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Fuel‐driven self‐assemblies are gaining ground for creating autonomous systems and materials, whose temporal behavior is preprogrammed by a reaction network. However, up to now there has been a lack of simple external control mechanisms of the transient behavior, at best using remote and benign light control. Even more challenging is to use different wavelengths to modulate the reactivity of different components of the system, for example, as fuel or building blocks. Success would enable such systems to navigate along different trajectories in a wavelength‐dependent fashion. Herein, we introduce the first examples of light control in ATP‐fueled, dynamic covalent DNA polymerization systems organized in an enzymatic reaction network of concurrent ATP‐powered ligation and restriction. We demonstrate concepts for light activation and modulation by introducing caged ATP derivatives and caged DNA building blocks, making it possible to realize light‐activated fueling, self‐sorting in structure and behavior, and transition across different wavelength‐dependent dynamic steady states., Tuning on multiple channels: Multiple components of the chemical reaction network in the ATP‐driven transient DNA polymerization systems can be caged by different photocleavable groups that respond to different colors of light. As a result, light can be used to modulate the non‐equilibrium behavior and multiple wavelength‐dependent dynamic steady states.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

There has been a recent upsurge in the study and application of approaches utilizing cyclotriphosphate 1 (cyclo‐TP, also known as trimetaphosphate, TMP) and/or proceeding through its analogues in synthetic chemistry to access modified oligo‐ and polyphosphates. This is especially useful in the area of chemical nucleotide synthesis, but by no means restricted to it. Enabled by new high yielding and easy‐to‐implement methodologies, these approaches promise to open up an area of research that has previously been underappreciated. Additionally, refinements of concepts of prebiotic phosphorylation chemistry have been disclosed that ultimately rely on cyclo‐TP 1 as a precursor, placing it as a potentially central compound in the emergence of life. Given the importance of such concepts for our understanding of prebiotic chemistry in combination with the need to readily access modified polyphosphates for structural and biological studies, this paper will discuss selected recent developments in the field of cyclo‐TP chemistry, briefly touch on ultraphosphate chemistry, and highlight areas in which further developments can be expected., Cyclotriphosphate (cyclo‐TP) and its analogues: Selected recent developments in the field of cyclo‐TP chemistry as well as ultraphosphate chemistry are discussed in this Review‐type article.

An iterative polyphosphorylation approach is described, which is based on a phosphoramidite (P-amidite) derived reagent (c-PyPA) obtained from the cyclization of pyrophosphate with a reactive diisopropylaminodichlorophosphine. This type of reagent is unprecedented as it represents a reactive P-amidite without protecting groups. The reagent proved to be stable in solution over several weeks. Its utility is described in the context of iterative monodirectional and bidirectional polyphosphorylations. The ensuing functionalized cyclotriphosphate can be opened with a variety of nucleophiles providing ready access to diverse functionalized polyphosphate chains of defined length with several tags, including both P-N and P-O labels. Their interaction with exo- and endopolyphosphatases is described.

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP-InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogues (“PCP-InsPs”). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively-charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as ‘suspension cables’. We demonstrate functionality for a triglycine peptide within a β-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP(6) as an inhibitor of catalytic activity.

Nucleoside triphosphates (NTPs) are essential biomolecules involved in almost all biological processes, and their study is therefore critical to understanding cellular biology. Here, we describe a chemical synthesis suitable for obtaining both natural and highly modified NTPs, which can, for example, be used as surrogates to probe biological processes. The approach includes the preparation of a reagent that enables the facile introduction and modification of three phosphate units: cyclic pyrophosphoryl P-amidite (c-PyPA), derived from pyrophosphate (PV ) and a reactive phosphoramidite (PIII ). By using non-hydrolyzable analogues of pyrophosphate, the reagent can be readily modified to obtain a family of non-hydrolyzable analogues containing CH2 , CF2 , CCl2 , and NH that are stable in solution for several weeks if stored appropriately. They enable the synthesis of NTPs by reaction with nucleosides to give deoxycyclotriphosphate esters that are then oxidized to cyclotriphosphate (cyclo-TP) esters. The use of different oxidizing agents provides an opportunity for modification at P-α. Furthermore, terminal modifications at P-γ can be introduced by linearization of the cyclo-TP ester with various nucleophiles. © 2020 The Authors. Basic Protocol 1: Synthesis of cyclic pyrophosphoryl P-amidite (c-PyPA) and derivatives (c-PyNH PA, c-PyCH2 PA, c-PyCCl2 PA, c-PyCF2 PA) Basic Protocol 2: Synthesis of 3'-azidothymidine 5'-γ-P-propargylamido triphosphates and analogues Basic Protocol 3: Synthesis of 2'-deoxythymidine 5'-γ-P-propargylamido triphosphate (15) Basic Protocol 4: Synthesis of adenosine 5'-γ-P-amido triphosphate (19) and adenosine 5'-γ-P-propargylamido triphosphate (20) Basic Protocol 5: Synthesis of d4T 5'-γ-propargylamido β,γ-(difluoromethylene)triphosphate Support Protocol: Synthesis of diisopropylphosphoramidous dichloride.

Inositol pyrophosphates (PP-InsPs) are “energetic” intracellular signals that are ubiquitous in animals, plants, and fungi. We are characterizing new proteins that regulate PP-InsP metabolism and/o...

Highly charged and unstable diphosphoinositol pentaphosphates (PP InsP5) are challenging but biologically relevant synthetic targets. A novel C2 symmetric phosphoramidite was developed that can be used to target all 4 unsymmetric PP InsP5. It does not only serve well in the desymmetrization but is also an excellent phosphate protecting group. With this approach the target structures were synthesized in few steps and high enantiomeric ratios. With the obtained compounds specificity of Ddp1 from yeast was studied.

The synthesis of the fluorescent thymidine nucleoside analog 5-methyl-pyrimidin-2-one nucleoside 1 (m5K) and that of its 2′,3′-dideoxy derivative 2 (dm5K) are described. Moreover, the conversion of 1 and 2 into the corresponding 3-methyl-cycloSal-phosphate triesters 11 and 12 or an enzyme-cleavable prodrug 5-acetoxymethylpropionate-cycloSal-phosphate triester 13, and the fluorescence and hydrolysis properties of these new lipophilic triesters are reported. Finally, the suitability of these cycloSal pronucleotides as probes is demonstrated in a cell-extract hydrolysis experiment and a model study for cell uptake. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

An extension of the cycloSal-pronucleotide approach is presented. Attachment of an enzyme-cleavable ester/acylal group to the cycloSal-d4TMP triesters should allow these compounds to be trapped intracellularly after cleavage. The ester/acylal groups were introduced in the 3- or 5-position of the cycloSal ring system, and surprising differences were observed in hydrolysis studies in CEM cell extracts with respect to the ester/acylal moiety. While acetyl and levulinyl esters were readily cleaved, alkyl esters of cycloSal-d4TMP acids proved to be resistant to enzymatic cleavage. In contrast, AM-, POM- and POC-acylals were rapidly cleaved in the extracts, leading to cycloSal-d4TMP acids. The antiviral activity of the compounds against HIV is also presented. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

1. N-type Ca(2+) channel modulation by an endogenous P2Y receptor was investigated by the whole-cell patch-clamp method in HEK 293 cells transfected with the functional rabbit N-type calcium channel. 2. The current responses (I(Ca(N))) to depolarizing voltage steps were depressed by ATP in a concentration-dependent manner. Inclusion of either guanosine 5'-O-(3-thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. 3. In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration-response curves of ADP and adenosine 5'-O-(2-thiodiphosphate) indicated a higher potency of these agonists than that of ATP, alpha,beta-methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 2'deoxy-N(6)-methyladenosine-3',5'-diphosphate or 2-methylthio AMP. 4. Using reverse transcription and polymerase chain reaction, mRNA for the P2Y(1), P2Y(4), P2Y(6), P2Y(11) and P2Y(13) receptor subtypes, but not the P2Y(2), and P2Y(12) subtypes, was detected in HEK 293 cells. 5. Immunocytochemistry confirmed the presence of P2Y(1), and to a minor extent that of P2Y(4), but not of P2Y(2) receptors. 6. Hence, it is tempting to speculate that P2Y(13) receptors may inhibit N-type Ca(2+) channels via the betagamma subunits of the activated G(i) protein.

The propagation of information inside cells is achieved in nature by small diffusible molecules. Many of these secondary messengers are highly charged species derived from myo -inositol. The diphosphoinositol polyphosphates (X-PP- myo -InsP 5 ) represent a novel group of secondary messengers that regulate various important biological processes. Depending on the phosphorylation pattern, these molecules can occur in symmetric or nonsymmetric forms. In a recent study, we have shown the potential of C 2 symmetric phosphoramidites as reagents for desymmetrizing myo -inositol and for obtaining all four possible nonsymmetric X-PP- myo -InsP 5 stereoisomers in enantiomerically pure form. These results and their potential implications for the design of tools for chemical biology are discussed.

The first enantioselective catalytic total synthesis of (–)-pyridovericin is reported. The key steps involve a modified HWE reaction under aqueous conditions and an asymmetric iridium-catalyzed hydrogenation. This resulted in a highly modular and stereoselective approach that delivered the target natural product in high yield and stereoselectivity.