Synthesis of a single G-quartet platform in water.

For over 50 years the G-quartet has been a defining self-assembled structure in biology and non-covalent synthesis. It is shown here for the first time that the G-quartet is isolatable in water in the absence of stabilizing G-quartet stacking or cations through the construction of a phosphate-linked template-assembled synthetic G-quartet. Synthetic design has facilitated preservation of the guanine base, ribose sugar, and phosphate components with correct linkage chemistry relative to G-quadruplex DNA. Thus, a minimal synthetic model of G-quadruplex DNA, as in that associated with human gene promoter or telomere regions, is represented by this system. An application as a probe for interactions between G-quadruplex DNA and potential anticancer therapeutical binding ligands is demonstrated. Binding constants of 10(5)-10(7) M(-1) magnitude and 1:1 stoichiometries for TMPyP4, piper, and azatrux ligands were determined, whereas perturbations in BSU1051 and BRACO19 ligand signal were not observed. These data suggest a unique test for critical end-stacking interactions at the exclusion of intercalative or looping interactions for G-quadruplex binding ligands.

A thymine tetrad assembly templated from thymidylic acid.

A template tetra-coupled with thymidylic acid through a phosphate linkage was characterized in methanol for emergent properties of nucleobase tetrad formation. Intramolecular hydrogen bonded base pairing in the absence of a cation was indicated for the thymidylic acid species supporting a monomeric template-assembled structure. Thus, an initial report of a stabilized individual thymine tetrad assembly is presented here. Consistent with previous investigations, a deoxyguanylic acid variant templated an analogous methanolic monomeric G-tetrad in comparison to the thymine species.

Ester hydrolysis by a histidine-containing cavitein.

We have used a template-assembled synthetic protein (TASP) to investigate catalytic function in ester hydrolysis. A histidine-containing cavitein has been found to catalyze ester hydrolysis with a rate increase of 18 times that of background.

A template-assembled synthetic U-quadruplex.

Fo(u)r U: A lipophilic cavitand with four dinucleoside (uridine) residues has been synthesized and characterized. NMR spectroscopy evidence suggests the self-assembly of a U-quadruplex by the uracil nucleobases in organic solution under ambient conditions.

Monomer-dimer control and crystal engineering in TASPs.

We have reported a template assembled synthetic protein (cavitein Q4) as an unexpected dimer in the solid state and as a monomer-dimer equilibrium in solution. We have since reported an ability to bias a cavitein's monomer-dimer equilibrium in solution by sequence design involving histidine metal chelation or disulfide incorporation. However, little remains known about the forces contributing to dimeric cavitein crystal nucleation and lattice stabilization. We, therefore, designed glutamine variants to probe factors involved in dimeric cavitein crystallization. It was found that a key glutamate hydrogen-bonding interaction between dimers is integral to crystal formation and stabilization. Additionally, we obtained a crystal structure of a cavitein (Q4-E3H) designed to bias the dimeric structure via histidine metal coordination. The resolved structure indicates a histidine cluster interaction that likely accounts for the biased dimeric form observed in solution.

Synthesis and characterization of a template-assembled synthetic U-quartet.

A lipophilic cavitand containing four triazole-linked uridine residues has been synthesized and characterized. Spectral evidence suggests a quartet arrangement of the uracil residues at both ambient and low temperatures. Treatment of the compound with Sr(2+) yields a homodimeric complex as evidenced by NMR spectroscopy.

Conformationally constrained sequence designs to bias monomer-dimer equilibriums in TASP systems.

We have designed template-assembled synthetic proteins (TASPs) with the intent of controlling their oligomeric state by stabilizing specific helical tertiary structures via histidine metal ion chelation or disulfide incorporation. In solution, cavitein Q4 was previously determined to interconvert between a four-helix bundle monomer and an eight-helix bundle dimer. In this paper, we show that judicious mutation of cavitein Q4 can stabilize either the monomeric parallel four-helix bundle or the dimeric antiparallel eight-helix bundle structure. Cavitein Q4-E3H, designed to be dimeric, is indeed biased toward dimerization as a result of incorporation of histidines. Moreover, the addition of nickel was found to further increase the association constant of dimerization. Similarly, a cavitein designed to stabilize the monomeric structure via histidine metal ion chelation (Q4-H) was found to favor a monomer in solution upon addition of nickel. Lastly, a cavitein intended to stabilize a monomeric structure via disulfide incorporation (Q4-C2) is reported. Surprisingly, this disulfide cavitein yielded two products upon oxidation suggesting disulfide formation both above the cavitand template and below may be possible. Nevertheless, the two disulfide caviteins were shown to exist as monomers as per their design.

Cation-complexation behavior of template-assembled synthetic G-quartets.

We report the preparation and solution study of a set of template-assembled synthetic G-quartets (TASQs) bound to different cations. These G-quartet baskets effectively extract cations of different sizes and valencies. They form isolated G-quartets with small cations such as Na+ and Sr(2+), and dimeric assemblies with larger cations such as Cs+. Their structures were determined by using (1)H NMR spectroscopy, and their sizes were evaluated by using a series of pulsed-field gradient NMR experiments. The effect of anion has been studied, and the cation selectivities have been investigated by a series of competition experiments.

X-ray crystal analysis of a TASP: structural insights of a cavitein dimer.

Cavitein Q4 is a template assembled synthetic protein designed for X-ray crystallographic analysis. It is based on a previous monomeric helical bundle cavitein (N1GG) that consists of four identical parallel helical peptides. Crystals that were grown in the presence of bromide ions were used to solve the initial phases via single-wavelength anomalous dispersion (SAD). A 1.4 A resolution data set was then refined starting with the SAD phases to provide the crystal structure of cavitein Q4. The crystal structure revealed cavitein Q4 as an asymmetric dimer, although the cavitein appears to be largely monomeric in solution. A comparative analysis is carried out to discern any intrinsic differences between Q4 and its parent cavitein N1GG. We present herein the first X-ray crystal structure of a TASP system and relate this structure to the solution data for both Q4 and its parent N1GG.

An investigation into the native-like properties of de novo designed cavitand-based four-helix bundle proteins.

We investigated the hydrophobic packing of two previously designed caviteins, LG2 and LG3, which differ by one Gly in the linker regions between the peptide sequence and the cavitand scaffold. We sought to diminish the putative native-like properties of LG2 and LG3, and see if we could diagnose a change in the conformational specificity of the hydrophobic core. We replaced the leucine residues with norleucine residues at the hydrophobic positions in LG2 and LG3, to create NG2 and NG3, respectively. LG2 exhibited more dispersion, but less sharp signals than LG3 in the amide region of its (1)H NMR spectrum. NG3 and NG2 were found to be slightly less helical and significantly less stable toward guanidine hydrochloride compared with their reference caviteins. The (1)H NMR spectrum of NG2 was very similar to that of LG2, whereas there was a noticeable loss in the number and sharpness of the amide signals of NG3 compared with LG3. These data suggest that LG3 is very well packed; a loss in conformational specificity resulted from replacement of the leucine residues with norleucine residues. In contrast, the packing and dynamics of the hydrophobic core in LG2 were similar to those in NG2 (both more modest than LG3), as their (1)H NMR spectra were virtually indistinguishable. Overall, substitution of leucine by norleucine provided an efficient, convenient, and informative probe of the packing and dynamics of our caviteins' hydrophobic cores.

The design, synthesis, and characterization of the first cavitand-based de novo hetero-template-assembled synthetic proteins (Hetero-TASPs).

The design, synthesis, and characterization of novel cavitand-based hetero-TASPs, TASPs having different peptide sequences within one bundle, are described. Three families of hetero-TASPs were designed: the LG3/LG2 family (different linker lengths), LG3/AG3 family (altering helix hydrophobicity), and the LG3/LG2C family (anti-parallel caviteins). These first generation hetero-TASPs were found to be alpha-helical, stable towards guanidine hydrochloride, and monomeric in solution. The LG3/LG2 caviteins exhibited primarily native-like properties. The remaining hetero-TASP families were found to exhibit less dispersion and broader signals in the amide regions of their (1)H NMR spectra than their respective reference caviteins. The success in the design of the LG3/LG2 hetero-TASPs suggests that subsequent hetero-TASPs may have potential to manifest superior native-like structure compared with homo-TASPs, and refinement of the linker and peptide sequences may accomplish this goal.

Analysis of peptide design in four-, five-, and six-helix bundle template assembled synthetic protein molecules.

Four-, five-, and six-helix bundle template assembled synthetic proteins (TASPs) have been synthesized using disulfide bonds between cavitand templates and peptides, and characterized in terms of stability and structural specificity. The peptide sequence (CGGGEELLKKLEE LLKKG) used was originally designed for a four-helix bundle. The TASPs were analyzed using CD spectroscopy, chemical denaturation studies, NMR spectroscopy, sedimentation equilibria studies, and hydrophobic dye binding studies to determine the effect of a single peptide sequence when incorporated into bundles with different numbers of helices. If the design was indeed idealized for a four-helix bundle, then the five- and six-helix bundles should be less stable and manifest lower conformational specificity. The TASPs all demonstrated high stability and cooperative unfolding. However, the four-helix bundle was found to be significantly more stable and nativelike compared to the five- and six-helix bundles. This suggests that the peptide sequence is specific to the four-helix bundle, as designed. This result demonstrates the ability to design de novo proteins with specified structure, not just generic stability.

Four-helix bundle cavitein reveals middle leucine as linchpin.

A template-assembled de novo four-helix bundle is used to examine the hydrophobic effect within the bundle interior. Leu to Ala variants of the basis sequence GG-EELLKKLEELLKKG were characterized by GuHCl denaturation, NMR dispersion, and N-H/D exchange experiments. The results show that the middle leucine (L7) is imperative in maintaining bundle stability. The average leucine was found to contribute 1.8 kcal mol(-1) toward stability, whereas the middle leucines contribute 2.7 kcal mol(-1) each. Substituting alanine into the middle position (7) constitutes a striking 95% reduction of the overall cavitein stability.

Optimal attachment position and linker length promote native-like character of cavitand-based template-assembled synthetic proteins (TASPs).

We have designed, synthesised and characterised a series of template-assembled de novo four-helix bundles, each differing in the linker length between the template and the peptides. The helix is based on an earlier peptide sequence: EELLKKLEELLKKLG (first-generation sequence), which was designed to link the hydrophilic/hydrophobic interface of the helices. Increasing or decreasing the linker length by one glycine residue had a significant effect on the structure and properties of the template-assembled synthetic proteins (TASPs). Here, the effect of the linker length is further probed by linking the peptides closer to the hydrophobic face by using the second-generation sequence, AEELLKKLEELLKKG, in an effort to improve the packing between the helices and to better understand the helical bundles. The peptides were synthesised with 0-4 Gly linker residues and linked onto a cavitand template. The proteins were found to be alpha-helical, stable to guanidine hydrochloride (GuHCl) and to unfold cooperatively. However, their stabilities toward GuHCl, propensity to self-aggregate and structural specificity differed. The two-glycine variant of the second-generation series demonstrated the highest stability and most native-like character of all the mononeric TASPs in both the first- and second-generation series. The structural specificity of this two glycine variant is comparable to that of other known native-like de novo proteins. Molecular dynamics simulations showed that the two-glycine variant contains helices that are tilted with respect to the cavitand template and may account for its unique properties.

Donald J. Cram.

Don Cram was definitely old school. Eccentric. Hard driven. Strong-willed. Spirited. Fearless. I was asked to provide personal reflections of Don Cram. I am sure these remembrances will bring back related memories to all who knew Don. But first, a brief background on DJC.

Characterization of de novo four-helix bundles by molecular dynamics simulations.

We have investigated the structure and dynamics of three cavitand-based four-helix bundles (caviteins) by computer simulation. In these systems, designed de novo, each of the four helices contain the identical basis sequence EELLKKLEELLKKG (N1). Each cavitein consists of a rigid macrocycle (cavitand) with four aryl linkages, to each of which is connected an N1 peptide by means of a linker peptide. The three caviteins studied here differ only in the linker peptide, which consist of one, two, or three glycine residues. Previous experimental work has shown that these systems exhibit very different behavior in terms of stability and oligomerization states despite the small differences in the linker peptide. Given that to date no three-dimensional structure is available for these caviteins, we have undertaken a series of molecular dynamics (MD) simulations in explicit water to try to rationalize the large differences in the experimentally observed behavior of these systems. Our results provide insight, for the first time, into why and how the cavitein with a single glycine linker forms dimers. In addition, our results indicate why although the two- and three-glycine-linked caviteins have similar stabilities, they have different native-like characteristics: the cavitein with three glycines can form a supercoiled helix, whereas the one with two glycines cannot. These findings may provide a useful guide in the rational de novo design of novel proteins with finely tunable structures and functions in the future.

A six-bowl carceplex that entraps seven guest molecules.

A six-bowl carceplex that entraps seven guest molecules, 5.(DMSO)7, was synthesized and characterized. The dynamics of the host shell was studied in solution in the absence and presence of water. A multiple-molecule template was found to drive the formation of 5.(DMSO)x.G(7-x) (G = DMA, DMF; x = 5-7). Higher selectivity was found for species containing greater numbers of DMSO molecules.

Ketonization of incarcerated acetophenone enol.

A free-standing simple enol has been generated inside a carceplex. Rates of ketonization under various conditions were determined; ketonization is extremely slow as compared to the rate in solution. Complexed water is required, and the mechanism proceeds via formation of an enolate followed by protonation at carbon by the same molecule of water that removed the proton from the enol. Acid or base retards ketonization by removing water from the cavity.