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  • Synthesis and Applications of Fluorinated -Amino Acids

    Overview and Recent Advances

    Sbastien CardinalMacMillan Group Meeting

    Princeton University11/15/2017

  • reference

    Introduction Interest of artificial amino acids Unique properties of the C-F bond Natural fluorinated amino acid

    Applications of fluorinated -amino acids Protein engineering Medicinal chemistry 19F-NMR 18F-PET

    Incorporation of fluorinated -amino acids

    Synthesis of fluorinated -amino acids Strategy I Strategy II Strategy III Strategy IV Strategy V Strategy VI

    Outlines

    reference

  • reference

    Peptides and proteins occupy innumerous biological functions Metabolic Structural

    Serve as inspiration in many research fields New therapeutic agents Biomaterials

    Appealing features Low toxicity Structural simplicity Low immune response

    Intrinsic limitations Enzymatic degradation (protease) Thermic stability Denaturation in organic solvents

    The use of non-canonical amino acids may overcome some of those limitations D--amino acids -amino acids Artificial amino acids

    Artificial amino acids expand the functional diversity and open the way to tailored protein design

    IntroductionInterest of Artificial -Amino Acids

    reference

  • referenceBuer, B. C.; Marsh, E. N. G. Protein Sci. 2012, 21, 453.Cametti, M.; Crousse, B.; Metrangolo, P.; Milani, R.; Resnati, G. Chem. Soc. Rev. 2012, 41, 31.

    Minor steric impact F: smallest Van der Waals radius after H and Ne

    Major electronic impact F: most electronegative element Inverted polarization vs C-H

    Unique features of the C-F bond Often described as the strongest organic bond (BDE up to 544 KJ/mol) Most polar covalent bond Can influence polarization on adjacent bonds Low polarizability of C-F can give rise to Fluorous effect

    Fluorous effect Auto-segregation of fluorine-rich organic species Intermolecular: fluorous phase Intermolecular: fluorophile domain on protein

    C-H C-F Bond Substitution

    reference

    Can those unique features be explored in peptide/protein engineering ?

    Fluorinated -amino acids (-AAF)

  • referenceOdar, C.; Winkler, M.; Wiltschi, B. Biotechnol. J. 2015, 10, 427.

    4-Fluoro-L-threonine (4F-Thr): only natural -AAF known Produced by Streptomyces cattleya (Gram-positive) Antibiotic agent Not identified in any peptide or protein so far

    Biosynthesis involves incorporation of fluoride by a fluorinase enzyme

    Natural Fluorinated -Amino Acid

    reference

    O

    HO OH

    NSN

    N N

    NH2

    NH2HO

    O O

    HO OH

    NN

    N N

    NH2F

    O

    HO OH

    FOPO32

    OPO32FOH

    OH

    O

    H

    OF

    O-

    O

    NH3+

    FOH

    OH

    OF

    L-Thr

    NAD+NADH

    4FT transaldolase

    Fluorinase

    F-acetyldehydedehydrogenase

    CH3CHO

    F- L-Met PO43- adenine

    Isomerase

    Aldolase

    FDA phosphorylase

  • referenceOdar, C.; Winkler, M.; Wiltschi, B. Biotechnol. J. 2015, 10, 427.

    Incorporation of Inorganic Fluoride by Fluorinase Enzyme

    reference

  • referenceMerkel, L.; Schauer, M.; Antranikian, G.; Budisa, N. ChemBioChem 2010, 11, 1505. Berger, A. A.; Vller, J.-S.; Budisa, N.; Koksch, B. Acc. Chem. Res. 2017, 50, 2093.

    Many properties can be influenced by the substitution of canonical -amino acids by fluorinated analogues Protein folding Protein-protein interactions Ribosomal translation Lipophilicity Acidity/basicity character Optimal pH Stability: temperature, protease, organic solvents

    Example: lipophilicity vs.VdW volume of side chains a-AAF can fill a desired space but with a different lipophilicity from -AA

    Often claimed to generally improve stability and properties in out-of-field literature

    But, not that simple !

    Application of -AAFProtein Engineering: General Considerations

    reference

    Fluorine has been shown to impart often favourable but seldom predictable properties to peptides and proteins

    -Beate Koksch (2017)-

  • (ProProGly)10(ProHypGly)10(ProFlpGly)10

    NH

    CO2H

    F

    Flp

    NH

    CO2H

    Hyp

    NH

    CO2H

    Pro

    HO

    referenceHolmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Nature 1998, 392, 666.

    Classic example: stabilization of the folded triple helix structure of collagen at high temperature

    Applications of -AAFProtein Engineering

    reference

  • referenceBerger, A. A.; Vller, J.-S.; Budisa, N.; Koksch, B. Acc. Chem. Res. 2017, 50, 2093.

    Interest for peptides in medicinal chemistry Low toxicity Simple structure / synthesis Low immune response

    Drawbacks of peptides in medicinal chemistry Biodisponibility Low membrane permeability Enzymatic cleavage

    Impact of fluorine in medicinal chemistry 150 fluorinated small molecules reaching the market since 1950 In 2010, 20% of all administered drugs 30% by 2017

    Applications of -AAFMedicinal Chemistry of Peptides

    reference

    Can -AAF help to overpass those limitations ?

    Can this impact be translated to peptides ?

  • referenceKollonitsch, J.; et al. Nature 1978, 274, 906.Seki, M.; Suzuki, M.; Matsumoto, K. Biosci. Biotechnol. Biochem. 1993, 57, 1024.

    Because of their resemblance with canonical a-amino acids, some -AAF can act as inhibitors Known action on amino acid decarboxylase enzymes Inhibition of the production of biologically relevant amines, including:

    -(difluoromethyl)ornithine (DMDO) Commercial name: Eflornithine Treatment of hirsutism and African trypanosomiasis (sleeping sickness) Inhibitor or ornithine decarboxylase Acts on putrescine biosynthesis

    Applications of -AAFInhibitors

    reference

    DMDO

    H2NO

    OH

    H2N CHF2

    NH2HO

    HODopamine

    HNN

    NH2 H2NNH2

    Histamine Putrescine

  • 19F-NMR spectra of a peptide bearing a Tyr(C4H9) at 500nM

    referenceSalwiczek, M.; Mikhailiuk, P. K.; Afonin, S.; Komarov, I. V.; Ulrich, A. S.; Koksch, B. Amino Acids 2010, 39, 1589.Tressler, C. M.; Zondlo, N. J. Org. Lett. 2016, 18, 6240.

    Widely used non-native NMR-active nucleus for interpretation of biological structure and functions

    Many advantages No marking necessary No background in biological systems Sensible Can be used at biorelevant concentrations

    Some -AAF specifically designed as NMR probes

    Applications of -AAF19F-NMR Spectroscopy

    NH2

    CO2H

    H

    F3C

    CF3-BpgUlrich

    Tyr(C4H9)Zondlo

    NH2

    CO2H

    H

    O

    F3C

    F3CF3C

  • referenceKotzerke, J.; et al. Eur. J. Nucl. Med. Mol. Imag. 2003, 30, 1004.Nodwell, M. B.; Britton, R.; et al. J. Am. Chem. Soc. 2017, 139, 3595.

    18F : t1/2 = 109.77 min

    18F-PET mainly used in oncology

    Main marker used: 18F-FDG (18F)-fluorodeoxyglucose Discrimination of cancer cells due to their higher metabolism

    Consumption of -amino acids also higher in cancer cells -AAF explored as new markers Better resolution in some cases

    -AAF could also be used for more refine analysis Marking based on active transport events Transport protein LAT1 upregulated in cancer cells

    Applications of -AAF18F-Positron Emission Tomography (18F-PET)

    reference

    O

    OH

    O

    NH218F

    [18F]-F-TYR

  • referenceCarpentier, C.; Godbout, R.; Otis, F.; Voyer, N. Tetrahedron Lett. 2015, 56, 1244.Devillers, E.; Pytkowicz, J.; Chelain, E.; Brigaud, T. Amino Acids 2016, 48, 1457.

    A lot of -AAF can be used in the context of SPSS

    Coupling in solution can overpass compatibility issues with problematic -AAF

    Incorporation of Fluorinated -Amino Acids in Peptides and ProteinsSynthetic Incorporation with Solid-Phase Peptide Synthesis (SPSS)

    reference

    O

    OFmocHN

    Me

    Me

    O

    ONH

    Me

    MeOHN

    FO

    FmocHN

    Me

    1) Piperidine/DMF 2) HATU, NMM, Fmoc-F-Ala-OH

    3) Piperidine/DMF 4) HATU, NMM, Fmoc-F-Ala-OH

    24% yield

    O

    O

    Cl

    Me

    Me

    NMM (2.2 equiv)(1.1 equiv)

    H2NO

    OHMeF3C

    Poorly Nu- amineNot suitable for standard SPPS

    +H3NO

    Ot-BuMeF3C

    Cl

    1)

    2)

    (0.9 equiv)

    NH

    O

    Ot-BuMeF3CO

    FmocHNOH

    OFmocHN

    Me

    Me

    81% yield

    Protected dipeptideSuitable for SPPS

    (R)--Tfm-Ala-OH

    Me

    Me

  • referenceHackenberger, C. P. R.; Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030.

    Limitations of SPSS

    Coupling of fragments in solution may be used for longer sequences

    Incorporation of Fluorinated -Amino Acids in Peptides and ProteinsSynthetic Incorporation Into Larger Peptide/Proteins

    reference

    Compatibility issues with some -AAFSpecific protecting groups required

    Not suitable for more than 50-100 -AA

  • Relies on native translational machinery of the cellIncorporates the modified -AA at every position occupied by the parental -AA

    Needs strains of bacteria that are auxotrophic for the parental -AA-AAF has to be sufficiently similar to its natural analog

    Particularly suitable for monoofluorated -AAF

    referenceOdar, C.; Winkler, M.; Wiltschi, B. Biotechnol. J. 2015, 10, 427.

    Residue specific

    Incorporation of Fluorinated -Amino Acids Into Pept

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