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Solid-phase synthesis of cyclic peptide chitinase inhibitors: SAR of the argifin scaffold.

Dixon MJ, Nathubhai A, Andersen OA, van Aalten DM, Eggleston IM - Org. Biomol. Chem. (2008)

Bottom Line: Introduction of the key N-methyl carbamoyl-substituted Arg side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the resin and side-chain deprotection.A severe aspartimide side-reaction observed upon final deprotection is circumvented by the use of a novel aqueous acidolysis procedure.The flexibility of the synthesis is demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of the natural product in complex with a representative family 18 chitinase.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA27AY, UK.

ABSTRACT
A new, highly efficient, all-solid-phase synthesis of argifin, a natural product cyclic pentapeptide chitinase inhibitor, is reported. The synthesis features attachment of an orthogonally protected Asp residue to the solid support and assembly of the linear peptide chain by Fmoc SPPS prior to cyclisation and side-chain manipulation on-resin. Introduction of the key N-methyl carbamoyl-substituted Arg side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the resin and side-chain deprotection. A severe aspartimide side-reaction observed upon final deprotection is circumvented by the use of a novel aqueous acidolysis procedure. The flexibility of the synthesis is demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of the natural product in complex with a representative family 18 chitinase.

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(a) HPLC of crude cyclisation precursor (lower trace) and crude cyclic peptide 10a (upper trace) following cleavage from the solid support with TFA/DCM (1:99). Conditions: Dionex C-18 column (see experimental), 5–95% solvent B in 10 min. (b) HPLC of crude cyclic peptide 10b following cleavage from the solid support with TFA/DCM (80:20). Conditions: as for Fig. 2a.
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fig2: (a) HPLC of crude cyclisation precursor (lower trace) and crude cyclic peptide 10a (upper trace) following cleavage from the solid support with TFA/DCM (1:99). Conditions: Dionex C-18 column (see experimental), 5–95% solvent B in 10 min. (b) HPLC of crude cyclic peptide 10b following cleavage from the solid support with TFA/DCM (80:20). Conditions: as for Fig. 2a.

Mentions: For our synthesis of 3 by this approach, Fmoc-Asp-OAll 5 was loaded onto 2-chlorotrityl chloride polystyrene resin to give the orthogonally protected Asp resin 6 with a loading of 0.4 mmol/g (Scheme 2). The desired resin-bound linear peptide 7 was assembled using standard Fmoc SPPS conditions, followed by removal of the C-terminal allyl ester (Pd(Ph3P)4/PhSiH3)25 and N-terminal Fmoc protection to give cyclisation precursor 8. Cyclisation was then effected upon the solid support by treatment with PyBOP/DIPEA for 2 × 2 h. Cleavage of a small sample of resin at this stage with TFA/DCM (1:99) and analysis by HPLC and ES-MS confirmed the success of this transformation, with essentially quantitative conversion to the expected cyclic peptide 10a being observed (Fig. 2a). Surprisingly, when the same material was exposed to a higher concentration of TFA, to effect simultaneous side-chain deprotection (Fig 2b), the product profile was more complex, mirroring the results obtained on attempted deprotection of the Arg side chain of the cyclic peptide in our original synthesis, and with ES-MS analysis also indicating the presence of aspartimide by-products (see ESI†). That these products were generated only upon acidolysis and not by base-induced aspartimide formation26,27 (Scheme 3) during Fmoc synthesis, was confirmed by the observation that cleavage of 7 with 80% TFA gave rise only to the expected linear pentapeptide in high purity. We had chosen tert-butyl protection for the non-resin-linked Asp residue specifically to eliminate aspartimide formation during linear assembly, based upon our previous observation that the use of Asp(OBn) was ineffective in this context.20 Similarly, elimination of the cyclised product from the solid support, via aspartimide formation during the basic conditions of the final acylation step (see below), was totally suppressed by the use of 2-chlorotrityl resin for the synthesis.28


Solid-phase synthesis of cyclic peptide chitinase inhibitors: SAR of the argifin scaffold.

Dixon MJ, Nathubhai A, Andersen OA, van Aalten DM, Eggleston IM - Org. Biomol. Chem. (2008)

(a) HPLC of crude cyclisation precursor (lower trace) and crude cyclic peptide 10a (upper trace) following cleavage from the solid support with TFA/DCM (1:99). Conditions: Dionex C-18 column (see experimental), 5–95% solvent B in 10 min. (b) HPLC of crude cyclic peptide 10b following cleavage from the solid support with TFA/DCM (80:20). Conditions: as for Fig. 2a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: (a) HPLC of crude cyclisation precursor (lower trace) and crude cyclic peptide 10a (upper trace) following cleavage from the solid support with TFA/DCM (1:99). Conditions: Dionex C-18 column (see experimental), 5–95% solvent B in 10 min. (b) HPLC of crude cyclic peptide 10b following cleavage from the solid support with TFA/DCM (80:20). Conditions: as for Fig. 2a.
Mentions: For our synthesis of 3 by this approach, Fmoc-Asp-OAll 5 was loaded onto 2-chlorotrityl chloride polystyrene resin to give the orthogonally protected Asp resin 6 with a loading of 0.4 mmol/g (Scheme 2). The desired resin-bound linear peptide 7 was assembled using standard Fmoc SPPS conditions, followed by removal of the C-terminal allyl ester (Pd(Ph3P)4/PhSiH3)25 and N-terminal Fmoc protection to give cyclisation precursor 8. Cyclisation was then effected upon the solid support by treatment with PyBOP/DIPEA for 2 × 2 h. Cleavage of a small sample of resin at this stage with TFA/DCM (1:99) and analysis by HPLC and ES-MS confirmed the success of this transformation, with essentially quantitative conversion to the expected cyclic peptide 10a being observed (Fig. 2a). Surprisingly, when the same material was exposed to a higher concentration of TFA, to effect simultaneous side-chain deprotection (Fig 2b), the product profile was more complex, mirroring the results obtained on attempted deprotection of the Arg side chain of the cyclic peptide in our original synthesis, and with ES-MS analysis also indicating the presence of aspartimide by-products (see ESI†). That these products were generated only upon acidolysis and not by base-induced aspartimide formation26,27 (Scheme 3) during Fmoc synthesis, was confirmed by the observation that cleavage of 7 with 80% TFA gave rise only to the expected linear pentapeptide in high purity. We had chosen tert-butyl protection for the non-resin-linked Asp residue specifically to eliminate aspartimide formation during linear assembly, based upon our previous observation that the use of Asp(OBn) was ineffective in this context.20 Similarly, elimination of the cyclised product from the solid support, via aspartimide formation during the basic conditions of the final acylation step (see below), was totally suppressed by the use of 2-chlorotrityl resin for the synthesis.28

Bottom Line: Introduction of the key N-methyl carbamoyl-substituted Arg side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the resin and side-chain deprotection.A severe aspartimide side-reaction observed upon final deprotection is circumvented by the use of a novel aqueous acidolysis procedure.The flexibility of the synthesis is demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of the natural product in complex with a representative family 18 chitinase.

View Article: PubMed Central - PubMed

Affiliation: Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA27AY, UK.

ABSTRACT
A new, highly efficient, all-solid-phase synthesis of argifin, a natural product cyclic pentapeptide chitinase inhibitor, is reported. The synthesis features attachment of an orthogonally protected Asp residue to the solid support and assembly of the linear peptide chain by Fmoc SPPS prior to cyclisation and side-chain manipulation on-resin. Introduction of the key N-methyl carbamoyl-substituted Arg side chain is achieved via derivatisation of a selectively protected Orn residue, prior to cleavage from the resin and side-chain deprotection. A severe aspartimide side-reaction observed upon final deprotection is circumvented by the use of a novel aqueous acidolysis procedure. The flexibility of the synthesis is demonstrated by the preparation of a series of argifin analogues designed from the X-ray structure of the natural product in complex with a representative family 18 chitinase.

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