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Computational Studies and Design of Biomolecular Diels-Alder Catalysis
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Brinck)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Diels-Alder reaction is one of the most powerful synthetic tools in organic chemistry, and asymmetric Diels-Alder catalysis allows for rapid construction of chiral carbon scaffolds. For this reason, considerable effort has been invested in developing efficient and stereoselective organo- and biocatalysts. However, Diels-Alder is a virtually unknown reaction in Nature, and to engineer an enzyme into a Diels-Alderase is therefore a challenging task. Despite several successful designs of catalytic antibodies since the 1980’s, their catalytic activities have remained low, and no true artificial ’Diels-Alderase’ enzyme was reported before 2010.

In this thesis, we employ state-of-the-art computational tools to study the mechanism of organocatalyzed Diels-Alder in detail, and to redesign existing enzymes into intermolecular Diels-Alder catalysts. Papers I–IV explore the mechanistic variations when employing increasingly activated reactants and the effect of catalysis. In particular, the relation between the traditionally presumed concerted mechanism and a stepwise pathway, forming one bond at a time, is probed. Papers V–X deal with enzyme design and the computational aspects of predicting catalytic activity. Four novel, computationally designed Diels-Alderase candidates are presented in Papers VI–IX. In Paper X, a new parameterization of the Linear Interaction Energy model for predicting protein-ligand affinities is presented.

A general finding in this thesis is that it is difficult to attain large transition state stabilization effects solely by hydrogen bond catalysis. In addition, water (the preferred solvent of enzymes) is well-known for catalyzing Diels- Alder by itself. Therefore, an efficient Diels-Alderase must rely on large binding affinities for the two substrates and preferential binding conformations close to the transition state geometry. In Papers VI–VIII, we co-designed the enzyme active site and substrates in order to achieve the best possible complementarity and maximize binding affinity and pre-organization. Even so, catalysis is limited by the maximum possible stabilization offered by hydrogen bonds, and by the inherently large energy barrier associated with the [4+2] cycloaddition.

The stepwise Diels-Alder pathway, proceeding via a zwitterionic intermediate, may offer a productive alternative for enzyme catalysis, since an enzyme active site may be more differentiated towards stabilizing the high-energy states than for the standard mechanism. In Papers I and III, it is demonstrated that a hydrogen bond donor catalyst provides more stabilization of transition states having pronounced charge-transfer character, which shifts the preference towards a stepwise mechanism.

Another alternative, explored in Paper IX, is to use an α,β -unsaturated ketone as a ’pro-diene’, and let the enzyme generate the diene in situ by general acid/base catalysis. The results show that the potential reduction in the reaction barrier with such a mechanism is much larger than for conventional Diels-Alder. Moreover, an acid/base-mediated pathway is a better mimic of how natural enzymes function, since remarkably few catalyze their reactions solely by non-covalent interactions.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , xii, 138 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2012:34
Keyword [en]
Computational chemistry, density functional theory, enzyme design, molecular modeling, organocatalysis, stepwise Diels-Alder, oxyanion hole
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-101706ISBN: 978-91-7501-435-7 (print)OAI: oai:DiVA.org:kth-101706DiVA: diva2:548763
Public defence
2012-09-21, K1, Teknikringen 56, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20120903

Available from: 2012-09-03 Created: 2012-08-31 Last updated: 2012-09-03Bibliographically approved
List of papers
1. Synergistic activation of the Diels-Alder reaction by an organic catalyst and substituents: a computational study
Open this publication in new window or tab >>Synergistic activation of the Diels-Alder reaction by an organic catalyst and substituents: a computational study
2009 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 7, no 7, 1304-1311 p.Article in journal (Refereed) Published
Abstract [en]

Density functional theory (DFT), using the hybrid functionals B3LYP and B2PLYP, has been employed to investigate the activation of the acrolein-butadiene Diels-Alder reaction, mediated by a thiourea catalyst. Effects due to electron-donating groups (EDGs) on the diene, as well as electron-withdrawing groups (EWGs) on the dienophile, have also been studied. Organic catalysts such as thioureas are known to lower the activation energy through hydrogen-bonding to the carbonyl oxygen, in a way that mimics the oxyanion holes of hydrolytic enzymes. EDGs and EWGs were found to further activate the reaction, and the catalyst showed a synergistic behavior towards the EDGs. Polar solvents were found to reduce the overall activation energy, but also the relative catalytic effect of the thiourea, in accordance with experimental studies. The substituent-mediated reactions displayed more asynchronous transition structures with lower activation energy, which led us to investigate the possibility of an alternative two-step, Michael-type route, similar to what has been found in macrophomate synthase. Although the concerted Diels-Alder route was found to be favored over the Michael route, the calculated activation energy difference is less than 1 kcal mol(-1), which suggests that the two mechanisms compete, and could be responsible for the particular stereochemical outcome of an experiment.

Identifiers
urn:nbn:se:kth:diva-18256 (URN)10.1039/b818655c (DOI)000264349200010 ()2-s2.0-67649349875 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. Mechanistic Insights into the Stepwise Diels-Alder Reaction of 4,6-Dinitrobenzofuroxan
Open this publication in new window or tab >>Mechanistic Insights into the Stepwise Diels-Alder Reaction of 4,6-Dinitrobenzofuroxan
2012 (English)In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 14, no 1, 118-121 p.Article in journal (Refereed) Published
Abstract [en]

The stepwise Diels-Alder reaction between 1-trimethylsiloxy-1,3-butadiene and 4,6-dinitrobenzofuroxan is explored using state-of-the-art computational methods. The results support a stepwise mechanism via a persistent Intermediate, however, not the one previously reported (Lakhdar et al., Chem. Eur. J. 2007, 16,5681) but a heterocyclic adduct. The novel OFT functional M062X and the SCS-MP2 method were essential to reproduce a reasonable potential energy surface for this challenging system.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-91138 (URN)10.1021/ol202913w (DOI)000298828500031 ()2-s2.0-84855483865 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20120309Available from: 2012-03-09 Created: 2012-03-08 Last updated: 2017-12-07Bibliographically approved
3. Stepwise Diels-Alder: More than Just an Oddity? A Computational Mechanistic Study
Open this publication in new window or tab >>Stepwise Diels-Alder: More than Just an Oddity? A Computational Mechanistic Study
2012 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 77, no 15, 6563-6573 p.Article in journal (Refereed) Published
Abstract [en]

We have employed hybrid DFT and SCS-MP2 calculations at the SMD-PCM–6-311++G(2d,2p)//6-31+G(d) level to investigate the relationship between three possible channels for forming a Diels–Alder adduct from a highly nucleophilic diene and moderately to highly electrophilic dienophiles. We discuss geometries optimized using the B3LYP and M06-2X functionals with the 6-31+(d) basis set. The transition states and intermediates are characterized on the basis of geometric and electronic properties, and we also address the possibility of predicting detectability of a zwitterionic intermediate based on its relative stability. Our results show that a conventional Diels–Alder transition state conformation yields intermediates in all four investigated cases, but that these are too short-lived to be detected experimentally for the less activated reactants. The stepwise trans pathway, beginning with a conjugate addition-like transition state, becomes increasingly competitive with more activated reactants and is indeed favored for the most electrophilic dienophiles. Addition of a trans diene leads to a dead-end as the trans intermediates have insurmountable rotation barriers that prohibit formation of the second bond, unless another, heterocyclic intermediate is formed. We also show that introduction of a hydrogen bond donating catalyst favors a stepwise pathway even for less activated dienophiles.

Keyword
Density-Functional Theory, Main-Group Thermochemistry, Noncovalent Interactions, Quantitative Characterization, Cycloaddition Reactions, Organic-Molecules, Metal-Free, M06 Suite, Energies, Butadiene
National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-101708 (URN)10.1021/jo301176t (DOI)000307037700021 ()2-s2.0-84864609647 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20120903

Available from: 2012-09-03 Created: 2012-08-31 Last updated: 2017-12-07Bibliographically approved
4. On the Method-Dependence of Transition State Asynchronicity in Diels-Alder Reactions
Open this publication in new window or tab >>On the Method-Dependence of Transition State Asynchronicity in Diels-Alder Reactions
(English)Manuscript (preprint) (Other academic)
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:kth:diva-101746 (URN)
Note

QS 2012

Available from: 2012-09-03 Created: 2012-09-03 Last updated: 2012-09-03Bibliographically approved
5. Racemase Activity of B. cepacia Lipase Leads to Dual-Function Asymmetric Dynamic Kinetic Resolution of alpha-Aminonitriles
Open this publication in new window or tab >>Racemase Activity of B. cepacia Lipase Leads to Dual-Function Asymmetric Dynamic Kinetic Resolution of alpha-Aminonitriles
Show others...
2011 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 50, no 29, 6592-6595 p.Article in journal (Refereed) Published
Abstract [en]

Applaudable promiscuity: Racemase-type activity discovered for B. cepacia lipase with N-substituted α-aminonitriles is proposed to involve a C-C bond-breaking/forming mechanism in the hydrolase site of the enzyme, as supported by experimental data and calculations. This promiscuous activity in combination with the transacylation activity of the enzyme enabled the asymmetric synthesis of N-methyl α-aminonitrile amides in high yield (see scheme).

Keyword
dynamic kinetic resolution, enzyme catalysis, racemase activity, secondary amines, Strecker reaction
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-37184 (URN)10.1002/anie.201007373 (DOI)000292644400026 ()2-s2.0-79959992526 (Scopus ID)
Note

Uppdated from Manuscript to Article. QC 20120903

Available from: 2011-08-03 Created: 2011-08-03 Last updated: 2017-12-08Bibliographically approved
6. Computational design of a lipase for catalysis of the Diels-Alder reaction
Open this publication in new window or tab >>Computational design of a lipase for catalysis of the Diels-Alder reaction
2011 (English)In: Journal of Molecular Modeling, ISSN 1610-2940, E-ISSN 0948-5023, Vol. 17, no 4, 833-849 p.Article in journal (Refereed) Published
Abstract [en]

Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like nearattack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the alpha,beta-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates (v (cat) /v (uncat) ) of up to 103 are predicted.

Keyword
CALB, DFT, Diels-Alder, Molecular dynamics, Rational design
National Category
Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-33221 (URN)10.1007/s00894-010-0775-8 (DOI)000289531300021 ()2-s2.0-79958143417 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20110512Available from: 2011-05-12 Created: 2011-05-02 Last updated: 2017-12-11Bibliographically approved
7. Designing a New Diels-Alderase: A Combinatorial, Semirational Approach Including Dynamic Optimization
Open this publication in new window or tab >>Designing a New Diels-Alderase: A Combinatorial, Semirational Approach Including Dynamic Optimization
Show others...
2011 (English)In: JOURNAL OF CHEMICAL INFORMATION AND MODELING, ISSN 1549-9596, Vol. 51, no 8, 1906-1917 p.Article in journal (Refereed) Published
Abstract [en]

A computationally inexpensive design strategy involving 'semirational' screening for enzymatic catalysis is presented. The protocol is based on well-established computational methods and represents a holistic approach to the catalytic process. The model reaction studied here is the Diels-Alder, for which a successful computational design has recently been published (Siegel, J. B. et al. Science 2010, 329, 309-313). While it is a leap forward in the field of computational design, the focus on designing only a small fraction of the active site gives little control over dynamics. Our approach explicitly incorporates mutagenesis and the analysis of binding events and transition states, and a promising enzyme substrate candidate is generated with relatively little effort. We estimate catalytic rate accelerations of up to 10(5).

Keyword
Chemistry, Multidisciplinary, Computer Science, Information Systems, Computer Science, Interdisciplinary Applications
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-39525 (URN)10.1021/ci200177d (DOI)000294081800017 ()2-s2.0-80052015812 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20110916

Available from: 2011-09-16 Created: 2011-09-12 Last updated: 2012-09-03Bibliographically approved
8. Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics
Open this publication in new window or tab >>Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics
Show others...
2012 (English)In: Journal of Computer-Aided Molecular Design, ISSN 0920-654X, E-ISSN 1573-4951, Vol. 26, no 9, 1079-1095 p.Article in journal (Refereed) Published
Abstract [en]

A novel computational Diels-Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus, is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels-Alderases with good results. The final designs in this work are predicted to have rate enhancements of a parts per thousand 10(3)-10(6) and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, 'ideal' model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme (T (opt) = 70 A degrees C).

Keyword
Diels-Alder, Computational enzyme design, DFT, Molecular dynamics
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:kth:diva-101711 (URN)10.1007/s10822-012-9601-y (DOI)000310089500009 ()2-s2.0-84869507791 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20121206

Available from: 2012-08-31 Created: 2012-08-31 Last updated: 2017-12-07Bibliographically approved
9. Envisioning an enzymatic Diels-Alder reaction by in situ acid-base catalyzed diene generation
Open this publication in new window or tab >>Envisioning an enzymatic Diels-Alder reaction by in situ acid-base catalyzed diene generation
Show others...
2012 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 48, no 45, 5665-5667 p.Article in journal (Refereed) Published
Abstract [en]

We present and evaluate a new and potentially efficient route for enzyme-mediated Diels-Alder reactions, utilizing general acid-base catalysis. The viability of employing the active site of ketosteroid isomerase is demonstrated.

Keyword
Delta(5)-3-Ketosteroid Isomerase, 3-Oxo-Delta-5-Steroid Isomerase, Noncovalent Interactions, Thermochemical Kinetics, Density Functionals, Antibody Catalysis, Design, Cycloadditions, Rearrangements, Energetics
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-96744 (URN)10.1039/c2cc31502e (DOI)000303889500037 ()2-s2.0-84862106071 (Scopus ID)
Note
QC 20120611Available from: 2012-06-11 Created: 2012-06-11 Last updated: 2017-12-07Bibliographically approved
10. "Adapted Linear Interaction Energy": A Structure-Based LIE Parametrization for Fast Prediction of Protein-Ligand Affinities
Open this publication in new window or tab >>"Adapted Linear Interaction Energy": A Structure-Based LIE Parametrization for Fast Prediction of Protein-Ligand Affinities
2013 (English)In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 9, no 2, 1230-1239 p.Article in journal (Refereed) Published
Abstract [en]

We present a structure-based parametrization of the Linear Interaction Energy (LIE) method and show that it allows for the prediction of absolute protein-ligand binding energies. We call the new model "Adapted" LIE (ALIE) because the a and beta coefficients are defined by system-dependent descriptors and do therefore not require any empirical gamma term. The best formulation attains a mean average deviation of 1.8 kcal/mol for a diverse test set and depends on only one fitted parameter. It is robust with respect to additional fitting and cross-validation. We compare this new approach with standard LIE by Aqvist and co-workers and the LIE + gamma SASA model (initially suggested by Jorgensen and co-workers) against in-house and external data sets and discuss their applicabilities.

Keyword
Monte-Carlo Simulations, Molecular-Dynamics Simulations, Binding Free-Energies, Hiv-1 Reverse-Transcriptase, Particle Mesh Ewald, Crystal-Structure, Continuum Solvent, Escherichia-Coli, Response Method, Efficient Generation
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-101710 (URN)10.1021/ct300783e (DOI)000315018300041 ()2-s2.0-84873671255 (Scopus ID)
Note

QC 20130322. Updated from manuscript to article in journal.

Available from: 2012-08-31 Created: 2012-08-31 Last updated: 2017-12-07Bibliographically approved
11. Computational enzyme design: Advances, hurdles and possible ways forward
Open this publication in new window or tab >>Computational enzyme design: Advances, hurdles and possible ways forward
2012 (English)In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, Vol. 2, no 3, e201209009Article, review/survey (Refereed) Published
Abstract [en]

 This mini review addresses recent developments in computational enzyme design. Successful protocols as well as known issues and limitations are discussed from an energetic perspective. It will be argued that improved results can be obtained by including a dynamic treatment in the design protocol. Finally, a molecular dynamics-based approach for evaluating and refining computational designs is presented.

Keyword
enzyme design, computational chemistry, enzyme catalysis
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:kth:diva-101709 (URN)10.5936/csbj.201209009 (DOI)2-s2.0-84902178089 (Scopus ID)
Note

QC 20150528. Updated from manuscript to article in journal.

Available from: 2012-08-31 Created: 2012-08-31 Last updated: 2015-05-28Bibliographically approved

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