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Foresight Update 28 (page 3)

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Table of Contents - Foresight Update 28

Recent Progress: Steps Toward Nanotechnology

by Jeffrey Soreff

Advances in Parallel Techniques

Advances in nanotechnology require techniques for creating complex, 3D, atomically precise structures. One general strategy towards forming these structures is to construct many molecules in parallel, synthesizing macroscopic amounts of materials which are designed to assemble themselves into the desired structures. The following four papers describe advances in these techniques.
Designing high-affinity ligands
In the first paper on techniques for synthesizing macroscopic quantities of materials, S. B. Shuker et. al., writing in [Science 274:1531-1534 29Nov96] describe a systematic method for designing high-affinity ligands using information from NMR. Their technique essentially builds up a composite ligand piece by piece, with excellent control of the detailed geometry of the protein/ligand interface. In this paper they built composite ligands out of two pieces, one of which bound to the protein with Kd = 2.0 mM and the other of which bound with Kd = 100 mM, to yield 5 composite ligands with affinities in the nanomolar range. Their procedure ensures that they can identify initial ligands that bind in two different places, then synthesize a link that will not interfere with the binding in either area. Their method has five steps:
  1. Screen compounds for a ligand that binds to the proteins. In all cases, binding is evaluated by using 15N labeled protein together with an NMR spectroscopy technique called 15N-heteronuclear single quantum correlation (HSQR). "These spectra can be rapidly obtained, making it possible to screen a large number of compounds." Because this technique only "sees" atoms near the 15N atoms, unbound ligand is invisible to it and even weak binding can be detected without prohibitive background signal from unbound ligand.
  2. Generate and evaluate derivative compounds to optimize the binding of the first ligand.
  3. In the presence of enough of the first ligand to saturate its binding site on the protein, screen for ligands that bind to another site on the protein. The saturation by the first ligand ensures that the second ligand will bind to a different place on the protein than the first one.
  4. Generate and evaluate derivative compounds to optimize the binding of the second ligand in the presence of saturating quantities of the first one.
  5. Use structural information from the NMR spectra of the doubly bound protein to find the relative positions of the ligands, allowing design of a link that spans nearby positions on them and that does not collide with the protein.
From a molecular engineering perspective, this technique allows expanding an existing stable structure step by step. At each step the atoms of the starting structure set up a coordinate system which the NMR spectra extend to the new ligands. This might allow an incremental solution to the fold design problem, by designing layer after layer of ligands to bind to an existing stable structure, introducing covalent links after the NMR spectra had determined the geometry of the noncovalent binding. This approach would not be sensitive to errors in fold prediction techniques, since it would rely on experimental data at each step.
Self-assembling cage accelerates Diels-Alder reaction
An article in [C&EN p50 20Jan97] describes the presentation of the James Flack Norris Award to Julius Rebek Jr. Amongst other notable work, the article describes Rebek's work on hydrogen bonded organic structures with cavities. C&EN says that "Such self-assembling superstructures are of tremendous interest for nanotechnology."

An example of the properties of one of these structures is described in a recent article by Rebek and Kang [Nature 385:50-52 2Jan97], the second article described in this section. They describe the acceleration of a Diels-Alder reaction by encapsulation of the reactants in a dimeric capsule. The capsule is assembled from a rather complex compound, containing a primarily linear array of 14 fused rings with quite a few functional groups, primarily containing amides but also containing two hydroquinones. In 3D space, the molecule curves into a "C" shape. Two of these molecules form the capsule, where "Intermolecular hydrogen bonds hold the two subunits together in much the same manner that the stitches along the seam hold a baseball together." Previous work by the authors had shown "that two molecules of solvent benzene are accommodated inside [the capsule], [which] raised the possibility of the use of these capsules as chambers for bimolecular reactions." The particular reaction discussed is the Diels-Alder addition of p-quinone to cyclohexadiene. When both reactants are present at 4mM concentrations and the capsules are present at 1mM concentration the p-quinone is rapidly detectable as an encapsulated species via NMR spectroscopy. Under these conditions the p-quinone reacts to form the Diels-Alder adduct with a time constant of about one day. By contrast, the same concentrations of the two reactants, in the absence of the capsules, have a reaction time constant of roughly a year. The reaction is accelerated roughly 200-fold.

"Self-assembling superstructures...of
tremendous interest for nanotechnology."

The authors did a number of control experiments to exclude several possible effects of the capsules other than geometrical confinement. They excluded the possibility of direct hydrogen bonding of one of the capsule molecule's hydroxyl groups to the reactants by synthesizing a molecule similar to the capsule molecule (with essentially the same functional groups) but with different global stereochemistry (an "S" shape instead of a "C" shape) that precluded formation of a capsule. This variant did not accelerate the reaction. The authors also methylated the hydroxyls of the capsule molecule, preventing the formation of the hydrogen bonds that held the capsule together and again suppressed the acceleration. A variant of the reactants was also examined. Replacing the p-quinone with napthoquinone (a larger molecule that does not fit in the capsule) eliminated the acceleration.

This work is applicable to nanotechnology because it illustrates the control of a reaction forming covalent bonds via nonbonded geometrical constraints on the reactants without the use of enzymes or other biopolymers. The authors write that the "interior of cage-like molecules can be considered to provide a new phase of matter, in which it become possible to stabilize reactive intermediates and to observe new forms of stereoisomerism." This "new phase" has an absence of solvent molecules or other non-reactant species and the presence of a constraining network around the reaction site. Both of these features are analogous to conditions during machine phase chemistry in molecular manufacturing.
Stable helical structures with beta peptides
The third article on parallel techniques discusses beta peptides. Natural proteins are composed of alpha amino acids, compounds where the amino nitrogen and carboxylic acid group are bound to the same carbon atom. Writing in [Nature 385:113-114 9Jan97], B. L. Iverson describes work by the research groups of S. Gellman and D. Seebach on oligomers of beta amino acids, compounds where the amino and acid groups are attached to adjacent carbons. Gellman's group found that a hexamer forms "well-defined helices in methanol solution and in the solid state." Seebach's group also found evidence of a helix in solution. The helices formed are analogous to alpha helices in natural proteins. In these oligomers they are "stabilized by hydrogen bonds between every third unit. This folding pattern results in 14-atom 'rings' being formed by the hydrogen bonds, so Gellman has called the structure the 14-helix." From an engineering viewpoint, the attractive feature of these compounds is the stability of their secondary structure even in very short polymers. By comparison, the shortest alpha peptide that I am aware of that forms a well-defined helix is 23 residues long [Science 271: 342-345 19Jan96]. Perhaps the fold prediction and fold engineering problems will prove to be intrinsically easier with beta peptides than with alpha peptides. These experimental results are "extraordinary, especially when one considers that the extra -CH2- group of the beta-amino acids might be expected to make the resulting resulting beta-peptides more flexible than alpha-amino-acid peptides, not more structured." Each of the two non-carboxylic carbons in a beta peptide residue can carry a side chain, so the potential design freedom for these peptides is higher than for alpha peptides. Further work will tell how much of this design freedom can be exploited while retaining the stable 3D structures demonstrated by Gellman's and Seebach's groups.
Trends in combinational chemistry
In the fourth article on parallel methods, J. C. Hogan, writing in [Nature (supp) 384:17-19 7Nov96] describes trends in combinational chemistry for drug development. He describes past attempts to generate drug leads from peptide and oligonucleotide libraries, but describes them as having been unproductive pharmocologically. He also mentions a wide variety of other oligomer libraries which have been produced by solid phase synthesis, writing: "the synthesis of oligomeric N-alkyl glycines shown in Fig. 1 is an excellent non-peptidic example. Using this [solid phase, with a single coupling chemistry] approach, libraries of oligocarbamates, peptide phosphonates, vinylogous polypeptides and other oligomeric scaffolds have been produced." This spectrum of oligomers would seem to provide a good selection of candidates for stably folded strands with well defined 3D structures for both drug and machine part applications, however Hogan goes on to write: "These molecules generally possess flexible backbones, which can weaken target binding and can also hinder the application of structure-guided techniques."

Hogan goes on to describe new techniques where "multiple variable groups are arranged about a central scaffold or core." In the examples that he shows, the cores appear to be fairly rigid in two of the three cases, with aromatic rings present and only two or three torsional degrees of freedom in the core. Unfortunately, the variable substituents are joined to the core with single bonds in all of the cases that he cites, so the molecules as a whole have quite a bit of flexibility. From a machine perspective, the current trend in synthetic libraries would become much more helpful if cyclization reactions between the substituents were incorporated.

Table of Contents - Foresight Update 28

Advances in Sequential Techniques

The other major strategy for building precise structures relies on sequential operations at some point in the techniques. This may include sequential scanning probe tip placements in a tunneling or force microscope or it may involve electron beam flashes in e-beam lithography. In general, these techniques allow more predictable geometrical control at the cost of slower fabrication than in the parallel methods. The three papers in this section describe advances in these sequential techniques.

STM positioning of buckyballs on a surface
In the first paper M. T. Cuberes, R. Schlittler, and J. K. Gimzewski, writing in [Appl. Phys. Lett. 69:3016-3018 11Nov96], describe the reversible positioning of individual C60 molecules adsorbed on to a step on a Cu(111) surface at room temperature. In a sense, this work is complementary to this group's earlier work on porphyrin manipulation. In that work the structure and flexibility of the mobile molecule, the porphyrin, helped control the binding to the surface, permitting controlled motion while avoiding thermal diffusion. In this case, the C60 molecules are rather rigid, but the Cu(111) surface contains a step edge that confines them to motion in one dimension. In addition "small kinks [in the step edge] are noticeable from the misalignment of the C60 molecules with respect to their neighbors ... The formation of kinks at the Cu steps around the C60 molecules increases the coordination of the molecule with Cu and hinders its diffusion along [emphasis added] the step edge." The authors demonstrated that an "STM tip can separate a C60 molecule from a molecular chain adsorbed at a monatomic Cu(111) [step] and shift it controllably and reversibly back and forth without significantly altering the position of the other atoms in the chain." This implies that STM tips can be remarkably clean, with sufficiently sharp surfaces on both sides that "pushing" molecules from either side can be done with little disturbance to adjacent molecules. This is not what one would expect, for instance, if STM tips always depended on a single critical atom on what otherwise was approximately a 100 nm sphere. This is a hopeful sign for many types of attempts to use scanning tips to build precise structures.

"...our results show promise for further advances in
bottom-up fabrication and operation of devices..."

There are limitations to the positioning in this system. The authors found that "only single molecules can be controllably repositioned in the current system. Attempts to move more than one molecule at a time distort the molecular rows." This, however, sounds like it is only a mechanical instability in a compressed row of spheres rather than a limitation on the precision with which the tip can apply forces. The description of the applicability to nanotechnology is best left in the original authors' words. They conclude: "Therefore, STM-aided manipulation can be used to fabricate a functional counting device based on the abacus mechanism at the molecular scale. Although the use of nanomechanics at the molecular level is still at an early stage of development, our results show promise for further advances in bottom-up fabrication and operation of devices with dimensions on the level of several nanometers."
Lithography-patterned template controls 3D assembly of particles
In the second paper, A. van Blaaderen, R. Ruel, and P. Wiltzius, writing in [Nature 385:321-323 23Jan97], describe a technique for controlling the 3D assembly of colloidal particles by initiating the assembly on a 2D patterned template. Their template was "a 500-nm-thick fluorescent polymer, with holes made with electron beam lithography." They deposited fluorescent silica spheres with a radius of 525 nm by gravity on their polymer template. In the absence of a template, "hard-sphere-like colloidal dispersions are known to crystallize with a random stacking of close packed planes." In this experiment, a (100) pattern of holes in the polymer caused the formation of a pure face centered cubic (FCC) crystal. The 2D template controlled the long range order of the 3D crystal. The (100) slice through the crystal was chosen rather than a denser (111) slice because close packed (111) layers can stack on top of each other in either of two possible positions, creating the possibility of twinning at each layer. In contrast, a (100) layer of hard spheres only permits one possible position for the next (100) layer. "In other words, there is no twinning possibility along this growth direction."

The authors also experimented with mismatched lattices, showing that templates with mismatched spacings generated defects which gradually converted the lattice to a random close packed one. Another experimental variation was to leave a gap between two patterned regions. When the gap was 11 diameters wide, a hexagonal region appeared between the two FCC regions. Manipulations like this might inject a considerable amount of information into the crystal. For example they might introduce twinning boundaries (at an angle to the template) at selected locations in the crystal. The authors suggest that their technique could be extended to charged spheres as well, using a charged template rather than purely hard-sphere-like repulsion.

While the present work does not create an atomically perfect structure, the substitution of virus particles, with well defined structures (and presumably with well defined interparticle contacts in a crystal) would permit lithographic control of a long range atomically perfect structure. This might permit molecular manufacturing to take advantage of the large investment that has been made in fine-line lithography.
Exponential replication of molecular bilayers
The third paper is not directly about an advance in a sequential technique, but rather describes an amplification technique which might improve our ability to exploit sequential techniques. Writing in [Nature 384:150-153 14Nov96], R. Maoz et. al. describe exponential replication of a stack of partially condensed alkyl siloxanol layers. At each step they have bilayers of n-octadecylsiloxanol (formally CH3-(CH2)17-Si(OH)3, but with partial dehydration of the -Si(OH)3 groups, forming lateral covalent Si-O-Si bridges within the layers). Alternate layers have their hydrophilic -Si(OH)3 groups pointing up and pointing down. Each replication is done in two steps. First the stack is treated with wet acetone. This places water molecules between the hydrophilic siloxanol groups. Next, the stack is treated with n-octadecyltrichlorosilane, CH3-(CH2)17-SiCl3. This enters the stack and reacts with the water to form additional n-octadecylsiloxanol, which inserts a new bilayer in between each existing pair of bilayers. The authors write that "A stack of preformed bilayers thus functions as a set of independent template units which define the discrete spatial distribution of the water incorporated into the film, while providing a succession of distinct polar interfaces, each of which is capable of sustaining the spontaneous self-assembly of a similarly structured bilayer." The authors also found that these layers were mechanically robust, with AFM examination showing "no defects, such as holes or steps", and finding that "no surface defects could be induced by the tip."

Ideally, it would be helpful if this work could be extended to allow the accurate complementary replication of layers containing mixed siloxanols. This would essentially yield a 2D analog to PCR. For example, if several different kinds of hydrophilic groups could bind selectively at the polar/polar interface of a bilayer, then an initial lateral pattern could be exponentially amplified to macroscopic quantities. This would greatly enhance the usefulness of the atomically precise patterns that can be produced sequentially with STMs today. One could program them and amplify them much as nucleic acids are handled today, but one would not be faced with predicting the folding of a 1D amino acid sequence into a 3D protein in order to produce a useful structure.

Table of Contents - Foresight Update 28

Foresight Update 28 was originally published 30 March 1997.
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