Oligocarbonate Molecular Transporters: Oligomerization-Based Syntheses and Cell-Penetrating Studies

Abstract

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...

Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells

“…As had been shown previously for the guanidinium-only carbonate oligomers, the carbonate backbone is shelf stable as a solid but, as desired for cargo release after cell entry, it hydrolyses with a half-life of about 8 h in Hepes-buffered saline (pH 7.4, 37°C) (26). We anticipated that the siRNA∶co-oligomer complexes would be similarly stable during cellular entry but subsequently degrade with the release of free siRNA.…”

“…We and others have since shown that these guanidinium-rich molecular transporters can enable or enhance the delivery of a variety of cargos, including small molecules, metals, imaging agents, peptides, plasmids, and proteins across biological barriers, such as cell membranes and the stratum corneum, the latter as part of a clinical trial (24,25). Recently, we developed an oligomerization strategy that generates unique guanidinium-rich homo-oligocarbonate molecular transporters in an exceptionally step-economical fashion (one to two steps) through a metal-free organocatalytic ring-opening oligomerization reaction (26). With this strategy, the length of the oligomer (degree of polymerization, DP) can be easily tuned by varying the ratio of monomer to initiator in the oligomerization step.…”

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…Contrasting their efficacy in delivering siRNA, complexes formed with guanidinium-functionalized oligocarbonates D 4 :G 4 12 and D 13 :G 12 13 resulted in no detectable EGFP expression. Relative to the rapid self-immolative rearrangement (t 1/2 = 2 min) of CARTs, oligocarbonates 12 and 13 degrade slowly by passive hydrolysis (t 1/2 = 8-12 h) (46,49), establishing a strong correlation between transporter degradation rate and mRNA expression. Collectively, the exceptional performance of the CARTs is consistent with our initial hypothesis that endosomal escape and cytosolic mRNA release can be attributed to the rapid charge-altering transformation of cationic amines to neutral amides.…”

Synthesis of Guanidinium‐Modified Hyaluronic Acid Hydrogel

“…[14] Recently, various synthetic polymers with guanidinium side chains have been developed and successfully used for cellular delivery. [14,15] The mode of delivery of the cargo molecules by such a delivery vehicle has been debated, however, now it is accepted that polyelectrolyte complexes bind to cell surface proteoglycans such as heparin sulfate and are internalised by macropinocytosis. [16] In this report, we present the synthesis of a nonviral, noncationic delivery vector that not only binds to DNA by ionic interactions but also has cell surface targeting proteoglycan (HA) which could facilitate gene delivery.…”