Development of Optimized Tissue-Factor-Targeted Peptide Amphiphile Nanofibers to Slow Noncompressible Torso Hemorrhage.

Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths. We report here on the development of injectable, targeted supramolecular nanotherapeutics based on peptide amphiphile (PA) molecules that are designed to target tissue factor (TF) and, therefore, selectively localize to sites of injury to slow hemorrhage. Eight TF-targeting sequences were identified, synthesized into PA molecules, coassembled with nontargeted backbone PA at various weight percentages, and characterized via circular dichroism spectroscopy, transmission electron microscopy, and X-ray scattering. Following intravenous injection in a rat liver hemorrhage model, two of these PA nanofiber coassemblies exhibited the most specific localization to the site of injury compared to controls (p < 0.05), as quantified using immunofluorescence imaging of injured liver and uninjured organs. To determine if the nanofibers were targeting TF in vivo, a mouse saphenous vein laser injury model was performed and showed that TF-targeted nanofibers colocalized with fibrin, demonstrating increased levels of nanofiber at TF-rich sites. Thromboelastograms obtained using samples of heparinized rat whole blood containing TF demonstrated that no clots were formed in the absence of TF-targeted nanofibers. Lastly, both PA nanofiber coassemblies decreased blood loss in comparison to sham and backbone nanofiber controls by 35-59% (p < 0.05). These data demonstrate an optimal TF-targeted nanofiber that localizes selectively to sites of injury and TF exposure, and, interestingly, reduces blood loss. This research represents a promising initial phase in the development of a TF-targeted injectable therapeutic to reduce preventable deaths from hemorrhage.

Through-Space Activation Can Override Substituent Effects in Electrophilic Aromatic Substitution.

Electrophilic aromatic substitution (EAS) represents one of the most important classes of reactions in all of chemistry. One of the "iron laws" of EAS is that an electron-rich aromatic ring will react more rapidly than an electron-poor ring with suitable electrophiles. In this report, we present unique examples of electron-deficient arenes instead undergoing preferential substitution in intramolecular competition with more electron-rich rings. These results were made possible by exploiting the heretofore unknown propensity of a hydrogen-bonding OH-arene interaction to switch to the alternative HO-arene interaction in order to provide activation. In an extreme case, this through-space HO-arene activation is demonstrated to overcome the deactivating effect of a trifluoromethyl substituent, making an otherwise highly electron-deficient ring the site of exclusive reactivity in competition experiments. Additionally, the HO-arene activation promotes tetrabromination of an increasingly more electron-deficient arene before the unactivated "control" ring undergoes monobromination. It is our hope that these results will shed light on biological interactions as well as provide new strategies for the electrophilic substitution of aromatic rings.

A C-F Bond Directed Diels-Alder Reaction.

We demonstrate a C-F bond driven Diels-Alder reaction of a fluorinated dienophile and a borole that shows remarkable diastereoselectivity. The product's structure was confirmed by X-ray crystallography, revealing an unusual conformation featuring a hypercoordinate boron. Calculations suggest that a B···F interaction instigates the reaction chemistry, the magnitude of which is maximized in the transition state-in essence, the B···F distance "yo-yos" from long to short in the transition state and back again to long in the product.

Positioning a Carbon-Fluorine Bond over the π†Cloud of an Aromatic Ring: A Different Type of Arene Activation.

It is known that the fluoro group has only a small effect on the rates of electrophilic aromatic substitutions. Imagine instead a carbon-fluorine (C-F) bond positioned tightly over the π cloud of an aryl ring-such an orthogonal, noncovalent arrangement could instead stabilize a positively charged arene intermediate or transition state, giving rise to novel electrophilic aromatic substitution chemistry. Herein, we report the synthesis and study of molecule 1, containing a rigid C-F⋅⋅⋅Ar interaction that plays a prominent role in both its reaction chemistry and spectroscopy. For example, we established that the C-F⋅⋅⋅Ar interaction can bring about a >1500 fold increase in the relative rate of an aromatic nitration reaction, affording functionalization on the activated ring exclusively. Overall, these results establish fluoro as a through-space directing/activating group that complements the traditional role of fluorine as a slightly deactivating aryl substituent in nitrations.

Search for a Symmetrical C-F-C Fluoronium Ion in Solution: Kinetic Isotope Effects, Synthetic Labeling, and Computational, Solvent, and Rate Studies.

Recently, we reported evidence for the generation of a symmetrical fluoronium ion (a [C-F-C](+) interaction) in solution from a cage-like precursor, relying heavily on a single isotopic-labeling experiment. Paraphrasing the axiom that a strong claim must be met by as much evidence as possible, we seek to expand upon our initial findings with comprehensive labeling studies, rate measurements, kinetic isotope effect (KIE) experiments, synthetic studies, and computations. We also chronicle the development of the system, our thought process, and how it evolved from a tantalizing indication of fluoronium ion assistance in a dibromination reaction to the final, optimized system. Our experiments show secondary KIE experiments that are fully consistent with a transition state involving fluorine participation; this is also confirmed by a significant remote isotope effect. Paired with DFT calculations, the KIE experiments are indicative of the trapping of a symmetrical intermediate. Additionally, starting with an epimeric in-triflate precursor that hydrolyzes through a putative frontside SNi mechanism involving fluorine participation, KIE studies indicate that an identical intermediate is trapped (the fluoronium ion). Studies also show that the rate-determining step of the fluoronium forming SN1 reaction can be changed on the basis of solvent and additives. We also report the synthesis of a nonfluorinated control substrate to measure a relative anchimeric role of the fluorine atom in hydrolysis versus µ-hydrido bridging. After extensive testing, we can make the remarkable conclusion that our system reacts solely through a "tunable" SN1 mechanism involving a fluoronium ion intermediate. Alternative scenarios, such as SN2 reactivity, do not occur even under forced conditions where they should be highly favored.

Synthesis of a Tight Intramolecular OH···Olefin Interaction, Probed by IR, (1)H NMR, and Quantum Chemistry.

We have synthesized a molecule containing a tight hydrogen-bonding interaction between an alcohol and a nonconjugated π-system. The strength of this hydrogen bond results in a large red shift, nearly 189 cm(-1), on the alcohol stretching frequency in the IR spectrum in comparison to a free alcohol control. The interaction is notable in that it possesses a better defined intramolecular hydrogen bond compared to the usual molecules for which it is noted, such as syn-7-norbornenol. This interaction was studied through the use of IR and NMR spectroscopy, X-ray crystallography, and molecular modeling calculations.

Search for a strong, virtually "no-shift" hydrogen bond: a cage molecule with an exceptional OH⋠⋠⋠F interaction.

Reported herein is the synthesis of a molecule containing an unusually strong hydrogen bond between an OH donor and a covalent F acceptor, a heretofore somewhat ill-defined if not controversial interaction. This unique hydrogen bond is to a large extent a product of the tight framework of the rigid caged system. Remarkably, the interaction shows little to no perceptible shift in the OH stretch of the IR spectrum relative to appropriate nonhydrogen-bound standards in fairly non-interactive solvents. This fascinating example of what has been termed a virtual "no-shift" hydrogen bond is investigated through NMR (coupling constants, isotopic chemical shift perturbations, proton exchange rates) and IR studies which all tell a consistent story.

Modulating "jousting" C-F---H-C interactions with a bit of hydrogen bonding.

We have synthesized a series of molecules wherein very close C-F---H-C σ-bond interactions, which we have termed "jousting", can be perturbed through both red- and blue-shifted hydrogen bonding effects. These interactions were induced by the placement of various functional groups geminal to the H-C bond. "Jousting" interactions appear to be an admixture of F---H hydrogen bonding and C-H bond compression. The associated electronic effects from changes in the functional group at the X-position were also studied.

Evidence for a symmetrical fluoronium ion in solution.

Halonium ions, in which formally positively charged halogens (chlorine, bromine, and iodine) are equivalently attached to two carbon atoms through three-center bonds, are well established in the synthetic chemistry of organochlorides, bromides, and iodides. Mechanistic studies of these ions have generated numerous insights into the origins of stereoselectivity in addition and displacement reactions. However, it has not been clear whether fluorine can form a halonium ion in the same manner. We present chemical and theoretical evidence for the transient generation of a true symmetrical fluoronium ion in solution from an appropriately configured precursor.