Faculty Bibliography

Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with approximately 200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.

Giant unilamellar vesicles (GUVs) represent a versatile model system to emulate the fundamental properties and functions associated with the plasma membrane of living cells. Deformability and shape transitions of lipid vesicles are closely linked to the mechanical properties of the bilayer membrane itself and are typically difficult to control under physiological conditions. Here, we developed a protocol to form cell-sized vesicles from an azobenzene-containing phosphatidylcholine (azo-PC), which undergoes photoisomerization on irradiation with UV-A and visible light. Photoswitching within the photolipid vesicles enabled rapid and precise control of the mechanical properties of the membrane. By varying the intensity and dynamics of the optical stimulus, controlled vesicle shape changes such as budding transitions, invagination, pearling, or the formation of membrane tubes were achieved. With this system, we could mimic the morphology changes normally seen in cells, in the absence of any molecular machines associated with the cytoskeleton. Furthermore, we devised a mechanism to utilize photoswitchable lipid membranes for storing mechanical energy and then releasing it on command as locally usable work.

Sinoracutine is a recently isolated alkaloid with unusual stereochemical and biological properties. It features an unprecedented tetracyclic 6/6/5/5 skeleton that bears an N-methylpyrrolidine ring fused to a cyclopentenone. Interestingly, both enantiomers of sinoracutine have been independently isolated from the same plant, yet the molecule does not appear to occur as a racemate. Here, we present a short synthesis of (-)-sinoracutine that relies on a highly diastereoselective Pauson-Khand reaction and a Mandai-Claisen reaction to install the quaternary stereocenter. Our work establishes the absolute configuration of the levorotatory isomer and suggests that the optical purity of sinoracutine varies in nature due to its gradual racemization. Experimental evidence supports this proposal, and a plausible mechanism for the racemization is provided.

An enantioselective total synthesis of the Lycopodium alkaloid lycoposerramine R is presented. It relies on a base-mediated cyclization that resembles the Conia-ene reaction of ynones and gold-catalyzed variants thereof. Thus, hydrindanones and other functionalized ring systems bearing an exocyclic alkene can be rapidly accessed at room temperature without noble metal catalysis or substrate preactivation.

A mild and general set of metalation procedures for the functionalization of unsymmetrical azobenzenes using a commercially available continuous-flow setup is reported. The metalations proceed with TMPLi under convenient conditions (0 degrees C, 20 s), and various classes of electrophiles can be used. With sensitive substrates, an in situ trapping metalation in which TMPLi is added to a mixture of the azobenzene and ZnCl2 or MgCl2.LiCl was very effective for achieving high yields.

Aplydactone (1) is a brominated ladderane sesquiterpenoid that was isolated from the sea hare Aplysia dactylomela together with the chamigranes dactylone (2) and 10-epi-dactylone (3). Given the habitat of A. dactylomela, it seems likely that 1 is formed from 2 through a photochemical [2 + 2] cycloaddition. Here, we disclose a concise synthesis of 1, 2, and 3 that was guided by excited state theory and relied on several highly stereoselective transformations. Our experiments and calculations confirm the photochemical origin of 1 and explain why it is formed as the sole isomer. Irradiation of 3 with long wavelength UV light resulted in a [2 + 2] cycloaddition that proceeded with opposite regioselectivity. On the basis of this finding, it seems likely that the resulting regioisomer, termed "8-epi-isoaplydactone", could also be found in A. dactylomela.

Two complex norditerpenoids, caribenols A and B, were accessed from a common building block. Our synthesis of caribenol A features the diastereoselective formation of the seven-membered ring through a Friedel-Crafts triflation and a late-stage oxidation of a furan ring. The first synthesis of caribenol B was achieved using an intramolecular organocatalytic alpha-arylation. An unusual intramolecular aldol addition was developed for the assembly of its cyclopentenone moiety, and the challenging trans-diol moiety was installed through a selective nucleophilic addition to a hydroxy 1,2-diketone. Our overall synthetic strategy, which also resulted in a second-generation synthesis of amphilectolide, confirms the usefulness of furans as powerful nucleophiles and versatile synthons.

Two amino acids play a key role in the first total synthesis of lycopalhine A. L-glutamic acid serves as a convenient chiral starting material for the 13-step synthesis, and l-proline promotes an unusual 5-endo-trig Mannich cyclization that generates the central pyrrolidine ring of the Lycopodium alkaloid. The bicyclo[3.3.0]octanol moiety of the molecule is formed through an intramolecular aldol addition that may occur spontaneously in nature.

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [gamma-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.

The arrangement of beta cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual beta cells in orchestrating these responses has long been suspected, but not directly demonstrated. We show here that the beta cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread beta cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.