WO2019033171A1 - Substrats de culture cellulaire - Google Patents

Substrats de culture cellulaire Download PDF

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Publication number
WO2019033171A1
WO2019033171A1 PCT/AU2018/050874 AU2018050874W WO2019033171A1 WO 2019033171 A1 WO2019033171 A1 WO 2019033171A1 AU 2018050874 W AU2018050874 W AU 2018050874W WO 2019033171 A1 WO2019033171 A1 WO 2019033171A1
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Prior art keywords
peptide
amino acid
cell culture
side chain
acid residues
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PCT/AU2018/050874
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English (en)
Inventor
Adam Martin
Lars Matthias ITTNER
Yazi Diana Ke
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Macquarie University
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Priority claimed from AU2017903293A external-priority patent/AU2017903293A0/en
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Publication of WO2019033171A1 publication Critical patent/WO2019033171A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • 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

Definitions

  • the present invention relates to a class of peptides and their use in cell culture applications.
  • Cell culture is an important scientific technique primarily used to maintain or grow cells in an artificial environment.
  • Cell culture substrates help provide the conditions necessary for the cells to adhere to surfaces, and survive and/or proliferate in such artificial environments.
  • cell culture substrates include both synthetic substrates (e.g. polylysine) and naturally derived substrates (e.g. Matrigel ® ) .
  • Polylysine is commonly used in cell culture as it enhances cell adherence to cultureware.
  • poly-D-lysine is commonly used as a coating agent in the production of poly-D-lysine coated petri dishes for growing cells. Owing to manufacturing constraints, the length of the polymeric chains in polylysine can vary from batch-to-batch, which can lead to inconsistencies in culture conditions and thus inconsistencies in experimental results.
  • Matrigel ® is a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma.
  • the chief components of Matrigel ® are structural proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans. Also present are growth factors like TGF-beta and EGF that prevent differentiation and promote proliferation of many cell types.
  • Matrigel ® contains other proteins in small amounts and, as a naturally derived product, its exact composition can vary from batch to batch. For this reason, Matrigel ® may not be appropriate for experiments that require precise knowledge of all proteins and their concentrations in the cell culture substrate.
  • Some cell types are more difficult to culture. For example, neurons require specific conditions to grow, limiting the cell culture substrates suitable for culturing such neuronal cells.
  • At least preferred embodiments of the present invention were able to provide an alternative cell culture substrate suitable for culturing neuronal cells including neurons. It would also be advantageous if at least preferred embodiments of the present invention were to provide compounds that can be used for preparing cell culture substrates under conditions that are suitable for cell adhesion, survival and/or proliferation.
  • the present invention provides a peptide comprising from 2 to 12 amino acid residues, wherein:
  • the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring;
  • At least two amino acid residues have a cation-containing side chain, or at least one amino acid residue has two or more cation-containing side chains, or at least one amino acid residue has one or more side chains comprising two or more cations;
  • the net charge of the peptide is positive at a pH of from about 4 to about 10.
  • the peptide comprises at least two amino acid residues having a cation-containing side chain.
  • At least one of the amino acid residues having a cation-containing side chain is selected from lysine, arginine and histidine residues.
  • At least one of the amino acid residues having a cation-containing side chain is a lysine residue.
  • the at least two amino acid residues having a cation-containing side chain are each selected independently from lysine, arginine and histidine residues.
  • the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan residues.
  • the at least one amino acid residue having a hydrophobic side chain is a phenylalanine residue.
  • the optionally substituted fused polycyclic group comprising an aromatic ring is linked to the N-terminus of the peptide by a linker group that is from 1 to 3 atoms in length.
  • the peptide comprises from 4 to 8 amino acid residues.
  • the 4 to 8 amino acid residues are selected from valine, isoleucine, leucine, methionine,
  • the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine,
  • the present invention provides a compound of Formula (I)
  • Ar is an optionally substituted fused polycyclic group comprising an aromatic ring
  • L is a bond or a linker that is 1, 2 or 3 atoms in length; n is an integer from 2 to 12; each A is independently selected from the amino acid residues (A' ) to ( G' )
  • each R' group in the compound is an independently selected radical, wherein at least one R' group is a hydrophobic moiety, and wherein at least two R' groups are moieties containing a cation or at least one R' group is a moiety containing 2 or more cations; and the net charge of the compound is positive at a pH of from about 4 to about 10.
  • the compound of Formula (I) has the Formula (la)
  • Ar is 9-fluorenyl.
  • Ar-L- has the chemical structure:
  • n 4.
  • the at least one R' group that is a hydrophobic moiety is -CH 2 Ph.
  • At least one of the R' groups that is a moiety containing a cation is a moiety containing a primary, secondary or tertiary amine.
  • the R' group is - CH 2 CH 2 CH 2 CH 2 NH 3 + .
  • the compound is selected from Fmoc-FkFk and Fmoc- FFkk and stereoisomers thereof.
  • the present invention provides a hydrogel comprising a peptide according to the first aspect of the present invention or a compound according to the second aspect of the present invention (a peptide according to the first aspect of the present invention or a compound according to the second aspect of the present invention is sometimes referred to below as a "compound of the present invention") .
  • the hydrogel comprises a compound of the present invention and an aqueous solution having a pH of from about 4 to about 10.
  • the aqueous solution has a pH of from about 6 to about 8.
  • the present invention provides a cell culture substrate comprising a compound of the present invention.
  • the cell culture substrate comprises a hydrogel according to the third aspect of the present invention.
  • the present invention provides a 3-dimensional cell culture substrate comprising a cell culture substrate according to the fourth aspect of the present invention, wherein all three dimensions of the cell culture substrate are greater than about 50 (am.
  • the present invention provides the use of a compound of the present invention, a hydrogel according to the third aspect of the present invention, or a cell culture substrate according to the fourth or fifth aspect of the present invention, for culturing a cell .
  • the present invention provides a method of making a cell culture substrate comprising mixing a compound of the present invention and an aqueous solution.
  • the aqueous solution has a pH of from about 6 to about 8. In an embodiment, the aqueous solution has a pH of from about 7.2 to about 7.5.
  • the method further comprises mixing the compound of the present invention and/or the aqueous solution with one or more cells .
  • the present invention provides a method of culturing a cell, comprising forming a cell culture substrate by the method of the seventh aspect of the present invention, and
  • the present invention provides a method of recovering one or more cells from a cell culture substrate, the method comprising contacting the cell culture substrate with a digestion enzyme to cause degelation, wherein the cell culture substrate comprises compound of the present invention, and wherein the compound comprises one or more natural amino acid residues.
  • Figure 1 shows the chemical structure of the two tetrapeptides described in Example 1: Fmoc-FFkk (1) and Fmoc-FkFk (2) .
  • Figure 2(a) and 2(b) are graphs of modulus (Pa) vs time (s) of time resolved rheology measurements, showing gelation of Fmoc-FFkk (a) and Fmoc-FkFk (b) over a time period of 5000 s, as described in Example 1.
  • Figure 2(c) and 2(d) are graphs of modulus (Pa) vs time (s) of thixotropy measurements of Fmoc-FFkk (a) and Fmoc-FkFk (b), showing the recovery of the network after application of a large external strain, as described in Example 1.
  • Figure 3(a) and 3(b) are graphs of modulus (Pa) versus frequency (Hz) for hydrogels of Fmoc-FFkk (a) and Fmoc-FkFk (b), showing frequency independent behaviour, typical of a hydrogel material.
  • Figure 3(c) and 3(d) are graphs of modulus (Pa) vs strain (%) for hydrogels of Fmoc-FFkk (a) and Fmoc-FkFk (b), showing deformation o the hydrogel network upon application of a large external strain.
  • Figure 4(a) and 4(b) are atomic force microscopy (AFM) images of a Fmoc-FFkk gel (a) and Fmoc-FkFk gel (b) (hydrogels were spread coated onto freshly cleaved mica substrates at 0.5% (w/v), as described in Example 1) .
  • Scale bars (a) 500 nm and (b) 1 ⁇ .
  • Figure 5(a) is a circular dichroism spectrum (CD spectrum; graph of ellipticity (mdeg) vs wavelength (nm)) of an Fmoc-FFkk gel (squares) and Fmoc-FkFk gel (triangles), as described in Example 1, indicative of their different secondary structures.
  • CD spectrum graph of ellipticity (mdeg) vs wavelength (nm)
  • Figure 5(b) is an ATR-IR spectra (graph of transmission (%) vs wavenumber (crrr 1 )) of an Fmoc-FFkk gel (grey; upper spectra) and Fmoc-FkFk gel (black; lower spectra), as described in Example 1, indicative of their different secondary structures.
  • Figure 7 is a series of images displaying primary neurons observed on coverslips coated with poly-D-lysine (PDL; top), Fmoc-FFkk gel (middle) and Fmoc-FkFk gel (bottom) at times DIV1 to DIV5.
  • DIV1 shows lamellipodia extending from cell body
  • DIV2 shows extension of lamellipodia
  • DIV3 shows generation of axons
  • DIV4 shows generation of dendrites
  • DIV5 shows the development of the network
  • Figure 8(d) is a graph showing the cell viability (%; y-axis) over time (10, 20, 30 and 40 days in vitro; x-axis) of primary neurons seeded on the peptide nanofibers Fmoc-FFkk (black) and Fmoc-FkFk (grey) .
  • Cell viability determined using an Alamar Blue colorimetric assay with PDL used as the positive control (100% viability), as described in Example 1.
  • Figure 9 is a series of images taken after 10 days in vitro (DIV10; top) and 14 days in vitro (DIV14; bottom) showing the transfection of primary neurons (indicated by arrows in images) cultured on coverslips coated with PDL (left), Fmoc-FFkk (centre) and Fmoc-FkFk (right), as described in Example 1. Neurons were transfected at DIV7, and stained after fixing with MAP2 and DAPI .
  • Figure 10 (c) is a graph showing the quantification of synaptic density (synapses/pm; y-axis) over time (20, 25 and 30 days in vitro; x-axis) for neurons cultured on PDL (grey), Fmoc-FFkk (black) or Fmoc-FkFk (white), as described in Example 1.
  • Figure 12 is a schematic diagram depicting the preparation of poly- D-lysine coverslips (top), a "2.5D" culture system (middle) and a 3D culture system for neuronal cultures (bottom) as described in
  • Figure 13 shows a photographic image of a 3D culture system before (left) and after (right) treatment with trypsin as described in
  • Example 3 for D-lysine containing peptide (top, no degradation) and L-lysine containing peptide (bottom, complete degradation) Images show treatment with trypsin causing the degradation of the L-lysine containing peptide, causing the hydrogel to liquefy and sink to the bottom of the vial.
  • Figure 14(a) is a series of still images taken from videos which show the electrical activity of primary neurons cultured on PDL (top), Fmoc-FFkk (middle) and Fmoc-FkFk (bottom), demonstrating synchronized firing at DIV15 (0 sec (left), 2 sec (centre) and 4 sec (right)) .
  • Primary neurons were cultured on PDL, Fmoc-FFkk or Fmoc- FkFk and transfected with GCaMP6s at DIV11 before imaging was undertaken four days later, as described in Example 1, section 1.4.
  • Scale bar 100 ⁇ .
  • Figure 15(a) and 15(b) are graphs of viscosity (cP) vs shear rate
  • FIG. 16(a) and 16(b) are graphs of I(q) (a.u.) vs q(A _1 ) showing the Small angle neutron scattering (SANS) patterns and associated fits obtained from 1% (w/v) solutions of Fmoc-FFkk (a) and Fmoc-FkFk (b) dissolved in D 2 0 as described in Example 1.
  • SANS Small angle neutron scattering
  • the present invention provides a peptide comprising from 2 to 12 amino acid residues, wherein:
  • the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring;
  • At least two amino acid residues have a cation-containing side chain, or at least one amino acid residue has two or more cation-containing side chains, or at least one amino acid residue has one or more side chains comprising two or more cations;
  • the net charge of the peptide is positive at a pH of from about 4 to about 10.
  • Peptides in accordance with the first aspect may be used to make hydrogels when combined with aqueous solutions at biologically relevant pHs.
  • these hydrogels are compatible with a wide variety of biological systems and are suitable for use as cell culture substrates.
  • at least preferred peptides of the first aspect are capable of forming a hydrogel that is capable of supporting the growth of primary hippocampal neurons, which are difficult to culture on many other substrates.
  • hydrogels formed using at least preferred peptides of the invention allow the long-term culturing of neurons in culture dishes.
  • these hydrogels are water soluble and do not require strongly basic or strongly acidic conditions and/or the use of additional agents such as traditional pH adjusters or buffers for solubilization.
  • pH adjusters and buffers have traditionally been problematic with prior art cell culture substrates, since these are generally toxic to cultured cells and lengthy washing procedures are required to remove them from the substrate prior to the addition of cells.
  • removal of pH adjusters and buffers from prior art media also lead to extra time and labour in preparing cell culture substrates. In the present invention, these additional steps (costing both time and labour) may be avoided or reduced by using the peptides of the present invention.
  • Some existing methods for culturing cells involve using poly-D- lysine coated plastic cultureware. It is technically challenging to control the length of D-lysine polymers which causes batch-to-batch variation. The variation of polymer length leads to differences in product stability, and can create inconsistencies in culture conditions and thus differences in experimental results. In addition, neuronal cell death is common when removing neuronal cells from the poly-D-lysine coated plastic cultureware using chemicals or enzymes .
  • the synthesis of the peptides of the invention can be controlled, which can give rise to more consistent properties compared to the commercial manufacture of poly-D-lysine or Matrigel ® .
  • the ability of the peptides of the invention to form hydrogels when added to aqueous solutions at biologically relevant pHs allows cells to be introduced before or during gel formation, thereby allowing the gel to form while the cells are dispersed, giving the ability to form three dimensional gels with cells dispersed therein. This is particularly advantageous for sensitive cells (such as neuronal cells including neurons), which are incompatible with the pHs required for gel formation using conventional cell culture substrates . Number of amino acid residues
  • the peptide of the present invention comprises from 2 to 12 amino acid residues (i.e. the peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues) . In some embodiments, the peptide comprises from 2 to 8 amino acid residues. In some embodiments, the peptide comprises from 4 to 8 amino acid residues. In some
  • the peptide comprises from 4 to 6 amino acid residues. In some embodiments, the peptide comprises 4 amino acid residues (i.e. is a tetrapeptide ) .
  • the peptide of the present invention comprises at least one amino acid residue that has a hydrophobic side chain.
  • a hydrophobic side chain is a side chain that repels water.
  • a hydrophobic side chain comprises one or more hydrophobic moieties (i.e. a moiety that repels water) . Hydrophobic moieties tend to be nonpolar (e.g.
  • hydrophobic moieties include straight or branched Ci-io-alkyl, C 3 - 6 -cycloalkyl , aryl and heteroaryl groups, wherein the Ci-io-alkyl, C 3 - 6 -cycloalkyl , aryl and heteroaryl groups may be optionally substituted with one, two or three Ci-6alkyl, C3-6- cycloalkyl, and/or halo (e.g. F, CI or Br) groups.
  • halo e.g. F, CI or Br
  • hydrophobic moieties include -C 6 H 5 , -C 6 H 4 Me, -C 6 H 3 Me 2 , -C 6 H 2 Me 3 , - CeH4Et, —CeH 3 Et2, — C63 ⁇ 4Et 3 , —C6H4F, C63 ⁇ 4F2, — CeH2F 3 , —C6H4CI, —CeH 3 Cl2, —
  • the hydrophobic side chain comprises one or more of these hydrophobic moieties (for example a straight or branched Ci-10-alkyl covalently bonded to an aryl or heteroaryl group) .
  • the hydrophobic side chain comprises an aryl or heteroaryl group.
  • the hydrophobic side chain is or comprises a straight or branched Ci-i 0 alkyl group substituted with an optionally substituted aryl or optionally substituted heteroaryl.
  • the hydrophobic side chain is or comprises Ci-6alkylphenyl,
  • the hydrophobicity of a compound there are different methods to determine the hydrophobicity of a compound.
  • An example of one such method is the partition coefficient as described in Hansch et al. [1975, Journal of Medicinal Chemistry, 865-868], where the distribution of a compound between two immiscible phases, with one phase being polar and the other phase non-polar, is used to approximate the compound's hydrophobicity.
  • the amino acid residue that has a hydrophobic side chain corresponds to an amino acid that has a LogP of at least 2.7 in its Fmoc-protected form, as measured by the method of Hansch et al . [1975, Journal of Medicinal Chemistry, 865-868] .
  • the amino acid residue has a side chain that has a calculated LogP of greater than 1.2 (for example, calculated using ChemDraw) .
  • the hydrophobic side chain is selected from optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-ioalkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted
  • heteroaryl optionally substituted straight or branched Ci-i 0 alkoxy, optionally substituted straight or branched C 2 -iothioether (e.g.
  • optionally substituted -CH 2 CH 2 SCH 3 optionally substituted straight or branched C 2 -ioalkenyl , optionally substituted straight or branched C2-ioalkynyl, optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C 3 - 6 cycloalkyl and optionally substituted C 3 - 6 cycloalkenyl , wherein the optional substituents are selected from halo (e.g. F, CI or Br), Ci-6alkyl, haloCi- 6 alkyl , C 3 - 6 cycloalkyl , optionally substituted aryl (e.g.
  • phenyl or -C 6 H 4 OH and optionally substituted heteroaryl (e.g.
  • the hydrophobic side chain has a molecular weight of less than about 500 g/mol (e.g. less than about 400 g/mol or less than about 300 g/mol) .
  • Amino acid residues that have a hydrophobic side chain may be "natural” amino acid residues or "non-natural” amino acid residues that have hydrophobic side chains.
  • “natural” amino acids that have a hydrophobic side chain include L-valine, L-isoleucine , L-leucine, L-methionine, L-phenylalanine , L-tyrosine and L- tryptophan.
  • L-Phenylalanine is preferred.
  • Examples of "non-natural” amino acids that have a hydrophobic side chain include D-valine, D- isoleucine, D-leucine, D-methionine, D-phenylalanine , D-tyrosine and D-tryptophan .
  • D-Phenylalanine is preferred.
  • Other examples of "non- natural" amino acids that have a hydrophobic side chain include L- norleucine, D-norleucine, L-norvaline and D-
  • amino acids having a hydrophobic side chain examples include amino acids (A-l) to (G-l)
  • each R 1 is hydrophobic and independently selected from optionally substituted straight or branched Ci-i 0 alkyl (e.g. straight or branched Ci-i 0 alkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted
  • heteroaryl optionally substituted straight or branched Ci-i 0 alkoxy, optionally substituted straight or branched C2-iothioether,
  • the at least one amino acid residue that has a hydrophobic side chain is an amino acid residue of any of the amino acids (A-l) to (G-l) described above .
  • Amino acid residues having a cationic side chain is an amino acid residue of any of the amino acids (A-l) to (G-l) described above .
  • the peptide of the present invention comprises at least two amino acid residues that have a cation-containing side chain, or at least one amino acid residue having two or more cation-containing side chains, or at least one amino acid residue having one or more side chains comprising two or more cations.
  • the amino acid residues that have a cation-containing side chain may be the same or different amino acid residues.
  • the amino acid residues may be the same or different amino acid residues .
  • a "cation” is a species that is cationic (i.e.
  • the cation exists as a cationic species when the peptide is in an aqueous solution having a pH of less than about 10 (e.g. less than about 9, less than about 8) .
  • the formation of a cation is typically an equilibrium process, with the ratio of cationic to neutral species being dependant on pH.
  • "cation" may refer to a species that is either wholly or partially cationic. For example, histidine in an aqueous solution near pH 7 exists as approximately 25% protonated (i.e. cationic) and 75% neutral. This would be considered "cationic" in the context of this specification.
  • the cation-containing side chain preferably contains a cation at biologically relevant pHs, e.g. a pH of 7.1 to 7.6 or 7.2 to 7.5, ideally 7.3 to 7.4.
  • a cation at biologically relevant pHs e.g. a pH of 7.1 to 7.6 or 7.2 to 7.5, ideally 7.3 to 7.4.
  • the cation-containing side chain may, in some
  • the cation-containing side chain is a side chain selected from -(CH 2 ) 6 NH 2 , -(CH 2 ) 5 NH 2 , -(CH 2 ) 4 NH 2 , -(CH 2 ) 3 NH 2 , -(CH 2 ) 2 NH 2 and -CH 2 NH 2 , especially -(CH 2 ) 3 NH 2 , -(CH 2 ) 4 NH 2 and -(CH 2 ) 5 NH 2 , more especially -(CH 2 ) 4 NH 2 .
  • Quaternary ammonium groups (-N + R 4 , where each R is independently alkyl or aryl) are cationic independent of pH. Accordingly, a moiety comprising a quaternary ammonium group is a moiety comprising a cation.
  • the cation-containing side chain is a straight or branched Ci-i 0 alkyl substituted with a quaternary ammonium group (e.g. Ci-ioalkylN + (Ci-6alkyl) (Ci-6alkyl) (Ci-6alkyl) ) .
  • Amino acid residues that have a cation-containing side chain may be "natural” amino acid residues or "non-natural” amino acid residues that have cation-containing side chains.
  • Examples of "natural" amino acids that have a cation-containing side chain (at biologically relevant pHs) include L-lysine, L-arginine and L-histidine.
  • L-lysine and L-arginine are preferred.
  • L-Lysine is particularly preferred .
  • the inventors believe that one of the cationic groups acts to negate or cancel out the effect of the free carboxylic acid of the peptide chain.
  • the second and subsequent cationic groups are beneficial for promoting the self-assembly of hydrogels and/or creating an environment in cell culture substrates that is beneficial for the adhesion, survival and/or proliferation of cells.
  • the cation-containing side chain is selected from optionally substituted straight or branched Ci-i 0 aminoalkyl (e.g.
  • the cation-containing side chain is straight or branched Ci-ioalkyl substituted with one or more groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl), -N ( optionally
  • the Ci-i 0 alkyl group is substituted with 1, 2, 3, 4, 5 or 6 groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl), -N ( optionally substituted Ci- 6 alkyl) (optionally substituted Ci-6alkyl) .
  • the Ci-ioalkyl substituted with 1, 2, 3, 4, 5 or 6 groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl), - N (optionally substituted Ci- 6 alkyl) (optionally substituted Ci- 6 alkyl), may optionally be further substituted with, for example, 1, 2, 3, 4, 5 or 6 substituents .
  • the optional further substituents may, for example, be independently selected from halo, haloCi- 6 alkyl and Ci-
  • a quaternary ammonium group e.g. -N + (alkyl) 4
  • imidazole group e.g. -C 3 H 3 N 2
  • the cation-containing side chain has a molecular weight of less than about 500 g/mol (e.g. less than about 400 g/mol or les than about 300 g/mol) .
  • amino acids having a cationic side chain examples include amino acids (A-2) to (G-2)
  • the R 2 groups may be the same or different.
  • the amino acid residue that has a cation-containing side chain is an amino acid residue of any of the amino acids (A-2) to (G-2) described above.
  • R 2 is selected from -(CH 2 ) 6 NH 2 , -
  • the peptide of the present invention may comprise amino acids in addition to the at least one amino acid residue that has a
  • hydrophobic side chain and the amino acid residue or residues that have a cation-containing side chain.
  • Additional amino acids that may be included in the peptide include “natural” and “non-natural” amino acids.
  • Examples of other amino acids that may be included in the peptide include L-glutamine, L-asparagine, L-serine, L-threonine, L-cysteine, L-alanine, L-valine, L-proline, L-aspartic acid, L-glutamic acid, glycine, beta-alanine, D-glutamine, D-asparagine, D-serine, D- threonine, D-cysteine, D-alanine, D-valine, D-proline, D-aspartic acid and D-glutamic acid.
  • less than 70% of the amino acid residues are other than amino acid residues having a hydrophobic side chain and amino acid residues having a cation-containing side chain (e.g. less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10%) .
  • there are no amino acid residues other than amino acid residues having a hydrophobic side chain and amino acid residues having a cation-containing side chain i.e. the peptide contains only amino acid residues having a hydrophobic side chain and amino acid residues having a cation- containing side chain
  • the ratio of charged amino acid residues to hydrophobic amino acid residues is from 1:3 ( charged : hydrophobic ) 5:1, for example, from about 1:2 to about 5:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1 or about 1:1.
  • N-terminus of the peptide is linked to an optionally
  • the fused polycyclic group comprises 2 or more fused rings, wherein at least one of the rings is aromatic.
  • the fused polycyclic group may contain 2, 3 or 4 rings that are fused together, at least one of the rings being aromatic.
  • the polycyclic group comprises 2, 3 or 4 fused rings, wherein 2 of the rings are aromatic.
  • the polycyclic group comprises 3 or 4 fused rings, wherein 3 of the rings are aromatic.
  • the polycyclic group comprises 4 fused rings, wherein 4 of the rings are aromatic.
  • One or more of the rings may contain a heteroatom (e.g. N, S, 0) and may therefore be heterocyclic or heteroaromatic .
  • each of the rings comprises from 5 to 10 ring atoms (e.g. is a 5-membered ring or a 6-membered ring) .
  • the fused polycyclic group comprising an aromatic ring is substituted.
  • the substituents are typically selected from Ci- 3 alkyl, Ci- 3 haloalkyl , halo (e.g. F, CI, Br), Ci- 3 alkoxy, C 2 -3alkenyl, C 2 -3alkynyl, N0 2 , NMe 2 or SMe .
  • the optionally substituted, fused polycyclic group comprising an aromatic ring include radicals of fluorene (e.g. 9- fluorenyl), phenothiazine (e.g. 10-phenolthiazinyl ) , indole (e.g.
  • the optionally substituted, fused polycyclic group comprising an aromatic ring may be directly bound to the N-terminus of the peptide or may be bound by a "linker".
  • the optionally substituted, fused polycyclic group comprising an aromatic ring is linked to the N-terminus of the peptide by a linker that is from 1 to 3 atoms in length.
  • the linker is typically alkyl, but may comprise heteroatoms such as 0, N, S.
  • the peptides of the first aspect of the present invention have a positive net charge at a pH of from about 4 to about 10.
  • the net positive charge may be determined by a number of different ways.
  • the net charge at a given pH can be calculated by a person skilled in the art by taking into account the theoretical or measured pKas of the constituent amino acids.
  • the net charge can be predicted using computer software.
  • the net charge at a given pH may be derived from the isoelectric point of the peptide. A person skilled in the art will be able to readily assess or predict the net charge of a peptide.
  • the peptide comprises from 4 to 8 amino acid residues selected from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, glutamine, asparagine, serine, threonine, cysteine, alanine, proline, glycine, and beta-alanine residues, wherein
  • the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine,
  • the at least two amino acid residues having a cation- containing side chain are independently selected from lysine, arginine and histidine residues.
  • the present invention provides a peptide comprising from 4 to 8 amino acid residues selected from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine,
  • peptide comprises at least one amino acid residue selected from valine, isoleucine, leucine,
  • amino acid residues referred to above may be, each independently, D- or L-amino acids.
  • the peptides of the present invention may be prepared by methods that are known to those skilled in the art. Examples include traditional solution phase synthesis, solid phase synthesis, native chemical ligation (an option often used in constructing larger peptides (e.g. more than 10 amino acid residues)) and automated synthesis .
  • the present invention provides a compound of Formula (I) -OH
  • Ar is an optionally substituted fused polycyclic group comprising an aromatic ring
  • L is a bond or a linker that is 1, 2 or 3 atoms in length; n is an integer from 2 to 12;
  • each A is independently selected from the amino acid residues (A' ) to ( G' )
  • each R' group in the compound is an independently selected radical, wherein at least one R' group is a hydrophobic moiety, and wherein at least two R' groups are moieties containing a cation or at least one R' group is a moiety containing 2 or more cations;
  • the net charge of the compound is positive at a pH of from about 4 to about 10.
  • the compound of Formula (I) has the Formula (la)
  • Ar comprises 2, 3 or 4 fused rings, wherein at least one of the rings is aromatic.
  • Ar is selected from 9-fluorenyl
  • Ar is 9- fluorenyl .
  • L is a linker that is three atoms in length.
  • Ar-L- has the chemical structure:
  • n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, n is from 4 to 8. In some embodiments, n is from 4 to 6. In some embodiments, n is 4.
  • each A in Formula (I) is an amino acid residue selected from the 20 "natural" amino acids or a stereoisomer thereof .
  • R' is a radical having a molecular weight of less than about 500 g/mol (e.g. less than about 400 g/mol or less than about 300 g/mol) .
  • each R' is selected from -H, optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-ioalkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted
  • heteroaryl optionally substituted straight or branched Ci-i 0 alkoxy, optionally substituted straight or branched C 2 -iothioether (e.g.
  • optionally substituted -CH2CH2SCH3 optionally substituted straight or branched C 2 -ioalkenyl , optionally substituted straight or branched C 2 -ioalkynyl, optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C 3 - 6 cycloalkyl , optionally substituted C 3 - 6 cycloalkenyl , optionally substituted straight or branched Ci-i 0 aminoalkyl , optionally substituted imines (e.g.,
  • the at least one R' group that is a hydrophobic moiety is selected from optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-i 0 alkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted heteroaryl), optionally substituted straight or branched Ci-ioalkoxy, optionally substituted straight or branched C 2 -i 0 thioether (e.g.
  • optionally substituted -CH 2 CH 2 SCH3 optionally substituted straight or branched C 2 -i 0 alkenyl , optionally substituted straight or branched C 2 -i 0 alkynyl , optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C3-
  • the at least one R' group that is a hydrophobic moiety is selected from optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-i 0 alkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted heteroaryl), optionally substituted straight or branched Ci-iohaloalkyl or optionally substituted -CH 2 Ph.
  • at least one of the moieties containing a cation is an optionally substituted straight or branched Ci- l oaminoalkyl .
  • at least two of the moieties containing a cation are optionally substituted straight or branched Ci-ioaminoalkyl, especially C 2 - 6 aminoalkyl .
  • Ar is 9-fluorenyl.
  • Ar-L- has the chemical structure:
  • n 4.
  • the at least one hydrophobic moiety is -CH 2 Ph.
  • At least one of the moieties containing a cation is -CH 2 CH 2 CH 2 CH 2 NH 3 + . In some embodiments, at least two of the moieties containing a cation are -CHzCHzCHzCHzNi .
  • the compound of Formula (I) is selected from:
  • the compound of Formula (I) is selected from Fmoc-FkFk and Fmoc-FFkk or a stereoisomer thereof.
  • Fmoc-FkFk (sometimes referred to as Fmoc- L Phe- D Lys- L Phe- D Lys ) has the structure
  • Fmoc-FFkk (sometimes referred to as Fmoc- L Phe- L Phe- D Lys- D Lys ) has the structure
  • the compound of Formula (I) is selected from Fmoc-FFKK ( SEQ ID NO: 24), Fmoc-FKFK (SEQ ID NO: 25) or a
  • the compound of Formula (I) is selected from Cbz-FFKK (SEQ ID NO: 26) and Cbz-FKFK (SEQ ID NO: 27) or a stereoisomer thereof (where Cbz is
  • the compound of Formula (I) is selected from Cbz-FFkk and Cbz-FkFk or a stereoisomer thereof.
  • the compound of Formula (I) is selected from Fmoc-WKK (SEQ ID NO: 28), Fmoc-VKVK (SEQ ID NO: 29), Fmoc-Wkk, Fmoc-VkVk, Fmoc-KFKFK (SEQ ID NO: 30), Fmoc-FFKKK (SEQ ID NO: 31), Cbz-WKK (SEQ ID NO: 32), Cbz-VKVK (SEQ ID NO: 33), Cbz-Wkk and Cbz-VkVk or a stereoisomer thereof (where Cbz is
  • the compounds of the present invention may be prepared by methods that are known to those skilled in the art. Examples include peptide synthesis from amino acids using traditional solution phase synthesis, solid phase synthesis, native chemical ligation (an option often used in constructing larger peptides (e.g. more than 10 amino acid residues) ) and automated synthesis. Methods are known for preparing various amino acids that are used in the compounds of the present invention. A person skilled in the art will be able to prepare various amino acids from common starting materials and use those to prepare the compounds of Formula (I) and Formula (la) .
  • the peptides and compounds of the present invention typically have good solubility at biologically relevant pHs .
  • at least preferred peptides /compounds are readily soluble in aqueous solutions having a pH of about pH 7.4.
  • the solubility is in excess of 5 mg/mL (e.g. >10 mg/mL or >15 mg/mL) and may be in excess of about 20 mg/mL.
  • Peptides previously used in cell cultures were typically only solubilised at high pH (e.g. pH>9) .
  • peptides/compounds of the present invention may have improved compatibility with biological systems, particularly those that are sensitive to high or low pH . This allows the use of the peptides/compounds of the present invention in biological systems that are sensitive to high or low pH.
  • the present invention provides a hydrogel comprising a peptide or compound of the present invention.
  • Hydrogels comprising a peptide or compound of the present invention are sometimes referred to herein as "networks" or similar (e.g.
  • the hydrogel comprises a peptide or compound of the present invention and an aqueous solution having a pH of from about 1 to about 10 (e.g. from about 2 to about 9, from about 3 to about 9, from about 4 to about 8, from about 5 to about 8, from about 6 to about 8, from about 7 to about 8 or from about 7.1 to about 7.6) .
  • the aqueous solution has a pH of from about 6 to about 8.
  • the peptides according to the first aspect and compounds according to the second aspect can form hydrogels at biologically relevant pHs (e.g. from about 6 to about 8, about 7.1 to about 7.6, about 7.2 to about 7.5, or about 7.3 to about 7.4.), cells may be added before or during gelation and survive owing to the relatively benign conditions of gel formation.
  • Prior art methods to form a gel typically require much lower or much higher pHs in order to form the gel. These pHs are generally not compatible with living cells or tissues.
  • the present invention provides a way of being able to include cells in a media whilst gelation occurs to thereby form a gel with cells distributed within the gel.
  • the present invention provides a cell culture substrate comprising a peptide or compound of the present invention or a hydrogel according to the third aspect.
  • the present invention provides a 3-dimensional cell culture substrate comprising a cell culture substrate according to the fourth aspect, wherein all three dimensions of the cell culture substrate are greater than about 50 ⁇ (e.g. more than about 100 ⁇ , 200 ⁇ , 500 ⁇ , 1 mm, 2 mm, 3mm, 5 mm, 10 mm, 15 mm or 20 mm) .
  • a 3-dimensional cell culture substrate may also sometimes be referred to as a medium, matrix or scaffold (or 3D medium, 3D matrix or 3D scaffold) .
  • Conditions e.g. nutrient formulations, pH and osmolality of cell culture media
  • Conditions can be varied depending on parameters such as cell type, cell density and culture system employed.
  • the present invention provides the use of a peptide or compound of the present invention for culturing a cell.
  • the present invention provides a method of making a cell culture substrate comprising mixing a peptide or compound of the present invention and an aqueous solution.
  • the aqueous solution may be, or comprise, a conventional cell culture medium used in prior art cell culturing systems.
  • the aqueous solution may be added to the peptide/compound or the
  • the peptide/compound may be added to the aqueous solution.
  • the pH of the aqueous solution may be between about 1 and about 10.
  • the pH is from about 6 to about 8.
  • a pH of from about 7.1 to about 7.6 e.g. from about 7.2 to about 7.5, about 7.3 to about 7.4 may be desired.
  • the peptide/compound forms a hydrogel.
  • a salt or other gelation agent e.g. divalent metal salt, or pH switching agent (such as a weak acid or base, depending on the peptide to be used) may be included in the aqueous solution to initiate or promote the formation of a hydrogel.
  • the cells that are to adhere, survive and/or proliferate in or on the substrate are mixed with the aqueous solution before the gelation occurs.
  • the cells may be added to the aqueous solution before the aqueous solution is mixed with the peptide or compound of the present invention.
  • the cells that are to adhere, survive and/or proliferate in or on the substrate are mixed with the aqueous solution during the gelation.
  • the cells that are to adhere, survive and/or proliferate in or on the substrate are added to the hydrogel after the gelation occurs .
  • the present invention provides a method of culturing a cell, comprising forming a cell culture substrate by the method of the seventh aspect of the present invention, and
  • the present invention provides a method of recovering one or more cells from a cell culture substrate, comprising contacting the cell culture substrate with a digestion enzyme to cause degelation, wherein the cell culture substrate comprises a peptide or compound according to the present invention, and wherein the peptide or compound comprises one or more natural amino acid residues.
  • the digestion enzyme should be a digestion enzyme capable of digesting the one or more natural amino acids.
  • peptide refers to compound comprising a chain of two or more amino acid residues (sometimes referred to as amino acid monomers or simply amino acids) linked by peptide bonds (amide bonds ) .
  • Amino acids are organic compounds containing both an amine group and a carboxylic acid group. Amino acids also typically have a side chain (i.e. a moiety bonded to a carbon atom between the amine and the carboxylic acid) .
  • amino acid may refer to any amino acid (i.e. an organic compound containing both an amine group and a carboxylic acid group) and typically refers to an organic compound containing both an amine and a carboxylic acid and having a molecular weight of from about 70 to about 700 g/mol (e.g. from about 70 to about 400 g/mol or from about 70 to about 200 g/mol) .
  • the peptides of the present invention typically comprise -amino acid residues.
  • the amine group is bonded directly to the carbon atom alpha to the carboxyl group (this carbon atom is sometimes referred to as the a-carbon) .
  • the side chain if present, is also bonded directly to the a-carbon.
  • the peptides of the present invention may also comprise ⁇ -amino acid residues.
  • ⁇ -Amino acids are amino acids in which the amine group is covalently bonded to a carbon atom which is beta to the carboxyl group ( ⁇ -carbon) .
  • the side chain if present, may be covalently bonded to either the ⁇ -carbon or the ⁇ -carbon.
  • peptide of the present invention may also comprise amino acid residues of such amino acids.
  • Amino acids typically contain a chiral centre (asymmetric centre) and may therefore give rise to stereoisomers.
  • the peptides of the present invention may contain any combination of stereoisomers.
  • the peptides of the present invention may comprise D- amino acid residues, L-amino acid residues, amino acid residues that are neither D- nor L-, or a mixture thereof.
  • amino acids are encoded directly by the codons of the universal genetic code.
  • Amino acids other than these 20 amino acids are commonly referred to as “non-standard” or “non-canonical” amino acids. These amino acids are typically non-proteinogenic (i.e. they cannot be incorporated into proteins during translation) .
  • Two known non-standard proteinogenic amino acids are selenocysteine and pyrrolysine .
  • Non-natural amino acids are commonly referred to as “non-natural” amino acids, regardless of whether or not they are present in nature.
  • “Non-natural” amino acids are typically not naturally encoded or found in the genetic code of any organisms.
  • “Non-natural” amino acids include ⁇ -, ⁇ - and ⁇ - amino acids; the D-enantiomers of the 20 "natural” amino acids referred to above; and a-amino acids having side chains that are not found in nature.
  • examples exist of “non-natural” amino acids that occur in nature e.g. ⁇ -aminobutyric acid (GABA) and ⁇ -alanine
  • GABA ⁇ -aminobutyric acid
  • ⁇ -alanine examples exist of “non-natural” amino acids that occur in nature
  • -Amino-acid residues are therefore structures that lack a hydrogen atom of the amino group (e.g. -NH-CHR-COOH) , or the hydroxyl moiety of the carboxyl group (e.g. NH 2 -CHR-CO- ) , or both (e.g. -NH-CHR-CO- ) . All units of a peptide chain are therefore amino-acid residues.
  • amino acid is sometimes used to describe an "amino acid residue".
  • N-terminal The residue in a peptide that has an amino group that is not acylated by another amino-acid residue is called the N-terminal (or N-terminus) .
  • C-terminal The residue that has a carboxyl group that does not acylate another amino-acid residue is called the C-terminal (or C- terminus ) .
  • alkyl refers to “alkyl” as well as the “alkyl” portions of “arylCi-6alkyl”, “heteroarylCi-6alkyl” etc.
  • alkyl refers to a straight chain or branched chain saturated hydrocarbyl group. Preferred are Ci-6alkyl and Ci-4alkyl groups.
  • C x - y alkyl refers to an alkyl group having x to y carbon atoms,
  • Ci- 6 alkyl refers to an alkyl group having 1 to 6 carbon atoms.
  • Ci-6alkyl examples include methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, hexyl and the like.
  • alkyl also encompasses alkyl groups containing one less hydrogen atom such that the group is attached via two positions, i.e. divalent.
  • alkenyl refers to a straight chain or branched chain hydrocarbyl group having at least one double bond of either E- or Z- stereochemistry where applicable. Preferred are C 2 - 6 alkenyl and C 2 - 4 alkenyl groups.
  • C x - y alkenyl refers to an alkenyl group having x to y carbon atoms. Examples of C 2 - 6 alkenyl include vinyl, 1- propenyl, 1- and 2-butenyl and 2-methyl-2-propenyl . Unless the context requires otherwise, the term “alkenyl” also encompasses alkenyl groups containing one less hydrogen atom such that the group is attached via two positions, i.e. divalent.
  • alkynyl refers to a straight chain or branched chain hydrocarbyl group having at least one triple bond. Preferred are C 2 - 6 alkynyl and C 2 - 4 alkynyl groups.
  • C x - y alkynyl refers to an alkynyl group having x to y carbon atoms. Examples of C2-6alkynyl include ethynyl, 1-propynyl, 1- and 2-butynyl, 2-pentynyl, 3- pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl and the like. Unless the context indicates otherwise, the term “alkynyl” also encompasses alkynyl groups containing one less hydrogen atom such that the group is attached via two positions, i.e. divalent.
  • halo refers to a halogen, such as F, CI or Br.
  • haloalkyl refers to an alkyl substituted with one or more halo groups .
  • alkoxy refers to an alkyl group as defined above covalently bound via an 0 linkage, such as methoxy, ethoxy, propoxy, isoproxy, butoxy, tert-butoxy and pentoxy.
  • Preferred are Ci- 6 alkoxy, Ci- 4 alkoxy and Ci- 3 alkoxy groups.
  • carboxylate or “carboxyl” refers to the group -COO ⁇ or -COOH.
  • Ci-i 0 aminoalkyl refers to a Ci-i 0 alkyl group (e.g. a Ci- 6alkyl group) substituted with one or more groups selected from -NH2, -NH ( optionally substituted Ci- 6 alkyl) and -N ( optionally substituted Ci- 6 alkyl) (optionally substituted Ci- 6 alkyl) .
  • the Ci-ioalkyl is substituted with 1, 2, 3, 4, 5 or 6 groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl) and -N ( optionally substituted Ci-6alkyl) (optionally substituted Ci-6alkyl) .
  • amino refers to the primary group -NH 2 , as well as substituted amino groups (both monosubstituted and disubstituted) .
  • substituted amino refers to an amino group having a hydrogen of the primary group -NH 2 replaced with, for example, an alkyl group ( “alkylamino” ) , an aryl or aralkyl group ( “arylamino", “aralkylamino” ) and so on.
  • Ci- 6 alkylamino and Ci- 3 alkylamino groups are preferred, such as for example, methylamino ( -NHMe ) , ethylamino (-NHEt) and propylamino (-NHPr) .
  • disubstituted amino or "tertiary amino” refers to an amino group having the two hydrogens of the primary group -NH 2 replaced with, for example, an alkyl group, which may be the same or different ( “di ( alkyl ) amino” ) , an aryl and alkyl group
  • Ci- 3 alkyl amino groups are preferred, such as, for example, dimethylamino (-NMe2), diethylamino (-NEt 2 ), dipropylamino (-NPr 2 ) and variations thereof (e.g. -N(Me) (Et) and so on) .
  • substituted amido or “substituted amide” refers to an amido group having a hydrogen replaced with, for example, an alkyl group ( “alkylamido” or “alkylamide” ) , an aryl ( “arylamido” ) , aralkyl group ( “aralkylamido” ) and so on.
  • alkyl group “alkylamido” or “alkylamide”
  • aryl “arylamido”
  • aralkyl group “aralkylamido”
  • methylamide (-C(O)NHMe), ethylamide (-C(O)NHEt) and propylamide (-C(O)NHPr) and reverse amides thereof (e.g. -NHC(0)Me, -NHC(0)Et and -NHC(O)Pr) .
  • disubstituted amido or “disubstituted amide” refers to an amido group having the two hydrogens replaced with, for example, an alkyl group ( “di ( alkyl ) amido” or “di ( alkyl ) amide” ) , an aralkyl and alkyl group ( “alkyl ( aralkyl ) amido” ) and so on.
  • Di ( Ci- 3 alkyl ) amide groups are preferred, such as, for example, dimethylamide
  • aryl refers to a carbocyclic (non-heterocyclic) aromatic ring or mono-, bi- or tri-cyclic ring system.
  • the aromatic ring or ring system is generally composed of 6 to 10 carbon atoms.
  • aryl groups include but are not limited to phenyl, biphenyl, naphthyl and tetrahydronaphthyl . 6-membered aryls such as phenyl are preferred.
  • arylalkyl or “aralkyl” refers to an arylCi- 6 alkyl- such as benzyl.
  • heterocyclyl refers to a moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound which moiety has from 3 to 10 ring atoms (unless otherwise specified) , of which 1, 2, 3 or 4 are ring heteroatoms, each heteroatom being independently selected from 0, S and N, and the remainder of the ring atoms are carbon atoms.
  • heterocycloalkyl refers to a heterocyclyl moiety comprising a saturated cyclic group comprising one or more ring carbons and one or more ring heteroatoms .
  • Heterocycloalkenyl refers to a heterocyclyl moiety comprising a cyclic group comprising at least one carbon-carbon double bond and one or more ring heteroatoms.
  • Heterocycloalkynyl refers to a heterocyclyl moiety comprising a cyclic group comprising at least one carbon-carbon triple bond and one or more ring heteroatoms.
  • the prefixes 3-, 4-, 5-, 6-, 7-, 8-, 9- and 10- membered denote the number of ring atoms, or range of ring atoms, whether carbon atoms or heteroatoms.
  • the term "3-10- membered heterocylyl”, as used herein, refers to a heterocyclyl group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms.
  • heterocylyl groups include 5-6-membered monocyclic heterocyclyls and 9-10 membered fused bicyclic heterocyclyls.
  • monocyclic heterocyclyl groups include, but are not limited to, those containing one nitrogen atom such as aziridine (3- membered ring), azetidine (4-membered ring), pyrrolidine
  • pyrroline e.g., 3-pyrroline, 2,5- dihydropyrrole ) , 2if-pyrrole or 3if-pyrrole (isopyrrole, isoazole) or pyrrolidinone (5-membered rings), piperidine, dihydropyridine, tetrahydropyridine (6-membered rings), and azepine (7-membered ring) ; those containing two nitrogen atoms such as imidazoline, pyrazolidine (diazolidine ) , pyrazoline (dihydropyrazole ) (5-membered rings), piperazine (6-membered ring); those containing one oxygen atom such as oxirane (3-membered ring), oxetane (4-membered ring), oxolane (tetrahydrofuran) , oxole (dihydrofuran) (5-membered rings), ox
  • dihydroisoxazole (5-membered rings), morpholine, tetrahydrooxazine, dihydrooxazine, oxazine (6-membered rings); those containing one nitrogen and one sulfur atom such as thiazoline, thiazolidine (5- membered rings), thiomorpholine (6-membered ring); those containing two nitrogen and one oxygen atom such as oxadiazine (6-membered ring) ; those containing one oxygen and one sulfur such as: oxathiole (5-membered ring) and oxathiane (thioxane) (6-membered ring); and those containing one nitrogen, one oxygen and one sulfur atom such as oxathiazine (6-membered ring) .
  • heterocyclyl encompasses aromatic heterocyclyls and non- aromatic heterocyclyls .
  • aromatic heterocyclyl may be used interchangeably with the term “heteroaromatic” or the term “heteroaryl” or “hetaryl”.
  • heteroatoms in the aromatic heterocyclyl group may be independently selected from N, S and 0.
  • Heteroaryl is used herein to denote a heterocyclic group having aromatic character and embraces aromatic monocyclic ring systems and polycyclic (e.g. bicyclic) ring systems containing one or more aromatic rings.
  • aromatic heterocyclyl also encompasses pseudoaromatic heterocyclyls.
  • the term “pseudoaromatic” refers to a ring system which is not strictly aromatic, but which is stabilized by means of delocali zation of electrons and behaves in a similar manner to aromatic rings.
  • aromatic heterocyclyl therefore covers polycyclic ring systems in which all of the fused rings are aromatic as well as ring systems where one or more rings are non- aromatic, provided that at least one ring is aromatic.
  • polycyclic systems containing both aromatic and non-aromatic rings fused together the group may be attached to another moiety by the aromatic ring or by a non-aromatic ring.
  • heteroaryl groups are monocyclic and bicyclic groups containing from five to ten ring members .
  • the heteroaryl group can be, for example, a five membered or six membered monocyclic ring or a bicyclic structure formed from fused five and six membered rings or two fused six membered rings or two fused five membered rings .
  • Each ring may contain up to four heteroatoms selected from nitrogen, sulphur and oxygen.
  • the heteroaryl group will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2 heteroatoms.
  • the heteroaryl group contains at least one ring nitrogen atom.
  • the nitrogen atoms in the heteroaryl group can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen.
  • the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.
  • Aromatic heterocyclyl groups may be 5-membered or 6-membered mono ⁇ cyclic aromatic ring systems.
  • 5-membered monocyclic heteroaryl groups include but are not limited to furanyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl
  • 6-membered monocyclic heteroaryl groups include but are not limited to pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, pyranyl, oxazinyl, dioxinyl, thiazinyl, thiadiazinyl and the like.
  • 6-membered aromatic heterocyclyls containing nitrogen include pyridyl (1 nitrogen), pyrazinyl, pyrimidinyl and pyridazinyl (2 nitrogens) .
  • Aromatic heterocyclyl groups may also be bicyclic or polycyclic heteroaromatic ring systems such as fused ring systems (including purine, pteridinyl, naphthyridinyl , lif-thieno [ 2 , 3- c] pyrazolyl , thieno [ 2 , 3-b] furyl and the like) or linked ring systems (such as oligothiophene, polypyrrole and the like) .
  • fused ring systems including purine, pteridinyl, naphthyridinyl , lif-thieno [ 2 , 3- c] pyrazolyl , thieno [ 2 , 3-b] furyl and the like
  • linked ring systems such as oligothiophene, polypyrrole and the like
  • Fused ring systems may also include aromatic 5-membered or 6-membered heterocyclyls fused to carbocyclic aromatic rings such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl and the like, such as 5-membered aromatic heterocyclyls containing nitrogen fused to phenyl rings, 5- membered aromatic heterocyclyls containing 1 or 2 nitrogens fused to phenyl ring.
  • aromatic 5-membered or 6-membered heterocyclyls fused to carbocyclic aromatic rings such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl and the like, such as 5-membered aromatic heterocyclyls containing nitrogen fused to phenyl rings, 5- membered aromatic heterocyclyls containing 1 or 2 nitrogens fused to phenyl ring.
  • groups include, for example, -CH 2 -S-Ci- 6 alkyl , -CH 2 CH 2 - S-Ci- 6 alkyl, -CH (CH 3 ) CH 2 -S-Ci- 6 alkyl, -CH 2 CH 2 -S-CH 3 etc.
  • the term "optionally substituted” as used herein indicates a group may or may not be substituted with 1, 2, 3, 4 or more groups, preferably 1, 2 or 3 groups, more preferably 1 or 2 groups, independently selected from the group consisting of alkyl (e.g. Ci- 6 alkyl), alkenyl (e.g. C 2 - 6 alkenyl ) , alkynyl (e.g. C 2 - 6 alkynyl), cycloalkyl (e.g. C 3 - 8 cycloalkyl ) , hydroxyl, oxo, alkoxy (e.g.
  • alkyl e.g. Ci- 6 alkyl
  • alkenyl e.g. C 2 - 6 alkenyl
  • alkynyl e.g. C 2 - 6 alkynyl
  • cycloalkyl e.g. C 3 - 8 cycloalkyl
  • Ci- 6 alkoxy , aryloxy, arylCi- 6 alkoxy, thioalkyl (e.g. -S-Ci- 6alkyl), halo, haloCi-6alkyl (such as -CF 3 and -CHF 2 ), haloCi-6alkoxy (such as -OCF 3 and -OCHF 2 ), carboxyl, esters, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketones, amides, aminoacyl, substituted amides, disubstituted amides, aryl,
  • arylCi- 6 alkyl arylCi- 6 alkyl , heterocyclylCi- 6 alkyl , arylC 2 - 6 alkenyl ,
  • Optional substituents in the case of heterocycles containing N may also include but are not limited to Ci- 6 alkyl i.e. N-Ci_ 6 alkyl.
  • the optional substituent or substituents are, unless otherwise defined, preferably selected from C 3 - 8 cycloalkyl, amino, substituted amino, disubstituted amino, aryl, heteroaryl, halo (e.g. F, CI, Br, I), haloCi- 6 alkyl , heterocyclyl, Ci- 6 alkoxy, oxo, aryloxy, carboxyl and esters.
  • halo e.g. F, CI, Br, I
  • haloCi- 6 alkyl e.g. F, CI, Br, I
  • haloCi- 6 alkyl e.g. F, CI, Br, I
  • haloCi- 6 alkyl e.g. F, CI, Br, I
  • Ci- 6 alkoxy e.g. F, CI, Br, I
  • oCi- 6 alkyl e.g. F, CI, Br, I
  • peptides are typically capable of forming salts (e.g. addition salts with suitable acids and/or bases) .
  • Salts of peptides are well known in the art and a person skilled in the art will be able to choose suitable salts of the peptides/compounds of the present invention, person skilled in the art will also be able to prepare suitable salts of the compounds of the present invention.
  • Common salts of peptides include Li + , Na + , K + , Mg 2+ , Ca 2+ , CI “ , Br, I " , " OAc, + NH 4 . Contemplated herein are all suitable salts of the peptides and compounds of the present invention.
  • the initial pH of 4 or 5 may be neutralized (e.g. to biologically relevant pHs) upon the addition of Neurobasal media.
  • the inventors characterise the sol and gel state of the tetrapeptides (sometimes referred to herein as “nanofiber networks”, “nanofiber substrates”, “peptide nanofibers” and the like), before using them to support the growth of notoriously sensitive primary hippocampal neurons. It is shown that primary neurons cultured on thin hydrogel layers exhibit spreading, neurite extension and synapse formation, comparable with the currently used gold standard, poly-D-lysine . Long term survival of neurons is also demonstrated, with electrical activity of mature neurons confirmed using a GCaMP reporter assay (section 1.4) .
  • Example 1 The peptides in Example 1 may be prepared using solid phase peptide synthesis using well-known techniques (e.g. Merrifield et al . , Journal of the American Chemical Society, 1963, pp2149-2154) .
  • Functionalised resin beads may be used as a solid support to build up the peptide sequences through iterative coupling and deprotection reactions.
  • cleavage of the peptide from the resin may be effected through the use of tri fluoroacetic acid, before removal of the solvent and lyophilisation . Further to this, the peptides may then be purified using semi-preparatvie HPLC before lyophilisation.
  • Example 1 The peptides in Example 1 were typically prepared using the process described below. Solid phase peptide synthesis of tetrapeptides
  • 2-chlorotrityl chloride resin (100-200 mesh; 1% DVB; 1.1 mmol/g) (500 mg, 0.55 mmol) was weighed into a 10 mL polypropylene syringe equipped with a porous polypropylene frit (Torviq SF-1000), which was used as the reaction vessel. The resin was washed with
  • dichloromethane (3 x 5 mL) before being allowed to swell in dichloromethane (5 mL) for at least 0.5 h prior to the loading of the first amino acid.
  • Fmoc-protected amino acids (3 equiv. ) were dissolved in a 0.45 M DMF solution of 1-hydroxybenzotriazole hydrate (HOBt'H20) / ⁇ , ⁇ , ⁇ " ,N “ - tetramethyl-O- (IH-benzotriazol-l-yl) uronium hexafluorophosphate (HBTU) (3 equiv.) and DIPEA (6 equiv., 0.6 mL) and this coupling solution added to the resin and stirred for 45 mins using an orbital shaker. The solution was expelled and the resin washed with DMF (5 x 4 mL) .
  • HOBt'H20 1-hydroxybenzotriazole hydrate
  • DIPEA 6 equiv., 0.6 mL
  • dichloromethane tri fluoroacetic acid with one drop of water was then added to the resin, and the resin stirred for 2 hours using an orbital shaker. The cleavage solution was then expelled, the resin washed with dichloromethane (2 x 4 mL) and the solvents evaporated under a stream of nitrogen. The resulting residue was lyophilised and purified by semi-preparative HPLC using an acetonitrile/water gradient, giving a white fluffy solid.
  • Fmoc-FFkk IR: 3281 (m) , 3063 (w) , 3029 (w) , 2939 (m) , 1690 (m) , 1631 (s), 1534 (s), 1451 (m) , 1397 (m) , 1320 (w) , 1261 (m) , 1202 (m) , 1134 (m) , 1086 (w) , 1039 (m) , 990 (w) , 836 (m) , 799 (m) , 756
  • Fmoc-FkFk IR: 3289 (m) , 3061 (w) , 3029 (w) , 2935 (m) , 1638 (s), 1528 (s), 1451 (m) , 1394 (m) , 1339 (w) , 1256 (m) , 1202 (s), 1177 (m) , 1133 (m) , 1086 (w) , 1034 (m) , 836 (m) , 799 (m) , 756 (m) , 739 (s), 721 (m) , 699 (s); 3 ⁇ 4 NMR (400 MHz, DMSO-d 6 ) ⁇ 8.70 (br.
  • Fmoc-diphenylalanine (Fmoc-FF) and related derivatives self-assemble into hydrogels, however the majority of these gelators must be dissolved in basic conditions using dilute sodium hydroxide
  • gelation typically requires a pH switch using either a mineral acid or lactone which slowly
  • hydrolyses into an acid hydrolyses into an acid.
  • These hydrogel formation methods are often not compatible with cell culture, due to the extremes of pH that are utilised, and once the hydrogel is formed the pH must be adjusted for the hydrogel to be used for cell culturing.
  • tetrapeptides 1 and 2 were observed to self-assemble into fibres or wormlike micelles at neutral pH, as evidenced through viscosity and small angle neutron scattering (SANS) measurements (described below) .
  • SANS small angle neutron scattering
  • tetrapeptides 1 and 2 can form hydrogels through the more biocompatible route method of charge screening at room temperature.
  • gels were formed through addition of NeuroBasal medium, with a view towards their applications for culturing primary neurons.
  • the fibrous network of the gels was then investigated through atomic force microscopy measurements. In both cases, a dense network of fibres is formed, with the compact arrangement of fibres precluding measurement of their diameter. The amorphous, globular deposits in the images are likely due to the presence of additives in the NeuroBasal medium used to induce gelation.
  • the arrangement of the tetrapeptide molecules within the fibres was investigated through circular dichroism and ATR-IR spectroscopy. Gels of Fmoc-FFkk show a negative peak in the CD centred around 220 nm ( Figure 5a), typical of the ⁇ -sheet structure previously observed for short peptides based upon the Fmoc-diphenylalanine motif.
  • Viscosity measurements were performed on an Anton Paar MCR 302 rheometer using a 25 mm stainless steel parallel plate geometry configuration and analysed using RheoPlus v3.61 software. Typical viscosity measurements involved casting 550 ]iL of a 0.5% (w/v) peptide solution onto one of the stainless steel plates, lowering the other plate to the measurement position, and monitoring viscosity over a range of shear rates (0.1 - 100 s _1 ) . A Peltier temperature control hood and solvent trap was used to reduce evaporation and maintain a temperature of 25 °C.
  • Peptide solutions were prepared at a concentration of 1% (w/v) transferred to a demountable titanium cell of 2 mm path length before being measured at detector distances of 2 and 14 m.
  • Data was corrected for the background, empty cell scattering and the sensitivity of the individual detector pixels. The data was reduced using IgorPro software employing NIST macros specific to QUOKKA to an absolute intensity scale.
  • Fmoc-FFkk Small angle neutron scattering (SANS) patterns and associated fits obtained from 1% (w/v) solutions of Fmoc-FFkk (a) and Fmoc-FkFk (b) dissolved in D 2 0 are shown in Figure 16 (a) -(b) .
  • Fmoc-FFkk can be fit to a flexible cylinder model with a diameter of 6.5 nm, a Kuhn length of 5.9 nm and fibre length of 45 nm.
  • Fmoc-FkFk can be fit to a fractal model with diameter 4 nm and fractal dimension of 2.7, consistent with a self-assembled fibre network.
  • PDL coated coverslips For poly-D-lysine (PDL) coated coverslips, a solution of PDL in borate buffer (0.2 mg/mL) was prepared and 100 ]iL added to freshly flamed glass coverslips in 24 well plates and incubated overnight.
  • borate buffer 0.2 mg/mL
  • peptides 1 and 2 are viable candidates for the long term culturing of neurons. Especially notable is the lack of degradation of the substrate, suggesting that the D-lysine is not susceptible to proteolytic degradation.
  • Cell viability was also further investigated. Primary hippocampal neurons were cultured on coverslips coated with Fmoc-FFkk, Fmoc- FkFk, or PDL for 10, 20, 30, or 40 days. Cell viability was determined using an Alamar Blue assay. Coverslips coated with PDL were used as a positive (100% viability) control. At DIV10 and DIV20, the viability of neurons cultured on peptide nanofibers showed a trend toward reduction. However, at longer time points, no significant differences in viability relative to PDL were observed (Figure 8(d)) . These results demonstrate the viability of long term cultures . Each experiment was repeated three times . At the
  • Neurons were cultured on peptide or PDL coated coverslips for up to thirty days, fixed at various intervals ranging from 10 to 30 days and then stained for the synaptic markers synaptophysin (a marker for pre-synaptic vesicles; green) and PSD-95 (post-synapse; red) alongside the nuclear stain DAPI (blue) as shown in Figure 10(a) .
  • synaptophysin a marker for pre-synaptic vesicles; green
  • PSD-95 post-synapse; red
  • red nuclear stain DAPI
  • AAV adeno-associated virus
  • transduction or liposome-mediated trans fections implying that the presence of the cationic peptide does not interfere with the transduction process. This suggests the lysine residues do not interfere with the cationic liposomes of the Lipofectamine reagent or the AAV.
  • GCaMP construct AAV-hSynl-GCaMP6s-nls-dTomato .
  • Adeno-associated virus vectors for neuronal expression (hSynl) of the GCaMP6s calcium sensor were cloned by conventional restriction enzyme cloning. All plasmids were amplified in E. coli DH5 . AAV vectors were propagated in E. coli Stbl3 to avoid recombination events. All constructs were verified by sequencing. Upon binding of calcium, GCaMP undergoes a conformational change which results in fluorescence emission.
  • Adeno-associated virus vectors for neuronal expression (hSynl) of the GCaMP6s calcium sensor were cloned by conventional restriction enzyme cloning. All plasmids were amplified in E. coli DH5 . AAV vectors were propagated in E. coli Stbl3 to avoid recombination events. All constructs were verified by sequencing.
  • Lipofectamine 300 ]iL of conditioned media was removed from each well, and 100 ]iL of a transfection mix containing 0.6 ]iL
  • Lipofectamine LTX reagent and 0.2 ]ig GFP DNA in Neurobasal was carefully added to each well. After lh incubation, media was aspirated from wells and the wells supplemented with the initially removed conditioned media.
  • AAV A 10 solution containing 0.3 pL either AAV-hSynl-GFP or AAV- hSynl-GCaMP6s-P2A-nls-dTomato (corresponding to approximately 10 11 viral particles) was added to wells containing primary neurons at appropriate timepoints .
  • AAV-hSynl-GCaMP6s-P2A-nls-dTomato was a gift from Jonathan Ting (Addgene plasmid # 51084) . Transduction was confirmed and imaging undertaken on an Olympus SZ61 with a 20x air obj ective .
  • the comparative examples were prepared by a similar process to that described in Example 1.
  • a flamed coverslip is coated with not more than 150 ]iL of poly-lysine dissolved in borate buffer (pH 8) at a concentration of 0.5 mg/mL.
  • the coated coverslip is incubated overnight, followed by aspiration of any remaining liquid and washing at least three times with sterile water .
  • the preparation of a 2.5D culture system involves dissolving the peptide in water at a concentration of 5 mg/ml before coating the flamed coverslip with not more than 100 ]iL of this solution and incubating overnight. Following this, any excess solution is aspirated.
  • the peptide of choice is dissolved in water at a concentration of 10 mg/mL, followed by the addition of an equal volume of cell culture media (here Neurobasal) .
  • cell culture media here Neurobasal
  • a cell suspension in culture media should be used.
  • At least 50 ]iL of the peptide-cell suspension mixture is then added to a flamed coverslip, before incubation overnight, resulting in formation of a hydrogel.
  • first hydrogels are prepared as above through mixing a peptide solution and a cell suspension in cell culture media.
  • trypsin-EDTA 5x solution
  • the hydrogel is then gently agitated to assist with disassembly, thus releasing the suspended cells and converting the hydrogel into a solution.

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Abstract

La présente invention concerne une classe de peptides comprenant de (2) à (12) résidus d'acides aminés : l'extrémité N-terminale du peptide étant liée à un groupe polycyclique fusionné éventuellement substitué comprenant un cycle aromatique ; au moins un résidu d'acide aminé a une chaîne latérale hydrophobe ; au moins deux résidus d'acides aminés ont une chaîne latérale contenant des cations, ou au moins un résidu d'acide aminé a au moins deux chaînes latérales contenant des cations, ou au moins un résidu d'acide aminé a une ou plusieurs chaînes latérales comprenant deux cations ou plus ; et la charge nette du peptide est positive à un pH d'environ (4) à environ (10). La présente invention concerne également des hydrogels et des substrats de culture cellulaire comprenant les peptides, ainsi que l'utilisation des substrats de culture cellulaire pour la culture de cellules.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2518041A1 (fr) * 2009-12-25 2012-10-31 Ajinomoto Co., Inc. Composé de benzyle

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2518041A1 (fr) * 2009-12-25 2012-10-31 Ajinomoto Co., Inc. Composé de benzyle

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BARLOS, K. ET AL.: "Fmoc/Trt-amino acids: comparison to Fmoc/tBu-ammo acids in peptide synthesis", JOURNAL OF PEPTIDE RESEARCH, vol. 51, no. 3, March 1998 (1998-03-01), pages 194 - 200, XP055572271 *
BAYAT, S. ET AL.: "Rational design of mimetic peptides based on aldo-ketoreductase enzyme as asymmetric organocatalysts in aldol reactions", RSC ADVANCES, vol. 4, 2014, pages 38859 - 38868, XP055572351, Retrieved from the Internet <URL:https://pubs.rsc.org/en/content/articlepdf/2014/ra/c4ra04866k?page=search> *
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