Iakovos Saridakis

The macrocyclic immunosuppressive natural product (−)-FR252921 was isolated in 2003 from a liquid culture of Pseudomonas fluorescens. Despite promising preliminary immunological insights, in-depth studies that advance our understanding of structure–activity relationship (SAR) have been scarce. Herein we document a detailed SAR mapping of (−)-FR252921 by the syntheses of 14 analogs and their subsequent evaluation. The composed library of analogs was designed to address crucial questions regarding the roles of explicit molecular features of the natural product while following a target-agnostic efficacy approach. Whereas previous efforts have focused on murine T cell assays, our endeavors transit to a primary human cell-based immunological platform using peripheral blood mononuclear cells (PBMCs), enabling direct assessment of immunosuppressive activity in B, natural killer (NK), and T cells via cytokine suppression. We emphasize that a new fully synthetic (−)-FR252921 analog (fs-FR4) profoundly outperformed the natural product in suppressing the response of both stimulated B cells (21-fold) and NK cells (16-fold) within the context the biological assays used, validating the power of our platform and standing as a lead candidate for further preclinical development. Lastly, we highlight that our studies included significant improvement in the synthetic efficacy of previously reported building blocks and key synthetic steps towards (−)-FR252921 and analogs.

Cross-linking mass spectrometry (XL-MS) has emerged as a powerful approach for probing protein structure and conformational dynamics. Conventional cross-linkers typically contain two N-hydroxysuccinimide (NHS) ester groups that primarily target lysine residues. Here, we report the optimization of the in-solution reactivity of disuccinimidyl sulfoxide carbamate (DSSO-carbamate), an analogue of DSSO in which the two NHS ester groups are replaced by NHS carbamates. The enhanced stability of the carbamate functionality reduces the degradation of DSSO through retro-ene sulfoxide elimination under standard XL-MS buffer conditions, thereby improving cross-linking efficiency. We further characterized the gas-phase dissociation behavior of DSSO-carbamate and optimized the collision energy (CE) parameters for automated data analysis with XL-MS search engines. Mapping of cross-linking sites for bovine serum albumin revealed an unexpectedly high frequency of cross-links involving the protein N-terminus, suggesting increased N-terminal reactivity of NHS carbamates relative to NHS esters. This hypothesis was corroborated by comparative cross-linking of nonacetylated and N-terminally acetylated α-synuclein using DSSO-carbamate and the NHS ester-based disuccinimidyl dibutyric urea (DSBU). We observed the same reactivity trend for the NHS carbamate-based cross-linker NNP9. Proteome-wide XL-MS analysis confirmed a higher propensity of NHS carbamate-based reagents to form cross-links with protein N-termini compared to NHS ester-based cross-linkers. Together, these results show that NHS carbamate-based reagents provide complementary XL-MS restraints to NHS ester-based cross-linkers and are particularly useful for investigating systems where N-terminal interactions are functionally relevant. We anticipate that this unique N-terminal selectivity of NHS carbamates will find broader applications in bioconjugation and chemical proteomics.

We deploy a new class of fluorophores, PyrAtes, in the first-time use of small-molecule fluorophore–drug conjugates in imaging endogenous SERT ex vivo.

Chemical cross-linking/mass spectrometry (XL-MS) has emerged as a complementary tool for mapping interaction sites within protein networks as well as gaining moderate-resolution native structural insight with minimal interference. XL-MS technology mostly relies on chemoselective reactions (cross-linking) between protein residues and a linker. DSSO represents a versatile cross-linker for protein structure investigation and in-cell XL-MS. However, our assessment of its shelf life and batch purity revealed decomposition of DSSO in anhydrous solution via a retro-Michael reaction, which may reduce the active ingredient down to below 90%. To mitigate the occurrence of this degradative mechanism, we report the rational design and synthesis of DSSO-carbamate, which contains an inserted nitrogen atom in the DSSO backbone structure. This modification to DSSO yielded remarkably favorable stability against such decomposition, which translated to higher cross-link and monolink recovery when performing XL-MS on monomeric flexible proteins. Recently, XL-MS has been leveraged against AlphaFold2 and other protein structure prediction algorithms for improved prediction of flexible monomeric multiconformational proteins. To this end, we demonstrate that our novel cross-linker, termed DSSO-carbamate, generated more accurate protein structure predictions when combined with AlphaFold2, on account of its increased recovery of cross-links and monolinks, compared to DSSO. As such, DSSO-carbamate represents a useful addition to the XL-MS community, particularly for protein structure prediction.

Cellular activity is spatially organized across different organelles. While several structures are well-characterized, many organelles have unknown roles. Profiling biomolecular composition is key to understanding function but is difficult to achieve in the context of small, dynamic structures. Photoproximity labeling has emerged as a powerful tool for mapping these interaction networks, yet maximizing catalyst localization and reducing toxicity remains challenging in live cell applications. Here, we disclose a new intracellular photocatalyst with minimal cytotoxicity and off-target binding, and we utilize this catalyst for HaloTag-based microenvironment-mapping (μMap) to spatially catalog subnuclear condensates in living cells. We also specifically develop a novel RNA-focused workflow (μMap-seq) to enable parallel transcriptomic and proteomic profiling of these structures. After validating the accuracy of our approach, we generate a spatial map across the nucleolus, nuclear lamina, Cajal bodies, paraspeckles, and PML bodies. These results provide potential new insights into RNA metabolism and gene regulation while significantly expanding the μMap platform for improved live-cell proximity labeling in biological systems.

Crosslinking mass spectrometry (XL-MS) is a powerful technology that recently emerged as an essential complementary tool for elucidating protein structures and mapping interactions within a protein network. Crosslinkers which are amenable to post-linking backbone cleavage simplify peptide identification, aid in 3D structure determination and enable system-wide studies of protein-protein interactions (PPIs) in cellular environments. However, state-of-the-art cleavable linkers are fraught with practical limitations, including extensive evaluation of fragmentation energies and fragmentation behaviour of the crosslinker backbone. We herein introduce DiSPASO as a lysine-selective, MS-cleavable cross-linker with an alkyne handle for affinity enrichment. DiSPASO was designed and developed for efficient cell membrane permeability and crosslinking while securing low cellular perturbation. We tested DiSPASO employing three different copper-based enrichment strategies using model systems with increasing complexity (Cas9-Halo, purified ribosomes, live cells). Fluorescence microscopy in-cell crosslinking experiments revealed a rapid uptake of DiSPASO into HEK 293 cells within 5 minutes. While DiSPASO represents progress in cellular PPI analysis, its limitations and low crosslinking yield in cellular environments require careful optimisation of the crosslinker design, highlighting the complexity of developing effective XL-MS tools and the importance of continuous innovation in accurately mapping PPI networks within dynamic cellular environments. Graphical

Hydride shuttle catalysis has emerged as a powerful synthetic platform, enabling the selective formation of C-C bonds to yield sp³-rich structures. By virtue of the compelling reactivity of sterically encumbered Lewis acids from the frustrated Lewis pair regime, hydride shuttle catalysis enables the regioselective functionalization of alkyl amines at either the α- or β-position. In contrast to classical Lewis acid reactivity, the increased steric hindrance prevents interaction with the Lewis basic amine itself, instead leading to reversible abstraction of a hydride from the amine α-carbon. The created positive charge facilitates the occurrence of transformations before hydride rebound or a similar capture event happen. In this Perspective, we outline a broad selection of transformations featuring hydride shuttle catalysis, as well as the recently developed approach of inverse hydride shuttle catalysis. Both strategies give rise to a wide array of functionalized amines and offer elegant approaches to otherwise elusive bond formations.

Linear diazirines can produce relatively long-lived diazoalkanes which are also capable of labeling proteins whereas cyclobutanediazirines are proposed to give minimal diazo product and labeling occurs predominantly via a carbene mechanism.

Der Einsatz kleiner fluoreszierender Moleküle spielt in der Medizin und Arzneimittelentwicklung eine immer größere Rolle. Mit diesem Beitrag ergänzen wir das Portfolio leistungsstarker Fluorophore durch die zufällige Entdeckung und Entwicklung einer neuen Klasse mit einem Imidazo[1,2‐a]pyridiniumtriflat‐Kern, die wir „PyrAt‐Verbindungen“ nennen. Diese Fluorophore werden in einem einzigen Schritt aus leicht verfügbaren Materialien synthetisiert (>60 Beispiele) und zeigen Stokes‐Verschiebungen bis zu 240 nm, während sie gleichzeitig NIR−I‐Emissionen bei λ_max bis zu 720 nm erreichen. Computergestützte Studien ermöglichen die Entwicklung einer Plattform für die Vorhersage von λ_max und λ_Em. Darüber hinaus demonstrieren wir die Kompatibilität dieser neuartigen Fluorophore mit der Lebendzellbildgebung in HEK293‐Zellen, was darauf hindeutet, dass sie potente intrazelluläre Marker sind.

The deployment of small‐molecule fluorescent agents plays an ever‐growing role in medicine and drug development. Herein, we complement the portfolio of powerful fluorophores, reporting the serendipitous discovery and development of a novel class with an imidazo[1,2‐a]pyridinium triflate core, which we term PyrAtes. These fluorophores are synthesized in a single step from readily available materials (>60 examples) and display Stokes shifts as large as 240 nm, while also reaching NIR−I emissions at λ_max as long as 720 nm. Computational studies allow the development of a platform for the prediction of λ_max and λ_Em. Furthermore, we demonstrate the compatibility of these novel fluorophores with live cell imaging in HEK293 cells, suggesting PyrAtes as potent intracellular markers.