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The strategic engagement of innate immunity is a promising avenue for cancer treatment. Antibody-recruiting molecules (ARMs) direct endogenous antibodies to target tumor sites, eliciting innate immune effector killing responses. In this study, we report the synthesis of ARMs by employing solid-phase peptoid synthesis to construct three libraries of antibody-recruiting oligomers. Using dinitrophenyl (DNP) as a model hapten and alkyl lipid chains for cell surface anchoring, we tailored oligomers with variations in valency and spatial configuration. Among these, an oligomer design featuring DNP connected to the oligomer backbone through an extended PEG linker and flanked by two lipid motifs emerged as the most effective in antibody recruitment in vitro. This oligomer was further functionalized to include an imidazoquinoline, creating a trifunctional hapten-lipid-TLR7/8 agonist oligomer, and a parallel variant was conjugated with rhodamine, resulting in a trifunctional hapten-lipid-dye oligomer. Upon intratumorally administration in a murine model, these oligomers induced localized immune activation within tumors. Subsequent ex vivo analysis of single-cell suspensions from excised tumors confirmed the enhanced binding of anti-DNP antibodies. These findings underscore the potential of custom-designed ARMs in orchestrating precise immune-mediated tumor targeting and highlight the adaptability of solid-phase synthesis in oligomer design for the design of multifunctional antibody recruiting molecules.

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An extended library of hybrids that combined a penicillin derivative with a peptoid moiety was designed and synthetized using either a solid-phase or a mixed solid-phase/solution-phase strategy. The library was further evaluated for antiproliferative activity. While none of the different synthesized compounds showed significant cytotoxicity against a normal cell line, tumor cell results drew several conclusions, when comparing with our reference, the highly active triazolylpeptidyl penicillin derivative, TAF7f. Thus, when the 1,2,3-triazole group was exchanged by its "retro-inverse" analogue, no change was noted in the activity of the hybrids; however, better performance was generally obtained if the triazole is replaced by a glycine moiety. Additionally, the absence of hydrogen bond donor groups decreased the compounds activity, which could explain that, in general, this set of derivatives were less active than their peptide-containing analogues. From this study, is indisputable that, regardless of the type of chain (peptide, peptoid or mixture) attached to penicillin, an isobutyl side chain placed in the position closest to penicillin and a benzyl in the next position are determinant for the activity.

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A Pro-Gly-typed cyclopeptide, cyclosenegalin A, was firstly isolated from Annona senegalensis and Annona scleroderma. In this study, we reported synthesis the cycloheptapeptide with a combination of solid- and solution-phase synthetic methods. This study also compared the effectiveness of ß-turn inducer type I and II in cyclosenegalin A to facilitate the cyclization process. The synthesis of cyclosenegalin A were prepared using two different sequences of linear peptides for cyclization. First sequence employed ß-turn type I inducer (Ser-Ala-Val-Thr) as turning point and second sequence employed ß-turn type II inducer (Thr-Pro-Gly-Leu). The successful cyclization was obtained using the linear sequence of NH2-Ala-Val-Thr-Pro-Gly-Leu-Ser-OH with ß-turn type II. The final product was obtained in 8.2 % yield with PyBOP/DIEA act as coupling agent. The synthetic cyclosenegalin A were characterized with HR-ToFMS, 1H NMR, 13C NMR, HSQC, HMBC, TOCSY, and ROESY. The synthetic product was also evaluated for its antimicrobial activity.

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DNA nanotechnology has revolutionized the ability to position matter at the nanoscale, but the preparation of DNA-based architectures remains laborious. To facilitate the formation of custom structures, a fully automated method is reported to produce sequence- and size-defined DNA nanotubes. By programming the sequential addition of desired building blocks, rigid DX-tile-based DNA nanotubes and flexible wireframe DNA structures are attained, where the total number of possible constructs increases as a power function of the number of different units available. Using single-molecule fluorescence imaging, the kinetics and yield of each synthetic step can be quantitatively determined, revealing differences in self-assembly dynamics as the nanotube is built up from the solid support and providing new insights into DNA self-assembly. The exploitation of automation for both assembly and analysis (through an ad-hoc developed K-means clustering algorithm) facilitates a workflow wherein the synthesis parameters may be iteratively improved upon, demonstrating how a single-molecule "assembly-analysis-optimization" sequence can be used to generate complex, noncovalent materials in good yield. The presented synthetic strategy is generalizable, making use of equipment already available in most standard laboratories and represents the first fully automated supramolecular assembly on a solid support.

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Lipophosphoglycans (LPGs) are found on the surface of Leishmania, a protozoan parasite, and are immunologically important. Herein, disaccharide 1-phosphate repeating units of LPGs were successfully synthesized on a solid support with high anomeric purity using a disaccharide α-1-phosphoramidite building block. To enhance solubility in the reaction solvent, hydroxy-protecting groups in the form of para-t-butylbenzoyl were introduced to the building block. The saccharide chain was elongated via stable glycosyl boranophosphate linkages, followed by the conversion of inter-sugar linkages to phosphodiester counterparts using an oxaziridine derivative. The addition of a silylating reagent post-reaction with the oxaziridine derivative efficiently facilitated the conversion of boranophosohodiesters to phosphodiesters. This method enabled the α-selective synthesis of up to 15 repeating units, marking the longest homogeneous repeating units of LPGs  synthesized chemically.  Given the chain length equivalence to native LPGs, the method developed herein holds promise for advancing anti-Leishmanial pharmaceuticals and vaccines.

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Synthetic peptides are important as drugs and in research. Currently, the method of choice for producing these compounds is solid-phase peptide synthesis. Here, we describe the scope and limitations of Fmoc solid-phase peptide synthesis. Furthermore, we provide a detailed protocol for Fmoc peptide synthesis.

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In the immunoassay process, for fulfilling the need to identify multiple analytes in a small amount of complex sample matrix, it is desirable to develop highly efficient and specific multiplex suspension array technology. Raman coding strategy offers an attractive solution to code the suspension arrays by simply combing narrow spectral bands with stable signal intensities through solid-phase synthesis on the resin beads. Based on this strategy, we report the bead-based spontaneous Raman codes for multiplex immunoassay. The study resulted in superior selectivity of the Raman-encoded beads for binding with single and multiple analytes, respectively. With the use of mixed types of Raman-encoded immunoassay beads, multiple targets in small amounts of samples were identified rapidly and accurately. By confirming the feasibility of bead-based spontaneous Raman codes for multiplex immunoassay, we anticipate this novel technology to be widely applied in the near future.

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Glycosaminoglycans (GAGs) are a family of structurally complex heteropolysaccharides that play pivotal roles in biological functions, including the regulation of cell proliferation, enzyme inhibition, and activation of growth factor receptors. Therefore, the synthesis of GAGs is a hot research topic in drug development. The enzymatic synthesis of GAGs has received widespread attention due to their eco-friendly nature, high regioselectivity, and stereoselectivity. The enhancement of the enzymatic synthesis process is the key to its industrial applications. In this review, we overviewed the construction of more efficient in vitro biomimetic synthesis systems of glycosaminoglycans and presented the different strategies to improve enzyme catalysis, including the combination of chemical and enzymatic methods, solid-phase synthesis, and protein engineering to solve the problems of enzyme stability, separation and purification of the product, preparation of structurally defined sugar chains, etc., and discussed the challenges and opportunities in large-scale green synthesis of GAGs.

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Due to their resemblance to the fibrillar structure of the extracellular matrix, electrospun nanofibrous meshes are currently used as porous and mechanically stable scaffolds for cell culture. In this study, we propose an innovative methodology for growing peptide sequences directly onto the surface of electrospun nanofibers. To achieve this, electrospun fibers were produced from a poly(acrylic acid)/poly(vinyl alcohol) blend that was thermally crosslinked and subjected to a covalent coating of branched poly(ethylenimine). The exposed amino functionalities on the fiber surface were then used for the direct solid-phase synthesis of the RGD peptide sequence. In contrast to established strategies, mainly involving the grafting of pre-synthesized peptides onto the polymer chains before electrospinning or onto the nanofibers surface, this method allows for the concurrent synthesis and anchoring of peptides to the substrate, with potential applications in combinatorial chemistry. The incorporation of this integrin-binding motive significantly enhanced the nanofibers' ability to capture human cervical carcinoma (HeLa) cells, selected as a proof of concept to assess the functionalities of the developed material.

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While foldamers have been extensively studied as protein mimics and especially as α-helix mimics, their use as capping motif to enhance α-helix propensity remains comparatively much limited. In this study, we leverage the structural similarities between urea-based helical foldamers and α-helix to investigate the efficacy of oligoureas as N- or C-caps for reinforcing α-helical structures in water. Short oligoureas, comprising 3 to 4 residues, were strategically introduced at the N- or C-terminus of two peptide sequences (S-peptide and an Ala-rich model sequence). The impact of these foldamer insertions on peptide conformation was examined using electronic circular dichroism (ECD) and solution NMR. This research identifies specific foldamer sequences capable of promoting α-helicity when incorporated at either terminus of the peptides. Not only does this work broaden the application scope of foldamers, but it also provides valuable insights into novel strategies for modulating peptide conformation in aqueous environments. The findings presented in this study may have implications for peptide design and the development of bioactive foldamer-based peptide mimics.

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Organoboron compounds have a wide range of applications in numerous research fields, and methods to incorporate them in biomolecules are much sought after. Here, on-resin chemical syntheses of aliphatic and vinylogous peptide boronic acids are presented by transition metal-catalyzed late-stage hydroboration of alkene and alkyne groups in peptides and peptoids, for example on allyl- and propargylglycine residues, using readily available chemicals. These methods yield peptide boronic acids with much shorter linkers than previously reported on-resin methods. Furthermore, the methods are regio- and stereoselective, compatible with all canonical amino acid residues and can be applied to short, long, and in part even "difficult" peptide sequences. In a feasibility study, the protected peptide vinylboronic acids are further derivatized by the Petasis reaction using salicylaldehyde derivatives. The ability of the obtained peptide boronic acids to reversibly bind to carbohydrates is demonstrated in a catch-release model experiment using a fluorescently labeled peptide boronic acid on cross-linked dextran beads. In summary, this highlights the potential of the target compounds for drug discovery, glycan-specific target recognition, controlled release, and diagnostics.

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Tubulysins are among the most recent antimitotic compounds to enter into antibody/peptide-drug conjugate (ADC/PDC) development. Thus far, the design of the most promising tubulysin payloads relied on simplifying their structures, e. g., by using small tertiary amide N-substituents (Me, Et, Pr) on the tubuvaline residue. Cumbersome solution-phase approaches are typically used for both syntheses and functionalization with cleavable linkers. p-Aminobenzyl quaternary ammonium (PABQ) linkers were a remarkable advancement for targeted delivery, but the procedures to incorporate them into tubulysins are only of moderate efficiency. Here we describe a novel all-on-resin strategy permitting a loss-free resin linkage and an improved access to super potent tubulysin analogs showing close resemblance to the natural compounds. For the first time, a protocol enables the integration of on-resin tubulysin derivatization with, e. g., a maleimido-Val-Cit-PABQ linker, which is a notable progress for the payload-PABQ-linker technology. The strategy also allows tubulysin diversification of the internal amide N-substituent, thus enabling to screen a tubulysin library for the discovery of new potent analogs. This work provides ADC/PDC developers with new tools for both rapid access to new derivatives and easier linker-attachment and functionalization.

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Here we report the efficient synthetic access to 13C/15N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNAGly CCmnm5U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids.

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Dichloroacetic acid or trichloroacetic acid are commonly used in the detritylation reaction of the automated solid-phase synthesis of oligonucleotides. Dichloroacetic acid, however, is often contaminated with trichloroacetaldehyde (chloral), leading to the formation of inseparable impurities in the final oligonucleotide product. In this work, three different sequences, namely T18, d(TAA)6, and an 18-mer mixed sequence, were used as models to compare the deprotection efficiency of three acids: trichloroacetic acid, dichloroacetic acid, and difluoroacetic acid. Comparable purities of full-length products were obtained for the synthesis of the three model sequences when dichloroacetic acid or difluoroacetic acid were used during the detritylation reaction, however, conditions need to be optimized for the synthesis of purine-rich sequences. Therefore, difluoroacetic acid is a potential alternative to dichloroacetic acid in the solid-phase synthesis of oligonucleotides to avoid the impurity formation due to presence of chloral.

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Self-promoted glycosylations with trichloroacetimidate glycosyl donors are demonstrated on solid-phase-anchored peptides orthogonally deprotected and tosylcarbamoylated on the side-chains of cysteine and serine, respectively. The donor scope included glucosyl as well as mannosyl trichloroacetimidates, carrying benzyl, acetyl, or isopropylidene protecting groups. Isopropylidene groups were found to be removed under the acidic conditions used for release of the neoglycopeptides from the solid support, yielding neoglycopeptides with unprotected hydroxyl groups. Glycosylation of a peptide containing a carbamoylated tyrosine was attempted as well, but the desired neoglycopeptide could not be synthesized due to thermal instability of the carbamate.

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The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases.

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Ribonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner-both in vitro and in vivo-are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site-specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid-phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N4-acetylcytidine, 5-hydroxymethylcytidine, 3-methylcytidine, 2'-OCF3, and 2'-N3 ribose modifications. We also discuss the all-chemical synthesis of 5'-capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single-molecule FRET studies. Finally, we highlight promising developments in RNA-catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities.

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The development of miniaturized high-throughput in situ screening platforms capable of handling the entire process of drug synthesis to final screening is essential for advancing drug discovery in the future. In this study, an approach based on combinatorial solid-phase synthesis, enabling the efficient synthesis of libraries of proteolysis targeting chimeras (PROTACs) in an array format is presented. This on-chip platform allows direct biological screening without the need for transfer steps.  UV-induced release of target molecules into individual droplets facilitates further on-chip experimentation. Utilizing a mitogen-activated protein kinase kinases (MEK1/2) degrader as a template, a series of 132 novel PROTAC-like molecules is synthesized using solid-phase Ugi reaction. These compounds are further characterized using various methods, including matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging, while consuming only a few milligrams of starting materials in total. Furthermore, the feasibility of culturing cancer cells on the modified spots and quantifying the effect of MEK suppression is demonstrated. The miniaturized synthesis platform lays a foundation for high-throughput in situ biological screening of potent PROTACs for potential anticancer activity and offers the potential for accelerating the drug discovery process by integrating miniaturized synthesis and biological steps on the same array.

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The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases.

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This study presents an efficient method for on-resin dimer generation through self-condensation of 3,3-dimethoxypropionic acid-modified molecules, resulting in 2-pyridones. The approach demonstrated remarkable versatility by producing homodimers of peptides, peptoids, and non-peptidic ligands. Its ease of application, broad utility, and mild reaction conditions not only hold significance for peptide and peptoid research but also offer potential for the on-resin development of a wide range of bivalent ligands.

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