reflections on biosystems motivating supramolecular engineering

Biosensors & Bioelectronics 9 (1994) 707-717

Reflections on biosystems motivating supramolecular engineering

Hans Kuhn

Ringoldswilstrasse 50, CH-3656 Tschingel, Switzerland

Abstract: Some goals of bioelectronics-interfacing biology and electronics - are the understanding of supramolecular bioprocesses and the construction of supramolecular devices. The principles for the design and fabrication of machineries with functional components of molecular size are inspired by reflecting on biosystems, and it seems important to consider such principles. We first discuss attempts to construct supramolecular machines, and then we consider the bacterial reaction centre as an example where supramolecular engineering helps to elucidate a bioprocess. We then discuss possible mechanisms leading to the emergence of life-like systems in the light of the basic principles used to design supramolecular devices. Finally, we reflect on prospects in molecular engineering inspired by studying the emergence of life- like systems.

Keywords: molecular engineering, supramolecular devices, bacterial reaction centre, photoinduced electron pump, electron transfer, lock-and-key principle, programmed-environmental-change concept, origin of life, organized molecular assemblies.

1. SUPRAMOLECULAR ENGINEERING

STRATEGIES

Present-day attempts to construct supramolecular machines are based on synthesising molecules that contain regions that are complementary: they ‘recognise’ each other, forming specifically planned aggregates acting as functional units. They interlock like the parts of a jig-saw puzzle. This interaction can take place by spontaneous interaction, interlocking and specific bonding, and it can be further assisted by exposing the molecules to a programmed sequence of environmental changes.

0956-5663/94/$07.00 @ 1994 Elsevier Science Ltd.

Strategies based on the lock-and-key principle

A great variety of supramolecular structures have been created in recent years, beginning with the synthesis of endoreceptor molecules recognising cations by forming cryptate-complexes (Lehn, 1988). Cryptates accepting increasingly sophisticated molecules were obtained. Concave receptors with two receptor sites were planned and realised in which guest molecules are recepted and forced to interlink (e.g. the synthesis of a pyrophosphate by bonding two acetylphosphate molecules). Supramolecular functional units were constructed by complexing several functional components with a cryptate, e.g. systems showing photo-induced energy and electron transfer (Lehn, 1988, 1990; Vogtle et al., 1991).

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Cleft structures were made showing specific binding of, e.g., CAMP by a combination of H-bonding, Coulomb and stacking interactions (Deslongchamps et al., 1992). A self-replicating molecule was obtained based on the idea that adenine, bound to an acceptor for adenine, should constitute a molecule that recognises an identical molecule and may be used as a template to synthesise an identical molecule from the two components. This is a very exciting case of an autocatalytic process (Rebek, 1990). Tennis-ball- like dimers were obtained by interlocking self- complementary molecules (Wyler et al., 1993).

Catenanes were obtained by threading an acceptor chain through a donor ring and subsequently interlinking the ends of the chain (Ashton et al., 1989). In another case, catenanes with three interlinked rings were the result of a template effect of copper ions. (Dietrich- Buchecker et al., 1993). Catenanes also self- organised, beginning by threading a chain into the hole of cyclodextrine (Armspach et al., 1993). Rotaxanes were obtained in a self-organising process by threading a donor chain through an acceptor ring and binding bulky groups to the ends of the chain (Ballardini et al., 1993). These mechanically interlocked structures exhibit remarkable properties of electronic switches of molecular size.

Appropriately constructed molecules self- organise into aggregates of particular architecture (Kunitake, 1992), e.g. molecule stacks forming columns with holes conducting ions (Lehn, 1988; Ahlers et al., 1990). Double helical strands can be constructed by, e.g., binding copper ions to bis-bipyridinium ligands (Lehn, 1990). Purposely functionalised vesicles, micelles and liquid crystals also emerge by self-organisation (Ringsdorf et al., 1988).

Strategies based on the lock-and-key and the programmed-environmental-change principles

Complex assemblies with planned functionalities can be constructed with relatively simple interlocking molecular components if interlocking is facilitated by external influences. In this case, the intention is to construct complex machinery by intelligently planning the nature and sequence of external influences instead of using intelligently planned components leading to machinery by self-organisation. The goal is creating increasingly sophisticated systems by using increasingly com-

plex molecular components as well as more and more intricate procedures for assembling.

A useful way to assist self-assembly is by spreading molecules with a hydrophilic head and a hydrophobic tail on a water surface to produce a monolayer of interlocking component molecules by pushing the molecules together with appropriate speed. Layers of increasing complexity are obtained by co-spreading two or more kinds of molecules (Kuhn & Mobius, 1993). For instance, the amphiphil dimyristoylphosphatidic acid and the nonamphiphil cyclic bisbipyridinium tetracation, in the appropriate ratio, form a well organised and densely packed monolayer (Ahuja et al., 1993a). The cyclic tetracation is below the densely packed amphiphil, with the plane of the ring parallel to the air/water interface. This arrangement acts as receptor for corresponding electron-rich guest molecules.

Such differently constructed monolayers can be manipulated in many ways to form increasingly complex arrangements. They can be transferred to a solid support in a programmed sequence. By including different kinds of chromophores, electron donating and accepting moieties in precise geometry, functional units to study,

e.g., energy transfer, exciton motion, electron tunneling and electron conduction through molecular wires can be obtained. Complex monolayer assemblies deposited on a glass slide can be cleaved between distinct monolayers, e.g. by covering the assembly with a poly(vinylalcoho1) film and lifting the film which serves as a transfer vehicle for the assembly. It can be contacted with a planned target surface. The polymer film on top can be removed again by dissolving it. Then the assembly surface is exposed to the water subphase and further manipulations can take place (Kuhn & Mobius, 1993).

Highly specific reactions can be performed in the planned superstructure of a well-designed monolayer assembly (in situ syntheses, in situ polymerisations, in-plane and interplane connections) (Ruaudel-Teixier, 1988). This is important in aiming to construct distinctly func- tioning machinery.

Monolayers with functional groups exposed to the water subphase can act as receptors, recognising specific groups or molecules dissolved in the subphase. For instance, a monolayer consisting of a barbituric acid lipid can specifically bind 2,4,6_triaminopyrimidine by forming six hydrogen bonds. This process triggers the cleav-

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Biosensors & Bioelectronics Reflections on biosystems motivating supramolecular engineering

age of the C=C bond linking barbituric acid to the lipid (Ahuja et al., 1993b). Binding sfreptavidin to a monolayer of a biotin lipid forming a well- ordered two-dimensional crystal of the protein is another case of specific interaction. This crystal has open binding sites for biotin facing the water subphase. This allows binding of an additional protein linking biotin to this layer, thus producing another layer acting as a programmed functional component (Ahlers ef al., 1990).

Another route to constructing assemblies of monolayers is by chemisorption of appropriate molecules from a solution to a solid support. The end groups of such chemisorbed monolayers can then be chemically modified to reactive

groups, allowing chemisorption of a second monolayer on top of the preceding one, etc. (Sagiv, 1980; Bain et al., 1989; Bain & Whitesides, 1989). Layered structures are fabricated which can be intercalated by, e.g., copper ions through lateral transport along the hydrophilic planes (Maoz & Sagiv, 1992).

In the present context, monolayer assemblies constituting light-induced electron transfer through a spacer layer are of particular interest (Kuhn & Mobius, 1993). The rate of electron transfer from the excited dye to the acceptor decreases exponentially with the spacer layer thickness, which is indicative of quantum mechanical tunnelling.

2. LIGHT-INDUCED ELECTRON PUMP IN THE REACTION CENTRE OF PURPLE BACTERIA. MECHANISM OF ELECTRON MOTION

Studies on electron transfer in monolayer assemblies lead to views on optimal arrangements of the chromophores in light-driven electron pumps (Kuhn, 1986) which are in close agreement with what is actually observed in bacterial reaction centres (Deisenhofer et al., 1984) (Fig. 1). The photoexcited electron must be removed from P (the ‘special pair’ of bacteriochlorophyll molecules) in about one picosecond, i.e. fast compared to the time for de-excitation (150 ps). It must be stored in QL (a quinone) for about one millisecond. In order to prevent the pair of separated charges from subsequent recombination during that time, the distance separating them must be sufficiently large to avoid recombination by quantum mechanical electron tunneling (3

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nm), and the level of Qr- must be sufficiently below P’ in order to avoid recombination by thermal activation (0.5 eV), but not so low that energy is lost unnecessarily.

A n-electron system acting as a molecular wire (Polymeropoulos et al., 1978) is required to shift the electron from P to Qr_. In the reaction centre, this wire is represented by bacteriochlorophyll BL and bacteriopheophytin Hr. The electron stays in Hr_ for about 200 ps before it moves to QL (Zinth & Kaiser, 1993), so the level of Hr- must be about 0.15 eV below the level of P’ to avoid charge recombination by thermal activation. A secondary donor (Cyt) must be present to restore (reduce) oxidised P.

A basic problem is how it is possible to shift the electron with the required high speed from P’ to Br_ and further to Hr_. Assuming an optimised device, Br_- is at the energy level of P’ and the rigidity of the environment prevents interaction with the environment during this early process. Then the time to transfer the electron from P’ to BL is roughly h/2npn = 0.3 ps (uncertainty principle; the matrix element &pg depends upon the overlap of the wave functions, and we find &PB = 6.4 X lop3 eV (see below)). The electron moves rurther to Hr_ and is trapped there by vibronic relaxation within a few picose- conds. The approximate energetic match of P’ and Br- is assumed to be due to evolutionary optimisation.

Considering the situation at T = 0, the electron, after exciting P (time t = 0), should oscillate between P and BL, according to quantum mech- anics. The probability that it is in BL is:

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Ham Kuhn Biosensors & Bioelectronics

. * &pBt PB = sm ( 1 h (1)

Even small fluctuations in the distance between P and BL (and therefore of &pg) will strongly damp the oscillation. pn will soon converge to the average value of 0.5. We assume an equal distribution between &pu-r) and &pnfq for Sim- plicity. Then:

(2)

A distance-fluctuation of 5% (0.05 nm) corre- sponds to a fluctuation of &pu of 20%, and this results in a damping time of 1 ps (eqn. 2). The electron is further transferred to HL at rate rnH = &kBH, where kgH = 2 ps-’ (see below), Pu = 0.5, and thus rnH = 1 PC’.

When the energetic match is not strictly fulfilled and b is the amount by which BL- is above or below the level of P’, then

41pB2 PB = 4epB2 + b2

sin’ ( J4Ppn2 + b2$ (3)

In the situation at room temperature, b will fluctuate in the range ?kT = +26 x 10e3 eV = *8~pu (k=B It o zmann constant). By averaging (assuming an equal distribution between b = +kT and -kT for simplicity) we find:

and thus

rBH T 2&PB L = -arctan rBH,T=O

The measured rate of formation of HL- at T = 300 K is a third of the rate at low temperature (Fleming et al., 1988, Zinth & Kaiser, 1993), and from eqn. (5) we then obtain &pu = 6.4 x 10m3 eV. This value is in the range of estimates from quantum mechanical calculations (10m2 to 10m3 eV, Kuhn, 1986). The oscillation period at low temperature (eqn. 1) is then hl _&pg = 0.65 ps, and the time constant of formation of BL- is roughly half of this, i.e. 0.3 ps.

Measurements of the transient absorption at low temperature show a change in the absorption of P and BL, indicating a time constant of

formation of BL- of 0.3 ps (Zinth & Kaiser, 1993), and a small oscillation with a period of 0.65-0.7 ps (Vos et al., 1992; Zinth & Kaiser, 1993) and a damping time of 0.8 ps (Vos et al., 1992). The agreement with the theoretical expectation supports the present stochastically perturbed adiabatic model for the very first step. The conventional non-adiabatic picture (Marcus & Sutin, 1985; Bixon & Jortner, 1986) does not predict an oscillation. At room temperature, the observed time constant of formation of Br_- is 1 ps, and an oscillation is not seen (Vos et al., 1992; Zinth & Kaiser, 1993). This could be due to stronger fluctuations in &pu with increasing temperature, causing an increased damping of the oscillation. This may slow down the initial speed of formation of B,_- to the measured speed of 1 ps-’ at 300 K. Pn rises to the maximum (l/2)(0.5) = 25% at T = 300 K (eqn. 4). The measured amount is 16% (Zinth & Kaiser, 1993).

The second step, the transfer from Br- to Hi_, can be non-adiabatic in the conventional sense. Then rnH= (27~/h)c~(l/ho Ptib where UAW =5.9 eV-l (w = CC valence vibration) for a glassy medium (Kitzing & Kuhn, in preparation). In the present case (-AGO = hw), &+=(PB+PH) exp(-&-PH) with Bu = BH = 0.48 (as obtained from quantum mechanical modeling n-electrons, Kuhn, 1986). We set cgH = lo-2eV(as suggested by INDO calculations) and obtain rgH = 2

ps-l, as mentioned above. With corresponding assumptions a non-adiabatic first step would be too slow (rpn = (21~/h) c&+(l/h~ Pvib with -AGO z 0, thus pvib = exp(-&-8H) = exp(-0.24 - 0.48) and, therefore, rpu = 0.3 ps-1).

In our model, the importance, of the exact match of the energy levels of P’ and BL- explains the unidirectionality of electron transfer in the reaction centre (restriction to the L branch). The level of BM- is about 0.2 eV above the level of P’ (Moser et al., 1992), and thereforep, (eqn. 3) is only low3 in the case of BM, as compared to 0.5 (T = 0 K) and l/6 (T = 300 K) for Br_. The necessary fast removal of the electron from P is restricted to the L branch. The model is supported by mutants in which the energetic position of BL- is changed. The rate of electron transfer from BL- to H,_ is strongly reduced, indicating the diminished value of &. Changing the energetic position of HL- is much less effective (Finkele et al., 1990, 1992; Schmidt et al. 1994).

We assume that the optimal arrangement of

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the chromophores, and the energy levels balanced to the optimum, are due to continuing selection in the course of evolution. The evolutionary pressure to do so exists only in the L branch and not in the M branch. This is seen by reflecting on how the reaction centre might have evolved (Kuhn, 1986). An early light-induced electron pump is considered to have only one branch, P being a single bacteriochlorophyll molecule and Q being loosely bound and acting as a high- energy electron carrier leading the charge to the pool by diffusion. Dimerisation by forming the special pair must be a great advantage since the special pair acts as an exciton trap, resulting in a highly increased quantum yield in the production of high-energy compounds. The symmetric arrangement has the disadvantage that both QA and Qn are frequently in the pool, and in this case no electron acceptor is present. This is avoided by a change in the protein resulting in a fixation of Qa. So the acceptor QA is always present and stores the high-energy electron until Qn binds, accepts the electron and carries it to the pool. By this division between the functions of QA and Qg, the electron transfer in the M branch must become disadvantageous, so by evolutionary pressure the energetic balance must be kept in the L branch and explicitly avoided in the M branch.

This example indicates the close connection of supramolecular engineering and theoretical modeling in the search for the design principles of supramolecular devices.

3. ORIGIN OF LIFE-LIKE SYSTEMS VIEWED AS A MOLECULAR ENGINEERING PROBLEM

The most challenging problem in molecular engineering is the elucidation of how the bio- machinery started and in what way reflections on this process are useful in future attempts to construct supramolecular machines.

In Section 1, we considered the lock-and- key and the programmed-environmental-change principles as the basic concepts in attempts to design and fabricate supramolecular machines. Our approach to rationalising the origin of life is based on the idea that the two concepts are also the guiding principles in the processes leading to the origin of life-like systems. In the present context, these two principles may be stated as follows:

(i) The lock-and-key principle. Organised molecular assemblies are formed by interlocking precisely matching molecular part- ners like the parts in a jig-saw puzzle. Molecules that do not match are rejected and replaced with correct copies. This effect - self-assembly, precise interlocking, and rejection of erroneous components - constitutes a constructing and repair process of crucial importance for fabricating machinery on all hierarchic levels.

(ii) The programmed-environmental-change concept. Organised molecular assemblies are exposed to distinctly changing external conditions, such as alternation between phases where molecules assemble forming functional entities exposed to competition, and phases of disassembly and component copying. Furthermore, the organised molecular assemblies are exposed to an evolutionary gradient that requires the pres- ence of a regime that cannot be populated by any of the reproducing devices existing at the time. If there occurs a rare beneficial random error somewhere that enables the resulting copy to utilise opportunities existing in a hitherto uncolonised region, and to survive and multiply in that region, the region will be colonised. Thus, increasingly intricate systems populating increasingly inhospitable regions emerge.

The content of these concepts will be illustrated in the following section by modeling the evolution of a translation machine. We shall begin with a simple case (Fig. 2a) and consider the effect of gradually tightening specifications (Fig. 2bg), thus trying to elucidate the basic mechanisms leading to the evolution of life-like systems (Kuhn, 1972; Kuhn & Waser, 1994).

In Fig. 2(a) we consider a solution of some monomers which exist in compartmentalised spaces, e.g. porous rocks. By some exceptional event (say, evaporation of solvent), short strands of oligomers are formed by the linking of monomers with strong inter-monomer bonds. A particular strand has the capability of forming copies under the given conditions. Each monomer of the strand interlocks with a free monomer in such a way that these monomers align at the template strand. In this way, they are induced to form a daughter strand by strongly bonding

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to each other. Subsequently, the two strands separate, e.g. due to rising temperature. Con- ditions will change periodically. Then an increasing number of strands will be formed by being copied in each subsequent period. Depending on the interlocking conditions, the daughter strand can have the same sense of direction (parallel) or the reverse sense (anti-parallel).

Figure 2(b) shows that longer strands evolve by occasional fusion of short strands. They are kept back in regions with larger pores and colonise such regions, in contrast to shorter strands. The populated area extends by the colonisation of larger pores with longer strands, but this lengthening process is limited by mismatch accumulation which prevents copying. Thus, strands consisting of a single kind of monomer cannot evolve further. We must begin our model- ling with different monomers.

Figure 2(c) shows complementary building blocks exposed to the same environmental conditions as in case (b). Strands evolve with sequences which favour the formation of folding structures. Hairpins are the most compact forms and should therefore be selected. For topological reasons, they can only be formed in the case of strands interlocking in an anti-parallel direction.

In Fig. 2(d) the monomers are attributed with the additional property that hairpins bind each other laterally. Aggregates of hairpins evolve. They constitute a supramolecular functional unit. A machinery is present to preserve the know- how of making hairpins.

Figure 2(e) shows that monomers form hairpins that can bind to an assembler strand. More compact aggregates are formed and selected. A strand forming a hairpin and the complementary strand obtained in the replication process ((+) and (-) strands, respectively) have an interesting topological property: they are identical except where complementary elements exist in the middle of the hairpin (where it binds to the assembler strand) and at the open end.

In Fig. 2(f) a second kind of monomer is assumed to be present in two subspecies (symbolised by a circle and a square). These molecules are able to bind to the open ends of (+) and (-) h al ‘rp’ ms, respectively. They can also link together to form an oligomer of the second kind. Oligomers are formed as the assembler hairpins aggregate. The sequence of monomers in the oligomer is determined by the sequence in the assembler. Thus, a translation device

evolves as a by-product of the events described above.

In Fig. 2(g) an oligomer with a distinct sequence intervenes in the replication process and lowers the probability of a mismatch to such an extent that this sequence of the translation product is conserved. Thus, a genetic apparatus evolves.

It should be emphasised that this pattern is both very simple and very intricate. Small variations in specifying the conditions can prevent evolution. For instance, an evolutionary barrier is sur- mounted only in the case where interlocking strands have reverse senses (Fig. 2c), and gener- ally it is important to assume that the spontaneous formation of a strand acting as a template for replication is a rare event, otherwise strands obtained by de nova production and copying would overrun strands evolving gradually by multiplication, variation and selection. Such con- siderations should be helpful in the search for future possibilities in molecular engineering.

The logic pattern of further evolutionary steps can be developed (Kuhn & Waser, 1994). They lead to a reorganisation of the genetic apparatus and to the evolution of a genetic code. The code evolves by the introduction of new amino acids in the approximate order of their availability. This consideration leads logically to the present- day genetic code. The insight thus afforded supports the assembler-hairpins model of the very first translation device and its refinement.

The programmed change in environmental conditions is fundamental, in the present view, to rationalise the emergence of life-like systems. The environmentally driven process considered here should be clearly distinguished from the process leading to complex dissipative structures by the self-imposed organisation of random assemblies of molecules.

4. IDEAS ON FUTURE DEVELOPMENTS

Prospects for the current paradigm

The present-day paradigm for constructing supramolecular machines appears very promising for future attempts to synthesise more and more complex molecules that recognise each other and organise into increasingly sophisticated assemblies. Assisting interlocking by a programmed sequence of external manipulations will be

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Hans Kuhn Biosensors & Bioelectronics

increasingly important with the increasing intricacy of planned machineries.

Manipulating packages of organised assemblies by cleaving and joining with molecular accuracy and the use of biomolecules as interlocking functional components, as well as constructing composites of bio- and synthetic molecules, should play an important role in developing increasingly sophisticated machineries. Using planned solid superstructures as matrices for chemical reactions-highly specific syntheses or reactions linking discrete parts of a superstructure-should also be of interest.

The development of surface treatments (e.g. changing a surface from hydrophilic to hydrophobic) that allow patterns to be imprinted at subnanometer size, on solid supports acting as templates, to form monolayers that have a well-planned in-plane organisation should be most relevant in developing information processing devices. Templates that allow the positioning of individual macromolecules and thus the formation of assemblies with in-plane organisation at the molecular level will be a great challenge. Assemb- ling modules on a two-dimensional base to form three-dimensional complex structures will also be of interest in future supramolecular engineering strategies.

Search for new paradigms

One key feature of the mechanism leading to the emergence of life-like systems is not mimicked in present-day attempts at molecular engineering, i.e. the emergence of increasing complexity and intricacy by multiplication, variation and selection, and it seems important to consider the molecular engineering aspects of the origin of life that might inspire future activities in constructing supramolecular devices. Basic to any attempt in this direction is the availability of a copying mechanism for a blueprint constituting the template to build the machinery. The blueprint may be a one-dimensional or a two-dimensional array. Developments attempting to realise one-dimensional or two-dimensional artificial replicating systems will be important in tracing such perspectives. Another, probably much faster and already promising, approach is using biosystems to synthesise the components of artificial machineries, which are, however, restricted due to their confinement to proteins and nucleotides.

ACKNOWLEDGMENTS

I am grateful to Professors W. Zinth and W. Kaiser for stimulating discussions and important information on bacterial reaction centres. Figure 2 and the explanation in Section 3 are reprinted with permission from IEEE Engineering in Medi- cine and Biology Magazine.

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reflections on biosystems motivating supramolecular engineering

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