The development of novel antimicrobial agents having high selectivity toward bacterial cells over mammalian cells is urgently required to curb the widespread emergence of infectious diseases caused by pathogenic bacteria. Toward this end, we have developed a set of cationic dimeric amphiphiles (bearing cleavable amide linkages between the headgroup and the hydrocarbon tail with different methylene spacers) that showed high antibacterial activity against human pathogenic bacteria (Escherichia coli and Staphylococcus aureus) and low cytotoxicity. The Minimum Inhibitory Concentrations (MIC) were found to be very low for the dimeric amphiphiles and were lower or comparable to the monomeric counterpart. In the case of dimeric amphiphiles, MIC was found to decrease with the increase in the spacer chain length (n = 2 to 6) and again to increase at higher spacer length (n > 6). It was found that the compound with six methylene spacers was the most active among all of the amphiphiles (MICs = 10-13 μM). By fluorescence spectroscopy, fluorescence microscopy, and field-emission scanning electron microscopy (FESEM), it was revealed that these cationic amphiphiles interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thus killing the bacteria. All of the cationic amphiphiles showed low hemolytic activity (HC(50)) and high selectivity against both gram-positive and gram-negative bacteria. The most active amphiphile (n = 6) had a 10-13-fold higher HC(50) than did the MIC. Also, this amphiphile did not show any cytotoxicity against mammalian cells (HeLa cells) even at a concentration above the MIC (20 μM). The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.30-0.11 mM) and were 10-27 times smaller than the corresponding monomeric analogue (CMC = 2.9 mM). Chemical hydrolysis and thermogravimetric analysis (TGA) proved that these amphiphiles are quite stable under both acidic and thermal conditions. Collectively, these properties make the newly synthesized amphiphiles potentially superior disinfectants and antiseptics for various biomedical and biotechnological applications.

The micellar aggregation of different amide bearing cleavable gemini surfactants with varying methylene spacer chain length (m = 2, 4, 6, 8, and 12) along with the corresponding monomeric surfactant in aqueous media has been investigated by conductometric and small angle neutron scattering (SANS) studies. The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.08-0.19 mM) and were 23-55 times lesser than the corresponding monomeric analogue (CMC = 4.4 mM). With increase in the spacer chain length, CMC was found to decrease whereas the degree of ionization was found to increase. SANS data have been analyzed by considering the screened coulombic interactions between the micelles to compute the interparticle structure factor S(Q). The extent of micellar growth and the variation of shapes of the micelles formed by these new surfactants in aqueous solution have been found to depend strongly on the spacer chain length. It was observed that the extent of micellar growth and variation of micellar shapes are more pronounced for surfactants with short spacer chain length (m ≤ 4), whereas the surfactants with a long spacer chain length (m ≥ 6) showed slight variation of these properties in aqueous solution. The effects of the variation of the concentration and temperature on the SANS spectra (and hence on the microstructure) of the gemini surfactant (m = 4) were also examined. With an increase in concentration the aggregation number (N) and size of the micelles (the ratio of semimajor axis (a) to semiminor axis (b = c)) increased whereas opposite phenomena was observed with an increase in temperature.

Strategies to visualize cellular membranes with light microscopy are restricted by the diffraction limit of light, which far exceeds the dimensions of lipid bilayers. Here, we describe a method for super-resolution imaging of metabolically labeled phospholipids within cellular membranes. Guided by the principles of expansion microscopy, we develop an all-small molecule approach that enables direct chemical anchoring of bioorthogonally labeled phospholipids into a hydrogel network and is capable of super-resolution imaging of cellular membranes. We apply this method, termed lipid expansion microscopy (LExM), to visualize organelle membranes with precision, including a unique class of membrane-bound structures known as nuclear invaginations. Compatible with standard confocal microscopes, LExM will be widely applicable for super-resolution imaging of phospholipids and cellular membranes in numerous physiological contexts.

Bioorthogonal ligations have been designed and optimized to provide new experimental avenues for understanding biological systems. Generally, these optimizations have focused on improving reaction rates and orthogonality to both biology and other members of the bioorthogonal reaction repertoire. Less well explored are reactions that permit control of bioorthogonal reactivity in space and time. Here we describe a strategy that enables modular control of the cyclopropene-tetrazine ligation. We developed 3-N-substituted spirocyclopropenes that are designed to be unreactive towards 1,2,4,5-tetrazines when bulky N-protecting groups sterically prohibit the tetrazine's approach, and reactive once the groups are removed. We describe the synthesis of 3-N spirocyclopropenes with an appended electron withdrawing group to promote stability. Modification of the cyclopropene 3-N with a bulky, light-cleavable caging group was effective at stifling its reaction with tetrazine, and the caged cyclopropene was resistant to reaction with biological nucleophiles. As expected, upon removal of the light-labile group, the 3-N cyclopropene reacted with tetrazine to form the expected ligation product both in solution and on a tetrazine-modified protein. This reactivity caging strategy leverages the popular carbamate protecting group linkage, enabling the use of diverse caging groups to tailor the reaction's activation modality for specific applications.

We describe a modular activation strategy for cyclopropene–tetrazine ligation. This activation strategy uses chemically diverse enzyme‐ or photolabile protecting groups as cyclopropene reactivity cages. The linkages between the caging groups and cyclopropene are through carbamates, thus permitting the application of diverse cages to allow bioorthogonal reactivity by administering enzymes or light.

Activatable cyclopropenes are unreactive toward their inverse electron demand Diels–Alder reaction partner (e.g., s-tetrazines) until they are activated. The activation strategy is highly modular due to the cyclopropene’s ability to be caged by various light- and enzyme-activatable groups. This work describes the next generation of activatable cyclopropenes with a new core scaffold that maintains the activation modularity of the first generation but improves upon the ligation kinetics with s-tetrazines by ≤270-fold.

Cyclic enamines are important synthons for many synthetic and pharmacological targets. Here, we report an inexpensive, catalyst-free, multigram-scale synthesis for cyclic enamines with exocyclic double bonds and four- to seven-membered rings. This strategy is more conducive to scale up, permissive of functionalization around the cyclic system, and less sensitive to the nature of the N-protecting group than previously-described methods for cyclic enamine synthesis. Further, we explore application of these enamines to the synthesis of highly-strained spirocyclic 3N-cyclopropyl scaffolds.

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