Syntheses,Receptor Bindings,in vitro and in vivo Stabilities and Biodistributions of DOTA‐Neurotensin(8–13) Derivatives Containing β‐Amino Acid Residues – A Lesson about the Importance of Animal Experiments

Neurotensin(8–13) (NTS(8–13)) analogs with C‐ and/or N‐terminal β‐amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1 – 6 ). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) derivatives, 6a , into a crystallographically identified receptor NTSR1 (Fig. 1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell‐membrane homogenates, while, with NTSR1‐exhibiting cancer tissues, affinities in the single‐digit nanomolar range can be observed (Table 2). Most of the β‐amino acid‐containing NTS(8–13) analogs (Table 1 and Fig. 2), including the ⁶⁸Ga complexes of the DOTA‐substituted ones ( 6 ; Figs. 2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two ⁶⁸Ga complexes (of 6a and 6b ) in HT29 tumor‐bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET‐imaging experiments with the tumor‐bearing mice (Fig. 6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10–15 min) of the two ⁶⁸Ga complexes shows that they are rapidly cleaved in the animals (Fig. 5).     

Synthesis,Structure, and Biological Applications of α‐Fluorinated β‐Amino Acids and Derivatives

This review gives a broad overview of the state of play with respect to the synthesis, conformational properties, and biological activity of α‐fluorinated β‐amino acids and derivatives. General methods are described for the preparation of monosubstituted α‐fluoro‐β‐amino acids (Scheme 1). Nucleophilic methods for the introduction of fluorine predominantly involve the reaction of DAST with alcohols derived from α‐amino acids, whereas electrophilic sources of fluorine such as NFSI have been used in conjunction with Arndt? Eistert homologation, conjugate addition or organocatalyzed Mannich reactions. α,α‐Difluoro‐β‐amino acids have also been prepared using DAST; however, this area of synthesis is largely dominated by the use of difluorinated Reformatsky reagents to introduce the difluoro ester functionality (Scheme 9). α‐Fluoro‐β‐amino acids and derivatives analyzed by X‐ray crystal and NMR solution techniques are found to adopt preferred conformations which are thought to result from stereoelectronic effects associated with F located close to amines, amides, and esters (Figs. 2–6). α‐Fluoro amide and β‐fluoro ethylamide/amine effects can influence the secondary structure of α‐fluoro‐β‐amino acid‐containing derivatives including peptides and peptidomimetics (Figs. 7–9). α‐Fluoro‐β‐amino acids are also components of a diverse range of bioactive anticancer (e.g., 5‐fluorouracil), antifungal, and antiinsomnia agents as well as protease inhibitors where such fluorinated analogs have shown increased potency and spectrum of activity.     

Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

β‐amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII–ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 β‐amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC‐MS analysis, respectively. The β‐amino acid‐substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure‐activity profiles were observed corresponding to substitutions in the N‐terminus, the central region and the C‐terminal region of AngII. The results show that β‐substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. β‐amino acid substitution in the N‐terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased β‐turn structure and enhanced substrate cleavage. β‐amino acid substitution in the C‐terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The β‐AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease. Copyright © 2010 John Wiley & Sons, Ltd.     

Interaction of β3/β2‐Peptides,Consisting of Val‐Ala‐Leu Segments,with POPC Giant Unilamellar Vesicles (GUVs) and White Blood Cancer Cells (U937) – A New Type of Cell‐Penetrating Peptides,and a Surprising Chain‐Length Dependence of Their Vesicle‐ and Cell‐Lysing Activity

Many years ago, β²/β³‐peptides, consisting of alternatively arranged β²‐ and β³h‐amino‐acid residues, have been found to undergo folding to a unique type of helix, the 10/12‐helix, and to exhibit non‐polar, lipophilic properties (Helv. Chim. Acta 1997 , 80, 2033). We have now synthesized such ‘mixed’ hexa‐, nona‐, dodeca‐, and octadecapeptides, consisting of Val‐Ala‐Leu triads, with N‐terminal fluorescein (FAM) labels, i.e., 1 – 4 , and studied their interactions with POPC (=1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow‐cytometry assay, a membrane‐toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β³/β²‐peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β³/β²‐, β³‐, and β²‐nonamers, 2, 5 , and 6 , respectively, the derivatives 5 and 6 consisting exclusively of β³‐ or β²‐amino‐acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β³/β²‐nonamer and/or the β³/β²‐dodecamer derivative, 2 and/or 3 , respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host‐defense antimicrobial peptides. Possible sources of the chain‐length‐dependent destructive potential of the β³/β²‐nona‐ and β³/β²‐dodecapeptide derivatives, and a possible relationship with the phosphate‐to‐phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of ‘mixed’ β³/β²‐peptides with membranes and to evaluate possible biomedical applications.     

Synthesis and Biological Evaluation of Gramicidin S‐Inspired Cyclic Mixed α/β‐Peptides

Via a Mannich reaction involving a dibenzyliminium species and the titanium enolates of Evans' chiral acylated oxazolidinones the β²‐amino acids (R)‐ and (S)‐Fmoc‐β²homovaline and (R)‐Fmoc‐β²homoleucine are synthesized. These building blocks were used, in combination with commercially available α‐ and β³‐amino acids, for the synthesis of the cyclo‐(αβ³αβ²α)₂ peptide 2 and the cyclo‐(αβ²αβ³α)₂ peptides 3 – 5 . The peptides 2 – 5 were screened for their ability to inhibit a small panel of Gram‐negative and Gram‐positive bacterial strains.     

Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

β‐Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β‐aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β³‐ and/or β²‐amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β‐peptide substrates, β‐aminopeptidases have potential to be used as biocatalysts for β‐peptide synthesis and for the resolution of enantiomerically pure β‐amino acids from racemic substrate mixtures. β‐Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N‐terminus of the newly formed β‐polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N‐terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β‐aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β‐aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β‐peptide conversion.     

Total synthesis,structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A ( 1a ) was reported to inhibit nitric oxide production in LPS‐stimulated murine macrophage RAW 264.7 cells. In the current study, solid‐phase total synthesis of stylissatin A was performed by using a safety‐catch linker and yielded the peptide with a trans‐Phe⁷‐Pro⁶ linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d ‐allo‐Ile in place of l ‐Ile and a different site for macrocyclization. The resulting product (d ‐allo‐Ile²)‐stylissatin A ( 1c ) was also found to have the trans‐Phe⁷‐Pro⁶ peptide conformations like rotamer 1b . Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a , 1b , and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2–7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product ( 1a ), the rotamer ( 1b ), and (d ‐allo‐Ile²)‐stylissatin A ( 1c ) exhibited significant inhibition of NO^.. Further investigations were focused on 1b , which also inhibited proliferation of T‐cells and inflammatory cytokine IL‐2 production. The analogs 2–7 weakly inhibited NO^. production, but strongly inhibited IL‐2 cytokine production compared with synthetic peptide 1b . All analogs inhibited the proliferation of T‐cells, with analog 7 having the strongest effect. In the analogs, the Pro⁶ residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Enantiomeric and Diastereoisomeric (Mixed) L/ D‐Octaarginine Derivatives – A Simple Way of Modulating the Properties of Cell‐Penetrating Peptides

Cell‐penetrating peptides (CPPs) are promising vehicles for delivery of drugs, antibiotics, proteins, nucleic acid derivatives, etc. into eukaryotic and prokaryotic target cells. To prevent premature degradation, CPPs consisting of D ‐ or β‐amino acid residues have been used. We present simple models for the various modes of delivery of physiologically active cargoes by CPPs, depending on the nature of their conjugation (Fig. 1), and we describe the plasma stability of oligoarginines (OAs) 1 – 4 , the most common unnatural CPPs. Fluorescein‐labeled L ‐octaarginine 1 was found to have a half‐life (t_1/2) of <0.5 min, the D ‐enantiomer ( 2 ) of >7 d (Fig. 2). For possible medicinal applications, the former type of derivative would be too unstable, and the latter one undesirably persistent. Thus, seven of the 256 possible ‘mixed’ Flua‐L /D ‐octaarginine amides, 4a – 4g , were synthesized and shown to have half‐lives in heparine‐stabilized human plasma between 8 min and 5.5 h (Figs. 3 and 4). The cell penetration of the new OAs was investigated with ‘healthy’ and with apoptotic HEK cells (Figs. 5–8), and their interactions with phospholipid bilayers were studied, using anionic lipid vesicles (Figs. 9 and 10). There are surprisingly large differences in the rates of cell penetration and binding to vesicle walls between the various stereoisomeric octaarginine derivatives 1, 2 , and 4a – 4g (Figs. 5 and 7). – The role of D ‐amino acids and D ‐peptides in nature and in drug design is briefly discussed and referenced.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the Selective Trifluoroacetolytic Scission of Native Peptaibols and Model Peptides Using HPLC and ESI‐CID‐MS

Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)₁₀‐O^tBu and Z‐(Aib)₇‐O^tBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)_10–5‐OH from Z‐(Aib)₁₀‐O^tBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)_7–3‐OH from Z‐(Aib)₇‐O^tBu were observed. Moreover, concomitant release of the series of H‐(Aib)_10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded.     

Structure‐kinetic relationship analysis of the therapeutic complement inhibitor compstatin

Compstatin is a 13‐residue peptide that inhibits activation of the complement system by binding to the central component C3 and its fragments C3b and C3c. A combination of theoretical and experimental approaches has previously allowed us to develop analogs of the original compstatin peptide with up to 264‐fold higher activity; one of these analogs is now in clinical trials for the treatment of age‐related macular degeneration (AMD). Here we used functional assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to assess the effect of modifications at three key residues (Trp‐4, Asp‐6, Ala‐9) on the affinity and activity of compstatin and its analogs, and we correlated our findings to the recently reported co‐crystal structure of compstatin and C3c. The K_D values for the panel of tested analogs ranged from 10^?6 to 10^?8 M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4‐fold range in k_on values (2–10 × 10⁵ M^?1 s^?1) and a k_off variation of >35‐fold (1–37 × 10^?2 s^?1) being observed. The stability of the C3b‐compstatin complex seemed to be highly dependent on hydrophobic effects at position 4, and even small changes at position 6 resulted in a loss of complex formation. Induction of a β‐turn shift by an A9P modification resulted in a more favorable entropy but a loss of binding specificity and stability. The results obtained by the three methods utilized here were highly correlated with regard to the activity/affinity of the analogs. Thus, our analyses have identified essential structural features of compstatin and provided important information to support the development of analogs with improved efficacy. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3‐homo‐amino acids

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β³‐homo‐amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β³‐homo‐Tyr in place of Tyr (Tyr‐d ‐Ala‐Phe‐Gly‐β³‐homo‐Tyr‐NH‐)₂ showed good μ receptor affinity and selectivity (IC₅₀ = 0.302, IC₅₀ ratio μ/δ = 68) and enzymatic stability in human plasma. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The Impact of Model Peptides on Structural and Dynamic Properties of Egg Yolk Lecithin Liposomes – Experimental and DFT Studies

Electron spin resonance (ESR), ¹H‐NMR, voltage and resistance experiments were performed to explore structural and dynamic changes of Egg Yolk Lecithin (EYL) bilayer upon addition of model peptides. Two of them are phenylalanine (Phe) derivatives, Ac‐Phe‐NHMe ( 1 ) and Ac‐Phe‐NMe₂ ( 2 ), and the third one, Ac‐(Z)‐ΔPhe‐NMe₂ ( 3 ), is a derivative of (Z)‐α,β‐dehydrophenylalanine. The ESR results revealed that all compounds reduced the fluidity of liposome's membrane, and the highest activity was observed for compound 2 with N‐methylated C‐terminal amide bond (Ac‐Phe‐NMe₂). This compound, being the most hydrophobic, penetrates easily through biological membranes. This was also observed in voltage and resistance studies. ¹H‐NMR studies provided a sound evidence on H‐bond interactions between the studied diamides and lecithin polar head. The most significant changes in H‐atom chemical shifts and spin‐lattice relaxation times T₁ were observed for compound 1 . Our experimental studies were supported by theoretical calculations. Complexes EYL? Ac‐Phe‐NMe₂ and EYL? Ac‐(Z)‐ΔPhe‐NMe₂, stabilized by NH???O or/and CH???O H‐bonds were created and optimized at M06‐2X/6‐31G(d) level of theory in vacuo and in H₂O environment. According to our molecular‐modeling studies, the most probable lecithin site of H‐bond interaction with studied diamides is the negatively charged O‐atom in phosphate group which acts as H‐atom acceptor. Moreover, the highest binding energy to hydrocarbon chains were observed in the case of Ac‐Phe‐NMe₂ ( 2 ).     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Identifying N‐terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO₂·nH₂O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N‐terminal peptides were not identified. We found the sequences of these N‐terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4‐bromine‐phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N‐terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.     

An extracellular hydrophilic carboxy‐terminal domain regulates the activity of TaALMT1, the aluminum‐activated malate transport protein of wheat

Al³⁺‐resistant cultivars of wheat (Triticum aestivum L.) release malate through the Al³⁺‐activated anion transport protein Triticum aestivum aluminum‐activated malate transporter 1 (TaALMT1). Expression of TaALMT1 in Xenopus oocytes and tobacco suspension cells enhances the basal transport activity (inward and outward currents present in the absence of external Al³⁺), and generates the same Al³⁺‐activated currents (reflecting the Al³⁺‐dependent transport function) as observed in wheat cells. We investigated the amino acid residues involved in this Al³⁺‐dependent transport activity by generating a series of mutations to the TaALMT1 protein. We targeted the acidic residues on the hydrophilic C‐terminal domain of TaALMT1 and changed them to uncharged residues by site‐directed mutagenesis. These mutant proteins were expressed in Xenopus oocytes and their transport activity was measured before and after Al³⁺ addition. Three mutations (E274Q, D275N and E284Q) abolished the Al³⁺‐activated transport activity without affecting the basal transport activity. Truncation of the hydrophilic C‐terminal domain abolished both basal and Al³⁺‐activated transport activities. Al³⁺‐dependent transport activity was recovered by fusing the N‐terminal region of TaALMT1 with the C‐terminal region of AtALMT1, a homolog from Arabidopsis. These findings demonstrate that the extracellular C‐terminal domain is required for both basal and Al³⁺‐dependent TaALMT1 activity. Furthermore, we identified three acidic amino acids within this domain that are specifically required for the activation of transport function by external Al³⁺.     

Synthesis of rigid tryptophan mimetics by the diastereoselective Pictet–Spengler reaction of β3‐homo‐tryptophan derivatives with chiral α‐amino aldehydes

The Pictet–Spengler (PS) cyclizations of β³‐hTrp derivatives as arylethylamine substrates were performed with L‐α‐amino and D‐α‐amino aldehydes as carbonyl components. During the PS reaction, a new stereogenic center was created, and the mixture of cis/trans 1,3‐disubstituted 1,2,3,4‐tetrahydro‐β‐carbolines was obtained. The ratio of cis/trans diastereomers depends on the stereogenic centre of used amino aldehyde and the size of substituents. It was confirmed by 1H and 2D NMR (ROESY) spectra. The conformations of cyclic products were studied by 2D NMR ROESY spectra. Products of the PS condensation after removal of protecting group(s) can be incorporated into a peptide chain as tryptophan mimetics with the possibility of the β‐turn induction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and binding potencies of cyclohexapeptide somatostatin analogs containing naphthylalanine and arylalkyl peptoid residues

We report the synthesis, binding affinities to the recombinant human somatostatin receptors, and structure‐activity relationship studies of compounds related to the cyclic hexapeptide, c‐[Pro⁶‐Phe⁷‐D‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹], L‐363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with the arylalkyl peptoid residues Nphe (N‐benzylglycine), (S)βMeNphe [(S)‐N‐[(α‐methyl)benzyl]glycine] or (R)βMeNphe [(R)‐N‐[(α‐methyl)benzyl]glycine] and l ‐1‐naphthylalanine is incorporated into either position 7 or 11 of the parent compound. The synthesis and binding data of the Nnal⁶ ([N‐naphthylmethyl]glycine) analog of L‐363,301 is also reported. The incorporation of the Nnal residue into position 6 of L‐363,301 resulted in an analog with weaker binding affinities to all hsst receptors but enhanced selectivity towards the hsst2 receptor compared with the parent compound. The other compounds bind effectively to the hsst2 receptor but show some variations in the binding to the hsst3 and hsst5 receptors resulting in different ratios of binding affinities to the hsst5 and hsst2 or hsst3 and hsst2, respectively. The incorporation of the Nphe residue into position 6 and the Nal residue into position 7 of L‐363,301 led to a compound which binds potently to the hsst2 and has increased selectivity towards this receptor (weaker binding to hsst3 and hsst5 receptors) compared with the parent compound. The analogs with β‐methyl chiral substitutions in the aromatic peptoid side chain and Nal in position 7 or 11 bind effectively to the hsst2 and hsst5 receptors. They exhibit similar ratios of binding affinities to the hsst5 and hsst2 receptors as observed for L‐363,301. There are however minor differences in binding to the hsst3 receptor among these analogs. These studies allow us to investigate the influence of additional hydrophobic groups on the binding activity to the isolated human somatostatin receptors and the results are important for the design of other somatostatin analogs. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.