Pramodkumar D. Jadhav

Detection, isolation and characterisation of cyclolinopeptides J and K in ageing flax

Methionine sulfone containing peptides CLs J (11) and K (12) may be produced from their reduced forms by oxidation but it is not known if these compounds occur in foods that contain flax. These compounds have been reported to possess greater immunosuppressive activity than their reduced methionine sulfoxide peptide forms 4 and 6, respectively. Since 11 and 12 have not been detected in commercial flax oil and milled flax seed, we tested for their presence in flax food products. Here we report that 11 and 12 accumulate in ground flaxseed that is exposed to air and heat (100°C) for more than 4h. Standards of 11 and 12 were prepared, isolated and extensively characterised using HPLC-MS/MS, 1D and 2D NMR methods. We also report the excellent thermal and oxidative stability of these peptides. Due to the harsh conditions required to produce 11 and 12, it is expected that their levels in flax based foods would be low and therefore their presence could serve as an indicative measure of severe oxidation of a food product.

Evaluating the cytotoxicity of flaxseed orbitides for potential cancer treatment

Flaxseed as well as its oil component possess antitumor activities against different types of cancer and have been used by some patients as complementary and/or alternative medicine. Linoorbitides (LOBs) are one family of flaxseed compounds that has implications for anticancer and antioxidant activity. The cytotoxicity of [1-9-NαC]-linusorb-B3 (LOB3), [1-9-NαC]-linusorb-B2 (LOB2), [1-9-NαC],[1-Rs,Ss-MetO]-linusorb-B2 ([MetO]-LOB2) and [1-8-NαC],[1-Rs,Ss-MetO]-linusorb-B1 ([MetO]-LOB1) was measured against human breast cancer Sk-Br-3 and MCF7 cell lines and melanoma A375 cell line. Overall cytotoxicity is cell-type specific. It scales as the hydrophobicity and concentration of the LOBs with the most abundant LOB3 being the most cytotoxic. Oral administration of LOB3 as a potential therapeutic agent might not be applicable as a much too high and/or frequent dose would be required to achieve a serum concentration of 400–500 μg/mL due to bioavailability and pharmacokinetic factors. However, LOB3 may be suitable for topical treatment formulations or as a lead compound in developing anticancer LOB derivatives.

Glycine-containing flaxseed orbitides.

Five new orbitides, cyclolinopeptides 21-25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z 1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H](+) corresponded to the molecular formulae C59H89N10O10, C58H87N10O10S, C58H87N10O11S, C53H80N9O9S, and C53H80N9O10S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1-10-NαC]-GILVPPFFLI, [1-10-NαC]-GMLIPPFFVI, [1-10-NαC]-GOLIPPFFVI, [1-9-NαC]-GMLVFPLFI, and [1-9-NαC]-GOLVFPLFI for cyclolinopeptides 21-25, respectively. Previously reported orbitides, [1-9-NαC]-ILVPPFFLI (1), [1-9-NαC]-MLIPPFFVI (2), [1-9-NαC]-OLIPPFFVI (3), [1-8-NαC]-MLVFPLFI (7), and [1-8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24 also produced orbitides 1, 2, and 7 by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25 contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23 was isolated for characterization via 1D ((1)H and (13)C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.

Cyclo-linopeptide K butanol disolvate monohydrate.

The title compound, C56H83N9O11S·2C4H10O·H2O, is a butanol–water solvate of the cyclolinopeptide cyclo(Metsulfone1-Leu2–Ile3–Pro4–Pro5–Phe6–Phe7–Val8–Ile9) (henceforth referred to as CLP-K) which was isolated from flax oil. All the amino acid residues are in an l configuration based on the CORN rule. The cyclic nonapeptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α- and a β-turn, each containing an N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) and the third residue (β-turn), repectively. In the crystal, the components of the structure are linked by intermolecular N—H⋯O and O—H⋯O hydrogen bonds into a two-dimensional network parallel to (001). The –C(H2)OH group of one of the butanol solvent molecules is disordered over two sets of sites with refined occupancies of 0.863 (4) and 0.137 (4).

Self-assembled fibrillar networks comprised of a naturally-occurring cyclic peptide—LOB3

To the best of our knowledge, this is the first report of a self-assembling orbitide that is capable of forming 1D nano-fibers and ultimately 3D molecular gel networks. LOB3 (a.k.a. cyclolinopeptide A), extracted from Linum usitatissimum L. (flaxseed), forms molecular gels in acetonitrile. LOB3 molecular gels, illustrate that cyclic peptides may be comprised of more complex amino acid sequences than have been currently reported. It appears that cyclization, to form orbitides, imparts conformational aspects to the molecule facilitating self-organization into crystalline nano-fibers. These nanoscale fibers, ∼300 nm in diameter and >100 μm in length, aggregate into bundles of fibers which may exceed micron dimensions. Within the nano-fibers, the orbitides adapt an antiparallel β-sheet-like conformation with high molecular periodicity, as illustrated by CD and XRD.

Nomenclature of homodetic cyclic peptides produced from ribosomal precursors: an IUPAC Task Group interim report

In 2015, an International Union of Pure and Applied Chemistry (IUPAC) Task Group was formed to develop nomenclature recommendations for homodetic cyclic peptides produced from ribosomal precursors. Delegates of the 2015 International Conference on Circular Proteins (ICCP) were presented with the strengths and weaknesses of four published approaches to homodetic cyclic peptide nomenclature, and a summary of the ensuing discussion is presented here. This interim report presents a potentially novel suggestion—the use of Cahn‐Ingold‐Prelog rules to specify amino acid priority in homodetic peptides for consistent numbering. Indeed, this might be the first extension of the Cahn‐Ingold‐Prelog rules in five decades. The authors invite interested parties to contact the corresponding author with suggestions for the improvement of the proposed nomenclature; these ideas will be discussed and considered for inclusion in the final report.

Site Covalent Modification of Methionyl Peptides for the Production of FRET Complexes

Flax cyclic peptides (orbitides, linusorbs (LOs)) [1-8-NαC],[1-MetO2]-linusorb B1 ([MetO2]-LO1) and [1-9-NαC],[1-MetO2]-linusorb B2 ([MetO2]-LO2) are biologically active. These LOs lack active nuclei commonly used in peptide modification. We have developed reactions to activate methionine methyl sulphide to produce stable derivatives. In these reactions, LOs are converted to sulfonium intermediates and subsequently to derivatives containing active nuclei while preserving their fundamental structures. The reaction conditions preserved cyclic peptide fundamental structure and organic solvent solubility. [Met]-LO1 and [Met]-LO2 analogues containing activated groups (-CN, -COOEt, and -NH2 ) in the form of methionine, methionine (S)-oxide, and methionine (S,S)-dioxide amino acids were synthesized and characterized by LCMS and 1D and 2D NMR spectroscopy. Coumarin orbitide complexes produced in this manner bind Eu(3+) yielding FRET compounds that absorb energy through coumarin antennae and emit photons at lanthanide wavelengths.

Characterization of metal-functionalized flax orbitide as a new candidate for light-emitting semiconductor

Organic materials display promise in numerous electronic applications, complimentary to traditional semi-conducting materials. Cyclolinopeptides show promise in light-emitting applications as an organic semiconductor. Photoluminescence measurements indicate charge transfer between the peptide and the metal, resulting in an increase in intensity of the emission from around the metal in the Cyclolinopeptide complex. Complementary X-ray absorption near-edge spectroscopy (XANES) shows a change in occupation of energy states in the peptide when complexed with the metal, indicating charge transfer, but peak positions show the peptide is not chemically changed by the metal. Combining X-ray emission and XANES provides element specific partial density of states, to estimate the element specific energy gap which is the proposed emission range for the peptide material. Organic light emitting diode devices have been fabricated, although no measurable emission has been seen as of yet. The devices have diode like current-voltage characteristics showing the peptide is semi-conducting with a threshold voltage of approximately 2.5 V.

Affinity binding of chicken apolipoprotein A1 to a novel flax orbitide (linusorb)

Bioactive orbitides (linusorbs, LOs) from flaxseed (Linum usitatissimum L.) were ligated through methionine with resin to form an affinity column. The affinity resin was characterized using elemental analysis and the resin bound 70% of its weight in LOs. Chicken serum was passed over the column and washed to remove non-binding materials. The column was eluted with unbound orbitide to competitively release bound protein. A single 28 kDa protein was found in the affinity binding pool. The protein MW and sequence were identical to apolipoprotein A1 (Apo A1), a major serum protein. Its role includes reverse cholesterol transport and cholesterol efflux. The affinity technique allowed convenient and rapid isolation of Apo A1 with a recyclable affinity column. LO binding to a cholesterol carrier molecule might also help us to understand the mechanism of action of LOs in health and the biological activity of flaxseed products.

Metal binding novel flaxseed peptides (linusorbs)

Linusorbs (LOs; a.k.a. cyclolinopeptides) are naturally occurring orbitides derived from flaxseed. These compounds consist of 8–10 amino acid residues, which are linked via an Nto C-terminal peptide bond with molecular masses of approximately 1 kDa. The LO circular structure makes them candidates for metal binding studies. Flaxseed extracts are known to suppress Pb and Cd toxicity. Hence, four metal salts surveyed include Zn(CH3COO)2, ZnSO4, Pb(CH3COO)2 and Cd(NO3)2 with pure LOs 1–5. Proton NMR spectra indicated interaction of LOs with metal salts in solution and were used to determine impacts of methionine oxidation on interactions with metal ions. The methyl group of methionine S,Sdioxide of related LOs did not show the same shift in the presence of Zn(CH3COO)2 and Pb(CH3COO)2 observed in their methionine S-oxide analogues. Metal complexes were observed forming at 10 2 M to 10 4 M but not at lower concentrations (10 5 M to 10 8 M). Mass spectrometry data confirmed that metal binding strength varied by metal in the order Zn(CH3COO)2