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Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

Cromm PM, Schaubach S, Spiegel J, Fürstner A, Grossmann TN, Waldmann H - Nat Commun (2016)

Bottom Line: The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand.This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far.The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.

ABSTRACT
Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

No MeSH data available.


Solid phase synthesis of bicyclic peptides by means of the RCM/RCAM method.(a) General scheme for the synthesis of bicyclic peptides obtained by means of RCM and RCAM of the acyclic precursor peptides. (j=2, number of C-terminal amino acids; R=side chain of a proteinogenic amino acid except Cys or Met). (b) Sequence of bicyclic test peptide 16 bearing an i,i+4 olefin crosslink (eight C-atoms) and an i,i+4 alkyne crosslink (nine C-atoms). Chromatograms of crude reaction mixtures of peptide 16 before macrocyclization (13, top), after RCAM (14, second) and RCM (15, third), respectively, and after simultaneous (one-pot) RCM and RCAM (16, bottom). Corresponding product peaks are highlighted: fully open (13, blue), alkyne monocycle (14, red), olefin monocycle (15, orange) and bicyclic peptide (16, green). Chromatograms were obtained after deprotection and cleavage of resin-bound intermediates. ‘l' represents Complex 5, dry toluene, 40 °C, 2 × 1.5 h; ‘m' represents Grubbs first-generation catalyst, DCE, 3 × 2 h; ‘n' represents Complex 5, Grubbs first-generation catalyst, dry toluene, 40 °C, 2 × 1.5 h.
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f3: Solid phase synthesis of bicyclic peptides by means of the RCM/RCAM method.(a) General scheme for the synthesis of bicyclic peptides obtained by means of RCM and RCAM of the acyclic precursor peptides. (j=2, number of C-terminal amino acids; R=side chain of a proteinogenic amino acid except Cys or Met). (b) Sequence of bicyclic test peptide 16 bearing an i,i+4 olefin crosslink (eight C-atoms) and an i,i+4 alkyne crosslink (nine C-atoms). Chromatograms of crude reaction mixtures of peptide 16 before macrocyclization (13, top), after RCAM (14, second) and RCM (15, third), respectively, and after simultaneous (one-pot) RCM and RCAM (16, bottom). Corresponding product peaks are highlighted: fully open (13, blue), alkyne monocycle (14, red), olefin monocycle (15, orange) and bicyclic peptide (16, green). Chromatograms were obtained after deprotection and cleavage of resin-bound intermediates. ‘l' represents Complex 5, dry toluene, 40 °C, 2 × 1.5 h; ‘m' represents Grubbs first-generation catalyst, DCE, 3 × 2 h; ‘n' represents Complex 5, Grubbs first-generation catalyst, dry toluene, 40 °C, 2 × 1.5 h.

Mentions: To determine whether RCM and RCAM can be performed orthogonally within one peptide sequence (Fig. 3a), peptide 16 was synthesized which embodies two alkyne-functionalized building blocks (1 and 2) in i,i+4-position at the carboxy (C) terminus, and two olefin-containing amino acids (6, Fig. 1b) in i,i+4-position at the amino (N) terminus (Fig. 3b). In peptide 16, an olefin macrocycle can be formed next to an alkyne-bearing macrocycle (Fig. 3b). The treatment of immobilized precursor peptide 13 (blue peak) with either complex 5 or Grubbs first-generation catalyst leads to selective formation of the alkyne (14, red peak) and olefin macrocycle (15, orange peak), respectively (Fig. 3b, Supplementary Figs 9 and 10). HPLC-MS analyses of the alkyne and olefin crosslinked intermediates (14 and 15) reveal highly selective formation of the desired macrocycle without formation of an alternative cyclization product (Supplementary Fig. 10). Both monocycles can be converted into the bicyclic product 16 by means of the second metathesis reaction. This result is remarkable since previous attempts of orthogonal macrocycle formation within peptides failed44, but were successful only for the assembly of simple building blocks47. In an even more demanding set-up, the simultaneous closure of both macrocycles in a one-pot reaction was tested (instead of the previous sequential synthesis). Strikingly, treatment of the open peptide precursor 13 with a mixture of complex 5 and Grubbs first-generation catalyst also yields the desired bicyclic peptide 16 (green peak, Fig. 3b and Supplementary Fig. 10).


Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides.

Cromm PM, Schaubach S, Spiegel J, Fürstner A, Grossmann TN, Waldmann H - Nat Commun (2016)

Solid phase synthesis of bicyclic peptides by means of the RCM/RCAM method.(a) General scheme for the synthesis of bicyclic peptides obtained by means of RCM and RCAM of the acyclic precursor peptides. (j=2, number of C-terminal amino acids; R=side chain of a proteinogenic amino acid except Cys or Met). (b) Sequence of bicyclic test peptide 16 bearing an i,i+4 olefin crosslink (eight C-atoms) and an i,i+4 alkyne crosslink (nine C-atoms). Chromatograms of crude reaction mixtures of peptide 16 before macrocyclization (13, top), after RCAM (14, second) and RCM (15, third), respectively, and after simultaneous (one-pot) RCM and RCAM (16, bottom). Corresponding product peaks are highlighted: fully open (13, blue), alkyne monocycle (14, red), olefin monocycle (15, orange) and bicyclic peptide (16, green). Chromatograms were obtained after deprotection and cleavage of resin-bound intermediates. ‘l' represents Complex 5, dry toluene, 40 °C, 2 × 1.5 h; ‘m' represents Grubbs first-generation catalyst, DCE, 3 × 2 h; ‘n' represents Complex 5, Grubbs first-generation catalyst, dry toluene, 40 °C, 2 × 1.5 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4834642&req=5

f3: Solid phase synthesis of bicyclic peptides by means of the RCM/RCAM method.(a) General scheme for the synthesis of bicyclic peptides obtained by means of RCM and RCAM of the acyclic precursor peptides. (j=2, number of C-terminal amino acids; R=side chain of a proteinogenic amino acid except Cys or Met). (b) Sequence of bicyclic test peptide 16 bearing an i,i+4 olefin crosslink (eight C-atoms) and an i,i+4 alkyne crosslink (nine C-atoms). Chromatograms of crude reaction mixtures of peptide 16 before macrocyclization (13, top), after RCAM (14, second) and RCM (15, third), respectively, and after simultaneous (one-pot) RCM and RCAM (16, bottom). Corresponding product peaks are highlighted: fully open (13, blue), alkyne monocycle (14, red), olefin monocycle (15, orange) and bicyclic peptide (16, green). Chromatograms were obtained after deprotection and cleavage of resin-bound intermediates. ‘l' represents Complex 5, dry toluene, 40 °C, 2 × 1.5 h; ‘m' represents Grubbs first-generation catalyst, DCE, 3 × 2 h; ‘n' represents Complex 5, Grubbs first-generation catalyst, dry toluene, 40 °C, 2 × 1.5 h.
Mentions: To determine whether RCM and RCAM can be performed orthogonally within one peptide sequence (Fig. 3a), peptide 16 was synthesized which embodies two alkyne-functionalized building blocks (1 and 2) in i,i+4-position at the carboxy (C) terminus, and two olefin-containing amino acids (6, Fig. 1b) in i,i+4-position at the amino (N) terminus (Fig. 3b). In peptide 16, an olefin macrocycle can be formed next to an alkyne-bearing macrocycle (Fig. 3b). The treatment of immobilized precursor peptide 13 (blue peak) with either complex 5 or Grubbs first-generation catalyst leads to selective formation of the alkyne (14, red peak) and olefin macrocycle (15, orange peak), respectively (Fig. 3b, Supplementary Figs 9 and 10). HPLC-MS analyses of the alkyne and olefin crosslinked intermediates (14 and 15) reveal highly selective formation of the desired macrocycle without formation of an alternative cyclization product (Supplementary Fig. 10). Both monocycles can be converted into the bicyclic product 16 by means of the second metathesis reaction. This result is remarkable since previous attempts of orthogonal macrocycle formation within peptides failed44, but were successful only for the assembly of simple building blocks47. In an even more demanding set-up, the simultaneous closure of both macrocycles in a one-pot reaction was tested (instead of the previous sequential synthesis). Strikingly, treatment of the open peptide precursor 13 with a mixture of complex 5 and Grubbs first-generation catalyst also yields the desired bicyclic peptide 16 (green peak, Fig. 3b and Supplementary Fig. 10).

Bottom Line: The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand.This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far.The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.

ABSTRACT
Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets.

No MeSH data available.