Palladium in the Chemical Synthesis and Modification of Proteins

The field of site‐specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium‐mediated C−C bond‐forming and C−O bond‐cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.     

Native chemical ligation of thioamide-containing peptides: development and application to the synthesis of labeled α-synuclein for misfolding studies

Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.     

The dehydroalanine effect in the fragmentation of ions derived from polypeptides

The fragmentation of peptides and proteins upon collision‐induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non‐proteinogenic amino acid with an unsaturated side‐chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c‐ and z‐ions. Because these fragment ion types are not commonly observed upon activation of positively charged even‐electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S‐alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas‐phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S‐alkyl cysteine residues and conversion of multiply‐protonated peptides to radical cations. In the latter case, loss of radical side‐chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue‐specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.     

Serine‐Selective Aerobic Cleavage of Peptides and a Protein Using a Water‐Soluble Copper–Organoradical Conjugate

The site‐specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine‐selective peptide‐cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water‐soluble copper–organoradical conjugate. The method is applicable to the site‐selective cleavage of polypeptides that possess various functional groups. Peptides comprising D ‐amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site‐selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.     

A novel ring opening reaction of peptide N-terminal thiazolidine with 2,2′-dipyridyl disulfide (DPDS) efficient for protein chemical synthesis

In the protein chemical synthesis via native chemical ligation (NCL) method with three peptide segments, the N-terminal cysteine residue of middle segment is generally protected by thiazolidine ring. In this paper, we show the novel method for thiazolidine ring opening using 2,2′-dipyridyl disulfide (DPDS). The N-terminal thiazolidine was converted into S-pyridylsulfenylated cysteine residue with DPDS under acidic conditions, and this N-terminally Cys peptide protected with disulfide was applicable to NCL reaction without purification and deprotection steps. DPDS treatment did not remove other Cys protecting groups generally used for regioselective disulfide bond formation reactions. These results indicate that this thiazolidine ring opening reaction is quite useful for the protein chemical synthesis with three-segment NCL strategy.     

Loss of PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein

SDS‐PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein‐containing conjugates, mostly pegylated proteins. PEG‐maleimide (MAL) is frequently used to site‐specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C–S linkage between protein and PEG‐MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein was observed. It is a thiol‐independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG‐MAL modified conjugates although PEG‐vinylsulfone conjugates to thiol‐containing proteins also through a C–S linkage. Cleavage kinetics of PEG40k‐MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide‐thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG‐MAL modified protein/peptide by SDS‐PAGE analysis.     

Protein Release from Biodegradable PolyHPMA–Lysozyme Conjugates Resulting in Bioactivity Enhancement

A novel biodegradable thiazolidine‐2‐thione functional chain transfer agent was synthesized and employed as a reversible additional fragmentation chain transfer agent to prepare well‐defined semitelechelic poly‐N‐(2‐hydroxypropyl) methacrylamides (polyHPMAs) with predetermined molecular weights and narrow polydispersities. The protein reactive group, thiazolidine‐2‐thione, was located at the polymer chain ends fixed by biodegradable disulfide bonds. The functional polyHPMA chains were subsequently conjugated to protein (lysozyme) by exploiting reactions between the thiazolidine‐2‐thione functionality and amine residues on the protein surface to form covalent amide linkages. The in vitro bioactivities of the lysozyme–polyHPMA conjugates were assessed by using Micrococcus lysodeikticus cells as substrates. The lysozyme bioactivity was significantly reduced following the conjugation procedure. However, cleavage of the polymer chains from the bioconjugates (under reducing conditions) yielded free protein and a remarkable recovery of bioactivity. In vivo tests were performed by subcutaneous injection into mice and clearly demonstrated decreased proteolytic degradation for the protein–polymer conjugate when compared with native protein, indicating effective protein protection through a conjugation strategy. This bioreversible approach to conjugation allows for a balance to be made between protein protection and effective bioactivity maintenance.     

Multivalent Chelators for In Vivo Protein Labeling

With the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.     

Charge dependent behavior of PNA/DNA/PNA triplexes in the gas phase

Intact noncovalent complexes were studied in the gas phase using negative ion nano-ESI mass spectrometry. Among various noncovalent systems studied in the gas phase, the interaction of DNA strands with peptide nucleic acids (PNAs) presents a strong interest as biologically relevant systems. PNAs originally described by Nielsen are used as DNA mimics as possible medical agents by imprisoning DNA single strands into stable noncovalent complexes. Two types of PNAs were investigated in the PNA/DNA multiplex: the original Nielsen's PNA and a modified backbone PNA by the introduction of syn- and anti-(aminoethyl)thiazolidine rings. We first investigated the stoichiometry of PNA/DNA multiplexes formed in solution and observed them in the gas phase via qualitative kinetics of complementary strand associations. It resulted in observing PNA2/DNA triplexes (ts) as the multiply deprotonated species, most stable in both the solution and gas phase. Second, charge-dependant decompositions of these species were undertaken under low-energy collision conditions. It appears that covalent bond cleavages (base releasing or skeleton cleavage) occur from lower ts charge states rather than ts unzipping, which takes place from higher charge states. This behavior can be explained by considering the presence of zwitterions depending on the charge state. They result in strong salt-bridge interactions between the positively charged PNA side chain and the negatively charged DNA backbone. We propose a general model to clearly display the involved patterns in the noncovalent triplex decompositions. Third, the relative stability of three PNA2/DNA complexes was scrutinized in the gas phase by acquiring the breakdown curves of their ts(6-) form, corresponding to the ts unzipping. The chemical structures of the studied PNAs were chosen in order to evidence the possible influence of backbone stereochemistry on the rigidity of PNA2/DNA complexes. It provided significantly different stabilities via V(m) measurements. The relative gas-phase stability order obtained was compared to that found in solution by Chassaing et al., and shows qualitative agreement.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Precision Biopolymers from Protein Precursors for Biomedical Applications

The synthesis of biohybrid materials with tailored functional properties represents a topic of emerging interest. Combining proteins as natural, macromolecular building blocks, and synthetic polymers opens access to giant brush‐like biopolymers of high structural definition. The properties of these precision polypeptide copolymers can be tailored through various chemical modifications along their polypeptide backbone, which expands the repertoire of known protein‐based materials to address biomedical applications. In this article, the synthetic strategies for the design of precision biopolymers from proteins through amino acid specific conjugation reagents are highlighted and the different functionalization strategies, their characterization, and applications are discussed.     

The case of the missing ring: radical cleavage of a carbon-carbon bond and implications for GFP chromophore biosynthesis

The green fluorescent protein (GFP) creates its fluorophore by promoting spontaneous peptide backbone cyclization and amino acid oxidation chemistry on its own Ser65, Tyr66, Gly67 tripeptide sequence. Here we use high-resolution crystallography and mutational analyses to characterize GFP variants that undergo backbone cyclization followed by either anticipated chromophore synthesis via Y66F Calpha-Cbeta double-bond formation or unprecedented loss of a Y66F benzyl moiety via Calpha-Cbeta bond cleavage. We discovered a Y66F cleavage variant that subsequently incorporates an oxygen atom, likely from molecular oxygen, at the Y66 Calpha position. The post-translational products identified from these Y66F GFP structures support a common intermediate that partitions between Calpha-Cbeta oxidation and homolytic cleavage pathways. Our data indicate that Glu222 is the branchpoint control for this partitioning step and also influences subsequent oxygen incorporation reactions. From these results, we propose mechanisms for Y66F Calpha-Cbeta cleavage, oxygen incorporation, and chromophore biosynthesis with shared features that include radical chemistry. By revealing how GFP and RFP protein environments steer chemistry to favor fluorophore biosynthesis and disfavor alternative reactivity, we identify strategies for protein design. The proposed, common, one-electron oxidized, radical intermediate for post-translation modifications in the GFP family has general implications for how proteins drive and control spontaneous post-translational chemical modifications in the absence of metal ions.     

Nutation frequency modulation on NMR signal of nuclear spins in chemical exchange with solvent water under the BEST conditions

Solvent exchange properties of protein backbone amide protons provide valuable residue‐specific information on protein solvent accessibility, structure stability and flexibility and hence are of significant interest in structural biology. NMR has served as a unique means for the characterization of chemical exchange including proton amide exchange with solvent water at residue‐specific levels across a broad range of exchange rates. One of the methods used for the characterization of protein backbone amide exchange by NMR involves the use of progressive selective irradiation of the water resonance. Here, we report the experimental observation of the nutation frequency (strength of RF field used for the irradiation of water resonance) modulation on amide proton signals for those in exchange with the solvent water under the band‐selective excitation short transient (BEST) conditions. Compared with conventional saturation transfer of water magnetization experiments, this nutation frequency modulation observed on signal of nuclear spins under the BEST conditions potentially offers a quick identification of protein backbone amides in rapid exchange with solvent water. Copyright © 2014 John Wiley & Sons, Ltd.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA–protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top‐down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single‐residue level of RNA. Our data show that the electrostatic interactions between HIV‐1 TAR RNA and a peptide comprising the arginine‐rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top‐down MS. Moreover, the MS data reveal time‐dependent 1:2 and 1:1 stoichiometries of the TAR–tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide.     

Selective cleavage enhanced by acetylating the side chain of lysine

Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision‐induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N‐terminal of the peptide. After acetylation of the lysine side‐chain, ions were the most abundant primary fragment products and the Lys_(Ac)–Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side‐chain of lysine promoted the selective cleavage of Lys–Xxx amide bond and generated much more information of the peptide backbone sequence. The results re‐evaluate the selective cleavage due to the lysine basic side‐chain and provide information for studying the post‐translational modification of proteins and other bio‐molecules containing Lys residues. Copyright © 2013 John Wiley & Sons, Ltd.     

Dissociation energies of X-H bonds in amino acids

In biochemistry, free radicals are versatile species which can perform diverse functions including: signaling, synthesis, and destructive modification. It is of interest to understand how radicals behave within all biomolecules and specifically within peptides and proteins. The 20 standard amino acids contain a wide range of chemical structures, which give proteins their complexity and ultimately their functionality. Many factors influence how radicals interact with these complex molecules, including the bond dissociation energies (BDEs) for homolytically cleaving any X-H bonds. The BDEs provide a simple measure for comparing the thermodynamic favorability of abstracting hydrogen atoms from various sites within a protein. BDEs for abstractable hydrogen atoms have been calculated for each amino acid, the peptide backbone, and peptide termini in order to compile a roadmap of the relative thermodynamics which influence protein radical chemistry. With this information it is possible to gain insight into what contributions both kinetics and thermodynamics will make to various radical mediated reaction pathways.     

Collision‐induced dissociation of oligonucleotide anions fully modified at the 2'‐position of the ribose: 2'‐F/‐H and 2'‐F/‐H/‐OMe mix‐mers

Gas‐phase dissociation of various 2'‐position modified oligonucleotide anions has been studied as a function of precursor ion charge state using ion trap and low energy beam‐type collision‐induced dissociation (CID). For a completely 2'‐O‐methyl modified 6‐mer, all possible dissociation channels along the phosphodiester linkage, generating complementary (a‐B)/w‐, b/x‐, c/y‐, d/z‐ion series, were observed with no single dominant type of dissociation pathway. Full sequence information was generated from each charge state via ion trap CID. More sequential fragmentation was noted under beam‐type CID conditions. Comparison with model DNA, in which all 2'‐OH groups are converted to 2'‐H, and RNA anions suggests that the 2'‐OMe substitution stabilizes the phosphodiester linkage with respect to fragmentation relative to both DNA and RNA oligomers. For modified mix‐mer anions, comprised of DNA nucleotides and 2'‐F substituted nucleotides or a mixture of DNA nucleotides and 2'‐O‐methyl (2'‐OMe) and 2'‐F substituted nucleotides, 3'‐side backbone cleavage was found to be inhibited by the 2'‐OMe or 2'‐F modification on the nucleotides under ion trap CID conditions. Thus, the sequence information was limited to the a‐Base/w‐fragments from the cleavage of the 3' C‐O bond of the 2'‐H (DNA) nucleotides. Under beam‐type CID conditions, limited additional cleavage adjacent to 2'‐OMe substituted nucleotides was noted but 2'‐F modified residues remained resistant to cleavage. Copyright © 2012 John Wiley & Sons, Ltd.     

Chemoenzymatic approaches to glycoprotein synthesis

The construction of homogeneous glycoproteins presents a formidable challenge to the synthetic chemist. Over the past few years there has been an explosion in the number of methods developed to address this problem. These methods include the development of novel ligation technologies for the synthesis of the protein backbone, as well chemical and enzymatic approaches for introducing complex glycans into the peptide backbone. This tutorial review discusses the application of these techniques to the synthesis of peptides and proteins possessing well defined glycans.     

Computational study on nonenzymatic peptide bond cleavage at asparagine and aspartic acid

Nonenzymatic peptide bond cleavage at asparagine (Asn) and glutamine (Gln) residues has been observed during peptide deamidation experiments; cleavage has also been reported at aspartic acid (Asp) and glutamic acid (Glu) residues. Although peptide backbone cleavage at Asn is known to be slower than deamidation, fragmentation products are often observed during peptide deamidation experiments. In this study, mechanisms leading to the cleavage of the carboxyl-side peptide bond of Asn and Asp residues were investigated using computational methods (B3LYP/6-31+G**). Single-point solvent calculations at the B3LYP/6-31++G** level were carried out in water, utilizing the integral equation formalism-polarizable continuum (IEF-PCM) model. Mechanism and energetics of peptide fragmentation at Asn were comparatively analyzed with previous calculations on deamidation of Asn. When deamidation proceeds through direct hydrolysis of the Asn side chain or through cyclic imide formationvia a tautomerization routeit exhibits lower activation barriers than peptide bond cleavage at Asn. The fundamental distinction between the mechanisms leading to deamidationvia a succinimideand backbone cleavage was found to be the difference in nucleophilic entities involved in the cyclization process (backbone versus side-chain amide nitrogen). If deamidation is prevented by protein three-dimensional structure, cleavage may become a competing pathway. Fragmentation of the peptide backbone at Asp was also computationally studied to understand the likelihood of Asn deamidation preceding backbone cleavage. The activation barrier for backbone cleavage at Asp residues is much lower (approximately 10 kcal/mol) than that at Asn. This suggests that peptide bond cleavage at Asn residues is more likely to take place after it has deamidated into Asp.