Enzyme-catalyzed Transetherification of Alkoxysilanes

We report the first evidence of an enzyme-catalyzed transetherification of model alkoxysilanes. During an extensive enzymatic screening in the search for new biocatalysts for silicon-oxygen bond formation, we found that certain enzymes promoted the transetherification of alkoxysilanes when tert-butanol or 1-octanol were used as the reaction solvents.


Introduction 24
Biotransformations are chemical processes which occur under the influence of biological materials 25 such as peptides and proteins. Amongst the myriad examples of bio-mediated transformations we have 26 focused our attention on enzyme-catalyzed reactions at a silicon centre. 27 In the literature, there are several examples of organo-silicon biotransformations, such as the 28 selective synthesis of organosilicon esters under mild reaction conditions [1], enzymatic silicone 29 oligomerization catalyzed by a lipid-coated lipase [2], and the hydrolysis of silatranes catalyzed by an 30 esterase obtained from the yeast Rhodotorula mucilaginosa [3]. In addition, nature provides the many 31

OPEN ACCESS
examples reactions from simple to very complex with Si-substrates, where peptides and proteins are 1 generally considered to be the undisputed arbiters [4,5]. Examples include silica formation in diatoms 2 and other silica-forming organisms [4,5]. 3 Our group has extensively studied silica precipitation [6] and the enzyme-catalyzed hydrolysis and 4 condensation of alxokysilanes [7,8,9] under mild conditions. We have discovered several enzymatic 5 candidates which were able to perform such reactions at room temperature and neutral pH and have 6 investigated the potential involvement of their respective active sites in the biocatalyzed organo-silicon 7 transformations. 8 In this contribution, we report a new enzyme-mediated reaction, namely the transetherification of 9 alkoxysilanes, under mild conditions. 10

Results and Discussion 11
During our previous work on enzyme-catalyzed organo-silicon transformations, we were interested 12 in siloxane-bond formation in the presence of biocatalysts both in aqueous and aqueous-organic media. 13 Several monophasic and biphasic aqueous-organic systems were investigated as reaction solvents. One 14 of the biphasic-aqueous organic systems employed during the alkoxysilane studies consisted of 1-15 octanol saturated with tris-buffered water. In addition to the enzyme-catalyzed hydrolysis and 16 condensation of alkoxysilanes [9], the formation of the octylsilyl ethers as a result of 17 transetherification and/or silanol-alcohol condensation/exchange was observed in this solvent whereas 18 no equivalent reaction was observed in the negative control reactions (Scheme 1). The "side-product" 19 octyl-silyl ether was identified as a new peak which appeared in the gas-chromatogram following 20 reaction and work-up when compared to our standard set of peaks which arise from solvents (THF,  21 ethanol and 1-octanol), unreacted starting material, hydrolyzed silanol, and the disiloxane 22 condensation product. 23 The identity of the structure was further investigated by means of GC-MS (not shown) and was 24 confirmed to be the alkoxyoctylsilyl ether. 25 Scheme 1. Enzyme-catalyzed transetherification and/or silanol-alcohol condensation. 26 The reactions were formulated with approximately 5:1 alkoxysilane to enzyme weight ratio in wet 1 (water-saturated) 1-octanol (5:1 solvent to alkoxysilane weight ratio) and conducted in inert glass 2 vials. After 24 hours of stirring at room temperature, the reactions were filtered and analyzed by GC-3 FID. The gas chromatography analysis was performed with an Agilent 6890 Series injector on an 4 Agilent 6890 plus gas chromatograph with a flame ionization detector. Dodecane was used as an 5 internal standard to quantitate the chromatographic analyses. The samples were prepared at ~1% (w/w) 6 product in a THF solution containing 1% (w/w) dodecane. Based on triplicate measurements, the 7 response factors for the analytes were calculated, and determined to be linear as a function of 8 concentration over four orders of magnitude (i.e. 0.01-10% w/w). 9 Scheme 2: Chemical synthesis of the octyl-silylethers. 10

11
In order to chromatographically quantify the trimethyloctyloxysilane and 12 phenyldimethyloctyloxysilane products, the two compounds had to be synthesized, as they were not 13 commercially available. The synthetic procedures are detailed in the Experimental Section. In general, 14 the appropriate chlorosilane was refluxed with 1-octanol in THF in the presence of triethylamine 15 (Scheme 2). The products were subsequently purified by vacuum distillation, characterized and used as 16 standard references in the gas-chromatography analyses. 17 As shown in Figure 1 and detailed in Tables 1-2, selected enzymes such as trypsin, Rhizopus 18 Oryzae lipase (ROL) and lysozyme were able to catalyze the formation of the octyltrimethyl-silyl ether 19 ( Figure 1, top) and/or the octylphenyldimethylsilyl ether ( Figure 1, bottom) after 24 hours at room 20 temperature. In similar conditions, no condensation was observed in the negative control reactions. 21   data, the mass balance being completed by either unreacted alkoxysilane, silanol formed by simple 8 hydrolysis, or the corresponding disiloxane from the condensation product with another molecule of 9 silanol (see Scheme 1). Notably, in the absence of any biocatalyst (negative control), no octyl-silyl 10 ether was observed, denoting the critical role of the enzyme in the alkoxysilane transetherification 11 transformation. 12 13 1  To our knowledge, this is the first case of an enzyme-mediated transetherification reaction of an 8 organo-silicon substrate under mild conditions. It is apparent that there is advantage in using an 9 enzyme at room temperature over the conventional synthetic procedure the synthesis of the octyl-silyl 10 products by avoiding the use of harsh chemicals and elevated reaction temperature. 11 Interestingly, the enzymatic screening conducted during the hydrolysis and condensation study of 12 monoalkoxysilanes in wet tert-butanol (see [9] and Figure1) did not lead to any tert-butylsilyl ether 13 product formation. This may be due to the steric hindrance of tert-butyl groups, as opposed to the 14 longer but more flexible octyl chains, which may be more accessible to the enzyme cavities. Notably, 15 ROL was observed to catalyze both octyl ether formation. Lipases normally interact with long-chain 16 alcohols and/or carboxylic acids as natural substrates. Our results show that ROL catalyzes the 17 formation of octyl-silyl ethers. Conversely, the lipase was not able to catalyze the formation of tert-18 butyl silyl ethers. This is in agreement with the natural substrate-selectivity of the ROL, and suggests 19 the involvement of the active site during the catalysis. Trypsin was a good biocatalyst for 20 trimethyloctyl silyl ether formation, and this is in line with our previous studies on the alkoxysilane 21 hydrolysis and condensation reactions [7,9]. Lysozyme, which was already observed to be a good 22 siloxane-bond biocatalyst [8], produced the highest yield of the phenyldimethyloctyl silyl ether in this 23 study. The reason for the unusual selectivity of this glycoside hydrolase is not yet understood and will 24 be the subject of further investigations. The work proves the potential of the use of (bio)-25 macromolecules as catalytic aids on unusual substrates under facile and mild reaction conditions. 26

Enzyme-Catalyzed Transetherification Reactions 1
The reactions were formulated with a 5:1 alkoxysilane (100mg) to enzyme (20mg) weight ratio in 2 0.5 g of alcohol (water-equilibrated 1-octanol or tert-butanol containing 5% (v/v) buffered water). 3 Prior to analysis, the reactions were filtered through a Whatman Autovial® 5 0.45-µm Teflon® filter. 4 The closed (screw capped) two-phase reactions were conducted in inert glass vials at 25 °C with 5 magnetic stirring for 24 hours. The reaction products were isolated and analyzed by GC-FID 6 (quantitative) and GC-MS (qualitative). 7 Control reactions are defined as non-enzymatic reactions. Specifically, experiments conducted in 8 the absence of a protein are defined as negative control reactions. 9

Gas Chromatography-Flame Ionization Detection 10
The gas chromatography (GC) analyses were performed with an Agilent 6890 Series injector on an 11 Agilent 6890 plus gas chromatograph (GC) with a flame-ionization detector (FID). 12 The system was configured as detailed in Table 1. Dodecane was used as an internal standard to 13 gravimetrically quantitate the chromatographic analyses. The samples were prepared at ~1% (w/w) 14 product in a THF solution containing 1% (w/w) dodecane. Based on triplicate measurements, the 15 response factors for the analytes were calculated (Equation 1), and found to be linear as a function of 16 concentration over four orders of magnitude (i.e. 0.01-10% (w/w) ( Table 3). (2) 2

Gas Chromatography-Mass Spectrometry 3
The gas chromatography-mass spectrometry (GC-MS) analyses were performed with an Agilent 4 6890 Series injector on an Agilent 6890 plus gas chromatograph with a 5973 MS detector. The MS 5 detector was autotuned with perfluorotributylamine (PFTBA) prior to analysis. The system was 6 configured as detailed in Table 4. 7 Table 3: GC-FID analyte retention times and response factors.