Bruno Linclau

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells.

Activation of invariant NK T (iNKT) cells with the glycolipid α-galactosylceramide promotes CD8+ cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8+ T cells occur on the same APCs. We demonstrate that injection of α-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8+ T cell response and abolishes the efficacy of α-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8+ T cells.

6′-Derivatised α-GalCer Analogues Capable of Inducing Strong CD1d-Mediated Th1-Biased NKT Cell Responses in Mice

α-Galactosyl ceramide (α-GalCer, also known as KRN 7000) is known as the prototypical antigen for invariant natural killer T (NKT) cells. Stimulation of NKT cells by CD1d-mediated α-GalCer presentation leads to rapid release of Th1 and Th2 cytokines. Since Th1 and Th2 cytokines antagonize each other’s effects, α-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. With the exception of a C-glycoside (α-C-GalCer), most of the known α-GalCer analogues able to induce polarized cytokine responses are characterized by modifications of the phytosphingosine or fatty acyl chains, expected to alter the affinity for CD1d. Herein we describe the synthesis of 6′-modified α-GalCer analogues with an intact phytoceramide moiety that are capable to skew the cytokine release profile to Th1, while maintaining strong antigenic activity. These analogues are characterized by the presence of an aromatic moiety that is connected via an amide or an urea linkage to C′-6 of the galactopyranose ring.

An Unexpected and Significantly Lower Hydrogen‐Bond‐Donating Capacity of Fluorohydrins Compared to Nonfluorinated Alcohols

The success of fluorination in improving molecular properties over a wide range of applications (including pharmaceuticals,1 agrochemicals,2 materials,3 and crystal engineering4) has been remarkable. Up to 20 % of the pharmaceuticals prescribed or administered in the clinic, and a third of the leading 30 blockbuster drugs, contain at least one fluorine atom1a and 30–40 % of currently marketed agrochemicals contain fluorine.5

Organic-Fluorous Phase Switches: A Fluorous Amine Scavenger for Purification in Solution Phase Parallel Synthesis

The synthesis of the fluorous amine scavenger [(C(6)F(13)CH(2)CH(2))(3)SiCH(2)CH(2)CH(2)](2)NH and its successful application in the automated solution phase parallel synthesis of a urea library are described. Ureas were made by robotic synthesis from organic amines and excess isocyanates. The amine scavenger reacts with excess isocyanate, and the fluorous tag serves to solubilize the resulting adduct in the fluorous phase so it can be removed by fluorous-organic extraction. Organic urea products are isolated in high yields and purities after liquid-liquid extraction. Preliminary biological evaluation shows that several of the ureas have ion channel modulation abilities. In contrast to polymer and ionic quenching methods, the fluorous quench works whether the product is soluble or insoluble in the reaction medium, and ionizable functional groups are tolerated in the products.

Structural basis of ligand binding to UDP-galactopyranose mutase from Mycobacterium tuberculosis using substrate and tetrafluorinated substrate analogues.

UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His6-MBP-tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxy-tetrafluorinated analogues of both UDP-Galp (UDP-F4-Galp) and UDP-Galf (UDP-F4-Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogues bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show an overall binding mode similar to those reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active-site closure. In addition, the complex structure of MtUGM with UDP-F4-Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high-energy conformation ((4)E) within the catalytic pocket. Moreover, these investigations provide structural insights into the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogues. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities for the use of polyfluorination in the design of carbohydrate mimetics.

Tetrafluorination of Sugars as Strategy for Enhancing Protein–Carbohydrate Affinity: Application to UDP-Galp Mutase Inhibition

Tetrafluorinated analogues of both UDP-galactopyranose and UDP-galactofuranose have been synthesized and assayed against UDP-galactopyranose mutase, a key enzyme for Mycobacterium tuberculosis cell wall biosynthesis. Competition assays and STD-NMR spectroscopy techniques have evidenced not only the first unambiguous case of affinity enhancement through local sugar polyfluorination, but also showed that tetrafluorination can still have a beneficial effect on binding when monofluorination at the same position does not.

Synthesis and in vitro Evaluation of α-GalCer Epimers

α‐GalCer (also known as KRN7000) is an immunomodulatory glycolipid that is known to potently activate invariant natural killer T (NKT) cells upon CD1d‐mediated stimulation. Because Th1 and Th2 cytokines, which are released after α‐GalCer presentation, antagonize each others effects, α‐GalCer analogues that induce a biased Th1/Th2 response are highly awaited. In this context, we report the synthesis and in vitro evaluation of α‐Gal‐D‐xylo‐Cer and two α‐Gal‐L‐lyxo‐Cer analogues, one with the natural acyl chain, the other with a truncated chain.

Enantioselective synthesis of tetrafluorinated ribose and fructose

A perfluoroalkylidene lithium mediated cyclisation approach for the enantioselective synthesis of a tetrafluorinated aldose (ribose) and of a tetrafluorinated ketose (fructose), both in the furanose and in the pyranose form, is described.

Investigating the influence of (Deoxy)fluorination on the lipophilicity of non-UV-active fluorinated alkanols and carbohydrates by a new log P determination method

Abstract Property tuning by fluorination is very effective for a number of purposes, and currently increasingly investigated for aliphatic compounds. An important application is lipophilicity (log P) modulation. However, the determination of log P is cumbersome for non‐UV‐active compounds. A new variation of the shake‐flask log P determination method is presented, enabling the measurement of log P for fluorinated compounds with or without UV activity regardless of whether they are hydrophilic or lipophilic. No calibration curves or measurements of compound masses/aliquot volumes are required. With this method, the influence of fluorination on the lipophilicity of fluorinated aliphatic alcohols was determined, and the log P values of fluorinated carbohydrates were measured. Interesting trends and changes, for example, for the dependence on relative stereochemistry, are reported.

Synthesis and in vitro evaluation of ?-GalCer epimers

α‐GalCer (also known as KRN7000) is an immunomodulatory glycolipid that is known to potently activate invariant natural killer T (NKT) cells upon CD1d‐mediated stimulation. Because Th1 and Th2 cytokines, which are released after α‐GalCer presentation, antagonize each others effects, α‐GalCer analogues that induce a biased Th1/Th2 response are highly awaited. In this context, we report the synthesis and in vitro evaluation of α‐Gal‐D‐xylo‐Cer and two α‐Gal‐L‐lyxo‐Cer analogues, one with the natural acyl chain, the other with a truncated chain.