MSc Chemistry
Master Thesis
Supramolecular Rhodium
Complexes with Small
Peptides and Application in
Asymmetric Hydrogenation
by
Kasper van den Hurk
5681944
Supervisor:
Examiner:
Van ’t Hoff Institute for Molecular Sciences
Homogeneous, Supramolecular and Bio-Inspired Catalysis
Summary
The need for more efficient synthesis procedures for biologically active compounds calls for catalysts with high activity and enantioselectivity. Over the last decade, significant efforts have been made to merge the attractive properties of natural and synthetic catalysts by introducing non-natural transition metals into proteins. These artificial metalloenzymes have shown great potential in
achieving enzyme-like activity and enantioselectivity for transition metal catalysts by using the second coordination sphere of the protein. This thesis describes a novel anchoring technique for the
supramolecular binding of a catalytically active transition metal center to a small peptide. This technique makes use of a bidentate phosphorus sulfonanmide ligand and two adjacent histidine residues in the peptide, exploiting their hydrogen bond donor and acceptor abilities, respectively. This technique was used to make supramolecular rhodium complexes with three different small peptides. Results in asymmetric catalysis are still fairly modest, but there is real potential.
Samenvatting
Door de groeiende wereldbevolking en de uitputting van grondstoffen wordt het steeds belangrijker om productieprocessen efficiënter te maken, waardoor er minder energie en grondstoffen nodig zijn voor de productie van bijvoorbeeld voedsel en medicijnen. Ook in de chemische industrie ligt daarom steeds meer de nadruk op het verbeteren van processen waardoor de productie van afvalstoffen en het gebruik van energie worden verlaagd.
Om die reden wordt er in de chemische industrie vaak gebruik gemaakt van katalysatoren. Deze stoffen helpen de energiebarrière voor de productie van de gewenste stof te verlagen, waardoor er minder toevoer van energie nodig is en er minder afvalstoffen worden geproduceerd.
Vaak kunnen tijdens een chemische reactie twee spiegelbeelden van hetzelfde molecuul ontstaan. Meestal is daarvan maar één bruikbaar. Dit komt omdat veel bouwstenen van de natuur, zoals aminozuren en suikers, maar als één van de twee spiegelbeelden voorkomen. Bij toepassingen in het leven – denk aan landbouw, medicijnen en geur- en smaakstoffen bijvoorbeeld – is het daarom vaak zo dat het spiegelbeeld van een bepaald molecuul niet de gewenste uitwerking heeft.
Bij de productie van chemische stoffen is het vaak moeilijk om effectief te sturen in de richting van een van de twee spiegelbeelden, omdat de stoffen en de weg ernaartoe chemisch gezien heel weinig van elkaar verschillen. Veel onderzoek naar nieuwe katalysatoren richt zich daarom juist op dat onderscheid.
Bijna alle chemische processen die in levende organismen voorkomen, worden ook gestuurd door katalysatoren. Deze natuurlijke katalysatoren bestaan meestal uit eiwitten en hebben zich in de loop van de evolutie zo ontwikkeld dat ze juist heel goed zijn in het maken van dat onderscheid. Ook kunnen ze vaak stoffen veel sneller omzetten dan kunstmatige katalysatoren, en dat alles in water bij normale temperaturen. De natuur is daarom een belangrijke inspiratiebron voor chemici bij de ontwikkeling van nieuwe katalysatoren.
Wel is het zo dat we met kunstmatige katalysatoren veel meer soorten reacties kunnen doen dan in de natuur voorkomen, maar de efficiëntie van natuurlijke processen is nog altijd onovertroffen. Er wordt sinds enige tijd geëxperimenteerd met kunstmatige katalysatoren die worden gebonden aan eiwitten, om zo te proberen iets van de gunstige eigenschappen van die eiwitten een rol te laten spelen in processen die in de natuur niet voorkomen.
In dit onderzoek zijn beschrijven we een nieuwe manier om een kunstmatige katalysator te verbinden aan een klein stukje eiwit (peptide) van twee of drie aminozuren lang door middel van
waterstofbruggen. De ontstane verbinding hebben we getest als katalysator voor de hydrogenering van verschillende stoffen. Voor een van de geteste stoffen bleek de katalysator erg actief en is het ook gelukt om een kleine overmaat van het ene spiegelbeeld ten opzichte van het andere te laten
ontstaan. De resultaten zijn nog zeer bescheiden, maar er zijn zeker aanwijzingen dat met verder onderzoek daar nog verbetering in kan komen.
Index
Summary...1 Samenvatting...2 Index...3 Abbreviations...4 1. Introduction...5 1.1 Asymmetric Catalysis...51.1.1 Transition Metal Catalysis...5
1.1.2 Biocatalysis...5
1.2 Artificial Metalloenzymes...6
1.2.1 Suitable Proteins...6
1.2.2 Anchoring Strategies...7
1.2.3 Artificial Metalloenzymes in Catalysis...8
1.3 Aim and Outline...8
2. Peptide Design and Synthesis...10
2.1 Peptide Design...10
2.2 Dipeptide synthesis...10
2.3 Peptide expansion...12
3. Catalyst Preparation...14
3.1 Coordination of the ligand...14
3.2 Supramolecular binding of the Peptide...14
4. Hydrogenation...16
4.1 choice of substrates...16
4.2 Substrate screening...16
4.3 Varying peptide concentration...17
4.4 Catalysis with tripeptide...18
5. Conclusion...20
6. Future Prospects...21
7. Experimental...22
Boc: tert-butyloxycarbonyl NMR: Nuclear Magnetic Resonance ee: Enantiomeric excess
DCM: Dichloromethane GC Gas Chromatography RT Room Temperature DIPEA Diisopropylethylamine THF Tetrahydrofuran MTBE Methyl tert-butyl ether
1. Introduction
Increasing the efficiency of the production of food, energy and medicine is of crucial importance to securing our society’s level of welfare as the global population increases and natural resources become scarcer. Great efforts are made to decrease waste production and energy consumption in chemical processes, in order to make better use of the available resources. In most of the chemical conversions in industry a catalyst is used1, which allows for milder reaction conditions and higher
atom efficiency.
1.1
Asymmetric Catalysis
Many of the building blocks of nature, such as amino acids and sugars, occur as one single enantiomer. As a result, the biological activity of a given compound is usually different with the various stereoisomers. Discrimination between enantiomers in chemical processes is not an easy task, since they are identical in terms of enthalpy and entropy. Asymmetric catalysis allows for differentiation in reaction pathways to both enantiomers, by introducing a chiral feature in the catalyst. This can cause one enantiomer to be kinetically preferred over the other as a result of a difference in energy barriers.
Optimizing the activity and enantioselectivity of the catalysts involved in these processes lowers the consumption of energy and reduces the production of the generally useless mirror image. Despite the growing insight in the mechanisms involved in catalyzed reactions, designing a new catalyst with optimal activity and selectivity still remains a challenge.
Transition metal catalysis and biocatalysis are two fundamentally different approaches to achieve enantiomeric discrimination, both having their respective strengths and weaknesses.
1.1.1 Transition Metal Catalysis
In transition metal catalysis, designing an appropriate catalyst usually involves the optimization of the steric and electronic properties of the first coordination sphere provided by the ligands coordinated to a catalytically active transition metal.2 The dominant strategy towards obtaining enantiomerically
pure compounds involves forcing an incoming reagent to approach from one prochiral face of a substrate, by sterically blocking the other side. The growing insight in organometallic chemistry and constant efforts to rationalize the mechanisms of catalyzed reactions have provided us with very effective transition metal catalysts.
Despite the accumulated knowledge, transition metal catalysts remain inadequate for numerous chemical conversions for which high activity and selectivity cannot be reached.
1.1.2 Biocatalysis
Enzymes often outperform synthetic catalysts by employing highly efficient second-sphere substrate interactions provided by the biomolecular scaffold, such as hydrogen bonding and hydrophobic
natural catalysts by selecting particular substrates, thereby increasing the local substrate
concentration, and by positioning the substrates and stabilizing intermediates.3 Moreover, they are
able to transform matter in a clean way at ambient temperature and pressure.
Although enzymes are increasingly applied in the chemical industry and have proven to be very successful4, they evolved to catalyse very specific reactions with specific substrates and their scope
cannot always be expanded to the desired conversions. Furthermore, it is not always possible to produce sufficient amount of enzyme for practical application and they are not always stable outside of their original organism.5
1.2
Artificial Metalloenzymes
Until recently, transition metal catalysis and biocatalysis have developed in parallel as separate approaches towards achieving enantioselective transformations. Over the last decade, however, significant efforts have been made to merge the attractive properties of natural and synthetic catalysts by introducing non-natural transition metals into proteins. This field seeks to achieve enzyme-like activity and selectivity for transition metal catalysts by using the second coordination sphere provided by the protein.6
1.2.1 Suitable Proteins
The number of proteins that can be used as a biomolecular scaffold for artificial metalloenzymes is limited. Despite the improving methods for the synthesis of polypeptides, it is not yet possible to construct a polypeptide sequence which folds precisely into a well-defined three-dimensional structure. Our current knowledge of protein folding limits the number of de novo designed proteins to only a few types.7 For this reason, the design of artificial metalloenzymes has focused on native
proteins.
Important considerations are the chemical properties of the protein, such as the overall charge, pH and temperature stability, and the tolerance to organic solvents. They should be readily available at reasonable costs and in large quantities. The rigidity of the structure is important for the
enantioselectivity of the catalyst and they should be amenable to modification. They should have a structure that is large enough to accommodate the desired transition metal complex and the substrates, but the catalytic centre should be in close proximity to the biomolecular scaffold for efficient chirality transfer to take place. The stability and tertiary structure of the host protein should not be effected by the structure of the transition metal complex or the chemistry used to introduce it. When using a native protein, an existing active site and/or binding pocket can be reengineered to introduce a transition metal complex. Pioneering work in this field of research was reported by Wilson and Whitesides and makes use of an existing binding pocket in avidin.8 Other examples that
have either been used or investigated include, apart from avidin and streptavidin, the oxygen
transport protein Myoglobin9, bovine serum albumin10, papain11 and bovine β-lactoglobulin12. Another
Class of possible scaffolds consists of proteins like ferritin, containing a large vacant space that allows for the incorporation of unnatural metal cofactors.13
An alternative is creating an active site in a polypeptide that does not yet have a binding pocket. This method can be applied to a greater number of proteins, but it is less clear how the structure and stability will be affected, as it may disrupt some important intramolecular interactions. An example of this approach is the binding of a CuII ion in the peptide hormone bovine pancreatic polypeptide.14
1.2.2 Anchoring Strategies
There are different strategies for the incorporation of the catalytic transition metal centres into the polypeptide scaffolds: dative, covalent and supramolecular anchoring (figure 1).
Figure 1. Representation of the different anchoring strategies: (a) dative, (b) covalent and (c)
supramolecular. M denotes the catalytically active transition metal centre.24
Dative anchoring involves direct coordination of the transition metal to the protein in existing cavities or on the outer sphere. Kaiser and co-workers were the first to modify the active site of an enzyme datively by substitution of zinc by copper in carboxypeptidase A15, affording novel catalytic properties.
More recently, zinc has been replaced by manganese16 and rhodium17 in the active site of carbonic
anhydrase.
Since proteins can have multiple residues present which can act as a metal binding site (His, Asp, Glu, Lys, etc.), it can be difficult to create a well-defined transition metal centre in the catalyst. Site-directed mutagenesis and chemical modification can lead to more precise positioning of the transition metal in the protein. For example, Reetz et al. have been able to increase the
enantioselectivity of a copper-coordinated protein by removing several native histidines, which could act as alternative copper binding sites.18
The covalent anchoring approach involves the covalent incorporation of a transition metal complex via the ligand to a predetermined position in the target protein. A cysteine residue is often used as the anchoring site. Many different metal complexes have been incorporated using the same covalent attachment procedure.19 Disappointingly however, the catalytic potential of the resulting artificial
metalloproteins remains very modest and the optimization techniques are not straightforward. The supramolecular approach makes use of the strong and highly specific non-covalent interactions between the protein and a small molecule. By exploiting the high affinity of an inhibitor for a protein, for example, an organometallic moiety can be introduced within a protein environment, resulting in the formation of a stable supramolecular complex. The interaction between avidin and biotin was used by Wilson and Whitesides to incorporate a rhodium-diphosphane complex into the protein.8 The
metal was introduced by connecting the ligand to the biotin molecule via a spacer (figure 2). Ward and co-workers found that changing the host protein to streptavidin, which has a less cationic
character and a deeper binding pocket, but similar affinity to biotin, improved the performance of the catalyst drastically.20
Figure 2. Schematic representation of artificial metalloproteins based on biotin-(strept)avidin technology.21
Additional supramolecular anchoring strategies have been applied. The transport proteins serum albumin display the ability to bind a variety of hydrophobic guests tightly22 and myoglobin can include
a metal complex or a modified heme instead of native heme in the active site.23
A great advantage of this strategy is that both the protein host and the transition metal catalyst can be optimized separately and joined together afterwards. This allows one to improve the performance of the catalyst either by varying the ligand, or by mutating the gene of the host protein.21
1.2.3 Artificial Metalloenzymes in Catalysis
Artificial metalloenzymes have been tested as catalysts for numerous types of reactions, including hydrogenations21, transfer hydrogenations24, sufoxidations25, epoxidations26 and various C-C bond
forming reactions27. Good results have been achieved in terms of enantioselectivity and in some cases
the host protein has a positive effect on the reaction rate. 28
The distance between the catalytically active metal centre and the second coordination sphere provided by the protein seems to have a great influence on the enantioselectivity of the catalyst. Transition metal centres incorporated in large vacant spaces of a protein have proven to be very active in catalysis and very successful in selecting substrates based on shape and size.13 However, no
significant enantiomeric discrimination has been observed using this type of catalyst.
The best results thus far with metalloproteins have been achieved in hydrogenation reactions using the biotin-(strept)avidin technology. Strong binding and the possibility to optimize the catalyst in different ways are great contributors to its success.
1.3
Aim and Outline
The need for more efficient synthesis procedures for biologically active compounds calls for catalysts with high activity and enantioselectivity. Artificial metalloenzymes have shown great potential in achieving enzyme-like activity and enantioselectivity for conversions which are not possible in traditional enzyme catalysis.
The best results have been achieved by introducing a transition metal catalyst using a
supramolecular anchoring technique, exploiting the affinity between (strept)avidin and biotin. However, this technique is limited only to the binding pocket of (strept)avidin. The development of new supramolecular anchoring techniques with high binding constants could allow us to increase the number of possible binding sites for transition metals in proteins and peptides and explore the possibilities of artificial metalloenzymes even further.
The aim of this project is to use a novel anchoring technique for the supramolecular binding of a catalytically active transition metal center to a small peptide. This technique makes use of a bidentate phosphorus sulfonanmide ligand [L] and two adjacent histidine residues in the peptide, exploiting their hydrogen bond donor and acceptor abilities, respectively (figure 3). For this technique to be useful in asymmetric catalysis, it is important that the chirality of the peptide results in enantiomeric discrimination during catalysis.
The central question of this research project is whether this technique can be used to successfully transfer the chirality of the peptide to cause enantiomeric discrimination during catalysis.
Figure 3. Supramolecular binding of dipeptide [His(L)-His(L)] to phosphorus sulfonamide ligand [L], exploiting their hydrogen bond donor and acceptor abilities, respectively. M represents a catalytically active transition
metal center.
For this purpose, one dipeptide and two diastereomeric tripeptides have been synthesized, all containing two adjacent histidine residues for the supramolecular binding to the phosphorus sulphonamide ligand [L]. At first, the dipeptide has been used to form a supramolecular complex containing a rhodium center. This supramolecular complex has been tested in the hydrogenation of multiple substrates, exploring the possibilities in asymmetric catalysis. Finally, the tripeptides have been used to see how a third residue in the peptide chain might influence these results.
Chapter 2 describes the synthesis of one dipeptide and two diastereomeric tripeptides. Chapter 3 describes the formation of the supramolecular rhodium catalyst. The results in catalysis will be discussed in chapter 4. The conclusion and future prospects are presented in chapter 5 and 6, respectively. Finally, detailed experimental procedures and data are described in chapter 7.
2. Peptide Design and Synthesis
2.1
Peptide Design
As pointed out in the introduction, the formation of the catalyst relies on the supramolecular binding of the peptide to the ligand, through the formation of hydrogen bonds between the π-nitrogens of the imidazole groups on the histidine residues in the peptide, and the –NH groups of the phosphorus sulfonamide ligand [L]of the rhodium complex (figure 3). Therefore, the peptide should have two adjacent histidine residues present.
The τ-nitrogens of the Histidine’s imidazole groups are protected by methyl groups. The termini of the resulting peptide are orthogonally protected, which allows for selective deprotection and therefore facile extension of the peptide. In this chapter, the synthesis route towards obtaining the protected dipeptide and tripeptides will be discussed.
2.2
Dipeptide synthesis
Starting from the commercially available (L)-Histidine methyl ester dihydrochloride 1, the most challenging in the synthesis of the desired dipeptide [His(L)-His(L)]are the protection groups on the N-terminus and the τ-nitrogen of the imidazole. Due to the different resonance structures of the imidazole ring in histidine, direct alkylation of the imidazole nitrogens on histidine often leads to mixtures of the π and τ alkylated species (scheme 1).29 Furthermore, using conventional methods for
the Boc-protection of The N-terminus of amino acids leads, in case of histidine, to overprotection of the imidazole nitrogens, so another step of selective deprotection is necessary.30
Scheme 1. Due to histidine’s two resonance structures, methylation can lead to regioisomers.
The methodology used to overcome these issues is one that allows for complete regioselective methylation of the τ nitrogen of histidine and selective Boc-protection of the N-terminus. The reaction involves the formation of a cyclic urea 2 to protect both the primary amine and the π nitrogen of the imidazole ring (scheme 2). This technique was first reported by Hodges and Chivkas31
and later optimized by Brégeon et al.32 This procedure only requires the heating of the two starting
materials without any solvent under mechanic stirring, providing the cyclic urea 2 in good yield. The methylation of the cyclic urea 2was then performed using methyliodide in acetonitrile, providing imidazolium salt 3 in good yield and high purity after crystallization. The subsequent urea ring opening was carried out using t-BuOH. Due to the relatively poor nucleophilicity of the sterically hindered alcohol, only a moderate yield could be achieved for this reaction. However, it leads directly to the orthogonally protected compound4, leaving a Boc-group on the N-terminus.
Orthogonally protected compound4could then be used to obtain both starting materials for the peptide coupling, simply by selectively deprotecting the N- and C-termini. Removal of the methyl ester could be done by treatment with Tesser’s base, providing sodium salt 5in nearly quantitative yield. The Boc-group was removed using HCl, which led to full conversion of 4 into 6.
Scheme 2. Synthetic route from (L)-Histidine methyl ester to dipeptide [His(D)-His(D)]
The structure of dipeptide [His(L)-His(L)]was confirmed by 1H NMR and 13C NMR experiments. In
order to determine how successful the chirality in the dipeptide can be transferred to give enantiomeric discrimination in catalysis, it is important to be sure that the peptide itself is enantiomerically pure. The enantiomeric purity of [His(L)-His(L)]was ascertained by chiral HPLC (chiralcel AD-H, heptane/isopropanol). For this purpose, all 4 diastereomers of the dipeptide were synthesizes. The enantiomers of 5 and 6 were synthesized using the same procedure, with (D)-Histidine methyl ester dihydrochloride as starting material. They were then coupled to give the 4 diastereomers(Figure 4).
Figure 4. The 4 diastereomers of the dipeptide.
2.3
Peptide expansion
Dipeptide [His(L)-His(L)] was extended by coupling a benzyl-protected (L)-phenylalanine, to form tripeptide [His(L)-His(L)-Phe(L)]. The dipeptide was treated with Tesser’s base in order to hydrolyze the methyl ester and form sodium salt7 (scheme 3).
Scheme 3. Hydrolyzation of the methyl ester to form a sodium salt.
The sodium salt was then extended in a peptide coupling reaction with commercially available (L)-phenylalaninebenzyl ether 8 to form tripeptide[His(L)-His(L)-Phe(L)] (scheme 4).
Scheme 4. Peptide coupling with (L)-phenylalanine benzyl ether to give the tripeptide.
A second tripeptide was made using the same procedure, however starting with the mirror image of the dipeptide [His(L)-His(L)], [His(D)-His(D)], in order to make tripeptide [His(D)-His(D)-Phe(L)], a
diastereomer of [His(L)-His(L)-Phe(L)] (figure 5). The structure of [His(L)-His(L)-Phe(L)] was
confirmed by 1H NMR.
3. Catalyst Preparation
The supramolecular binding of the peptides to the catalytically active rhodium center relies on the formation of hydrogen bonds between the peptide and the bidentate phosphorus sulphonamide ligand [L], exploiting the hydrogen bond acceptor abilities of the ligand and the hydrogen bond donor abilities of the side chains of the adjacent histidine residues of the peptide. Rhodium is used as the metal, to allow the complex to be used as a precatalyst for hydrogenation reactions.
3.1
Coordination of the ligand
The formation of the desired rhodium complex [Rh][L] was achieved by mixing bidentate sulfonamide ligand [L] with Rhodium precursor [Rh], followed by crystallization from DCM/Pentane (scheme 5).
Scheme 5. Coordination of the sulfonamide ligand to the rhodium center
Coordination of the ligand was evidenced by the doublet observed in 31P NMR at 72.6 ppm, as
opposed to a singlet at 33.4 ppm for the free ligand. Mass spectroscopy also confirmed the formation of complex [Rh][L] (m/z = 659.08).
3.2
Supramolecular binding of the Peptide
The next step in the preparation of the catalyst is the supramolecular binding of the dipeptide
[His(L)-His(L)]to the ligand via hydrogen bonding. A solution of dipeptide [His(L)-His(L)] in DCM was added to a DCM solution of complex [Rh][L] to form supramolecular complex [Rh][L][His(L)-His(L)](scheme 6).
Scheme 6. Supramolecular binding of the peptide to the sulfonamide ligand
31P NMR shows a shift from 74.2 in[Rh][L] to 62.7 in [Rh][L][His(L)-His(L)]. This indicates that
4. Hydrogenation
In chapter 3 it was shown that two adjacent histidine residues can be used as an anchoring site for the supramolecular binding of a rhodium complex, using a bidentate sulfonamide ligand for the formation of hydrogen bonds. Now we wanted to investigate whether the chirality of the histidine residues can be transferred to give enantiomeric discrimination during catalysis.
For this purpose we chose to do hydrogenation reactions, since they have proven to be the most successful reactions with artificial metalloenzyme type catalysts.21 Furthermore, hydrogenation
reactions have a low risk of undesired side reactions and other impurities in the reaction mixture, which allows for facile analysis of the conversion and enantiomeric discrimination.
4.1
choice of substrates
To explore the potential in asymmetric hydrogenation, a diverse library of industrially relevant substrates with various functional groups and coordination abilities was used (figure 6). In this way, we could investigate which type of substrate would be suitable for this type of catalyst. This would enable us to focus further experiments on those substrates.
Figure 6. Industrially relevant substrates subject to asymmetric hydrogenation
4.2
Substrate screening
Hydrogenation experiments were performed at room temperature at 20 bars of hydrogen pressure for 16 hours in DCM in the presence of 1 mol% of the Rhodium precursor [Rh], 1.1 mol% of
sulfonamide ligand [L] and 5.5 mol% of dipeptide [His(L)-His(L)] (scheme 7).
We chose to use an excess of peptide in the reaction mixture, because at this point we did not know how strong the binding between the ligand and the peptide is. With too little of the peptide present in the reaction mixture, we would risk to have too much of the complex [Rh][L] present which is not bound to the peptide. This would result in lower, perhaps undetectable ee numbers. Too much peptide, on the other hand, might block the catalytically active center and oppose conversion.
At this point, the five equivalents of peptide relative to the ligand was merely a guess. In section 4.3, the influence of the peptide concentration in terms of ee and conversion will be discussed in more detail.
Scheme 7. Standard conditions for hydrogenation reactions
Substrate F (methyl 2-acetamidoacrylate) is the only one to give full conversion under the given conditions. Substrate C gives 27% conversion and substrate A gives 4% conversion. The other substrates did not yield any detectable conversion (table 1).
Substrate A B C D E F G H I
Conversion (%) 4 0 27 0 0 >99 0 0 0
ee (%) 0 0 11
Table 1. Substrate screening
Furthermore, F is the only substrate to give ee (11%) in the presence of supramolecular complex [Rh] [L][His(L)-His(L)]. This result shows that the chirality of the peptide can lead to enantiomeric
discrimination. It encouraged us to do further experimentation on the hydrogenation of substrate F with this catalyst, in order to find out how we should interpret these results.
4.3
Varying peptide concentration
As [Rh][L][His(L)-His(L)] is a supramolecular complex, held together by hydrogen bonds, there is an equilibrium in the reaction mixture between the bound state and the unbound state of the catalyst (scheme 8).
Scheme 8. Equilibrium of the catalyst between a bound peptide and an unbound peptide.
Rhodium complex [Rh][L] in the absence of peptide [His(L)-His(L)] is also active as a hydrogenation catalyst but, as there is no chiral feature present, does not effectuate enantiomeric discrimination (table 2). As a result, catalysis takes place both at [Rh][L][His(L)-His(L)] – effectuating enantiomeric discrimination – and at [Rh][L] – not effectuating enantiomeric discrimination.
In order to determine the ee with the peptide bound catalyst [Rh][L][His(L)-His(L)], we wanted to investigate at which point the catalyst reaches near saturation with peptide. Furthermore, we wanted to investigate how the peptide concentration influences the conversion. For this purpose, we did the hydrogenation of substrate F with varying peptide concentration (scheme 9).
Scheme 9. Hydrogenation of substrate F with varying peptide concentration
In table 2, the conversion and ee of the hydrogenation reaction are displayed as a function of the peptide concentration relative to the concentration of ligand in the reaction solution.
n ee conv 0 0 100 1 3 99 5 11 99 10 10 54 25 11 32 50 10 24 100 10 16
Table 2. Conversion and ee as a function of peptide concentration, where n is the number of equivalents of dipeptide [His(L)-His(L)] relative to the concentration of ligand [L].
The enantiomeric discrimination of the catalyst increases with the concentration of peptide and remains roughly constant with peptide concentrations of n > 5. These results suggest that the catalyst reaches near saturation of peptide at around n = 5. Unfortunately, we have not been able to determine an association constant for the supramolecular binding of the peptide to the ligand and we cannot draw any hard conclusions from these numbers. Nonetheless, these results show that we can reach maximum ee at five equivalents of peptide.
This is useful for further hydrogenation experiments, as the conversion numbers drop at higher peptide to ligand ratio’s. A large excess of peptide may block the catalytically active metal center, thereby impeding the access of incoming substrates.
4.4
Catalysis with tripeptide
In section 4.2 it was shown that a supramolecular complex with a dipeptide containing two adjacent histidine residues acting as an anchoring site for phosphorus sulfonamide ligand [L] can effectuate enantiomeric discrimination when used as a hydrogenation catalyst. Next, we wanted to investigate what the influence of a third amino acid in the peptide chain, directly next to the anchoring site, can be on the ee of the reaction.
We chose to use the same conditions for the hydrogenation of substrate F (scheme 10), since an evaluation of the influence of the concentration of peptide had shown that five equivalents of peptide relative to the ligand results in maximum enantiomeric discrimination without reducing the conversion under the given conditions (see section 4.3).
Both [His(L)-His(L)] and its enantiomer [His(D)-His(D)] were expanded by coupling a benzyl protected phenylalanine residue to give the two diastereomeric tripeptides [His(L)-His(L)-Phe(L)] and
we can expect it to favor the other enantiomer when applied in catalysis with rhodium complex [Rh]
[L]. However, it is interesting to see what the influence of the phenylalanine is on the ee.
Scheme 10. Hydrogenation in the presence of a tripeptide.
Both diastereomers of the tripeptide gave full conversion under the given conditions. The ee values are represented in table 3.Unsurprisingly, tripeptide [His(D)-His(D)-Phe(L)], bearing the His(D)-His(D) core, favors another enantiomer than tripeptide[His(L)-His(L)-Phe(L)] and dipeptide [His(L)-His(L)],
that both have the His(L)-His(L) core. Interestingly, both diastereomers of the tripeptide give rise to higher ee values than dipeptide [His(L)-His(L)] with the same substrate under the same conditions.
tripeptide ee (%) [His(L)-His(L)-Phe(L)] 26 [His(D)-His(D)-Phe(L)] -16
Table 3. ee values of hydrogenation reactions in the presence of tripeptides [His(L)-His(L)-Phe(L)] and [His(D)-His(D)-Phe(L)], respectively. the negative value represents an excess of the other enantiomer.
These results show that the addition of a third amino acid residue to the peptide chain of the supramolecular catalyst can have a substantial influence on the enantiomeric discrimination, although the ee is still fairly modest.
5. Conclusion
A dipeptide consisting of two histidine residues has been successfully attached to a rhodium complex to form a supramolecular complex, exploiting the hydrogen bond donor abilities of the phosphorus sulfonamide ligand [L] and the hydrogen bond acceptor abilities of the π-nitrogens in the imidazole rings of the histidine residues. This method looks promising for the incorporation of transition metal complexes in peptides, using two adjacent histidine residues as an anchoring site.
It was shown that the chirality of the peptide can effectuate enantiomeric discrimination when a supramolecular complex based on this technique is used as a catalyst in hydrogenation reactions, although this was achieved with only one of the tested substrates (F) and the ee values are still fairly modest (11%).
The addition of a third amino acid residue (phenylalanine) in the peptide chain of the supramolecular catalyst showed to have a substantial influence on the enantiomeric discrimination achieved in hydrogenation. This result suggests that there is still room for further optimization.
6. Future Prospects
For this anchoring strategy to be useful for the incorporation of transition metal complexes in larger peptides and proteins and the development of artificial metalloenzymes, strong binding to the host or significant rate accelerations are necessary in order to prevent unbound catalyst from producing a racemic product and consequently lowering ee values. Therefore, in order to explore the possibilities of this anchoring strategy, it will be important to determine an association constant and explore the kinetics of the catalysis.
As the addition of the third amino acid residue in the peptide chain significantly influenced the enantiomeric discrimination of the catalyst, it would be very interesting to see what further extension and further optimization of the peptide chain could accomplish in terms of ee.
Furthermore, as this research only covers hydrogenation experiments for 9 different substrates, it will be necessary to test this strategy with other transition metal complexes and substrates, to further explore the potential in catalysis.
7. Experimental
All reactions involving rhodium were carried out in dry glassware under argon atmosphere. Every solution addition or transfer involving rhodium was performed via syringes or in a glovebox. The ligand and rhodium precursor were weighed under air. All solvents were dried and distilled using standard procedures, unless stated otherwise. Chromatography purifications were carried out using Silicycle Silia-P Flash Silica Gel (40-63 μm) and technical grade solvents.
Nuclear Magnetic resonance experiments were performed on a Brucker AMX 400 (1H: 400.1 MHz, 13C:
100.6 MHz and 31P: 162.0 MHz) or a Varian Mercury (1H: 300 MHz). Chemical shifts are referenced to
the solvent signal. All conversions and ee values were determined by gas Chromatography on an InterscienceFocus GC Ultra with a chiralsil DEX-CB column (25m x 0.32mm). Unless otherwise stated, reactions were performed at room temperature.
methyl (R)-5-oxo-5,6,7,8-tetrahydroimidazo[1,5-c]pyrimidine-7-carboxylate
10.0 g L-histidine methyl ester dihydrochloride (41.30 mmol) and 8.04 g 1,1-carbonyldiimidazole (49.56 mmol) were heated to 80 °C under argon with mechanical stirring for 45 minutes. The mixture was then quenched with 5 ml H2O and allowed to cool to RT. The mixture was extracted with DCM
(12 x 20 ml). Organic fractions were combined, dried with anhydrous MgSO4 and concentrated until a
white precipitate appears. The crude product was washed with Et2O (200 ml) and dried in vacuo
providing a white solid (5.55 g, 69%). 1H NMR (400 MHz, Chloroform-d) δ 8.15 (d, J = 2.7 Hz, 1H,
NCHN), 6.91 – 6.86 (m, 1H, CCHN), 6.40 (s, 1H, NH), 4.37 (ddd, J = 9.3, 5.4, 2.3 Hz, 1H, α-CH), 3.81 (d, J = 2.6 Hz, 3H, OCH3), 3.38 (dd, J = 15.8, 5.1 Hz, 1H, β-CHH), 3.21 – 3.09 (m, 1H, β-CHH).
(R)-7-(methoxycarbonyl)-2-methyl-5-oxo-5,6,7,8-tetrahydroimidazo[1,5-c]pyrimidin-2-ium iodide
5.3 ml of Methyl iodide (84,5 mmol) was added to a solution of 2 (5.50 g, 28.2 mmol) in 120ml acetonitrile. Then the reaction mixture was heated to reflux for 20h, cooled to RT and concentrated. Then recrystallized from MeOH/t-Bu-Methyl ether to give a white/yellow solid (6.61 g, 70%). 1H NMR
(300 MHz, Deuterium Oxide) δ 9.33 (dd, J = 1.7, 0.9 Hz, 1H, NCHN), 7.37 (d, J = 1.3 Hz, 1H, CCHN), 4.69 (td, J = 5.6, 0.9 Hz, 1H, α-CH), 3.92 (t, J = 0.8 Hz, 3H, CH3), 3.73 (d, J = 0.8 Hz, 3H, CH3), 3.49 – 3.40
(m, 2H, β-CH2).
6.55 g (19.4 mmol) of 3 was dissolved in 100 ml of t-BuOH. 6.4 ml (38.8 mmol) of DIPEA was added and the reaction mixture was heated to 80 °C and left stirring. After 20h the solvent was removed and the residue was dissolved in DCM (50 ml), washed with water (2x 40 ml) and concentrated. The product was purified by column chromatography (EtOAc), washed with water (2x 50 ml), dried with MgSO4 and concentrated to give a sticky, slightly yellow oil (2,81 g, 51%) 1H NMR (300 MHz, CDCl3) δ
7.32 (s, 1H, NCHN), 6.63 (d, J = 1.3 Hz, 1H, CCHN), 5.91 (d, J = 8.3 Hz, 1H, NH), 4.52 (dt, J = 8.2, 5.1 Hz, 1H, α-CH), 3.70 (s, 3H, CH3), 3.61 (s, 3H, CH3), 3.03 (qd, J = 14.6, 5.1 Hz, 2H, β-CH2), 1.43 (s, 9H, Boc).
sodium Na-(tert-butoxycarbonyl)-Nt-methyl-L-histidinate
500 mg (1.77 mmol) of 4 was dissolved in a mixture of 3.30 ml MeOH and 9.20 ml dioxane at RT. 0.66 ml (2.66 mmol) of NaOH was added to the solution and the reaction mixture was left stirring. After 6h the solvent was evaporated and the residue was extracted with CHCl3 (3x 10 ml) and concentrated to
provide 496 mg of a slightly yellow solid (96 %)
.
1H NMR (300 MHz, DMSO-d6) δ 7.31 (s, 1H, NCHN),
6.67 (s, 1H, CCHN), 5.86 (d, J = 6.8 Hz, 1H, NH), 3.53 (s, 3H, CH3), 2.87 – 2.76 (m, 1H, α-CH), 2.81 –
2.64 (m, 2H, β-CH2), 1.32 (s, 9H, Boc).
methyl Nt-methyl-L-histidinate dihydrochloride
10 ml of 4M HCl in dioxane .was added dropwise to a solution of 1.08 g of 4 in 40 ml of DCM at RT under argon atmosphere. The solution was stirred for 4h and then concentrated to provide 962 mg of a slightly yellow powder (99%).1H NMR (300 MHz, DMSO-d
6) δ 8.98 (s, 1H, NCHN), 8.75 (bs, 2H, NH2),
7.50 (s, 1H, CCHN), 4.41 (d, J = 7.4 Hz, 1H, α-CH), 3.81 (s, 3H, CH3), 3.74 (d, J = 1.0 Hz, 3H, CH3), 3.30 –
1000 mg (3.43 mmol) of 5 was dissolved in 10 ml of THF. 463 mg (3.43 mmol) of benzotriazol-1-ol was added and the reaction mixture was cooled to 0°C. A solution of 780 mg (3.78 mmol)
dicyclohexylcarbodiimide in 10 ml THF was added and the reaction mixture was left stirring for 1h. A solution of 879 mg (3.43 mmol) of 6 and 1.43 ml (10.3 mmol) of trimethylamine in THF was then added in small portions and the reaction mixture was left stirring for 3 days at RT. Then a precipitate was filtered off and the solution was concentrated, dissolved in 120 ml of EtOAc, washed with saturated NaHCO3/NaCl 1:1 (2x 50 ml), dried over MgSO4 and concentrated. The residue was then
grinded with ether and purified by column chromatography (DCM/EtOH 9:1) to give a light brown solid after concentration (445 mg, 30%)
.
1H NMR (300 MHz, CDCl3) δ 7.72 (s, 1H, CNHC=O), 7.29
(dddd, J = 3.5, 1.5, 1.0, 0.5 Hz, 2H, NCHN), 6.70 (s, 1H, CCHN), 6.59 (s, 1H, CCHN), 6.05 (s, 1H, CNHBoc), 4.77 – 4.62 (m, 1H, α-CH), 4.42 (s, 1H, α-CH), 3.67 (d, J = 0.5 Hz, 3H, OCH3), 3.61 (s, 3H,
NCH3), 3.59 (s, 3H, NCH3), 3.12 – 2.82 (m, 4H, β-CH2), 1.42 (s, 9H, Boc). 13C NMR (101 MHz, CDCl3) δ
171.68 (C=O), 171.47 (C=O), 155.53 (C=O), 138.04 (ImC), 137.27 (NCHN), 137.21 (NCHN), 136.92 (ImC), 117.99 (CCHN), 117.66 (CCHN), 79.32 (BocC), 54.38 (NHCHC=O), 52.14(OCH3), 51.98
(NHCHC=O), 33.15(NCH3), 33.12 (NCH3), 30.53 (CH2), 29.91 (CH2), 28.12 (Boc).
benzyl Na-(Na-(tert-butoxycarbonyl)-Nt-methyl-L-histidyl)-Nt-methyl-L-histidyl-L-phenylalaninate
55 mg (0.127 mmol) of [His(L)-His(L)] was dissolved in a mixture of 1.41 ml dioxane and 0.51 ml MeOH. 100 µl of 4N NaOH was added and the reaction mixture was left stirring at RT for 2h, concentrated, extracted with CHCl3 and concentrated. The residue was dissolved in 1ml THF. 17 mg
(0.124 mmol) of benzotriazol-1-ol was added and the reaction mixture was cooled to 0°C. A solution of 28 mg (0.137 mmol) dicyclohexylcarbodiimide in 2 ml THF was added and the reaction mixture was left stirring for 1h at RT. A solution of 17 mg (0.124 mmol) of L-Phenylalanine benzyl ether and 19 µl (0.137 mmol) of trimethylamine in THF was then added in small portions and the reaction mixture was left stirring for 3 days at RT. Then a precipitate was filtered off and the solution was concentrated, dissolved in 15 ml of EtOAc, washed with saturated NaHCO3/NaCl 1:1 (2x 6 ml), dried over MgSO4 and
concentrated. The residue was then grinded with ether and purified by column chromatography (DCM/EtOH 9:1) to give a light brown solid after concentration (35 mg, 43%)
.
1H NMR (300 MHz,CDCl3) δ 8.65 (d, J = 7.8 Hz, 1H, NHC=OO), 7.95 (d, J = 7.6 Hz, 1H, CNHC=O), 7.58 – 6.87 (m, 12H,
NCHN and Bz-ArH), 6.71 (s, 1H, CCHN), 6.54 (s, 1H, CCHN), 6.09 (d, J = 5.6 Hz, 1H, NHBoc), 5.14 – 4.87 (m, 2H, Bz-CH2), 4.72 (q, J = 7.5 Hz, 1H, α-CH), 4.61 (dt, J = 7.5, 4.9 Hz, 1H, α-CH), 4.20 (q, J = 5.9 Hz, 1H, α-CH), 3.59 (s, 3H, NCH3), 3.50 (s, 3H, NCH), 3.25 – 2.59 (m, 6H, CH2), 1.42 (s, 9H, Boc).
5.13 mg (0.0137 mmol) of [L] and 6.20 mg (0.0137 mmol) of [Rh(nbd)
2]BF
4were dissolved
in 0.5 ml of THF under argon atmosphere and left stirring for 45 minutes. The solvent was
then evaporated and the residue was crystallized from DCM/MTBE. m/z calculated for
C
31H
30N
2O
2P
2RhS 659.05583, found 659.08227.
31P NMR (162 MHz, CD2Cl2) δ 72.56 (d, J = 168.2Hz).
5.00 mg (0.0133 mmol) of [L] and 6.04 mg (0.0133 mmol) of [Rh(nbd)
2]BF
4were dissolved in
0.5 ml of THF under argon atmosphere and left stirring for 45 minutes. The solvent was then
evaporated and a solution of 5.78 mg (0.0133 mmol) [His(L)-His(L)] in o.6 ml CD
2Cl
2was
added.
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