Would the Piano Stools please stand up?

Figure 1. A d6-piano stool complex for Iridium.

I have recently read an article from Chem. Commun. on the chemistry-biology interface [1]. It should be fascinating to both researchers in chemistry and biology alike, as the research is about the application of an artificial metalloenzyme in catalysis.

Modular in nature, the metalloenzyme consists of a host protein and the synthetic metal complex. In order to incorporate the metal catalyst to the protein host, a well-established way, for which the researchers have adopted, is the biotin-streptavidin technology.

The metal complex the researchers were using belonged to the class of d6-piano stool complex, and in this particular case, the metal was iridium (Figure 1). This type of piano-stool complexes seemed to be stable in biological environment, thus providing a benefit for the development of bio-catalysis. In order to make their catalyst more active in a biological environment, they have devised a ‘shielding strategy’, where they coated silicon nanoparticles as a protective layer on the metalloenzyme (Figure 2).

Figure 2. The design of the catalyst architecture.

The synthesis of the iridium complex involved the reaction of a protected TFA salt of the precursor di-amine with [Cp*IrCl₂]₂ in the presence of triethylamine at room temperature, resulting in the iridium catalyst with satisfactory yield (Figure 3).

Figure 3. Synthesis of the iridium complex.

The metalloenzyme, henceforth known as an ATHase, was covalently anchored onto the silicon nanoparticles (Figure 2). After self-assembly and the poly-condensation of silanes, a protective layer could be formed and embedded the ATHase inside the 'silica core'. The nanoparticles were visualized by scanning electron microscopy.

Figure 4. Catalysis of imine reduction by ATHase.

The ATHase was used to catalyze an imine reduction reaction (Figure 4). Depending on the specific mutant employed, it could give rise to a specific enantiomer as the product. In a nutshell, the conversion, enantioselectivity and turnover number of the silica-protected ATHase were impressive. What was notable was that, when the researchers compared the data of the catalysis of the metalloenzyme with or without silica-protection, the results were more or less comparable, signifying that the extra silica protection has not led to a negative impact to the catalysis. The silica-protected catalyst could be recycled and re-used, with almost the same enantiomeric excess in subsequent runs.

In terms of the context of the research aim, the authors would like to explore the applications of their ATHase in biological scenarios. Thus, they used the ATHase to carry out a reaction well known to anyone in biochemistry : the conversion of NAD⁺ to NADH. They have used absorbance spectroscopy (340 nm) – another obvious technique for biochemists – to monitor the conversion. They observed that the silica-protected ATHase had a somewhat better turnover number as compared to the non-protected version.

Another interesting aspect was that the researchers attempted to test their ATHase in vivo, which meant they did the reduction in a cellular ‘soup’. Using cell lysate of E. coli and other cellular media, satisfactory turnover numbers could be achieved, and the results were indeed better than the homogeneous metalloenzyme. They rationalized that, since the homogeneous metalloenzymes did not possess any protective layers, the catalytic activity was lost in the presence of cellular debris in the cellular ‘soup’. Thus, the silica ‘shell’ was able to protect the ATHase in the cellular media, while at the same time allowing the ATHase to carry out its mission. Even an imine reduction in urine (!!) could be achieved (TON= 4500)! 

The methodology has the added advantages that it could be conducted in physiological conditions – room temperature, pH 7, making it a stunning approach for bio-catalysis.

Impressive!

by Ed Law
20/8/2016

Reference:

1. Immobilization of an artificial imine reductase within silica nanoparticles improves its performance

Martina Hestericová, M. Rita Correro, Markus Lenz, Philippe F.-X. Corvini, Patrick Shahgaldian and Thomas R. Ward

Chem. Commun., 2016, asap

DOI: 10.1039/c6cc04604e

Doping the Iron out

The use of recyclable catalysts in chemical reactions seems to be a hot research area! I have looked the a recent issue of Chem. Commun. and have read another nice paper about the immobilization of a ferrocene-containing phosphine onto iron nanoparticles, through the use of dopamine as a linker [1]. The corresponding rhodium and palladium complexes of the magnetic ferrocenyl-phosphine ligands were prepared, and they have been shown to be great and recyclable (hence re-usable) catalysts for hydroformylation (Rh) and Heck coupling reactions (Pd) respectively.

Figure 1. Synthesis of the ligand and preparation of the metal complexes.

Let me show you the design of this modular ligand (Figure 1). First, the ligand was a ferrocenyl phosphine known as BPPFA. This phosphine ligand was first conjugated to dopamine. When the dopamine-linked phosphine was sonicated with Fe₃O₄, the phosphine was thus functionalized with magnetic nanoparticles, and the structure was designated as Fe₃O₄@dop-BPPF. These magnetite nanoparticles were formed as a black precipitate, and it was notable that they could be collected and separated from the reaction mixture through the use of a piece of magnet.

When  Fe₃O₄@dop-BPPF was allowed to react with a slight excess of [Rh(nbd)Cl]₂ or [Pd(C₃H₅)Cl]₂, the resulting rhodium or palladium complexes were afforded respectively.

Why did the researchers use dopamine as a linker? They rationalized that the 2 hydroxyl groups on dopamine, which were arranged in a 1,2 relationship, was great as a bidentate ligand and thus could provide tighter binding to the iron oxide, leading to higher stability for the resulting catalyst.

Before submitting to rounds of catalysis, the chemical and magnetic properties of these compounds were first investigated with a number of techniques. Besides NMR and FTIR spectroscopy, transmission electron microscopy, selected area electron diffraction, and X-ray diffraction studies have also been employed to access the appearances of these ligands and metal complexes.

One aspect I find particularly fascinating is the investigations of the magnetic properties of the nanoparticles. It is related to a phenomenon known as ‘magnetic hysteresis’, and the concept of hysteresis is actually important in the field of Nonlinear Dynamics, something I am also highly fascinated in.

The researchers have found that, the dop-BFFP phosphine’s magnetic properties, namely its coercivity (H_c) and remanence (M_r), were not affected by the functionalization with iron nor complexation to rhodium or palladium. While the saturation magnetization value was decreased slightly upon the iron nanoparticle functionalization and complexation with metals, the bulk magnetization has not been affected, which meant that the magnetic properties were retained even if the iron particles were functionalized with the phosphine and complexed to the metal center, and this would benefit the separation process at the end of a catalytic trial through the use of a magnet. Indeed, if we look at the magnetic hysteresis loops of  Fe₃O₄@dop-BPPF, Fe₃O₄@dop-BPPF-Pd and Fe₃O₄@dop-BPPF-Rh at room temperature were almost overlapping with each other, confirming the above arguments.

Figure 2. Hydroformylation catalysis.

Next, the researchers used their newly-prepared metal complexes to do 2 catalytic reactions – Rh-catalyzed hydroformylation and Pd-catalyzed Heck coupling reaction. Not only they wanted to investigate the conversion and substrate scope, a prime aim was to see whether these nanoparticle catalysts could be recycled and recused efficiently.

For the hydroformylation (Figure 2), they have focused much of their studies on the substrate styrene. They have been able to optimize the reaction conditions and the branch to linear selectivity was reasonably high, with the branched product as the predominant. They have also found that styrene substituted with electron withdrawing groups (such as NO₂ and Br) gave a very high branch to linear selectivity of 99:1. Linear alkenes, such as 1-octene, gave the opposite selectivity, with the normal isomer as the predominant product (b : l = 0 : 100).

The hydroformylation catalyst could be recycled and re-used for 3 further times, and then a gradual loss of reactivity was observed, and the researchers attributed to a higher pressure in the reaction system, leading to catalyst leaching.

Figure 3. Heck coupling catalysis.

The Pd catalyst was subject to Heck coupling reaction, with styrene and iodobenzene as the 2 coupling partners. Reaction temperature, choice of base, and solvent system were optimized. When the less reactive bromobenzene was employed instead of iodobenzene, full conversion could be achieved through a longer reaction time, and this signified the stability of the new catalyst.

With the styrene / iodobenzene system, the Pd catalyst could also be recycled and re-used, by simply using an external magnet to separate the catalyst from the reaction mixture. The clean catalyst could be re-used for at least 9 more times, which was very impressive.

Figure 4. Cobalt-phosphine catalyzed hydroarylation - a potential reaction to explore?

This is great research, and the authors are working on other catalytic reactions to explore the scope of their novel ligands. Since they have been using styrene as a substrate, that fact rings a bell on me as I remember reading an article about a cobalt-catalyzed hydroarylation of styrene from J. Am. Chem. Soc. [2]. That could also be an interesting and potentially useful reaction to try, too!

by Ed Law

19/7/2016

Reference:

1. Magnetic nanoparticle-supported ferrocenylphosphine: a reusable catalyst for hydroformylation of alkene and Mizoroki–Heck olefination

M. Nasiruzzaman Shaikh, Md. Abdul Aziz, Aasif Helal, Mohamed Bououdina, Zain H. Yamani and Tae-Jeong Kim

RSC Adv., 2016, 6, 41687–41695

2. Regioselectivity-Switchable Hydroarylation of Styrenes

Ke Gao and Naohiko Yoshikai

J. Am. Chem. Soc., 2011, 133, 400–402

Fatten up a Few Gear

Ever since my undergraduate years in biochemistry, I have developed an intense fascination with the chemistry and biology of membranes. While most elementary textbooks on biology have often portrayed the cell membrane as a pair of lines (or curves in some cases), this organelle is actually one of the most dynamic components of the cell, and the chemistry and molecular biology behind it serve as the driving force for many wonderful molecular phenomena. 

Now, I am working in the field of bi-phasic catalysis, and it is quite gratifying to see that there are parallels between my field and that of membrane science. If possible in the future, membrane chemical biology is certainly a research field I would love to explore! This week, I have read a great article in Journal of American Chemical Society about lipid membrane and hydrogel formation [1], and I would like to share with you here.

Figure 1. Hydrogel network formation through the catalysis of a lipid membrane.

The paper is about the use of a negative-charged lipid membrane as a catalyst to facilitate the formation of a hydrogel network (Figure 1). A hydrogel is defined as a polymeric, gel-like macromolecular structure, which is made by the cross-linking of polymer chains. Hydrogels have been used in drug delivery, tissue engineering, supramolecular catalysis [2c], and many other fields.  The self-assembly and aggregation of the resulting fibrous hydrogel network has to be designed in a way so that the material properties of the resulting gel fiber can be controlled. On the other hand, the spatial position of hydrogel formation has to be carefully defined, to serve the aim of controlled release or delivery of, for example, drug molecules.

The researchers have found that a negatively-charged lipid membrane can be used as a catalyst to form supramolecular hydrogel networks. The negatively-charged liposomes can be used to catalyze the formation of a gelator molecule 3, and they have been able to achieve spatial control – the gelator molecule 3 is formed near the membrane, so that the resulting hydrogel networks can be formed in this well-defined area. The mechanism involves the generation of a local proton (H⁺) gradient - due to the prevalence of the negative charges. The high proton concentration facilitates the acid-catalyzed formation of hydrazone– the functional group in the gelator molecule 3.

For the chemistry, the first stage to form the hydrogel is a reaction between a hydrazide 1 and an aldehyde 2 to form the hydrazone derivative 3. At neutral pH, this reaction is very slow. Yet, the researchers have established that, when an acid or aniline is added, the reaction rate improves a lot. The structure of the hydrazone enables itself to self-assemble and forms a fiber-like structure, and aggregates towards a cross-linked network, resulting in gelation of the surrounding solvent.

The researchers are interested to see whether a negatively-charged lipid membrane can somehow catalyze the formation of hydrazone 3 and also the formation of the final hydrogel network. Their rationale is that the negative charges on the membrane can induce an increase in the proton concentration, leading to a decrease of pH and renders the chemical environment more acidic. They believe this acidic environment may catalyze the formation of the hydrazone stucture and subsequent hydrogelation.

The reaction is carried out in the following way. The hydrazide 1 and the aldehyde 2 are mixed in a defined ratio in a buffered solution at neutral pH. After that, the negative-charged liposome solution is added, and the reaction is carried out at room condition. As observed, the gelator molecule 3 is formed and eventually gelation of solvent occurs, signifying the complete formation of the hydrogel network.

How did the researchers access whether catalysis occurred from the liposome? They employed a parameter known as minimum gelation concentration (MGC). They have first measured a control value – where no liposome is added to 3 at pH 7. When a liposome from a lipid with negative charges, DPPG (Figure 2), is added to the reaction mixture with 1 and 2, a decrease in MGC is evident. Of course, if an acid or aniline is added to the reaction mixture instead, due to their catalytic effects, the MGC should also decrease. Indeed, the researchers have found that the negatively charged liposomes work even better – they lead to lower MGC as compared to the acid / aniline scenarios. As another control experiment, the addition of a positive charged liposome sample does not lead to a decrease of MGC. With the use of an UV-active hydrazide substrate, the researchers can monitor the formation of hydrazone using absorbance spectroscopy for different reaction conditions. Thus, the logical conclusion from these trials is that a negative charged membrane is essential for catalysis to occur.

Figure 2. Structure of DPPG and DOPG.

Yet, there is one important thing to note before we jump to simple conclusions. The researchers have found that, while negatively charged membrane is likely to catalyze the formation of hydrogelator network, not all negative charged membranes can achieve that. The one missing piece in the puzzle is the melting temperature, T_m, of the liposome. When the researchers added the liposome from the negative charged lipid DOPG (Figure 2, T_m -20^oC, a liquid phase membrane at room temperature), to the reaction mixture, no hydrogelation occurs even at high liposome concentration. Yet, when the researchers increase the rigidity of the DOPG membrane through the addition of cholesterol (this should be familiar to anyone doing biochemistry or membrane biology), the DOPG-cholesterol hybrid membrane can then catalyze hydrogelation, suggesting the T_m of this hybrid liposome has increased.

Thus, the researchers have summarized that, in order for the liposome to catalyze hydrogelation, 2 criteria have to be fulfilled:

(1)   a negatively charged membrane surface

(2)   a solid phase at room temperature

The researchers have also established, from oscillatory rheology, that lipid concentration can control the physical properties of the hydrogel network. They also believe that liposomes are serving as nucleation points for the formation of fibrous network. I can think of a similar analogy in the case of cytoskeleton biology, where accessory proteins can serve as nucleation centers for actin polymerization.

By using confocal microscopy and a fluorescent aldehyde substrate, the researchers can also visualize the formation of the hydrogel network. When no liposomes are present, the resulting structure is very slack and un-connected. In the presence of liposomes, by contrast, the resulting hydrogel network becomes well-organized and dense, and the effect is enhanced when the liposome concentration is increased.

A very interesting aspect of the gel fiber formation occurs from the ‘underdog’, DOPG, which does not meet up to potential at the catalytic tests only until cholesterol comes to help. Rather curiously, because the T_m of DOPG is low, that meant the membrane it forms is more fluid than the membrane from DPPG. If we look at the chemical structure of DOPG and DPPG, it may shed light on this observation, and this concept is also familiar to biochemistry students. DOPG contains carbon-carbon double bonds, while DPPG is totally saturated on the carbon chains. The presence of double bonds will provide kinks and prevent a close-packing of the hydrocarbon chains, which by contrast is facile when only saturated chains are present. Thus, the unsaturated DOPG is more fluid than the saturated DPPG, and this also explains why the T_m of DOPG is lower.

DOPG has a higher affinity for the gel fibers. The affinity of the hydrogel fiber for the lipid membrane is related to the phase behavior of the hydrocarbon chains of the lipids, thus, a more fluid membrane should favor this interaction. Thus, DOPG-derived membrane seems to interact better with the hydrogel fiber than DPPG-type membrane.

What I am particularly impressed is that, by carrying out so many control experiments, the researchers draw together all the clues and provide a coherent explanation for the different performance of the DPPG and DOPG in catalysis. They propose that, because the DPPG membrane has less affinity to the hydrogel fiber, so the fiber is not blocking the way for DPPG to effect catalysis on its surface, therefore an efficient catalysis occurs and it goes on and on. In contrast, DOPG-type membrane, which is fluid and ‘loves’ the hydrogel fiber,  interacts with the gel fiber with such a high affinity that it is literally blocking the way for further rounds of catalysis. The researchers also draw analogy to the product blocking phenomena in heterogeneous catalytic systems, and I find this as an impressive explanation!

At the biological side, the researchers have also generated hydrogel fiber formations on HeLa cell systems.

All in all, this is a wonderful paper on membrane chemical biology. I have learnt a number of new techniques from it, and I am particularly impressed by the mechanistic insights, both in terms of catalytic and material, from all the great experiments they have carried out to arrive at the conclusions. Brilliant!

by Ed Law

13/7/2016

Reference:

1. Negatively Charged Lipid Membranes Catalyze Supramolecular Hydrogel Formation

Frank Versluis, Daphne M. van Elsland, Serhii Mytnyk, Dayinta L. Perrier, Fanny Trausel, Jos M. Poolman, Chandan Maity, Vincent A. A. le Sage, Sander I. van Kasteren, Jan H. van Esch and Rienk Eelkema

J. Am. Chem. Soc., 2016, asap, DOI: 10.1021/jacs.6b03853

2. Originally, I plan to talk about a self-emulsifying system which is used in the hydroformylation of lipid substrates. When I read Ref. [1], I decide to talk about that instead. It is interesting to see there are some connections regarding the two topics. Here are the references:

(a) A self-emulsifying catalytic system for the aqueous biphasic hydroformylation of triglycerides

T. Vanbésien, A. Sayede,   E. Monflier and    F. Hapiot 

Catal. Sci. Technol., 2016,6, 3064-3073

DOI: 10.1039/C5CY01758K

(b) Supramolecular Emulsifiers in Biphasic Catalysis: The Substrate

Drives Its Own Transformation

Théodore Vanbésien, Eric Monflier, and Frédéric Hapiot

ACS Catal. 2015, 5, 4288−4292

DOI: 10.1021/acscatal.5b00861

(c) Thermoresponsive Hydrogels in Catalysis

Frédéric Hapiot, Stéphane Menuel, and Eric Monflier

ACS Catal. 2013, 3, 1006−1010

dx.doi.org/10.1021/cs400118c