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An investigation into the effect of modified β-lysines on the growth rates of eubacterial cells is reported. It is shown that the effects observed are due to the post translational modification of Elongation Factor P (EFP) with these compounds catalysed by PoxA. PoxA was found to be remarkably promiscuous, which allowed the activity of a wide range of exogenous β-lysines to be examined. Two chain-elongated β-lysine derivatives which differ in aminoalkyl chain length by only 2 carbon units exhibited opposing biological activities - one promoting growth and the other retarding it. Both compounds were shown to operate through modification of EFP., (Copyright © 2022. Published by Elsevier Ltd.)

Histone lysine methyltransferases and acetyltransferases are two classes of epigenetic enzymes that play pivotal roles in human gene regulation. Although they both recognise and posttranslationally modify lysine residues in histone proteins, their difference in histone peptide-based substrates and inhibitors remains to be firmly established. Here, we have synthesised lysine mimics that posses an amide bond linker in the side chain, incorporated them into histone H3 tail peptides, and examined synthetic histone peptides as substrates and inhibitors for human lysine methyltransferases and acetyltransferases. This work demonstrates that histone lysine methyltransferases G9a and GLP do catalyse methylation of the most similar lysine mimic, whereas they typically do not tolerate more sterically demanding side chains. In contrast, histone lysine acetyltransferases GCN5 and PCAF do not catalyse acetylation of the same panel of lysine analogues. Our results also identify potent H3-based inhibitors of GLP methyltransferase, providing a basis for development of peptidomimetics for targeting KMT enzymes.

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABL T315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells., (© 2021 Wiley-VCH GmbH.)

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme's function in various diseases. In this context, the radiosynthesis and detailed in vitro radiopharmacological evaluation of an 18 F-labeled N ε -acryloyllysine piperazide are reported. Robust and facile detection of the radiotracer-TGase 2 complex by autoradiography of thin layer plates and polyacrylamide gels after chromatographic and electrophoretic separation owing to irreversible covalent bond formation was demonstrated for the isolated protein, cell lysates, and living cells. By use of this radiotracer, quantitative data on the expression profile of activatable TGase 2 in mouse organs and selected tumors were obtained for the first time by autoradiography of tissue sections.

Gaining a fundamental insight into the biomolecular recognition of posttranslationally modified histones by epigenetic reader proteins is of crucial importance to understanding the regulation of the activity of human genes. Here, we seek to establish whether trimethylthialysine, a simple trimethyllysine analogue generated through cysteine alkylation, is a good trimethyllysine mimic for studies on molecular recognition by reader proteins. Histone peptides bearing trimethylthialysine and trimethyllysine were examined for binding with five human reader proteins employing a combination of thermodynamic analyses, molecular dynamics simulations and quantum chemical analyses. Collectively, our experimental and computational findings reveal that trimethylthialysine and trimethyllysine exhibit very similar binding characteristics for the association with human reader proteins, thereby justifying the use of trimethylthialysine for studies aimed at dissecting the origin of biomolecular recognition in epigenetic processes that play important roles in human health and disease.

Two myo-inositol derivatives having an Nα,Nε-diacetyl-l-lysine (Ac2Lys) moiety linked to the inositol 1-O-position through a self-cleavable linker and a metabolically stable 2-azidoethyl group linked to the inositol 3-O- and 4-O-positions, respectively, were designed and synthesized. The Ac2Lys moiety blocking the inositol 1-O-position required for GPI biosynthesis was expected to be removable by a combination of two enzymes, histone deacetylase (HDAC) and cathepsin L (CTSL), abundantly expressed in cancer cells, but not in normal cells, to transform these inositol derivatives into biosynthetically useful products with a free 1-O-position. As a result, it was found that these inositol derivatives could be incorporated into the glycosylphosphatidylinositol (GPI) biosynthetic pathway by cancer cells, but not by normal cells, to express azide-labeled GPIs and GPI-anchored proteins on cell surfaces. Consequently, this study has established a novel strategy and new molecular tools for selective metabolic labeling of cancer cells, which should be useful for various biological studies and applications.

The treatment of bacterial infections is hindered by the presence of biofilms and metabolically inactive persisters. Here, we report the synthesis of an enantiomeric block co-beta-peptide, poly(amido-D-glucose)-block-poly(beta-L-lysine), with high yield and purity by one-shot one-pot anionic-ring opening (co)polymerization. The co-beta-peptide is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA), including replicating, biofilm and persister bacterial cells, and also disperses biofilm biomass. It is active towards community-acquired and hospital-associated MRSA strains which are resistant to multiple drugs including vancomycin and daptomycin. Its antibacterial activity is superior to that of vancomycin in MRSA mouse and human ex vivo skin infection models, with no acute in vivo toxicity in repeated dosing in mice at above therapeutic levels. The copolymer displays bacteria-activated surfactant-like properties, resulting from contact with the bacterial envelope. Our results indicate that this class of non-toxic molecule, effective against different bacterial sub-populations, has promising potential for the treatment of S. aureus infections.

A novel series of lysine-based ampholytic amphiphiles, with alkylsuccinic anhydrides of varying chain lengths as hydrophobic acylating agents, were synthesized in medium to high yield (50.23-90.15%) based on a facile, catalyst-free method in water medium; and structurally confirmed by mass spectrometry (MS), Fourier transform infra-red (FTIR) spectroscopy, and 1 H/ 13 C nuclear magnetic resonances (NMR) analysis. The resulting compounds were subjected to pH-dependent amphiphilic property, ferrous ion chelating, DPPH antioxidant capacity, and cytotoxicity analyses. Results showed that CMC values decrease, γ value increase, and emulsion stability increase with the increase of medium pH, suggesting that the surface activity of synthetic compounds at air/water and oil/water interface under neutral and alkaline conditions was remarkably higher than that under acidic condition. Lauryl O-acylated malic lysine (compound 4b) presented excellent foaming ability close to commercial detergent sodium dodecyl sulphate; dodecyl succinic lysine (compound 4a) afforded highly stable o/w nanoemulsion. Moreover, compound 4b displayed comparable ferrous ion chelating property to lysine and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidative capacity similar to a commercial food ingredient, diacetyl tartaric acid esters of mono- and di-glycerides (DATEM), indicating its multi-faceted functionalities. A cytotoxicity study of compounds 3b &4b showed that they were non-toxic. Thus, these novel ampholytic amphiphiles may find multi-purpose applications in food, detergent, pharmaceutical, and cosmetic industry., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Anhydrouridines react with aliphatic amines to give N-alkyl isocytosines, but reported procedures often demand very long reaction times and can be low yielding, with narrow scope. A modified procedure for such reactions has been developed, using microwave irradiation, significantly reducing reaction time and allowing facile access to a diverse range of novel nucleosides on the gram scale. The method has been used to prepare a precursor to a novel analogue of lysidine, a naturally occurring iminonucleoside found in t RNA.

Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B., (© 2019 Elsevier Inc. All rights reserved.)

The commercially available antibody-drug conjugate (ADC) product, Kadcyla ® is synthesized using a 2-step reaction, wherein the linker is conjugated to native lysines on the mAb in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine-conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla ® using a 1-step reaction. Mass spectrometry of both products revealed a subpopulation of Kadcyla ® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the 1-step reaction ADC product. Differential scanning calorimetry thermograms showed that the drug and linker conjugation significantly reduced the thermal stability and energies of activation for the denaturation of the C H 2 domain of the ADCs. The heating induced aggregation events started as early as ∼57°C and ∼45°C for Kadcyla ® and Syn-ADC, respectively, compared with 71°C for Herceptin ® . The colloidal stability measurements clearly showed that the hydrophobic drug payload on ADCs significantly reduced the repulsive interprotein interactions when compared to the unconjugated antibody under formulation buffer conditions (pH 6.0). Attaching hydrophobic drug and linker moieties onto the antibody lowered the thermal and colloidal stabilities and increased the aggregation propensity of the ADCs., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Lumbrokinase (LK) is an enzyme complex with antithrombotic, antioxidant, antitumor, and immunomodulatory effects. It has been extensively studied and used in clinical anti-tumor therapy. However, its half-life is short, its bioavailability is low, and its toxicity and side effects are great, which greatly limit its clinical application. Therefore, LK is often combined with other drugs (such as immune agents, hormones, or Chinese herbal medicine) to reduce its dosage and side effects and to improve its anti-tumor effects., Methods and Results: Here, we described an LK/paclitaxel (PTX) nanocarrier based on poly(ethylene glycol)- b -(poly(ethylenediamine l-glutamate)- g -poly(ε-benzyoxycarbonyl-l-lysine)- r -poly(l-lysine)) (PEG- b -(PELG- g -(PZLL- r -PLL))). In the present study, LK and PTX were loaded by electrostatic and/or hydrophobic effects under mild conditions, thereby increasing the half-life and bioavailability of the drugs via the sustained release and enhancement of tumor site enrichment by the LK/PTX/PEG- b -(PELG- g -(PZLL- r -PLL)) complex through passive targeting. In this study, using bladder cancer cells (J82 cells) and rat bladder cancer model as the object, the structure of the nanocarrier, the relationship between drugs composition and antitumor properties were systematically studied., Conclusion: We propose that the block copolymer PEG- b -(PELG- g -(PZLL- r -PLL)) may function as a potent nanocarrier for augmenting anti-bladder cancer pharmacotherapy, with unprecedented clinical benefits., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

DNA-protein cross-links (DPCs) are super-bulky DNA adducts induced by common chemotherapeutic agents, reactive oxygen species, and aldehydes, and also formed endogenously as part of epigenetic regulation. Despite their presence in most cells and tissues, the biological effects of DPCs are poorly understood due to the difficulty of constructing site-specific DNA-protein conjugates. In the present work, a new approach of conjugating proteins to DNA using oxime ligation was used to generate model DPCs structurally analogous to lesions formed in cells. In our approach, proteins and peptides containing an unnatural oxy-Lys amino acid were cross-linked to DNA strands functionalized with 5-formyl-dC or 7-(2-oxoethyl)-7-deaza-dG residues using oxime ligation. The conjugation reaction was site-specific with respect to both protein and DNA, provided excellent reaction yields, and formed stable DPCs amenable to biological evaluation.

Reactive dicarbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are common intermediates in protein damage, leading to the formation of advanced glycation end products (AGEs) through nonenzymatic glycation. (+)-Catechin, a natural plant extract from tea, has been evaluated for its ability in trapping GO and MGO. However, (+)-catechin is also reported to have both antioxidant ability and pro-oxidant properties. Until now, whether (+)-catechin can inhibit the formation of nonenzymatic glycation and the mechanism of the inhibition in nucleoprotein nonenzymatic glycation is still unclear. In the present study, histone H1 and MGO were used to establish an in vitro (100 mM phosphate buffer solution (PBS), pH 7.4, 37 °C) protein glycation model to study the trapping ability of (+)-catechin. Our data show that MGO caused dose-dependent protein damage, and the content of MGO-induced Schiff base formation was inhibited by (+)-catechin when the molecular ratio of catechin:MGO was 1:6. The formation of N ε -carboxymethyllysine (CML) was reduced significantly when the ratio of (+)-catechin and MGO was 1:1, which was similar to the inhibition effect of aminoguanidine (AG). The formation of CML under in vitro conditions can be inhibited by low concentration (12.5-100 μM) of (+)-catechin but not with high concentration (200-800 μM) of (+)-catechin. The reason is that the high concentration of (+)-catechin did not inhibit CML formations due to H 2 O 2 produced by (+)-catechin. In the presence of catalase, catechin can inhibit MGO-induced CML formation. In conclusion, the trapping ability of (+)-catechin may be more effective at the early stage of nonenzymatic glycation. However, a high concentration (200-800 μM) of (+)-catechin may not inhibit the formation of CML because it induced the increase of H 2 O 2 formation.

Glu-Urea-Lys (GUL) derivatives have been reported as prostate-specific membrane antigen (PSMA) agent. We developed derivatives of GUL conjugated with NOTA or DOTA via a thiourea linker and tested their feasibility as PSMA imaging agents after labeling with 68 Ga. NOTA-GUL and DOTA-GUL were synthesized and labeled with 68 Ga using generator-eluted 68 GaCl 3 in 0.1 M HCl in the presence of 1 M NaOAc at pH 5.5. The stabilities of 68 Ga-labeled compounds in human serum were tested at 37.5 °C. A competitive binding assay was performed using the PSMA-positive prostate cancer cell line 22Rv1 and [ 125 I]MIP-1072 (PSMA-specific binding agent) as a tracer. Biodistribution and micro-PET studies were performed using 22Rv1-xenograft BALB/c nude mice. The radiolabeling efficiency of NOTA-GUL (>99%) was higher than that of DOTA-GUL (92%). The IC 50 of Ga-NOTA-GUL was 18.3 nM. In the biodistribution study, tumor uptake of 68 Ga-NOTA-GUL (5.40% ID/g) was higher than that of 68 Ga-DOTA-GUL (4.66% ID/g) at 1 h. Tumor/muscle and tumor/blood uptake ratios of 68 Ga-NOTA-GUL (31.8 and 135, respectively) were significantly higher than those of 68 Ga-DOTA-GUL (16.1 and 31.1, respectively). The tumor/kidney uptake ratio of 68 Ga-NOTA-GUL was 3.4-fold higher than that of 68 Ga-DOTA-GUL. 68 Ga-NOTA-GUL showed specific uptake to PSMA positive tumor xenograft and was blocked by co-injection of the cold ligand. In conclusion, we successfully synthesized 68 Ga-NOTA-GUL and 68 Ga-DOTA-GUL for prostate cancer imaging. 68 Ga-NOTA-GUL showed better radiochemical and biodistribution results. 68 Ga-NOTA-GUL may be a promising PSMA targeting radiopharmaceutical., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The solvatochromic IR responsivity of small side chain -NCS in two unexplored unnatural amino acids, isothiocyanyl alanine ( NCS Ala = Ita) and lysine ( NCS Lys = Itl), without perturbing the conformation is demonstrated in two designed short tripeptide (BocAla- NCS Ala-Ala-OMe) and hexapeptide (BocLeu-Val-Phe-Phe- NCS Lys-Gly-OMe). Demonstration of site specific fluorescent labeling in both the peptides and ligation type reaction in NCS Lys indicates the novelty of these two amino acids as alternative to the available canonical amino acids., (Copyright © 2018. Published by Elsevier Ltd.)

We report a strategy to introduce a reactive electrophile into proteins through the conversion of a chemically inert group into a bioreactive group in response to an inducer molecule. This strategy was demonstrated by oxidation-induced and proximity-enhanced protein-protein crosslinking in the presence of a large excess of free nucleophile.

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with β,β-difluorovinyl phenyl sulfone, a new (1'-fluoro)vinyl cation equivalent, and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biologically relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addition/elimination, the resulting transoid α-(1'-fluoro)-β-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-β-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson analysis reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (K i = 630 ± 20 μM; t 1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (K i = 470 ± 30 μM; t 1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.

Effects exerted by heavy isotope substitution in biopolymers on the functioning of whole organisms have not been investigated. We report on the decrease of permissive temperature of nematodes fed with bacteria containing 5,5-D2-lysine. We synthesized 5,5-dideuterolysine and, taking advantage of lysine being an essential amino acid, showed that C. elegans with modified lysine poorly develop from larvae into fertile adult hermaphrodites. This effect occurs only at high temperature within the permissible range for C. elegans (25 °C) and completely vanishes at 15 °C. The only known metabolic involvement of C5 in lysine is in post-translational modification through lysyl hydoxylases. Indeed, siRNA experiments showed that deficiency of let-268/plod lysyl-hydroxylase/glycosydase further amplifies the isotope effect making it apparent even at 20 °C, whereas control siRNAs as well as another lysyl-hydroxylase (psr-1/jmjdD) siRNA do not. We report for the first time that a site-specific deuteration may strongly affect the development of the whole animal organism especially under the conditions of deficiency of the corresponding enzyme. These findings provide the basis for our ongoing efforts to employ isotope effects for fine tuning of metabolic pathways to mitigate pathological processes.

A total of 48 analogues of the natural product myxochelin A were prepared and evaluated for their inhibitory effects on human 5-lipoxygenase in both cell-free and cell-based assays. Structure-activity relationship analysis revealed that the secondary alcohol function and only chiral center of myxochelin A is not required for biological activity. By expanding the diaminoalkane linker of the two aromatic residues it was possible to generate a myxochelin derivative with superior activity against 5-lipoxygenase in intact cells., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The radiosynthesis of [(18) F]DCFPyL on 2 distinct automated platforms with full regulatory compliant quality control specifications is described. The radiotracer synthesis was performed on a custom-made radiofluorination module and the Sofie Biosciences ELIXYS. The radiofluorination module synthesis was accomplished in an average of 66 minutes from end of bombardment with an average specific activity at end of synthesis (EOS) of 4.4 TBq/μmol (120 Ci/μmol) and an average radiochemical yield of 30.9% at EOS. The ELIXYS synthesis was completed in an average of 87 minutes with an average specific activity of 2.2 TBq/μmol (59.3 Ci/μmol) and an average radiochemical yield of 19% at EOS. Both synthesis modules produced large millicurie quantities of [(18) F]DCFPyL while conforming to all standard US Pharmacopeia Chapter <823> acceptance testing criteria., (Copyright © 2016 John Wiley & Sons, Ltd.)

Antimicrobial peptides (AMPs) have shown potential as alternatives to traditional antibiotics for fighting infections caused by antibiotic-resistant bacteria. One promising example of this is gramicidin A (gA). In its wild-type sequence, gA is active by permeating the plasma membrane of Gram-positive bacteria. However, gA is toxic to human red blood cells at similar concentrations to those required for it to exert its antimicrobial effects. Installing cationic side chains into gA has been shown to lower its hemolytic activity while maintaining the antimicrobial potency. In this study, we present the synthesis and the antibiotic activity of a new series of gA mutants that display cationic side chains. Specifically, by synthesizing alkylated lysine derivatives through reductive amination, we were able to create a broad selection of structures with varied activities towards Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Importantly, some of the new mutants were observed to have an unprecedented activity towards important Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Psuedomonas aeruginosa., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid (pLysAAm/HA) interpenetrating network (IPN) hydrogels were successfully fabricated through the combination of hydrazone bond crosslinking and photo-crosslinking reactions. The HA hydrogel network was first synthesized from 3,3'-dithiodipropionate hydrazide-modified HA and polyethylene glycol dilevulinate by hydrazone bond crosslinking. The pLysAAm hydrogel network was prepared from Nɛ-acryloyl-L-lysine and N,N'-bis(acryloyl)-(L)-cystine by photo-crosslinking. The resultant pLysAAm/HA hydrogels had a good shape recovery property after loading and unloading for 1.5 cycles (up to 90%) and displayed a highly porous microstructure. Their compressive moduli were at least 5 times higher than that of HA hydrogels. The pLysAAm/HA hydrogels had an equilibrium swelling ratio of up to 37.9 and displayed a glutathione-responsive degradation behavior. The results from in vitro biocompatibility evaluation with pre-osteoblasts MC3T3-E1 cells revealed that the pLysAAm/HA hydrogels could support cell viability and proliferation. Hematoxylin and eosin staining indicated that the pLysAAm/HA hydrogels allowed cell and tissue infiltration, confirming their good in vivo biocompatibility. Therefore, the novel pLysAAm/HA IPN hydrogels have great potential for bone tissue engineering applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 μM were obtained for 1a-c, respectively., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We report a selective ruthenium catalyzed reduction of tertiary amides on the side chain of Fmoc-Gln-OtBu derivatives, leading to innovative unnatural α,β or γ-amino acids functionalized with tertiary amines. Rapid and scalable, this process allowed us to build a library of basic unnatural amino acids at the gram-scale and directly usable for liquid- or solid-phase peptide synthesis. The diversity of available tertiary amines allows us to modulate the physicochemical properties of the resulting amino acids, such as basicity or hydrophobicity.

Phosphorylation is a key process for changing the activity and function of proteins. The impact of phospho-serine (pSer), -threonine (pThr) and -tyrosine (pTyr) is certainly understood for some proteins. Recently, peptides and proteins containing N-phosphorylated amino acids such as phosphoarginine (pArg), phosphohistidine (pHis) and phospholysine (pLys) have gained interest because of their different chemical properties and stability profiles. Due to its high intrinsic lability, pLys is the least studied within this latter group. In order to gain insight into the biological role of pLys, chemical and analytical tools, which are compatible with the labile P(=O)-N bond, are highly sought-after. We recently reported an in-solution synthetic approach to incorporate pLys residues in a site-specific manner into peptides by taking advantage of the chemoselectivity of the Staudinger-phosphite reaction. While the in-solution approach allows us to circumvent the critical TFA cleavage, it still requires several transformations and purification steps to finally deliver pLys peptides. Here we report the synthesis of site-specific pLys peptides directly from a solid support by using a base labile resin. This straightforward and highly efficient approach facilitates the synthesis of various site-specific pLys-containing peptides and lays the groundwork for future studies about this elusive protein modification.

We report the synthesis and genetic encoding of a recently discovered post-translational modification, 2-hydroxyisobutyryl-lysine, to the genetic code of E. coli. The production of homogeneous proteins containing this amino acid will facilitate the study of modification in full-length proteins.

β-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, β-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the β-hairpin motif.

An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/t-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (R)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and t-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides.

Background: Despite recent advances, major hurdles still need to be cleared for widespread application of therapeutic antisense technologies. In particular, pharmacokinetic properties and efficient cellular uptake need to be improved through chemical derivatization or bioconjugation., Results: The 2'-O-thioethylene nucleotide building block affords easy implementation into standard oligonucleotide synthesis protocols and was used to attach oligolysine chains to phosphodiester oligonucleotides by direct reaction with S-sulfonate protected peptides. Efficient gene silencing was induced in a cell culture model after transfection reagent-free application of the conjugates., Conclusion: A facile optimized procedure for generating oligonucleotide-peptide conjugates was established. The attachment of short basic peptides via a labile linker is sufficient to enhance membrane permeability of oligonucleotides and result in successful gene silencing.

L-lysine 4-nitrophenolate monohydrate (LLPNP) has been synthesized and grown by solution growth method at room temperature using deionised water as a solvent. The crystal structure of the materials was solved by single crystal X-ray diffraction analysis and it was found that the material has orthorhombic system. The crystallinity of the grown crystals was studied by the powder X-ray diffraction analysis. Molecular structure of the grown crystal was investigated by 1H NMR spectroscopy. The various functional groups of the sample were identified by Fourier transform infrared and Fourier transform-Raman spectroscopic analyses. Thermal stability of the grown crystal has been studied by Thermogravimetric and Differential thermal (TG&DTA) analysis. The optical absorption of the grown crystals has been ascertained by UV-Vis-NIR absorption studies. Second harmonic generation (SHG) efficiency of the material has been determined by Kurtz and Perry technique and the efficiency was found to be 4.45 and 1.4 times greater than that of standard KDP and urea samples, respectively., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo(L-alanine) and oligo(L-lysine). Using (1)H NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems.

Copper catalyzed coupling of Mannich reagents to aza-propargylglycine residues has been employed to synthesize constrained aza-lysine peptides. Employing growth hormone releasing peptide-6 (GHRP-6) as a model peptide and a variety of secondary amines, 18 aza-Lys analogs were synthesized by this so-called A(3)-coupling reaction. This effective method for making constrained aza-Lys-peptides offers strong potential for exploring various recognition events implicating lysine residues including post-translational peptide modification.

Background: An impact on glycation, and possibly on diabetic complications, is attributed to fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) because they degrade Amadori compounds in vivo. Little is known about individual differences in FN3K-RP activity, which might contribute to an individual risk for diabetic complications., Methods: An HPLC-based activity assay for FN3K-RP in erythrocytes with the substrate N-α-hippuryl-N-ε-psicosyllysine was developed. The activities of FN3K and FN3K-RP were also analysed in erythrocytes of 103 consecutive participants of a health-care survey amongst a high-risk group for diabetes. The potential associations of these activities with the subjects' health background (anthropometric data, glucose tolerance and HbA1c, blood lipids, history of metabolic diseases in the subjects and their families, and medication) were examined., Results: The interindividual variability of FN3K-RP is less pronounced than that of FN3K [60-135 vs. 2.8-12.5 mU/g haemoglobin (Hb)]. No correlations with age, sex, body weight, blood cholesterol, or plasma glucose in an oral glucose tolerance test were observed. Subjects with kidney disease had higher activity of mainly FN3K-RP [111±15 vs. 98±18 mU/g Hb, mean±standard deviations (SDs), n=16 vs. 87, p=0.009], whereas subjects whose parents or siblings had a stroke showed lower FN3K activity (6.2±1.6 vs. 7.1±1.8 mU/g Hb, mean±SD, n=24 vs. 66, p=0.040)., Conclusions: There is a likely impact of FN3K and FN3K-RP on the glycation cascade in vivo with potential positive and negative effects. The new screening method enables further studies to elucidate the function and importance of FN3K-RP.

Polycations have good potential as carriers of proteins and genetic material. However, poor control over the release rate and safety issues currently limit their use as delivery vehicles. Here we introduce a new lysine-based polycation, poly(ethylene lysinylaspartate diglyceride) (PELD), which exhibits high cytocompatibility. PELD self-assembles with the biological polyanion heparin into a coacervate that incorporates proteins with high loading efficiency. Coacervates of varying surface charge were obtained by simple alteration of the PELD:heparin ratio and resulted in diverse release profiles of the model protein bovine serum albumin. Therefore, coacervate charge represents a direct means of control over release rate and duration. The PELD coacervate also rapidly adsorbed onto a porous polymeric scaffold, demonstrating potential use in tissue engineering applications. This coacervate represents a safe and tunable protein delivery system for biomedical applications., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Advanced Glycation Endproducts (AGEs) are modified amino acids that form on proteins and are known to be implicated in the pathogenesis of diabetes and related diseases. Ready access to synthetic stable isotope-labelled AGEs allows for quantitative mass spectrometry studies to be undertaken, providing key insights into the roles AGEs play in the progression of such diseases. However, the majority of current syntheses of these compounds suffer from poor yields and lengthy procedures and are not suitable for the purposes required here. Here, we report robust syntheses of stable isotope-labelled monolysyl AGEs, N(ε)-(carboxymethyl)lysine, N(ε)-(carboxyethyl)lysine and pyrraline, that provide straightforward access to these compounds for quantitative amino acid analysis. This work will facilitate future investigations with these compounds and lead to a better understanding of the roles they play in diabetes and related diseases.

Reduction and pH dual-sensitive reversibly core-crosslinked polypeptide micelles were developed from lipoic acid (LA) and cis-1,2-cyclohexanedicarboxylic acid (CCA) decorated poly(ethylene glycol)-b-poly(L-lysine) (PEG-P(LL-CCA/LA)) block copolymers for active loading and triggered intracellular release of doxorubicin (DOX). PEG-P(LL18-CCA4/LA14) and PEG-P(LL18-CCA8/LA10) (M(n PEG) = 5.0 kg/mol) formed nano-sized micelles that were readily crosslinked in the presence of a catalytic amount of dithiothreitol (DTT) in phosphate buffer (pH 7.4, 10 mM). PEG-P(LL18-CCA4/LA14) micelles displayed an elevated DOX loading over PEG-P(LL14-LA14) controls likely due to presence of ionic interactions between DOX and CCA. These core-crosslinked polypeptide micelles while exhibiting high stability against extensive dilution and high salt concentration were quickly dissociated into unimers in the presence of 10 mM DTT. The in vitro release studies showed that DOX release from PEG-P(LL18-CCA4/LA14) micelles at pH 7.4 and 37 °C was significantly inhibited by crosslinking (i.e. less than 20% release in 24 h). The release of DOX was, however, doubled under endosomal pH of 5.0, possibly triggered by cleavage of the acid-labile amide bonds of CCA. In particular, rapid DOX release was observed under a reductive condition containing 10 mm glutathione (GSH), in which 86.0% and 96.7% of DOX were released in 24 h at pH 7.4 and 5.0, respectively, under otherwise the same conditions. MTT assays demonstrated that these core-crosslinked polypeptide micelles were practically non-toxic up to a tested concentration of 1.0 mg/mL, while DOX-loaded micelles caused pronounced cytotoxic effects to HeLa and HepG2 tumor cells with IC50 (inhibitory concentration to produce 50% cell death) of ca. 12.5 μg DOX equiv/mL following 48 h incubation. Confocal microscopy observations revealed that DOX-loaded crosslinked PEG-P(LL18-CCA4/LA14) micelles more efficiently delivered and released DOX into the nuclei of HeLa cells than PEG-P(LL14-LA14) counterparts. These dual-bioresponsive core-crosslinked polypeptide micelles have appeared as an advanced platform for targeted cancer therapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Organocatalysis: A concise synthesis of L-pyrrolysine has been accomplished in six steps from simple starting materials. The facile synthetic strategy relies on an organocatalytic Michael addition, an efficient amide coupling, and a challenging method for the imine-bond construction., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A general and efficient route towards the synthesis of three derivatives of structurally and functionally important amino acid, lysine is reported. Chemoselective reduction of aldehydic functionality in C-3-azido conjugated aldehyde 4, under Luche condition, is the key step in the synthetic sequence. The lysine derivative, (2S,3R)-2,6-diazido-3-hydroxy-hex-4-ene-oic acid 9 could be used to prepare switch peptide using Staudinger reaction, while the unprotected (2S,3R)-2,6-diamino-3-hydroxy-hexanoic acid hydrochloride 10 is a proven reaction intermediate towards the synthesis of natural product (-)-Balanol., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

What's the catch? A pyrrolysine analogue bearing a terminal alkyne and an ester functionality can be incorporated into recombinant proteins and render them amenable to capture by the click reaction and subsequent release through ester hydrolysis. The utility of this pyrrolysine-inspired technology is demonstrated for the identification of SUMOylation sites., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe the fabrication of asymmetric nanopores sensitive to ultraviolet (UV) light, and give a detailed account of the divalent ionic transport through these pores using a theoretical model based on the Nernst-Planck equations. The pore surface is decorated with lysine chains having pH-sensitive (amine and carboxylic acid) moieties that are caged with photo-labile 4,5-dimethoxy-2-nitrobenzyl (NVOC) groups. The uncharged hydrophobic NVOC groups are removed using UV irradiation, leading to the generation of hydrophilic "uncaged" amphoteric groups on the pore surface. We demonstrate experimentally that polymer membranes containing single pore and arrays of asymmetric nanopores can be employed for the pH-controlled transport of ionic and molecular analytes. Comparison between theory and experiment allows for understanding the individual properties of the phototriggered nanopores, and provides also useful clues for the design and fabrication of multipore membranes to be used in practical applications.

1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [(99m)Tc(CO)(3)](+) core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys(Dap) in which the ε-NH(2) group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH(2) group of the α-amino acid portion is Fmoc- and the -NH(2) of Dap are Boc-protected. Fmoc-l-Lys(Dap(Boc)) was either conjugated to the N- and C-terminus of bombesin BBN(7-14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced the native lysine in a cyclic RGD peptide with l-Lys(Dap). For all peptides, quantitative labeling with the [(99m)Tc(CO)(3)](+) core at a 10 μM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from [Re(OH(2))(3)(CO)(3)](+) and Lys(Dap)-BBN(7-14) or cyclo-(RGDyK(Dap)), respectively. Determination of integrin receptor binding showed low to medium nanomolar affinities for various receptor subtypes. The IC(50) of cyclo-(RGDyK(Dap[Re(CO)(3)])) for α(v)β(3) is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude mice showed reasonable α(v)β(3)-integrin specific tumor uptake. Altogether, orthogonally protected l-Lys(Dap) represents a highly versatile building block for integration in any peptide sequence. Lys(Dap)-precursors allow high-yield (99m)Tc-labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+), forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics.

Single molecule Fluorescence Resonance Energy Transfer (FRET) has been widely applied to study structure, function and dynamics of complex biological systems. Labeling of proteins at specific positions with fluorescent dyes is a challenging and key step for any single molecule FRET measurement. Genetic code expansion has facilitated site specific incorporation of unnatural amino acids into proteins. These unnatural amino acid bears bioorthognal functional groups that provide opportunity to install a unique chemical handle into proteins. Propargyllysine is an unnatural amino acid which, when incorporated into a protein, can be exploited to attach commercially available fluorescent azide dyes through copper-catalyzed alkyne-azide cycloaddition click reaction (also known as click reaction). We describe here an optimized strategy to combine synthesis of propargyllysine, its genetic incorporation in the protein and click reaction to site-specifically label the protein with azide derivative of Alexa® 488. Later the protein is labeled at unique cysteine residue via maleimide coupling chemistry with acceptor Alexa® 594 dye to yield double labeled protein as required for any single molecule FRET experiments., (Copyright © 2013 Elsevier Inc. All rights reserved.)

N(ε)-(carboxymethyl) lysine (CML) is the most abundant advanced glycation end product (AGE), and frequently selected as an AGEs marker in laboratory studies. In this paper, the formation and inhibition of N(ε)-(carboxymethyl)lysine in saccharide-lysine model systems during microwave heating have been studied. The microwave heating treatment significantly promoted the formation of CML during Maillard reactions, which was related to the reaction temperature, time and type of saccharide. The order of CML formation for different saccharides was lactose > glucose > sucrose. Then, the inhibition effect on CML by five inhibitors was further examined. According to the results, ascorbic acid and tocopherol did not affect inhibition of CML, in contrast, thiamin, rutin and quercetin inhibited CML formation, and the inhibitory effects were concentration dependent.

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pentosidine, a biologically important advanced glycation endproduct, has been accessed in a rapid, high-yielding manner. The synthesis was accomplished via a six-step sequence starting with 3-amino-2-chloropyridine and features a palladium-catalyzed tandem cross-coupling/cyclization to construct the imidazo[4,5-b]pyridine core.

An efficient route for the synthesis of 2,4-diaminopyrimidine ribosides from cytidine is described consisting of six steps with overall yields >50% and only one chromatographic step. The key amine addition step utilizes LiCl and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to ensure clean conversion to a single tautomeric product. This route has been used to prepare the modified tRNA nucleosides lysidine and agmatidine in quantities suitable for structural characterization.

The class III lysine deacylases (KDACs), also known as the sirtuins, have emerged as interesting drug targets for therapeutic intervention in a variety of diseases. To gain a deeper understanding of the processes affected by sirtuins, the development of selective small molecule modulators of individual isozymes has been a longstanding goal. Essential for the discovery of novel modulators, however, are good screening protocols and mechanistic insights with regard to the targets in question. We therefore evaluated the activities of the seven human sirtuin hydrolases against a panel of fluorogenic substrates. Both commonly used, commercially available substrates and novel chemotypes designed to address recent developments in the field of lysine post-translational modification were evaluated. Our investigations led to the discovery of two new fluorogenic ε-N-succinyllysine-containing substrates that enable highly efficient and enzyme-economical screening employing sirtuin 5 (SIRT5). Furthermore, optimized protocols for facile kinetic investigations were developed, which should be valuable for enzyme kinetic investigations. Finally, these protocols were applied to a kinetic analysis of the inhibition of SIRT5 by suramin, a potent sirtuin inhibitor previously shown by X-ray crystallography to bind the substrate pocket of the human SIRT5 KDAC enzyme.