In this paper we investigate the effect of three different types of surfactants, on the hydrolysis of Cytochrome c (Cyt c), a predominantly α helical protein containing a heme group, promoted by [Ce(α PW11O39)2]10- (CeK) and [Zr(α PW11O39)2]10- (ZrK) polyoxometalates. In the presence of SDS, Zw3 12, or CHAPS surfactants, which are commonly used for solubilizing hydrophobic proteins, the specificity of CeK or ZrK toward hydrolysis of Cyt c does not change. However, the hydrolysis rate of Cyt c by CeK was increased in the presence of SDS, but decreased in the presence of CHAPS, and was nearly inhibited in the presence of Zw3 12. The Circular dichroism and Tryptophan fluorescence spectroscopy have shown that the structural changes in Cyt c caused by surfactants are similar to those caused by POMs, hence the same specificity in the absence or presence of surfactants was observed. The results also indicate that for Cyt c hydrolysis to occur, large unfolding of the protein is needed in order to accommodate the POMs. While SDS readily unfolds Cyt c, the protein remains largely folded in the presence of CHAPS and Zw3 12. Addition of POMs to Cyt c solutions in CHAPS results in unfolding of the structure allowing the interaction with POMs to occur and results in protein hydrolysis. Zw3 12, however, locks Cyt c in a conformation that resists unfolding upon addition of POM, and therefore results in nearly complete inhibition of protein hydrolysis.

The development of artificial proteases is challenging, but important for many applications in modern proteomics and biotechnology. The hydrolysis of hydrophobic or unstructured proteins is particularly difficult due to their poor solubility, which often requires the presence of surfactants. Herein, it is shown that a zirconium(IV)‐substituted Keggin polyoxometalate (POM), (Et2NH2)10[Zr(α‐PW11O39)2] (1), is able to selectively hydrolyze β‐casein, which is an intrinsically unstructured protein at pH 7.4 and 60 °C. Four surfactants (sodium dodecyl sulfate (SDS), N‐dodecyl‐N,N‐dimethyl‐3‐ammonio‐1‐propanesulfonate (ZW3‐12), 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS), and polyethylene glycol tert‐octylphenyl ether (TX‐100)), which differ in the nature of their polar groups, were investigated for their role in influencing the selectivity and efficiency of protein hydrolysis. Under experimental conditions, β‐casein forms micellar structures in which the hydrophilic part of the protein is water accessible and able to interact with 1. Identical fragmentation patterns of β‐casein in the presence of 1 were observed through SDS poly(acrylamide) gel electrophoresis both in the presence and absence of surfactants, but the rate of hydrolysis varied, depending on the nature of surfactant. Whereas TX‐100 surfactant, which has a neutral polar head, caused only a slight decrease in the hydrolysis rate, stronger inhibition was observed in the presence surfactants with charges in their polar heads (CHAPS, ZW3‐12, SDS). These results were consistent with those of tryptophan fluorescencequenching studies, which showed that the binding between β‐casein and 1 decreased with increasing repulsion between the POM and the polar heads of the surfactants. In all cases, the micellar structure of β‐casein was not significantly affected by the presence of POM or surfactants, as indicated by circular dichroism spectroscopy.

The reactivity and selectivity of Wells-Dawson type polyoxometalate (POM), K16[Hf(α2-P2W17O61)2]·19H2O (Hf1-WD2), have been examined with respect to the hydrolysis of ovalbumin (OVA), a storage protein consisting of 385 amino acids. The exact cleavage sites have been determined by Edman degradation experiments, which indicated that Hf1-WD2 POM selectively cleaved OVA at eight peptide bonds: Phe13-Asp14, Arg85-Asp86, Asn95-Asp96, Ala139-Asp140, Ser148-Trp149, Ala361-Asp362, Asp362-His363, and Pro364-Phe365. A combination of spectroscopic methods including 31P NMR, Circular Dichroism (CD), and Tryptophan (Trp) fluorescence spectroscopy were employed to gain better understanding of the observed selective cleavage and the underlying hydrolytic mechanism. 31P NMR spectra have shown that signals corresponding to Hf1-WD2 gradually broaden upon addition of OVA and completely disappear when the POM-protein molar ratio becomes 1:1, indicating formation of a large POM/protein complex. CD demonstrated that interactions of Hf1-WD2 with OVA in the solution do not result in protein unfolding or denaturation even upon adding an excess of POM. Trp fluorescence spectroscopy measurements revealed that the interaction of Hf1-WD2 with OVA (Kq = 1.1 × 105 M−1) is both quantitatively and qualitatively slightly weaker than the interaction of isostructural Zr-containing Wells-Dawson POM (Zr1-WD2) with human serum albumin (HAS) (Kq = 5.1 × 105 M−1).

Development of catalysts for the selective hydrolysis of proteins is challenging, yet important for many applications in biotechnology and proteomics. The hydrolysis of hydrophobic proteins is particularly challenging, as due to their poor solubility, the use of surfactants is often required. In this study, the proteolytic potential of catalyst systems based on the Zr(IV)-substituted Keggin polyoxometalate (Et2NH2)10[Zr(PW11O39)2] (Zr-K 1:2) and three different surfactants (ionic SDS (sodium dodecyl sulfate); zwitterionic Zw3-12 (n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate); and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)), which differ in structure and polarity, has been investigated. Hydrolysis of ovalbumin (OVA) was examined in the presence of Zr-K 1:2 and surfactants by sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), which showed the appearance of new polypeptide fragments at lower molecular weight, indicating that selective hydrolysis of OVA took place for all three catalyst systems. The same fragmentation pattern was observed, showing that the selectivity was not affected by surfactants. However, the surfactants influenced the performance of the catalyst. Hence, the interactions of OVA with surfactants and Zr-K 1:2 were investigated using different techniques such as tryptophan fluorescence, Circular Dichroism, and Dynamic Light Scattering. The speciation of the catalyst in surfactant solutions was also followed by 31P Nuclear Magnetic Resonance spectroscopy providing insight into its stability under reaction conditions.

The interaction between the 1:2 Zr(IV) :Wells-Dawson complex, K15 H[Zr(α2 -P2 W17 O61 )2] (1), and a range of surfactants was studied in detail with the aim of developing metal-substituted POMs as potential artificial proteases for membrane proteins. The surfactants include the positively charged cetyl(trimethyl)ammonium bromide (CTAB), the negatively charged sodium dodecyl sulfate (SDS), the neutral Triton X-100 (TX-100), and zwitterionic 3-[dodecyl(dimethyl)ammonio]-1-propanesulfonate (Zw3-13) and 3-[dimethyl(3-{[(3α,5β,7α,12α)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)ammonio]-1-propanesulfonate (CHAPS). A combination of multinuclear (1)H, (13)C, and (31) P NMR spectroscopy, (1)H diffusion-ordered NMR spectroscopy ((1)H DOSY), and nuclear Overhauser effect spectroscopy (NOESY) was used to examine the interaction between 1 and each surfactant on the molecular level. Cationic surfactant CTAB caused precipitation of 1 due to strong electrostatic interactions, while the anionic SDS and neutral TX-100 surfactants did not exhibit any interaction at neutral pD. (1)H DOSY NMR spectroscopy indicated an interaction between 1 and zwitterionic surfactants Zw3-12 and CHAPS, which occurs via the positively charged ammonium group in the surfactant molecule. In the presence of anionic, neutral, and zwitterionic surfactants, 1 preserves its catalytic activity towards the hydrolysis of the peptide bond in the dipeptide glycyl-l-histidine (GH). The fastest hydrolysis was observed at pD 7.0 and could be rationalized by taking into account pD-dependent speciation of 1 and coordination properties of GH.

A new postfunctionalization method was developed for the Anderson-type POM based on a nucleophilic substitution reaction occurring at an electrophilic sp hybridized carbon localized on the hybrid POM. Using this method, several types of different nucleophiles including primary and secondary amines, carboxylates, and thiolates were efficiently coupled to a chloride-functionalized Anderson-type POM in high yields and purity. The heterogeneous acetonitrile-NaCO conditions were found to be superior over other bases and solvents for the coupling of amines and thiolates to the chloride-functionalized POM. Moreover, the addition of 1 equiv of tetrabutylammonium iodide as a catalyst drastically decreased the reaction times to 24 h for the complete coupling of amines and only a couple of hours for thiolates. In the case of carboxylic acids as substrates, using tetrabutylammonium hydroxide as the base for the reaction proved to be beneficial. This is because the resulting tetrabutylammonium carboxylates were found to be much more reactive than the corresponding sodium carboxylates and allowed homogeneous reaction conditions. Using sodium carbonate, only 25% of N-acetyl glycylglycine could be coupled after 24 h at 80 °C, while full conversion was achieved after the same reaction time when using tetrabutylammonium hydroxide as a base.

The catalytic fragmentation of hydrophobic proteins by polyoxometalates (POMs) requires the presence of surfactants in order to increase the solubility of the protein. Depending on the nature of the surfactant, different effects on the kinetics of protein hydrolysis are observed. As the molecular interactions between the POMs and surfactants in solutions have been scarcely explored, in this study, the interaction between the catalytically active Keggin polyoxometalate [Zr(α-PW11O39)2]10− and four different surfactants—sodium dodecyl sulfate (SDS), dodecyldimethyl(3-sulfopropyl)ammonium (Zw3-12), dodecyldimethyl(3-sulfopropyl) ammonium (CHAPS), and polyethylene glycol tert-octylphenyl ether (TX-100)—have been studied in aqueous media. The effect of polyoxometalate on the self-assembly of surfactant molecules into micelles and on the critical micellar concentration (CMC) has been examined by fluorescence spectroscopy and diffusion ordered NMR spectroscopy (DOSY).

A mild post-functionalization method, based on a copper(i) catalyzed azide-alkyne cycloaddition, was developed for the fragile [PWV] (PWV). PWV was functionalized with an azide and used as a platform to attach various alkyne functionalized compounds.