Computational Study of the Addition of Methanethiol to 40+ Michael Acceptors as a Model for the Bioconjugation of Cysteines.

A long series of Michael acceptors are studied computationally as potential alternatives to the maleimides that are used in most antibody-drug conjugates to link Cys of mAbs with cytotoxic drugs. The products of the reaction of methanethiol (CH3SH/MeSH, as a simple model of Cys) with N-methylated ethynesulfonamide, 2-ethynylpyridinium ion, propynamide, and methyl ethynephosphonamidate (that is, with HC≡C-EWG) are predicted by the M06-2X/6-311+G(d,p) method to be thermodynamically more stable, in relation to their precursors, than that of MeSH with N-methylmaleimide and, in general, with H2C═CH-EWG; calculations with AcCysOMe and tBuSH are also included. However, for the addition of the anion (MeS-), which is the reactive species, the order changes and N-methylated 2-vinylpyridinium ion, 2,3-butadienamide, and maleimide may give more easily the anionic adducts than several activated triple bonds; moreover, the calculated ΔG⧧ values increase following the order HC≡C-SO2NHMe, N-methylmaleimide, HC≡C-PO(OMe)NHMe, and HC≡C-CONHMe. In other words, MeS- is predicted to react more rapidly with maleimides than with ethynephosphonamidates and with propynamides, in agreement with the experimental results. New mechanistic details are disclosed regarding the advantageous use of some amides, especially of ethynesulfonamides, which, however, are more prone to double additions and exchange reactions.

(Z)-Oxopropene-1,3-diyl, a Linker for the Conjugation of the Thiol Group of Cysteine with Amino-Derivatized Drugs.

We have developed a conjugation reaction based on the thia-Michael addition to activated triple bonds, which can be an alternative to maleimides, the most commonly used reagents to link thiol groups (of Cys) to drugs and labels. An amino group is converted into its propynamide and, in aqueous media at 37 °C and pH 7.4, Cys derivatives are added. The oxopropene-1,3-diyl linker is formed with excellent Z selectivity without secondary reactions. No exchange with other thiols is observed.

Formal Total Synthesis of Amphidinolide E.

A formal total synthesis of the cytotoxic macrolide amphidinolide E is reported. The strategic steps are three Julia-Kocienski reactions (J-K), for the formation of the C5-C6, C9-C10, and C17-C18 double bonds, a Suzuki-Molander C21-C22 bond formation reaction, and a Kita-Trost macrolactonization. The "instability" of the two dienic systems and of the stereocenter at C2 (allylic methine, α to the carboxy group) and the protecting groups at C17-OH and C18-OH have posed difficult challenges. Each Julia-Kocienski olefination has been systematically optimized to provide the highest possible E/Z ratios.

The Performance of Several Docking Programs at Reproducing Protein-Macrolide-Like Crystal Structures.

The accuracy of five docking programs at reproducing crystallographic structures of complexes of 8 macrolides and 12 related macrocyclic structures, all with their corresponding receptors, was evaluated. Self-docking calculations indicated excellent performance in all cases (mean RMSD values ≤ 1.0) and confirmed the speed of AutoDock Vina. Afterwards, the lowest-energy conformer of each molecule and all the conformers lying 0-10 kcal/mol above it (as given by Macrocycle, from MacroModel 10.0) were subjected to standard docking calculations. While each docking method has its own merits, the observed speed of the programs was as follows: Glide 6.6 > AutoDock Vina 1.1.2 > DOCK 6.5 >> AutoDock 4.2.6 > AutoDock 3.0.5. For most of the complexes, the five methods predicted quite correct poses of ligands at the binding sites, but the lower RMSD values for the poses of highest affinity were in the order: Glide 6.6 ≈ AutoDock Vina ≈ DOCK 6.5 > AutoDock 4.2.6 >> AutoDock 3.0.5. By choosing the poses closest to the crystal structure the order was: AutoDock Vina > Glide 6.6 ≈ DOCK 6.5 ≥ AutoDock 4.2.6 >> AutoDock 3.0.5. Re-scoring (AutoDock 4.2.6//AutoDock Vina, Amber Score and MM-GBSA) improved the agreement between the calculated and experimental data. For all intents and purposes, these three methods are equally reliable.

Importance of the Electron Correlation and Dispersion Corrections in Calculations Involving Enamines, Hemiaminals, and Aminals. Comparison of B3LYP, M06-2X, MP2, and CCSD Results with Experimental Data.

While B3LYP, M06-2X, and MP2 calculations predict the ΔG° values for exchange equilibria between enamines and ketones with similar acceptable accuracy, the M06-2X/6-311+G(d,p) and MP2/6-311+G(d,p) methods are required for enamine formation reactions (for example, for enamine 5a, arising from 3-methylbutanal and pyrrolidine). Stronger disagreement was observed when calculated energies of hemiaminals (N,O-acetals) and aminals (N,N-acetals) were compared with experimental equilibrium constants, which are reported here for the first time. Although it is known that the B3LYP method does not provide a good description of the London dispersion forces, while M06-2X and MP2 may overestimate them, it is shown here how large the gaps are and that at least single-point calculations at the CCSD(T)/6-31+G(d) level should be used for these reaction intermediates; CCSD(T)/6-31+G(d) and CCSD(T)/6-311+G(d,p) calculations afford ΔG° values in some cases quite close to MP2/6-311+G(d,p) while in others closer to M06-2X/6-311+G(d,p). The effect of solvents is similarly predicted by the SMD, CPCM, and IEFPCM approaches (with energy differences below 1 kcal/mol).

Total Synthesis of Amphidinolide K, a Macrolide That Stabilizes F-Actin.

The total synthesis of (-)-amphidinolide K (1) based on asymmetric addition of allylsilane C1-C8 to enal C9-C22 is reported. The 1,9,18-tris-O-TBDPS ether was converted into the desired 9,18-dihydroxy acid. Its macrolactonization was accomplished by the Shiina method. Compound 1 together with some of its stereoisomers and analogues were subjected to evaluation of the possible disruption of the α,ß-tubulin-microtubule and/or G-actin-F-actin equilibria. Compound 1 behaves as a stabilizer of actin filaments (F-actin) in vitro.

Tosvinyl and besvinyl as protecting groups of imides, azinones, nucleosides, sultams, and lactams. Catalytic conjugate additions to tosylacetylene.

The use of the 2-(4-methylphenylsulfonyl)ethenyl (tosvinyl, Tsv) group for the protection of the NH group of a series of imides, azinones (including AZT), inosines, and cyclic sulfonamides has been examined. The Tsv-protected derivatives are obtained in excellent yields by conjugate addition to tosylacetylene (ethynyl p-tolyl sulfone). The stereochemistry of the double bond can be controlled at will: with only 1 mol % of Et3N or with catalytic amounts of NaH, the Z stereoisomers are generated almost exclusively, while the E isomers are obtained using a stoichiometric amount of DMAP. Analogous phenylsulfonylvinyl-protected groups (with the besvinyl or Bsv group instead of Tsv) are obtained stereospecifically by reaction with (Z)- or (E)-bis(phenylsulfonyl)ethene. For lactams and oxazolidinones, this last method is much better. The Tsv and Bsv groups are stable in the presence of non-nucleophilic bases and to acids. They can be removed highly effectively via a conjugate addition-elimination mechanism using pyrrolidine or sodium dodecanethiolate as nucleophiles.

Nucleophile-catalyzed additions to activated triple bonds. Protection of lactams, imides, and nucleosides with MocVinyl and related groups.

Additions of lactams, imides, (S)-4-benzyl-1,3-oxazolidin-2-one, 2-pyridone, pyrimidine-2,4-diones (AZT derivatives), or inosines to the electron-deficient triple bonds of methyl propynoate, tert-butyl propynoate, 3-butyn-2-one, N-propynoylmorpholine, or N-methoxy-N-methylpropynamide in the presence of many potential catalysts were examined. DABCO and, second, DMAP appeared to be the best (highest reaction rates and E/Z ratios), while RuCl3, RuClCp*(PPh3)2, AuCl, AuCl(PPh3), CuI, and Cu2(OTf)2 were incapable of catalyzing such additions. The groups incorporated (for example, the 2-(methoxycarbonyl)ethenyl group that we name MocVinyl) serve as protecting groups for the above-mentioned heterocyclic CONH or CONHCO moieties. Deprotections were accomplished via exchange with good nucleophiles: the 1-dodecanethiolate anion turned out to be the most general and efficient reagent, but in some particular cases other nucleophiles also worked (e.g., MocVinyl-inosines can be cleaved with succinimide anion). Some structural and mechanistic details have been accounted for with the help of DFT and MP2 calculations.

Computational Study of the Stability of Pyrrolidine-Derived Iminium Ions: Exchange Equilibria between Iminium Ions and Carbonyl Compounds.

The tendency of carbonyl compounds to form iminium ions by reaction with pyrrolidine or chiral pyrrolidine derivatives (in other words, the relative stability to hydrolysis of these iminium ions) has been computationally examined, mainly using the M06-2X/6-311+G(d,p) method. We have thus obtained the equilibrium positions for R-CH=O + CH2=CH-CH=N+R2* → R-CH=N+R2* + CH2=CH-CH=O reactions and for related exchanges. In these exchanges, there is a transfer of a secondary amine between two carbonyl compounds. Their relative energies may be used to predict which iminium species can be predominantly formed when two or more carbonyl groups are present in a reaction medium. In the catalytic Michael additions of nucleophiles to iminium ions arising from conjugated enals, dienals, and trienals, if the formation of the new Nu-C bond is favorable, the chances of amino-catalyzed reactions to efficiently proceed, with high conversions, depend on the calculated energy values for these exchange equilibria, where the iminium tetrafluoroborates of the adducts (final iminium intermediates) must be more prone to hydrolysis than the initial iminium tetrafluoroborates. The density functional theory (DFT) calculations indicate that the MacMillan catalysts and related oxazolidinones are especially suitable in this regard.

The structure of the human tRNALys3 anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs.

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

Amino-Catalyzed Reactions of Aldehydes with Chiral Nitroalkenes.

Chiral nitroalkenes are used for the first time in Michael additions of aldehydes, catalyzed by pyrrolidine derivatives. They yield the same major stereoisomer with either (S)-proline or (R)-proline, but this asymmetric induction does not overcome the effect of sterically more congested catalysts. Nitrocyclobutane intermediates are often formed, which are more stable than those from (E)-1-nitro-2-phenylethene. The cyclobutanes and final products were characterized by 2D NMR and chemical correlations.

BMS Derivatives C7-Linked to ß-Cyclodextrin and Hyperbranched Polyglycerol Retain Activity against R5-HIV-1NLAD8 Isolates and Can Be Deemed Potential Microbicides.

Amides from indole-3-glyoxylic acid and 4-benzoyl-2-methylpiperazine, which are related to entry inhibitors developed by Bristol-Myers Squibb (BMS), have been synthesized with aliphatic chains located at the C7 position of the indole ring. These spacers contain an azido group suitable for the well-known Cu(I)-catalyzed (3+2)-cycloaddition or an activated triple bond for the nucleophilic addition of thiols under physiological conditions. Reaction with polyols (ß-cyclodextrin and hyperbranched polyglycerol) decorated with complementary click partners has afforded polyol-BMS-like conjugates that are not cytotoxic (TZM.bl cells) and retain the activity against R5-HIV-1NLAD8 isolates. Thus, potential vaginal microbicides based on entry inhibitors, which can be called of 4th generation, are reported here for the first time.

(15)N Double-labeled guanosine from inosine through ring-opening-ring-closing and one-pot Pd-catalyzed C-O and C-N cross-coupling reactions.

[N,1-(15)N(2)]-Guanosine, or [1,NH(2)-(15)N(2)]-guanosine, and derivatives were prepared from tri-O-acetylinosine, via N-nitration and reaction with (15)NH(2)OH, followed by conversion of the (15)N-labeled 1-hydroxyinosine to the corresponding 2,6-dichloropurine riboside. The sequential one-pot C-O and C-N key couplings of this dichloro derivative with PhCH(2)OH and PhCO(15)NH(2) or (i)PrCO(15)NH(2) was achieved in good overall yields, with Pd(0)-Xantphos as the best choice of five different catalytic systems examined.

Catalytic Staudinger-Vilarrasa reaction for the direct ligation of carboxylic acids and azides.

2,2'-Dipyridyl diselenide (PySeSePy) is the catalyst or activator of choice for the direct reaction of carboxylic acids with azides and trimethylphosphine at room temperature. The mechanism of the process, which is not an aza-Wittig reaction, has been elucidated.

NMR and Computational Studies on the Reactions of Enamines with Nitroalkenes That May Pass through Cyclobutanes.

The addition of aldehyde enamines to nitroalkenes affords cyclobutanes in all solvents, with all of the pyrrolidine and proline derivatives tested by us and with all of the substrates we have examined. Depending on the temperature, concentration of water, solvent polarity, and other factors, the opening and hydrolysis of such a four-membered ring may take place rapidly or last for several days, producing the final Michael-like adducts (4-nitrobutanals). Thirteen new cyclobutanes have now been characterized by NMR spectroscopy. As could be expected, s-trans-enamine conformers give rise to all-trans-(4S)-4-nitrocyclobutylpyrrolidines, while s-cis-enamine conformers afford all-trans-(4R)-4-nitrocyclobutylpyrrolidines. These four-membered rings can isomerize to adduct enamines, which should be hydrolyzed via their iminium ions. MP2 and M06-2X calculations predict that one iminium ion is more stable than the other iminium species, so that protonation of the adduct enamines can be quite stereoselective; in the presence of water, the so-called syn adducts (e.g., OCH-*CHR-*CHPh-CH2NO2, with R and Ph syn) eventually become the major products. Why one syn adduct is obtained with aldehydes, whereas cyclic ketones (the predicted ring-fused cyclobutanes of which isomerize to their enamines more easily) produce the other syn adduct, is also explained by means of molecular orbital calculations. Nitro-Michael reactions of aldehyde enamines that "stop" at the nitrocyclobutane stage and final enamine stage do not work catalytically, as known, but those of cyclic ketone enamines that do not work stop at the final enamine stage (if their hydrolysis to the corresponding nitroethylketones is less favorable than expected). These and other facts are accounted for, and the proposals of the groups led by Seebach and Hayashi, Blackmond, and Pihko and Papai are reconciled.

Michael addition-elimination reactions of chiral enolates with ethyl 3-halopropenoates.

Key dienoic or dienal substructures of cytotoxic macrolides amphidinolide E and dictyostatin have been prepared via a Michael addition (followed by elimination of X-) of chiral enolates on beta-halo derivatives of ethyl acrylate, with full retention of the initial E or Z configuration. Evans oxazolidin-2-ones and our related thiazolidin-2-ones, as well as a fine-tuning of the reaction conditions, have been essential. Many chiral building blocks are accessible from these adducts.

Further Insight into the Interactions of the Cytotoxic Macrolides Laulimalide and Peloruside A with Their Common Binding Site.

The binding site of the macrolides laulimalide and peloruside A, which is different from that of the clinically useful drugs paclitaxel/taxol and ixabepilone (tax site), is known to be between two adjacent ß-tubulin units (ext site). Here, we report our study of the binding of these molecules to an α1ß1/α2ß2-tubulin "tetramer" model. AutoDock 4.2.6//AutoDock Vina dockings predicted that the affinities of laulimalide and peloruside A for the tax site are quite similar to those for the ext site. However, molecular dynamics (MD) simulations indicated that only when these two ligands are located at the ext site, there are contacts that help stabilize the system, favoring the ß1/ß2 interactions. The binding affinity of laulimalide for this site is stronger than that of peloruside A, but this is compensated for by additional ß1/ß2 contacts that are induced by peloruside A. MD studies also suggested that epothilones at the tax site and either laulimalide or peloruside A at the ext site cause similar stabilizing effects (mainly linking the M-loop of ß1 and loop H1-B2 of ß2). In a "hexamer" model (3 units of αß-tubulin), the effects are confirmed. Metadynamics simulations of laulimalide and peloruside A, which are reported for the first time, suggest that peloruside A produces a stronger change in the M-loop, which explains the stabilization of the ß1/ß2 interaction.

Efficient preparation of N-phenylsulfenyl ketimines from oximes or nitro compounds without racemization of alpha-stereocenters.

As N-sulfenyl imines (e.g., RR'C=N-SAr) can be readily transformed to their N-sulfinyl imines (RR'C=N-SOAr), N-sulfonyl imines (RR'C=N-SO2Ar), and N-sulfonyl oxaziridines, the very mild procedure developed to convert ketoximes and secondary nitro derivatives to N-arenesulfenyl ketimines constitutes a new and efficient route to all these series of compounds. The configuration of the alpha-stereocenters is retained.

Toward a total synthesis of amphidinolide X and Y. The tetrahydrofuran-containing fragment C12-C21.

The key THF derivative (9a) for an enantioselective synthesis of amphidinolide X/Y was obtained from 1a via a selenoetherification reaction. In fact, among the cyclization methods investigated, the highest yield and stereocontrol were achieved at -78 degrees C with PhSeCl/EtiPr2N from diols 1a (anti-Z) and 1b (anti-E) and with PhSeCl/ZnBr2 from diols 1c (syn-Z) and 1d (syn-E). Also, surprisingly, use of protecting groups (on the allylic OH) was detrimental in the cases studied. The diverse THF-tetrasubstituted stereoisomers will provide a series of amphidinolide X/Y analogues. [structure: see text]

Collision-induced dissociation of cytidine and its derivatives.

The collision-induced dissociation of adenosine, uridine and guanosine, and their corresponding nucleobases has been published previously.1-3 Here we report the collision-induced dissociation of cytidine and the elucidation of its fragmentation pathways using stable isotope-labeled cytidines, through a quadrupole ion trap for tandem mass spectrometry up to MS(4). Furthermore, we investigated the collision-induced dissociation of five cytidine derivatives: 3-methylcytidine, N(4)-methyl-2'-deoxycytidine, 5-methylcytidine, 2-thiocytidine and N(4)-acetylcytidine. The primary fragmentation pathway was the neutral loss of ribose. MS(3) on the retained nucleobase generally resulted in an intense signal from the elimination of ammonia, but also in fragment ions characteristic of the different cytosine derivatives. On the basis of the MS(n) data, fragmentation pathways and plausible mechanisms are suggested.