Solution-phase Peptide Synthesis Research Articles

Alanine Scan of the Peptide Antibiotic Feglymycin: Assessment of Amino Acid Side Chains Contributing to Antimicrobial Activity

The antibiotic feglymycin is a linear 13-mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution-phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di- and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D-Hpg1, L-Hpg5, and L-Phe12, the inhibitory activity against MurA and MurC depends mainly on L-Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin.

Ethyl 2‐(tert‐Butoxycarbonyloxyimino)‐2‐cyanoacetate (Boc‐Oxyma) as Coupling Reagent for Racemization‐Free Esterification, Thioesterification, Amidation and Peptide Synthesis

AbstractHere we report the synthesis and utility of ethyl 2‐(tert‐butoxycarbonyloxyimino)‐2‐cyanoacetate (Boc‐Oxyma) as an efficient coupling reagent for racemization‐free esterification, thioesterification, amidation reactions and peptide synthesis that uses equimolar amounts of acids and alcohols, thiols, amines or amino acids, respectively. Its application to solid phase as well as solution phase peptide synthesis is also demonstrated and a mechanistic investigation is discussed. Boc‐Oxyma is similar to the well known coupling agent COMU {1‐[1‐cyano‐2‐ethoxy‐2‐oxoethylideneaminooxy)‐dimethylaminomorpholino]uronium hexafluorophosphate} in terms of its high reactivity and mechanism of action. However, it is not only much easier to prepare, but also to recover and reuse, thereby generating far less chemical waste.

C → N and N → C solution phase peptide synthesis using the N-acyl 4-nitrobenzenesulfonamide as protection of the carboxylic function

In this paper we describe a solution phase peptide synthesis strategy using the 4-nitrobenzenesulfonamido/N-methyl-4-nitrobenzenesulfonamido group as a protecting/activating system of the carboxyl function. The 4-nitrobenzenesulfonamido group is stable during peptide chain elongation (Fmoc chemistry). The N-aminoacyl or N-dipeptidyl-4-nitrobenzensulfonamides, when activated by methylation, can be easily coupled with another amino acid or reconverted into the free-carboxyl function amino acids or peptides. This activatable protecting group allows both the C → N and the N → C direction solution phase peptide synthesis. We also verified that the absolute configuration at the chiral centers does not change during the coupling reactions.

Microwave-assisted solution phase peptide synthesis in neat water

An environmentally benign protocol for solution phase peptide synthesis has been developed in neat water using TBTU/HOBt/DIEA as a coupling combination under microwave irradiation. Key features of this procedure are the replacement of commonly used toxic organic solvents like DMF and NMP, the use of lower amounts of reactants, compatibility with both N-α-Boc- and N-α-Fmoc-protected amino acids and all commonly used side-chain protective groups, short reaction time, and racemization-free synthesis in high yield and purity.

Rapid microwave-assisted solution-phase peptide synthesis

Rapid microwave-assisted solution-phase peptide synthesis protocol has been developed. The reaction temperature of 50°C, 40W and short coupling time of 28min gave racemization-free synthesis of peptides in 50–80% yield. The method is applicable with equal ease in coupling both side-chain protected and unprotected amino acids indicating reactive-functional group tolerance and high atom-economy. In the case of dipeptides, DIC/HONB-mediated coupling rendered the best results. In the case of tri- and tetrapeptides, HATU/HOAt/DIEA emerged as the best coupling combination.

Facile synthesis and anticancer activity of C-10 non-acetal deoxoartemisinin dimers

In this research, N-(2-aminoethyl)glycine-linked C-10 non-acetal deoxoartemisinin dimers were synthesized by using solution phase peptide synthesis approach. In addition, chemical modification of the C-10 non-acetal deoxoartemisinin monomers and dimers by adding a lysine unit to the N-terminus has been performed. The biological activities of all synthesized compounds were evaluated against the colon cancer cell line (Caco-2). The non-acetal deoxoartemisinin monomers 12a, 15a–c and dimers 13a, 16a–c were active against Caco-2 cells and more potent than dihydroartemisinin.

Development and characterization of anionic liposaccharides for enhanced oral drug delivery

The aim of this study was to synthesize charged amphoteric molecules, which after complexation with poorly bioavailable drugs would have the potential to improve their oral uptake. Novel anionic liposaccharide derivatives containing d-glucose and lipoamino acids were synthesized by solution phase peptide synthesis. High sensitivity isothermal titration microcalorimetry was used to determine the critical aggregation concentration and the thermodynamic profiles. Hemolytic and cytotoxic activities of the liposaccharides were studied and they revealed that the liposaccharides were non-toxic at the concentration used for oral administration. Mixing a model drug, tobramycin, with the liposaccharide containing two lipids formed aggregates around 200nm, which increased tobramycin partitioning between n-octanol/water. The results suggested that the studied liposaccharide with two lipids was safe to apply biologically and may have an absorption enhancing activity on hydrophilic, orally poorly available drugs.

S-2-Amino-4-cyanobutanoic acid (β-cyanomethyl-l-Ala) as an atom-efficient solubilising synthon for l-glutamine

Glutamine (Gln) is often a difficult amino acid to incorporate during solution-phase peptide synthesis, owing to poor solubility and unwanted dehydrations as side-reactions. Current approaches to solving these problems are highly atom-inefficient. Nα-Cbz-β-cyanomethyl-l-Ala is readily accessible by dehydration of Cbz-l-Gln. β-Cyanomethyl-l-Ala can be incorporated into short peptides easily by conventional methods. The nitrile is stable to the hydrogenolysis conditions used to remove Cbz and to acidic deprotection but is quantitatively hydrated to the γ-carboxamide of l-Gln with hydrogen peroxide. Thus β-cyanomethyl-l-Ala may represent a new, soluble, perfectly atom-efficient synthon for l-Gln.

Applications of Propargyl Esters of Amino Acids in Solution-Phase Peptide Synthesis

Propargyl esters are employed as effective protecting groups for the carboxyl group during solution-phase peptide synthesis. The propargyl ester groups can be introduced onto free amino acids by treating them with propargyl alcohol saturated with HCl. The reaction between propargyl groups and tetrathiomolybdate is exploited to deblock the propargyl esters. The removal of the propargyl group with the neutral reagent tetrathiomolybdate ensures that most of the other protecting groups used in peptide synthesis are untouched. Both acid labile and base labile protecting groups can be removed in the presence of a propargyl ester. Amino acids protected as propargyl esters are employed to synthesize di- to tetrapeptides in solution-phase demonstrating the possible synthetic utilities of the methodology. The methodology described here could be a valuable addition to currently available strategies for peptide synthesis.

Peptide synthesis ‘in water’ by a solution‐phase method using water‐dispersible nanoparticle Boc‐amino acid

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in-water solution-phase peptide synthesis method using Boc-amino acids. It is based on a coupling reaction utilizing suspended water-dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity.

Abstract P4-08-11: Surface Plasmon Resonance (SPR) Analysis of COX-2 Expression for Early Detection and Progression of Breast Cancer and Evaluation of a Novel Class of Peptides as Its Inhibitor

Abstract Background: Cyclooxygenase-2 (COX-2) over expression has been observed in different cancers. Till date, COX-2 expression in breast cancer has been studied using RT-PCR and immunohistochemistry. The aim of this study was to use surface plasmon resonance (SPR) technology for determining the expression levels of COX-2 in breast cancer and design peptide inhibitors to evaluate their enzyme inhibition in vitro. Patients and methods: This case-controlled study was performed on 84 biopsy proven breast cancer patients having invasive ductal carcinoma and 40 age, sex and ethnicity matched healthy subjects. Blood and tissue samples were collected and serum and tissue lysate was prepared. Serum and tissue levels of COX-2 were evaluated using SPR where samples were allowed to flow over anti-COX-2 antibody immobilized sensor chip to obtain the respective RUs. Similarly, standard curve of COX-2 was prepared from cloned, expressed and purified protein to deduce the concentrations in different samples. To evaluate enzyme inhibition, fifteen peptides were synthesized using the solid and solution-phase peptide synthesis based on the active site structure of COX-2 and enzyme activity was evaluated spectrophotometrically. Results: A significant increase in COX-2 levels in breast cancer patients 11.23-30.77μg/ml (Mean±SD=14.55±5.74) as compared to normal controls 2.13-5.16µg/ml (Mean±SD=3.71±0.86) (P>0.0001) was observed. Serum COX-2 levels were significantly higher (P<0.0061) in patients with lymph node involvement. More than 65% patients showed significantly (P<0.0025) reduced COX-2 levels after chemotherapy. This was confirmed by western blot to check tissue level expression and spectrophotometrically to determine the activity of COX-2 at different stages. In addition, the peptides showed significant in vitro enzyme inhibition, and had a KD ranging from 1 X 10-8 to 1X10-10. Conclusion: SPR is a useful proteomic technique for early detection and monitoring progession of breast cancer and peptides are a novel class of COX-2 inhibitors that can be further developed for anti-tumor therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-11.

Organic Solvent Nanofiltration: A New Paradigm in Peptide Synthesis

Solid-phase synthesis is the dominant paradigm for peptide synthesis, used ubiquitously from discovery to production scale. However, the solid-phase approach produces coupling steps that may not be quantitative, introducing errors in amino acid sequences. It also entails an excess of reagents to overcome mass transfer limitations and restrictions on solvent, coupling chemistry, and protecting groups. Organic solvent nanofiltration (OSN) is a newly emerging technology capable of molecular separations in organic solvents. This contribution reports a new technology platform which advantageously combines OSN with solution-phase peptide synthesis, Membrane Enhanced Peptide Synthesis (MEPS). A first amino acid is linked to a soluble polyethylene glycol anchor. Through subsequent repeated coupling and deprotection steps, the peptide is extended to the desired length. The residual byproducts and excess reagents after each reaction are removed by diafiltration through a solvent-stable membrane which retains the peptide. Two pentapeptides are produced using this new technology. The purity of the peptides produced by MEPS is higher than that of peptides produced by solid-phase synthesis, under the same conditions. This illustrates clearly that MEPS benefits from the advantages of solution-phase synthesis, while avoiding the purification steps that have until now made solution-phase synthesis practically difficult.

Ethyl 2-Cyano-2-(hydroxyimino)acetate (Oxyma): An Efficient and Convenient Additive Used with Tetramethylfluoroformamidinium Hexafluorophosphate (TFFH) to Replace 1-Hydroxybenzotriazole (HOBt) and 1-Hydroxy-7-azabenzotriazole (HOAt) during Peptide Synthesis

Abstract The appropriateness of ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) as a substitute for benzotriazole-based additives, for use in the TFFH approach for peptide synthesis, is discussed in terms of its capacity to control racemization, its coupling efficiency in difficult couplings either for stepwise or segment coupling in solution- and solid-phase coupling. In addition, Boc-based solution-phase peptide synthesis and its stability in the presence of growing peptide chains were studied. Oxyma displayed remarkable results in terms of racemization depression together with impressive coupling efficiency in both solution- and solid-phase synthesis. Furthermore, Oxyma suggests a lower risk of explosion than HOBt and HOAt.

ChemInform Abstract: Synthesis of the Simple Peptide Model Ac-Abu(PO3H2)-NHMe.

The simple model substrate AC-L-Abu(PO3H2)- NHMe was prepared by the use of the protected 4( diethylphosphono ) butanoic acid derivative Boc -Abu(PO3Et2)-OH in the Boc mode of solution phase peptide synthesis. The protected peptide model Ac-Abu(P03Et2)- NHMe was prepared by initial reaction of the isobutoxycarbonyl mixed anhydride of Boc-Abu(PO3Et2)-OH with N-methylamine followed by cleavage of the Boc group from Boc -Abu(PO3Et2)- NHMe with 4 M HCl/dioxan and N- acetylation of H-Abu(PO3Et2)-NHMe.HCl with the isobutoxycarbonyl mixed anhydride of acetic acid. Cleavage of the phosphonate ethyl groups was effected with 33% hydrogen bromidelacetic acid or 10% bromotrimethylsilane/acetonitrile to give AC-L-Abu(PO3H2)-NHMe in near-quantitative yield.

Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide δ- l-α-aminoadipoyl- l-cysteinyl- d-valine ( lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme. δ- l-α-Aminoadipoyl- l-cysteinyl- d-2-amino-3,3-dideuteriobutyrate ( lld-AC d 2Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer δ- l-α-aminoadipoyl- l-cysteinyl- l-2-amino-3,3-dideuteriobutyrate ( lll-AC d 2Ab) has the opposite configuration at its third amino acid. lll-ACV has previously been shown to inhibit IPNS turnover of its substrate lld-ACV; the all-protiated tripeptide δ- l-α-aminoadipoyl- l-cysteinyl- d-2-aminobutyrate ( lld-ACAb) is a substrate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the crystal structures of the IPNS:Fe(II): lld-AC d 2Ab and IPNS:Fe(II): lll-AC d 2Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.

Cyclic Peptide Synthesis with Thioacids

C-Terminal amino acid 9-fluorenylmethylthioesters may be carried through Boc chemistry solution phase peptide synthesis sequences. After insertion of the final residue in the form of an Fmoc carbamate, treatment with piperidine releases a seco-peptide as a C-terminal thioacid that on treatment with Sanger's reagent undergoes cyclization to a cyclic peptide. Cyclic penta- and hexapeptides have been synthesized in this manner, as has a cyclic glycopeptide. Functional group compatibility with alcohols and carboxylic acids is demonstrated.

Foldamers of bifunctional diketopiperazines displaying a β-bend ribbon structure

The synthesis of two oligomers, of a bifunctional diketopiperazine scaffold DKP-1, formally derived from the cyclization of l-aspartic acid and ( S)-2,3-diaminopropionic acid, is reported. A trimeric and a tetrameric structure were prepared by solution-phase peptide synthesis (Boc strategy). Conformational analysis of these derivatives was carried out by a combination of 1H NMR spectroscopy, CD spectroscopy, and molecular modeling, and revealed the formation of β-bend ribbon involving 10-membered H-bonded rings and a reverse turn of the growing peptide chain.

Synthesis and electrochemical studies of disubstituted ferrocene/dipeptide conjugates with sulfur-containing side chains

A series of 1,1′-disubstituted ferrocenoyl peptides incorporating dipeptide sidearms has been synthesized and studied electrochemically. The target peptides include ferrocene as an electrochemical reporter, sulfur-containing amino acids ( l-methionine, S-methyl- l-cysteine, S-trityl- l-cysteine, S-benzhydryl- l-cysteine) as metal binding agents, and amino acids with non-polar side chains ( l-alanine, l-valine, l-phenylalanine) as spacers between reporter and metal binding groups. Ferrocene/dipeptide conjugates were prepared using solution phase peptide synthesis methods employing a BOC-protecting group strategy and HBTU- ( O-(benzotriazol-1-yl)- N, N, N′, N′-tetramethyluronium hexafluorophosphate) mediated peptide coupling. The electrochemical properties of these 1,1′-substituted ferrocenoyl peptides have been characterized using cyclic voltammetry. All exhibit fully reversible one electron oxidation steps; forward sweep half wave peaks ( E F), reverse sweep half wave peaks ( E R), peak separations (Δ E P) and half wave potentials ( E 1/2) are reported. Finally, towards the goal of utilizing ferrocenoyl peptides to detect heavy metals in solution, the response of these ferrocene/dipeptide conjugates to metal cations (zinc(II), mercury(II), cadmium(II), lead(II), silver(I)) has been examined. Monitoring changes in the potential of the Fe(II)/Fe(III) redox couple to follow peptide/metal interactions, we have probed the influence of the spacer unit between the redox reporter and the metal-binding amino acid, and shown that these systems respond to mercury(II) more strongly than to other heavy metal ions.

Sugar amino acid based peptide epoxyketones as potential proteasome inhibitors

This paper describes the synthesis and biological evaluation of nine epoxomicin-derived sugar amino acid containing peptide epoxyketones. The title compounds are assembled from six sugar amino acid dipeptide isosteres and are synthesized using solution-phase peptide synthesis protocols. Although neither of the compounds displays inhibitory activity towards any of the proteasome active sites, our approach holds promise towards the development of structurally new proteasome inhibitors. It is likely that the central sugar amino acid dipeptide isoster needs to be designed such that it closely resemble dipeptides at position P2 and P3 in proteasome substrates inhibitors, such as the Thr-Ile dipeptide present in the lead compound, epoxomicin.

Preparation of the caspase-3/7 substrate Ac-DEVD-pNA by solution-phase peptide synthesis

This protocol describes the gram-scale solution-phase synthesis of the colorimetric caspase-3/7 substrate Ac-DEVD-pNA. The caspase enzymes are integral to cellular inflammation and apoptotic cascades, and are commonly studied by cell biologists, medicinal chemists and chemical biologists. In particular, the assessment of caspase enzymatic activity is a standard method to evaluate cell death pathways and new apoptosis-modulating agents. Caspase enzymatic activity can be conveniently monitored with peptidic chromogenic or fluorogenic substrates, with certain peptide sequences imparting selectivity for certain caspases. The synthesis of these peptide substrates is typically carried out by solid-phase synthesis, a method that is not ideal for production of the gram quantities needed for high-throughput screening. Described herein is a facile method for the synthesis of the Ac-DEVD-pNA caspase-3/7 substrate using solution-phase peptide synthesis. This protocol, involving iterative PyBOP-mediated couplings and Fmoc deprotections, is rapid (about 5 d), operationally simple and can be used to generate over 1 g of product at a fraction of the cost of the commercial substrate.