Enantioselective synthesis of protected L-4-[sulfonamido(difluoromethyl)]phenylalanine and L-4-[sulfonamido(methyl)]phenylalanine and an examination of hexa- and tripeptide platforms for evaluating pTyr mimics for PTP1B inhibition

The first enantioselective syntheses of L-4-(sulfonamidomethyl)phenylalanine and L-[sulfonamido(difluoromethyl)]phenylalanine suitably protected for peptide syntheses are described. A key step in the synthesis of L-(sulfonamidomethyl)phenylalanine was an oxidative chlorination on Ac-L-Phe(4-CH2SCOCH3)-OEt to give crude Ac-L-Phe(4-CH2SO2Cl)-OEt, which could be reacted with amines to give the corresponding sulfonamides. Key to the preparation of L-[sulfonamido(difluoromethyl)]phenylalanine was a highly enantioselective reaction involving William's auxiliary and a benzylic bromide intermediate. These amino acids were incorporated into two peptide sequences, DADE-X-LNH2 and FmocGlu(OBn)-X-LNH2, which have previously been employed as platforms for assessing pTyr mimics for inhibition of protein tyrosine phosphatase 1B (PTP1B). Inhibition studies with these and other peptides and PTP1B revealed that good inhibition could be obtained using the tripeptide platform, although the presence of a pTyr mimic was not required for good inhibition. These results suggest that the FmocGlu(OBn)-X-LNH2 tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition.     

A self-immobilizing and fluorogenic unnatural amino acid that mimics phosphotyrosine

Synthesis of the first self-immobilizing, fluorogenic unnatural amino acid that mimics phosphotyrosine (pTyr) is reported. By using solid-phase peptide synthesis, it was subsequently incorporated into peptide-based probes which found applications in bioimaging and fluorescence-activated cell sorting (FACS).     

Calorimetric and structural studies of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide inhibitors of Src SH2 domain binding.

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.     

Serine-cis-proline and serine-trans-proline isosteres: stereoselective synthesis of (Z)- and (E)-alkene mimics by Still-Wittig and Ireland-Claisen rearrangements

Two new amide isosteres of Ser-cis-Pro and Ser-trans-Pro dipeptides were designed and stereoselectively synthesized to be incorporated into potential inhibitors of the phosphorylation-dependent peptidylprolyl isomerase Pin1, an essential regulator of the cell cycle. The cis mimic, the (Z)-alkene isomer, was formed through the use of a Still-Wittig [2,3]-sigmatropic rearrangement, while the trans mimic, the (E)-alkene, was synthesized through the use of an Ireland-Claisen [3,3]-sigmatropic rearrangement. Starting from N-Boc-Ser(OBn)-N(OMe)Me, both mimics were synthesized in Boc-protected form suitable for peptide synthesis with an overall yield of 20% in 10 steps for the cis mimic and 13% in eight steps for the trans mimic.     

Efficient synthesis of a side-chain extended diaminodiacid for solid-phase synthesis of peptide disulfide bond mimics

Solid-phase incorporation of diaminodiacids is one of the most effective approaches for synthesis of peptide disulfide bond mimics. One of a limitation of current diaminodiacid toolbox is that only four-atom linkage mimics are available that may not fully meet the activity optimization requirement. In this work, we developed a new diaminodiacid that contains a five-atom thioether (C–C–S–C–C) bridge for the first time. With this diaminodiacid in hand, we successfully obtained oxytocin containing new disulfide bond mimic by solid phase peptide synthesis.     

Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.     

Synthesis and Evaluation of Non-Hydrolyzable Phospho-Lysine Peptide Mimics

The intrinsic lability of the phosphoramidate P−N bond in phosphorylated histidine (pHis), arginine (pHis) and lysine (pLys) residues is a significant challenge for the investigation of these post-translational modifications (PTMs), which gained attention rather recently. While stable mimics of pHis and pArg have contributed to study protein substrate interactions or to generate antibodies for enrichment as well as detection, no such analogue has been reported yet for pLys. This work reports the synthesis and evaluation of two pLys mimics, a phosphonate and a phosphate derivative, which can easily be incorporated into peptides using standard fluorenyl-methyloxycarbonyl- (Fmoc-)based solid-phase peptide synthesis (SPPS). In order to compare the biophysical properties of natural pLys with our synthetic mimics, the pK_a values of pLys and analogues were determined in titration experiments applying nuclear magnetic resonance (NMR) spectroscopy in small model peptides. These results were used to compute electrostatic potential (ESP) surfaces obtained after molecular geometry optimization. These findings indicate the potential of the designed non-hydrolyzable, phosphonate-based mimic for pLys in various proteomic approaches.     

Photo-induced hydrogen production in a helical peptide incorporating a [FeFe] hydrogenase active site mimic

There is growing interest in the development of hydrogenase mimics for solar fuel production. Here, we present a bioinspired mimic designed by anchoring a diiron hexacarbonyl cluster to a model helical peptide via an artificial dithiol amino acid. The [FeFe]-peptide complex catalyses photo-induced production of hydrogen in water.     

Utilization of 3'-carboxy-containing tyrosine derivatives as a new class of phosphotyrosyl mimetics in the preparation of novel non-phosphorylated cyclic peptide inhibitors of the Grb2-SH2 domain

A new class of phosphotyrosyl (pTyr) mimetics, distinct from the conventional pTyr mimetic design of adding non-hydrolyzable acidic functionalities to the 4'-position of phenylalanine, was created by introducing carboxy-containing groups to the 3'-position of tyrosine. The effect of the chain length of the carboxy substituent was examined. Reported herein is the chiral pool synthesis of the new pTyr mimetics, and their first use in a novel non-phosphorylated Grb2-SH2 domain binding motif with the 5-amino-acid sequence Xx1-Leu-(3'-substituted-Tyr)-Ac6c-Asn. The highest affinity was exhibited by the 3-L-(2-carboxyethyl)tyrosine-containing sulfoxide-cyclized peptide , with an IC50 = 1.1 microM, providing a promising new template for further development of potent Grb2-SH2 domain inhibitors with reduced charge and peptidic nature, but improved selectivity and bioavailability.     

Novel Bicyclic Lactams as XaaPro Type VI beta Turn Mimics: Design, Synthesis, and Evaluation

The design, enantioselective synthesis, and structural characterization of novel bicyclic lactams as peptide mimics of the type VI beta turn is described. The mimics duplicate the conformation of the backbone and disposition of the side-chain atoms of the central two residues of the turn. The Gly L-Pro mimic, lactam 6, was prepared in good overall yield starting from (S)-2-(2'-propenyl)proline. (1)H NMR spectroscopy defined the relative stereochemistry of the substituents and conformational characteristics of the six-membered ring of the lactam; X-ray crystallographic analysis confirmed the conformational and stereochemical assignment. Examination of the crystal structure of lactam 6 revealed that the central amide bond was twisted appreciably out of planarity. The twisting of the amide bond was attributed to angle strain resulting from the presence of the sp(2)-hybridized nitrogen atom at the junction of the two rings. Alkylation of the enolate of the N,N-dimethylformamidine derivative of lactam 6 with benzyl bromide afforded stereoselectively the formamidine 11, a mimic of an L-Phe L-Pro dipeptide in the type VI turn conformation. The efficient synthetic route to highly functionalized peptidomimetics such as 11 will prove highly useful in peptide structure-function studies.     

Structural and energetic effects in the molecular recognition of protonated peptidomimetic bases by 18-crown-6

Absolute 18-crown-6 (18C6) affinities of nine protonated peptidomimetic bases are determined using guided ion beam tandem mass spectrometry techniques. The bases (B) included in this work are mimics for the n-terminal amino group and the side chains of the basic amino acids, i.e., the favorable sites for binding of 18C6 to peptides and proteins. Isopropylamine is chosen as a mimic for the n-terminal amino group, imidazole and 4-methylimidazole are chosen as mimics for the side chain of histidine (His), 1-methylguanidine is chosen as a mimic for the side chain of arginine (Arg), and several primary amines including methylamine, ethylamine, n-propylamine, n-butylamine, and 1,5-diamino pentane as mimics for the side chain of lysine (Lys). Theoretical electronic structure calculations are performed to determine stable geometries and energetics for neutral and protonated 18C6 and the peptidomimetic bases, as well as the proton bound complexes comprised of these species, (B)H(+)(18C6). The measured 18C6 binding affinities of the Lys side chain mimics are larger than the measured binding affinities of the mimics for Arg and His. These results suggest that the Lys side chains should be the preferred binding sites for 18C6 complexation to peptides and proteins. Present results also suggest that competition between Arg or His and Lys for 18C6 is not significant. The mimic for the n-terminal amino group exhibits a measured binding affinity for 18C6 that is similar to or greater than that of the Lys side chain mimics. However, theory suggests that binding to n-terminal amino group mimic is weaker than that to all of the Lys mimics. These results suggest that the n-terminal amino group may compete with the Lys side chains for 18C6 complexation.     

A triply templated artificial beta-sheet.

This paper describes the design, synthesis, and structural evaluation of a compound (4) comprising three molecular templates and a peptide strand that mimics a three-stranded protein beta-sheet. Two of the templates mimic the hydrogen-bonding functionality of peptide beta-strands and serve as the top and bottom strands by embracing the peptide strand, which is located in the middle of the sheet. The remaining template holds the three strands next to each other. The synthesis of artificial beta-sheet 4 begins with the bottom template and involves the sequential addition of the middle and top strands. (1)H NMR chemical shift and NOE studies establish that this compound folds to adopt a hydrogen-bonded beta-sheetlike structure in CDCl(3) solution. Chemical shift studies indicate that triply stranded artificial beta-sheet 4 is more tightly folded than its smaller doubly stranded homologue, artificial beta-sheet 1.     

Can collision‐induced negative‐ion fragmentations of [M–H]– anions be used to identify phosphorylation sites in peptides?

A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.     

Structural examination of ring-closing metathesis-derived 15-member macrocycles as Grb2 SH2 domain-binding tetrapeptide mimetics

Ring-closing metathesis (RCM) was employed to join carboxy-terminal alkenyl glycine side chains together with vinyl- and allyl-functionality appended to the beta-methylene of amino-terminal phosphotyrosyl (pTyr) mimetics. This required the synthesis of a variety of new pTyr mimetics, including a novel aza-containing analogue. Many of the resulting 15-member macrocyclic tetrapeptide mimetics exhibited low nanomolar Grb2 SH2 domain-binding affinities in spite of the fact that differing ring junction stereochemistries and geometries of the RCM-derived double bond were employed. The finding that significant latitude exists in the structural requirements for ring closure may facilitate the development of therapeutically relevant macrocyle-based Grb2 SH2 domain-binding antagonists. The synthetic approaches used in this study may also find application to peptide mimetics directed at other biological targets.     

Crystal structure of a transition state mimic for Tdp1 assembled from vanadate,DNA, and a topoisomerase I-derived peptide

Tyrosyl-DNA phosphodiesterase (Tdp1) is a member of the phospholipase D superfamily and acts as a DNA repair enzyme that removes stalled topoisomerase I- DNA complexes by hydrolyzing the bond between a tyrosine side chain and a DNA 3' phosphate. Despite the complexity of the substrate of this phosphodiesterase, vanadate succeeded in linking human Tdp1, a tyrosine-containing peptide, and a single-stranded DNA oligonucleotide into a quaternary complex that mimics the transition state for the first step of the catalytic reaction. The conformation of the bound substrate mimic gives compelling evidence that the topoisomerase I-DNA complex must undergo extensive modification prior to cleavage by Tdp1. The structure also illustrates that the use of vanadate as the central moiety in high-order complexes has the potential to be a general method for capturing protein-substrate interactions for phosphoryl transfer enzymes, even when the substrates are large, complicated, and unusual.     

The energy landscape of a selective tumor-homing pentapeptide

Recently, a potentially powerful strategy based on phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified simulated annealing combined with molecular dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu, and Lys, which induces a beta-turn motif in the backbone observed in all simulated CREKA systems.     

CH/pi hydrogen bonds determine the selectivity of the Src homology 2 domain to tyrosine phosphotyrosyl peptides: an ab initio fragment molecular orbital study

The CH/pi hydrogen bond is a weak molecular force occurring between CH groups (soft acids) and pi-systems (soft bases), and has been recognized to be important in the interaction of proteins with their specific ligands. For instance, it is well known that Src homology-2 protein (SH2) recognizes its specific pTyr peptide in two key regions, pTyr-binding region and specificity-determining region, by the use of attractive molecular forces, including the CH/pi hydrogen bond. We hypothesized that the CH/pi hydrogen bond plays a key role in determining the selectivity of SH2 proteins, and studied this issue by the ab initio fragment molecular orbital (FMO) method. The FMO calculations were carried out, at the HF/6-31G* and MP2/6-31G* level, for SH2 domains of Src, Grb2, P85alpha(N), Syk, and SAP, in complex with corresponding pTyr peptides. CH/pi hydrogen bonds have in fact been found to be important in stabilizing the structure of the complexes. We conclude that the CH/pi hydrogen bond plays an indispensable role in the recognition of SH2 domains with their specific pTyr peptides, thus playing a vital role in the signal transduction system.     

Chiral dipeptide mimics possessing a fluoroolefin moiety: a relevant tool for conformational and medicinal studies

The replacement of the amide bond in a peptide backbone is a promising strategy in peptidomimetic drug research. Over the various amide bond surrogates, the fluoroolefin moiety has been successfully developed as an effective mimic. Today, fluorine-containing compounds account for a large proportion of new active molecules in life sciences. The synthesis of fluoroolefin peptide mimics is not a trivial task and innovative approaches often need to be addressed, in particular for the stereocontrol of the double bond configuration and the chiral centres adjacent to the fluoroalkene. These fluorinated peptidomimetics have been synthesised and evaluated as metabolically stable and/or conformationally constrained analogs of enzyme inhibitors, and as tools for probing the function, structure, and binding process of receptors.