A Disulfide Intercalator Toolbox for the Site‐Directed Modification of Polypeptides

A disulfide intercalator toolbox was developed for site‐specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole–SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live‐cell imaging.     

Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.     

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA–Protein Cross‐Linking

N‐(3‐Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross‐linking of biomolecules through copper(I)‐catalyzed azide–alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene‐containing DNA or protein and then reacts with cysteine‐containing peptides or proteins to form covalent cross‐links. Several examples of bioconjugations of ethynyl‐ or octadiynyl‐modified DNA with peptides, p53 protein, or alkyne‐modified human carbonic anhydrase with peptides are given.     

Guanidine Additive Enabled Intermolecular ortho-Phthalaldehyde-Amine-Thiol Three-Component Reactions for Modular Constructions

Recently, ortho-phthalaldehyde (OPA) is experiencing a renascence for the modification of proteins and peptides through OPA-amine two-component reactions for bioconjugation and intramolecular OPA-amine-thiol three-component reactions for cyclization. Historically, small thiol molecules were used in large excess to allow for the intermolecular OPA-amine-thiol reaction forming 1-thio-isoindole derivatives. In this study, we discovered that guanidine could serve as an effective additive to switch the intermolecular OPA-amine-thiol three-component reaction to a stoichiometric process and enable the modular construction of peptide-peptide, and peptide-drug conjugate structures. Thus, 12 model peptide-peptide conjugates have been synthesized from unprotected peptides featuring all proteinogenic residues. Besides, 6 peptide-drug conjugates have been prepared in one step, with excellent conversions and isolated yields. In addition, a conjugate product has been further functionalized by utilizing a premodified OPA derivative, demonstrating the versatility and flexibility of this reaction.     

Continuous Flow Bioconjugations of NIR-AZA Fluorophores via Strained Alkyne Cycloadditions with Intra-Chip Fluorogenic Monitoring**

The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in-flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2-amino-2-deoxy-α-D-glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS-OH and c(RGDfK) respectively. The catalyst and reagent-free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3-dipolar cycloadditions. Reaction completion was achieved with residence times of 5 min at 40 °C for tetrazine versus 10 min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra-chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels-Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real-time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.     

Constrained Cyclopeptides: Biaryl Formation through Pd‐Catalyzed C−H Activation in Peptides—Structural Control of the Cyclization vs. Cyclodimerization Outcome

A series of short tryptophan‐phenylalanine peptides containing an iodo substituent on the phenyl ring was subjected to Pd‐catalyzed CH activation reactions to give the corresponding aryl‐indole coupled products. Two types of adducts were generated: cyclomonomer and cyclodimeric peptides; no evidence of oligo‐ or polymerization products was detected. Contrary to standard peptide macrocyclizations, the factors controlling the fate of the reaction are the number of amino acids between the aromatic residues and the regiochemistry of the parent iodo derivative, independent of both the concentration and the cyclization mode. The method is general and allows access to novel biaryl peptidic topologies, which have been fully characterized.     

One-Pot Dual C−C Coupling Reaction via Site Selective Cascade Formation by PdII-Cryptate of an Amino-Ether Heteroditopic Macrobicycle

Selectivity of aryl iodo over ethynyl iodo toward the Suzuki cross coupling reaction is explored by utilizing a palladium complex of amino-ether heteroditopic macrobicycle. Subsequently, unreacted ethynyl iodide undergoes homocoupling reaction in the same catalytic atmosphere, thereby representing a cascade dual C−C coupling reaction. Furthermore, this approach is extended for novel one-pot synthesis of unsymmetrical 1,3-diynes.     

Synthesis of Novel (Phenylalkyl)amines for the Investigation of Structure?Activity Relationships,Part 3

An easy and efficient pathway for the preparation of 4‐ethynyl‐2,5‐dimethoxyphenethylamine (=4‐ethynyl‐2,5‐dimethoxybenzeneethanamine; 2C‐YN; 1 ) was developed, an ethynyl analogue of the potent 5‐HT_2A/C agonists, e.g., 4‐iodo‐2,5‐dimethoxy‐amphetamine (DOI; 2b ). The ethynyl moiety was introduced by a Pd‐catalyzed Sonogashira reaction of (trimethylsilyl)ethyne with N‐(trifluoroacetyl)‐protected 4‐iodo‐2,5‐dimethoxyphenethylamine ( 7 ) in almost quantitative yield within only 1 h. Removal of the Me₃Si group was accomplished with Bu₄NF. Final N‐deprotection by NaOH treatment afforded the novel phenethylamine 1 in an overall yield of 88%.     

Fast surface immobilization of native proteins through catalyst-free amino-yne click bioconjugation

Surface immobilization provides a useful platform for biosensing, drug screening, tissue engineering and other chemical and biological applications. However, some of the used reactions are inefficient and/or complicated, limiting their applications in immobilization. Herein, we use a spontaneous and catalyst-free amino-yne click bioconjugation to generate activated ethynyl group functionalized surfaces for fast immobilization of native proteins and cells. Biomolecules, such as bovine serum albumin (BSA), human IgG and a peptide of C(RGDfK), could be covalently immobilized on the surfaces in as short as 30 min. Notably, the bioactivity of the anchored biomolecules remains intact, which is verified by efficiently capturing target antibodies and cells from the bulk solutions. This strategy represents an alternative for highly efficient surface biofunctionalization.

Fast surface immobilization of native bioconjugates through a spontaneous amino-yne click reaction is realized.     

Bioinspired Nitroalkylation for Selective Protein Modification and Peptide Stapling

Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by ¹⁹F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples.     

Isoindolium-Based Allenes: Reactivity Studies and Applications in Fluorescence Temperature Sensing and Cysteine Bioconjugation

The reaction of a series of electron-deficient isoindolium-based allenes with sulfhydryl compounds has been studied, leading to the formation of isoindolium-based vinyl sulfides. The vinyl sulfides generated could be readily converted into the corresponding indanones and amines upon heating at 30–70 °C with good yields up to 61 %. The thermal cleavage reaction of vinyl sulfides was further studied for developing temperature-sensitive systems. Notably, a novel FRET-based fluorescent temperature sensor was designed and synthesized for temperature sensing at 50 °C, giving a 6.5-fold blue fluorescence enhancement. Moreover, chemoselective bioconjugation of cysteine-containing peptides with the isoindolium-based allenes for the construction of multifunctional peptide bioconjugates was investigated. Thermal cleavage of isoindoliums on the modified peptides at 35–70 °C gave indanone bioconjugates with up to >99 % conversion. These results indicated the biocompatibility of this novel temperature-sensitive reaction.     

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.     

An efficient one‐pot access to poly(arylene ethynylene) homopolymers: Use of the Bu3Sn?moiety as a recyclable carrier to introduce the ethynyl unit into the chain

The palladium‐catalyzed Stille coupling of an aromatic diiodide with half an equivalent of tributyl(ethynyl)tin results in a complex mixture containing the diethynylaromatic compound, the iodo(ethynyl)aromatic compound, unreacted diiodide and the tributyltin iodide side product. Addition of LDA to this mixture, while leaving the iodoaromatic moieties unaffected, causes deprotonation of the ethynyl functionalities, which immediately recombine with tributyltin iodide to form a mixture of bis(tributylethynyltin)aromatic, iodo(tributylethynyltin)aromatic, and unchanged diiodide. Being the palladium catalyst still active, it is sufficient to warm up this mixture to obtain the coupling of the tributylethynyltin and iodo moieties resulting in the formation of a poly(arylene ethynylene) polymer. Isolation of the polymer is easily and rapidly achieved by precipitation, while distillation of the mother liquor allows recovery of the tributyltin iodide side product. The latter can be utilized for the preparation of new tributyl(ethynyl)tin, thus allowing a convenient turnover of the tributyltin moiety. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2603–2621, 2000     

Rapid chemoselective bioconjugation through oxidative coupling of anilines and aminophenols

A highly efficient protein bioconjugation method is described involving addition of anilines to o-aminophenols in the presence of sodium periodate. The reaction takes place in aqueous buffer at pH 6.5 and can reach high conversion in 2-5 min. The major product was characterized using X-ray crystallography, which revealed that an unprecedented oxidative ring contraction occurs after the coupling step. The compatibility of the reaction with protein substrates has been demonstrated through attachment of small molecules, polymer chains, and peptides to p-aminophenylalanine residues introduced into viral capsids through amber stop codon suppression. Coupling of anilines to o-aminophenol groups derived from tyrosine residues is also described. The compatibility of this method with thiol modification chemistry is shown through attachment of a near-IR fluorescent chromophore to cysteine residues inside the viral capsid shells, followed by attachment of integrin-targeting RGD peptides to anilines on the exterior surface.     

Interfacial interactions of hydrophobic peptides with lipid bilayers

Four hydrophobic laminin-related peptides and their corresponding parent peptides were synthesized to use them to target liposomes to tumoral cells. The peptide sequence was YIGSR((NH(2))), and hydrophobic residues linked to the alpha-amino terminal end were decanoyl, myristoyl, stearoyl, and cholesteryl-succinoyl. Before use in biological systems, a physicochemical study was carried out in order to determine their interaction with DPPC bilayers that could compromise both the toxicity and the stability of liposomal preparations. The experiments were based on DSC, fluorescence polarization, outer-membrane destabilization, and vesicle leakage. These peptides showed in general a low interaction with the vesicles, promoting in all cases the rigidification of bilayers. This lack of strong disturbances in the ordered state of phospholipid molecules seems more likely due to the similarity of peptide acyl chains with those of lipids than to the absence of interactions. The bulkiness of cholesteryl derivative as well as its tendency toward aggregation resulted in weak interaction levels except in thermograms. The binding of peptides to the surface of liposomes loaded with doxorubicin resulted in preparations with good entrapment yields and small size, required for long circulating vesicles (especially for the myristoyl derivative). The alternative method based on the reaction of parent peptide to the surface of liposomes through an amide linkage was slightly more efficient when the peptide was linked to the carboxy-terminal end of the DSPE-PEG-COOH-containing liposomes. Nevertheless, the final decision must be made with the simplicity of the procedure and reduction in losses during all the steps of the processes taken into consideration.     

Investigation of rational syntheses of heteroleptic porphyrinic lanthanide (europium, cerium) triple-decker sandwich complexes

The use of lanthanide triple-decker sandwich molecules containing porphyrins and phthalocyanines in molecular information storage applications requires the ability to attach monomeric triple deckers or arrays of triple deckers to electroactive surfaces. Such applications are limited by existing methods for preparing triple deckers. The reaction of a lanthanide porphyrin half-sandwich complex ((Por)M(acac)) with a dilithium phthalocyanine (PcLi2) in refluxing 1,2,4-trichlorobenzene (bp 214 degrees C) affords a mixture of triple deckers of composition (Pc)M(Pc)M(Por), (Por)M(Pc)M(Por), and (Pc)M(Por)M(Pc). We have investigated more directed methods for preparing triple deckers of a given type with distinct metals in each layer. Application of the method of Weiss, which employs reaction of a (Por)M(acac) species with a lanthanide double decker in refluxing 1,2,4-trichlorobenzene, afforded the desired triple decker in some cases but a mixture of triple deckers in others. The approach we developed employs in situ formation of the lanthanide reagent EuCl[N(SiMe3)2]2 or CeI[N(SiMe3)2]2, which upon reaction with a porphyrin affords the half-sandwich complex (Por)EuX or (Por)CeX' (X = Cl, N(SiMe3)2; X' = I, N(SiMe3)2). Subsequent reaction with PcLi2 gives the double decker (Por)M(Pc). The (Por(1))EuX half-sandwich complex gave the desired triple decker upon reaction with (Pc)Eu(Pc) but little of the desired product upon reaction with (Por(2))Eu(Pc). The (Por(1))CeX' half-sandwich complex reacted with europium double deckers (e.g., (tBPc)Eu(Por(2)), (tBPc)2Eu) to give the triple deckers (Por(1))Ce(tBPc)Eu(Por(2)) and (Por(1))Ce(tBPc)Eu(tBPc) in a rational manner (tB = tetra-tert-butyl). The reactions yielding the half-sandwich, double-decker, and triple-decker complexes were performed in refluxing bis(2-methoxyethyl) ether (bp 162 degrees C). The porphyrins incorporated in the various triple deckers include meso-tetrapentylporphyrin, meso-tetra-p-tolylporphyrin, octaethylporphyrin, and meso-tetraarylporphyrins bearing iodo, ethynyl, or iodo and ethynyl substituents. The triple deckers bearing iodo and/or ethynyl substituents constitute useful building blocks for information storage applications.     

De novo design, synthesis, and function of semiartificial myoglobin conjugated with coiled-coil two-alpha-helix peptides

The introduction of a flavin chromophore on the myoglobin (Mb) surface and an effective electron-transfer (ET) reaction through the flavin were successfully achieved by utilizing the self-assembly of heterostranded coiled-coil peptides. We have prepared a semiartificial Mb, named Mb-1alphaK, in which an amphiphilic and cationic alpha-helix peptide is conjugated at the heme propionate (Heme-1alphaK). Heme-1alphaK has a covalently bound iron-protoporphyrin IX (heme) at the N terminus of a 1alphaK peptide sequence. This sequence was designed to form a heterostranded coiled-coil in the presence of a counterpart amphiphilic and anionic 1alphaE peptide sequence in a parallel orientation. Two peptides, Fla(1)-1alphaE and Fla(31)-1alphaE, both incorporating a 10-methylisoalloxazine moiety as an artificial flavin molecule, were also prepared (Fla=2-[7-(10-methyl)isoalloxazinyl]-2-oxoethyl). Heme-1alphaK was successfully inserted into apomyoglobin to give Mb-1alphaK. Mb-1alphaK recognized the flavin-modified peptides and a two-alpha-helix structure was formed. In addition, an efficient ET from reduced nicotinamide adenine dinucleotide to the heme center through the flavin unit was observed. The ET rate was faster in the presence of Fla(1)-1alphaE than in the presence of Fla(31)-1alphaE or the equivalent molecule that has no peptide chain. These results demonstrate that the introduction of a functional chromophore on the Mb surface can be achieved by using specific peptide-peptide interactions. Moreover, the dependence of the ET rate on the position of the flavin indicated that the distance between the heme active site and the flavin chromophore was regulated by the three-dimensional structure of the designed polypeptide.     

Chemo‐ and Regioselective Ethynylation of Tryptophan‐Containing Peptides and Proteins

Ethynylation of various tryptophan‐containing peptides and a single model protein was achieved using Waser's reagent, 1‐[(triisopropylsilyl)ethynyl]‐1,2‐benziodoxol‐3(1 H)‐one (TIPS‐EBX), under gold(I) catalysis. It was demonstrated by NMR that the ethynylation occured selectively at the C2‐position of the indole ring of tryptophan. Further, MS/MS showed that the tryptophan residues could be modified selectively with ethynyl functionalities even when the tryptophan was present as a part of the protein. Finally, the terminal alkyne was used to label a model peptide with a fluorophore by means of copper‐catalyzed click chemistry.     

Cleavable and tunable cysteine-specific arylation modification with aryl thioethers

Cysteine represents an attractive target for peptide/protein modification due to the intrinsic high nucleophilicity of the thiol group and low natural abundance. Herein, a cleavable and tunable covalent modification approach for cysteine containing peptides/proteins with our newly designed aryl thioethers via a S_NAr approach was developed. Highly efficient and selective bioconjugation reactions can be carried out under mild and biocompatible conditions. A series of aryl groups bearing different bioconjugation handles, affinity or fluorescent tags are well tolerated. By adjusting the skeleton and steric hindrance of aryl thioethers slightly, the modified products showed a tunable profile for the regeneration of the native peptides.

A cleavable and tunable covalent modification approach for cysteine by aryl thioethers via a S_NAr approach was developed. The highly efficient and selective bioconjugation reactions can proceed under the mild and biocompatible conditions.     

Using high-concentration trypsin-immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature

We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 microg/microL) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly.