Traceless Preparation of C‐Terminal α‐Ketoacids for Chemical Protein Synthesis by α‐Ketoacid–Hydroxylamine Ligation: Synthesis of SUMO2/3

A novel protecting group for enantiopure α‐ketoacids delivers C‐terminal peptide α‐ketoacids directly upon resin cleavage and allows the inclusion of all canonical amino acids, including cysteine and methionine. By using this approach, SUMO2 and SUMO3 proteins were prepared by KAHA ligation with 5‐oxaproline. The synthetic proteins containing homoserine residues were recognized by and conjugated to RanGAP1 by SUMOylation enzymes.     

De Novo Design of a βαβ Motif

A designer monomeric protein with a βαβ fold—two parallel β strands connected by an α helix (see structure)—was constructed solely from coded amino acids. The high thermal stability of the structure is due to a large extent to tryptophan–tryptophan interactions between the two β strands.

     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

A Hybrid Ring‐Opening Metathesis Polymerization Catalyst Based on an Engineered Variant of the β‐Barrel Protein FhuA

A β‐barrel protein hybrid catalyst was prepared by covalently anchoring a Grubbs–Hoveyda type olefin metathesis catalyst at a single accessible cysteine amino acid in the barrel interior of a variant of β‐barrel transmembrane protein ferric hydroxamate uptake protein component A (FhuA). Activity of this hybrid catalyst type was demonstrated by ring‐opening metathesis polymerization of a 7‐oxanorbornene derivative in aqueous solution.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometry characterization of 3‐OH butyrated β‐cyclodextrin

Oligo(3‐OH butyrate)‐β‐cyclodextrin esters (PHB‐CD) were obtained through ring opening of β‐butyrolactone (β‐BL) in the presence of β‐cyclodextrin (CD) and (‐)‐sparteine (SP) as nucleophilic activator. The resulted reaction mixture was first separated in two fractions and then investigated through a deep mass spectrometry (MS) study performed on a liquid chromatography‐electrospray ionization‐quadrupole time of flight (LC‐ESI‐QTOF) instrument. LC MS and tandem MS structural assignment of the reaction products was completed by NMR. The performed analysis revealed that poly(3‐OH butyrate) homopolymers (PHB) are formed together with the PHB‐CD products. Secondary reactions resulting in the formation of crotonates were also proved to occur. A comparison between MS and NMR results demonstrated that more than one PHB oligomer is attached to the CD in the PHB‐CD product. The tandem MS fragmentation studies validated the proposed structure of CD derivatives. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Resistance of Cu(Aβ4–16) to Copper Capture by Metallothionein‐3 Supports a Function for the Aβ4–42 Peptide as a Synaptic CuII Scavenger

Aβ4‐42 is a major species of Aβ peptide in the brains of both healthy individuals and those affected by Alzheimer's disease. It has recently been demonstrated to bind Cu^II with an affinity approximately 3000 times higher than the commonly studied Aβ1‐42 and Aβ1‐40 peptides, which are implicated in the pathogenesis of Alzheimer's disease. Metallothionein‐3, a protein considered to orchestrate copper and zinc metabolism in the brain and provide antioxidant protection, was shown to extract Cu^II from Aβ1‐40 when acting in its native Zn₇MT‐3 form. This reaction is assumed to underlie the neuroprotective effect of Zn₇MT‐3 against Aβ toxicity. In this work, we used the truncated model peptides Aβ1‐16 and Aβ4‐16 to demonstrate that the high‐affinity Cu^II complex of Aβ4‐16 is resistant to Zn₇MT‐3 reactivity. This indicates that the analogous complex of the full‐length peptide Cu(Aβ4‐42) will not yield copper to MT‐3 in the brain, thus supporting the concept of a physiological role for Aβ4‐42 as a Cu^II scavenger in the synaptic cleft.     

Effective Assignment of α2,3/α2,6‐Sialic Acid Isomers by LC‐MS/MS‐Based Glycoproteomics

Distinct structural changes of the α2,3/α2,6‐sialic acid glycosidic linkages on glycoproteins are of importance in cancer biology, inflammatory diseases, and virus tropism. Current glycoproteomic methodologies are, however, not amenable toward high‐throughput characterization of sialic acid isomers. To enable such assignments, a mass spectrometry method utilizing synthetic model glycopeptides for the analysis of oxonium ion intensity ratios was developed. This method was successfully applied in large‐scale glycoproteomics, thus allowing the site‐specific structural characterization of sialic acid isomers.     

Electronic Structure Analysis of the Oxygen‐Activation Mechanism by FeII‐ and α‐Ketoglutarate (αKG)‐Dependent Dioxygenases

α‐Ketoglutarate (αKG)‐dependent nonheme iron enzymes utilize a high‐spin (HS) ferrous center to couple the activation of oxygen to the decarboxylation of the cosubstrate αKG to yield succinate and CO₂, and to generate a high‐valent ferryl species that then acts as an oxidant to functionalize the target C? H bond. Herein a detailed analysis of the electronic‐structure changes that occur in the oxygen activation by this enzyme was performed. The rate‐limiting step, which is identical on the septet and quintet surfaces, is the nucleophilic attack of the distal O atom of the O₂ adduct on the carbonyl group in αKG through a bicyclic transition state (^{5, 7}TS1). Due to the different electronic structures in ^{5, 7}TS1, the decay of ⁷TS1 leads to a ferric oxyl species, which undergoes a rapid intersystem crossing to form the ferryl intermediate. By contrast, a HS ferrous center ligated by a peroxosuccinate is obtained on the quintet surface following ⁵TS1. Thus, additional two single‐electron transfer steps are required to afford the same Fe^IV–oxo species. However, the triplet reaction channel is catalytically irrelevant. The biological role of αKG played in the oxygen‐activation reaction is dual. The αKG LUMO (C?O π*) serves as an electron acceptor for the nucleophilic attack of the superoxide monoanion. On the other hand, the αKG HOMO (C1? C2 σ) provides the second and third electrons for the further reduction of the superoxide. In addition to density functional theory, high‐level ab initio calculations have been used to calculate the accurate energies of the critical points on the alternative potential‐energy surfaces. Overall, the results delivered by the ab initio calculations are largely parallel to those obtained with the B3LYP density functional, thus lending credence to our conclusions.     

Intermediates Formed in the Reactions of Organocuprates with α,β‐Unsaturated Nitriles

Conjugate additions of organocuprates are of outstanding importance for organic synthesis. To improve our mechanistic understanding of these reactions, we have used electrospray ionization mass spectrometry for the identification of the ionic intermediates formed upon the treatment of LiCuR₂ ? LiCN (R=Me, Bu, Ph) with a series of α,β‐unsaturated nitriles. Acrylonitrile, the weakest Michael acceptor included, did not afford any detectable intermediates. Fumaronitrile (FN) yielded adducts of the type Li_n?1Cu_nR_2n(FN)_n^?, n=1–3. When subjected to fragmentation in the gas phase, these adducts were not converted into the conjugate addition products, but re‐dissociated into the reactants. In contrast, the reaction with 1,1‐dicyanoethylene furnished the products of the conjugate addition without any observable intermediates. Tri‐ and tetracyanoethylene proved to be quite reactive as well. The presence of several cyano groups in these substrates opened up reaction pathways different from simple conjugate additions, however, and led to dimerization and substitution reactions. Moreover, the gas‐phase fragmentation behavior of the species formed from these substrates indicated the occurrence of single‐electron transfer processes. Additional quantum‐chemical calculations provided insight into the structures and stabilities of the observed intermediates and their consecutive reactions.     

Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation

The chemical synthesis of the 184‐residue ferric heme‐binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α‐ketoacid‐hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild‐type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well‐suited for the preparation of hydrophobic protein targets.     

Stabilization of an α Helix by β‐Sheet‐Mediated Self‐Assembly of a Macrocyclic Peptide

Protein roll call : Peptide‐based building blocks, in which both an α‐helix‐forming segment and a β‐sheet segment are located within a single macrocyclic structure, self‐assemble into α‐helix‐decorated artificial proteins. This approach provides a starting point for developing artificial proteins that can modulate α‐helix‐mediated interactions occurring in a multivalent fashion.

     

Hydrolysis Reaction of N-Phosphoryl-α-, β- and γ-amino Acids Studied by HPLC

The hydrolysis reactions of N-（O,O＇diisopropyl）phosphoryl-L-α-alanine （DIPP-L-α-Ala）, N-（O,O＇diisopropyl）- phosphoryl-D-α-alanine （DIPP-D-α-Ala）, N-（O,O＇-diisopropyl）phosphoryl-β-alanine （DIPP-β-Ala） and N-（O,O＇-diisopropyl）phosphoryl-γ-amino butyric acid （DIPP-γ-Aba）, were studied by HPLC and their hydrolysis reaction kinetic equations were obtained. Under acid conditions, the reaction rate of DIPP-L-α-Ala was close to that of DIPP-D-α-Ala and the same rule was true between DIPP-β-Ala and DIPP-γ-Aba. Meantime, the reaction rate of DIPP-L/D-α-Ala was as 10 times as that of DIPP-β-Ala or DIPP-γ-Aba. Under basic conditions, the hydrolysis reactions of DIPP-β-Ala and DIPP-γ-Aba almost did not take place and the reaction rate of DIPP-L/D-α-Ala was about 1/10 of that under acid conditions. Moreover, theoretical calculation further illuminated the differences of the hydrolysis rate from the view of energy. The results would provide some helpful clues to why nature chose a-amino acids but not other kinds of analogs as protein backbones.     

Synthesis and Crystal Structure of α‐ThTe3

The binary thorium tritelluride, α‐ThTe₃, was synthesized by solid‐state methods at 1223 K. From a single‐crystal X‐ray diffraction study the material crystallizes in the TiS₃ structure type with two formula units in space group C²_2h–P2₁/m of the monoclinic system in a cell with lattice constants a = 6.1730 (4) Å, b = 4.3625(3) Å, c = 10.4161(6) Å, and β = 97.756(3)° (at 100 K). The asymmetric unit of this compound comprises one Th atom and three Te atoms each with site symmetry m. Each Th atom is coordinated to eight Te atoms in a bicapped trigonal‐pyramidal arrangement. Th–Te distances range from 3.1708(4) Å to 3.2496(6) Å. The structure features a Te–Te interaction 2.7631(8) Å in length, which is typical for a Te–Te single bond. Thus α‐ThTe₃ may be charge balanced and formulated as Th⁴⁺Te^2–Te₂^2–.     

Sequestration of a β‐Hairpin for Control of α‐Synuclein Aggregation

The misfolding and aggregation of the protein α‐synuclein (α‐syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson’s disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β‐wrapins (β‐wrap proteins) with affinity for α‐synuclein (α‐syn). The NMR structure of an α‐syn:β‐wrapin complex reveals a β‐hairpin of α‐syn comprising the sequence region α‐syn(37–54). The β‐wrapin inhibits α‐syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.     

carbo‐Naphthalene: A Polycyclic carbo‐Benzenoid Fragment of α‐Graphyne

A ring carbo‐mer of naphthalene, C₃₂Ar₈ (Ar=p‐n‐pentylphenyl), has been obtained as a stable blue chromophore, after a 19‐step synthetic route involving methods inspired from those used in the synthesis of carbo‐benzenes, or specifically devised for the present target, like a double Sonogashira‐type coupling reaction. The last step is a SnCl₂/HCl‐mediated reduction of a decaoxy‐carbo‐decalin, which is prepared through successive [8+10] macrocyclization steps. Two carbo‐benzene references are also described, C₁₈Ar₆ and o‐C₁₈Ar₄(C≡C‐SiiPr₃)₂. The carbo‐naphthalene bicycle is locally aromatic according to structural and magnetic criteria, as revealed by strong diatropic ring current effects on the deshielding of ¹H nuclei of the Ar groups and on the negative value of the DFT‐calculated NICS at the center of the C₁₈ rings (?12.8 ppm). The stability and aromaticity of this smallest fused molecular fragment of α‐graphyne allows prediction of the same properties for the carbon allotrope itself.     

The Molecular Basis of the Interaction of Cyclophilin A with α‐Synuclein

Peptidylprolyl isomerases (PPIases) catalyze cis/trans isomerization of prolines. The PPIase CypA colocalizes with the Parkinson's disease (PD)‐associated protein α‐synuclein in cells and interacts with α‐synuclein oligomers. Herein, we describe atomic insights into the molecular details of the α‐synuclein/CypA interaction. NMR spectroscopy shows that CypA catalyzes isomerization of proline 128 in the C‐terminal domain of α‐synuclein. Strikingly, we reveal a second CypA‐binding site formed by the hydrophobic sequence ⁴⁷GVVHGVATVA⁵⁶, termed PreNAC. The 1.38 Å crystal structure of the CypA/PreNAC complex displays a contact between alanine 53 of α‐synuclein and glutamine 111 in the catalytic pocket of CypA. Mutation of alanine 53 to glutamate, as found in patients with early‐onset PD, weakens the interaction of α‐synuclein with CypA. Our study provides high‐resolution insights into the structure of the PD‐associated protein α‐synuclein in complex with the most abundant cellular cyclophilin.     

Cyclization of Zincated α‐N‐Homoallylamino Nitriles: A New Entry to Enantiopure 2,3‐Methanopyrrolidines

Stereoselective cyclization of zincated α‐N‐homoallylamino nitriles has been developed. Following treatment with lithium diisopropylamide (LDA) and transmetalation with zinc bromide, α‐N‐(1‐phenylethyl)‐N‐homoallylamino nitriles lead to 2,3‐methanopyrrolidines in moderate to good yields (up to 66 %) and excellent selectivities (up to >98:2). With substrates derived from α‐branched homoallylic amines, a stereospecific inversion of the homoallylic stereogenic center was observed. To account for this, a mechanistic rationale involving the formation of zincioiminium ions from zincated α‐amino nitriles is put forward. 2,3‐Methanopyrrolidines should then arise from a sequence involving an aza‐Cope rearrangement providing a configurationally stable (2‐azoniaallyl)zinc species that then undergoes a [3+2] cycloaddition reaction.