Genetically Encoded Libraries of Constrained Peptides

Many therapeutic peptides can still be improved with respect to target specificity, target affinity, resistance to peptidases/proteases, physical stability, and capacity to pass through membranes required for oral delivery. Several modifications can improve the peptides’ properties, in particular those that impose (a) conformational constraint(s). Screening of constrained peptides and the identification of hits is greatly facilitated by the generation of genetically encoded libraries. Recent breakthrough bacterial, phage, and yeast display screening systems of ribosomally synthesized post-translationally constrained peptides, particularly those of lanthipeptides, are earning special attention. Here we provide an overview of display systems for constrained, genetically encoded peptides and indicate prospects of constrained peptide-displaying phage and bacterial systems as such in vivo.     

Detecting Cysteine in Bioimaging with a Near-Infrared Probe Based on a Novel Fluorescence Quenching Mechanism

Near-infrared (NIR) fluorescent probes are very significant for detecting cysteine in biological systems. Herein, we report a highly selective and sensitive NIR turn-on fluorescent probe (BDP-NIR) based on BODIPY with large Stokes shift (105 nm) for detecting Cys. We clarified the sensing mechanism based on the different thiol-induced S_NAr substitution/rearrangement reaction of the probe with cysteine and homocysteine/glutathione, which leads to the corresponding amino- and thiol-BODIPY dyes with distinct photophysical properties. Moreover, a novel mechanism of fluorescence quenching was demonstrated by density functional theory calculation. The reason for the fluorescence quenching of the probe might be intersystem crossing (from singlet to triplet excited state). Moreover, BDP-NIR had a high linear dynamic range of 0–500 μM, which was promising for detecting cysteine quantificationally. Significantly, BDP-NIR was capable of sensing endogenous cysteine in living cells and in vivo.     

Synthesis of DNA-Encoded Disulfide- and Thioether-Cyclized Peptides

DNA-encoded chemical library technologies enable the screening of large combinatorial libraries of chemically and structurally diverse molecules, including short cyclic peptides. A challenge in the combinatorial synthesis of cyclic peptides is the final step, the cyclization of linear peptides that typically suffers from incomplete reactions and large variability between substrates. Several efficient peptide cyclization strategies rely on the modification of thiol groups, such as the formation of disulfide or thioether bonds between cysteines. In this work, we established a strategy and reaction conditions for the efficient chemical synthesis of cyclic peptide–DNA conjugates based on linking the side chains of cysteines. We tested two different thiol-protecting groups and found that tert-butylthio (S-tBu) works best for incorporating a pair of cysteines, and we show that the DNA-linked peptides can be efficiently cyclized through disulfide and thioether bond formation. In combination with established procedures for DNA encoding, the strategy for incorporation of cysteines may be readily applied for the generation and screening of disulfide- and thioether-cyclized peptide libraries.     

锁阳膳食纤维的制备及其物理性质

以锁阳为原料,制备锁阳膳食纤维。锁阳粉碎成黄豆大小颗粒,由体积分数95%乙醇提取去除脂溶性物质,残渣分别由半胱氨酸加维生素C水溶液或纯水在60℃下浸泡提取48h,清洗残渣、烘干粉碎过40目筛,测定制得的两种膳食纤维的物理特性。半胱氨酸酸溶液处理的膳食纤维其持水性为9.20g/g、溶胀性为7.5mL/g,水处理的膳食纤维这两种性质分别是5.83g/g和4.4mL/g;半胱氨酸酸溶液处理后所得膳食纤维的明显质量优于水处理所得膳食纤维。半胱氨酸酸溶液可将大分子缩合鞣质降解为可溶性的小分子,在水洗过程中被除去,得到更纯的膳食纤维。     

Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk

The rate of vitamin B₁₂ loss was 3X greater in heated, chocolate‐flavoured milk than in unflavoured milk. Studies of B₁₂ stability in the presence of cocoa powder components were performed to identify the reactive agents. Cocoa polyphenols with a strong capacity both for reduction and for peroxide generation accelerated B₁₂ destruction as much as 20‐fold vs. polyphenols without both capacities. Polyphenols with both capacities include the cocoa polyphenol (+)‐catechin and oligomers thereof, but also the structurally related polyphenols gallic acid, caffeic acid and (?)‐epigallocatechin gallate. The demonstrated physical affinity of cocoa powder for B₁₂ was a significant factor in the destructive process. B₁₂ may exhibit decreased stability in heated, neutral pH, polyphenol‐fortified foods.     

Late-Stage Hydrocarbon Conjugation and Cyclisation in Synthetic Peptides and Proteins

The conventional S-alkylation of cysteine relies upon using activated electrophiles. Here we demonstrate high-yielding and selective S-alkylation and S-lipidation of cysteines in unprotected synthetic peptides and proteins by using weak electrophiles and a Zn²⁺ promoter. Linear or branched iodoalkanes can S-alkylate cysteine in an unprotected 38-residue Myc peptide fragment and in a 91-residue miniprotein Omomyc, thus highlighting selective late-stage synthetic modifications. Metal-assisted cysteine alkylation is also effective for incorporating dehydroalanine into unprotected peptides and for peptide cyclisation via aliphatic thioether crosslinks, including customising macrocycles to stabilise helical peptides for enhanced uptake and delivery to proteins inside cells. Chemoselective and efficient late-stage Zn²⁺-promoted cysteine alkylation in unprotected peptides and proteins promises many useful applications.     

Chemical Synthesis of a Synthetic Analogue of the Sialic Acid‐Binding Lectin Siglec‐7

As a basis for the development of an artificial carbohydrate‐binding lectin, we chemically synthesized a domain of siglec‐7, a well‐characterized sialic‐acid‐binding lectin. The full polypeptide (127 amino acids) was constructed by sequential native chemical ligation (NCL) of five peptide segments. Because of poor cysteine availability for NCL, cysteine residues were introduced at suitable ligation sites; these cysteine residues were alkylated in order to mimic native glutamine or asparagine residues, or converted to an alanine residue by desulfurization after NCL. After folding the full‐length polypeptide, the sialic‐acid‐binding activity of the synthetic siglec‐7 was clearly demonstrated by STD NMR and ELISA experiments. We succeeded in the synthesis of siglec‐7 by installing three extra cysteine residues with side‐chain modifications and found that these modifications did not affect the binding activity.