Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A.

Backbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.

Chemical Synthesis of Interleukin-2 and Disulfide Stabilizing Analogues.

Chemical protein synthesis allows the construction of well-defined structural variations and facilitates the development of deeper understanding of protein structure-function relationships and new protein engineering strategies. Herein, we report the chemical synthesis of interleukin-2 (IL-2) variants on a multimilligram scale and the formation of non-natural disulfide mimetics that improve stability against reduction. The synthesis was accomplished by convergent KAHA ligations; the acidic conditions of KAHA ligation proved to be valuable for the solubilization of the hydrophobic segments of IL-2. The bioactivity of the synthetic IL-2 and its analogues were shown to be equipotent to recombinant IL-2 and exhibit improved stability against reducing agents.

Discovery of a crystalline sulforaphane analog with good solid-state stability and engagement of the Nrf2 pathway in vitro and in vivo.

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23 °C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.

Improved synthesis of (S)-N-Boc-5-oxaproline for protein synthesis with the α-ketoacid-hydroxylamine (KAHA) ligation.

We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA) ligation. The new synthetic pathway to the enantiopure oxaproline is based on a chiral amine mediated enantioselective conjugate addition of a hydroxylamine to trans-4-oxo-2-butenoate. This route is practical, scalable and economical and provides decagram amounts of material for protein synthesis and conversion to other protected forms of (S)-oxaproline.

Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.

KAHA ligations that form aspartyl aldehyde residues as synthetic handles for protein modification and purification.

Aldehydes are widely recognized as valuable synthetic handles for the chemoselective manipulation of peptides and proteins. In this report, we show that peptides and small proteins containing the aspartic acid semialdehyde (Asa) side chain can be easily prepared by a chemoselective amide-forming ligation that results in the formation of the Asa residue at the ligation site. This strategy employs the α-ketoacid-hydroxylamine (KAHA) ligation in combination with a new isoxazolidine monomer that forms a side-chain aldehyde upon ligation. This monomer is easily prepared on a preparative scale by a catalytic, enantioselective approach and is readily introduced onto the N-terminus of a peptide segment by solid phase peptide synthesis. The ligated product can be further functionalized by bioorthogonal reactions between the aldehyde residue and alkoxyamines or hydrazides. We demonstrated that glucagon aldehyde, an unprotected 29-mer peptide prepared by KAHA ligation, can be site specifically and chemoselectively modified with biotin, dyes, aliphatic oximes, and hydroxylamines. We further describe a simple and high recovery one-step purification process based on the capture of a 29-mer glucagon aldehyde and a 76-mer ubiquitin aldehyde by an alkoxyamine-functionalized polyethylene glycol resin. The peptide or protein was released from the resin by addition of a hydroxylamine to provide the corresponding oximes.

New chemistries for chemoselective peptide ligations and the total synthesis of proteins.

The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chemistry. The requirements of the native chemical ligation - an N-terminal cysteine and C-terminal thioesters - have encouraged a search for alternative amide-forming ligation reactions. Among successful alternatives to native chemical ligation, are the α-ketoacid-hydroxylamine ligation with 5-oxaproline and, serine/threonine ligation, and potassium acyltrifluoroborate (KAT) ligation. In addition, the KAT ligation, along with the non-amide forming alkyne-azide ligation, is very useful for synthetic conjugations. All of these recent ligation methods were applied to synthesize different proteins, and have allowed chemists to incorporate unnatural amino acids, or to modify the peptide backbone.