Cinchona Alkaloid-Squaramide Catalyzed Sulfa-Michael Addition Reaction: Mode of Bifunctional Activation and Origin of Stereoinduction

The mechanism of the enantioselective sulfa-Michael addition reaction catalyzed by a cinchona alkaloid-squaramide bifunctional organocatalyst was studied using density functional theory (DFT). Four possible modes of dual activation mechanism via hydrogen bonds were considered. Our study showed that Houk’s bifunctional Brønsted acid–hydrogen bonding model, which works for cinchonidine or cinchona alkaloid-urea catalyzed sulfa-Michael addition reactions, also applies to the catalytic system under investigation. In addition, we examined the origin of the stereoselectivity by identifying stereocontrolling transition states. Distortion–interaction analysis revealed that attractive interaction between the substrates and catalyst in the C–S bond forming transition state is the key reason for stereoinduction in this catalytic reaction. Noncovalent interaction (NCI) analysis showed that a series of more favorable cooperative noncovalent interactions, namely, hydrogen bond, π-stacking, and C–H···π interaction and C–H···F interactions, in the major <i>R</i>-inducing transition state. The predicted enantiometric excess is in good accord with the observed value

Analysis of GO classifications involving differentially co-expressed genes in three models of distinct and notable dynamic expression patterns in both sugarcane genotypes.

<p>Analysis of GO classifications involving differentially co-expressed genes in three models of distinct and notable dynamic expression patterns in both sugarcane genotypes.</p

Comparison of the expression stability of 13 reference genes in <i>Saccharum officinarum</i> as calculated by geNorm, Normfinder and deltaCt.

<p>*β-actin,</p><p>**β-tubulin.</p

Dynamic expression patterns of differentially expressed genes in “ROC”22 after <i>S. scitamineum</i> inoculation.

<p>Notes: 0, 1, 2, 3, <b>−</b>1, <b>−</b>2 and <b>−</b>3 do not refer to the actual expression of the differentially expressed genes, but for the classification mark of gene dynamic changes. Gene numbers represent the actual number of dynamic expression patterns of differentially expressed genes. T1, “ROC”22 sample under sterile water stress after 24 h; T2–T4, “ROC”22 sample under <i>S. scitamineum</i> stress for 24, 48, and 120 h, respectively.</p><p>Dynamic expression patterns of differentially expressed genes in “ROC”22 after <i>S. scitamineum</i> inoculation.</p

The optimal number of reference genes required for effective normalization in each of four experimental sets in sugarcane.

<p>The pairwise variation (V_n/V_n+1) was analyzed between normalization factors NF_n and NF_n+1 by geNorm program to determined the optimal number of reference genes for accurate normalization in samples from different sugarcane cultivar samples (1^st set), sugarcane hormone-treated (2^nd set), abiotic-treated (3^rd set) and treatments (hormone-& abiotic-treated, 4^th). <i>ACT</i> stand for “β-actin” and <i>TUB</i> stand for “β-tubulin”.</p

Cluster heatmap of expression patterns of differentially co-expressed genes in both sugarcane cultivars at different time points after <i>S. scitamineum</i> inoculation.

<p>T1, T2, T3 and T4 denote “ROC”22 at 24 h after water inoculation, and at 24, 48 and 120 h after <i>S. scitamineum</i> inoculation, respectively; T5, T6, T7 and T8 denote Yacheng05-179 at 24 h after water inoculation, and at 24, 48 and 120 h after <i>S. scitamineum</i> inoculation, respectively; k1∼k9 indicate nine distinct expression patterns of differentially co-expressed genes.</p

Gene expression stability of 13 candidate genes in sugarcane as predicted by geNorm.

<p>Average expression stability (M) following stepwise exclusion of the least stable gene across all the samples within an experimental set. The least stable gene is on the left, and the most stable on the right. The name <i>eIF-4a</i> in the figure stands for <i>eIF-4α. ACT</i> stands for “β-actin” and <i>TUB</i> stands for “β-tubulin”.</p

Assembly results of sugarcane transcriptome using trinity software.

<p>Notes: N50 length is an indicator of measuring assembly effect, which is calculated by the accumulated length of the assembled fragments from long to short. When the sum is greater than or equal to 50% of the total length, the final accumulated fragment length is the N50 value. Mean length = for the average assembly length.</p><p>Assembly results of sugarcane transcriptome using trinity software.</p

Correlation coefficients based on the visualizing reference genes ranked by geNorm, Normfinder and deltaCt.

<p>The p-value indicated by asterisks (***<0.0001; **<0.05).</p

Venn diagram showing the number of genes with sustained differential co-expression between both sugarcane cultivars.

<p>DR-up and DR-down denote continuously up-regulated/down-regulated gene sets in “ROC”22 samples at 24, 48 and 120 h after <i>S. scitamineum</i> inoculation compared to control sample 24 h after water inoculation, respectively; DY-up and DY-down denote continuously up-regulated/down-regulated gene sets in Yacheng05-179 samples at 24, 48 and 120 h after <i>S. scitamineum</i> inoculation compared to control sample 24 h after water inoculation, respectively.</p