Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Diverse Catalytic Reactions for the Stereoselective Synthesis of Cyclic Dinucleotide MK-1454.

As practitioners of organic chemistry strive to deliver efficient syntheses of the most complex natural products and drug candidates, further innovations in synthetic strategies are required to facilitate their efficient construction. These aspirational breakthroughs often go hand-in-hand with considerable reductions in cost and environmental impact. Enzyme-catalyzed reactions have become an impressive and necessary tool that offers benefits such as increased selectivity and waste limitation. These benefits are amplified when enzymatic processes are conducted in a cascade in combination with novel bond-forming strategies. In this article, we report a highly diastereoselective synthesis of MK-1454, a potent agonist of the stimulator of interferon gene (STING) signaling pathway. The synthesis begins with the asymmetric construction of two fluoride-bearing deoxynucleotides. The routes were designed for maximum convergency and selectivity, relying on the same benign electrophilic fluorinating reagent. From these complex subunits, four enzymes are used to construct the two bridging thiophosphates in a highly selective, high yielding cascade process. Critical to the success of this reaction was a thorough understanding of the role transition metals play in bond formation.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Raman hyperspectral imaging with multivariate analysis for investigating enzyme immobilization.

Directed enzyme evolution has led to significant application of biocatalysis for improved chemical transformations throughout the scientific and industrial communities. Biocatalytic reactions utilizing evolved enzymes immobilized within microporous supports have realized unique advantages, including notably higher enzyme stability, higher enzyme load, enzyme reusability, and efficient product-enzyme separation. To date, limited analytical methodology is available to discern the spatial and chemical distribution of immobilized enzymes, in which techniques for surface visualization, enzyme stability, or activity are instead employed. New analytical tools to investigate enzyme immobilization are therefore needed. In this work, development, application, and evaluation of an analytical methodology to study enzyme immobilization is presented. Specifically, Raman hyperspectral imaging with principal component analysis, a multivariate method, is demonstrated for the first time to investigate evolved enzymes immobilized onto microporous supports for biocatalysis. Herein we demonstrate the ability to spatially and spectrally resolve evolved pantothenate kinase (PanK) immobilized onto two commercially-available, chemically-diverse porous resins. This analytical methodology is able to chemically distinguish evolved enzyme, resin, and chemical species pertinent to immobilization. As such, a new analytical approach to study immobilized biocatalysts is demonstrated, offering potential wide application for analysis of protein or biomolecule immobilization.

Component Quantification in Solids with the Mixture Analysis Using References Method.

Quantifying components in solid mixtures composed of the same chemical species exhibiting different physical forms represents a difficult challenge in many areas of chemistry. The development of small-molecule active pharmaceutical ingredients (APIs) is a classic example. APIs predominantly exhibit polymorphism and the propensity to form solvates and hydrates. The various API phases typically display different physical properties affecting chemical stability, processability, and bioperformance. Accordingly, API development critically relies on characterizing and quantifying the relevant API forms in complex mixtures in the presence of each other and in the presence of excipients. Presented here is a new solid-state-NMR-based quantification method for components in solid mixtures: mixture analysis using references (MAR). The method utilizes weighted pure component reference spectra in a linear combination fitting procedure to reproduce the corresponding mixture spectrum. The results yield the respective component contributions to the mixture composition. Using several model systems of varying complexity, the applicability and performance of the MAR analysis utilizing 13C and 19F cross-polarization magic-angle-spinning data are evaluated. Finally, the MAR method is compared to one of the most commonly applied traditional quantification methods. The results demonstrate that MAR performs with the same high accuracy as conventional methods. However, MAR exhibits clear efficiency advantages over conventional methods by requiring significantly less overall time (experimental and computational) and displaying remarkable robustness and general applicability. The MAR quantification protocol as presented here can easily be applied to nonpharmaceutical molecular systems in other branches of chemistry.

Spin-selective charge transport pathways through p-oligophenylene-linked donor-bridge-acceptor molecules.

A series of donor-bridge-acceptor (D-B-A) triads have been synthesized in which the donor, 3,5-dimethyl-4-(9-anthracenyl)julolidine (DMJ-An), and the acceptor, naphthalene-1,8:4,5-bis(dicarboximide) (NI), are linked by p-oligophenylene (Ph(n)) bridging units (n = 1-5). Photoexcitation of DMJ-An produces DMJ(+*)-An(-*) quantitatively, so that An(-*) acts as a high potential electron donor, which rapidly transfers an electron to NI yielding a long-lived spin-coherent radical ion pair (DMJ(+*)-An-Ph(n)-NI(-*)). The charge transfer properties of 1-5 have been studied using transient absorption spectroscopy, magnetic field effects (MFEs) on radical pair and triplet yields, and time-resolved electron paramagnetic resonance (TREPR) spectroscopy. The charge separation (CS) and recombination (CR) reactions exhibit exponential distance dependencies with damping coefficients of beta = 0.35 A(-1) and 0.34 A(-1), respectively. Based on these data, a change in mechanism from superexchange to hopping was not observed for either process in this system. However, the CR reaction is spin-selective and produces the singlet ground state and both (3*)An and (3*)NI. A kinetic analysis of the MFE data shows that superexchange dominates both pathways with beta = 0.48 A(-1) for the singlet CR pathway and beta = 0.35 A(-1) for the triplet CR pathway. MFEs and TREPR experiments were used to measure the spin-spin exchange interaction, 2J, which is directly related to the electronic coupling matrix element for CR, V(CR)(2). The magnitude of 2J also shows an exponential distance dependence with a damping coefficient alpha = 0.36 A(-1), which agrees with the beta values obtained from the distance dependence for triplet CR. These results were analyzed in terms of the bridge molecular orbitals that participate in the charge transport mechanism.

Intersystem crossing mediated by photoinduced intramolecular charge transfer: julolidine-anthracene molecules with perpendicular pi systems.

Time-resolved electron paramagnetic resonance studies show that the primary mechanism of triplet formation following photoexcitation of julolidine-anthracene molecules linked by a single bond and having perpendicular pi systems is a spin-orbit, charge-transfer intersystem crossing mechanism (SOCT-ISC). This mechanism depends on the degree of charge transfer from julolidine to anthracene, the dihedral angle (theta1) between their pi systems, and the magnitude of the electronic coupling between julolidine and anthracene. We compare 4-(9-anthracenyl)-julolidine with the more sterically encumbered 4-(9-anthracenyl)-3,5-dimethyljulolidine and find that fixing theta1 congruent with 90 degrees serves to enhance SOCT-ISC by increasing the change in orbital angular momentum accompanying charge transfer. Given that the requirements for the SOCT-ISC mechanism are quite general, we expect it to occur in a variety of electron donor-acceptor systems.

Direct observation of the preference of hole transfer over electron transfer for radical ion pair recombination in donor-bridge-acceptor molecules.

Understanding how the electronic structures of electron donor-bridge-acceptor (D-B-A) molecules influence the lifetimes of radical ion pairs (RPs) photogenerated within them (D+*-B-A-*) is critical to designing and developing molecular systems for solar energy conversion. A general question that often arises is whether the HOMOs or LUMOs of D, B, and A within D+*-B-A-* are primarily involved in charge recombination. We have developed a new series of D-B-A molecules consisting of a 3,5-dimethyl-4-(9-anthracenyl)julolidine (DMJ-An) electron donor linked to a naphthalene-1,8:4,5-bis(dicarboximide) (NI) acceptor via a series of Phn oligomers, where n = 1-4, to give DMJ-An-Phn-NI. The photoexcited charge transfer state of DMJ-An acts as a high-potential photoreductant to rapidly and nearly quantitatively transfer an electron across the Phn bridge to produce a spin-coherent singlet RP 1(DMJ+*-An-Phn-NI-*). Subsequent radical pair intersystem crossing yields 3(DMJ+*-An-Phn-NI-*). Charge recombination within the triplet RP then gives the neutral triplet state. Time-resolved EPR spectroscopy shows directly that charge recombination of the RP initially produces a spin-polarized triplet state, DMJ-An-Phn-3*NI, that can only be produced by hole transfer involving the HOMOs of D, B, and A within the D-B-A system. After the initial formation of DMJ-An-Phn-3*NI, triplet-triplet energy transfer occurs to produce DMJ-3*An-Phn-NI with rate constants that show a distance dependence consistent with those determined for charge separation and recombination.

Photoinduced charge separation in self-assembled cofacial pentamers of zinc-5,10,15,20-tetrakis(perylenediimide)porphyrin.

A bichromophoric electron donor-acceptor molecule composed of a zinc tetraphenylporphyrin (ZnTPP) surrounded by four perylene-3,4:9,10-bis(dicarboximide)(PDI) chromophores (ZnTPP-PDI(4)) was synthesized. The properties of this molecule were compared to a reference molecule having ZnTPP covalently bound to a single PDI (ZnTPP-PDI). In toluene, ZnTPP-PDI(4) self-assembles into monodisperse aggregates of five molecules arranged in a columnar stack, (ZnTPP-PDI(4))(5). The monodisperse nature of this assembly contrasts sharply with previously reported ZnTPP-PDI(4) derivatives having 1,7-bis(3,5-di-t-butylphenoxy) groups (ZnTPP-PPDI(4)). The size and structure of this assembly in solution was determined by small angle X-ray scattering (SAXS) using a high flux synchrotron X-ray source. The ZnTPP-PDI reference molecule does not aggregate. Femtosecond transient absorption spectroscopy shows that laser excitation of both ZnTPP-PDI and (ZnTPP-PDI(4))(5) results in quantitative formation of ZnTPP(+*)-PDI(-*) radical ion pairs in a few picoseconds. The transient absorption spectra of (ZnTPP-PDI(4))(5) suggest that the PDI(-*) radicals interact strongly with adjacent PDI molecules within the columnar stack. Charge recombination occurs more slowly within (ZnTPP-PDI(4))(5)(tau= 4.8 ns) than it does in ZnTPP-PDI (tau= 3.0 ns) producing mostly ground state as well as a modest yield of the lowest triplet state of PDI ((3*)PDI). Formation of (3*)PDI occurs by rapid spin-orbit induced intersystem crossing (SO-ISC) directly from the singlet radical ion pair as evidenced by the electron spin polarization pattern exhibited by its time-resolved electron paramagnetic resonance spectrum.

Time-resolved EPR studies of photogenerated radical ion pairs separated by p-phenylene oligomers and of triplet states resulting from charge recombination.

Photoexcitation of a series of donor-bridge-acceptor (D-B-A) systems, where D = phenothiazine (PTZ), B = p-phenylene (Phn), n = 1-5, and A= perylene-3,4:9,10-bis(dicarboximide) (PDI) results in rapid electron transfer to produce 1(PTZ+*-Phn-PDI-*). Time-resolved EPR (TREPR) studies of the photogenerated radical pairs (RPs) show that above 150 K, when n = 2-5, the radical pair-intersystem crossing mechanism (RP-ISC) produces spin-correlated radical ion pairs having electron spin polarization patterns indicating that the spin-spin exchange interaction in the radical ion pair is positive, 2J > 0, and is temperature dependent. This temperature dependence is most likely due to structural changes of the p-phenylene bridge. Charge recombination in the RPs generates PTZ-Phn-3*PDI, which exhibits a spin-polarized signal similar to that observed in photosynthetic reaction-center proteins and some biomimetic systems. At temperatures below 150 K and/or at shorter donor-acceptor distances, e.g., when n = 1, PTZ-Phn-3*PDI is also formed from a competitive spin-orbit-intersystem crossing (SO-ISC) mechanism that is a result of direct charge recombination: 1(PTZ+*-Phn-PDI-*) --> PTZ-Phn-3*PDI. This SO-ISC mechanism requires the initial RP intermediate and depends strongly on the orientation of the molecular orbitals involved in the charge recombination as well as the magnitude of 2J.