Regioselective Acylation of Diols and Triols: The Cyanide Effect

Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective <i>O</i>-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-<i>cis</i>-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at −78 °C the thermodynamically unfavorable axial <i>O</i>-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-<i>O</i>-unproteced galacto- and fucopyranosides and 2,3-<i>O</i>-unprotected mannopyranosides. Even for 3,4,6-<i>O</i>-unprotected galactopyranosides as triols, axial 4-<i>O</i>-acylation is appreciably faster than <i>O</i>-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the “cyanide effect” is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial <i>O</i>-acylation

2‑Nitro-thioglycosides: α- and β‑Selective Generation and Their Potential as β‑Selective Glycosyl Donors

Michael-type addition of thiolates to 2-nitro-d-glucal or to 2-nitro-d-galactal derivatives readily provides 2-deoxy-2-nitro-1-thioglycosides. Kinetic and thermodynamic reaction control permitted formation of either the α- or preferentially the β-anomers, respectively. Addition of achiral and chiral thiourea derivatives to the reaction mixture increased the reaction rate; the outcome is substrate-controlled. The 2-deoxy-2-nitro-1-thioglycosides are excellent glycosyl donors under arylsulfenyl chloride/silver triflate (ArSCl/AgOTf) activation, and they provide, anchimerically assisted by the nitro group, mostly β-glycosides

Regioselective One-Pot Benzoylation of Triol and Tetraol Arrays in Carbohydrates

Protection of 2,3,4-<i>O</i>-unprotected α-galacto- and α-fucopyranosides with BzCN and DMAP/DIPEA as the base afforded directly and regioselectively the 3-<i>O</i>-unprotected derivatives. The rationale for these studies was to take advantage of the eventual cooperativity of the “cyanide effect” and “the alkoxy group mediated diol effect”. This way, even the totally unprotected α-galactopyranosides could be regioselectively transformed into the corresponding 2,4,6-<i>O</i>-protected derivatives. The great utility of these building blocks was demonstrated in efficient trisaccharide syntheses

Clonal variation in OCH-CD1d tetramer binding by human iNKT cells is not related to TCR expression levels.

<p>Flow cytometric analysis of one representative CD4+ human Vα24+/Vβ11+ iNKT line (A) and three representative CD4+ human Vα24+/Vβ11+ iNKT clones from different donors (B) demonstrates clonal variation in binding to OCH-CD1d (upper row), but not K7-CD1d (lower row) tetramers. (C) K7- and OCH-CD1d tetramer staining in pure human iNKT lines (<i>n</i> = 68) and clones (<i>n</i> = 256) was related to expression levels of iNKT TCR Vα24 and Vβ11. The intensity (MFI) of K7- but not OCH-CD1d tetramer staining was strongly associated with Vα24 and Vβ11 expression, as determined by Pearson correlation analysis, but not with CD4+ (blue markers) or CD4−CD8− double negative (red markers) phenotype.</p

The CDR3β loop strongly impacts on human iNKT TCR affinity to CD1d, independent of the CD1d-bound ligand.

<p>(A) Binding of two recombinant human iNKT TCRs, one OCH^HIGH (4C1369) and one OCH^LOW (4C12), to K7-, OCH-, βGC-, and LacCer-CD1d at equilibrium is shown (see also panel C and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000402#pbio-1000402-t002" target="_blank">Table 2</a>). (B) The affinity of the seven recombinant iNKT TCRs to OCH-CD1d, as determined by SPR, was linearly related to the staining intensity (MFI) of the original iNKT clone with OCH-CD1d tetramers. (C) The seven recombinant human iNKT TCRs followed a strict hierarchy of binding to ligand-CD1d complex, which was not affected by the specific CD1d-bound ligand. These iNKT TCRs differed only with regard to their CDR3beta sequence (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000402#pbio-1000402-t001" target="_blank">Table 1</a>).</p

Distinct iNKT cell subpopulations revealed by OCH-CD1d tetramer staining.

<p>OCH- and K7-CD1d tetramer stainings of (A) a representative K7-stimulated human iNKT line after 14 d in vitro culture and (B) a healthy human volunteer's PBMC ex vivo are shown. While K7-CD1d tetramer staining identifies a single homogeneous population of iNKT cells (upper row), OCH-CD1d tetramer staining reveals the presence of different distinct iNKT populations within these samples (lower row).</p

Differential binding of OCH^HIGH and OCH^LOW iNKT cells to βGC-CD1d tetramers.

<p>Ex vivo sorted human Vα24+/Vβ11+ iNKT clones were stained with different, α- or β-glycosylceramide loaded CD1d-tetramers. (A) A representative pair of CD4+ OCH^HIGH and OCH^LOW iNKT clones from one donor is shown. βGC-CD1d tetramers only stained OCH^HIGH but not OCH^LOW iNKT clones above background (as determined by PE-streptavidin binding). TCR Vα24 and Vβ11 surface expression levels for the two clones shown were very similar (for PE-conjugated anti-Vα24, MFI 2673 (OCH^HIGH) and 2710 (OCH^LOW); for FITC-conjugated anti-Vβ11, MFI 106 (OCH^HIGH) and 97 (OCH^LOW)). (B) βGC-CD1d tetramer staining intensity (MFI) of a panel of OCH-LOW (red markers), OCH-INT (green markers), and OCH-HIGH (blue markers) iNKT clones showed almost linear correlation with OCH-CD1d tetramer staining, but no correlation with either Vα24 or Vβ11 surface expression.</p

Binding of 7 human iNKT TCRs to different CD1d/ligand complexes.

<p>K_D, dissociation constant; T<b>½</b>, dissociation half-time; ND, not determined. All values given ± standard deviation.</p

Differential autoreactive functional responses by human OCH^HIGH and OCH^LOW iNKT clones.

<p>Matched pairs of human OCH^HIGH (red columns and markers) and OCH^LOW (blue columns and markers) iNKT clones were compared for their ability to proliferate, secrete cytokines, and exhibit cytotoxicity in response to lipid-pulsed or endogenous lipid presenting CD1d-positive antigen presenting cells. (A) Proliferation of three representative pairs of OCH^HIGH and OCH^LOW iNKT clones from different healthy donors in response to K7-, OCH-, or vehicle-pulsed human CD1d-expressing T2 cells (T2-CD1d) or to K7-pulsed CD1d negative T2 cells (T2-) is shown. OCH^HIGH clones consistently displayed greater proliferation than OCH^LOW clones in response to OCH or vehicle pulsed T2-CD1d. cpm, counts per minute. Mean values ± s.e.m. are shown. (B) Cytokine secretion profiles of a representative pair of matched OCH^HIGH and OCH^LOW iNKT clones in response to the strong agonist ligand K7 and the partial agonist ligand OCH, presented by T2-CD1d, are shown. OCH^HIGH iNKT clones exhibited much stronger cytokine secretion than OCH^LOW iNKT cells in response to OCH-pulsed T2-CD1d, while cytokine secretion was similar for both in response to K7-pulsed T2-CD1d. (C) Autoreactive cytokine release in response to T2-CD1d in the absence of added exogenous ligands is shown for four matched pairs of OCH^HIGH and OCH^LOW iNKT clones. OCH^HIGH but not OCH^LOW iNKT clones consistently exhibited substantial autoreactive cytokine secretion. (D) Specific lysis of K7- (filled markers) and OCH- (unfilled markers) pulsed T2-CD1d targets is shown for three matched pairs of OCH^HIGH and OCH^LOW iNKT clones from different donors.</p

Differential binding of OCH^HIGH and OCH^LOW iNKT clone derived TCR tetramers to endogenous lipid presenting CD1d molecules.

<p>PE-conjugated recombinant iNKT TCR tetramers derived from OCH^HIGH (4C1369; red lines) and OCH^LOW (4C12; blue lines) iNKT clones, at increasing concentrations, were used to stain T2-CD1d lymphoblasts. Clear staining of vehicle-pulsed T2-CD1d (unfilled markers) was only seen with the OCH^HIGH TCR tetramer, whereas both iNKT TCR tetramers strongly bound to K7-pulsed T2-CD1d (filled markers). The black bar shows background staining of T2- cells with iNKT TCR tetramers.</p