5-Hydroxypyrroloindoline Affords Tryptathionine and 2,2'-bis-Indole Peptide Staples: Application to Melanotan-II.

With peptides increasingly favored as drugs, natural product motifs, namely the tryptathionine staple, found in amatoxins and phallotoxins, and the 2,2'-bis-indole found in staurosporine represent unexplored staples for unnatural peptide macrocycles. We disclose the efficient condensation of a 5-hydroxypyrroloindoline with either a cysteine-thiol or a tryptophan-indole to form a tryptathionine or 2-2'-bis-indole staple. Judicious use of protecting groups provides for chemoselective stapling using α-MSH, which provides a basis for investigating both chemoselectivity and affinity. Both classes of stapled peptides show nanomolar Ki's, with one showing a sub-nanomolar Ki value.

Diphenylphosphinylhydroxylamine (DPPH) Affords Late-Stage S-imination to access free-NH Sulfilimines and Sulfoximines.

Sulfilimines, as potential aza-isosteres of sulfoxides, are valued as building blocks, auxiliaries, ligands, bioconjugation handles, and as precursors to versatile S(VI) scaffolds including sulfoximines and sulfondiimines. Here, we report a thioether imination methodology that exploits O-(diphenylphosphinyl)hydroxyl amine (DPPH). Under mild, metal-free, and biomolecule-compatible conditions, DPPH enables late-stage S-imination on peptides, natural products, and a clinically trialled drug, and shows both excellent chemoselectivity and broad functional group tolerance. This methodological report is extended to an efficient and high-yielding one-pot reaction for accessing free-NH sulfoximines with diverse substrates including ones of potential clinical importance. In the presence of a rhodium catalyst, sulfoxides are S-iminated in higher yields to afford free-NH sulfoximines. S-imination was validated on an oxidatively delicate amatoxin to give sulfilimine and sulfoximine congeners. Interestingly, these new sulfilimine and sulfoximine-amatoxins show cytotoxicity. This method is further extended to create sulfilimine and sulfoximine-Fulvestrant and buthionine analogues.

Toward tryptathionine-stapled one-bead-one-compound (OBOC) libraries: solid phase synthesis of a bioactive octretoate analog.

New somatostatin analogs are highly desirable for diagnosing and treating neuroendocrine tumors (NETs). Here we describe the solid-phase synthesis of a new octreotate (TATE) analog where the disulfide bond is replaced with a tryptathionine (Ttn) staple as part of an effort to prototyping a one-bead-one-compound (OBOC) library of Ttn-stapled peptides. Library design provides the potential for on- and off-bead screening. To validate our method, we labelled Ttn-TATE with a fluorescent dye to demonstrate binding to soluble somatostatin receptor subtype-2 and staining of Ar42J rat prostate cancer cells. By exploring this staple in the context of a ligand of known affinity, this method paves the way for an OBOC library construction of bioactive octreotate analogs and, more broadly speaking, tryptathionine-staped peptide macrocycles.

The "Ins and Outs" of Prostate Specific Membrane Antigen (PSMA) as Specific Target in Prostate Cancer Therapy.

Prostate-specific membrane antigen (PSMA) is expressed in epithelial cells of the prostate gland and is strongly upregulated in prostatic adenocarcinoma, with elevated expression correlating with metastasis, progression, and androgen independence. Because of its specificity, PSMA is a major target of prostate cancer therapy; however, detectable levels of PSMA are also found in other tissues, especially in salivary glands and kidney, generating bystander damage of these tissues. Antibody target therapy has been used with relative success in reducing tumor growth and prostate specific antigen (PSA) levels. However, since antibodies are highly stable in plasma, they have prolonged time in circulation and accumulate in organs with an affinity for antibodies such as bone marrow. For that reason, a second generation of PSMA targeted therapeutic agents has been developed. Small molecules and minibodies have had promising clinical trial results, but concerns about their specificity had arisen with side effects due to accumulation in salivary glands and kidneys. Herein we study the specificity of small molecules and minibodies that are currently being clinically tested. We observed a high affinity of these molecules for PSMA in prostate, kidney and salivary gland, suggesting that their effect is not prostate specific. The search for specific prostate target agents must continue so as to optimally treat patients with prostate cancer, while minimizing deleterious effects in other PSMA expressing tissues.

Imidazolium-methylene-trifluoroborate: A novel radioprosthetic group validated with preclinical 18 F-Positron Emission Tomography imaging of Prostate Specific Membrane Antigen in mice.

Organotrifluoroborates have gained acceptance as radioprosthetic groups for radiofluorination. Of these, the zwitterionic prosthetic group "AMBF3 " with a quaternary dimethylammonium ion dominates the trifluoroborate space. Herein, we report on imidazolium-methylene trifluoroborate (ImMBF3 ) as an alternative radioprosthetic group and report on its properties in the context of a PSMA-targeting EUK ligand that was previously been conjugated to AMBF3 . The ImMBF3 is readily synthesized from imidazole and conjugated via CuAAC "click" chemistry to give a structure similar to PSMA-617. 18 F-labeling proceeded in one step per our previous reports and imaged in LNCaP-xenograft bearing mice. The [18 F]-PSMA-617-ImMBF3 tracer proved to be less polar (LogP7.4 = -2.95 ± 0.03) while showing a significantly lower solvolytic rate (t1/2 = 8100 min) and slightly higher molar activity (Am) at 174 ± 38 GBq/µmol. Tumor uptake was measured at 13.7 ± 4.8%ID/g and a tumor:muscle ratio of 74.2 ± 35.0, tumor:blood ratio of 21.4 ± 7.0, tumor:kidney ratio of 0.29 ± 0.14, and tumor:bone ratio of 23.5 ± 9.5. In comparison with previously reported PSMA-targeting EUK-AMBF3 conjugates, we have altered the LogP7.4 value, tuned the solvolytic half-life of the prosthetic, and increased radiochemical conversion while achieving similar tumor uptake, contrast ratios, and molar activities compared with AMBF3 bioconjugates.

Salt Metathesis: Tetrafluoroborate Anion Rapidly Fluoridates Organoboronic Acids to give Organotrifluoroborates.

Tetrafluoroborate (BF4 - ) has long been used as a spectator counter anion. Herein, we report an unprecedented salt metathesis between a variety of BF4 - salts and a series of organoboronic acids yielding the corresponding organotrifluoroborates. We identified conditions for fast and efficient fluoridation (<1 h) with minimal workup. Fundamentally, this work discloses the proclivity of BF4 - to exchange fluoride atoms with organoboronates, highlighting the lability of BF4 - .

Rationally Designed Amanitins Achieve Enhanced Cytotoxicity.

For 70 years, α-amanitin, the most cytotoxic peptide in its class, has been without a synthetic rival; through synthesis, we address the structure-activity relationships to inform the design of new amatoxins and disclose analogues that are more cytotoxic than the natural product when evaluated on CHO, HEK293, and HeLa cells, whereas on liver-derived HepG2 cells, the same toxins show diminished cytotoxicity.

Synthesis and preliminary evaluation of octreotate conjugates of bioactive synthetic amatoxins for targeting somatostatin receptor (sstr2) expressing cells.

Targeted cancer therapy represents a paradigm-shifting approach that aims to deliver a toxic payload selectively to target-expressing cells thereby sparing normal tissues the off-target effects associated with traditional chemotherapeutics. Since most targeted constructs rely on standard microtubule inhibitors or DNA-reactive molecules as payloads, new toxins that inhibit other intracellular targets are needed to realize the full potential of targeted therapy. Among these new payloads, α-amanitin has gained attraction as a payload in targeted therapy. Here, we conjugate two synthetic amanitins at different sites to demonstrate their utility as payloads in peptide drug conjugates (PDCs). As an exemplary targeting agent, we chose octreotate, a well-studied somatostatin receptor (sstr2) peptide agonist for the conjugation to synthetic amatoxins via three tailor-built linkers. The linker chemistry permitted the evaluation of one non-cleavable and two cleavable self-immolative conjugates. The immolating linkers were chosen to take advantage of either the reducing potential of the intracellular environment or the high levels of lysosomal proteases in tumor cells to trigger toxin release. Cell-based assays on target-positive Ar42J cells revealed target-specific reduction in viability with up to 1000-fold enhancement in bioactivity compared to the untargeted amatoxins. Altogether, this preliminary study enabled the development of a highly modular synthetic platform for the construction of amanitin-based conjugates that can be readily extended to various targeting moieties.

Selection of M2+ -Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine.

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+ ) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dUga TP) and 5-aminoallyl-2'-dCTP (dCaa TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

Synthesis and evaluation of "Ama-Flash", a photocaged amatoxin prodrug for light-activated RNA Pol II inhibition and cell death.

Amanitin is used extensively as a research tool to inhibit RNA Pol II thereby implicating its role in mRNA transcription. Recently, amanitin has gained traction as a toxic payload for targeted therapy. Here we report the first-ever photocaged amanitin analog, that is non-toxic and can be pre-loaded into cells. Light provides a means to inhibit RNA Pol II and provoke cell death on-demand.

Chiral Benzothiazole Monofluoroborate Featuring Chiroptical and Oxygen-Sensitizing Properties: Synthesis and Photophysical Studies.

Advances in personalized medicine are prompting the development of multimodal agents, that is, molecules that combine properties promoting various diagnostic and therapeutic applications. General approaches exploit chemical conjugation of therapeutic agents with contrast agents or the design of multimodal nanoplatforms. Herein, we report the design of a single molecule that exhibits potential for different diagnostic modes as well as the ability to sensitize oxygen, thus offering potential for photodynamic therapy. Exceptionally, this work involves the synthesis and chiral resolution of an enantiomeric pair of chiral monofluoroborates that contain a stereogenic boron atom. Combining experimental and theoretical chiroptical studies allowed the unambiguous determination of their absolute configuration. Photophysical investigations established the ability of this compound to sensitize oxygen even in the absence of heavy atoms within its structure. The synthesis of a chiral benzothiazole monofluoroborate paves a way to multimodal diagnostic tools (fluorescence and nuclear imaging) while also featuring potential therapeutic applications owing to its ability to activate oxygen to its singlet state for use in photodynamic therapy.

Design, Synthesis, and Biochemical Evaluation of Alpha-Amanitin Derivatives Containing Analogs of the trans-Hydroxyproline Residue for Potential Use in Antibody-Drug Conjugates.

Alpha-amanitin, an extremely toxic bicyclic octapeptide extracted from the death-cap mushroom, Amanita phalloides, is a highly selective allosteric inhibitor of RNA polymerase II. Following on growing interest in using this toxin as a payload in antibody-drug conjugates, herein we report the synthesis and biochemical evaluation of several new derivatives of this toxin to probe the role of the trans-hydroxyproline (Hyp), which is known to be critical for toxicity. This structure activity relationship (SAR) study represents the first of its kind to use various Hyp-analogs to alter the conformational and H-bonding properties of Hyp in amanitin.

Scaling Amatoxin Synthesis with an Improved Route to (2S,3R,4R)-Dihydroxyisoleucine Exemplified by a Toxic, Clickable α-Amanitin Analogue.

Here we report a scalable synthesis of the key amino acid residue, (2S,3R,4R)-4,5-dihydroxyisoleucine (DHIle) in α-amanitin, that in turn enables the scalable synthesis of an equipotent analogue, Asn(N-ethylazide)-S,6'-dideoxy-α-amanitin, suitable for CuAAC conjugation to empower studies on therapeutic antibody-drug conjugates.

Rapid, High-Yielding Solid-Phase Synthesis of Cathepsin-B Cleavable Linkers for Targeted Cancer Therapeutics.

Antibody-drug conjugates (ADCs) constitute an emerging class of anticancer agents that deliver potent payloads selectively to tumors while avoiding systemic toxicity associated with conventional chemotherapeutics. Critical to ADC development is a serum-stable linker designed to decompose inside targeted cells thereby releasing the toxic payload. A protease-cleavable linker comprising a valine-citrulline (Val-Cit) motif has been successfully incorporated into three FDA-approved ADCs and is found in numerous preclinical candidates. Herein, we present a high-yielding and facile synthetic strategy for a Val-Cit linker that avoids extensive protecting group manipulation and laborious chromatography associated with previous syntheses and provides yields that are up to 10-fold higher than by standard methods. This method is easily scalable and takes advantage of cost-effective coupling reagents and high loading 2-chlorotrityl chloride (2-CTC) resin. Modularity allows for introduction of various conjugation handles in final stages of the synthesis. Facile access to such analogues serves to expand the repertoire of available enzymatically cleavable linkers for ADC generation. This methodology empowers a robust and facile library generation and future exploration into linker analogues containing unnatural amino acids as a selectivity tuning tool.

A hypothesis for examining dihydroxyacetone, the active component in sunless tanning products, as a topical prophylactic against SARS-COV-2 transmission.

This hypothesis raises the interesting prospect that dihydroxyacetone (DHA), the key ingredient in self-tanning creams, when applied daily to the face and hands may have prophylactic action against SARS-COV-2 transmission and infection. The scientific and mechanistic basis for this hypothesis is elaborated based on our understanding of the chemical reactivity of DHA with proteins to afford advanced glycation products. This piece ends with a proposal for doing key experiments that can be run to test this hypothesis. As more than 30 million people have been infected with this disease world-wide, a safe method for stopping spread is worthy of consideration. Publication of this hypothesis would enable the scientific community at large to test this in a clinically meaningful setting to address the potential for DHA-based prophylaxis. Given the calamity of this crisis, it is anticipated that the publication of this hypothesis, which is supported by key studies on protein and nucleoside glycation, can be disseminated to as many researchers as possible.

FlICk (fluorescent isoindole crosslinking) for peptide stapling.

The rigidification of peptide secondary structure via stapling is an important and enduring goal in the development of functional peptides for biochemical and pharmaceutical applications. In addition, the incorporation of fluorophores and chromophores has been a sought-after application for creating peptidic probes of cellular function and localization. The combined application of peptide stapling and fluorescent-readout is featured by the reaction of ortho-phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into fluorescent or visibly colored monocyclic and bicyclic products with noted biological activity. Given its user-friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry and we have featured this application on an array of high-affinity macrocyclic α-MSH derivatives as well as for late-stage intra-annular isoindole stapling furnished a bicyclic peptide mimic of α-amanitin that is cytotoxic to CHO cells. The synthetic methods for preparing substituted ortho-phthalaldehydes along with subsequent applications to FlICk stapling are detailed herein.

Pharmacokinetics, radiation dosimetry, acute toxicity and automated synthesis of [18F]AmBF3-TATE.

INTRODUCTION: [18F]AmBF3-TATE is a somatostatin agonist that selectively binds to somatostatin receptor subtype 2 (SSTR2). For clinical translation, pharmacokinetics, radiation dosimetry, and acute toxicity of [18F]AmBF3-TATE were assessed with good laboratory practice (GLP) standards. METHODS: ICR mice were intravenously administered 0.8-2.0 MBq of [18F]AmBF3-TATE, with one group pre-injected with 100 µg of [19F]AmBF3-TATE 30 min before radiopharmaceutical administration to assess uptake specificity. The mice were euthanized at 0.5, 1, 2, or 4 h post-injection (p.i.). Blood and tissues were collected, weighed, and counted on a gamma counter to determine percentage injected dose per gram (%ID/g). Dosimetry was calculated based on biodistribution data using the mouse and human phantoms included in OLINDA. Acute toxicity was assessed in Sprague-Dawley rats at the dose of 0.742 mg/kg [19F]AmBF3-TATE, with a 14-day observation/recovery period. Blood chemistry parameters, gross, and histopathology were evaluated. Body weight change and food consumption were monitored. The production of [18F]AmBF3-TATE was automated on a Trasis AllinOne synthesis module. RESULTS: [18F]AmBF3-TATE was cleared through the renal and hepatobiliary pathway. At 1 h p.i., the pancreas (F, 15.7 ± 3.72 and M 14.3 ± 1.61 %ID/g), stomach (F, 15.3 ± 3.63 and M, 19.0 ± 3.49 %ID/g), and lungs (F, 9.26 ± 2.24 and M, 6.17 ± 6.04 %ID/g) were the organs with the highest specific uptake. Pre-injection with [19F]AmBF3-TATE significantly reduced pancreatic uptake (F, 0.13 ± 0.03 and M, 0.18 ± 0.09 %ID/g) at 1 h p.i. For dosimetry extrapolated to the average adult human, the bladder (0.027-0.030 mGy/MBq), pancreas (0.018-0.028 mGy/MBq), and lungs (0.006-0.013 mGy/MBq) are expected to receive the highest doses. No test-item related effects were observed upon evaluation of clinical observations, body weights, food consumption, clinical pathology, gross pathology, and histopathology for acute toxicity. [18F]AmBF3-TATE was produced at activity yields of 15.6 ± 4.59 GBq, average molar activity of 435 ± 162 GBq/µmol, and radiochemical purity of 98.0 ± 1.73% with the automated synthesizer. CONCLUSION: [18F]AmBF3-TATE binds specifically to SSTR2. At 1000× clinical dose, [19F]AmBF3-TATE was well tolerated with no treatment-related adverse effects.

Toward 18 F-Labeled Theranostics: A Single Agent that Can Be Labeled with 18 F, 64 Cu, or 177 Lu.

We report a single-molecule radiotracer that can be labeled independently with 18 F-fluoride or radiometals (64 Cu, 177 Lu) in a single step. A prostate-specific membrane antigen (PSMA)-targeting ligand, armed with both an organotrifluoroborate and a metal-chelator (DOTA), was designed to optionally afford 18 F-, 64 Cu- or 177 Lu-labeled products that were injected into mice bearing prostate cancer (LNCaP) xenografts. PET/CT images and ex vivo biodistribution data show high, specific tumor uptake irrespective of which radionuclide is used, thereby demonstrating a new approach to combining, in a single molecule, 18 F-labeling capabilities for PET imaging with radiometalation for potential imaging and therapeutic applications.

Synthesis and 18F-radiolabeling of thymidine AMBF3 conjugates.

In pursuit of 18F-labeled nucleosides for positron emission tomography (PET) imaging, we report on the chemical and radiochemical synthesis of two thymidine (dT) analogs, dT-C5-AMBF3 and dT-N3-AMBF3, that are radiofluorinated by isotope exchange (IEX) and studied as PET imaging agents in mice with tumor xenografts. dT-C5-AMBF3 shows preferential, and tumor-specific, uptake over dT-N3-AMBF3. This work provides a new synthetic method in order to access new nucleoside tracers for PET imaging.

Meeting key synthetic challenges in amanitin synthesis with a new cytotoxic analog: 5'-hydroxy-6'-deoxy-amanitin.

Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5'-hydroxy-6'-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of α-amanitin, that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide.