Michael J. Palte

Boronate-Mediated Biologic Delivery

Inefficient cellular delivery limits the landscape of macromolecular drugs. Boronic acids readily form boronate esters with the 1,2- and 1,3-diols of saccharides, such as those that coat the surface of mammalian cells. Here pendant boronic acids are shown to enhance the cytosolic delivery of a protein toxin. Thus, boronates are a noncationic carrier that can deliver a polar macromolecule into mammalian cells.

Organocatalytic conversion of cellulose into a platform chemical

The search for a source of fuels and chemicals that is both abundant and renewable has become of paramount importance. The polysaccharide cellulose meets both criteria, and methods have been developed for its transformation into the platform chemical 5-(hydroxymethyl)furfural (HMF). These methods employ harsh reaction conditions or toxic heavy metal catalysts, deterring large-scale implementation. Here, we describe a low-temperature, one-pot route that uses ortho-carboxyl-substituted phenylboronic acids as organocatalysts in conjunction with hydrated magnesium chloride and mineral acids to convert cellulose and cellulose-rich municipal waste to HMF in yields comparable to processes that use toxic heavy metal catalysts. Isotopic labeling studies indicate that the key aldose-to-ketose transformation occurs via an enediol intermediate. The route, which also allows for facile catalyst recovery and recycling, provides a green prototype for cellulose conversion.

A Potent, Versatile Disulfide-Reducing Agent from Aspartic Acid

Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pKa values that are ∼1 unit lower than those of DTT and forms a disulfide with a similar E°′ value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.

Interaction of Nucleic Acids with the Glycocalyx

Mammalian cells resist the uptake of nucleic acids. The lipid bilayer of the plasma membrane presents one barrier. Here, we report on a second physicochemical barrier for uptake. To create a sensitive probe for nucleic acid-cell interactions, we synthesized fluorescent conjugates in which lipids are linked to DNA oligonucleotides. We found that these conjugates incorporate readily into the plasma membrane but are not retained there. Expulsion of lipid-oligonucleotide conjugates from the plasma membrane increases with oligonucleotide length. Conversely, the incorporation of conjugates increases markedly in cells that lack the major anionic components of the glycocalyx, sialic acid and glycosaminoglycans, and in cells that had incorporated highly cationic lipids into their plasma membrane. We conclude that anionic oligosaccharides provide a formidable barrier to the uptake of nucleic acids by mammalian cells. This conclusion has implications for genomic stability, as well as the delivery of genes and siRNAs into mammalian cells.

Ribonucleoside 3'-phosphates as pro-moieties for an orally administered drug.

Oral administration of chemotherapeutic agents is the mainstay for the treatment of disease. Sustained release formulations have been crucial for the safe and effective dosing of orally administered drugs.[1] Such formulations allow for the prolonged maintenance of therapeutic drug concentrations, reducing the required dosages per day and thereby enhancing patient compliance. Sustained release formulations also provide tighter control over the pharmacokinetics of a drug, thereby minimizing side effects.[1] Aqueous solubility is likewise a critical attribute for an orally available drug.[2] Robust absorption across the intestinal epithelium relies upon a high concentration of the drug to drive diffusion into enterocytes and, eventually, into the circulatory system. On average, 35–40% of lead compounds have aqueous solubilities of <5 mg/mL, which is defined by the U.S. Pharmacopeia as being slightly soluble or worse.[3] Accordingly, the bioavailability and consequent efficacy of many compounds relies on enhancing their aqueous solubility.[2c] The formation of a phosphomonoester can improve the oral bioavailability of poorly water-soluble chemotherapeutic agents.[2b,2c,4] Endogenous phosphatases near the surface of enterocytes can catalyze the hydrolysis of the phosphoryl group, releasing the lipophilic drug and allowing for its efficient absorption into the body. Several prodrugs approved by the U.S. Food and Drug Administration rely on this strategy, including estramustine, fosamprenavir, and prednisolone phosphate.[4] Recently, we reported on the potential utility of a phosphodiester as the pro-moiety for a drug administered intravenously.[5] Specifically, we found that the coupling of 4-hydroxytamoxifen to uridine 3′-phosphate enabled its timed-release in serum by human pancreatic ribonuclease (RNase 1[6]; EC 3.1.27.5). This modification also increased the aqueous solubility of 4-hydroxytamoxifen. RNase 1 is an ideal endogenous enzyme to elicit pro-moiety release. A major excreted enzyme, RNase 1 has a concentration of 6.4 mg/mL in human pancreatic juice and 0.2 mg/mL in saliva, according to a radioimmunoassay.[7] Moreover, like its renowned homologue bovine pancreatic ribonuclease (RNase A[8]), RNase 1 catalyzes the cleavage of RNA by a transphosphorylation reaction[9] with little specificity for its leaving group.[10] Herein, we report on the utility of several ribonucleoside 3′-phosphates as pro-moieties for a model orally available drug, metronidazole. Metronidazole is a commonly used antibiotic for a variety of protozoa and anaerobic bacterial infections, including Bacteroides fragilis, Helicobacter pylori, Clostridium difficile, Trichomonas vaginalis, and Entamoeba histolytica.[11] In 1997, Flagyl ER, an extended release formulation of metronidazole, was approved by the FDA as a superior treatment for bacterial vaginosis. Still, metronidazole has several common side effects, such as nausea, diarrhea, and metallic taste. Moreover, metronidazole therapy can occasionally cause more severe side effects, such as pancreatitis, neutropenia, neuropathies, or CNS toxicities.[12] These adverse effects could be attenuated with better control over the pharmacokinetics of metronidazole.[13] Hence, in this proof-of-concept study, we elected to attach metronidazole to ribonucleoside 3′-phosphates to assess the attributes of this promoiety for orally available drugs (Figure 1). Figure 1 Scheme showing catalysis of the cleavage of a ribonucleoside 3′-(metronidazole phosphate) (NpMet) by RNase 1 to yield a nucleoside 2′,3′-cyclic phosphate (N>p) and Met. Each ribonucleoside 3′-(metronidazole phosphate) (NpMet) was synthesized in four steps from commercially available metronidazole (Met) and a ribonucleoside phosphoramidite (Scheme 1). Briefly, Met was coupled to the phosphoramidite by using N-methylbenzimidazolium triflate (MBIT) as a catalyst.[14] The coupled product was oxidized with iodine and deprotected stepwise. The final products were purified by chromatography on silica gel. This route was used to synthesize three different NpMets: cytidine 3′-(metronidazole phosphate) (CpMet, 18% non-optimized yield), uridine 3′-(metronidazole phosphate) (UpMet, 80%), and adenosine 3′-(metronidazole phosphate) (ApMet, 64%). Scheme 1 Route for the synthesis of NpMets. We expected the ribonucleoside 3′-phosphate moiety of an NpMet to endow the prodrug with greater hydrophilicity than the parent drug, which could improve its oral bioavailability. To investigate this issue, we calculated the partition (log P) and distribution (log D) coefficients of Met, CpMet, UpMet, and ApMet.[15] The calculated log P and log D values for the NpMets were indeed significantly lower than those of the parent drug (Table 1), indicative of increased hydrophilicity and decreased tendency to aggregate. Table 1 Calculated Partition and Distribution Coefficients of Met and NpMets[15] To be the basis for an effective timed-release prodrug strategy, the pro-moiety needs to be released by the activating enzyme over time. Hence, we used 1H NMR spectroscopy to assess the rate at which RNase 1 catalyzed the release of Met from the prodrugs (Figure S1). We assumed that pancreatic juice is diluted in the intestine, which led us to use RNase 1 at concentrations of 0.1 mg/mL and 0.01 mg/mL in these assays. Because inorganic phosphate inhibits RNase A with a Ki value of 2.3 mM,[16] we initially investigated the effect of phosphate in simulated intestinal fluid (SIF) on the rates of UpMet unmasking (Figure 2A). Compared to a buffer with no inorganic phosphate (19.5 mM Tris–HCl, pH 7.4, 2.5% v/v D2O), the rate of Met release in SIF was only marginally slower. RNase 1 cleaves after pyrimidine residues more readily than after purine residues.[6,7] Accordingly, we predicted that RNase 1 would unmask CpMet and UpMet faster than ApMet. For both concentrations of RNase 1, we did indeed observe that the cytidine and uridine prodrugs were unmasked faster than the adenosine prodrug (Figure 2B). Moreover, unlike the uridine 3′-phosphate–4-hydroxytamoxifen conjugate that cleaved spontaneously in aqueous solutions lacking ribonucleases, the NpMet conjugates were stable in SIF, which has pH 7.5, and in simulated gastric fluid (SGF), which has pH 1.1. The absence of appreciable degradation (<5%) in either medium (Figure S2) is attributable to the alkoxyl group of metronidazole being a much worse leaving group than the aryloxyl group of 4-hydroxytamoxifen. Figure 2 Progress curves for the release of Met from NpMets under various conditions as determined by 1H NMR spectroscopy. (A) Comparison of UpMet cleavage rates in Tris–HCl buffer, pH 7.4, and simulated intestinal fluid (SIF). (B) Comparison of CpMet, ... Finally, we sought to assess the antimicrobial activity of our NpMet prodrugs on B. fragilis. This penicillin-resistant Gram-negative bacillus is common in anaerobic infections, like those that originate from the gastrointestinal tract. We determined the minimum inhibitory concentration (MIC) of UpMet and ApMet, as well as Met (Figure S3). We found that both UpMet and ApMet had considerably higher MIC values than did Met (Table 2), demonstrating that the prodrugs were relatively inactive. Incubating UpMet with 0.1 mg/mL RNase 1 overnight resulted in an MIC similar to that of Met. Finally, adding UpMet to a culture medium containing RNase 1 gave an intermediate MIC, demonstrating the in situ release of the drug. Table 2 MIC Values of NpMets for Bacteroides fragilis

Sub-picomolar Inhibition of HIV-1 Protease with a Boronic Acid

Boronic acids have been typecast as moieties for covalent complexation and are employed only rarely as agents for non-covalent recognition. By exploiting the profuse ability of a boronic acid group to form hydrogen bonds, we have developed an inhibitor of HIV-1 protease with extraordinary affinity. Specifically, we find that replacing an aniline moiety in darunavir with a phenylboronic acid leads to 20-fold greater affinity for the protease. X-ray crystallography demonstrates that the boronic acid group participates in three hydrogen bonds, more than the amino group of darunavir or any other analog. Importantly, the boronic acid maintains its hydrogen bonds and its affinity for the drug-resistant D30N variant of HIV-1 protease. The BOH···OC hydrogen bonds between the boronic acid hydroxy group and Asp30 (or Asn30) of the protease are short ( rO···O = 2.2 Å), and density functional theory analysis reveals a high degree of covalency. These data highlight the utility of boronic acids as versatile functional groups in the design of small-molecule ligands.

Development of a Commercial Reference Laboratory Elective Rotation for Residents in Clinical Pathology

Objectives To develop a curriculum for a commercial reference laboratory clinical pathology training elective. Methods A 4-day elective at Quest Diagnostics was developed. The elective included 32 sessions composed of interactive didactic sessions and laboratory tours/demonstrations. Ten residents who attended the elective completed a written evaluation and scored each component of the curriculum. Results Written comments were very positive and demonstrated the goals of the elective were achieved. Laboratory tours and one-on-one sessions with the medical directors were especially well received. Most of the residents stated that the rotation gave them exposure to an area of laboratory medicine that they were not familiar with. Conclusions The elective provided a resident training experience that was highly regarded and exposed residents to an area of laboratory medicine not encountered in most pathology training programs. Our curriculum could serve as a model for establishing a similar elective in other training programs.

Kayexalate (Sodium Polystyrene Sulfonate) as a Hallmark and Cause of Gastrointestinal Tract Injury: Two Case Reports and a Review of the Literature

Kayexalate (sodium polystyrene sulfonate) is a cation exchange resin that is commonly used for the treatment for hyperkalemia. With the increasing prevalence of type II diabetes mellitus in developed nations, there has been a concordant rise in renal disease and hyperkalemic patients receiving Kayexalate therapy. Gastrointestinal necrosis is a rare but major complication associated with Kayexalate use that has not been previously well characterized in the forensic literature. Kayexalate crystals have a distinctive “mosaic” appearance on hematoxylin and eosin stained sections and a characteristic coloration with periodic acid-Schiff (PAS) and acid-fast stains. As they are relatively easily identified in histologic sections, and may represent a primary or contributory cause of death, it is suggested that forensic pathologists become familiar with these features. We present two cases in which Kayexalate crystals were detected at autopsy. In one case, the crystals were located within the intestinal lumen and mucosa in areas of mucosal ulceration, while in the other, crystals were located on the intestinal serosal surface without apparent mucosal ulceration. We discuss how to interpret the significance of Kayexalate crystals, review prior literature on Kayexalate therapy-related gastrointestinal necrosis, and explain how to notify the Food and Drug Administration of an adverse Kayexalate reaction detected at autopsy.