Oxidative diversification of amino acids and peptides by small-molecule iron catalysis

Abstract

Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C-H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C-H oxidation strategy is demonstrated in its capacity for generating diversity: four 'chiral pool' amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C-H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C-H oxidation to one containing a linear unnatural amino acid.

Figure 1. NRPS-inspired strategy for iron-catalysed C-H oxidative functionalization of amino acids and peptides

(A) Oxidative tailoring iron enzyme pre- and post-assembly modifications in the biosynthesis of vancomycin. Iron enzymes diversify tyrosine into two unnatural amino acids hydroxyphenylglycine and β-hydroxytyrosine that are incorporated by the NRPS into a heptapeptide. Post-assembly oxidative tailoring by iron enzymes effects side-chain cross-linking to afford the vancomycin core. X = OH or peptidyl carrier protein; R = H or methyl (B) Small molecule non-heme iron C-H oxidation catalysts Fe(PDP) 1 and Fe(CF₃PDP) 2. PDP = [N,N′-Bis(2-pyridylmethyl)]-2,2′-bipyrrolidine. (C) Iron catalysts 1 and 2 catalyzed pre-assembly oxidative modification of proline to afford numerous classes of unnatural amino acids. Post-assembly oxidative modifications by 1 and 2 of proline-containing polypeptides to furnish UAA-functionalized polypeptides. AA = amino acid; UAA = unnatural amino acid; Ns = 4-nitrophenylsulfonyl; Bn = Benzyl.

Figure 2. Four amino acids transformed to twenty-one chiral unnatural amino acids via Fe(PDP)-catalysed C-H hydroxylations

(A) Oxidations to glutamic acid and 5-HP. Slow addition: AcOH (0.5–5 equiv.) was added to a MeCN solution of (−)-3. 1 (0.25 equiv. in CH₃CN, 0.2 M) and H₂O₂ (5–9 equiv. in CH₃CN, 0.4–0.72 M) were added via syringe pump (75 minutes) simultaneously. Iterative addition: (−)-3 in MeCN was cooled to 0 °C. 1 (5 mol%) and AcOH (0.5 equiv.) were added, followed by dropwise addition (3 min) of 0 °C MeCN solution of H₂O₂ (1.9 equiv.). The addition of 1, AcOH, and H₂O₂ was repeated twice, every 10 minutes. Crude 5-HP was passed through a silica gel plug and concentrated prior to (B) arylation (C) reduction, olefination, or reductive amination. (D) Aliphatic C-H oxidation. ^a Starting material recycled 1×. Yields in parentheses are average yield per step.

Figure 3. Direct oxidative modification of N-, C-terminal and internal proline residues in peptides by Fe(PDP) catalyzed C-H hydroxylation

(A) Chemoselective oxidative modifications of N- and C-terminal proline-containing peptides. (B) Diversification of a tetrapeptide via chemoselective oxidation / functionalization sequences. ^a, Starting material recycled 1×. (C) Direct oxidative opening of internal proline residues in tripeptides affords UAA- or glutamic acid-containing tripeptides. Yields in parentheses note the average yield per step. All slow additions run with AcOH (0.5 equiv.)/H₂O₂ (5 equiv.).

Figure 4. Fe(PDP)-catalyzed oxidative diversification of tripeptides and macrocycles

(A) Fe(PDP) 1 and Fe(CF₃PDP) 2 oxidative modifications of a single tripeptide enables synthesis of eight functionally diverse UAA-containing tripeptides. Slow addition with AcOH (0.5 equiv.)/H₂O₂ (5 equiv.). ^a Macrocycles 60–62 were prepared from tripeptides 52–54 using 5-step transformations involving: alkene appendage to the UAA residue, coupling of a fourth alkene-containing amino acid to the C-terminus, conversion of Nosyl to a Boc group, ring-closing metathesis, and hydrogenation. Individual routes vary in order. See the SI for full details. (B) Late-stage diversification of a proline-containing peptide macrocycle via post-assembly oxidation / reductive amination. RCM = ring closing metathesis. ^b Hoveyda-Grubbs Catalyst, second generation (5 mol%) ^c Dipotassium azodicarboxylate (40 equiv.), AcOH (80 equiv.). Values in parentheses indicate the average yield per step.