Peptide Synthesis Conditions Research Articles

A Convenient On-Site Oxidation Strategy for the N-Hydroxylation of Melanostatin Neuropeptide Using Cope Elimination

AbstractA convenient synthetic protocol for the unprecedented N-hydroxylation of proline residue in Melanostatin (MIF-1) neuropeptide is reported. This methodology is grounded on the incorporation of N-(cyanoethyl)prolyl residue followed by on-site oxidation by Cope elimination with m-chloroperbenzoic acid, exploring the unrecognized dual role of the cyanoethyl group as an effective N-protecting group under peptide synthesis conditions and as a suitable leaving group during the chemoselective on-site N-oxidation. Following this protocol N-hydroxy-MIF-1 is obtained in 78% global yield from N-(cyanoethyl)-l-proline. This synthetic approach opens a new avenue for access to N-hydroxylated Melanostatin analogues with direct application in neurochemistry and Parkinson’s research.

Synthesis of a Photo‐Caged DOPA Derivative by Selective Alkylation of 3,4‐Dihydroxybenzaldehyde

Natural and synthetic polymers containing the catechol moiety of noncoded amino acid 3,4‐dihydroxyphenylalanine (DOPA) are capable of metal‐coordination and adhesion under wet conditions. Masking the catechol subunit with a photo‐cleavable group would provide an opportunity to design tunable adhesion properties that are especially important for biomaterial and biomedicine applications. Herein, we report the regioselective synthesis of a photo‐caged DOPA bearing an ortho‐nitrobenzyl (oNB) group that is capable of undergoing cleavage upon irradiation with UV light. We developed a selective synthetic route towards a 3‐O‐oNB alkylated DOPA regioisomer that can be readily incorporated into proteins by using a previously developed bio‐expression platform. The synthesis is based on a regioselectivity switch in 3,4‐dihydrozybenzaldehyde alkylation upon application of different equivalents of deprotonating base. The enantiomerically pure 3‐O‐oNB‐DOPA was prepared on a gram scale and proved to be generally compatible with the solid‐phase peptide synthesis conditions. We also demonstrate the general applicability of the developed synthetic strategy by providing the synthesis of 3‐O‐methyl‐DOPA.

Enantioselective Synthesis of (2S,3S)‐epi‐Oxetin and Its Incorporation into Conformationally Constrained Pyrrolidinyl PNA with an Oxetane Backbone

AbstractFmoc‐protected (2S,3S)‐epi‐oxetin was synthesized from (E)‐4‐(benzyloxy)but‐2‐enal via enantioselective organocatalytic epoxidation, epoxide ring opening with azide, alcohol activation and ring closure, followed by functional‐group manipulation in eight steps with 12 % overall yield and 94 % ee. The amino acid was used as a building block for a new conformationally constrained pyrrolidinyl PNA with an oxetane‐containing backbone. The unexpected sensitivity of the oxetane backbone posed considerable synthetic challenges under standard Fmoc‐solid‐phase peptide synthesis conditions, and a mechanism for acid‐catalyzed degradation was proposed. In addition, the DNA‐ and RNA‐binding properties of the oxetane PNA were investigated. The presence of an oxetane ring decreased the stability of the PNA⋅DNA and PNA⋅RNA duplexes when compared to PNA with a cyclobutane‐containing backbone, which could be explained by the flattening of the oxetane ring, leading to a suboptimal torsional angle.

Microwave-assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis.

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use -Alloc and -OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and -OAllyl may be removed using Pd(PPh3 )4 and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent-intensive. Herein, we report the development of a method in which the -Alloc/-OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent-intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5-min deprotections at 38°C, all -Alloc and -OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis of PtIV‐Biomolecule Conjugates through Click Chemistry

AbstractThe azide‐functionalized PtIV complex {(OC‐6–34)‐chlorido(cyclobutane‐1,1′‐dicarboxylato)(cyclohexane‐1R,2R‐diamine)[2‐hydroxyethyl(2‐{2‐[2‐(2‐azidoethoxy)ethoxy]ethoxy}ethyl)carbamate]platinum(IV)} was synthesized and characterized to obtain a starting complex that was suitable for coupling with appropriate biological carriers in a drug targeting and delivery strategy. The following click reaction (CuI‐catalyzed Huisgen cycloaddition, CuAAC) was successfully applied for coupling with three different model biomolecules that can be exploited for selective targeting of the platinum conjugate toward tumor cells; i.e., the amino acid alanine and the dipeptide lysine‐alanine, both previously alkyne‐functionalized with pent‐4‐ynoic acid, and the hormone 17α‐ethynylestradiol. The title complex demonstrated very good compatibility with both CuAAC reaction and solid‐phase peptide synthesis conditions, making it a suitable antiproliferative fragment for the design of nanovectors.

Synthesis of 'head-to-tail' cyclized peptides on solid support using a chelating amide as new orthogonal protecting group.

The synthesis of 'head-to-tail' cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2-pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa-protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)2 and 2-(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of 'head-to-tail' cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides.

Facile Synthesis of N-(9-Fluorenylmethyloxycarbonyl)-3-amino-3-(4,5-dimethoxy-2-nitrophenyl)propionic Acid as a Photocleavable Linker for Solid-Phase Peptide Synthesis

A photocleavable linker, N -(9-fluorenylmethyloxycarbonyl)-3-amino-3-(4,5-dimethoxy-2-nitrophenyl)propionic acid was synthesized from veratraldehyde, with simple reaction and separation steps. This linker was stable under the normal solid-phase peptide synthesis conditions and rearranged to a labile form when irradiated with UV light (λ = 365 nm). A model peptide sequence (YGGFL) was synthesized and released from solid supports with high efficiency by UV irradiation.

ChemInform Abstract: Challenges in the Syntheses of Peptidic Natural Products

AbstractReview: ca. 100 refs.

Challenges in the Syntheses of Peptidic Natural Products

Peptidic natural products are commonly perceived as ‘simple’ molecules that are easily accessible by standard methods. This review sets out to demonstrate that the total synthesis of peptidic natural products is far from being ‘standard’ and highly elaborate methodologies paired with careful synthesis planning are required to attain their often complex structures. 1 Introduction 2 Peptide Synthesis with Amino Acids Incompatible with ‘Standard’ Peptide Synthesis Conditions: The Synthesis of Feglymycin and Tubulysin D 3 Macrocyclizations as Pitfalls in the Synthesis of Cyclo(depsi)peptides: The Synthesis of Largazole 4 Synthesis of Cyclo(depsi)peptides via Carbon–Carbon Bond-Forming Macrocyclizations: The Synthesis of Chondramide C and Syringolin A 5 Control of Atropisomerism in Side-Chain-Cyclized Peptidic Natural Products: The Synthesis of Chloropeptin, Complestatin (Chloropeptin II) and Isocomplestatin 6 Solid-Phase Syntheses of Peptidic Natural Products: The Synthesis of Oxathiocoraline and Symplocamide A 7 Structural Complexity as a Challenge: The Synthesis of Kapakahines 8 Conclusions

4-Methoxybenzyloxymethyl group as an Nπ-protecting group for histidine to eliminate side-chain-induced racemization in the Fmoc strategy

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the N π-position of histidine, and its utility was examined under the conditions for peptide synthesis by Fmoc strategy. The N π-MBom group proved to prevent the risk of racemization during incorporation of the His residue and to possess all of the chemical properties required for Fmoc chemistry. The side reaction associated with formaldehyde generated from the N π-MBom group upon acidolysis could be effectively prevented by performing the standard TFA treatment in the presence of methoxyamine·hydrochloride (MeONH 2·HCl).

Automated Maskless Photolithography System for Peptide Microarray Synthesis on a Chip

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.

Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Resin‐Bound Aminofluorescein for C‐Terminal Labeling of Peptides: High‐Affinity Polarization Probes Binding to Polyproline‐Specific GYF Domains

A polymer support for the solid-phase synthesis of C-terminally labeled carboxylic acids has been developed. Fluorophore-labeled peptides were constructed directly on the amino group of resin-bound aminofluorescein. Fmoc-protected aminofluorescein was coupled onto tritylpolystyrene, and the free phenolic hydroxyl positions of the fluorescein were blocked with suitable protecting groups. The mode of attachment was analyzed and found to be selective for the phenoxy ether linkage. The conditions for peptide synthesis on the labeling resin were investigated, and a small library of C-terminally labeled peptides was prepared. The fluorescence quantum yields of C-terminally labeled peptides were determined and indicated the suitability of the compounds for imaging and binding experiments. The obtained peptides were therefore investigated as fluorescence polarization probes. Two different proline-rich binding domains of the GYF family-CD2BP2 and PERQ2-were targeted by peptides labeled either C- or N-terminally. Reversible binding constants were determined by fluorescence polarization measurements and were verified by competition experiments with the corresponding unlabeled peptide. As a second control, the binding constants were measured by NMR titration experiments, recording the HSQC NMR spectra of (15)N-labeled proteins in the presence of the peptide polarization probes. Ligands with higher affinities than all others known previously were identified for both GYF domains. The competition assay with the developed fluorescent probe has a high statistical reliability and can thus be used for screening of GYF domain inhibitors.

A class of novel nitronyl nitroxide labeling basic and acidic amino acids: Synthesis, application for preparing ESR optionally labeling peptides, and bioactivity investigations

Aimed at optional ESR label 2-(4′-hydroxyl)phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl was introduced into the guanido of l-Arg-OH, the ω-amino group of l-Lys-OH with methylcarboxyl as a linker, and into the β-carboxyl of l-Asp-OH and the γ-carboxyl of l-Glu-OH with ethylamino as a linker. It was explored that the synthetic 30 novel ESR labeling amino acid derivatives were stable enough to the reaction conditions of peptide synthesis. Their incorporation led to 12 novel ESR optionally labeling PAK, RGDS, RGDV, and ECG. A series of NO related chemical tests, the in vitro and in vivo assays of these peptides confirmed that this strategy was practical.

Peptide ormosils as cellular substrates

Peptide-functionalized thin films exhibit significant potential for integration into implantable devices and cell-based technologies. A new type of neuroactive peptide-modified silica was developed using sol–gel reaction chemistry to produce thin films from four different peptide silane precursors. Peptide silanes containing binding sequences from laminin (YIGSR and KDI), fibronectin (RGD), and EGF repeats from laminin and tenascin (NID) were produced using standard solid-state FMOC peptide synthesis conditions and the covalent attachment of 3′-(aminopropyl)trimethoxysilane (APTMS), using carbonyldiimadazole (CDI) as a linking molecule. Precursor formation was confirmed with MALDI-MS. Thin films were produced by dip-coating using the peptide precursors combined with hydrolyzed tetramethoxysilane. Atomic force microscopy indicated that the surface topography was not affected by low concentrations of peptide precursor (0.0025 mol%), but higher concentrations of peptide precursor (0.01 mol%) resulted in features that were 50–75 nm in height. The height features observed were consistent in size with previously determined topographical morphology supportive of neuronal cell lines. The surfaces were biologically active and modulated the phenotype of the embryonic carcinoma stem cell line, P19. Combinations of the peptide silanes resulted in altered cell types after retinoic acid treatment. More neurons were observed on RGD/YIGSR and RGD/YIGSR/NID surfaces compared to tetramethoxysilane (TMOS) controls. More supporting cells were observed compared to collagen coated tissue culture plates. In addition, neurites were significantly longer on the peptide ormosils compared to controls. This work demonstrates a novel method for producing biologically active peptide ormosils using peptide-modified precursors.

Solid-Phase Synthesis of 1,3-Azole-Based Peptides and Peptidomimetics

[reaction: see text] We report highly efficient two-step procedures for the synthesis of 1,3-oxazole-, thiazole-, and imidazole-containing peptides on solid phase from dipeptides composed of C-terminal threonine, serine, cysteine, or diaminopropionic acid by using different cyclodehydration procedures followed or preceded by oxidation. The methods are compatible with Fmoc solid-phase peptide synthesis conditions and with N-Fmoc, N-Boc, N-Cbz, and N-Alloc protecting groups.

Cover Picture: A Micropatterned Hydrogel Platform for Chemical Synthesis and Biological Analysis (Adv. Mater. 5/2006)

AbstractA polymer hydrogel platform for peptide arrays compatible with both solid‐phase peptide synthesis conditions and the aqueous environment essential for biological assays is reported on p. 655 by Ulijn and co‐workers. Micropatterning followed by multiple‐step peptide synthesis produces the peptide‐functionalized poly(ethylene glycol)–polyamide “biochips” shown schematically on the cover. On‐chip biological assays involving cells and enzymes provide proof‐of‐concept.

Side-Chain-Anchored Nα-Fmoc-Tyr-OPfp for Bidirectional Solid-Phase Synthesis

[reaction: see text] A mild resin-immobilization strategy employing a readily prepared trityl bromide resin for anchoring building blocks via a phenol group has been developed. With N(alpha)-Fmoc-Tyr-OPfp as a starter building block, it was possible to prepare asymmetrically substituted hybrids of spider- and wasp-type polyamine toxins using solid-phase peptide synthesis conditions.

Photophysics and Biological Applications of the Environment-Sensitive Fluorophore 6-N,N-Dimethylamino-2,3-naphthalimide

We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biological probe with emission in the 500-600 nm range and a marked response to changes in the environment polarity. The 6DMN fluorescence is red-shifted in polar protic environments, with the maximum emission intensity shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Additionally, the fluorescence quantum yield decreases more than 100-fold from chloroform (Phi = 0.225) to water (Phi = 0.002). The scope and applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid derivative (5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides, demonstrating that it can be manipulated under standard solid-phase peptide synthesis conditions. Peptides incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified in studies with Src homology 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm) upon binding to selected SH2 domains (e.g., Crk SH2, Abl SH2, and PI3K SH2). The peptides can be used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable throughout the titrations.

Just add water: a new fluorous capping reagent for facile purification of peptides synthesized on the solid phase.

A novel fluorous capping reagent is introduced to facilitate purification during solid-phase peptide synthesis (SPPS). Reagent 1 is a trivalent iodonium salt that reacts vigorously with free amines to deliver a long-chain fluoroalkyl group. It has been used to tag all unreacted amines following the peptide coupling step in SPPS. The resulting fluoroalkylated amine is no longer able to couple in further peptide coupling steps and is also stable to standard peptide synthesis conditions. Deletion products are removed using flash fluorous chromatography to yield the pure, full-length peptide.