Synthesis and Evaluation of a Monomethyl Auristatin E─Integrin αvß6 Binding Peptide-Drug Conjugate for Tumor Targeted Drug Delivery.

Many anticancer drugs exhibit high systemic off-target toxicities causing severe side effects. Peptide-drug conjugates (PDCs) that target tumor-specific receptors such as integrin αvß6 are emerging as powerful tools to overcome these challenges. The development of an integrin αvß6-selective PDC was achieved by combining the therapeutic efficacy of the cytotoxic drug monomethyl auristatin E with the selectivity of the αvß6-binding peptide (αvß6-BP) and with the ability of positron emission tomography (PET) imaging by copper-64. The [64Cu]PDC-1 was produced efficiently and in high purity. The PDC exhibited high human serum stability, integrin αvß6-selective internalization, cell binding, and cytotoxicity. Integrin αvß6-selective tumor accumulation of the [64Cu]PDC-1 was visualized with PET-imaging and corroborated by biodistribution, and [64Cu]PDC-1 showed promising in vivo pharmacokinetics. The [natCu]PDC-1 treatment resulted in prolonged survival of mice bearing αvß6 (+) tumors (median survival: 77 days, vs αvß6 (-) tumor group 49 days, and all other control groups 37 days).

Preclinical Evaluation of 68Ga- and 177Lu-Labeled Integrin αvß6-Targeting Radiotheranostic Peptides.

The integrin αvß6, an epithelium-specific cell surface receptor, is overexpressed on numerous malignancies, including the highly lethal pancreatic ductal adenocarcinomas. Here, we developed and tested a novel αvß6-targeting peptide, DOTA-5G (1) radiolabeled with 68Ga, for PET/CT imaging and 177Lu for treatment. With the goal to develop a radiotheranostic, further modifications were made for increased circulation time, renal recycling, and tumor uptake, yielding DOTA-albumin-binding moiety-5G (2). Methods: Peptides 1 and 2 were synthesized on solid phase, and their affinity for αvß6 was assessed by enzyme-linked immunosorbent assay. The peptides were radiolabeled with 68Ga and 177Lu. In vitro cell binding, internalization, and efflux of 68Ga-1 and 177Lu-2 were evaluated in αvß6-positive BxPC-3 human pancreatic cancer cells. PET/CT imaging of 68Ga-1 and 68Ga-2 was performed on female nu/nu mice bearing subcutaneous BxPC-3 tumors. Biodistribution was performed for 68Ga-1 (1 and 2 h after injection), 68Ga-2 (2 and 4 h after injection), and 177Lu-1 and 177Lu-2 (1, 24, 48, and 72 h after injection). The 177Lu-2 biodistribution data were extrapolated for human dosimetry data estimates using OLINDA/EXM 1.1. Therapeutic efficacy of 177Lu-2 was evaluated in mice bearing BxPC-3 tumors. Results: Peptides 1 and 2 demonstrated high affinity (<55 nM) for αvß6 by enzyme-linked immunosorbent assay. 68Ga-1, 68Ga-2, 177Lu-1, and 177Lu-2 were synthesized in high radiochemical purity. Rapid in vitro binding and internalization of 68Ga-1 and 177Lu-2 were observed in BxPC-3 cells. PET/CT imaging and biodistribution studies demonstrated uptake in BxPC-3 tumors. Introduction of the albumin-binding moiety in 177Lu-2 resulted in a 5-fold increase in tumor uptake and retention over time. Based on the extended dosimetry data, the dose-limiting organ for 177Lu-2 is the kidney. Treatment with 177Lu-2 prolonged median survival by 1.5- to 2-fold versus controls. Conclusion: 68Ga-1 and 177Lu-2 demonstrated high affinity for the integrin αvß6 both in vitro and in vivo, were rapidly internalized into BxPC-3 cells, and were stable in mouse and human serum. Both radiotracers showed favorable pharmacokinetics in preclinical studies, with predominantly renal excretion and good tumor-to-normal-tissue ratios. Favorable human dosimetry data suggest the potential of 177Lu-2 as a treatment for pancreatic ductal adenocarcinoma.

Primary Hemiarthroplasty for the Treatment of Basicervical Femoral Neck Fractures.

Purpose Basicervical femoral neck fractures are uncommon injuries that occur at the extracapsular base of the femoral neck at its transition with the intertrochanteric line. Controversy remains in the orthopedic literature as to the optimal method of treatment for this fracture type given the inherent instability and greater rate of implant failure with traditional fixation constructs. The purpose of this study is to quantify the incidence and preferred treatment methods of basicervical hip fractures at a single, regional, Level 1 trauma center and to identify differences in postoperative complications between treatment options. Methods The present study is a retrospective case series from a single regional health network, including 316 patients with hip fractures. Basicervical femoral neck fractures were identified. Reoperation rates within 90 days, implant failures or nonunions, postoperative ambulation distances and range of motion, and discharge dispositions were compared across patients grouped by surgical treatment with either cephalomedullary nail, sliding hip screw, or hemiarthroplasty (HA). Results Basicervical femoral neck fractures represented 6.6% of this study population. The cephalomedullary nail group demonstrated rates of implant failure and return to the operating room within 90 days of 40% (4/10) and 20% (2/10), respectively. No patients who underwent hemiarthroplasty experienced a failure of fixation or return to the operating room. Conclusions This study suggests a much lower rate of fixation failure or need for reoperation with hemiarthroplasty treatment compared to cephalomedullary nail construct for basicervical femoral neck fractures and may be an underutilized treatment method for this fracture type. The promising results seen with this case series should encourage further investigation into HA as a primary treatment for these uncommon, yet challenging, fractures.

A Comparison of Evans Blue and 4-(p-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αvß6 Binding Peptide.

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when conjugated to an integrin αvß6 binding peptide (αvß6-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified αvß6-BP peptides were synthesized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) chelator for radiolabeling with copper-64 to yield [64Cu]Cu DOTA-EB-αvß6-BP ([64Cu]1) and [64Cu]Cu DOTA-IP-αvß6-BP ([64Cu]2). Both peptides were evaluated in vitro for serum albumin binding, serum stability, and cell binding and internalization in the paired engineered melanoma cells DX3puroß6 (αvß6 +) and DX3puro (αvß6 −), and pancreatic BxPC-3 (αvß6 +) cells and in vivo in a BxPC-3 xenograft mouse model. Serum albumin binding for [64Cu]1 and [64Cu]2 was 53−63% and 42−44%, respectively, with good human serum stability (24 h: [64Cu]1 76%, [64Cu]2 90%). Selective αvß6 cell binding was observed for both [64Cu]1 and [64Cu]2 (αvß6 (+) cells: 30.3−55.8% and 48.5−60.2%, respectively, vs. αvß6 (−) cells <3.1% for both). In vivo BxPC-3 tumor uptake for both peptides at 4 h was 5.29 ± 0.59 and 7.60 ± 0.43% ID/g ([64Cu]1 and [64Cu]2, respectively), and remained at 3.32 ± 0.46 and 4.91 ± 1.19% ID/g, respectively, at 72 h, representing a >3-fold improvement over the non-ABM parent peptide and thereby providing improved PET images. Comparing [64Cu]1 and [64Cu]2, the IP-ABM-αvß6-BP [64Cu]2 displayed higher serum stability, higher tumor accumulation, and lower kidney and liver accumulation, resulting in better tumor-to-organ ratios for high contrast visualization of the αvß6 (+) tumor by PET imaging.

Repurposing an atherosclerosis targeting peptide for tumor imaging.

Cancer and atherosclerosis are chronic diseases that share common characteristics at both early and advanced stages and can arise from multiple factors. Both diseases are characterized by uncontrolled cell proliferation, inflammation, angiogenesis and apoptosis. Herein we investigated the ability of a peptide (CTHRSSVVC), that was previously reported to bind atherosclerotic lesions to home in the tumor microenvironment. The CTHRSSVVC peptide was synthesized on solid phase and N-terminally labeled with a sulfo-Cy5 dye. The specific binding to macrophage was evaluated in vitro with flow cytometry and immunofluorescence and in vivo for tumor targeting in BALB/c mice bearing a 4T1 tumor using optical imaging. The sulfo-Cy5-CTHRSSVVC peptide was synthesized in greater than 99% purity. No selective binding of the sulfo-Cy5-CTHRSSVVC peptide to macrophages in vitro was observed, however in vivo the sulfo-Cy5-CTHRSSVVC peptide accumulated in the 4T1 tumor, with a tumor-to-normal tissue ratio of 7.21 ± 1.44 at 2 h post injection. Ex vivo analysis of tumor tissue by confocal microscopy suggested that the sulfo-Cy5-CTHRSSVVC peptide had accumulated in the stroma of the tumor specifically, in regions of spindle shaped cells. In conclusion, although the target for the sulfo-Cy5-CTHRSSVVC peptide remains to be identified, the Cy5-CTHRSSVVC peptide warrants further investigation as a tumor imaging agent.

Evaluation of Copper-64-Labeled αvß6-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics.

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [18F]AlF NOTA-K(ABM)-αvß6-BP, where αvß6-BP is a peptide with high affinity for the cell surface receptor integrin αvß6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [18F]AlF NOTA-K(ABM)-αvß6-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αvß6-targeted uptake with continuously improving detection of αvß6(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (t1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αvß6-BP with copper-64 (t1/2 = 12.7 h). Two peptides were synthesized without (1) and with (2) the ABM and radiolabeled with copper-64 to yield [64Cu]1 and [64Cu]2, respectively. The affinity of [natCu]1 and [natCu]2 for the integrin αvß6 was assessed by enzyme-linked immunosorbent assay. [64Cu]1 and [64Cu]2 were evaluated in vitro (cell binding and internalization) using DX3puroß6 (αvß6(+)), DX3puro (αvß6(-)), and pancreatic BxPC-3 (αvß6(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroß6/DX3puro or BxPC-3 xenograft tumors. [64Cu]1 and [64Cu]2 were synthesized in ≥97% radiochemical purity. In vitro, [natCu]1 and [natCu]2 maintained low nanomolar affinity for integrin αvß6 (IC50 = 28 ± 3 and 19 ± 5 nM, respectively); [64Cu]1 and [64Cu]2 showed comparable binding to αvß6(+) cells (DX3puroß6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αvß6(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [64Cu]2 showed a 6-fold higher binding to human serum protein than [64Cu]1. In vivo, selective uptake in the αvß6(+) tumors was observed with tumor visualization up to 72 h for [64Cu]2. A 3-5-fold higher αvß6(+) tumor uptake of [64Cu]2 vs [64Cu]1 was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [64Cu]2 at 48 h. Incorporation of an iodophenyl-based ABM into the αvß6-BP ([64Cu]2) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αvß6(+) tumors that enabled visualization of αvß6(+) tumors up to 72 h by PET/CT imaging.

αvß6-Targeted Molecular PET/CT Imaging of the Lungs After SARS-CoV-2 Infection.

The true impact and long-term damage to organs such as the lungs after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain to be determined. Noninvasive molecularly targeted imaging may play a critical role in aiding visualization and understanding of the systemic damage. We have identified αvß6 as a molecular target; an epithelium-specific cell surface receptor that is low or undetectable in healthy adult epithelium but upregulated in select injured tissues, including fibrotic lung. Herein we report the first human PET/CT images using the integrin αvß6-binding peptide (18F-αvß6-BP) in a patient 2 mo after the acute phase of infection. Minimal uptake of 18F-αvß6-BP was noted in normal lung parenchyma, with uptake being elevated in areas corresponding to opacities on CT. This case suggests that 18F-αvß6-BP PET/CT is a promising noninvasive approach to identify the presence and potentially monitor the persistence and progression of lung damage.

The Effects of an Albumin Binding Moiety on the Targeting and Pharmacokinetics of an Integrin αvß6-Selective Peptide Labeled with Aluminum [18F]Fluoride.

PURPOSE: The αvß6-BP peptide selectively targets the integrin αvß6, a cell surface receptor recognized as a prognostic indicator for several challenging malignancies. Given that the 4-[18F]fluorobenzoyl (FBA)-labeled peptide is a promising PET imaging agent, radiolabeling via aluminum [18F]fluoride chelation and introduction of an albumin binding moiety (ABM) have the potential to considerably simplify radiochemistry and improve the pharmacokinetics by increasing biological half-life. PROCEDURES: The peptides NOTA-αvß6-BP (1) and NOTA-K(ABM)-αvß6-BP (2) were synthesized on solid phase, radiolabeled with aluminum [18F]fluoride, and evaluated in vitro (integrin ELISA, albumin binding, cell studies) and in vivo in mouse models bearing paired DX3puroß6 [αvß6(+)]/DX3puro [αvß6(-)], and for [18F]AlF 2, BxPC-3 [αvß6(+)] cell xenografts (PET imaging, biodistribution). RESULTS: The peptides were radiolabeled in 23.0 ± 5.7 % and 22.1 ± 4.4 % decay-corrected radiochemical yield, respectively, for [18F]AlF 1 and [18F]AlF 2. Both demonstrated excellent affinity and selectivity for integrin αvß6 by ELISA (IC50(αvß6) = 3-7 nM vs IC50(αvß3) > 10 µM) and in cell binding studies (51.0 ± 0.7 % and 47.2 ± 0.7 % of total radioactivity bound to DX3puroß6 cells at 1 h, respectively, vs. ≤ 1.2 % to DX3puro for both compounds). The radiotracer [18F]AlF 1 bound to human serum at 16.3 ± 1.9 %, compared to 67.5 ± 1.0 % for the ABM-containing [18F]AlF 2. In vivo studies confirmed the effect of the ABM on blood circulation (≤ 0.1 % ID/g remaining in blood for [18F]AlF 1 as soon as 1 h p.i. vs. > 2 % ID/g for [18F]AlF 2 at 6 h p.i.) and higher αvß6(+) tumor uptake (4 h: DX3puroß6; [18F]AlF 1: 3.0 ± 0.7 % ID/g, [18F]AlF 2: 7.2 ± 0.7 % ID/g; BxPC-3; [18F]AlF 2: 10.2 ± 0.1 % ID/g). CONCLUSION: Both compounds were prepared using standard chemistries; affinity and selectivity for integrin αvß6 in vitro remained unaffected by the albumin binding moiety. In vivo, the albumin binding moiety resulted in prolonged circulation and higher αvß6-targeted uptake.

Identification, Characterization, and Optimization of Integrin αvß6-Targeting Peptides from a One-Bead One-Compound (OBOC) Library: Towards the Development of Positron Emission Tomography (PET) Imaging Agents.

The current translation of peptides identified through the one-bead one-compound (OBOC) technology into positron emission tomography (PET) imaging agents is a slow process, with a major delay between ligand identification and subsequent lead optimization. This work aims to streamline the development process of 18F-peptide based PET imaging agents to target the integrin αvß6. By directly identify αvß6⁻targeting peptides from a 9-mer 4-fluorobenzoyl peptide library using the on-bead two-color (OBTC) cell-screening assay, a total of 185 peptide beads were identified and 5 beads sequenced for further evaluation. The lead peptide 1 (VGDLTYLKK(FB), IC50 = 0.45 ± 0.06 µM, 25% stable in serum at 1 h) was further modified at the N-, C-, and bi-termini. C-terminal PEGylation increased the metabolic stability (>95% stable), but decreased binding affinity (IC50 = 3.7 ± 1 µM) was noted. C-terminal extension (1i, VGDLTYLKK(FB)KVART) significantly increased binding affinity for integrin αvß6 (IC50 = 0.021 ± 0.002 µM), binding selectivity for αvß6-expressing cells (3.1 ± 0.8:1), and the serum stability (>99% stable). Our results demonstrate the challenges in optimizing OBOC-derived peptides, indicate both termini of 1 are sensitive to modifications, and show that further modification of 1 is necessary to demonstrate utility as an 18F-peptide imaging agent.

Fully automated peptide radiolabeling from [18F]fluoride.

The biological properties of receptor-targeted peptides have made them popular diagnostic imaging and therapeutic agents. Typically, the synthesis of fluorine-18 radiolabeled receptor-targeted peptides for positron emission tomography (PET) imaging is a time consuming, complex, multi-step synthetic process that is highly variable based on the peptide. The complexity associated with the radiolabeling route and lack of robust automated protocols can hinder translation into the clinic. A fully automated batch production to radiolabel three peptides (YGGFL, cRGDyK, and Pyr-QKLGNQWAVGHLM) from fluorine-18 using the ELIXYS FLEX/CHEM® radiosynthesizer in a two-step process is described. First, the prosthetic group, 6-[18F]fluoronicotinyl-2,3,5,6-tetrafluorophenyl ester ([18F]FPy-TFP) was synthesized and subsequently attached to the peptide. The [18F]FPy-peptides were synthesized in 13-26% decay corrected yields from fluorine-18 with high molar activity 1-5 Ci µmol-1 and radiochemical purity of >99% in an overall synthesis time of 97 ± 3 minutes.

Preclinical Development and First-in-Human Imaging of the Integrin αvß6 with [18F]αvß6-Binding Peptide in Metastatic Carcinoma.

PURPOSE: The study was undertaken to develop and evaluate the potential of an integrin αvß6-binding peptide (αvß6-BP) for noninvasive imaging of a diverse range of malignancies with PET. EXPERIMENTAL DESIGN: The peptide αvß6-BP was prepared on solid phase and radiolabeled with 4-[18F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired αvß6-expressing and αvß6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired αvß6-expressing and αvß6-null cell xenografts. A first-in-human PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer. RESULTS: [18F]αvß6-BP displayed excellent affinity and selectivity for the integrin αvß6 in vitro [IC50(αvß6) = 1.2 nmol/L vs IC50(αvß3) >10 µmol/L] in addition to rapid target-specific cell binding and internalization (72.5% ± 0.9% binding and 52.5% ± 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [18F]αvß6-BP was rapid, primarily via the kidneys. In patients, [18F]αvß6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [18F]αvß6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung. CONCLUSIONS: The clinical impact of [18F]αvß6-BP PET imaging demonstrated in this first-in-human study is immediate for a broad spectrum of malignancies.

N,N,N-Trimethyl-5-[(2,3,5,6-tetrafluorophenoxy)carbonyl]pyridin-2-aminium trifluoromethanesulfonate a precursor for the synthesis of 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate.

The synthesis, recrystallization, and X-ray deterimination of N,N,N-trimethyl-5-[(2,3,5,6-tetrafluorophenoxy)carbonyl]pyridin-2-aminium trifluoromethanesulfonate (PyTFP-precursor), C15H13F4N2O2+·CF3SO3-, is described. This triflate salt precursor is required for the synthesis of 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate ([18F]FPyTFP), a prosthetic group used to radiolabel peptides for positron emission tomography (PET), as peptides are increasingly being used as PET-imaging probes in nuclear medicine. Radiolabeling of peptides is typically done using a `prosthetic group', a small synthon to which the radioisotope is attached in the first step, followed by attachment to the peptide in the second step. During the synthesis of the PyTFP-precursor, displacement of a Cl atom with trimethylamine gas and anion replacement with a triflate counter-ion is critical, as incomplete replacement would hinder radioisotopic incorporation of nucleophilic fluorine-18 and result in diminished radiochemical yields. The structural determination of the PyTFP-precursor by X-ray crystallography helped confirm the anion exchange of chloride with triflate.

In Vivo Tracking of Copper-64 Radiolabeled Nanoparticles in Lactuca sativa.

Engineered nanoparticles (NPs) are increasingly used in commercial products including automotive lubricants, clothing, deodorants, sunscreens, and cosmetics and can potentially accumulate in our food supply. Given their size it is difficult to detect and visualize the presence of NPs in environmental samples, including crop plants. New analytical tools are needed to fill the void for detection and visualization of NPs in complex biological and environmental matrices. We aimed to determine whether radiolabeled NPs could be used as a noninvasive, highly sensitive analytical tool to quantitatively track and visualize NP transport and accumulation in vivo in lettuce (Lactuca sativa) and to investigate the effect of NP size on transport and distribution over time using a combination of autoradiography, positron emission tomography (PET)/computed tomography (CT), scanning electron microscopy (SEM), and transition electron microscopy (TEM). Azide functionalized NPs were radiolabeled via a "click" reaction with copper-64 (64Cu)-1,4,7-triazacyclononane triacetic acid (NOTA) azadibenzocyclooctyne (ADIBO) conjugate ([64Cu]-ADIBO-NOTA) via copper-free Huisgen-1,3-dipolar cycloaddition reaction. This yielded radiolabeled [64Cu]-NPs of uniform shape and size with a high radiochemical purity (>99%), specific activity of  2.2 mCi/mg of NP, and high stability (i.e., no detectable dissolution) over 24 h across a pH range of 5-9. Both PET/CT and autoradiography showed that [64Cu]-NPs entered the lettuce seedling roots and were rapidly transported to the cotyledons with the majority of the accumulation inside the roots. Uptake and transport of intact NPs was size-dependent, and in combination with the accumulation within the roots suggests a filtering effect of the plant cell walls at various points along the water transport pathway.

Solid-phase synthesis and fluorine-18 radiolabeling of cycloRGDyK.

Solid-phase peptide synthesis, head-to-tail cyclization, and subsequent radiolabeling provided a reproducible, simple, rapid synthetic method to generate the cyclic peptide radiotracer cRGDyK([18F]FBA). Herein is reported the first on-resin cyclization and 18F-radiolabeling of a cyclic peptide (cRGDyK) in an overall peptide synthesis yield of 88% (cRGDyK(NH2)) and subsequent radiolabeling yield of 14 ± 2% (decay corrected, n = 4). This approach is generally applicable to the development of an automated process for the synthesis of cyclic radiolabeled peptides for positron emission tomography (PET).

Synthesis of cholesteryl-α-D-lactoside via generation and trapping of a stable ß-lactosyl iodide.

The generation of ß-lactosyl iodide was carried out under non-in situ-anomerization, metal free conditions by reacting commercially available ß-per-O-acetylated lactose with trimethylsilyl iodide (TMSI). The ß-iodide was surprisingly stable as evidenced by NMR spectroscopy. Introduction of octanol or cholesterol under microwave conditions gave high yields of α-linked glycoconjugates. Careful analysis of the reaction products and mechanistic considerations suggest an acid catalyzed rearrangement that provides α-linked glycosylation products with a free C2-hydroxyl. Accessibility to these compounds may further advance glycolipidomic profiling of immune modulating bacterial derived-glycans.

Synthesis and structural characterization of three unique Helicobacter pylori α-cholesteryl phosphatidyl glucosides.

Steryl glycosides produced by bacteria play important biological roles in the evasion and modulation of host immunity. Step-economical syntheses of three cholesteryl-6-O-phosphatidyl-α-D-glucopyranosides (αCPG) unique to Helicobacter pylori have been achieved. The approach relies upon regioselective deprotection of per-O-trimethylsilyl-α-D-cholesterylglucoside at C6 followed by phosphoramidite coupling. Global TMS ether deprotection in the presence of oxygen and subsequent deprotection of the cyano ethyl phosphoester afforded the target compounds in 16-21 % overall yield starting from D-glucose. The structures of these natural products were determined using a combination of 2D NMR methods and mass spectrometry. These robust synthesis and characterization protocols provide analogues to facilitate glycolipidomic profiling and biological studies.

Tandem glycosyl iodide glycosylation and regioselective enzymatic acylation affords 6-O-tetradecanoyl-α-d-cholesterylglycosides.

A generalized synthesis of α-d-cholesterylglycosides has been achieved using one-pot per-O-trimethylsilyl glycosyl iodide glycosidation. Both cholesteryl α-d-glucopyranoside (αCG) and cholesteryl α-d-galactopyranoside were prepared in high yield. These compounds were further esterified using regioselective enzymatic acylation with tetradecanoyl vinyl ester to afford 6-O-tetradecanoyl-α-d-cholesteryl glucopyranoside (αCAG) of Helicobacter pylori and the corresponding galactose analogue in 66-78% overall yields from free sugars. The tandem step-economy sequence provides novel analogues to facilitate glycolipidomic profiling.

Two-step functionalization of oligosaccharides using glycosyl iodide and trimethylene oxide and its applications to multivalent glycoconjugates.

Oligosaccharide conjugates, such as glycoproteins and glycolipids, are potential chemotherapeutics and also serve as useful tools for understanding the biological roles of carbohydrates. With many modern isolation and synthetic technologies providing access to a wide variety of free sugars, there is increasing need for general methodologies for carbohydrate functionalization. Herein, we report a two-step methodology for the conjugation of per-O-acetylated oligosaccharides to functionalized linkers that can be used for various displays. Oligosaccharides obtained from both synthetic and commercial sources were converted to glycosyl iodides and activated with I2 to form reactive donors that were subsequently trapped with trimethylene oxide to form iodopropyl conjugates in a single step. The terminal iodide served as a chemical handle for further modification. Conversion into the corresponding azide followed by copper-catalyzed azide-alkyne cycloaddition afforded multivalent glycoconjugates of Gb3 for further investigation as anti-cancer therapeutics.

A fused [3.3.0]-neoglycoside lactone derived from glucuronic acid.

The bridged next-generation aminoglycoside (neoglycoside), 1-deoxy-1-[(methoxy)methylamino)]-2,5-di-O-triethylsilyl-ß-D-glucofuranurono-γ-lactone {systematic name: (3S,3aS,5R,6R,6aS)-5-[methoxy(methyl)amino]-3,6-bis[(triethylsilyl)oxy]-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-2-one}, C20H41NO6Si2, was synthesized in a one-pot manner from commercially available D-glucuronic acid. This structure supports the properties associated with the anomeric effect for furanosides and can be employed to provide insight into the mechanisms by which alkoxyamine-appended natural products derive their enhanced biological activity. To the best of our knowledge, this is the first published crystal structure of a bicyclic neoglycoside and is the first neoglycoside to be completely and unambiguously characterized.

Chemoenzymatic synthesis of cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside: a product of host cholesterol efflux promoted by Helicobacter pylori.

In a three-step protocol involving regioselective enzymatic acylation, per-O-trimethylsilylation, and a one-pot α-glycosidation-deprotection sequence, cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (α-CAG) of Helicobacter pylori is afforded starting from glucose in an overall yield of 45%. The production of CAG can be scaled to make purified quantities available to the biological community for the first time.