N-methyl Amino Acids Research Articles

Glutathione metabolism links FOXRED1 to NADH:ubiquinone oxidoreductase (complex I) deficiency: A hypothesis

FOXRED1 mutations result in complex I (NADH:ubiquinone oxidoreductase) deficiencies and Leigh syndrome (subacute necrotizing encephalomyelopathy). FOXRED1 is a mitochondrial flavoprotein related to N-methyl amino acid dehydrogenases. How is FOXRED1 required for the biogenesis of complex I? I present a hypothesis that suggests FOXRED1 catalytic activity as a sarcosine oxidase protects the developing fetus from oxidative stress during pregnancy. Loss of FOXRED1, coupled with protein, choline and/or folate-deficient diets results in the depletion of glutathione, the dysregulation of nitric oxide metabolism and the peroxynitrite-mediated inactivation of complex I.

Solid-Phase Synthesis of Triostin A Using a Symmetrical Bis(diphenylmethyl) Linker System.

Triostin A is a symmetric bicyclic depsipeptide with very potent antitumoral activity because of its bisintercalation into DNA. In this study, we report a new synthetic strategy that exploits a structural symmetry of triostin A. First, we prepared a novel symmetric linker molecule that is labile under mildly acidic conditions and suitable for a solid-phase synthesis procedure. Two Cys units were attached to a linker-resin conjugate via their free thiol groups, and double deprotection and double coupling reactions were then applied to synthesize linear tetradepsipeptides. Subsequently, the key biscyclization of the tetradepsipeptides was performed on the resin, and the resulting cyclic octapeptide was detached from the linker-resin conjugate to give a peptide with two free thiols. Finally, triostin A was obtained by oxidizing the free thiols in solution to produce a disulfide. The yield was improved through exploration of two different solid-phase synthetic approaches under similar strategy. Mainly, this strategy was developed to enable the ease and rapid preparation of libraries of symmetric bicyclic depsipeptides. It also addresses several synthetic problems with our synthesis, including diketopiperazine (DKP) formation, poor cyclization yields and preparation of noncommercial N-methyl amino acids in good yields.

X-ray Crystallographic Structures of Oligomers of Peptides Derived from β2-Microglobulin.

Amyloid diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes share common features of toxic soluble protein oligomers. There are no structures at atomic resolution of oligomers formed by full-length amyloidogenic peptides and proteins, and only a few structures of oligomers formed by peptide fragments. The paucity of structural information provides a fundamental roadblock to understanding the pathology of amyloid diseases and developing preventions or therapies. Here, we present the X-ray crystallographic structures of three families of oligomers formed by macrocyclic peptides containing a heptapeptide sequence derived from the amyloidogenic E chain of β2-microglobulin (β2m). Each macrocyclic peptide contains the heptapeptide sequence β2m63-69 and a second heptapeptide sequence containing an N-methyl amino acid. These peptides form β-sheets that further associate into hexamers, octamers, and dodecamers: the hexamers are trimers of dimers; the octamers are tetramers of dimers; and the dodecamers contain two trimer subunits surrounded by three pairs of β-sheets. These structures illustrate a common theme in which dimer and trimer subunits further associate to form a hydrophobic core. The seven X-ray crystallographic structures not only illustrate a range of oligomers that a single amyloidogenic peptide sequence can form, but also how mutation can alter the size and topology of the oligomers. A cocrystallization experiment in which a dodecamer-forming peptide recruits a hexamer-forming peptide to form mixed dodecamers demonstrates that one species can dictate the oligomerization of another. These findings should also be relevant to the formation of oligomers of full-length peptides and proteins in amyloid diseases.

Evolution of FOXRED1, an FAD-dependent oxidoreductase necessary for NADH:ubiquinone oxidoreductase (Complex I) assembly

Complex I (NADH:ubiquinone oxidoreductase) is the major entry point for electrons into the respiratory chains of bacteria and mitochondria. Mammalian complex I is composed of 45 subunits and harbors FMN and iron–sulfur cluster cofactors. A heterogeneous disease profile is associated with complex I deficiency. In a large fraction of complex I deficiencies, the primary defect is not in any of the genes encoding a subunit. The proper assembly and function of complex I require the participation of at least 12 assembly factors or chaperones. FOXRED1 encodes a complex I-specific assembly factor and mutations in this gene result in complex I deficiency, infantile onset encephalomyopathy and Leigh syndrome. The human FOXRED1 protein is a mitochondria-targeted 486-amino acid FAD-dependent oxidoreductase. It is most closely related to N-methyl amino acid dehydrogenases. FOXRED1 orthologs are present in archaea, bacteria and eukaryotes. Fungal FOXRED1 orthologs were likely acquired from alphaproteobacteria by horizontal gene transfer. The phylogenetic profile of FOXRED1 orthologs does not parallel the phylogenetic profile of complex I, strongly suggesting that, at least in some organisms, FOXRED1 has a function unrelated to complex I. The only large clade where all members investigated contain both FOXRED1 and complex I is the metazoans. Some bacterial FOXRED1 genes are present in metabolic operons related to amino acid metabolism. FOXRED1 phylogenetic distribution and gene organization suggest a metabolic role for FOXRED1 in complex I biogenesis should be considered.

The effect of N-methylation of amino acids (Ac-X-OMe) on solubility and conformation: a DFT study.

N-Methylation has a significant impact on improving the oral bioavailability, lipophilicity and aqueous solubility of peptide-based lead drug structures. The selected mono-amino acid derivatives Ac-X-OMe, where X = Gly, Val, Leu, Ile, Phe, Met, Cys, Ser, Asp and His as well as their corresponding N-methylated analogues were studied. The clog P values of all N-methylated peptides are greater than those of native compounds. Quantum chemical calculations were performed to estimate the aqueous solubility of these lipophilic compounds using density functional theory (DFT). To confirm the contribution of dispersion forces on quantum chemical data, the long-range corrected (LC) hybrid density functional (ωB97X-D) was also probed for some amino acid derivatives. The ωB97X functional gave similar results. Our results reveal that after mono N-methylation of the peptide backbone, ΔGsolv becomes more negative (more water soluble) while polarizability and dipole moment are also increased. Natural atomic charges derived by natural bond orbital (NBO) analysis of N, C, and O atoms involved in amide functional group become more positive/(less negative) after N-methylation. All N-methylated amino acids have higher EHOMO (less negative) in comparison with the amino acid analogues, and in all cases N-methylation decreases EHOMO-LUMO. The calculated amide cis/trans activation energies (EA) of all the N-methylated amino acid derivatives were lower than that of native species. N-methylation of these compounds leads to an increase in lipophilicity, aqueous solubility, polarization, dipole moment and lowering of the cis/trans amide energy barrier (EA).

Silver acetate-assisted formation of amides from acyl chlorides

A mild method for the aminolysis of carboxylic acid chlorides to give amides is disclosed. Reactions are carried out in the presence of silver acetate in non-aqueous environments under heterogeneous phase conditions. Amides are easily recovered in very good to excellent yields and without racemization. The approach is successful in forming peptide bonds starting from N-(4-nitrobenzenesulfonyl)-amino acid chlorides and allows the formation of dipeptides also when N-methylated amino acid derivatives are used.

Synthesis of IB-01212 by multiple N-methylations of peptide bonds

There are many natural peptides with multiple N-methylamino acids that exhibit potent attractive biological activities. N-methylation of a peptide bond(s) is also one of the standard approaches in medicinal chemistry of bioactive peptides, to improve the potency and physicochemical properties, especially membrane permeability. In this study, we investigated a facile synthesis process of N-methylated peptides via simultaneous N-methylation of several peptide bonds in the presence of peptide bonds that were not to be methylated. As a model study, we investigated the synthesis of the antiproliferative depsipeptide, IB-01212. We used a pseudoproline to protect the non-methylated peptide bond during a simultaneous N-methylation with MeI–Ag2O. Using further manipulations including a dimerization/cyclization process, IB-01212 and its derivatives were successfully synthesized. A preliminary structure–activity relationship study demonstrated that the symmetric structure contributed to the potent cytotoxic activity of IB-01212.

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed.

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro

Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA(Pro), increases translation incorporation rates of all three studied NMAAs compared with tRNA(Phe)- and tRNA(Ala)-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.

Forcefield_NCAA: ab initio charge parameters to aid in the discovery and design of therapeutic proteins and peptides with unnatural amino acids and their application to complement inhibitors of the compstatin family.

We describe the development and testing of ab initio derived, AMBER ff03 compatible charge parameters for a large library of 147 noncanonical amino acids including β- and N-methylated amino acids for use in applications such as protein structure prediction and de novo protein design. The charge parameter derivation was performed using the RESP fitting approach. Studies were performed assessing the suitability of the derived charge parameters in discriminating the activity/inactivity between 63 analogs of the complement inhibitor Compstatin on the basis of previously published experimental IC50 data and a screening procedure involving short simulations and binding free energy calculations. We found that both the approximate binding affinity (K*) and the binding free energy calculated through MM-GBSA are capable of discriminating between active and inactive Compstatin analogs, with MM-GBSA performing significantly better. Key interactions between the most potent Compstatin analog that contains a noncanonical amino acid are presented and compared to the most potent analog containing only natural amino acids and native Compstatin. We make the derived parameters and an associated web interface that is capable of performing modifications on proteins using Forcefield_NCAA and outputting AMBER-ready topology and parameter files freely available for academic use at http://selene.princeton.edu/FFNCAA. The forcefield allows one to incorporate these customized amino acids into design applications with control over size, van der Waals, and electrostatic interactions.

Abstract C212: Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras.

Abstract Many well-validated oncology targets regulate signaling pathways through interactions with other proteins. Addressing these targets, which often lack suitable small molecule binding sites, requires larger and more complex molecules that are capable of binding relatively large or flat surfaces. We are testing whether this target class can be addressed by using in vitro selection to screen extremely large cyclic peptidomimetic libraries with novel design features. Peptide-mRNA display libraries comprised of approximately 1 trillion cyclic peptidomimetics were created by in vitro translation using a mixture of natural, unnatural, and N-methylated amino acids. Pooled libraries were subjected to multiple rounds of selection and amplification to identify sequences that bound with high affinity to immobilized Mcl-1 or Ras. Selected Mcl-1 cyclic peptidomimetics were synthesized and their binding and specificity were studied by surface plasmon resonance (SPR) using immobilized Mcl-1 and Bcl-2. Fluorescence polarization (FP) was used to demonstrate that isolated peptidomimetics competed with Bim-derived peptides (IC50≈100-400 nM), a measure of their ability to block the interactions between Mcl-1 and binding partners. The co-crystal structure of one Mcl-1 inhibitor revealed that the cyclic peptidomimetic bound the BH3 helix-binding grove by forming a β-hairpin, an unprecedented type of interaction with Bcl-2 family members. The cell permeability of analogs of this compound was verified by fluorescence microscopy and by induction of apoptosis. In a second example, a cyclic peptidomimetic selected against mutant H-Ras was shown to bind both H-Ras and K-Ras with high affinity by SPR (KD=2.5 nM) and FP (Ki≈£10 nM). Despite its high affinity, this compound did not significantly impact Ras activity in several in vitro assays (GST-Raf-RBD binding, GTPase, and GDP/GTP exchange). A co-crystal structure with mutant K-Ras, however, revealed that this compound binds a novel site in the C-terminus of the Ras G-domain. Remarkably, this compound also forms a β-hairpin, and like the Mcl-1 compound, also appears to enter cells. Experiments to test how this compound may affect Ras signaling or localization, and the function of this novel site in cells or in vitro with full-length lipid modified Ras are in progress. The target-bound conformation of both inhibitors reveled a network of intramolecular hydrogen bonds that are predicted to reduce interactions with water and may help to explain the intrinsic cell permeability of these molecules. The use of extremely large peptidomimetic libraries built with novel chemical diversity appears to be an extremely efficient platform for isolating high affinity binders for hard-to-target intracellular targets, yielding novel binding modes and binding sites that are unlikely to be identified by typical small molecule approaches or rational design. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C212. Citation Format: Kristopher Josephson, Zhong Ma, Zhaolin Wang, Yili Sun, Sylvia Tobe, Sarah Perlmutter, Rohit Vyasamneni, Ping Ye, Nicolas Boyer, Micelle Arata, Kelli Pattavina, Kathleen Seyb, Hong Zheng, Imelda Sollomoni, Ezekiel Nims, Emily de Koning, Alonso Ricardo, Douglas Treco. Discovery of high affinity cyclic peptidomimetics binding Mcl-1 and Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C212.

BioArena System for Knowing and Understanding the Biological World: A Review with New Experimental Results

A simple observation is the basis of the development of BioArena system: according to the first observations during the biological incubation after inoculation there is formaldehyde (HCHO) emission from the chromatographic spots; in this emission process, the level of HCHO molecules decreases time dependently. In fact, the antibiotic effect of an antibiotic-like compound decreases in parallel with the HCHO emission. The investigations demonstrated clearly a unique function and role of endogenous HCHO and its one main reaction product, ozone (O3), in the antiproliferative (e.g., antimicrobial) effect of different molecules with diverse chemical structures. The results in BioArena can be extended for in vivo conditions (e.g., greenhouse experiments), as well. For the pretreatment with different doses of inducers (immunostimulation-inducing molecules) there are always four bioequivalent immunostimulating response ranges (quadruple bioequivalent immune response system) in plants. The inducers (e.g., N-methylated basic amino acids, salicylic acid, cinnamic acid, and trace elements) do not participate directly in the induction of the immunostimulating effect. These new findings support a statement that HCHO and its reaction products (mainly O3), as bioreactive small molecules, are responsible for the immunostimulating activity (in vivo conditions), as well.

Dissociation of Antimicrobial and Hemolytic Activities of Gramicidin S through N‐Methylation Modification

β-Sheet antimicrobial peptides (AMPs) are well recognized as promising candidates for the treatment of multidrug-resistant bacterial infections. To dissociate antimicrobial activity and hemolytic effect of β-sheet AMPs, we hypothesize that N-methylation of the intramolecular hydrogen bond(s)-forming amides could improve their specificities for microbial cells over human erythrocytes. We utilized a model β-sheet antimicrobial peptide, gramicidin S (GS), to study the N-methylation effects on the antimicrobial and hemolytic activities. We synthesized twelve N-methylated GS analogues by replacement of residues at the β-strand and β-turn regions with N-methyl amino acids, and tested their antimicrobial and hemolytic activities. Our experiments showed that the HC50 values increased fivefold compared with that of GS, when the internal hydrogen-bonded leucine residue was methylated. Neither hemolytic effect nor antimicrobial activity changed when proline alone was replaced with N-methylalanine in the β-turn region. However, analogues containing N-methylleucine at β-strand and N-methylalanine at β-turn regions exhibited a fourfold increase in selectivity index compared to GS. We also examined the conformation of these N-methylated GS analogues using (1)H NMR and circular dichroism (CD) spectroscopy in aqueous solution, and visualized the backbone structures and residue orientations using molecular dynamics simulations. The results show that N-methylation of the internal hydrogen bond-forming amide affected the conformation, backbone shape, and side chain orientation of GS.

The unique crystallographic signature of a β-turn mimic nucleated by N-methylated phenylalanine and Aib as corner residue: conformational and self-assembly studies

The secondary α-amino acid, proline, having high β-turn forming propensity is known to stabilize discrete conformations in peptides. A similar influence is ascribed to N-methylated amino acids with the advantage of the introduction of a side chain functionality. In this report our effort lies to modulate the conformational analysis (solid phase: x-ray diffraction analysis; solution phase: NMR and CD measurements) of two terminally protected synthetic tripeptides Boc-N-MePhe-Aib-Xx-OMe, where Xx is Phe in peptide I and mABA (mABA: meta amino benzoic acid) in peptide II, forming β-turns stabilized by 4 → 1 intramolecular hydrogen bonding. Up to now, a significant amount of work has been dedicated to employing different strategies in stabilizing β-turn mimics. However, to the best of our knowledge, these novel examples represent a unique crystallographic evidence of a β-turn mimic nucleated by an N-methylated amino acid (N-methylated phenylalanine) and Aib as corner residues and stabilized by the conformational features of the acyclic tripeptides. The literature documentation further reveals that the aromatic residue containing the amyloidogenic peptide calcitonin, consists of ribbon like structures, which twist back upon themselves to form a tubular ensemble. Interestingly, the FESEM images of peptide II shows a ribbon like morphology, resulting due to the aggregation of supramolecular helices in higher order self-assembly. Importantly, peptide I which also produces supramolecular helices does not produce any type of morphology under similar conditions emphasizing the importance of the location of the aromatic residue in the peptide sequence in generating a particular morphological structure. This report may open a new avenue in introducing β-turn motifs, replacing proline turns, in the bioactive conformation of selected peptides. They may also assist in understanding the structure and function of misfolded disease causing peptides like prion and Alzheimer's amyloid.

Synthesis, Structure–Activity Analysis, and Biological Evaluation of Sanguinamide B Analogues

We report the first synthesis of sanguinamide B analogues. Substituting N-methylated (N-Me) amino acids, glycine (Gly), and L- or D-phenylalanine (Phe) into the backbone of sanguinamide B showed that only l- and d-Phe residues controlled the macrocycle conformation. The N-methylated and glycine analogues all had multiple conformations, whereas the L- and D-Phe derivatives only had a single conformation. Testing of all conformer analogues showed that inclusion of an L- or D-Phe was a superior design element than incorporating the N-Me moiety that is often utilized to control macrocyclic conformation. Finally, we show that there is an ideal Phe residue (in this case L-Phe) for generating compounds that have the greatest inhibitory effect on bacterial motility. Our data support the hypothesis that the macrocyclic conformation is dictated by the benzyl moiety requiring a "pseudoequatorial" position, and all other energy considerations are secondary.

Studies on the biosynthesis of chetomin: enantiospecific synthesis of a putative, late-stage biosynthetic intermediate

The enantiospecific synthesis of desthiochetomin, a putative biosynthetic intermediate of the epidithiodioxopiperazine natural product chetomin, is described. A diastereoselective N-alkylation was employed to form the key C3–N1′ bond of the heterodimeric indoline core, followed by peptide coupling and dioxopiperazine cyclization with the requisite N-methyl amino acids. A related sarcosine-derived dioxopiperazine was prepared in the same manner. The first proposed biosynthesis of chetomin is also detailed in the text.

Revisiting the enniatins: a review of their isolation, biosynthesis, structure determination and biological activities.

Enniatins are cyclohexadepsipeptides isolated largely from Fusarium species of fungi, although they have been isolated from other genera, such as Verticillium and Halosarpheia. They were first described over 60 years ago, and their range of biological activities, including antiinsectan, antifungal, antibiotic and cytotoxic, drives contemporary interest. To date, 29 enniatins have been isolated and characterized, either as a single compound or mixtures of inseparable homologs. Structurally, these depsipeptides are biosynthesized by a multifunctional enzyme, termed enniatin synthetase, and are composed of six residues that alternate between N-methyl amino acids and hydroxy acids. Their structure elucidation can be challenging, particularly for enniatins isolated as inseparable homologs; however, several strategies and tools have been utilized to solve these problems. Currently, there is one drug that has been developed from a mixture of enniatins, fusafungine, which is used as a topical treatment of upper respiratory tract infections by oral and/or nasal inhalation. Given the range of biological activities observed for this class of compounds, research on enniatins will likely continue. This review strives to digest the past studies, as well as, describe tools and techniques that can be utilized to overcome the challenges associated with the structure elucidation of mixtures of enniatin homologs.

Proton and potassium affinities of aliphatic and N-methylated aliphatic α-amino acids: Effect of alkyl chain length on relative stabilities of K+ bound zwitterionic complexes

The relative and absolute K+ affinities of a full series of aliphatic amino acids (Gly, Ala, Val, Leu, Ile and α-amino-isobutyric acid (α-AiB)) and N-methylated aliphatic amino acids (N-Me-(Gly/Ala/α-AiB/Val/Leu)) were investigated theoretically by density functional theory calculations at the B3–LYP/6-311+G(3df,2p)//B3-LYP/6-31G(d) level, and experimentally measured by the standard mass spectrometric kinetic method. Very good quantitative agreement was found between the theoretical and experimental results. The experimental K+ affinities were found to be consistent with the most stable charge-solvated CS1 binding mode (bidentate form with K+ bonded between two carboxylic oxygens, OC, OH) for aliphatic amino acids, and the zwitterionic ZW1 binding mode (bidentate form with K+ bonded between two carboxylate oxygens, COO−) for the N-methylated aliphatic amino acids. A steeper rising trend in ZW1 stabilities (affinities) relative to that of CS1 was found in the smaller aliphatic amino acids (Gly/Ala/α-AiB), but leveling off among the larger aliphatic amino acids (Val/Leu/Ile). This observed trend was rationalized in terms of greater deformation energy, Edef, found for the smaller Gly/Ala, and the leveling off of stabilizing ion-induced dipole (molecular polarizability) contributions from the non-interacting alkyl side chain in the larger aliphatic amino acids. The leveling off effect is aided by the relatively large size and soft, not so polarizing nature of the potassium cation. A good linear correlation (r2≥0.95) was found between the proton affinities (PA) and the ZW1/CS1K+ affinities, but because of the leveling off effect, the cross over or transition from CS1 to ZW1K+ binding mode was not found for isoleucine.

A Two‐Step Strategy for Structure–Activity Relationship Studies of N‐Methylated Aβ42 C‐Terminal Fragments as Aβ42 Toxicity Inhibitors

Neurotoxic Aβ42 oligomers are believed to be the main cause of Alzheimer's disease. Previously, we found that the C-terminal fragments (CTFs), Aβ(30-42) and Aβ(31-42) were the most potent inhibitors of Aβ42 oligomerization and toxicity in a series of Aβ(x-42) peptides (x=28-39). Therefore, we chose these peptides as leads for further development. These CTFs are short (12-13 amino acids) hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the N terminus resulted in toxic peptides. Therefore, we next incorporated N-methyl amino acids, which are known to increase the solubility of such peptides by disrupting the β-sheet formation. Focusing on Aβ(31-42), we used a two-step N-methyl amino acid substitution strategy to study the structural factors controlling inhibition of Aβ42-induced toxicity. First, each residue was substituted by N-Me-alanine (N-Me-A). In the next step, in positions where substitution produced a significant effect, we restored the original side chain. This strategy allowed exploring the role of both side chain structure and N-Me substitution in inhibitory activity. We found that the introduction of an N-Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aβ(31-42). In particular, N-Me amino acid substitution at position 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aβ42 toxicity by short peptides is highly structure-specific, providing a basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.

Anthelmintic PF1022A: stepwise solid-phase synthesis of a cyclodepsipeptide containing N-methyl amino acids

Cyclodepsipeptides of the enniation-, PF1022-, and verticilide-family represent a diverse class of highly interesting natural products with respect to their manifold biological activities. However, until now no stepwise solid-phase synthesis has been accomplished due to the difficult combination of N-methyl amino acids and hydroxycarboxylic acids. We report here the first stepwise solid-phase synthesis of the anthelmintic cyclooctadepsipeptide PF1022A based on an Fmoc/THP-ether protecting group strategy on Wang-resin. The standard conditions of our synthesis allow an unproblematic adaption to an automated peptide synthesizer.