Generation and reactivity studies of dehydrotropylium -cobalt hexa(carbon monoxide)complex: Aromaticity of tropyne-cobalt hexa(carbon monoxide) complexes

Despite a large amount of information on the application of Nicholas reactions in organic syntheses, little attention has been paid to the structural variation of cationic intermediates and the factors that may influence reactivities and stabilities of these species. The primary focus of this project was to design and generate a new type of Nicholas carbocation that possesses multiple sources of stabilization. For this matter, nominally aromatic cation 99 was chosen as the target compound. The effects of resonance stabilization on the stability and reactivity of the cation 99 were investigated both experimentally and by means of computational calculations. From reactivity studies of cation 99, a sharp switching of reaction pathway from electrophilic addition to dimerization was observed for the nucleophiles with N \u3c1. Non-aromatic, highly conjugated acyclic cation 127, was prepared as structural model and its reactivities in Nicholas reactions were investigated for comparison purposes. From experimental and computational studies cation 99 was found to be weakly aromatic with its NICS (1) value approximately 28% of tropylium ion.* Preliminary attempts were made to prepare the precursor to the benzo-fused derivative of dehydrotropylium cation (100). This has led to the formation of phosphonate substituted benzo-fused dehydrotropone ( 151). The scope and limitations of the method for synthesis of other Co2(CO)6-complexes of substituted benzo-fused dehydrotropone (152 and 153) were further investigated.* *Please refer to dissertation for diagrams

Dehydrotropylium-Co2(CO)6 Ion. Generation, Reactivity and Evaluation of Cation Stability

The dehydrotropylium–Co2(CO)6 ion was generated by the action of HBF4 or BF3⋅OEt2 on the corresponding cycloheptadienynol complex, which in turn has been prepared in four steps from a known diacetoxycycloheptenyne complex. The reaction of the cycloheptadienynol complex via the dehydrotropylium–Co2(CO)6 ion with several nucleophiles results in substitution reactions with reactive nucleophiles (N\u3e1) under normal conditions, and a radical dimerisation reaction in the presence of less reactive nucleophiles. Competitive reactions of the cycloheptadienynol complex with an acyclic trienynol complex show no preference for generation of the dehydrotropylium–Co2(CO)6 ion over an acyclic cation. DFT studies on the dehydrotropylium–Co2(CO)6 ion, specifically evaluation of its harmonic oscillator model of aromaticity (HOMA) value (+0.95), its homodesmotic-reaction-based stabilisation energy (≈2.8 kcal mol−1) and its NICS(1) value (−2.9), taken together with the experimental studies suggest that the dehydrotropylium–Co2(CO)6 ion is weakly aromatic

Generation and reactivity of the dehydrotropylium-Co(2)(CO)(6) ion

Dehydrotropylium-Co(2)(CO)(6) ion (2) has been generated by the Lewis acid mediated ionization of alcohol (3); it is attacked by relatively strong nucleophiles (N \u3e 1), but undergoes a radical homocoupling in the presence of weak nucleophiles (N \u3c 1)

Substrates and Inhibitors of SAMHD1

<div><p>SAMHD1 hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. In this paper, we evaluated the impact of 2' sugar moiety substitution for different nucleotides on being substrates for SAMHD1 and mechanisms of actions for the results. We found that dNTPs ((2'<i>R</i>)-2'-H) are only permissive in the catalytic site of SAMHD1 due to L150 exclusion of (2'<i>R</i>)-2'-F and (2'<i>R</i>)-2'-OH nucleotides. However, arabinose ((2'<i>S</i>)-2'-OH) nucleoside-5'-triphosphates analogs are permissive to bind in the catalytic site and be hydrolyzed by SAMHD1. Moreover, when the (2'<i>S</i>)-2' sugar moiety is increased to a (2'<i>S</i>)-2'-methyl as with the SMDU-TP analog, we detect inhibition of SAMHD1’s dNTPase activity. Our computational modeling suggests that (2'<i>S</i>)-2'-methyl sugar moiety clashing with the Y374 of SAMHD1. We speculate that SMDU-TP mechanism of action requires that the analog first docks in the catalytic pocket of SAMHD1 but prevents the A351-V378 helix conformational change from being completed, which is needed before hydrolysis can occur. Collectively we have identified stereoselective 2' substitutions that reveal nucleotide substrate specificity for SAMHD1, and a novel inhibitory mechanism for the dNTPase activity of SAMHD1. Importantly, our data is beneficial for understanding if FDA-approved antiviral and anticancer nucleosides are hydrolyzed by SAMHD1 <i>in vivo</i>.</p></div

Role of 3'-OH sugar moiety for SAMHD1’s substrate specificity.

<p>A-D) Using semi-quantitative HLPC analysis method, 2',3'-ddATP, 2',3'-ddGTP, 2',3'-ddCTP and 2',3'-ddITP are incubated with and without 1.6 μM of SAMHD1 enzyme plus dGTP (A1 site activator) to determine if they are substrates of SAMHD1. Data are presented as the percent compound remaining (y-axis), showing that none of these nucleoside analogs were hydrolyzed by SAMHD1. dGTP is also used as an internal positive control and is significantly hydrolyzed (p < 0.001) by SAMHD1 in the presence of all the 2'3'-ddNTP. Mean and SEM are plotted with significant or no significant (n.s.) differences determined by T test analysis.</p

Ara-CTP does not fit into the A2 site of SAMHD1.

<p>A) Evaluating ara-CTP hydrolysis in the presence of dGTP, using as A1site activator. When dGTP was present, ara-CTP and dCTP were significantly hydrolyzed (p < 0.001) by SAMHD1. Data are presented as the percent compound remaining (y-axis). B) Determining if ara-CTP is hydrolysis by SAMHD1 in the presence of GTP. GTP will only fit into the A1 site of SAMHD1, thus requiring ara-CTP to occupy the A2 and catalytic sites for ara-CTP hydrolysis to occur. The percentage of ara-CTP remained constant with and without SAMHD1, indicating that ara-CTP cannot occupy the A2 site. Reactions containing dCTP was conducted and led to hydrolysis of dCTP in the presence of SAMHD1. Mean and SEM are plotted with significant or no significant (n.s.) differences determined using T test analysis.</p

Examining the role of L150 for nucleotide specificity.

<p>A) dCTP, C) (2'<i>R</i>) 2'-F-dCTP and E) CTP nucleotides (in green) are modeled in the catalytic site of SAMHD1. L150 clashes with (2'<i>R</i>) 2'-F-dCTP and CTP, but not dCTP (see arrows) within the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, (2'<i>R</i>) 2'-F-dCTP and CTP can be hydrolyzed for SAMHD1 <i>in vitro</i>. Structures of the compounds are above the HLPC graphs. Using semi-quantitative HLPC analysis method, compounds were incubated with and without 1.6 μM of SAMHD1 enzyme plus dGTP (A1 site activator) to determine if they are substrates of SAMHD1. Data are presented as the percent compound remaining (y-axis). dCTP and dGTP were significantly hydrolyzed (p < 0.001; T test). No significant (n.s.) differences were detected between samples with and without SAMHD1 protein for (2'<i>R</i>) 2'-F-dCTP and CTP analogs. HPLC analysis of each nucleoside was done twice in triplicate. Mean and SEM are plotted with significant or no significant (n.s.) differences determined by T test analysis.</p

Determining nucleotide analog specificity for SAMHD1.

<p>A) Modification of the 2' sugar position of a nucleotide can lead to several different outcomes. First, (2'<i>R</i>)-2'-F and (2'<i>R</i>)-2'-OH sugar moieties have been shown not to be substrates for SAMHD1. Additional analogs with (2'<i>R</i>)-2'-F and (2'<i>R</i>)-2'-OH sugar moieties would be predicted not to be substrates for SAMHD1. Second, canonical dNTPs and the non-canonical dUTP are substrates for SAMHD1. Our data shows that ((2'<i>S</i>)-2'-OH) arabinose nucleoside-5'-triphosphates are also substrates for SAMHD1. Therefore, we also predict several other arabinose nucleoside analogs would be substrates for SAMHD1. Moreover, clofarabine-TP ((2'<i>S</i>)-2'-F) was reported hydrolyzed by SAMHD1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169052#pone.0169052.ref043" target="_blank">43</a>]. Finally, we found the SMDU-TP, (2'<i>R</i>)-2'-methyl sugar moiety, inhibited the triphosphohydrolase activity of SAMHD1. We postulate that the (2'<i>R</i>)-2'-methyl moiety may prevent the conformational change in the catalytic site of SAMHD1 due to the size of the methyl group clashing with Y374. Therefore, we predicted that nucleotides with a (2'<i>S</i>)-2'-cyano moiety may also inhibit dNTPase activity of SAMHD1. B) A SAMHD1 inhibitor has been reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169052#pone.0169052.ref034" target="_blank">34</a>]. The pppCH₂-dU analog has a 5'-methylene modification, making the analog non-hydrolysable in the catalytic site, but also was shown to block homotetramerization when present in the A2 site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169052#pone.0169052.ref034" target="_blank">34</a>]. C) Modification of the 3'-OH sugar moiety is not permissive. NRTIs and ddNTPs lack a 3'-OH moiety, making them chain terminators for DNA polymerases, are not substrates for SAMHD1. D) Base modifications for different nucleoside analogs are permissive substrates for SAMHD1.</p

Role of Y374 and C2' sugar moiety substitution in acting as substrates of SAMHD1.

<p>A) dCTP, C) ara-CTP and E) SMDU-TP nucleotides (in green) are modeled within the catalytic site of SAMHD1. Both dCTP and ara-CTP do not clash with Y374 (see arrow). However the model shows that the (2'<i>S</i>)-2'-methyl group of SMDU-TP clashes with Y374 in the catalytic pocket of SAMHD1. B, D and F) Determining if dCTP, ara-CTP and SMDU-TP can be hydrolyzed for SAMHD1 <i>in vitro</i>. Structures of the compounds are above the HLPC graphs with experimental conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169052#pone.0169052.g002" target="_blank">Fig 2</a>. Data are presented as the percent compound remaining (y-axis). dCTP and ara-CTP are significantly hydrolyzed (p < 0.001). SMDU-TP and dGTP, in the same reaction tube, had no significant hydrolysis in the presence of SAMHD1. HPLC analysis of each nucleoside was done twice in triplicate. Mean and SEM are plotted with significant or no significant (n.s.) differences determined using T test analysis.</p