Neighbor-directed histidine N (τ)-alkylation: A route to imidazolium-containing phosphopeptide macrocycles.

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation.

Mitsunobu mischief: neighbor-directed histidine N(τ)-alkylation provides access to peptides containing selectively functionalized imidazolium heterocycles.

There are few methodologies that yield peptides containing His residues with selective N(τ), N(π)-bis-alkylated imidazole rings. We have found that, under certain conditions, on-resin Mitsunobu coupling of alcohols with peptides having a N(π)-alkylated His residue results in selective and high-yield alkylation of the imidazole N(τ) nitrogen. The reaction requires the presence of a proximal phosphoric, carboxylic or sulfonic acid, and proceeds through an apparent intramolecular mechanism involving Mitsunobu intermediates.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics.

Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection.

Peptide-based inhibitors of Plk1 polo-box domain containing mono-anionic phosphothreonine esters and their pivaloyloxymethyl prodrugs.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group.

Design and synthesis of a reagent for solid-phase incorporation of the phosphothreonine mimetic (2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid (Pmab) into peptides in a bio-reversible phosphonyl-bis-pivaloyloxymethyl (POM) prodrug form.

Reported herein are the synthesis and solid-phase peptide incorporation of N-Fmoc-(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid bis-pivaloyloxymethyl phosphoryl ester [Fmoc-Pmab(POM)2-OH, 2] as a phosphatase-stable phosphothreonine (pThr) mimetic bearing orthogonal protection suitable for the synthesis of Pmab-containing peptides having bio-reversible protection of the phosphonic acid moiety. This represents the first report of a bio-reversibly protected pThr mimetic in a form suitable for facile solid-phase peptide synthesis.

Non-proteinogenic amino acids in the pThr-2 position of a pentamer peptide that confer high binding affinity for the polo box domain (PBD) of polo-like kinase 1 (Plk1).

We report herein that incorporating long-chain alkylphenyl-containing non-proteinogenic amino acids in place of His at the pT-2 position of the parent polo-like kinase 1 (Plk1) polo box domain (PBD)-binding pentapeptide, PLHSpT (1a) increases affinity. For certain analogs, approximately two orders-of-magnitude improvement in affinity was observed. Although, none of the new analogs was as potent as our previously described peptide 1b, in which the pT-2 histidine imidazole ring is alkylated at its π nitrogen (N3), our current finding that the isomeric His(N1)-analog (1c) binds with approximately 50-fold less affinity than 1b, indicates the positional importance of attachment to the His imidazole ring.

Peptoid-Peptide hybrid ligands targeting the polo box domain of polo-like kinase 1.

We replaced the amino terminal Pro residue of the Plk1 polo-box-domain-binding pentapeptide (PLHSpT) with a library of N-alkyl-Gly "peptoids", and identified long-chain tethered phenyl moieties giving greater than two-orders-of-magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low-nanomolar affinity N-acylated tetrapeptides. Binding of the N-terminal long-chain phenyl extension was demonstrated by X-ray co-crystal data.

Identification of high affinity polo-like kinase 1 (Plk1) polo-box domain binding peptides using oxime-based diversification.

In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein.

Construction of multiring frameworks by metal-free cascade reactions of stable isochromenylium tetrafluoroborate.

Efficient methodologies for constructing several multiring frameworks have been developed utilizing the cascade reactions of air-stable isochromenylium tetrafluoroborates with olefins. Successive additions of two equivalents of styrene-type olefins to isochromenylium tetrafluoroborate (ICTB) 1 have been observed and their mechanisms were proposed and discussed. Intramolecular capture of the early stage cationic intermediates by predevised heteroatoms or Friedel-Crafts donors provided two different types of bridged-ring systems.

Transformation of reactive isochromenylium intermediates to stable salts and their cascade reactions with olefins.

Transformation of reactive isochromenylium intermediates to the corresponding storable and stable reagents has been achieved, and a number of isochromenylium tetrafluoroborates (ICTBs, 1) have been conveniently prepared and characterized. Direct metal-free treatment of isochromenylium tetrafluoroborate 1a with olefins afforded a variety of polycyclic frameworks 4 via mild cascade reactions. Starting from the prefunctionalized o-alkynylbenzaldehydes, a one-pot metal-free procedure of intramolecular cascade annulation to 2,3-dihydrophenanthren-4(1H)-one derivatives was also developed.