Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.     

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]⁶-Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT₂R/AT₁R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2 , has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.     

Predicting 15N amide chemical shifts in proteins. I. An additive model for the backbone contribution

Because proteins adopt unique structures, chemically identical nuclei in proteins exhibit different chemical shifts. Amide ¹⁵N chemical shifts have been shown to vary over 20 ppm. The cause of these chemical shift inequivalencies is the different intra‐ and intermolecular interactions that individual nuclei experience at different locations in the protein structure. These chemical shift inequivalencies can be described as structural shifts, the difference between the actual chemical shift and the random coil chemical shift. As a first step toward the prediction of these amide ¹⁵N structural shifts, calculations have been carried out on acetyl‐glycine‐methyl amide to examine how a neighboring peptide group influences the amide ¹⁵N structural shifts. The ϕ,ψ dihedral angle space is completely surveyed, while all other geometrical variables are held fixed, to isolate the effect of the backbone conformation. Similar calculations for a limited number of conformations of acetyl‐glycine‐glycine‐methyl amide were carried out, where the effects of the two terminal peptide groups on the central amide ¹⁵N structural shift are examined. It is shown that the effect of the two adjacent groups can be accurately modeled by combining their individual effects additively. This provides a quite simple method to predict the backbone influence on amide ¹⁵N structural shifts in proteins. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 366–372, 2001     

Contributions of Various Noncovalent Bonds to the Interaction between an Amide and S‐Containing Molecules

N‐Methylacetamide, a model of the peptide unit in proteins, is allowed to interact with CH₃SH, CH₃SCH₃, and CH₃SSCH₃ as models of S‐containing amino acid residues. All of the minima are located on the ab initio potential energy surface of each heterodimer. Analysis of the forces holding each complex together identifies a variety of different attractive forces, including SH???O, NH???S, CH???O, CH???S, SH???π, and CH???π H‐bonds. Other contributing noncovalent bonds involve charge transfer into σ* and π* antibonds. Whereas some of the H‐bonds are strong enough that they represent the sole attractive force in several dimers, albeit not usually in the global minimum, charge‐transfer‐type noncovalent bonds play only a supporting role. The majority of dimers are bound by a collection of several of these attractive interactions. The SH???O and NH???S H‐bonds are of comparable strength, followed by CH???O and CH???S.     

Synthesis of Cyclopentapeptides with Three to Five Aib Units

Four new Aib‐containing cyclopentapeptides have been synthesized by cyclization of the corresponding linear pentapeptides using the diethyl phosphorocyanidate (DEPC)/EtN(ⁱPr)₂ method. The linear precursors were prepared via the ‘azirine/oxazolone method’, i.e., the Aib units were introduced by the reaction of amino acids or peptide acids with a 2,2‐dimethyl‐2H‐azirin‐3‐amine, followed by selective hydrolysis of the terminal amide function. Most remarkably, cyclo[(Aib)₅] exists in CDCl₃ solution in a symmetrical conformation, i.e., no intramolecular H‐bonds are detectable.     

Peptide cation-radicals. A computational study of the competition between peptide N-Calpha bond cleavage and loss of the side chain in the [GlyPhe-NH2 + 2H]+. cation-radical

Cation‐radicals and dications corresponding to hydrogen atom adducts to N‐terminus‐protonated N_α‐glycylphenylalanine amide (Gly‐Phe‐NH₂) are studied by combined density functional theory and Møller‐Plesset perturbational computations (B3‐MP2) as models for electron‐capture dissociation of peptide bonds and elimination of side‐chain groups in gas‐phase peptide ions. Several structures are identified as local energy minima including isomeric aminoketyl cation‐radicals, and hydrogen‐bonded ion‐radicals, and ylid‐cation‐radical complexes. The hydrogen‐bonded complexes are substantially more stable than the classical aminoketyl structures. Dissociations of the peptide N? C_α bonds in aminoketyl cation‐radicals are 18–47 kJ mol^?1 exothermic and require low activation energies to produce ion‐radical complexes as stable intermediates. Loss of the side‐chain benzyl group is calculated to be 44 kJ mol^?1 endothermic and requires 68 kJ mol^?1 activation energy. Rice‐Ramsperger‐Kassel‐Marcus (RRKM) and transition‐state theory (TST) calculations of unimolecular rate constants predict fast preferential N? C_α bond cleavage resulting in isomerization to ion‐molecule complexes, while dissociation of the C_α? CH₂C₆H₅ bond is much slower. Because of the very low activation energies, the peptide bond dissociations are predicted to be fast in peptide cation‐radicals that have thermal (298 K) energies and thus behave ergodically. Copyright © 2003 John Wiley & Sons, Ltd.     

Chemical Mimics of Aspartate-Directed Proteases: Predictive and Strictly Specific Hydrolysis of a Globular Protein at Asp−X Sequence Promoted by Polyoxometalate Complexes Rationalized by a Combined Experimental and Theoretical Approach

Creating efficient and residue-directed artificial proteases is a challenging task due to the extreme inertness of the peptide bond, combined with the difficulty of achieving specific interactions between the catalysts and the protein side chains. Herein we report strictly site-selective hydrolysis of a multi-subunit globular protein, hemoglobin (Hb) from bovine blood, by a range of Zr^IV-substituted polyoxometalates (Zr-POMs) in mildly acidic and physiological pH solutions. Among 570 peptide bonds in Hb, selective cleavage was observed at only eleven sites, each occurring at Asp−X peptide bonds located in the positive patches on the protein surface. The molecular origins of the observed Asp−X selectivity were rationalized by means of molecular docking, DFT-based binding, and mechanistic studies on model peptides. The proposed mechanism of hydrolysis involves coordination of the amide oxygen to Zr^IV followed by a direct nucleophilic attack of the side chain carboxylate group on the C-terminal amide carbon atom with formation of a cyclic anhydride, which is further hydrolyzed to give the reaction products. The activation energy for the cleavage of the structurally related Glu−X sequence compared to Asp−X was calculated to be higher by 1.4 kcal mol⁻¹, which corresponds to a difference of about one order of magnitude in the rates of hydrolysis. The higher activation energy is attributed to the higher strain present in the six-membered ring of glutaric anhydride (Glu−X), as compared to the five-membered ring of the succinic anhydride (Asp−X) intermediate. Similarly, the cleavage at X−Asp and X−Glu bonds are predicted to be kinetically less likely as the corresponding activation energies were 6 kcal mol⁻¹ higher_, explaining the experimentally observed selectivity. The synergy between the negatively charged polyoxometalate cluster, which binds at positive patches on protein surfaces, and selective activation of Asp−X peptide bonds located in these regions by Zr^IV ions, results in a novel class of artificial proteases with aspartate-directed reactivity, which is very rare among naturally occurring proteases.     

A Mechanistic Study of the Spontaneous Hydrolysis of Glycylserine as the Simplest Model for Protein Self‐Cleavage

A common feature of several classes of intrinsically reactive proteins with diverse biological functions is that they undergo self‐catalyzed reactions initiated by an N→O or N→S acyl shift of a peptide bond adjacent to a serine, threonine, or cysteine residue. In this study, we examine the N→O acyl shift initiated peptide‐bond hydrolysis at the serine residue on a model compound, glycylserine (GlySer), by means of DFT and ab initio methods. In the most favorable rate‐determining transition state, the serine ?COO^? group acts as a general base to accept a proton from the attacking ?OH function, which results in oxyoxazolidine ring closure. The calculated activation energy (29.4 kcal mol^?1) is in excellent agreement with the experimental value, 29.4 kcal mol^?1, determined by ¹H NMR measurements. A reaction mechanism for the entire process of GlySer dipeptide hydrolysis is also proposed. In the case of proteins, we found that when no other groups that may act as a general base are available, the N→O acyl shift mechanism might instead involve a water‐assisted proton transfer from the attacking serine ?OH group to the amide oxygen. However, the calculated energy barrier for this process is relatively high (33.6 kcal mol^?1), thus indicating that in absence of catalytic factors the peptide bond adjacent to serine is no longer a weak point in the protein backbone. An analogous rearrangement involving the amide N‐protonated form, rather than the principle zwitterion form of GlySer, was also considered as a model for the previously proposed mechanism of sea‐urchin sperm protein, enterokinase, and agrin (SEA) domain autoproteolysis. The calculated activation energy (14.3 kcal mol^?1) is significantly lower than the experimental value reported for SEA (≈21 kcal mol^?1), but is still in better agreement as compared to earlier theoretical attempts.     

Energetic switch of the proline effect in collision‐induced dissociation of singly and doubly protonated peptide Ala‐Ala‐Arg‐Pro‐Ala‐Ala

Suppression of the selective cleavage at N‐terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg‐Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision‐induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy‐dependent hypothesis. The results provide an explanation to the long‐term arguments between the steric conflict and the proton mobility mechanisms of proline effect.     

Parameters for molecular dynamics simulations of iron‐sulfur proteins

Iron‐sulfur proteins involved in electron transfer reactions have finely tuned redox potentials, which allow them to be highly efficient and specific. Factors such as metal center solvent exposure, interaction with charged residues, or hydrogen bonds between the ligand residues and amide backbone groups have all been pointed out to cause such specific redox potentials. Here, we derived parameters compatible with the AMBER force field for the metal centers of iron‐sulfur proteins and applied them in the molecular dynamics simulations of three iron‐sulfur proteins. We used density‐functional theory (DFT) calculations and Seminario's method for the parameterization. Parameter validation was obtained by matching structures and normal frequencies at the quantum mechanics and molecular mechanics levels of theory. Having guaranteed a correct representation of the protein coordination spheres, the amide H‐bonds and the water exposure to the ligands were analyzed. Our results for the pattern of interactions with the metal centers are consistent to those obtained by nuclear magnetic resonance spectroscopy (NMR) experiments and DFT calculations, allowing the application of molecular dynamics to the study of those proteins. © 2013 Wiley Periodicals, Inc.     

Antiparallel Self‐Association of a γ,α‐Hybrid Peptide: More Relevance of Weak Interactions

To learn how a preorganized peptide‐based molecular template, together with diverse weak non‐covalent interactions, leads to an effective self‐association, we investigated the conformational characteristics of a simple γ,α‐hybrid model peptide, Boc‐γ‐Abz‐Gly‐OMe. The single‐crystal X‐ray diffraction analysis revealed the existence of a fully extended β‐strand‐like structure stabilized by two non‐conventional C?H???O=C intramolecular H‐bonds. The 2D ¹H NMR ROESY experiment led us to propose that the flat topology of the urethane‐γ‐Abz‐amide moiety is predominantly preserved in a non‐polar environment. The self‐association of the energetically more favorable antiparallel β‐strand‐mimic in solid‐state engenders an unusual ‘flight of stairs’ fabricated through face‐to‐face and edge‐to‐edge Ar???Ar interactions. In conjunction with FT‐IR spectroscopic analysis in chloroform, we highlight that conformationally semi‐rigid γ‐Abz foldamer in appositely designed peptides may encourage unusual β‐strand or β‐sheet‐like self‐association and supramolecular organization stabilized via weak attractive forces.     

Synthesis and characterization of new aromatic polyamides bearing crown ethers or their dipodal counterparts in the pendant structure. I. Benzo‐12‐crown‐4 and ortho‐bis(2‐ethoxyethoxy)benzene

Two novel isophthalic diacid‐based monomers have been synthesized by inclusion in ring position 5 of a functionalized benzoylamine moiety. The functionalization includes a 12‐crown‐4 ether group fused with the benzene subunit and a dipodand substructure, formally a disubstitution of the benzene ring, with two sequences of ethyl‐terminated ethylene oxide units, which represent the open‐chain counterpart of the alicylic crown moiety. The polycondensation of the two diacids with five aromatic diamines yielded 10 new polyamides with crown or podand pendant substructures. The polyamides had previously been chemically characterized by NMR, IR, and elemental analysis. The polymers showed high glass transition temperatures of up to 349 °C, good thermal stability (T_{donset, N2} ≈ 400 °C), and improved solubility in organic solvents. The presence of acyclic or alicyclic oxyethylene sequences as crown ether or podand substructures and an additional amide side group per repeat unit made the polymers essentially amorphous and improved their water absorption ability in comparison with nonsubstituted polyamides. Water uptake values as high as 12% were observed at 65% relative humidity. All the polyamides showed a good film‐forming ability, and the mechanical properties of these films are considered to be satisfactory for experimental aromatic polyamides. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2270–2281, 2006     

Optically high transparency and light color of organosoluble polyamides containing trifluoromethyl and kink diphenylmethylene linkage

New polyamides were prepared directly from a diamine, bis[4‐(2‐trifluoromethyl 4‐aminophenoxy)phenyl] diphenylmethane, containing an electron‐withdrawing trifluoromethyl group and a kink diphenylmethylene linkage with various aromatic dicarboxylic acids having inherent viscosities ranging from 0.66 to 0.83 dL g^?1. All the polyamides showed outstanding solubility and could be easily dissolved in amide‐type polar aprotic solvents (e.g., N‐methyl‐2‐pyrrolidinone, N,N‐dimethylacetamide, and N,N‐dimethylformamide) and even dissolved in less polar solvents (e.g., pyridine, cyclohexanone, and tetrahydrofuran). The dielectric constants of the polyamide films were 3.37–3.87 (100 KHz) and decreased with an increase in the frequency, which ranged from 1 Hz to 100 KHz. A low coefficient of thermal expansion for the polyamides was observed in the range of 54–78 ppm/°C (by thermomechanical analysis). These polyamides showed excellent thermal stability, and the 10% weight loss temperatures were in the range of 484–507 °C in an atmosphere of nitrogen. The polymers had an initial modulus of 1.8–2.2 GPa. The polyamides with kink and electron‐withdrawing trifluoromethyl units afforded light‐color polymer films with high transmittance in the visible region (400–700 nm), and their cutoff wavelength was lower than 362 nm. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4559–4569, 2005     

Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. A general, reversible bicyclization strategy is now reported to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell‐penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell‐permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was applied to generate a cell‐permeable bicyclic peptidyl inhibitor against the NEMO‐IKK interaction.     

Evidence of Splitting 1,2,3‐Triazole into an Alkyne and Azide by Low Mechanical Force in the Presence of Other Covalent Bonds

The cycloaddition reaction of an alkyne and azide to form a 1,2,3‐triazole is widely used in many areas. However, the stability of the triazole moiety under mechanical stress is unclear. To see if a triazole could be selectively split into an alkyne and azide in the presence of other typical covalent bonds, a mica surface functionalized with a molecule containing a triazole moiety in the middle and an activated ester at the end was prepared. An atomic force microscope (AFM) tip with amino groups on its surface was ramped over the mica surface at predefined locations, which could temporarily link the tip to the surface through amide bond formation. During retraction, the triazole or another bond in the linkage broke, and a force was recorded. The forces varied widely at different ramps from close to 0 pN to 860 pN due to nonspecific adhesions and to the inherent inconsistency of single bond rupture. If some of the forces were from triazole cycloreversion, there would be alkynes at the predefined ramping locations. The surface was reacted with an azide carboxylic acid followed by labeling with amino Au nanoparticles (AuNPs). AFM imaging revealed AuNPs at the predicted locations, which provided evidence that under certain conditions triazole could be split selectively in the presence of other bonds at forces below 860 pN.     

聚酰胺类DNA识别分子质谱的碎裂机理

采用ESI-MS法研究了8个含有N-甲基吡咯(Py)和N-甲基咪唑(Im)杂环的聚酰胺质谱的特征和碎裂机理. MSⁿ数据表明, 聚酰胺化合物的主要碎裂路径是环与环间化学键的断裂, 即C—CO键、CO—NH键、HN—C键的断裂, 同时伴随着H原子的重排. 利用这些碎裂特征, 可以得到聚酰胺丰富的结构信息和区分它们的两种同分异构体.     

Highly covalent ferric-thiolate bonds exhibit surprisingly low mechanical stability

Depending on their nature, different chemical bonds show vastly different stability with covalent bonds being the most stable ones that rupture at forces above nanonewton. Studies have revealed that ferric-thiolate bonds are highly covalent and are conceived to be of high mechanical stability. Here, we used single molecule force spectroscopy techniques to directly determine the mechanical strength of such highly covalent ferric-thiolate bonds in rubredoxin. We observed that the ferric-thiolate bond ruptures at surprisingly low forces of ～200 pN, significantly lower than that of typical covalent bonds, such as C-Si, S-S, and Au-thiolate bonds, which typically ruptures at >1.5 nN. And the mechanical strength of Fe-thiolate bonds is observed to correlate with the covalency of the bonds. Our results indicated that highly covalent Fe-thiolate bonds are mechanically labile and display features that clearly distinguish themselves from typical covalent bonds. Our study not only opens new avenues to investigating this important class of chemical bonds, but may also shed new lights on our understanding of the chemical nature of these metal thiolate bonds.     

Improvement in semipermeable membrane performance of wholly aromatic polyamide through an additive processing strategy

A new concept for the method to provide semipermeability in ultrathin and single‐component wholly aromatic polyamide membranes has been developed for the first time. It was found that water molecules could permeate through the membrane prepared not from polyamides containing flexible ether, bulky binaphthyl, or fluorene rigid units, but one with carboxylic acid groups under a reverse osmosis mode. However, the enhancement of water transport properties by introducing the hydrophilic group of polyamide was not substantial. Therefore, polyamide membranes were prepared from the solution containing aqueous additives in order to weaken hydrogen bonds between polymer chains and thereby to suppress the aggregation of the polymer chains. As a result, water flux was dramatically improved with slightly improved NaCl rejection. Our analyses based on attenuated total reflectance Fourier transform infrared spectroscopy and solid‐state carbon polarization and magic angle spinning nuclear magnetic resonance (¹³C CPMAS NMR) spectroscopy confirmed that the aggregation of polymer chains due to the hydrogen bonds among the amide linkages was suppressed by the co‐ordination of the aqueous additives to the amide linkage. The state of water in the membranes analyzed by differential scanning calorimetry also supported the formation of pores. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1275–1281     

GaGa‐ und AlFe‐Mehrfachbindungen? Ein Interpretationsversuch auf der Grundlage von Kraftkonstanten

GaGa and AlFe Multiple Bonds? An Attempt of Interpretation on the Basis of Force Constants Comparison of force constant values given by DFT frequency calculations shows that GaGa‐bonds in Ga₂H₄^2– and Ga₂H₂^2– are only slightly strengthened with respect to the GaGa‐single bond in Ga₂H₆^2–. On the other hand FeAl bonds in compounds like CpAlFe(CO)₄ are interpreted as double bonds.