Synthesis and evaluation of analogues of the glycinocin family of calcium-dependent antibiotics

The glycinocins are a class of calcium-dependent, acidic cyclolipopeptide antibiotics that are structurally related to the clinically approved antibiotic daptomycin. In this article, we describe the synthesis of a small library of glycinocin analogues that differ by variation in the exocyclic fatty acyl substituent. The glycinocin analogues were screened against a panel of Gram-positive bacteria (as well as Gram-negative P. aeruginosa). These analogues exhibited similar calcium-dependent activity to the parent natural products against Gram-positive bacteria but showed no activity against P. aeruginosa. The length of the fatty acid was shown to be important for optimal biological activity, while the hybridisation at the α,β position and branching within the fatty acyl chain had only subtle effects on activity

CLE peptide tri-arabinosylation and peptide domain sequence composition are essential for SUNN-dependent autoregulation of nodulation in Medicago truncatula

-MtCLE12 and MtCLE13 encode CLAVATA3/EMBRYO‐SURROUNDING REGION RELATED (CLE) peptides which regulate autoregulation of nodulation (AON) in Medicago through the shoot receptor, SUNN (SUPER NUMERIC NODULES). Genetics suggests RDN1 (ROOT‐DETERMINED NODULATION 1) arabinosylates MtCLE12 to enable SUNN perception. The functional structures of MtCLE12 and MtCLE13 peptides, however, remain elusive. -We combined genetic and chemical synthesis approaches to determine if glyco‐modifications of three nodule‐expressed CLE peptides are essential for AON. We also examined how root and shoot applied AON‐CLEs inhibit nodulation. -MtCLE12, MtCLE13 and MtCLE42 peptides were synthesized with hydroxylation, mono‐arabinosylation or tri‐arabinosylation (TaP) at proline 7. Only MtCLE12‐TaP and MtCLE13‐TaP peptides induced AON in wild‐type (WT) and rdn1‐1, but not in sunn‐4. The application of MtCLE13‐TaP to cotyledons 1 d before rhizobial inoculation completely inhibited both rhizobial infection and nodulation. By contrast, MtCLE12‐TaP induced significant AON without abolishing rhizobial infection. -The results indicate that key CLE domain amino acids and TaP modifications to MtCLE12 and MtCLE13 are essential for SUNN‐dependent AON. We also show evidence that RDN1 does not tri‐arabinosylate MtCLE13. Finally, MtCLE13‐TaP can induce a strong AON response in shoots that inhibits the entire symbiotic processes in roots. We present a new model for AON in Medicago.An Australian Research Council (ARC) grant to M.A.D. (DP150104250) and an ARC Future Fellowship to R.J.P. (FT130100150) supported this work. N.P. was partly supported by an Endeavour Fellowship. The authors gratefully acknowledge the funding provided to L.C. by the John A. Lamberton research scholarship and the Agnes Campbell postgraduate prize

Synthesis of Rhamnosylated Arginine Glycopeptides and Determination of the Glycosidic Linkage in Bacterial Elongation Factor P

A new class of N-linked protein glycosylation – arginine rhamnosylation – has recently been discovered as a critical modification for the function of bacterial elongation factor P (EF-P). Herein, we describe the synthesis of suitably protected α- and β-rhamnosylated arginine amino acid “cassettes” that can be directly installed into rhamnosylated peptides. Preparation of a proteolytic fragment of Pseudomonas aeruginosa EF-P bearing both α- and β-rhamnosylated arginine enabled the unequivocal determination of the native glycosidic linkage to be α through 2D NMR and nano-UHPLC-tandem mass spectrometry studies

Triarabinosylation is required for nodulation-suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum

Legumes form root nodules to house beneficial nitrogen-fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism, called Autoregulation of Nodulation (AON), to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesised variants of nodulation-suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesised and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild-type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O-arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide-encoding genes, including orthologues of nodulation-suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation-suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity

Diverse peptide hormones affecting root growth identified in the Medicago truncatula secreted peptidome

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.This work was supported by ARC grants to MAD: DP150104050 and LP150100826. NP was partly supported by an Endeavor Fellowship. NAMR was supported by an ANU Ph.D. scholarship supported by DP120101893. AI was supported by an Australian Post-graduate Award and an AW Howard Memorial Award. LC was supported by the Bruce-Veness Chandler and the John A. Lamberton research scholarship

Stereoselective Synthesis of Plant and Animal-Derived O-linked Glycopeptides

This thesis describes efforts towards the stereoselective synthesis of homogeneous plant and animal-derived O-linked glycopeptides for the purposes of interrogating structure-function relationships. The first section of this thesis (chapter 2) details the synthesis of novel CLE and CEP glycopeptide plant hormones which bear the plant-specific β1,2-triarabinosylated hydroxyproline motif. The hormones which were synthesised have been linked to the root growth programme in legumes (Medicago truncatula and Soybean), but have not previously been structurally characterised. One of these glycopeptides, MtCLE13, was biologically evaluated and was shown to possess nodulation inhibiting properties only when triarabinosylated at the key hydroxyproline residue, thus confirming the critical functional role of the carbohydrate moiety. The second section of this thesis (chapters 3-5) describes efforts towards the stereoselective synthesis of human cell membrane-derived MUC1 glycopeptides containing α-sialylated tumour-associated carbohydrate antigens (sialyl Tn, 2,6-sialyl T, 2,3-sialyl T and glycophorin antigens). The work described in these chapters culminated in the development of a divergent and highly stereoselective synthetic route to all of the key glycosylamino acids required for the construction of a library of homogeneous glycopeptide cancer vaccine candidates

Stereoselective Synthesis of Sialylated Tumor-Associated Glycosylamino Acids

Suitably protected sialyl TN and 2,6-sialyl T tumor-associated carbohydrate antigen-derived amino acids have been prepared stereoselectively using an oxazolidinone-derived sialoside donor. These glycosylamino acids can be employed directly in the solid-phase synthesis of glycopeptides, as demonstrated by the efficient preparation of tumor-associated MUC1 glycopeptide fragments

Synthesis of a GlcNAcylated arginine building block for the solid phase synthesis of death domain glycopeptide fragments

Herein we describe the synthesis of glycopeptide fragments from the death domains of TRADD and FADD bearing the recently discovered Nω-GlcNAc-β-arginine post-translational modification. TRADD and FADD glycopeptides were accessed through the use of a suitably protected synthetic glycosylamino acid ‘cassette’ that could be directly incorporated into conventional solid phase peptide synthesis (SPPS) protocols

Stereoselective Synthesis of Sialylated Tumor-Associated Glycosylamino Acids

Suitably protected sialyl T_N and 2,6-sialyl T tumor-associated carbohydrate antigen-derived amino acids have been prepared stereoselectively using an oxazolidinone-derived sialoside donor. These glycosylamino acids can be employed directly in the solid-phase synthesis of glycopeptides, as demonstrated by the efficient preparation of tumor-associated MUC1 glycopeptide fragments

Corrigendum: CLE peptide triarabinosylation and peptide domain sequence composition are essential for SUNN-dependent autoregulation of nodulation in Medicago truncatula (vol 218, pg 73, 2018)

Since its publication it has been brought to our attention that the affiliation listed for Nijat Imin is incomplete. Nijat Imin is affiliated to the Division of Plant Science, Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia and the School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand. We apologize to our readers for this mistake