Conspectus All living systems require biochemical barriers. As a consequence, all drugs, imaging agents, and probes have targets that are either on, in, or inside of these barriers. Fifteen years ago, we initiated research directed at more fully understanding these barriers and at developing tools and strategies for breaching them that could be of use in basic research, imaging, diagnostics and medicine. At the outset of this research and to a lesser extent now, the “rules” for drug design biased the selection of drug candidates to mainly those with an intermediate and narrow log P. At the same time, it was becoming increasingly apparent that Nature had long ago developed clever strategies to circumvent these “rules”. In 1988, for example, independent reports appeared documenting the otherwise uncommon passage of a protein (HIV-Tat) across a membrane. A subsequent study called attention to a highly basic domain in this protein (Tat49–57) being responsible for its cellular entry. This conspicuously contradictory behavior, i.e., a polar, highly charged peptide passing through a non-polar membrane, set the stage for learning how Nature had gotten around the current “rules” of transport. As elaborated in our studies and discussed herein, the key strategy used in Nature rests in part on the ability of a molecule to change its properties as a function of microenvironment, being a polarity chameleon – i.e., being polar in a polar milieu and relatively non-polar in a non-polar environment. Because this research originated in part with the protein Tat and its basic peptide domain, Tat49–57, the field focused heavily on peptides, even limiting its nomenclature to names such as ‘cell-penetrating peptides,’ ‘cell-permeating peptides,’ ‘protein transduction domains,’ and ‘membrane translocating peptides’ to note a few. Starting in 1997, through a systematic reverse engineering approach, we established that the ability of Tat49–57 to enter cells is not a function of its peptide backbone, but rather the number and spatial array of its guanidinium groups. These function-oriented studies allowed one to design more effective peptidic agents and to think beyond the confines of peptidic systems to new and even more effective non-peptidic agents. Because the function of passage across a cell membrane is not limited to or even best achieved with the peptide backbone, we referred to these agents by their shared function, i.e., ‘cell-penetrating molecular transporters’. The scope of this molecular approach to breaching biochemical barriers has expanded remarkably in the past 15 years, enabling or enhancing the delivery of a wide range of cargos into cells and across other biochemical barriers; creating new tools for research, imaging, and diagnostics; and introducing new therapies into clinical trials.

A new family of guanidinium-rich molecular transporters featuring a novel oligocarbonate backbone with 1,7-sidechain spacing is described. Conjugates can be rapidly assembled irrespective of length in a one step oligomerization strategy that can proceed with concomitant introduction of probes (or by analogy drugs). The new transporters exhibit excellent cellular entry as determined by flow cytometry and fluorescence microscopy, and the functionality of their drug delivery capabilities was confirmed by the delivery of the bioluminescent small molecule probe luciferin and turnover by its intracellular target enzyme.New strategies, devices and agents that enable or enhance the passage of drugs or probes across biological barriers are required to address a range of major challenges in chemotherapy, imaging, diagnostics, and mechanistic chemical biology. 1 In 2000, we reported that the cellular uptake of the Tat 49-57 peptide could be mimicked by homooligomers of arginine. 2 Uptake was shown to be a function of the number and array of guanidinium groups, observations that led to the design and synthesis of the first guanidinium-rich (GR) peptoids, 2 GR-spaced peptides, 3 GR-oligocarbamates 4 and GR-dendrimeric molecular transporters (MoTrs). 5 Noteworthy subsequent studies from several groups showed that a variety of other scaffolds, including betapeptides, carbohydrates, heterocycles, and peptide nucleic acids, upon perguanidinylation, exhibit cell-penetrating activity. 6 GR MoTrs have been shown to carry a variety of cargos into cells, including small molecules, probes, metals, peptides, proteins, siRNA, morpholinoRNAs, and DNA plasmids. 7 Activatable MoTrs have been reported for targeted therapy and imaging, 8 a releasable octaarginine-drug conjugate has been shown to overcome Pgp-mediated resistance in animal models of cancer, 9 and a drug-heptaarginine conjugate has been advanced to phase II human clinical trials. 10Correspondence to: James L. Hedrick; Robert M. Waymouth; Paul A. Wender, wenderp@stanford.edu. Supporting Information Available: Experimental procedures, flow cytometry and concentration dependent uptake data, NMR data and fluorescence microscopy images. This material is available free of charge via the internet at http://pubs.acs.org. While octaarginine MoTrs have been made on scale under GMP conditions and a step-saving segment doubling approach has been introduced, 11 the length and associated costs of these syntheses preclude some anticipated applications. A solid phase synthesis of octaarginine requires ≥16 steps, while the segment doubling approach involves 9 steps. 11 We report herein a new family of oligocarbonate GR MoTrs that can be flexibly and efficiently assembled in a one-step organocatalytic ring opening oligomerization process that also allows for concomitant probe (or drug) attachment and control over transporter length. NIH Public AccessWe have previously shown that a metal-free, organocatalytic ring-opening polymerization (ROP) 12 of cyclic carbonates 13 initiated by a var...

The development of abiological catalysts that can function in biological systems is an emerging subject of importance with significant ramifications in synthetic chemistry and the life sciences. Herein we report a biocompatible ruthenium complex [Cp(MQA)Ru(C3H5)]+PF6–2 (Cp = cyclopentadienyl, MQA = 4-methoxyquinoline-2-carboxylate) and a general analytical method for evaluating its performance in real time based on a luciferase reporter system amenable to high throughput screening in cells and by extension to evaluation in luciferase transgenic animals. Precatalyst 2 activates alloc-protected aminoluciferin 4b, a bioluminescence pro-probe, and releases the active luminophore, aminoluciferin (4a), in the presence of luciferase-transfected cells. The formation and enzymatic turnover of 4a, an overall process selected because it emulates pro-drug activation and drug turnover by an intracellular target, is evaluated in real time by photon counting as 4a is converted by intracellular luciferase to oxyaminoluciferin and light. Interestingly, while the catalytic conversion (activation) of 4b to 4a in water produces multiple products, the presence of biological nucleophiles such as thiols prevents byproduct formation and provides almost exclusively luminophore 4a. Our studies show that precatalyst 2 activates 4b extracellularly, exhibits low toxicity at concentrations relevant to catalysis, and is comparably effective in two different cell lines. This proof of concept study shows that precatalyst 2 is a promising lead for bioorthogonal catalytic activation of pro-probes and, by analogy, similarly activatable pro-drugs. More generally, this study provides an analytical method to measure abiological catalytic activation of pro-probes and, by analogy with our earlier studies on pro-Taxol, similarly activatable pro-drugs in real time using a coupled biological catalyst that mediates a bioluminescent readout, providing tools for the study of imaging signal amplification and of targeted therapy.

Interest in algae has significantly accelerated with the increasing recognition of their potentially unique role in medical, materials, energy, bioremediation, and synthetic biological research. However, the introduction of tools to study, control, or expand the inner-workings of algae has lagged behind. Here we describe a general molecular method based on guanidinium-rich molecular transporters (GR-MoTrs) for bringing small and large cargos into algal cells. Significantly, this method is shown to work in wild-type algae that have an intact cell wall. Developed using Chlamydomonas reinhardtii , this method is also successful with less studied algae including Neochloris oleoabundans and Scenedesmus dimorphus thus providing a new and versatile tool for algal research.