Oxidation of the 5′-hydroxymethylene of nucleosides to 5′-carboxylates is an essential step in the preparation of a number of biologically active molecules. 1 There are relatively few methods describing the general preparation of such nucleoside-5′-carboxylic acids. Herein, we report a mild procedure for the oxidation of 2′,3′-isopropylideneprotected purine-and pyrimidine-containing nucleosides to their respective 5′-carboxylic acids. This method gives high yields and has a very simple isolation procedure.One of the most widely applied methods for affecting the oxidation of the 5′-hydroxyl of unprotected nucleosides employs molecular oxygen and a platinum catalyst. 2 However, this method affords relatively low yields when applied to 2′,3′-isopropylidene-protected nucleosides. 3 Instead, these nucleosides are most often oxidized using potassium permanganate under strongly alkaline reaction conditions. 4 This limits the potassium permanganate methodology to purine-containing nucleosides. Two other systems used to affect these conversions are CrO 3 /acetic acid 5 and a two-step method involving the generation of the aldehyde followed by oxidation with m-CPBA. 6 However, these methods have not been generally applied.Recently, a method utilizing ruthenium trichloride and sodium periodate under Sharpless conditions was used to obtain the 5′-carboxylic acids of 2′,3′-isopropylidenepurine-containing nucleosides in high yield. 7 Unfortunately, the methodology cannot be used with pyrimidinecontaining nucleosides, as the reaction conditions cause loss of the nucleoside base. Extension of ruthenium trichloride-mediated oxidation to 2′,3′-isopropylidenepyrimidine-containing nucleosides requires the use of both alkaline conditions and potassium persulfate. 8 Thus, many available methods for the generation of nucleoside-5′-carboxylic acids require relatively basic conditions that limit their utility.A recent publication described the oxidation of alcohols to ketones and aldehydes using catalytic amounts of 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) and stoichiometric amounts of an organic oxidant, [bis(acetoxy)-iodo]benzene (BAIB). 9 The method drew our attention because of its mildness and efficiency. The active oxidant is an N-oxoammonium salt generated by dismutation of TEMPO; BAIB is necessary to regenerate TEMPO by oxidizing the corresponding hydroxylamine of TEMPO. The reaction generates acetic acid and iodobenzene as byproducts and is different from most other TEMPOmediated oxidations in that it avoids inorganic salt contaminants. 9 In addition, N-oxoammonium salt-mediated oxidations are compatible with double and triple bonds, esters, ethers, acetals, epoxides, amides, halides, and azides. Finally, protecting groups such as TBDMS, THP, MOM, Boc, Cbz, benzyl, and acetyl are also stable to the reaction conditions. 10 In the presence of high concentrations of water, Noxoammonium salts convert aliphatic alcohols to their respective carboxylic acids. 10 Since the TEMPO-BAIB system seemed to be a particularly convenient m...

No abstract

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.

A comprehensive approach to the synthesis of sulfate esters was developed. This approach permits the direct and high-yielding synthesis of protected sulfate monoesters. Subsequent deblocking to reveal sulfate monoesters is accomplished in near-quantitative yield. The exceptionally stable neopentyl protecting group and the labile isobutyl protecting group were utilized in the synthesis of aromatic and aliphatic sulfate monoesters. Strategies for tuning protecting group reactivity were also explored and developed.

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation.

No abstract

Summary Sulfation of tyrosine is a common posttranslational modification of secreted proteins that influences numerous physiological and pathological processes. Studies of tyrosine sulfation have been hindered by the difficulty of introducing sulfate groups at specific positions of peptides and proteins. Here we report a general strategy for synthesis of peptides containing sulfotyrosine at one or more specific position(s). The approach provides a substantial improvement in both yield and convenience over existing methods. Using synthetic sulfopeptides derived from the chemokine receptor CCR3, we demonstrate that sulfation enhances affinity for the chemokine eotaxin by ∼7-fold or more than 28-fold, depending on which of two adjacent tyrosine residues is sulfated. The new synthetic methodology will substantially enhance efforts to understand the functional and structural consequences of protein tyrosine sulfation.

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.