Optimization of a biomimetic transamination reaction

For a range of protein substrates, N-terminal transamination offers a convenient way to install a reactive ketone or aldehyde functional group at a single location. We report herein the effects of the identity of N-terminal residues on the product distribution generated upon reaction with pyridoxal 5'-phosphate (PLP). This study was accomplished through the combination of solid-phase peptide synthesis with detailed liquid chromatography-mass spectrometry analysis. Many N-terminal amino acids provided high yields of the desired transaminated products, but some residues (His, Trp, Lys, and Pro) generated adducts with PLP itself. N-terminal Cys and Ser residues were observed to undergo beta-elimination in addition to transamination, and the transamination product of N-terminal Gln was resistant to subsequent oxime formation attempts. The information generated through the screening of peptide substrates was successfully applied to a protein target, changing an initially unreactive terminus into one that could be modified in high (70%) yield. Thus, these studies have increased our predictive power for the reaction, both in terms of improving conversion and suppressing reaction byproducts. An initial set of guidelines that may be used to increase the applicability of this reaction to specific proteins of interest is provided.     

Visible-Light-Induced Cysteine-Specific Bioconjugation: Biocompatible Thiol–Ene Click Chemistry

Bioconjugation methods using visible-light photocatalysis have emerged as powerful synthetic tools for the selective modification of biomolecules under mild reaction conditions. However, the number of photochemical transformations that allow successful protein bioconjugation is still limited because of the need for stringent reaction conditions. Herein, we report that a newly developed water-compatible fluorescent photosensitizer Q_PEG can be used for visible-light-induced cysteine-specific bioconjugation for the installation of Q_PEG by exploiting its intrinsic photosensitizing ability to activate the S−H bond of cysteine. The slightly modified Q_CAT enables the effective photocatalytic cysteine-specific conjugation of biologically relevant groups. The superior reactivity and cysteine selectivity of this methodology was further corroborated by traceless bioconjugation with a series of complex peptides and proteins under biocompatible conditions.     

A three-component Mannich-type reaction for selective tyrosine bioconjugation

A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic aromatic substitution pathway. The reaction takes place under mild pH and temperature conditions and can modify protein substrates at concentrations as low as 20 muM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a number of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymatic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.     

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.     

Olefin cross-metathesis on proteins: investigation of allylic chalcogen effects and guiding principles in metathesis partner selection

Olefin metathesis has recently emerged as a viable reaction for chemical protein modification. The scope and limitations of olefin metathesis in bioconjugation, however, remain unclear. Herein we report an assessment of various factors that contribute to productive cross-metathesis on protein substrates. Sterics, substrate scope, and linker selection are all considered. It was discovered during this investigation that allyl chalcogenides generally enhance the rate of alkene metathesis reactions. Allyl selenides were found to be exceptionally reactive olefin metathesis substrates, enabling a broad range of protein modifications not previously possible. The principles considered in this report are important not only for expanding the repertoire of bioconjugation but also for the application of olefin metathesis in general synthetic endeavors.     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

Cysteine‐Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide‐modified building block reacts first with an ethynylphosphonite through a Staudinger‐phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine‐selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.     

Coumarin‐Based Fluorogenic Probes for No‐Wash Protein Labeling

A fluorescent protein‐labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.     

Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

Formylglycine‐generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site‐specific oxidation of a cysteine residue to the aldehyde‐containing amino acid C^α‐formylglycine (FGly). This non‐canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine‐generating enzyme (FGE) and the iron‐sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.     

Predicting in vivo protein peptide interactions with random phage display

Binding sites in protein complexes occasionally map to small peptides within one or more proteins. Random peptide display methods simulate binding interactions by providing all possible peptide combinations with an equal opportunity to bind a protein of interest. The natural substrates for the protein are typically known in advance. However, it is often the case that such substrates are identified as putative partner proteins by using in vivo methods such as yeast two hybrid screening. Unfortunately, such methods often produce lengthy datasets of protein sequences and offer little mechanistic insight into how such interactions might take place in vivo. Here, we review an approach that addresses this problem. First, sequence alignment tools identify and characterize blocks of conserved sequences among peptides recovered during random peptide display. Next, searching programs detect similar blocks of conserved sequences within naturally occurring proteins to predict partner proteins. Finally, the significance of an interaction is tested using site specific mutagenesis, binding competition or co-immunoprecipitation experiments. This strategy should become increasingly powerful with the growing popularity of interaction studies, sequencing projects and microarray analyses in modern biology.     

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

Stable Pyrrole-Linked Bioconjugates through Tetrazine-Triggered Azanorbornadiene Fragmentation

An azanorbornadiene bromovinyl sulfone reagent for cysteine-selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole-linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene-tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro-Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site-selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.     

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.     

Stable Pyrrole‐Linked Bioconjugates through Tetrazine‐Triggered Azanorbornadiene Fragmentation

An azanorbornadiene bromovinyl sulfone reagent for cysteine‐selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole‐linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene‐tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro‐Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site‐selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.     

Kinetic Assay of the Michael Addition‐Like Thiol–Ene Reaction and Insight into Protein Bioconjugation

The chemical modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chemical modification of proteins. The Michael addition‐like thiol–ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent‐exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic analysis was performed. New vinyl‐substituted pyridine derivatives were designed and synthesized. The reactivity of these vinyl tags with L ‐cysteine was evaluated by UV absorption and high‐resolution NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent‐exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.     

Bioactive unnatural somatostatin analogues through bioorthogonal iodo- and ethynyl-disulfide intercalators

Iodo- and ethynyl-containing bisalkylating bioconjugation agents 5 and 8 were achieved and allow the introduction of reactive unnatural substituents into proteins and peptides whilst the bioactive 3D structure is retained. Derivatives of the peptide hormone somatostatin bearing a single iodo or ethynyl group were prepared through intercalation into the disulfide bridge. For the first time, the exact reaction mechanism of the intercalation was elucidated by applying 2D NMR experiments and it was shown that, during the reaction, somatostatin diastereomers were formed. Site-directed modification of the ethynyl-modified peptide with a coumarin chromophore was achieved through a [1,3] dipolar Huisgen cycloaddition reaction; this suggests that such a derivative could serve as an attractive platform to prepare artificial somatostatin compound libraries. The biological activity and specificity of a representative modified somatostatin derivative was demonstrated and efficient receptor-mediated cell uptake occurred in a dose-dependent manner into receptor positive cells only. The iodo and ethynyl bioconjugation reagents presented herein could be applied for introducing such substituents into alternative peptides and proteins and, in principle, could facilitate the efficient design of a broad variety of artificial protein and peptide analogues with previously unknown bioactivities.     

A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry

The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.     

A method for N-terminal de novo sequencing of Nα-blocked proteins by mass spectrometry

A method for de novo sequencing of N(α)-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N(α)-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two N(α)-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.     

Metal‐Mediated Functionalization of Natural Peptides and Proteins: Panning for Bioconjugation Gold

Selective modification of natural proteins is a daunting methodological challenge and a stringent test of selectivity and reaction scope. There is a continued need for new reactivity and new selectivity concepts. Transition metals exhibit a wealth of unique reactivity that is orthogonal to biological reactions and processes. As such, metal‐based methods play an increasingly important role in bioconjugation. This Review examines metal‐based methods as well as their reactivity and selectivity for the functionalization of natural proteins and peptides.     

A novel tyrosine hyperoxidation enables selective peptide cleavage

A novel tyrosine hyperoxidation enabling selective peptide cleavage is reported. The scission of the N-terminal amide bond of tyrosine was achieved with Dess–Martin periodinane under mild conditions, generating a C-terminal peptide fragment bearing the unprecedented hyperoxidized tyrosine motif, 4,5,6,7-tetraoxo-1H-indole-2-carboxamide, along with an intact N-terminal peptide fragment. This reaction proceeds with high site-selectivity for tyrosine and exhibits broad substrate scope for various peptides, including those containing post-translational modifications. More importantly, this oxidative cleavage was successfully applied to enable sequencing of three naturally occurring cyclic peptides, including one depsipeptide and one lipopeptide. The linearized peptides generated from the cleavage reaction significantly simplify cyclic peptide sequencing by MS/MS, thus providing a robust tool to facilitate rapid sequence determination of diverse cyclic peptides containing tyrosine. Furthermore, the highly electrophilic nature of the hyperoxidized tyrosine unit disclosed in this work renders it an important electrophilic target for the selective bioconjugation or synthetic manipulation of peptides containing this unit.

A Tyr-selective peptide cleavage was reported using Dess–Martin periodinane. The cleavage generates an unprecedented hyperoxidized tyrosine motif in the C-terminal fragment and showed excellent site-specificity and broad scope for various peptides.