Two-dimensional (2D) crystalline materials possess unique structural, mechanical, and electronic properties1,2, which have rendered them highly attractive in many applications3-5. Although there have been advances in preparing 2D materials that consist of one or few atomic/molecular layers6,7, bottom-up assembly of 2D crystalline materials remains a considerable challenge and an active area of development8-10. Even more challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamicity in porous 3D crystalline solids has been exploited for stimuli-responsive functions and adaptive behavior11-13. As in the case of such 3D materials, integrating flexibility/adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns through a readily accessible design strategy. Three single- or double-point mutants of the C4 symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single disulfide, double disulfide and metal coordination) into crystalline 2D arrays. Owing to the flexibility of the single disulfide interactions, the lattices of one of the variants (C98RhuA) are essentially defect-free and undergo substantial but fully correlated changes in molecular arrangement, giving coherently dynamic 2D molecular lattices. Notably, C98RhuA lattices possess a Poisson's ratio of −1, the lowest thermodynamically possible value for an isotropic material.

A series of lithium salts of boryl anion, boryllithiums, were synthesized and characterized by NMR spectroscopy and crystallographic analysis. In addition to the parent boryllithium compound 35a, structural modification of boryllithium, using saturated C-C and benzannulated C=C backbones in the five-membered ring and mesityl groups on the nitrogen atoms, also allowed generation of the corresponding boryllithium. The solid state structures of boryllithium showed that the boron-lithium bond is polarized where the boron atom is anionic in all (35a x DME)(2), 35a x (THF)(2), 35b x (THF)(2), and 35c x (THF)(2) when compared to the structures of hydroborane 38a-c and optimized free boryl anion opt-46a-c. Dissolution of the isolated single crystals of (35a x DME)(2) and 35a x (THF)(2) in THF-d(8) showed that the boron-lithium bond remained in solution and free DME or THF molecules were observed. Temperature-dependent (11)B NMR chemical shift changes of 35a were observed in THF-d(8) or methylcyclohexane-d(14), suggesting a change of chemical shift anisotropy around the boron center. The HOMO of opt-35a x (THF)(2) had a lone pair character on the boron atom, as observed for phenyllithium, whereas the HOMO of hydroborane 38a corresponds to the pi-orbital of the boron-containing five-membered heterocycle. The polarity of the B-Li bond, estimated by AIM analysis, was similar to that of alkyllithium. Boryllithiums 35a and 35b behave as a base or a boron nucleophile in reaction with organic electrophiles via deprotonation, S(N)2-type substitution, halogen-metal exchange or electron-transfer, 1,2-addition to a carbonyl group, and S(N)Ar reaction. In the case of the reaction with CO(2), intramolecular cyclization followed by CO elimination from borylcarboxylate anion and subsequent protonation gave hydroxyboranes 64a and 64b. The characters of the carbonyl groups in the borylcarbonyl compounds 60a, 60b, 61, 62, and 63a, which were obtained from the reaction of boryllithiums 35a and 35b, were investigated by X-ray crystallography, IR, and (13)C NMR spectroscopy to show that the boryl substituent weakened the C=O bond when compared to carbon substituted analogues.

Fluorine is a valuable probe for investigating the interactions of biological molecules because of its favorable NMR characteristics, its small size, and its near total absence from biology. Advances in biosynthetic methods allow fluorine to be introduced into peptides and proteins with high precision, and the increasing sensitivity of NMR spectrometers has facilitated the use of (19)F NMR to obtain molecular-level insights into a wide range of often-complex biological interactions. Here, we summarize the advantages of solution-state (19)F NMR for studying the interactions of peptides and proteins with other biological molecules, review methods for the production of fluorine-labeled materials, and describe some representative recent examples in which (19)F NMR has been used to study conformational changes in peptides and proteins and their interactions with other biological molecules.

Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. To gain further insight into the effect of these highly fluorinated amino acids on helix formation exclusively, we measured the helix propensity of three highly fluorinated amino acids: (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), and (S)-pentafluorophenylalanine (Pff). We have developed a short chemoenzymatic synthesis of Hfl with extremely high enantioselectivity (>99%). To measure the helix propensity (w) of the amino acids, alanine-based peptides were synthesized, purified, and investigated by circular dichroism spectroscopy (CD). On the basis of the CD data, the helix propensity of hydrocarbon amino acids can decrease up to 24-fold (1.72 kcal.mol-1.residue-1) upon fluorination. This difference in helix propensity has previously been overlooked in estimating the magnitude of the fluoro-stabilization effect (which has been estimated to be 0.32-0.83 kcal.mol-1.residue-1 for Hfl), resulting in a gross underestimation. Therefore, the full potential of the fluoro-stabilization effect should provide even more stable proteins than the fluoro-stabilized proteins to date.

Telomeric repeat-containing RNA (referred to as TERRA), a noncoding RNA molecule, has recently been found in mammalian cells. The detailed structural features and function of the TERRA RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In the present studies, we investigated the structural features of human TERRA RNA in living cells. Using a light-switching pyrene probe, we found that human TERRA RNA forms a parallel G-quadruplex structure in living cells, providing the in vivo evidence for the presence of the G-quadruplex in human TERRA RNA. Furthermore, imaging experiments clearly show that TERRA RNA G-quadruplex localizes to telomere DNA at cell nuclei. These results provide valuable information to allow understanding of the structure and function of human TERRA RNA.telomere biology | telomere RNA | RNA folding | chemical probe

In the commonly used nucleation-dependent model of protein aggregation, aggregation proceeds only after a lag phase in which the concentration of energetically unfavorable nuclei reaches a critical value. The formation of oligomeric species prior to aggregation can be difficult to detect by current spectroscopic techniques. By using real-time 19F NMR along with other techniques, we are able to show that multiple oligomeric species can be detected during the lag phase of Aβ1-40 fiber formation, consistent with a complex mechanism of aggregation. At least 6 types of oligomers can be detected by 19F NMR. These include the reversible formation of large β-sheet oligomer immediately after solubilization at high peptide concentration; a small oligomer that forms transiently during the early stages of the lag phase; and 4 spectroscopically distinct forms of oligomers with molecular weights between ~30–100 kDa that appear during the later stages of aggregation. The ability to resolve individual oligomers and track their formation in real-time should prove fruitful in understanding the aggregation of amyloidogenic proteins and in isolating potentially toxic non-amyloid oligomers.

Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real-time. We show that alternative pathways of aggregation can be distinguished by using 19F NMR to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable build up of non-fibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of 19F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using 19F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes non-fibrillar large aggregates during fibrillogenesis.

Contents S1-S5 Experimental Section S6 Table S1 S7-S8 Figures S1-S4 S9-S11 1 H, 11 B, and 13 C NMR spectra of 3 inTHF-d 8 S12-S15 1 H, 11 B, 13 C, 7 Li NMR spectra of 4 inTHF-d 8 S16 7 Li NMR spectrum of LiBr in THF-d 8 S17-S19 1 H, 11 B, and 13 C NMR spectra of 5 inTHF-d 8 General. All manipulations were carried out in the argon-filled glovebox (Miwa MFG). The 1 H, 7 Li{ 1 H}, 11 B{ 1 H}, and 13 C{ 1 H} NMR spectra were recorded on 500 MHz spectrometer with residual protiated solvent for 1 H, external LiCl in D 2 O for 7 Li{ 1 H}, external BF 3 •OEt 2 for 11 B{ 1 H}, and deuterated solvent for 13 C{ 1 H} used as reference.THF, hexane, and pentane for the reaction of borylmagnesium were purified by passing through a solvent purification system (Grass Contour) and were further dried by stirring with Na/K alloy at room temperature in the glovebox prior to use unless otherwise noted. THF-d 8 was dried by stirring with Na/K alloy at room temperature in the glovebox prior to use. Lithium powder was made by washing commercial lithium oil dispersion with hexane.Large scale synthesis of 1. A suspension of (2,6-(i-Pr) 2 C 6 H 3 )N=CHCH=N-(2,6-(i-Pr) 2 C 6 H 3 ) (34.6 g, 91.9 mmol) and lithium granule (1.89 g, 272 mmol) in ether (400 mL) was stirred at room temperature for 2 days under argon atmosphere to afford a dark yellow suspension. After removing excess lithium, NEt 3 HCl (26.2 g, 190 mmol) was added to the filtrate, and the resulting white suspension was stirred at room temperature for 1 h. The suspension was filtered through a Celite® pad to remove excess NEt 3 HCl, the solvent and NEt 3 were evaporated and the resulting residue was dissolved in CH 2 Cl 2 (100 ml). BBr 3 solution (1M CH 2 Cl 2 , 100 mL, 100 mmol) was added dropwise to the resulting solution at 0 ºC and stirred at room temperature for 12 h. Reaction was quenched with i Pr 2 EtN (28.2 g, 218 mmol), and the solvents and excess i Pr 2 EtN were removed at reduced pressure. Hexane (400 mL) was added to the resulting residue, the suspension was filtered through a Celite® pad and the insoluble salts were washed with hexane. After volatiles ware removed from the filtrate, recrystallization of the residue from hexane at -45 ºC gave colorless crystals of 1 (32.9 g, 77%). Chemical propaties of the crystal was identical to 1 which we previously reported. S1