m, 1H, Ar-H), 7.52 – 7.44 (m, 3H, 3 ^ Ar-H), 7.24 – 7.14 (m, 4H, 8-H, 10-H + 2 ^ Ar-H), 7.07 – 7.02 (m, 1H, 11-H), 6.94 – 6.92 (m, 1H, Ar-H), 6.70 – 6.63 (m, 2H, 2 ^ Ar-H), 4.45 (d, J = 13.6 Hz, 1H, 6- H), 3.90 (dd, J = 8.3, 3.4 Hz, 1H, 27-H), 3.85 (d, J = 13.6 Hz, 1H, 6′-H), 3.61 – 3.52 (m, 1H, 5-H), 3.49 – 3.45 (m, 2H, 2-H + 4-H), 2.87 – 2.80 (m, 1H, 3-H), 2.62– 2.51 (m, 1H, 3′-H), 2.19– 1.96 (m, 5H, 4′-H, 5′-H, 28-H + 32-H), 1.93 (t, J = 2.6 Hz, 1H, 34-H), 1.66 – 1.55 (m, 3H, 28′-H + 30-H), 1.51– 1.44 (m, 2H, 31-H), 1.33– 1.18 (m, 2H, 29-H). 13C NMR: (101 MHz, CDCl3) ^ 180.0 (C=O), 179.3 (C=O), 170.3 (C=N), 161.6 (d, J = 247.9 Hz, Ar-C), 142.2 (Ar-C), 134.2 (d, J = 3.1 Hz Ar-C), 133.8 (Ar-CH), 133.3 (Ar-CH), 132.2 (Ar- CH), 131.2 (d, J = 8.4 Hz, Ar-CH), 129.7 (Ar-CH), 129.0 (Ar-C + Ar-CH), 127.5 (Ar-CH), 127.2 (Ar-CH), 126.6 (Ar-CH), 124.5 (d, J = 3.4 Hz, Ar-CH), 123.7 (Ar-CH), 120.8 (Ar-C), 120.4 (d, J = 14.5 Hz, Ar-CH), 116.1 (d, J = 22.1 Hz, Ar-CH), 84.3 (C), 70.4 (CH), 70.3 (CH), 68.4 (C), 56.8 (CH2), 55.6 (CH2), 35.2 (CH2), 30.7 (3-CH2), 28.4 (CH3), 28.1 (CH2), 24.9 (CH2), 23.7 (CH2), 18.3 (CH2). 19F NMR: (377 MHz, CDCl3) ^ –113.9. 008528580 Mp 168–170 °C. [α]D25 = +2494 (c 0.05 in MeOH). IR (υmax/cm-1, neat) 3240 (C≡C-H) 2934 (C-H), 1670 (C=O), 1634 (C=N). HRMS-EI (calcd for C34H34FN3NiO3 [M + Na]+) 632.1835, found 632.1840 (Δ = 0.8 ppm). Peptide Sequences The sequences of the key peptides used in the following examples is shown in Table 2. 008528580 e = d i = ] - 5 3-) e e emS o e l n a y n y n y l K L n i l a & m e e p p a t l k l k l k a a a e i n R Z a V y x l p a t s s e 8- 8 8 5 -5 -5 l p m r Z o a t n 8 8 e 8 a t et X - N N b r s e e k l p s C X $ a n e c e n e l a N N Q Q a t N Q e n e = Z (-2 R ` 2 e l k k a- -s ZN Z s N ZN i y 2 2 l pH P = l a i s n a 2 X 2 X e n 2 X d a H H a t N , C ' O ] 2 n 2 c 2 r t H e U H e U i y H e U N N s i d 1# 7 H c ' S X N a m [ - a H N- a H N- a N- ` K R N- ` d K R a N ` - R m r - o K -K e n f A A i y c ' i $ , K m r K m r K m r i d 2o E A I o A I o A I o A I Y ' m A I Y ' m m K Y r A I ' I I m o t E E d n # c f f f a H a 5 E E M c N e 8 - K o t E d K o t E d K o t K T E d L ] V Z K T o b L ] V Z f r E K T b e L ] V Z d K K b e L k &] &L ] a H H m r x = i o K = L 5 ] e k = L 5 ] e k = L 5 ] e 8 k o 8 N 8 U o 8 N h U t e 8o N n 7 7 U i l S S o fh o R n X y A I S n S n S n X R X g 8 e X [ X [ o ti n [ , -8 m e - n s E K X [ il X [ il X [ il [ [ R o X [ R r H H H e H e H e H a a n i V H a t ] H a a ] d e = n o n n x l a ) o oi L t A G r a G r a G r a G Q le _ G V Q le _8o V Q 8 G _8 G G o 8 l e l e k n h l a i n a R l e c N= 5 ] l S e N= 5 ] l S e N= 5 ] R ^ R ^ l e R ^ S N N N i l [ N [ X [ N [ X [ e , i G R V Z R V Z o R V Z o R A R A r A y t m e a fi l e R A X [ R A X [ R A X [ A A A a ] t u b y a x d o N R Y ] d n Y ] d n Y ] d n 8Y ] YN 8Y ] YN w t Yn l o e Y ] N w t e &Y ] &Y ] & #"a c y y o b M A= Y8 R a ] = 8 R a ] = 8 R a o ] 8 Z o N 8 Z 8 N h t o 8 Z H 7 H 7 $ N h t S S X [ c c- r a o c i L (- d n A X[ = 8 R X[ = 8 R X[ = 8 X R [ X K V _ [ K V _ d n X[ V _ d X n [ X K [ K o h t = o 2 a -) a K ec A K - A- X [ K e A- X [ K e A- X [ e A- 8M x o 8 . A- 8M in x o a K 8 , i n A- 8M o a 8 , A- A- w t i n x x e i s b S - ( x h x h 5 C h x h h x h h h X h X x h X h h h-n , = n e A- A- t t t e i e r A- e r A- e r A- [ a e h c A- [ a e h [ a c A- e h c A- A- t ee i n &] # u q C T C T e h C T e h C T e c h o r e c h e d o r eh e d C r T e c h e o c o r ec c u "$ e S - - I F I F I I m i m i I d i m m h s X w F w F w F w s F w s F w s F F we r l e o r [ , u o l f n d - i c = L a C = i c T I l e o F N n , y-. n d 8-i a e t n o h a l c y t n -l e e y d c h i t s a e K d = K K K K K K K K K K c m - I A I A I A I A I A I A I e A 2 E E E E E E E I A E I A I i fi A - d , o ) ) o E d L S K m e i n A T L K L K L K K K K E K E K v m H L A X X L X L X L X L X L X L X R i Q H H H H H H H Hh t a a l e y x G A G X G X G X G X G X G X G G G w c ( i o e d c M X X X R W Y c n b r n a i s a e A R A R A R A R A R A R A R A R A R A o e r c( u Y H A E Y A Y X Y X Y X Y Y Y Y Y i n -f - 2 q F X X X X X f -) e E T K K K K K K K K Km o r S A L A A A A A A A A A a S o ( = ) = X " &] - " . ) $ s H . d e ( K L c i n N c - &R a e ( c u n q i V Z i c s Z o s s i n - ar e n o e - c-s e r X N n - N a c 2 - t 2 -2 e o c i y H7 H7 i n X $ p d o e o r -t Z p - S -S - - - S e d- n e R S Sp e R ` C C C n H7 H7 e b Z y x 1 i - H- H H y d i y A d T S S P y R o e - - - - S - - - : a 2 \ ' n d R A R A R A R A R A R A R A T- R A R A e m . l b h # ' y- c 4 i - t i m' p T P S T T T T T T $ t n C T T e S - T S- S- S- S- S S T S S a h T w F # e p P T A T T T T - T - T I F - T - T 5 e l l e e p l l e p pa a t a t s s p e a t ts s e n ek e e n ek e 2 n e l e l e l H l k l a n - e s 2 l k l a p 2 -s 2 a p p t a t a t N a- n a- n s 2 s s - K i s a r H i s H a H r e n H e n e n AI c t a N- c N- t N - i y N - i y i y E K 2 2 2 a A A a A A a 2 A A d A H H m r H m r m r m r S ] 2 a A d a 2 2 d H H a O 7 L ] N- N- o f N o f = 5 S ] o f = 5 S ] o f H N&" H N m r &S "] m r N- N m r S 2 A A o - t A o t S X[ o t S X o t - $ A X - [ A o f $ X o - f A A o f X[ H A S A ] S d A e S d e a d [ e a d e A S a b A S o t [ a o t A S A ] S ] o ts n H N- = 5 = 5] k n = 5] k b n C k b n C k n &"] C&"] d b o G e C d &" &" dit l e 2 S S S T X X i l S i l Q i l Q i l $ Q$ e $ $ e A k n Q k n X X k n a c N H L [ a [ a e r X [ a e r A e r A e r X [ X a [ i W a i l A i l [ a [ a i lfi R A N- Q d o OY A b b C C a ] b C a ] W Y a ] W Y a ] b Y b e r W Y e r b b C C e r ] S = Q Q 5 = S Q 5 = S E ] 5 = S E ] 5 C S Q&E ] C Q a ] E ] a ] Q Q a ] M 7 T S L W Y A A X [ A X [ = 8 = R X [ 8 X [ A" $ A&" & &" $ " A A&" $ $d X Q n [ A H E W Y W Y d W Y d X [ d R X[ d W Y X [ W Y X$ [ X [ X [ W Y W Y X [ a K F e A- W Y T E ] E n n ] a c x h H L- = 8 = 8 R R = 8] = 8 E ] a = 8] F n T a F n L = 8] T a E ] F T E ] o F L = 8] &" L- &" w t T o E ] E ] o L w t &" &" w t E x X [ XR R XR - x R - x R $ X x h $ X e - x e $ $ X[ X [ e n h [ e A- F A- F F X [ [ F X [ h A X [ h A X [ [ F A - [ F h t h A h t A A h tu q c o T L C T L T L e r T L e r - C e r - C e r T L c o T L e r - C e r T L T e r e S m - F c T A I - F c - A c e A h - w c e A h T w I e F h T w I e F h - w c - A m F c e A h T e h - L w I F w c - A c e A h w K AI A A A A A A A A A A EK A A A A L S S A S A S A S A S A S A S A S A S S X T S L T L X X X X X X X X X X X X X X X S X X X X X X H X Q Q Q Q Q Q Q Q e G X A A A A A A Q A Q A Q A Q A Q A A A c n e R W A Y W Y W W W W W WWW W W W Y u E Y E Y E Y E Y E Y E Y E Y E Y Y E Y E q Y H H X E E X X X X X X X X E X X X e K F T F T F T F T F T F F F F F F A A S A L L L L L T L T L T L T L T L T L T L T L ) 8 s a 4 8 o i s n c a 0 r 6 o-l c- t 1 ( o l r o r o- ) 3- - - - t n t n r a e 5 P e 4 S n e n e n e n e n o c o c S T e k - 1 ( l A k 8 t l e k a l e k l y k l y k l e e - a- a- a- a- i v i v ) 4 n e 1 4 1 4 1 4 1 1 t a t a a 4e a - 8 d i R t A 3- c s 0 0 0 4 4 g g 6 ) 0 0 e e 1 p T S P S e r ) o b 7- 7 5 4 P S ) - 25 P ) S a 7- 3 P ’ S a ’ 7- 7 3 b 3 b 3 a 9 b 9 P - S P ) S b n 0 e n ) a n 2 e n , a 3e u 5 5 6 i y 6 i y 6 P - T T A l F 1 ( T A 1 ( T A 1 ( T A 1 ( 5 1 5 1 5 1 5 1 T A T A 1 ( D 1 ( D 1 ( e l e p l 2 a p e l t a p s t s 2 a t H s H N- e n e n i y N- e n i 2 A i y y A d 2 2 d A A d H S a H H a S ] a N- &"] m N- N- m r O 7 m r G $ r X[ o o S o G f A a o A A A f X f K t S S o t [ a o t K s b n C d e O ] 7 O ] S 7 S d b e C d e W K oit Q a A k n X [ X [ k n i l Q A k n i M R c i W l e a b a b l e We Rifi Y r d a C C r a Y r N ] a o &E "] &"] Q QO E ] O ] Q F M$ $ A A7 O S 7 7 S S X[ X [ W Y W Y X [ X X [ W I d n A a T o E ] E ] o [ F o K I Q e c L- w t O 7 O 7 w t T w t x e S S e L- R x e - xn h e u A- h t X [ X [ h t h h t h q C e r F T F T e r A- e A- T e L- L- e C r T e C e h w c c h h T S I F A A w I F w I F G G KK A A A A A W S A K X S A X S A X S M X X X X X R Q Q Q Q R A A A N e A c W Q n e Y WW Wu E Y F E Y Y W I q X e A X E F X E F X F K I S T L T L T L T L Q R e i vt a ge n e n i y d ) t b n 4 e e 6 c 1 ( n e d i s t p e l r o P-e o r u t n a 9 a 0 b 0 C T P l F o c 6 1 7 1 7 1 I F Example 1: Application of Diyne Staple for Disruption of Sam68 Dimerisation The STAR proteins are a family of RNA binding proteins implicated in alternative splicing. All proteins in this family contain an extended KH RNA binding domain of around two hundred amino acids, unlike other proteins which contain multiple KH domains. The KH domain is situated between an N-terminal QUA1 domain that is responsible for dimerisation and a C-terminal QUA2 domain which has been proposed to participate in RNA binding (Artzt et al., 2010). The first STAR protein determined to be involved in alternative splicing was Sam68 (Hartmann et al., 1999), and will be the focus in this Example. Sam68 (the Src-Associated substrate in Mitosis of 68 kDa) belongs to the STAR family of RNA binding proteins. Unlike other hnRNP K homology proteins, the STAR proteins contain a single RNA binding KH domain. Post-translational modifications occur throughout the protein influencing its localisation, its ability to interact with other proteins and binding to RNA (Frisone et al., 2015). At the end of the C-terminus there is a twenty-four amino acid nuclear localisation sequence (NLS) that means Sam68 predominantly localises to the nucleus. A second member of this KH domain-containing family is known as T-STAR. T-STAR is a tissue specific analogue of Sam68, mainly expressed in neurons. T-STAR differs by the absence of a one hundred amino acid N-terminal region in comparison with Sam68, however it retains 77% sequence homology at the KH domain. Sam68 has been reported to be involved in the alternative splicing of oncogenes, typically favouring the most oncogenic form, with Sam68 levels found to be upregulated in a variety of cancers. The protein has been associated with poor prognosis when linked to cancer due to its implications in tumour progression (Frisone et al., 2015; Busà et al., 2007). The exact mechanism of the protein within the splicing machinery however, remains poorly understood and whether or not RNA binding is the specific cause of the oncogenic characteristics of Sam68 is not known. Another important question is if there is a correlation between the RNA binding ability of Sam68 and its role in signal transduction. A peptide inhibitor strategy was used to probe these interactions. In addition to understanding the core function of Sam68 in alternative splicing, concomitant design of a therapeutic to target Sam68 would both inform on the mechanism of action and potentially be a method of disrupting its oncogenic function. Using information gained from the crystal structure of T-STAR and due to the similarities with Sam68 (Feracci et al., 2016), synthesis of a T-STAR truncate was carried out to determine whether stapled peptides could be employed to outcompete the dimerisation of Sam68. As α-helix 3 of the KH domain is responsible for its dimerisation, this sequence was chosen as a starting point to develop analogues for synthesis. 008528580 Comparative Study Using an Alkene Staple with His to Arg Mutation In an attempt to overcome solubility issues, the crystal structure of T-STAR was studied for any potential sites for mutation. Upon further investigation, a His residue (H146) was identified on the back face of the α-helical interaction domain with potential for mutation. It was hypothesised that mutation of this residue for a more polar and less hydrophobic amino acid such as Arg would increase the peptide solubility. In addition to this, Arg is often found in CPPs and could overcome aggregation. Arg also possesses a guanidinium group that has the potential to form bidentate hydrogen bonding and electrostatic interactions with negatively charged glycolipids of the cell membrane. This could aid its ability to penetrate cells. Synthesis of Native Peptide Firstly, an analogue of the native peptide, T-STAR-1, was synthesised containing the H167 to R167 replacement for initial experiments as a control for FP experiments. Synthesis of the peptide proceeded well with 10 min couplings at 75 °C for all amino acids. The synthesis was carried out in automation up to and including the unnatural linker, Ahx (6-aminohexanoic acid). After manual Fmoc deprotection for 2 × 15 mins, FITC (2 equiv.) was coupled overnight with shielding from light. A test cleavage after this reaction revealed excellent crude purity (84%, determined by RP-HPLC), and after subsequent purification via semi-preparative RP-HPLC, the peptide was obtained in 10% yield and 99% purity. Synthesis of Alkene Stapled Peptide The synthesis of the alkene stapled peptide was achieved with positioning of the R8 and S5 unnatural amino acids in i, i + 7 positions. The synthesis was carried out with coupling reactions employing unnatural amino acids (2 equiv.), combined with double coupling and extending couplings (20 mins) for the amino acids directly following the bulky α-methyl-disubstituted alkenyl amino acids. The peptide was synthesised up to and including the Ahx linker and then a test cleavage of the linear peptides was performed. Analytical RP-HPLC and LC-MS revealed a deletion corresponding to Tyr making up around 20% of the crude material. An on-resin RCM 008528580 reaction was performed using Grubbs 1st generation catalyst in DCE for 2 × 2 h. To determine conversion, a test cleavage was performed with complete conversion to the stapled product observed with a 50/50 mixture of cis/trans alkene isomers. The N-terminal Fmoc was removed manually and FITC coupled using standard protocols. After global deprotection/cleavage from the resin and purification via semi-preparative RP-HPLC, each isomer was isolated. After a further purification, each isomer was obtained in >95% purity by analytical RP-HPLC in an overall yield of 5%. Both isomers were isolated to compare their activity, since differences in activity have previously been reported for isomers of i, i + 7 staples (Yuen et al., 2019). NMR experiments were performed which revealed the earlier eluting isomer by RP-HPLC as a cis isomer, and the later eluting isomer was assumed to be the trans isomer. Analysis of Alkene Stapled Peptide To determine whether there is a difference in activity between the two isomers formed during RCM, it is important to identify which isomer is cis and which is trans. NMR spectroscopy is a useful technique to distinguish between the two through a decoupling experiment. Decoupling can be performed by irradiating the signal of adjacent methylene protons coupling to the alkene. Through this process, the multiplet corresponding to the alkene protons can be simplified allowing an accurate J value for the isomer to be calculated. By deconvoluting the coupling constants to only show the 3JHH coupling constant, the size of the coupling constant can be correlated to each isomer. Experiments were performed with each isomer dissolved in d6-DMSO and homo-decoupling of the ^ and δ protons by irradiating the signal at a chemical shift of 1.95 ppm. The earlier eluting isomer by RP-HPLC was successfully decoupled to form two doublets with a 3JHH = 11.5 Hz. This corresponds with a previous report of a cis isomer assigned with a 3JHH = 11 Hz and trans isomer with a coupling constant of 3JHH = 16 Hz. Attempts to decouple the later eluting isomer were unsuccessful and thus through process of elimination this was assumed to be the trans isomer. To assess the degree of α-helicity induced by the alkene constraint, CD spectra of the native (T-STAR-1) and stapled mimetics (T-STAR-HCS-2-cis and T-STAR-HCS-2-trans) were acquired (Figure 1). Samples were dissolved in H2O at a peptide concentration of 50 µM scanning from 190 to 260 nm. Data produced from T-STAR-1 showed no defined secondary structure, with a curve matching that of a random coil (Lopes et al., 2014). The alkene stapled peptidomimetics showed a characteristic α-helical curve. Both isomers had a very similar curve with calculated helicities of 71% and 73% for T-STAR-HCS-2-cis and T-STAR-HCS-2-trans respectively. In order to test binding affinity of the peptides synthesised, bacterial expression of the NKKH domain of Sam68 was undertaken. This domain was selected as it is the portion involved in 008528580 dimerisation and is more stable than the KH domain alone (Foot et al., 2014). The gene of interest was cloned into a pBR322 plasmid containing a gene for kanamycin resistance, at the University of Leicester Protex facility. Rosetta BL21 DE3 E. Coli with resistance to chloramphenicol and kanamycin were transformed with the Sam68 plasmid. Selection for the desired bacteria was then achieved by plating the resulting cells onto agar containing kanamycin and chloramphenicol antibiotics. Colonies were picked and transferred to 20 mL of 2 × TY medium containing kanamycin and chloramphenicol. These starter cultures were then transferred to 1.6 L of 2 × Y medium for protein expression. Protein expression is controlled in BL21 DE3 cells by the presence of a T7 promotor next to the gene that is to be expressed. This promotor itself is under the control of the lac operon that is restricted by the presence of lac binding proteins. With these proteins present, the cells are prevented from generating T7 polymerase which therefore prevents transcription of the DNA of the gene of interest to RNA and thus inhibits protein expression. In practice, a change in the conformation of the lac promotor can be caused by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) as it binds to the lac repressor protein. This process causes release of the T7 promotor and allows for T7 polymerase and subsequent protein expression to occur. In this case, protein expression was induced once the cell cultures had reached an optical density of 0.6, within the exponential growth phase. After protein expression, purification of the desired protein was carried out. The desired gene had been engineered to contain a 6-His tag at the C-terminus of the sequence. A 6-His tag provides a facile method of purification as His has a high affinity for nickel. Cells were lysed by sonication; cell debris was removed by centrifugation and the resulting protein purified by affinity chromatography. The protein mixture was passed through a column packed with Ni-NTA agarose resin, with 6-His tag containing proteins binding to the nickel. Non-specifically bound proteins were then removed by a gradient of imidazole (25-250 mM) which competes with His to bind the nickel. Once a sufficient concentration of imidazole was present in the eluent, the 6-His tagged protein is eluted. After this, dialysis of the soluble protein was carried out to remove the high concentrations of imidazole used in the purification. Expression of NKKH-Sam68 was confirmed by taking samples at various stages during the expression process. A sample was taken after cell lysis and the soluble protein content was measured by centrifugation of a cell lysis sample followed by analysis of the supernatant. Some target protein was observed in the wash fractions of the His-tag purification due to over-loading of the resin but only a minimal amount was observed on the gel. A band was observed at the correct mass in each of the fractions from the His-trap purification. From 1.6 L of culture, 150 mg of protein was obtained (7 µM) as determined by a Bradford Assay. 008528580 Under the denaturing conditions of an SDS-PAGE, the protein mass corresponds to that of monomeric NKKH-Sam68. However, previous experiments using single angle X-ray scattering show that the NKKH region of Sam68 exists as a dimer (Feracci et al., 2016). To determine whether T-STAR-HCS-2-cis and T-STAR-HCS-2-trans retain activity for binding to Sam68 with the His to Arg mutation, FP experiments were carried out. Conditions for the experiment were repeated as before with serial dilutions of the NKKH domain of Sam68 across a 96-well plate and final concentrations of peptide 166 nM. Interestingly the native peptide T-STAR-1 showed no binding to the protein suggesting the peptide does not fold into its bioactive conformation during the experiment. Both isomers of the alkene stapled peptide T-STAR-HCS-2 showed the standard sigmoidal curve expected for binding to NKKH-Sam68 with a KD of 3.5 µM for the cis isomer and 1.8 µM for the trans isomer. From the binding data it was concluded that the His to Arg mutation did not have a significant impact on binding to Sam68. Cellular uptake experiments were performed to determine whether the His to Arg mutation is able to increase solubility and cellular uptake. Experiments were performed in poly-D-lysine coated 24-well plates to aid with cell adhesion. Cells were plated out at a density of 60,000 cells/mL with 500 µL of cell containing media added to each well. Media was replaced with serum free media before addition of each peptide to the well (10 µM), as well as a nuclear stain. Experiments were performed in triplicate with DMSO as a vehicle control, FITC-Pen as a positive control, T-STAR-1 and T-STAR-HCS-2 (cis and trans). After an overnight treatment with the peptides, media was removed and the cells washed several times with PBS before fixation using a 4% PFA solution and imaging under a fluorescent microscope. No cellular uptake was observed in the FITC channel for the DMSO control and uptake is visible from the FITC channel for the positive control FITC-Pen. Both T-STAR-HCS-2-cis and T-STAR-HCS-2-trans show cellular uptake in the cells from visualisation of the FITC channel and overlay with the nuclear dye. No aggregation was observed in this experiment and thus the mutation of His to Arg was included in the design of further analogues. Diyne Staple The alkene stapled T-STAR peptides synthesised have been shown to have increased binding for Sam68 compared to the native, unconstrained peptide and are found to be cell penetrant. This peptide sequence was therefore taken on as a model system for optimisation of the diyne staple. Synthesis of a variety of different alkyne containing amino acids bearing different chain lengths and stereochemistry allows for a comparison between a series of different staples. A comparison between the different chain lengths and stereochemistries of the bridge would be analysed using Glaser conversion by analytical RP-HPLC and helicity calculated from CD. 008528580 To date there are no examples in the literature comparing differences in helicity or activity between i, i + 7 alkene stapled peptides bearing differing stereochemistries of the alkene unnatural amino acids. Therefore, determining any differences between the different combinations could also be informative for the traditional alkene stapling, in addition to developing the diyne staple. Synthesis of Alkyne Amino Acids via Ni(II) Schiff Base Complex Synthesis of Iodo-alkyne A series of iodo-alkynes were synthesised ranging from five carbons to eight carbons starting from the appropriate alkyn-ol. The terminal alcohol was converted to a mesylate and displaced with the more reactive iodide using a Finklestein reaction with NaI to produce an iodo-alkyne, as shown in Scheme 1. Reaction conditions were as described herein for the synthesis of Compound 155 Amino Acid Synthesis section above). Scheme 1: Conversion of terminal hydroxyl to iodide. 1. MsCl, Et3N DCM, rt, 16 h 2. NaI, acetone, Iodo-alkynes may also be synthesised from the appropriate chloro-compound, with reaction conditions as described herein for the synthesis of Compound 166. Yields were generally excellent with a 96% yield for both C7 and C8 iodo-alkynes (Table 3). A slight decrease was observed for C6 and C5 analogues. This decrease in yield is a result of increased volatility for the smaller compounds and thus, some material was lost during evaporation of solvent after the reaction. 008528580 Table 3. Yields for Finkelstein reaction of carbon chain lengths between five and eight. Starting Material Product Yield (%) 78 86 96 96 Alkylation of Ni(II) Schiff Base Complex A series of R- and S- configured alkylated Ni(II) Schiff base complexes at the α-position derived from Ala were synthesised under conditions as described for the synthesis of Compound 156 (Amino Acid Synthesis section above). An SN2 reaction with the appropriate iodo-alkyne gave alkylated complexes 85a-d and 98a-d. The reaction proceeded at rt for 1 h using sodium t-butoxide as a base with yields ranging from 69-76% for the alkylated products (Scheme 2). The dr of each crude reaction was determined using 19F NMR spectroscopy with a single diastereomer isolated in each case after purification. 008528580 Scheme 2: Alkylation of S-complex 73 and R-complex 92 with iodo-alkynes 86, 111, 113 and 115 to generate alkylated complexes 85a-d and 98a-d. 85d R = (CH2)3C≡CH, 72%, dr 86:14 98d R = (CH2)3C≡CH, 70%, dr 84:16 Decomplexation and Fmoc Protection Decomplexation of alkylated complexes 85a-d and 98a-d was achieved by heating under acidic conditions with Ni removed via chelation with EDTA. The conditions used are as described for Compound 157 in the Amino Acid Synthesis section above. Fmoc protection proceeded using Fmoc-OSu and 6% NaCO3 (aq) in acetonitrile. Fmoc protected amino acids 99a-d and 100a-d were obtained in yields ranging from 60-77% for R- and S- configured amino acids incorporating alkynyl side-chains ranging from five to eight carbons (Scheme 3). Purity of all amino acids was calculated using analytical RP-HPLC and deemed to be >95% for all cases. 008528580 Scheme 3: Decomplexation and Fmoc protection forming the terminal alkyne S- and R- Fmoc protected amino acids 99a-d and 100a-d. 99d R = (CH2)3C≡CH, 60% 100d R = (CH2)3C≡CH, 62% Synthesis of Diyne Peptides To determine the requirements for the optimal diyne bridge, eight combinations of alkyne containing T-STAR peptides were synthesised using the same staple incorporation site as described above (T-STAR-HCS-2). Initially, peptides containing two C8 or two C7 bridges were synthesised with four stereoisomeric combinations of each bridge length: SS, SR, RR and RS. This would provide information on any stereochemical requirements for the staple which could then be applied to determine the shortest staple that is required. The linear alkyne containing peptides were prepared using microwave-assisted SPPS with rink amide ChemMatrix resin. Standard 10 min couplings at 75 °C using HCTU followed by piperidine-meditated deprotections were employed (Scheme 4). Unnatural alkyne containing amino acids were coupled using half of the equivalents of amino acids (2 equiv.), HCTU (2 equiv.) and DIPEA (4 equiv.). Linear peptides were synthesised up to and including the Ahx linker with the N-terminal Fmoc group still attached to the peptide. 008528580 Scheme 4: Synthesis of linear T-STAR-diyne-o via SPPS. Glaser reactions was performed as described in General Method B on 0.01 mmol scale using CuCl (5 equiv.), bpy-diol 58 (7.5 equiv.), DIPEA (20 equiv.) and DMF for 3 h at 60 ºC using microwave irradiation (Scheme 5). Conversion to the stapled product was determined using analytical RP-HPLC and HRMS, with stapled products typically eluting later. Scheme 5: Reaction conditions to form diyne stapled analogues of T-STAR. Complete conversion was observed for combinations R8S8, S8S8 and S7S7, with excellent conversion (>90%) for R8R8 and R7S7 (Figure 2). For S8R8 and S7R7, slightly lower conversions of 68% and 52% respectively were obtained. This could be a result of the alkyne side-chains pointing in a less favourable orientation in space for the macrocyclisation to be easily achieved. Based on these results, it is clear that both a C16 and C14 bridge are feasible for this system. The SS combination of amino acids gave the best conversion for both bridge lengths and would only require the synthesis of one unnatural amino acid rather than two. To determine what the optimal bridge length would be, a C10 and C12 bridge were 008528580 then synthesised from two S5 and two S6 amino acids. This would determine the optimal bridge length and at what point the system is too strained to form the stapled product. A decrease in conversion was observed for both the C12 and C10 bridge (Figure 3). From this result, it was concluded that a C14 bridge is the optimum for this system as it is the shortest constraint able to provide high Glaser conversion. In addition, S stereochemistry for both unnatural alkynyl amino acids also yielded optimal Glaser conversion with requirement of the synthesis of only one amino acid. To determine how this diyne system compares with other types of diyne peptides in the literature, two other analogues of the diyne were synthesised (Silvestri et al., 2017; Verlinden et al., 2015; Verlinden et al., 2019). Firstly, to determine the importance of the α-methyl group on the amino acid, a monosubstituted amino acid was synthesised as a direct comparison to the α, α -disubstituted alkyne amino acids. In addition, introduction of O-alkylated amino acids of the same chain length was probed as an alternative strategy with a shorter synthesis. Synthesis of Monosubstituted Alkyne Amino Acids Alkylation of Glycine Derived Ni(II) Schiff Base Complex Synthesis of a S7H monosubstituted alkynyl amino acid proceeded via a Gly derived Ni(II) Schiff base complex 116 and C7 iodo-alkyne 115 (Scheme 6), under reaction conditions as described in the Amino Acid Synthesis section above (Compound 117). An SN2 reaction proceeded using NaOH (5 equiv.) as the base and iodo-alkyne 115 (3 equiv.) at rt for 30 mins. Analysis by 19F NMR spectroscopy and LC-MS showed that 11% of the crude material corresponded to the bis-alkylated complex. The reaction was repeated with 1.1 equiv. of iodo-alkyne 115 and pleasingly no bis-alkylation was observed by LC-MS. A crude dr of 97:3 was calculated from 19F NMR spectroscopy, which yielded 117 as a single diastereomer after purification. Scheme 6. Alkylation of S-Gly complex 116 with iodo-alkyne 115 to generate alkylated complex 117. 008528580 Decomplexation of C7H Alkylated Complex and Fmoc Protection Decomplexation of 117 was carried out as before using 3 M HCl at 70 °C for 30 mins (Scheme 7). The conditions used are as described for Compound 157 in the Amino Acid Synthesis section above. EDTA was added to chelate Ni, with the ligand recovered in the organic layer during work-up. Fmoc protection was performed using Fmoc-OSu and 6% Na2CO3 to generate amino acid 118. Scheme 7. Decomplexation of 117 and Fmoc protection forming the terminal alkyne S- Fmoc protected amino acid 118. The S7H alkyne amino acid was incorporated into the T-STAR sequence using the same conditions used for the disubstituted alkynyl amino acids to provide T-STAR-S7HS7H-o (Scheme 8). Scheme 8. Synthesis of linear T-STAR-S7HS7H-o via SPPS. Glaser reactions were trialled using the conditions described in General Method B. The reaction was performed on 0.1 mmol scale using CuCl (5 equiv.), bpy-diol 58 (7.5 equiv.), DIPEA (20 equiv.) and DMF for 3 h at 60 °C using microwave irradiation. After 3 h, a 38% conversion was observed to the stapled diyne product T-STAR-S7HS7H-c. A second treatment under the same conditions was attempted, however no significant increase in conversion was observed. The lower conversion obtained for the peptide containing monosubstituted alkyne amino acids in comparison with the peptide containing disubstituted amino acids could be due to 008528580 the absence of the α-methyl substituent. As this is the only difference between the two peptides, it is likely that the presence of the methyl group helps to facilitate the Glaser reaction by placing the alkyne groups closer to each other in space. Synthesis of Alkylated Serine Amino Acids Synthesis of a C7 alkyne amino acid derived from Ser was achieved via an approach used by Cistrone et al. Commencing from L-Boc-Ser-OH 119, an SN2 reaction with iodo-alkyne 111 (1.4 equiv.) and NaH (2.4 equiv.) as base was carried out (Scheme 9). Compound 120 was obtained in a 42% yield with no purification required as residual Boc-Ser could be removed by washing with H2O during the work-up. The Boc protecting group was removed with TfA and the free amine protected with Fmoc-OSu (1.1 equiv.). The Fmoc protected amino acid 121 was generated in 62% yield. Scheme 9. Alkylation of L-Boc-Ser-OH 119 to generate 120. Boc deprotection and subsequent Fmoc protection yielded 121. The S7 alkyne amino acids derived from Ser were incorporated into the T-STAR sequence using the same conditions used for the disubstituted alkynyl amino acids (Scheme 10). 008528580 Scheme 10. Synthesis of linear T-STAR-alk-Ser-o via SPPS. Glaser reactions were trialled using the conditions described in General Method B above. The reaction was performed on 0.1 mmol scale using CuCl (5 equiv.), bpy-diol 58 (7.5 equiv.), DIPEA (20 equiv.) and DMF for 3 h at 60 °C using microwave irradiation. After 3 h, no conversion was observed to the stapled diyne product. A second treatment for 3 h at 60 °C again yielded no product. These results show that the reactivity of alkyne-modified serine residues in the Glaser reaction is reduced compared to the alkynes of the present invention, and no stapled product can be obtained under the same conditions for the serine analogues. Deprotection and FITC Coupling All diyne analogues successfully synthesised were labelled with FITC to allow for binding studies via FP and cellular uptake experiments. The N-terminal Fmoc protecting group was removed manually using 2 × 15 mins treatments of 20% piperidine in DMF. FITC (2 equiv.) was added to the resin bound peptide in a solution with DIPEA (4 equiv.) in DMF and left to react overnight at rt (Scheme 11). Coupling was confirmed using HRMS before the peptides were cleaved from resin and protecting groups removed using a cleavage cocktail of TFA/TIS/H2O (95:2.5:2.5, v/v/v) for 3 h. After trituration using Et2O, crude peptides were dissolved in 50:50 H2O/acetonitrile and lyophilised before purification. Scheme 11. Manual Fmoc deprotection and FITC coupling on resin. Peptides were cleaved from resin using a standard cleavage cocktail. 008528580 All peptides were purified using a linear gradient of H2O and acetonitrile spiked with 0.1% TFA and fractions combined with a purity >95% by analytical RP-HPLC. To compare whether there was a structural difference between the different diyne peptides, all peptides synthesised were taken on for analysis using CD. Circular Dichroism CD analysis was undertaken for each diyne peptide at a concentration of 50 µM, scanning from 185 to 260 nm as described in General Method E. For the C16 bridges synthesised from two C8 alkyne amino acids, combinations SS, SR and RS also showed very similar results in terms of helicity (Figure 4). The RR configured analogue was much lower in helicity. For the C14 bridge, similar helicity was observed for SS (77%) and RS (70%) combination (Figure 5). The SR bridge had a lower helicity of 54% and the RR had the lowest helicity of 40%. A comparison between the C14, C12 and C10 bridged peptides showed a decreasing trend in terms of helicity (Figure 5). This is most apparent at 190 nm where the MRE value for C12 and C10 is around half that of C14. This result suggests that both the C12 and C10 constraints are changing the conformation of the peptide due to increased strain from the shorter bridge. It was concluded from these results that an S7S7 bridge is the optimum in terms of helicity and with the added benefit of requiring only a single unnatural amino acid. Comparing this S7S7 bridge with the analogue lacking methyl groups at the α-position, S7HS7H, an almost identical CD curve was observed with very similar calculated helicity 008528580 (76% – S7S7 and 74% – S7HS7H, Figure 5). This result suggests that the main function of the α-methyl group is to facilitate the Glaser reaction by forcing the alkyne groups closer together, and the impact on the structure of the peptide is modest. A graph summarising peptide helicity of all analogues is shown in Figure 6. Proteolytic Stability To establish whether incorporation of the staple increased the peptides stability to protease enzymes, a stability study was undertaken. Chymotrypsin was chosen for the experiment as it typically cleaves at the C-terminus of aromatic residues. Since there is a single Tyr located within the centre of the peptide sequence this would provide a good model for resistance due to increased secondary structure. Cleavage site of chymotrypsin (red) in T-STAR sequence: After 4 h, T-STAR-1 was completely degraded by the enzyme as confirmed by analytical RP-HPLC and LC-MS (Figure 7). Of the stapled analogues tested, both alkene and diyne based constraints were extremely stable with >92% of all peptides remaining after the last time point. This result is consistent with CD data which showed very similar helicity levels between T-STAR-HCS-2-cis, T-STAR-HCS-2-trans, T-STAR-S7S7 and T-STAR-S7HS7H. The constraints synthesised are effective in locking the peptide into a helical structure making enzymatic degradation by proteases difficult. Additional Assays Methods Cell permeability fluorescence assay: HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high glucose with GlutaMAX, Gibco) supplemented with 10 % (v/v) foetal bovine serum (Gibco) and 1 % (v/v) penicillin/streptomycin (10,000 units/mL penicillin, 10,000 μg/mL streptomycin, Gibco). Cultured cells were maintained in a humidified incubator at 37°C, 5 % CO2 and passaged twice weekly in T-25 flasks (Corning). For cell counting, an aliquot (10 μL) of cell solution in media was added to a haemocytometer slide which was viewed using a microscope for manual inspection and counting. For experiments, 300,000 cells were seeded into 6-well plates (CytoOne) on 30 mm cover glass slides pre-treated with 0.1 mg/mL poly-D-lysine and left to grow for two days to reach ca.80 % confluency before compound incubation. Media was removed and the cells were washed with PBS prior to treatment with compound in PBS (2 h, 20 μM, 37°C). Cells were then 008528580 washed again with PBS twice, fixed with a 4% (w/v) solution of formaldehyde in PBS (10 min, 37°C) and washed with PBS twice prior to analysis. Fluorescence imaging: Images were acquired on a MetaMorph/Metafluor fluorescence imaging microscope system equipped with a 40 × Superfluor objective for an exposure time of 1000 ms. Excitation for fluorescein was conducted at 495 nm. Image analysis and processing was performed using MetaMorph microscopy software. Raman spectroscopy experiments: Raman spectroscopy was performed using the Horiba Jobin Yvon LabRAM HR system with a Ventus CD laser at 532.02 nm, 100 mW. The hole width was 200 μM with a diffraction grating of 600 g/mm using an Olympus x50LWD objective lens. The recorded spectral range was 600–4000 cm-1 and data acquisition was performed during 5 seconds with 3 repeats and collected with the Synapse OCD detector. 100% power was used for T-STAR-1 peptide and 50 % power was used for the stapled peptide T-STAR-S7S7. Data was analysed using the Labspec 5 software. Cell Permeability Fluorescence Assay Fluorescence microscopy was used to investigate the cellular uptake of the diyne stapled T-STAR-S7S7, compared to the unmethylated T-STAR-S7HS7H, the alkene stapled peptides T-STAR-HCS-2-cis, and T-STAR-HCS-2-trans, and the native T-STAR-1 peptide. HEK- 293 cells were treated with either 20 μM fluorescein stapled peptides or fluorescein native peptide and imaged 3 hours post--treatment (Figure 10). The stapled analogues showed a diffused intracellular localization, confirming efficient cellular penetration (Figure 10A–D). The native peptide showed no cellular internalisation (Figure 10E). Brightfield images were also acquired and overlaid with the fluorescence channel images for all experiments (data not shown). Raman Spectroscopy Traditionally, cell-uptake experiments are performed using fluorescently labelled analogues of peptides that have different physicochemical properties compared to the unlabelled pep- tide. A conformational constraint that also acts as a chromophore for imaging would provide a unique advantage. Alkyne groups are Raman chromophores and have been used extensively as molecular probes for cell imaging. It was hypothesised that the diyne constraint would act as a Raman chromophore and facilitate the visualisation of peptides in cells using Raman microscopy. To test this hypothesis, unlabelled analogues of native T-STAR-1 and a preferred stapled analogue, T-STAR-S7S7 were prepared for analysis using Raman spectroscopy. Raman imaging of the solid diyne stapled T-STAR-1 peptide was conducted using Raman spectroscopy with a laser at 532.02 nm. Figure 11(A) shows the spectra from the diyne stapled peptide. A 008528580 significant peak can be observed in the cell-silent region at ~ 2,255 cm-1. Raman was also performed on the native T-STAR-1 peptide to confirm the absence of this peak at ~ 2,255 cm-1 (Figure 11B). These data demonstrate that the diyne stapled peptide shows a significant peak in the cell- silent region (1,800–2,800 cm-1), and thus has potential application in confocal cellular Raman imaging. Conclusions A variant of the T-STAR native sequence (T-STAR-1) was synthesised to improve solubility, in addition to an all-hydrocarbon i, i + 7 stapled peptide (T-STAR-HCS-2). By mutating a His residue to an Arg, aggregation issues observed during cell work were overcome. Two isomers were produced during the RCM reaction that were assigned using homo-decoupling 1H NMR experiments to decouple adjacent protons to the alkene. CD analysis showed that both T-STAR-HCS-2-cis and T-STAR-HCS-2-trans were significantly more helical than the native sequence, T-STAR-1. The NKKH domain of Sam68 was expressed and purified for FP analysis of the peptides. Results showed that T-STAR-1 did not bind to Sam68, whereas both T-STAR-HCS-2-cis and T-STAR-HCS-2-trans retained binding to Sam68. An in vitro splicing assay was performed to determine whether the peptides synthesised had an impact on the splicing of the Nrxn3 minigene which is a common splicing target for Sam68. No difference in splicing was observed for the peptides in comparison with control experiments, however the experimental protocol requires further optimisation. A series of alkyne containing α, α-disubstituted amino acids were synthesised bearing five-eight carbon side-chains and both S and R stereochemistry via a Ni(II) Schiff base complex. These amino acids were incorporated into the T-STAR sequence to compare both the length of the diyne bridge and stereochemistry of the unnatural amino acids in the bridge. Conversion of the Glaser reaction was followed using analytical RP-HPLC and helicity calculated from CD measurements. A C14 bridge bearing SS stereochemistry was deemed the optimal system based on conversion, helicity and the requirement of only one unnatural amino acid. As a comparison, an S-configured monosubstituted amino acid with a seven carbon side-chain group was synthesised via a Gly Ni(II) Schiff base complex. After incorporation into the T-STAR sequence, a lower conversion to the stapled product was observed during the Glaser reaction with respect to the staple containing α-methyl groups. An analogous O-alkylated Ser analogue was also synthesised with no conversion observed to the stapled product during the Glaser reaction. A proteolytic study with chymotrypsin was undertaken to compare between T-STAR-1, T-STAR-HCS-2-cis, T-STAR-HCS-2-trans, T-STAR-S7S7 and T-STAR-S7HS7H. After 4 h of incubation, T-STAR-1 was completely degraded as observed by analytical RP-HPLC. 008528580 Enhanced stability was observed for all stapled analogues tested, with each showing >92% peptide remaining after 4 h. Example 2: Application of the Diyne Staple to p53 The p53/MDM2 interaction is a very well researched target, especially for α-helical peptides. Therefore, this application was selected to trial the diyne staple type on, and compare the conformational properties and biological activity to the alkene stapled peptide (ATSP-7041). ATSP-7041 is the precursor peptide of ALRN-6924 and was chosen for this study. To compare alkene and diyne stapled ATSP-7041 analogues, both peptides were synthesised (Scheme 12). A linear analogue reported by Chang et al., 2013 (ATSP-3848) which was based on native p53 was synthesised as a linear control to assess the peptides. The synthesis of the linear peptide was performed first to identify any problem residues before including the unnatural amino acids needed for the stapling. The stapled peptides were then synthesised with an acetyl group at the N-terminus to mask the positive charge, to closer replicate the segment from the WT protein. N-terminal fluorescently labelled analogues were also synthesised to facilitate FP studies. Scheme 12: Target peptides (A) A linear analogue (ATSP-3848 developed by Chang et al.; (B) ATSP-7041 featuring the alkene staple; and (C) The diyne analogue of ATSP-7041. The interacting residues are shown in pink. Synthesis of Linear Peptides 008528580 The synthesis of the linear peptide 145 (ATSP-3848) was achieved using standard microwave-assisted SPPS methods and a tBu/Fmoc protection strategy. Synthesis was performed using ChemMatrix© Rink Amide resin (0.45 mmol/g), a PEG based resin (Garcia Martin et al., 2006). Microwave heating was used for all coupling reactions in the synthesis of the peptide as couplings are more efficient at elevated temperature with shorter reaction times required. Coupling reactions were achieved using Fmoc protected amino acids (5 equiv.), Oxyma Pure and DIC in DMF with heating to 90 °C for 2 mins followed by washing the resin using DMF. Deprotection of N-terminal Fmoc groups was achieved using 20% morpholine/DMF for 1 min at 90 °C. Unlabelled (145a) and fluorescently labelled (145b) peptides were synthesised for the selected assays. For the unlabelled peptides, the N-terminus was acetyl capped using acetic anhydride in DMF (5 M) for 10 mins at rt (Scheme 13). The fluorescently labelled peptides were synthesised with an Ahx linker between the last N-terminal amino acid and the FITC, to avoid the formation of a fluorescein thiohydantoin. 008528580 .i nse rei dm Ak i n R © i xr ta M meh C hi t w g i nt r a ts, SPP S g i ns u eug l oanar ae i n l a54 1 d e t l a y te c a dn a b54 1 del leba l i necs e ro l u f e h tf o i sse h tny S:3 1 e mehc S After synthesis of the linear sequences, a test cleavage was carried out on a small portion of resin (1-2 mg) using a cleavage cocktail containing TFA:TIS:H2O (95:2.5:2.5 v/v/v) for 1 h. After evaporation of the TFA and precipitation using Et2O, the pellet was analysed using LC- MS. After full cleavage of the peptide from resin, LC-MS analysis showed that a carbamate functional group (+44) from the Boc protecting group was still present on the peptide. Tryptophan is an electron-rich amino acid, hence it can undergo various side reactions, especially during TFA peptide cleavage. One potential side reaction is the incomplete removal of the Boc protecting group with the carbamate (CO2) group still attached to the indole nitrogen. A final procedure was used to sufficiently remove the carbamate adduct utilising Le Chatelier’s Principle. Firstly, a larger volume of cleavage cocktail was used and the cleavage time increased to 4 hours. After cleavage, the peptide was dissolved in acetonitrile (ACN) and H2O, spiked with 0.1% TFA, and left in solution overnight. LC-MS analysis revealed the successful removal of the adduct and the peptide was freeze-dried ready for purification. Synthesis of the Alkene Stapled Peptide After successful synthesis of the linear peptide 152, the synthesis of the alkene-stapled peptide 153a/153a’ was achieved using standard microwave-assisted SPPS methods on resin as described above for the linear sequence. Two equivalents were used for the unnatural amino acids. The amino acids after the unnatural amino acids were double coupled, with coupling times also doubled due to the increased steric demand of the α,α-disubstituted unnatural amino acids. Unlabelled and fluorescein labelled analogues were synthesised. After synthesis of the linear sequence, a test cleavage was carried out on a small portion of resin (1-2 mg) to confirm the successful synthesis of the linear sequence. 008528580 Scheme 14: The overall synthesis of the acetylated alkene stapled peptide 153 starting with ChemMatrix© Rink Amide resin. (a and a’ denote the cis and trans isomers). The linear peptide 152 containing R8 and S5 was cyclised by RCM using Grubbs catalyst, excluding light (Scheme 14) using conditions as described in General Method B (see Ring Closing Metathesis section). A test cleave was performed and HPLC and LC-MS analysis was used to determine the success of the RCM reaction. During the RCM reaction, removal of two carbon atoms and four hydrogens results in a mass difference of 28, which was observed using LC-MS. Furthermore, a shift in retention time was observed between the linear and closed peptide product using HPLC analysis. As expected, the product peptide resulted in two separate peaks with the same mass by LC- MS, which is indicative of the isomers produced from the alkene staple. 008528580 The geometric configuration of the two isomers was determined using 1H NMR spectroscopy. The earlier eluting isomer 153a (isomer 1, TR (50 min) = 42.098) gave a proton signal at 5.13- 5.30 ppm with a centrosymmetric doublet of triplets splitting pattern with 3J(-CH=CH-) = 11.5 Hz and 7.0 Hz and was assigned as the cis isomer. The later eluting isomer 153a’ (isomer 1, TR (20 min) = 42.846) was assigned as the trans isomer with an unresolved broad peak in the 1H NMR. This data is in agreement with Yuen et al.2019, who also used 1H NMR to identify the isomers of ATSP-7041. The cis (153a) and trans (153a’) isomers were separated using RP-HPLC, however, due to the proximity of the two peaks, the prepped mixture was re-subjected to purification several times to ensure complete separation and >95% purity. The two isomers were isolated and tested separately. Synthesis of the Diyne Stapled Peptides Synthesis of the Alkyne Amino Acid A Ni(II) complex 154 was used for the synthesis of the Fmoc protected alkyne amino acid 157 based on methods reported by Aillard et al., 2014 and Mahindra et al., 2019. Scheme 15 shows the synthesis route to afford the Fmoc protected alkyne amino acid 157. 008528580 Scheme 15: The synthesis of an alkyne amino acid using a Nickel-Schiff base method. The Nickel complex was alkylated with an alkyne electrophile with the desired carbon length needed to generate the diyne stapled peptide. To generate the alkyne amino acid 157 with the correct carbon chain length, synthesis began from the commercially available 6-heptyn-1-ol 155i (Scheme 16) and Amino Acid Synthesis section above). The primary alcohol was converted to a mesylate 155ii and displaced with the more reactive iodide via a Finklestein reaction using NaI. Conversion of alcohol to iodide was confirmed by 13C NMR (signal from CH2-OH at 63.0 ppm vs CH2-I signal at 6.7 ppm). Scheme 16: The conversion of a primary alcohol 155i to an iodide 155 using a Finklestein reaction. The formation of insoluble sodium methanesulfonate as a byproduct in the reaction drives the equilibrium to favour conversion to the product. 008528580 Diastereoselective alkylation of the S-Ni(II) Schiff base complex 154 was achieved using sodium hydroxide as base and iodo-alkyne as the electrophile. After formation of a planar enolate intermediate, attack is preferred at the Si face of the complex as the 2-fluorobenzyl moiety sterically blocks attack from the Re face facilitating formation of the S-alkylated complex. The diastereomeric ratio (dr) of the crude mixture 156 was determined using 19F NMR spectroscopy with a ratio of 96:4. Upon purification via column chromatography, one diastereomer was isolated, as assessed again using 19F NMR spectroscopy. Stereochemistry of the major diastereomer has previously been confirmed by X-ray crystallography (Aillard et al., 2014). Decomplexation of the alkylated complex 156 under acidic conditions yielded the amino acid 157. EDTA was used to remove the Ni by-product. Fmoc protection was achieved using Fmoc-OSu and 6% Na2CO3 (aq.) as base to generate the Fmoc alkyne amino acid 157. Synthesis of the Diyne Stapled Analogue of ATSP-7041 With the desired alkyne amino acid 157 in hand, the diyne stapled peptides were synthesised (Scheme 17). Synthesis of a diyne stapled analogue of ATSP-7041 was achieved by microwave-assisted, automated SPPS using a Fmoc/tBu protection strategy, ChemMatrix© Rink Amide resin to afford the C-terminal amide and using Oxyma Pure/DIC for coupling reactions. Two equivalents of Fmoc alkyne amino acid were used to couple the unnatural amino acid. Amino acids after the unnatural amino acids were double coupled, with coupling times also doubled due to the increased steric demand of the α,α-disubstituted unnatural amino acid. Linear unlabelled 159a and Ahx spaced 159b (for fluorescent labelling) peptides were synthesised. 008528580 sa w b951rekn i lxh A n ahi t wedi t peprae i n l A .sn i o i tso p 7+i, i n is i dc a o i n m a eny l k a gni t a ro p roc i n a95 1 edi tpe p d e t l a . y t g e i n c l l a e r b a a e l i n e l cn e e h t c f s o e r S ou P l f P r S o e f h de T: i s 7 s 1 e e h tn m y eh s c o S l s a Peptide Stapling using the Glaser Reaction With the linear alkyne peptide 159 in hand, appropriate conditions for performing a Glaser reaction to form the stapled peptide were applied to prepare the macrocyclised peptide. Conditions for the Glaser type reaction were trialled based on the work by Cistrone et al. 2018. The highest conversion to the cyclised product 160 was achieved using CuCl and a 4,4’-bis(hydroxymethyl)-2,2’-bipyridine ligand at 60 ºC (microwave heating) for 3 hours (Scheme 18 and General Method B). Scheme 18: Macrocyclisaton on resin was achieved using a Glaser coupling. The Ahx linear peptide 159b was cyclised using the same Glaser conditions as for 159a, followed by Fmoc deprotection and coupling with FITC to afford the fluorescently labelled diyne stapled peptide 160b (Scheme 19). 008528580 Scheme 19: The cyclisation to achieve the fluorescently labelled diyne stapled peptide 160b was performed on resin using a Glaser coupling. Upon completion of the Glaser reaction, a shift in retention time was observed differentiating the linear peptide 159a (TR (50 min) = 42.167 min) and the cyclised peptide 160a (TR (50 min) = 41.902 min) using RP-HPLC analysis. LC-MS confirmed the mass difference of -2, signifying the loss of two protons. After complete conversion of the linear peptide 159 to the closed diyne peptide 160, the peptide was cleaved from the resin using TFA peptide cleavage conditions as described in General Method B. The crude peptide was purified using RP-HPLC which was significantly easier than purification of the alkene analogue due to the cis/trans isomers. The diyne stapled peptide was isolated in good yield (10%) and good purity (97%). Synthesis of the Negative Control (Phe-Ala) Diyne Stapled Peptide Chang et al.2013 have previously used an amino acid substitution in the sequence of ATSP-7041 to display the importance of the interacting residues. Changing the interaction residue Phe to Ala, drastically reduced the binding of the peptide to MDM2 and MDMX. 008528580 However, they showed that the stapled peptide analogue was still able to penetrate cells using fluorescence microscopy experiments. A similar diyne analogue was synthesised as a negative control. The peptide was synthesised in the same way as described above but the Phe residue was substituted for an Ala (Scheme 20). Unlabelled (164a) and fluorescently labelled (164b) analogues were synthesised. Complete conversion of the linear to cyclised peptide was achieved in 3 hours as monitored by RP-HPLC and LC-MS. 008528580 n i n wohs ( 0 h eu idse r eh P gni tc ar etn i eh T .a46 1 edi tpe p d lep at s en iydlor tno c evi tage n e htf o isse htny s ehT:0 2 e meh . ) c d S e r 5 Synthesis of the Alkylated Serine Diyne Stapled Peptide One disadvantage of the synthesis of the diyne stapled peptide 160 is the long alkyne amino acid synthesis route. Therefore, to further develop the stapling technique, a route was explored to achieve the diyne stapled peptides faster. A synthesis route was designed which incorporated a diyne staple without the need to synthesise the unnatural alkyne amino acid 157 via the Ni(II) Schiff base method. Alkylation of naturally occurring serine has been previously performed to create diyne stapled peptides (Cistrone et al., 2018, Verlinden et al., 2015). Synthesis of a C7 alkyne amino acid derived from Ser was achieved via an approach used by Cistrone et al., 2018. A Williamson ether synthesis was performed between L-Boc-Ser-OH 165, iodo-alkyne 166 and NaH as base (Scheme 21). Compound 167 was obtained in a 45% yield. The Boc protecting group was removed using TFA and then the amine was re-protected using Fmoc-OSu. The Fmoc protected amino acid 168 was generated in 40% yield. Scheme 21: The synthesis route to afford the alkylated serine amino acid 168. COOH 167 168 008528580 The synthesis of the linear peptide 169a was achieved using ChemMatrix© Rink Amide resin to afford the C-terminal amide and a standard Fmoc/tBu SPPS approach using Oxyma Pure/DIC for coupling reactions (Scheme 22). The same conditions were used as described above for the synthesis of Diyne stapled analogue of ATSP-7041, and fluorescently labelled analogues were synthesised. Scheme 22: SPPS of the linear peptide 169a before cyclisation on resin. With the linear peptide in hand 169a, conversion to the cyclised product 170a was achieved using CuCl and a 4,4’-bis(hydroxymethyl)-2,2’-bipyridine ligand using 60 °C microwave conditions for 3 hours (Scheme 23 and General Method B). 008528580 Scheme 23: Macrocyclisation on resin using a Glaser coupling. The Glaser reaction was performed on-resin as described General Method B, however, the reaction did not progress to completion (HPLC traces after one round of Glaser reaction showed that the conversion of linear 169a to closed 170a was 40:60). The resin was re- subjected to the Glaser reaction conditions for a second time with fresh reagents. The second round of treatment led to a 50:50 conversion. The 50:50 mix was taken on to full cleavage of the peptide from the resin in the hope that the open and closed peptides could be separated upon purification. Interestingly, upon cleavage of the peptide, the product was incredibly insoluble in most solvents (DMF, DMSO, ACN, H2O and methanol (MeOH)). Thus, this strategy was abandoned as purification of the peptide was not possible and any further experiments would have been challenging. One of the differences and disadvantages of this technique is the absence of the α-methyl carbons that are present in the unnatural alkyne amino acid (Scheme 24). The α-methyl carbons are proposed to aid in the success of the Glaser reaction and in the induction of peptide helicity through the Thorpe-Ingold effect. 008528580 Scheme 24: The chemical structure of the α,α-disubstituted unnatural alkyne amino acid 157 synthesised via the nickel Schiff-base method and the chemical structure of the alkylated serine amino acid 168. The α-methyl carbon is absent. Testing of Peptides Synthesis Comparison With the alkene 153 and diyne stapled 160 peptides in hand, experiments were designed and set up to compare the properties of the stapled peptides. Namely the two peptides were assessed in terms of conformational analysis and bioactivity. The alkene stapled peptide 153 has a lower yield due to the production of cis and trans isomers during the RCM reaction (Yields; Diyne: 10%, Alkene cis: 4%, Alkene trans: 2%). The crude purity of the alkene vs diyne stapled peptide was compared and quantified using RP-HPLC upon cleavage of the crude peptides before purification. The results show that the cis isomer in 51% and the trans isomer in 31%. The crude purity of the diyne 160a is significantly improved at 79%. Conformational Analysis The effectiveness of the diyne staple as an α-helical constraint was assessed using CD. Experiments were set out to measure how much of an effect the diyne staple has on inducing α-helicity in the peptide compared to the alkene staple. CD experiments were performed using two concentrations which shows concentration dependence, as coiled-coil structures can increase helicity. Peptide concentrations were quantified using a NanodropOne using UV absorption of peptides at 280 nm. The absorption and the extinction coefficients of Trp and Tyr residues were used in the Beer-Lambert Law equation (Equation D) to calculate the peptide concentration. 008528580 Equation D: The Beer-Lambert Law equation. A = absorption (at 280 nm), ^ = extinction coefficient in M-1cm-1 (of Trp and Tyr residues at 280 nm), c = concentration in M and l = the path length in cm. ^ = ^ c l Each peptide was analysed at 50 µM (Figure 8) and 25 µM (Figure 9) in phosphate buffered saline (PBS, pH 7.4). The peptides were not soluble at 100 µM. CD spectra were measured from 190 - 260 nm in order to observe the characteristic α-helical minima at 208 nm and 222 nm. The CD experiments confirmed that alkene hydrocarbon stapling stabilises the sequence into an α-helix, relative to the linear peptide which is random coil. Interestingly, the diyne stapled peptide 160 showed an enhanced helicity compared to the alkene, this was seen with both the 50 µM and 25 µM concentrations. Percentage Helicities The output of CD is typically converted from machine units (millidegrees) to MRE with units of deg∙cm2∙dmol-1. This unit is useful as it takes into account the concentration and length of the peptide which normalises the output for comparison with other systems (Sreerama et al., 2004). The raw CD data was converted to MRE and the value at 222 nm was used to calculate the % helicities using Equations B and C (see General Method E). The diyne stapled peptide 160a was the most helical with a 59% helicity, in comparison to the cis (153a) and trans (153a’) alkene stapled peptides with 48% helicity (Table 4). As such, the diyne conformational constraint in this example peptide clearly has an enhanced effect on inducing the helical bioactive conformation. Table 4: The stapled peptide % helicities calculated using Equations B and C. Peptide % Helicities Cis Alkene 153a 48 Trans Alkene 153a’ 48 Diyne 160a 59 Determination of the Binding Affinity to MDM2 The diyne stapled peptide 160 was synthesised in improved crude purity compared to the alkene 153 and conformational analysis displayed that it is more effective at inducing α-helicity. However, an improvement in α-helical conformation is not always directly correlated to the binding and bioactivity of the peptide. Biophysical techniques can be used to study the structure, properties, dynamics or function of biomolecules at an atomic or molecular level. Common methods for probing peptide binding include FP, SPR and ITC. 008528580 FP analysis using MDM2 (1-138) Protein Fluorescently labelled peptides were first tested for binding with MDM2 sequence 1-138 according to General Method D. Chang et al. reported the binding of the linear peptide (ATSP-3848) and ATSP-7041 to MDM2 (1-138) with low nM binding affinity, using SPR and FP. A comparable FP assay was set up to test the binding affinity of the diyne stapled peptide 160b in comparison to the alkene stapled 153b/b’ and linear peptide. A 10 μM stock concentration of MDM2 (1-138) was prepared in Tris buffer. A fixed concentration of fluorescently labelled peptides (10 nM) and varying concentrations of protein (10 μM to 9 pM) were serially diluted in the 384-well plate. The samples were left to incubate at room temperature for 3 hours to allow the interaction between the peptides and the protein. Each FP measurement was repeated three times for statistical significance. The results are shown in Table 5. Table 5: FP binding data for the linear and stapled analogues to MDM2 (1-138). Peptide number Kd (nM) Linear 145b (ATSP-3848) 1.49 ^ 0.29 Alkene cis 153b (ATSP-7041) 0.33 ^ 0.19 Alkene trans 153b’ (ATSP-7041) 0.31 ^ 0.06 Diyne 160b 1.39 ^ 0.08 Diyne negative control 164b 3680 ^ 0.15 The FP binding data confirmed the low nM binding of the linear 145b and alkene stapled peptides 153b/b’ as seen by Chang et al. (Table 5). Likewise, the diyne stapled analogue 160b was comparable in binding. The slight difference in values are within the experimental error of the assay, therefore it was concluded that all the peptides showed comparable binding to MDM2 (1-138). As expected, the diyne negative control 164b with the Phe to Ala substitution showed a significant loss in binding compared to the diyne stapled peptide 160b, this was expected due to the substitution of the binding residue. Cellular Uptake Experiments using Fluorescence Microscopy To investigate the cellular uptake of the diyne stapled peptide 160 compared to alkene ATSP-7041153, fluorescence microscopy was used. Importantly, fluorescently labelled ATSP-7041 has been found to be equipotent to the unlabelled analogue in terms of MDM2 and MDMX binding and cellular potency. Indicating that addition of the fluorophore does not significantly alter the biophysical/biological properties of ATSP-7041 (Paronetto et al., 2007). HCT-116 cells were treated with either 20 μM fluorescein diyne stapled peptide 160b, fluorescein ATSP-7041153b/b’ or fluorescein native peptide 145b stained with Hoechst 33342 nuclei stain and imaged 4.5 hours post-treatment. 008528580 Fluorescein ATSP-7041153b/b’ and fluorescein diyne stapled peptide 160b showed a diffused intracellular localization, confirming efficient cellular penetration. The native peptide 145b showed no cellular internalisation. Conclusions An alkyne amino acid 157 was synthesised with the optimal stereochemistry and carbon length to generate a diyne staple with 14 carbons in the bridge. The diyne stapled peptide 160 was compared to the alkene analogue ATSP-7041153 using different experiments; conformational analysis using CD confirmed that the diyne showed a 10% improvement in helicity. Synthetically, the diyne peptide 160 was isolated in improved yield, due to the elimination of the isomers generated from the alkene staple. FP was used to measure the binding affinities to MDM2 and MDMX. It was observed that the alkene 153, linear 145 and diyne stapled peptide 160 had comparable binding affinities to MDM2 (low nM), thus demonstrating the benefits of the diyne staple. Fluorescence microscopy was used to confirm the cell permeability of the diyne stapled peptide 160 compared to the alkene analogue 153. The diyne peptide was confirmed as cell permeable. References A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein. Artzt et al., Adv. Exp. Med. Biol., 2010, 693, 1–24. Aillard et al., Org. Biomol. Chem., 2014, 12, 8775-8782. Akhtar et al., Synth. Commun., 2020, 50, 3337-3368. Busà et al., Oncogene, 2007, 26, 4372–4382. Chang et al., Proc. Natl. Acad. Sci. U. S. A., 2013, 110, E3445-3454. Cistrone et al., Chembiochem, 2018, 19, 1031-1035. Feracci et al., Nat. Commun., 2016, 7, 1–12. Foot et al., Methods, 2014, 65, 288–301. Frisone et al., Biomed Res. Int., 2015, 2015, 1–14. Garcia-Martin et al., J. Comb. Chem., 2006, 8, 213-220. Hartmann et al., Mol. Biol. Cell, 1999, 10, 3909–3926. Kim et al., Nat. Protoc., 2011, 6, 761-771. 008528580 Lopes et al., Protein Sci., 2014, 23, 1765–1772. Mahindra et al., Org. Lett., 2019, 21, 3178-3182. Paronetto et al., J. Cell Biol., 2007, 176, 929–939. Silvestri et al., Angew. Chemie - Int. Ed., 2017, 56, 10438–10442. Sreerama et al., Methods Enzymol., 2004, 383, 318-351. Verlinden et al., Org. Biomol. Chem., 2015, 13, 9398-9404. Verlinden et al., J. Pept. Sci., 2019, 25, 1–9. Walensky et al., Science, 2004, 305, 1466-1470. Yuen et al., Chem. Sci., 2019, 10, 6457-6466. WO 2017/040990 008528580

Claims

Claims: 1. A polypeptide comprising a group of Formula (I) wherein -R1 and -R2 are each independently selected from optionally substituted C1-4 alkyl and -H; -R3 and -R4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue; -L1- and -L2- are each independently an optionally substituted C2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof. 2. The polypeptide of claim 1, wherein -L1- and -L2- are each independently C5-10 alkylene. 3. The polypeptide of claim 1, wherein -L1- and -L2- are both C5 alkylene or both C6 alkylene. 4. The polypeptide of claim 1, wherein the total number of carbon atoms in -L1-(C≡C-C≡C)-L2- is in the range from 14 to 16. 5. The polypeptide of any one of claims 1 to 4, wherein each amino acid residue -A- is independently selected from a natural amino acid or a non-natural amino acid, such as an α-amino acid, including a proteinogenic amino acid, such as wherein the amino acid residue is selected from: Alanine [Ala, A], Aminobenzoic acid [PABA], Aminobutyric acid [Abu], Aminohexanoic acid [Ahx], Aminoisobutyric acid [Aib], Arginine [Arg, R], Asparagine [Asn, N], Aspartic acid [Asp, D], Butylglycine, Citrulline [Cit], Cyclohexylalanine [Cha], Cysteine [Cys], Diaminobutanoic acid [Dab], Diaminopropionic acid [Dpr or Dap], Dihydroxyphenylalanine [DOPA], Glutamic acid [Glu, E], Glutamine [Gln, Q], Glycine [Gly, G], Histidine [His, H], Homoserine [Hse], Hydroxyproline [Hyp], Isoleucine [Ile, I], Isonipecotic acid [Isn], Leucine [Leu, L], Lysine [Lys, K], Methionine [Met, M], Norleucine [Nle], Norvaline [Nva], Ornithine [Orn], Phenylalanine [Phe, F], 008528580 Phenylglycine [Phg], Proline [Pro, P], Sarcosine [Sar], Serine [Ser, S], Statine and derivatives thereof [Sta], Tetrahydroisoquinoline-3-carboxylic acids [Tic], Thienylalanine [Thi], Threonine [Thr, T], Tryptophan [Trp, W], Tyrosine [Tyr, Y], and Valine [Val, V]. 6. The polypeptide of any one of claims 1 to 5, wherein n is an integer from 2 to 8, such as from 3 to 8, such as 6. 7. The polypeptide of any one of claims 1 to 6, wherein (i) -R1 and -R2 are each independently selected from methyl and -H; and/or (ii) -R3 and -R4 are each independently -H. 8. The polypeptide of any one of claims 1 to 7, comprising a group of Formula (IRS), (ISR) or (ISS): wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as defined in any one of claims 1 to 7. 008528580 9. The polypeptide of any one of claims 1 to 8, comprising a group of Formula (ISS): 10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide is of Formula (Ia) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described in any one of claims 1 to 7, and: each -A’- is independently an amino acid residue or an N-methylated amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200, such as from 2 to 50. 11. The polypeptide of any one of claims 1 to 9, wherein the polypeptide is of Formula (Ib) 008528580 wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described in claim any one of claims 1 to 7, each -A’- is independently an amino acid residue or an N-methylated amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200, such as from 2 to 50; and z is an integer from 1 to 4. 12. The polypeptide of claim 11, wherein z is 1 or 2. 13. A polypeptide comprising a group of Formula (II) wherein -R1 and -R2 are each independently selected from optionally substituted C1-4 alkyl and -H; -R3 and -R4 are each independently selected from -H and methyl; each -A- is independently an amino acid residue; -L1- and -L2- are each independently an optionally substituted C2-10 alkylene; and n is an integer from 2 to 10, and the salts, solvates and protected forms thereof. 008528580 14. The polypeptide of claim 13, wherein: (i) -L1- and -L2- are each independently C5-10 alkylene, such as wherein -L1- and -L2- are both C5 alkylene or both C6 alkylene; and/or (ii) n is an integer from 2 to 8, such as from 3 to 8, such as 6; (iii) -R1 and -R2 are each independently selected from methyl and -H; and/or (iv) -R3 and -R4 are each independently -H. 15. The polypeptide of claim 13 or claim 14, comprising a group selected from Formula (IIRS), (IISR) and (IISS): wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as defined in claim 13 or claim 14. 008528580 16. The polypeptide of any one of claims 13 to 15, comprising a group of Formula (IISS): 17. The polypeptide of any one of claims 13 to 16 having the Formula (IIa) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2- and n are as described in claim 13 or claim 14, and wherein each -A’- is independently an amino acid residue or an N-methylated amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200, such as from 2 to 50. 008528580 18. The polypeptide of any one of claims 13 to 16 having the Formula (IIb) wherein -R1, -R2, -R3, -R4, -A-, -L1-, -L2-, and n are as described in claim 13 or 14, each -A’- is independently an amino acid residue or an N-methylated amino acid residue; -RA is selected from -H, a protecting group, acyl, C1-4 alkyl, a protected linker, and a tag optionally with a linker; -RB is selected from hydroxyl, alkoxy, benzyloxy, amino, protected amino, a protected linker, a solid phase optionally with a linker, and a tag optionally with a linker; and m and m’ are each independently an integer from 0 to 200, such as from 2 to 50; and z is an integer from 1 to 4. 19. The polypeptide of claim 18, wherein z is 1 or 2. 20. A method of preparing a diyne stapled peptide, comprising the steps of: (i) providing a polypeptide according to any one of claims 13 to 19; and (ii) coupling the terminal alkynyl groups of the polypeptide to provide a 1,3-diyne linkage. 21. The method of claim 20, wherein step (ii) is performed in the presence of a metal salt, such as a transition metal salt. 22. The method of claim 21, wherein step (ii) is performed in the presence of a ligand selected from 4,4’-bis(hydroxymethyl)-2,2’-bipyridine, 2,2’-bipyridine and tetramethylethylenediamine. 23. A pharmaceutical composition comprising a polypeptide of one of claims 1 to 12 and a pharmaceutically acceptable carrier. 24. A polypeptide of any one of claims 1 to 12, or a pharmaceutical composition of claim 23, for use in a method of treatment or prophylaxis. 008528580 25. A polypeptide according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 23, for use in a method of treating cancer. 26. A polypeptide according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 23, for use in a method of treating a disease with altered Sam68 activity or a disease with altered p53 and/or MDM2 activity. 27. A compound of Formula (III): wherein -LX- is optionally substituted C5-10 alkylene; -R1 is selected from optionally substituted C1-4 alkyl and -H; -R3 is selected from -H and methyl; -RN is selected from -H, a protecting group, C1-4 alkyl, acyl and a tag optionally with a linker, such as -H, Fmoc, Boc, methyl, ethyl, iso-propyl, acetyl, farnesyl and geranyl; and -RC is selected from -OH and a protecting group, or -RC together with the carbonyl group to which it is attached forms an activated acid, and the salts and solvates thereof. 28. The compound of claim 27, wherein (i) -LX- is optionally substituted C6-10 alkylene; (ii) -R1 is selected from methyl and -H, preferably wherein -R1 is methyl; and/or (iii) -R3 is -H. 008528580
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