Dendritic Oligoglycerol Regioisomer Mixtures and Their Utility for Membrane Protein Research

Abstract Dendrons are an important class of macromolecules that can be used for a broad range of applications. Recent studies have indicated that mixtures of oligoglycerol detergent (OGD) regioisomers are superior to individual regioisomers for protein extraction. The origin of this phenomenon remains puzzling. Here we discuss the synthesis and characterization of dendritic oligoglycerol regioisomer mixtures and their implementation into detergents. We provide experimental benchmarks to support quality control after synthesis and investigate the unusual utility of OGD regioisomer mixtures for extracting large protein quantities from biological membranes. We anticipate that our findings will enable the development of mixed detergent platforms in the future.

OGD regioisomer mixtures 1, 2, and 3. Shorter retention times were obtained for the isomers 1a, 2a, and 3a, thus indicating that these isomers exhibit a less hydrophobic character. Information about the mobile phase composition (H2O, MeOH), detector system, e.g., UV detector or RI detector, and flow rate (mL/min) are given. The injection peak is labelled with Inj. Figure S8. Utility of [G1] OGDs for the purification of the membrane protein AmtB-MBP. Increasing the size of the head and length of the tail among 3a and 3b reduces the obtainable protein yields. Higher yields of AmtB-MBP were obtained from the [G1] OGD regioisomer mixture 3. Tables   Table S1. Information about oligoglycerol mixtures, including Table S4. Summary of detergents, [G1] OGD regioisomer ratios (a:b), relative protein quantities [P], and standard deviations (±SD) obtained from the extraction of AqpZ-GFP and AmtB-MBP from native E. coli membranes. As described above, the protein quantities were obtained from a previously published paper. [1] Protein

General Information about Synthesis
Synthesis of [pG1]-OH and [pG2]-OH regioisomers were conducted in analogy to previously published procedures. [2] The acetal protection of oligoglycerol mixtures was done as described previously. [2a] The syntheses of regioisomer mixtures and individual regioisomers have been subjects of a previous invention. [3] Synthesis protocols that led to the obtainment of [G1] OGD batches 1 and 3 as well as [G2] OGD batches 5, 8, and 9 have been published before. [1,4] Synthesis protocols which led to the obtainment of 2, 4, 6, and 7 have not been published before in journal format. The starting materials, lab equipment, and work-flows used to obtain the here-described compounds were similar to those from previously published protocols: [1,[4][5] Chemicals were purchased from Sigma-Aldrich (Germany), Acros Organics (Germany), Alfa Aesar (Germany), Fluka (Germany), Fisher Scientific (Germany), Merck (Germany), TCI (Germany).
Chemicals were used as supplied. Ethyl acetate (EtOAc), n-hexane, and n-pentane were distilled before they were used. Other solvents, such as methanol (MeOH), dimethylformamide (DMF), tertbutanole ( t BuOH), and dichloromethane (DCM) were used as supplied. Dry solvents were purchased in bottles sealed with a septum or tapped from a solvent purification system (MS-SPS-800) that was purchased from M. Braun (Germany). Deionized water used for synthesis was provided by a deionization system installed in the Freie Universität's Institute of Chemistry and Biochemistry. Argon was purchased from Linde (Germany). For working under dry and oxygen-free reaction conditions, chemicals and solvents were handled under argon atmosphere. To support dry conditions the glassware was evacuated, heated up to 300 °C with a heat gun, and filled with argon.
Monitoring of reactions and purification procedures was achieved by normal-phase thin-layer chromatography (TLC) analysis. TLC plates (DC-Fertigfolien ALUGRAM® Xtra SIL G/UV254) based on silica (SiO2) were purchased from Macherey-Nagel (Germany

HPLC
For analytical normal-phase HPLC analysis, a Nucleosil column from Macherey Nagel was used as stationary phase (pore size: 50 Å, particle size: 5 μm, length: 250 mm, diameter: 4 mm). Mixtures of n-hexane and isopropanol were used (v:v) as mobile phase. The NP HPLC system was equipped with a Smartline pump 1050, a Smartline UV detector 2550, and a Smartline RI detector 2300, which were purchased from Knauer. The system was operated with a flow rate of 1 mL/min. Data processing and analysis was performed with ChromeGate Client Viewer (v.3.3.2) from Knauer. For preparative normal-phase HPLC, a Nucleosil column from Macherey Nagel was used as stationary phase (pore size: 50 Å, particle size: 5 μm, length: 250 mm, diameter: 32 mm). The preparative HPLC system consisted of a Smartline pump 1800, UV variable wavelength monitor from Knauer, and a Smartline RI detector 2400. The system was operated with a flow rate of 64 mL/min. The normal-phase HPLC system was operated by Marleen Selent.
For analytical reversed-phase HPLC, a system from Knauer was used, which was equipped with two Smartline 1000 pumps, a variable wavelength UV detector 2500, RI detector, and an Autosampler 3950. As stationary phase, a pre-packed C18 column was used (RSC-Gel, C18ec, pore size: 100 Å, particle size: 5 μm, length: 250 mm, diameter: 4 mm). The flow rate was 1 mL/min. Data processing and analysis was performed with ChromeGate Client Viewer (v.3.3.2) from Knauer. For reversedphase HPLC purification, a setup from Knauer was used, which consisted of a Smartline Manager 5000 (+ interface-module), two Smartline Pumps 1000, a 6-port-3-channel-injection valve, a sample loop (10 mL), UV Detektor 2500, RI detector, and a high pressure gradient mixer. Spectra were recorded with a x-y-plotter from Knauer. As stationary phase, a pre-packed column was used (RSC-Gel, C18ec, 5 μm). The setup was constructed by Dr. Carlo Fasting. The mobile phase was degassed prior usage. The flow rate was adjusted to 20 mL/min and the detection wavelength was 240 nm.
[1a] DL-1,2-Isopropylidenglycerol (5.62 g, 42.5 mmol) was dissolved in THF (125 mL) and NaH (60w%, 5.10 g, 128 mmol) was added. Catalytic amounts of 15-crown-5 were added and the mixture was stirred at 50 °C for 1 h. Methallyl dichloride (2.65 g, 21.2 mmol), catalytic amounts of 18-crown-6, and potassium iodide were added and the mixture was stirred at 80 °C for 12 h. The reaction mixture was then allowed to cool down to room temperature and water (10 mL

[pG1]-OH (b)
[pG0]-OBn. D,L-Isopropylidenglycerol (2.00 g, 15.1 mmol) was dissolved in DMF (80 mL) and the flask was cooled in an mixture of water and ice (50:50). NaH (60w%, 2.42 g, 60.5 mmol) was added in small portions, while the mixture was stirred and allowed to warm up to room temperature. Benzyl bromide (3.10 g, 18.2 mmol) was added and the mixture was stirred for 12 hours at room temperature.
Subsequently, water (10 mL) was added drop wise and the solvent was removed under reduced pressure. The remaining material was suspended with Brine (200 mL) and water (100 mL) and the aqueous layer was extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over [pG2]-OH (aa,ab,bb). [1] The starting material [pG2]-ene (8.00 g, 11.5 mmol, aa:ab:bb, 4:4:1) was dried was dried under reduced pressure (~ 10 -2 mbar) and dissolved in a mixture of dry DCM (35 mL) and dry MeOH (35 mL). The mixture was cooled down to -78 °C and ozone was passed through the reaction mixture until its colour changed to deep blue. Oxygen was then passed through the solution until it became colourless. Sodium borohydride (4.36 g, 115 mmol) was added slowly and the mixture was allowed to heat up to RT overnight. A saturated aqueous solution of NH4Cl (100 mL) was added and the mixture was extracted with DCM (5 x 50 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. Column chromatography (SiO2, DCM/EtOAc, 97/3 + 3% MeOH) gave the desired product (6.40 g, 9.18 mmol, aa:ab:bb, 4:4:1, 80%
Methallyl dichloride (0.17 mL, 1.48 mmol), potassium iodide (catalytic amounts), and 18-crown-6 (catalytic amounts) were added. The reaction was stirred at 80 °C for 12 hours. The reaction mixture was allowed to cool down to room temperature and water (6 mL) was added drop wise. The solvent was removed under reduced pressure and the crude product was mixed with water (60 mL), Brine (60 mL), and DCM (60 mL

[G1] OGD Regioisomer
[pG1]-OMes. was added and the mixture was stirred at 120 °C for 16 hours. The mixture was allowed to cool down to room temperature. Water (20 mL) and a saturated aqueous solution of NH4Cl (20 mL) were added.

Collision Cross Section Calculation
Conformational search and optimization of the head groups of 4a and 4b were achieved using the MM2 force field as implemented in ChemBio3D v14.0 (PerkinElmer). The pdb data of the head groups are listed below. Theoretical collision cross section (CCS) values were calculated from the pdb data using the projection approximation algorithm. [6] pdb data of the head group 4a:

Pendant Drop Method
Pendant drop experiments were performed as described before. [5,7] Briefly, a dilution series was prepared and the samples were investigated using a contact angle tensiometer OCA 20 (DataPhysics Instruments GmbH, Germany). To reduce the impact of evaporation effects, a wet filter paper was placed in a watch glass on the bottom of the measurement chamber. The dispensed droplet (volume = 18 -20 µL) was equilibrated between 30 and 90 minutes at room temperature (22 -23 °C) before a constant interfacial surface tension (IFT) was obtained. IFT values were determined for every concentration from three independent droplets. The IFT values were averaged and plotted with their standard deviation against the logarithm of the concentration (see Figure 3, manuscript). The error bars are smaller than the size of the plotted dots.

Estimation of logP Values
The logP values were estimated from the molecular structures of individual regioisomers using ChemDraw Professional (v19.1.1.21).

Critical Aggregation Concentration
Critical aggregation concentration (cac) values were determined by dynamic light scattering (DLS) using previously published procedures: [1,4,8] Serial dilutions with OGD concentrations between 10 −8 and 10 −2 M were prepared in MilliQ water. The samples were filtered (RC, 0.2 µm) and equilibrated for 16 hours at room temperature prior to their analysis. The samples were transferred into a quartz cuvette (Quartz Suprasil, width × length: 2 mm × 10 mm) and analysed with a Zetasizer Nano-ZS ZEN3600 (Malvern, UK). The instrumental parameters were as follows: material (polystyrene latex), dispersant (water), sample viscosity parameters (use dispersant viscosity as sample viscosity), temperature (22.5 °C), equilibration time (120 s), cell type (quartz cuvettes), measurement angle (173° backscatter), measurement duration (manual), number of runs (11), run duration (10 s), number of measurements (3), delay between the measurements (0 s), data processing (general purpose, normal resolution). The derived count rate values obtained from three measurements per concentration were averaged. The unit of the derived count rate is kilo counts per second (kcps). The logarithm of the derived count rate was plotted against the logarithm of the concentration. The double logarithmic plots showed two characteristic regions: (1) a flat region with low count rates at lower concentrations and (2) a linear growth of the count rate at higher concentrations. Both regions were fitted to linear functions and the intersection was taken as the cac value. An image visualizing the fitting procedure is provided in Figure S9 (Supporting information) of another articlesee Ref. [4].

Membrane Protein Purification
The relative protein quantities obtained upon IMAC purification discussed throughout this paper have been obtained from a previously published paper (Table S4). [1] Briefly, the membrane proteins were expressed in Escherichia Coli (E. coli) and purified from bacterial membranes using n-dodecyl-ß-Dmaltoside (DDM) and [G1] OGD batches 3 (= 3a + 3b), 3a, and 3b. Protein solutions obtained upon IMAC were concentrated to equal volumes and relative protein quantities were determined by UV/VIS spectroscopy. Absorbance values (A485 for AqpZ-GFP, A280 for AmtB-MBP) were normalized to those obtained from DDM, averaged (n = 3), and plotted with standard deviation (±SD) against the detergent abbreviation ( Figure 3, Figure S8). The data are summarized in Table S4. For further information about the experimental procedure see Urner et al. [1] 3.9 Monitoring the Activity of Outer Membrane Protease T The outer membrane protease was refolded as described before. [1] The activity assay was also performed in analogy to a previously published procedure: [1] The activity of OmpT was assessed by monitoring the time-dependent cleavage of a self-quenching fluorescent peptide Abz-ARRAY-Tyr(NO2)-NH2 (Biomatik, custom synthesis) in which "Abz" abbreviates o-aminobenzoyl and "Tyr(NO2)" abbreviates 3-nitrotyrosine [9] . The following components were mixed in chambers of a 96 well plate