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.

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.

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.

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.

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.

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.

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.

18F-Labeled Cyclized α-Melanocyte-Stimulating Hormone Derivatives for Imaging Human Melanoma Xenograft with Positron Emission Tomography.

Since metastatic melanoma is deadly, early diagnosis thereof is crucial for managing the disease. We recently developed α-melanocyte-stimulating hormone (αMSH) derivatives, [68Ga]Ga-CCZ01048 and [18F]CCZ01064, that target the melanocortin 1 receptor (MC1R) for mouse melanoma imaging. In this study, we aim to evaluate [18F]CCZ01064 as well as a novel dual-ammoniomethyl-trifluoroborate (AmBF3) derivative, [18F]CCZ01096, for targeting human melanoma xenograft using µPET imaging. The peptides were synthesized on solid phase using Fmoc chemistry. Radiolabeling was achieved in a one-step 18F-19F isotope-exchange reaction. µPET imaging and biodistribution studies were performed in NSG mice bearing SK-MEL-1 melanoma xenografts. The MC1R density on the SK-MEL-1 cell line was determined to be 972 ± 154 receptors/cell (n = 4) via saturation assays. Using [18F]CCZ01064, moderate tumor uptake (3.05 ± 0.47%ID/g) and image contrast were observed at 2 h post-injection. Molar activity was determined to play a key role. CCZ01096 with two AmBF3 motifs showed comparable sub-nanomolar binding affinity to MC1R and much higher molar activity. This resulted in improved tumor uptake (6.46 ± 1.42%ID/g) and image contrast (tumor-to-blood and tumor-to-muscle ratios were 30.6 ± 5.7 and 85.7 ± 11.3, respectively) at 2 h post-injection. [18F]CCZ01096 represents a promising αMSH-based µPET imaging agent for human melanoma and warrants further investigation for potential clinical translation.

A Metal-Free DOTA-Conjugated 18F-Labeled Radiotracer: [18F]DOTA-AMBF3-LLP2A for Imaging VLA-4 Over-Expression in Murine Melanoma with Improved Tumor Uptake and Greatly Enhanced Renal Clearance.

DOTA is commonly used for radiometal chelation in molecular imaging. Yet in the absence of a radiometal, DOTA is hypothesized to promote renal clearance of 18F-labeled peptide tracers. In light of an increasing interest in the use of F18 for PET, here the effect of DOTA is evaluated for the first time with an 18F-labeled tracer and is found to significantly improve the quality of images acquired through positron emission tomography (PET). We chose to image the peptide LLP2A that recognizes the transmembrane protein very-late antigen 4 (VLA-4) that is overexpressed by many cancers. Since it is known that [18F]RBF3-PEG2-LLP2A derivatives gave low tumor uptake values and significant GI tract accumulation, this ligand thus represents an ideal means of testing the additive effects of a DOTA group on clearance while permitting a facile, user-friendly, one-step 18F-labeling. A newly designed RBF3-LLP2A bioconjugate with an appended DOTA moiety increased tumor uptake nearly 3-fold and reduced GI accumulation by more than 10-fold. The DOTA-AMBF3-PEG2-LLP2A was radiolabeled by isotope exchange and was purified by semiprep HPLC and C18 cartridge elution. Male C57BL/6J mice bearing B16-F10 melanoma tumors that overexpress the VLA-4 target were used to evaluate [18F]DOTA-AMBF3-PEG2-LLP2A using a combination of static and dynamic PET scans, biodistribution studies, and blocking controls at 1 h post injection (p.i.). The precursor peptide was synthesized and 18F-labeled to provide formulations with mean (±SD) radiochemical purities of 95.9 ± 1.8%, in radiochemical yields of 4.8 ± 2.9% having molar activities of 131.7 ± 50.3 GBq/µmol. In vivo static PET images of [18F]DOTA-AMBF3-PEG2-LLP2A provided clear tumor visualization, and biodistribution studies showed that tumor uptake was 9.46 ± 2.19% injected dose per gram of tissue (%ID/g) with high tumor/muscle and tumor/blood contrast ratios of ∼8 and ∼10, respectively. Blocking confirmed the specificity of [18F]DOTA-AMBF3-PEG2-LLP2A to VLA-4 in the tumor and the bone marrow. Dynamic PET showed clearance of [18F]DOTA-AMBF3-PEG2-LLP2A mainly via the renal pathway, wherein accumulation in the intestines was reduced 10-fold compared to our previously investigated LLP2A's, while spleen uptake was at levels similar to previously reported LLP2A-chelator radiotracers. [18F]DOTA-AMBF3-PEG2-LLP2A represents a promising VLA-4 radiotracer and provides key evidence as to how a DOTA appendage can significantly reduce GI uptake in favor of urinary excretion. Implications for the development of dual-isotope theranostics that exploit the use fluorine-18 for imaging and DOTA to chelate therapeutic metal cations for therapy are discussed.

One-Step 18F-Labeling and Preclinical Evaluation of Prostate-Specific Membrane Antigen Trifluoroborate Probes for Cancer Imaging.

After the identification of the high-affinity glutamate-ureido scaffold, the design of several potent 18F- and 68Ga-labeled tracers has allowed spectacular progress in imaging recurrent prostate cancer by targeting the prostate-specific membrane antigen (PSMA). We evaluated a series of PSMA-targeting probes that are 18F-labeled in a single step for PET imaging of prostate cancer. Methods: We prepared 8 trifluoroborate constructs for prostate cancer imaging, to study the influence of the linker and the trifluoroborate prosthetic on pharmacokinetics and image quality. After 1-step labeling by 19F-18F isotopic exchange, the radiotracers were injected in mice bearing LNCaP xenografts, with or without blocking controls, to assess specific uptake. PET/CT images and biodistribution data were acquired at 1 h after injection and compared with 18F-DCFPyL on the same mouse strain and tumor model. Results: All tracers exhibited nanomolar affinities, were labeled in good radiochemical yields at high molar activities, and exhibited high tumor uptake in LNCaP xenografts with clearance from nontarget organs. Most derivatives with a naphthylalanine linker showed significant gastrointestinal excretion. A radiotracer incorporating this linker with a dual trifluoroborate-glutamate labeling moiety showed high tumor uptake, low background activity, and no liver or gastrointestinal track accumulation. Conclusion: PSMA-targeting probes with trifluoroborate prosthetic groups represent promising candidates for prostate cancer imaging because of facile labeling while affording high tumor uptake values and contrast ratios that are similar to those obtained with 18F-DCFPyL.

Melanoma Imaging Using 18F-Labeled α-Melanocyte-Stimulating Hormone Derivatives with Positron Emission Tomography.

Melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, a leading cause of death related to skin cancers. Accurate staging and early detection is crucial in managing melanoma. Based on the α-melanocyte-stimulating hormone (αMSH) sequence, MC1R-targeted peptides have been studied for melanoma imaging, predominately for use with single-photon emission computed tomography, with few attempts made for positron emission tomography (PET). 18F is a commonly used PET isotope due to readily available cyclotron production, pure positron emission, and a favorable half-life (109.8 min). In this study, we aim to design and evaluate αMSH derivatives that enable radiolabeling with 18F for PET imaging of melanoma. We synthesized three imaging probes based on the structure of Nle4-cyclo[Asp5-His-d-Phe7-Arg-Trp-Lys10]-NH2 (Nle-CycMSHhex), with a Pip linker (CCZ01064), an Acp linker (CCZ01070), or an Aoc linker (CCZ01071). 18F labeling was enabled by an ammoniomethyl-trifluoroborate (AmBF3) moiety. In vitro competition binding assays showed subnanomolar inhibition constant ( Ki) values for all three peptides. The 18F radiolabeling was performed via a one-step 18F-19F isotope exchange reaction that resulted in high radiochemical purity (>95%) and good molar activity (specific activity) ranging from 40.7 to 66.6 MBq/nmol. All three 18F-labeled peptides produced excellent tumor visualization with PET imaging in C57BL/6J mice bearing B16-F10 tumors. The tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38, and 5.46 ± 2.64% injected dose per gram of tissue (%ID/g) for [18F]CCZ01064, [18F]CCZ01070, and [18F]CCZ01071 at 1 h post-injection (p.i.), respectively. Minimal background activity was observed except for kidneys at 4.99 ± 0.20, 4.42 ± 0.54, and 13.55 ± 2.84%ID/g, respectively. The best candidate [18F]CCZ01064 was further evaluated at 2 h p.i., which showed increased tumor uptake at 11.96 ± 2.31%ID/g and further reduced normal tissue uptake. Moreover, a blocking study was performed for CCZ01064 at 1 h p.i., where tumor uptake was significantly reduced to 1.97 ± 0.60%ID/g, suggesting the tumor uptake was receptor mediated. In conclusion, [18F]CCZ01064 showed high tumor uptake, low normal tissue uptake, and fast clearance and is therefore a suitable and promising candidate for PET imaging of melanoma.

Organotrifluoroborates as prosthetic groups for Single-Step F18-Labeling of Complex Molecules.

To meet the long-standing challenge of a one-step, last-stage aqueous radiofluorination method for labeling large, water-soluble molecules such as peptides, we sought a mild aqueous fluorination reaction that uses organoboron compositions, which are readily fluorinated in mild aqueous acid to give 18F-labeled organotrifluoroborates. This robust reaction now provides new 18F-labeled radioprosthetic groups that can be pre-conjugated to peptides and other polar molecules, and then labeled with aqueous no-carrier-added (NCA) [18F]fluoride ion in a single step. A key application facilitated by this method is the production of 18F-labeled fluorescent-PET tracers. The development of these radioprosthetics, their elaboration onto peptides, and their extension to fluorescent bioconjugates is the focus of this review.

Preliminary evaluation of 18F-labeled LLP2A-trifluoroborate conjugates as VLA-4 (α4ß1 integrin) specific radiotracers for PET imaging of melanoma.

INTRODUCTION: The transmembrane α4ß1 integrin receptor, or very-late antigen 4 (VLA-4), is associated with tumor metastasis and angiogenesis, the development of chemotherapeutic drug resistance, and is overexpressed in multiple myelomas, osteosarcomas, lymphomas, leukemias, and melanomas. The peptidomimetic, LLP2A, is a high-affinity ligand with specificity for the extracellular portion of VLA-4 and several conjugates have been evaluated in vivo by NIR-fluorescence, 111In-SPECT and 68Ga- and 64Cu-PET imaging, but to date, not with 18F-PET. METHODS: Using two highly stable organotrifluoroborate prosthetic groups: ammoniumdimethyl-trifluoroborate (AMBF3) and a new N-pyridinyl-para-trifluoroborate (N-Pyr-p-BF3), both capable of facile aqueous 18F-labeling by isotope exchange (IEX), we present the first PET imaging evaluations of two [18F]R-BF3--PEG2-LLP2A tracers using VLA-4 overexpressing B16-F10 murine melanoma tumor mouse models. RESULTS: Here, we demonstrate successful one-step 18F-labeling of both conjugates with wet NCA [18F]F- in radiochemical yields of up to 11.6% within 75 min at molar activities of 40-100 GBq/µmol. Average tumor uptake values based on ex vivo biodistribution values were 4.4%ID/g (11) and 2.8%ID/g (12) using 18F-labeled LLP2A-conjugates with the two prosthetic groups: N-Pyr-p-BF3 (5) and alkyl-N,N-dimethylammonio-BF3 (AMBF3) (7), respectively, and was found to be target-specific as evidenced by in vivo blocking controls. Dynamic PET scanning and biodistribution studies revealed slow clearance of the [18F]R-BF3--PEG2-LLP2A tracers from the tumors, and also substantial uptake in the intestines, gall bladder, liver and bladder. Observed bone uptake was blockable, consistent with known VLA-4 expression in hematopoietic stem cells found in bone marrow. CONCLUSIONS: These studies show that these [18F]R-BF3--PEG2-LLP2A conjugates (11 and 12) are promising VLA-4 targeting radiotracers, yet, further optimization will be required to reduce uptake in the gastro-intestinal tract.

Systematic study of constraints imposed by modified nucleoside triphosphates with protein-like side chains for use in in vitro selection.

Successful selection of modified DNAzymes depends on the potential for modified nucleoside triphosphates (dNTPs) to replace their unmodified counterparts in enzyme catalyzed primer extension reactions and, once incorporated, to serve as template bases for information transfer prior to PCR amplification. To date, the most densely modified DNAzymes have been selected from three modified dNTPs: 8-histaminyl-deoxyadenosine (dAimTP), 5-guanidinoallyl-deoxyuridine (dUgaTP), and 5-aminoallyl-deoxycytidine (dCaaTP) to provide several RNA-cleaving DNAzymes with greatly enhanced rate constants compared to unmodified counterparts. Here we report biophysical and enzymatic properties of these three modified nucleosides in the context of specific oligonucleotide sequences to understand how these three modified nucleobases function in combinatorial selection. The base-pairing abilities of oligonucleotides bearing one or three modified nucleosides were investigated by thermal denaturation studies and as templates for enzymatic polymerization with both modified and unmodified dNTPs. While we address certain shortcomings in the use of modified dNTPs, we also provide key evidence of faithful incorporation and enzymatic read-out, which strongly supports their continued use in in vitro selection.

Efficient oxidation of N-protected tryptophan and tryptophanyl-dipeptides by in situ generated dimethyldioxirane provides hexahydropyrroloindoline-containing synthons suitable for peptide synthesis and subsequent tryptathionylation.

A series of hydroxypyrroloindoline (Hpi) containing dipeptides along with the corresponding monomeric Hpi-α-amino acid (Hpi-2-carboxylate), were prepared by reacting a series of N α-protected-tryptophans in aqueous or biphasic [water/cyclopentyl methyl ether (CPME)] solutions containing Oxone® (potassium peroxymonosulfate) and acetone. This procedure avoids the tedious distillation of unstable dimethyldioxirane (DMDO), which is commonly used to oxidize indoles. Monomers N α-Boc-Hpi-OH and N α-Fmoc-Hpi-OH were readily incorporated by solid-phase peptide synthesis (SPPS) into a peptide containing a cysteine; in trifluoroacetic acid (TFA), the Hpi underwent intramolecular dehydrative condensation with the cysteine thiol to afford the anticipated tryptathionine crosslink. This eco- and user-friendly oxidative methodology greatly simplifies the synthesis of Hpi derivatives while enabling the synthesis of tryptathionine crosslinks characteristic of phalloidin and amanitin, two potent peptide toxins of present interest.

Facile synthesis and 18F-radiolabeling of α4ß1-specific LLP2A-aryltrifluoroborate peptidomimetic conjugates.

The peptidomimetic, LLP2A, is a specific, high-affinity ligand for α4ß1 integrin receptors. Previously, several PEGylated LLP2A conjugates were evaluated in vivo as imaging agents for the detection of lymphoma, leukemia, multiple myeloma and melanoma tumours via NIR-fluorescence, 111In-SPECT, and 64Cu- and 68Ga-PET imaging. Despite these successes, to date there is no report of an 18F-labeled LLP2A conjugate. Notably, fluorine-18 is a preferred radionuclide for PET imaging, yet its short half-life and general inactivity under aqueous conditions present challenges for peptide labeling. A simple method for labeling complex biomolecules can be achieved with arylboronic acids that readily capture aqueous [18F]-fluoride ion resulting in an 18F-labeled aryltrifluoroborate ([18F]-ArBF3-) radioprosthetic group. Herein, we present the first radiosynthesis of an 18F-labeled LLP2A conjugate by both one-step 18F-labeling and one-pot two-step 18F-labeling post-'click' conjugation of the 18F-alkynyl-ArBF3- prosthetic. Competition with a fluorescent conjugate of LLP2A demonstrated specific binding of the non-radioactive isotopolog ArBF3--PEG2-LLP2A to α4ß1 integrin-expressing MOLT-4 leukemia cells, as evidenced and confirmed by fluorescence microscopy. This work provides a key first step in the development of an expanding library of [18F]-R-BF3--LLP2A radiotracers for PET imaging.