Application of Simulation and Theory to ... - ACS Publications

Chapter 6

Application of Simulation and Theory to Biocatalysis and Biomimetics Adel M . Naylor and William A. Goddard III

Biocatalysis and Biomimetics Downloaded from pubs.acs.org by FUDAN UNIV on 11/19/16. For personal use only.

Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, C A 91125 Examples are given for the role of simulation and theory in biocatalysis and biomimetics. Simulations on the novel Starburst Dendrimer polymers are used to suggest a design for encapsulating and delivering dopamine to the kidney for cardiovascular therapies. For Dihydrofolate Reductase (DHFR), the simulations (i) indicate why a particular mutation (Phe-31 to Tyr-31) causes a significant change in the catalytic rate, (ii) explain the high degree of kinetic similarity between two dissimilar forms of DHFR, and (iii) suggest that the stable form of the enzyme/substrate complex is not the reactive one.

A goal of our research efforts is to develop the theoretical tools useful i n designing artificial biocatalytic systems for the production of fuels a n d chemical feedstocks. In the long t e r m , one w o u l d envision the development of biocatalysts that could convert methane into more valuable (or more transportable) fuels or t o convert syngas ( C O + H2) i n t o various fuels a n d feedstocks. In the short t e r m , we are focusing o n systems capable of selective oxygenations or reductions. W e w o u l d hope to develop artificial systems w i t h the selectivity of cytochrome P 450 enzymes that play a role i n selective oxidation of long chain alkanes a n d selective epoxidation of alkenes (1). A l t h o u g h selective, the various cytochrome P-450 enzymes involve complex assemblies (cofactors, co-enzymes) that w o u l d be difficult to reproduce a n d control i n an artificial system (see F i g u r e 1). T h u s , we w o u l d hope to incorporate catalytic control procedures as effective as those i n P-450, yet delete the excess baggage special to biological systems (e.g., cofactor, bulk protein necessary to ellicit stability a n d to create substrate b i n d i n g pockets, etc.) T o that e n d we have focused o u r research o n two aspects of this complex problem: (i) the design of artificial systems that possess precision sites capable of selectively encapsulating a n d delivering smaller molecules, a n d (ii) understanding the detailed structural a n d chemical aspects of enzyme active 0097-6156/89/0392-0065$06.75/0 ©1989 American Chemical Society


Figure 1. Schematic representation of cytochrome P-450 indicating the excess baggage carried by many biological catalysts.

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sites involved i n a n d affecting the i m p o r t a n t catalytic processes, i.e., substrate b i n d i n g , product release, rate of catalysis.


Starburst Dendrimers :

Molecular Encapsulation

A recent synthetic development has led to a new class of polymers called "starburst dendrimers" (2) that provide the opportunity for design of precise encap sulation strategies. These novel materials (i) start w i t h an i n i t i a t o r unit (termed the "core") that possesses multiple sites for monomer condensation a n d (ii) use monomer subunits terminating i n functional groups that allow for regularized growth a n d multiple branching at the next generation. W i t h the appropriate use of protecting groups a n d synthetic strategies, each generation of monomer condensation can be completed before embarking o n a new one. Because of the unique chemical requirements for dendrimer formation, these polymers grow i n a systematic manner, producing materials w i t h a well-defined number of monomer subunits a n d a quantized number of terminal groups (Figure 2). W i t h precise topology a n d chemical properties suitable for the encapsulation of specific target molecules that could be delivered to particular organs (by recognition of surface molecules), one c o u l d , i n principle, change the monomer f r o m generation to generation, developing onion-like molecules where every layer of the onion has a different chemical composition, thickness, or number of branching sites. T h u s , one can imagine designing and synthesizing complex polymer structures where (i) i n t e r n a l layers are suitable for encapsulation of the desired molecule, (ii) outer layers are suitable for recognizing b i n d i n g sites on the organ to w h i c h the target molecule is to be delivered, a n d (iii) intermediate layers protect the target molecule i n the b l o o d stream but release t h e m (a) u p o n b i n d i n g of the outer layer to recognition sites, (b) u p o n radiation w i t h external light or charged particles or (c) u p o n change i n acidity or solvent associated w i t h the target organ. β — A l a n i n e Dendrimers. T o illustrate this application, consider the alanine dendrimers where the core unit is a m m o n i a ( w i t h three condensation sites) a n d the monomer u n i t is an amino amide ( w i t h two b r a n c h sites) as depicted below.

Η κ

Ν

Ο -CH -CH -C-N-CH -CH -l/

χ

Η Core

H

2

Η

2

2

Η

2

H

Monomer

E x p e r i m e n t a l l y it has been possible to develop complete dendrimers u p through the tenth generation. However, no experimental structural d a t a are available (these materials are fractal a n d do not crystallize). W e have been carrying out molecular dynamics simulations on these /^-alanine dendrimers u p through generation 9 (3) using the molecular simulation facilities of P O L Y G R A F (from B i o d e s i g n , Inc., Pasadena, C A 91101). These investigations i n d i cate a dramatic change i n the overall structural properties for the /?-alanine sys tems as these polymers grow past generation 4. T h e early generation dendrimers


68

BIOCATALYSIS AND BIOMIMETICS

CORE

GENERATION 1

GENERATION 3

GENERATION 2

GENERATION 4

Figure 2. Cascade growth pattern for the /^-alanine type dendrimers from the core ammonia unit up to generation 4.

6. NAYLOR AND GODDARD


69


(generations 1 through 3) possess very open a n d well-extended structures. These structures are hemispherical disks ( w i t h the core nitrogen responsible for the cur vature) but there is no real inside to the polymer. T h i s topology for the early generation systems is strikingly different f r o m earlier proposals (4), but these hemispheric structures provide a simple interpretation of observed nuclear m a g netic resonance relaxation times (Naylor, A . M . ; G o d d a r d III, W . Α.; Keifer, G . E . ; T o m a l i a , D . A . J . A m . C h e m . S o c , i n press 1988). W e f i n d a distinct change i n structural properties for those polymers above generation 5. T h e overall structure of the polymers is more sphere-like, w i t h ample internal hollows connected by channels that r u n the length of the poly mer assembly. These interior cavities should be suitable for sequestering guest molecules. T h u s , i n F i g u r e 3, w h i c h limns a generation 6 dendrimer, we find an overall spherical structure containing a channel r u n n i n g through the inte rior of the polymer f r o m one side to the other! T o better illustrate the space available i n the interior, we show i n Figure 4 the solvent-accessible surface for the polymer (generation 6). [The solvent-accessible surface is constructed us i n g the approach developed by M i c h a e l C o n n o l l y (University of C a l i f o r n i a , S a n Diego) a n d is based o n the definitions for molecular surfaces proffered by Frederic Richards (5). T h e molecular surfaces are composed of dots located i n terms of a probe sphere (radius = 1.4 Â for water) rolling along the van der W a a l s spheres of the outer (accessible) atoms of the polymer.] Here we have used graphical slabbing to remove f r o m view the front a n d rear portions of the image. T h e channels a n d cavities i n this structure are representative of those found i n the higher generation β-alanine dendrimers. D o p a m i n e — Sequestered Dendrimers. A s an illustration of the dimensions of these channels a n d of how dendrimers might be used to sequester s m a l l molecules, we have used the molecular simulation capabilities of POLYGRAF to predict the o p t i m u m conformations for several dopamine 1 molecules

H N-CH -CH -/

y~

DOPAMINE 1

OH

2

2

2

O H

inside this starburst polymeric m a t r i x . D o p a m i n e was chosen because it is a good candidate for effective sequesterization i n a /^-alanine type dendrimer. Its size a n d shape are suitable for the cavities a n d channels found to exist i n the higher generation /9-alanine systems. F r o m a chemical standpoint, dopamine possesses b o t h the hydrogen b o n d donor a n d acceptor sites needed for favorable b i n d i n g interactions to the polymer's c a r b o n y l , amide, a n d a m i n o substituents. W e estimate that a generation 6 polymer should be capable of holding 15-20 molecules of dopamine. Figure 5 illustrates a dopamine/dendrimer complex w i t h examples of the conformations adopted by the sequestered molecules a n d the polymeric m a t e r i a l .

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Pharmacologically, the ability (i) to selectively deliver dopamine to peripheral kidney receptors without eliciting complications due to the presence of dopamine receptors i n the central nervous system ( C N S ) a n d (ii) to m a i n t a i n a supply of nonmetabolized dopamine w o u l d be advantageous i n cardiovascular hypertension therapy (6). T h e additional issues presented by this type of a p p l i cation include the dimensions of the dendrimer encapsulated dopamine a n d the target t i n g of the unit to the appropriate dopamine receptor sites. T h e dimensions of the higher generation ^ - a l a n i n e dendrimers (see F i g u r e 6 w i t h the m i n i m u m diameter plotted as a function of generation) are such that these d o p a m i n e / d e n d r i m e r complexes w o u l d not readily cross the C N S b l o o d b r a i n barrier. In order to ensure the delivery to kidney dopamine receptors, we suggest m o d i f y i n g the t e r m i n a l amine sites of the polymer w i t h catechol fragments, as shown i n 2

DENDRIMER

CATECHOL 2

T h i s leads to surface features resembling dopamine a n d m a y lead to selective b i n d i n g at dopamine receptor sites (Figure 7). T h e polymer encapsulation m a t r i x should protect the catecholamine f r o m r a p i d metabolic inactivation. These studies provide structural models that should be useful for analyzing the d o p a m i n e / d e n d r i m e r systems. T h e next step is to test the effectiveness of these modified materials for encapsulation of dopamine (and related materials) a n d to determine how effectively they are delivered to the kidney centers. A s such experiments proceed, continuing simulation w i l l be useful i n p r o v i d i n g a quantitative framework for understanding various results. W e expect that the judicious selection of a core u n i t , internal monomer subunits, a n d t e r m i n a l monomer fragments w i l l allow the design of dendrimers to complex w i t h specific guest molecules a n d to deliver t h e m to specific sites. It w i l l , of course, be essential to develop synthetic schemes that allow for chemical control a n d fidelity. Dihydrofolate Reductase ;

Roles of Precise Structure i n Catalysis

In designing new catalysts, it is essential to understand how the character of the active site (including cofactor) controls catalytic activity since one w i l l want to modify this active site or cofactor. A s a prototype for such studies, we have carried out a series of molecular simulations o n the Dihydrofolate Reductase ( D H F R ) system.


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Figure 3. A representative structure for a generation 6 β-alanine dendrimer where generations 1 to 4 are colored red, generation 5 is shown in light blue, and generation 6 is yellow. .

Figure 4. A view of a generation 6 dendrimer with its solvent-accessible surface displayed and clipped to reveal the nature of the internal cavities and channels found in the higher generation β-alanine dendrimers.


Figure 5. A view of various dopamine molecules (magenta) bound in the inner regions of a alanine dendrimer (generations 1 through 5 in light blue; generation 6 in yellow). 75 ι

1

60

MIN


72

45

Û

Ο

>

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