Kaiser’s test - Solid-phase-assisted synthesis

3.2 Methods

3.2.3 Solid-phase-assisted synthesis

3.2.3.9 Kaiser’s test

DBU. After everything was prepared, the synthesizer was started, and desired sequences were synthesized according to the scheme above. At the end, Kaiser’s test (See Kaiser’s test) was performed to check if the deprotection was successful.

3.2.3.7 Cleavage off the resin

The syringe reactor with a dry resin inside, which was dried in an exsiccator under vacuum overnight, was filled in the fume hood with a cleavage cocktail that comprises TFA, TIS, Millipore water in the ratio of 95: 2.5: 2.5 (10 mL/g resin), respectively, and then incubated for 90 min at RT and 65 rpm (Heidolph Reax 2). For the oligoaminamides containing cysteine, the cleavage cocktail comprised of TFA, TIS, Millipore water, EDT in the ratio of 94: 2.5: 2.5: 1 (10 mL/g resin), respectively, was prepared. Meantime the precipitation cocktail was prepared which, composing of 30 mL of n-hexane and 10 ml of MTBE in the 50 mL disposable centrifuge test tube and cooled down to -7 °C. The cleaved solution was slowly injected into the precipitation cocktail after 90 min and briefly shaken. The centrifuge tube was inserted into the centrifuge (Megafuge 1.0R, Heraeus (Hanau, Germany)) and balanced with Millipore water that had the same mass as the sample and centrifuged for 10 min at 4 °C and 4000 rcf. The supernatant was discarded, and the precipitate was dried by nitrogen until it was completely dry. The centrifuge tube was stored in fridge at 7 °C.

3.2.3.8 Mini cleave

To check if the coupling steps were executed successful, a mini cleave was performed. A small sample of dry resin (around 20 mg) was added into a 2 mL syringe reactor and cleaved with 1.5 mL of the cleavage cocktail. For the following steps see Cleavage off the resin. The structure was identified by MALDI-TOF MS (See MALDI-TOF MS).

3.2.3.9 Kaiser’s test

After the deprotection of amino acids on the resin, the free amines were qualitatively determinated by Keiser’s test. A small sample of resin, which was previously washed with DCM, was transferred into a 1.5 mL Eppendorf reaction tube. Three reagent solutions were prepared which consisted of 80 % of phenol in EtOH (w/v), 5 % of ninhydrin in EtOH (w/v) and 20 μM potassium cyanide (KCN) in mixture of 1 mL aqueous 0.001 M KCN solution and 49 mL pyridine. One drop of each was added into the Eppendorf tube and the mixture was then incubated under steady shaking at 600 rpm and 99 °C for 3 min. The presence of free amines was indicated by a deep blue coloring. Otherwise, the mixture would have been colored yellow.

Figure 11: Reaction of ninhydrin in Keiser’s test 3.2.4 Synthesis of four-arm oligoaminoamides

The arm branching core was loaded on Fmoc-Ala-Wang resin (See Loading of the four-arm branching core on Fmoc-Ala-Wang resin). 0.01 mmol of the branching core was volumetrically split using the Vac-Man® Laboratory Vacuum Manifold into 10 mL syringe reactors and swollen in DCM for 5 min. According to the chapter Automated synthesis, following sequences were synthesized automatically; [Stp-H-R]3, [Stp-H]3R3, [Stp-H-W]3, [Sph-H]3Y3, [Sph-H]3W3, [Sph-H]3R3, [Sph-H-R]3, [Sph-H-K]3, [Sph-H-W]3, and [Sph-H]3

twice. The resins were volumetrically transferred using the Vac-Man® Laboratory Vacuum Manifold into the new 10 mL syringe reactors. Four resins were swollen for 20 min in DCM containing 1 % Triton™ X-100 (v/v). The above mentioned motifs were coupled first with Fmoc-L-Cys(Trt)-OH and second with Fmoc-L-Lys(N3)-OH, except for one motif [SphH]3

that was coupled only with Fmoc-L-Cys(Trt)-OH. Coupling and deprotection steps were performed according to the Manual synthesis and Fmoc deprotection with some exceptions.

DMF and DCM contained 1 % Triton™ X-100 (v/v) and equivalents of amino acid, DIPEA, PyBOP, HOBt were multiplied by 8. After all the steps were performed the resins were dried in the exsiccator under a vacuum overnight. Next day, cleavage off the resin was performed

(See Cleavage off the resin) with slight modification of reagent ratio 93.5: 2.5: 2.5: 2.5 and after centrifugation the resins were dried and stored in the fridge at 7 °C.

3.2.5 Size-exclusion chromatography

All four-arm oligoaminoamides were purified by size-exclusion chromatography using an ÄKTApurifier 10 FPLC system (GE Healthcare Bio-Sciences AB (Uppsala, Sweden)) that was using Sephadex G-10 column in fraction range of 0–700 Da. The column consists of a 60 cm high hollow tube packed with micron-scale dextran polymer beads (Sephadex G-10).

3.2.5.1 Colum packing

The hollow tube was scrupulously cleaned. All materials were equilibrated at RT, avoiding exposure to sunlight to prevent the heating of the column and subsequent formation of bubbles. 100 g of Sephadex G-10 was dissolved in 500 mL of Millipore water and swollen for 3 h at RT. The bottom filter was wet with Millipore water. The bottom end piece and filter were mounted onto the column. The column was fastened vertically on the ring stand with utility clamps and Millipore water was poured into the column, approximately 2 cm above the column end piece. The suspension of Sephadex G-10 was taken with a pipette controller at the bottom as it was possible to obtain its highest density and the column was filled with suspension of Sephadex G-10. The pump outlet was connected to the inlet of the packing reservoir and the column outlet was opened. 2 to 3 column volumes of Millipore water were passed with the 1.1 mL/min flow rate through the column to stabilize the bed and equilibrate. The packing suspension was added until a constant bed height was obtained, and the column outlet was closed. Millipore water was added to fill the column and the adapter was inserted into the column so that there were no trapped air bubbles. The column was stored in the fridge at 7 °C.

3.2.5.2 Execution of size-exclusion chromatography

For the mobile phase 70 % of 0.01 M HCl and 30 % of acetonitrile (v/v) was used. 2 L of mobile phase was prepared in two 1 L reservoirs. 0.693 L of Millipore water was poured into each reservoir and 7 mL of 1 M HCl was added. Later, 0.3 L of acetonitrile was added to each reservoir, and both were shaken. In the program, the flow was set to 1 mL/min. The column was fastened onto the ring stand with utility clamps and the tube was installed on the upper outlet of the column after some drops of the mobile phase had dripped off. When some drops fell out from the column, another tube on the lower outlet of the column was installed to avoid air entering the column. Before starting SEC, the following parameters

were set in the program; fraction size to 0.75 mL and wavelength to 280 nm, 254 nm, and 214 nm. Samples of four arms were dissolved in 4 mL of Millipore water and two runs were performed using 2 mL sample for each run. The program was executed and after approximately one third of the peak’s slope showed up, the sample was collected in a 50 mL centrifuge tube (after 15–20 min). Also, the last third of the slope was discarded. At the end the column was washed until the UV signal returned to the starting state. After that, the next run was executed. Collected samples were frozen with liquid nitrogen, caps were perforated, lyophilized overnight using Alpha 1-4 LSCbasic and stored in the refrigerator at - 7 °C.

3.2.6 Formation of polyplexes with pDNA

Lyophilized oligomers were dissolved in Millipore water in a 5 mL Eppendorf tube, so that 10 mg/mL solution was prepared and diluted in a 1:10 ratio with Millipore water. The stock solution of the plasmid pCMV-luc that encodes gene for firefly luciferase under control of CMV promoter, was diluted with HBG buffer from 1 mg/mL to 20 μg/mL with a 1.2 excess.

The corresponding volume of oligomer solution that included adequate number of mole as in Table 7: N/P ratios for formation of polyplexes according to the N/P ratio (ratio of polymer’s positively-chargeable amine groups compared to the negatively-charged phosphate groups of nucleic acid) was taken with an automatic pipette into a 1.5 mL Eppendorf tube and diluted with HBG buffer to 60 μL. Subsequently, 60 μL of 20 μg/mL pCMV-luc solution was added, the mixture was rapidly pipetted 15-times and incubated for 40 min at RT.

Table 7: N/P ratios for formation of polyplexes

N/P protonable amines [nmol] phosphate [nmol]

6 22.397 3.732

12 44.784 3.732

20 74.64 3.732

3.2.6.1 HBG buffer pH 7.4

2.38 g of HEPES was weighed into the 0.5 L bottle and dissolved in 0.3 L of Millipore water.

A magnetic stir bar was added into the bottle and solution was stirred on the magnetic stirrer at 500 rpm. After everything was dissolved, pH meter electrode (Lab 875, SI Analytics) was inserted into the bottle and the pH of the solution was adjusted to 7.4 at RT with 3 M aqueous

sodium hydroxide solution that was added dropwise with an automatic pipette. 27.5 g of D-(+)-glucose monohydrate was added and stirred for 1 h at RT and 500 rpm. Next, the bottle was filled with Millipore water to 0.5 L mark, stirred for one additional hour and stored overnight in the fridge at 7 °C. The next day, the pH was checked, and in the case, it changed, was readjusted again to 7.4. The HBG buffer was stored in a 15 mL disposable centrifuge test tube in the refrigerator at -7 °C.

3.2.7 Measurement of polyplex size and zeta potential

The size and zeta potential of polyplexes was measured by dynamic laser-light scattering (DLS) using the Zetasizer Nano ZS with backscatter detection (Malvern Instruments, Worcestershire, UK). After 40 min of incubation, 120 μL of the polyplex solution was added with an automatic pipette into the folded capillary cell which was washed four-times with ethanol before first use and then three-times with Millipore water before every measurement of a new sample. There should not be any air bubbles in the cuvette. Three measurements with 6 runs were performed. Following parameters were chosen for measurements of size (z-average) and polydispersity index (PDI); equilibration time 0 minutes, temperature 25 °C, refractive index 1.330, viscosity 0.8872 mPas. Each sample was measured three times with six sub runs each.

For the measurement of zeta potential, 700 μL of HBG buffer (pH 7.4) was added to the sample into the folded capillary cell and rapidly pipetted 3-times. Three measurements with 15 sub runs lasting 10 seconds each at 25 °C were performed.

3.2.8 Electrophoretic mobility shift assay

First, 1 % (w/v) of agarose gel was prepared. 1.5 g of agarose was dissolved in 150 mL of TBE buffer. The solution was heated up to 100 °C in the microwave until agarose was completely dissolved. When the solution cooled down to 50 °C, 150 μL of GelRed™ was added and the solution was poured into the electrophoresis unit (Sub-Cell GT Horizontal Electrophoresis System, 15 x 10 cm tray, BIORAD) and solidified at RT. The agarose gel was overlaid with a TBE-buffer (approximately 1 L), 5 μL of loading buffer were placed into the agarose gel pockets and subsequently 20 μL of polyplexses formed with 200 ng of pCMV-luc after 40 min of incubation. In the first pocket control free pCMV-luc was placed.

Electrophoresis was performed at 120 V for 70 min at RT. Picture of the electrophoresis assay was taken in the Dark Hood DH-40 using Canon EOS 2000 Kit.

37 3.2.9 Analytical methods

3.2.9.1 ¹H-NMR

1H-NMR spectra of all synthesized structures were recorded using an AVANCE III HD 500 (500 MHz) by Bruker with a 5 mm CPPBBO probe. Spectra were recorded without TMS as internal standard. Therefore, all signals were calibrated to the residual proton signal of the deuterium oxide (D2O) and chloroform-d, respectively. All chemical shifts were calibrated to the residual proton signal of the solvent (deuterium oxide; δ = 4.79 ppm and chloroform-d; δ = 7.26 ppm) and reported in ppm. The spectra were analyzed using the software MestreNova (Ver.11.0.4; Mestrelab Research). Integrals were set manually and normalized to the succinic acid peaks due to its high resolution.

3.2.9.2 ESI-MS

ESI-MS was carried out using Thermo Finnigan LTQ FT Ultra Fourier Transform ion cyclotron resonance mass spectrophotometer with resolution set to 100.000 at 400 m/z.

Depending on the sample, mass ranges from 50 to 2000 u were measured. The spray capillary voltage at the IonMax ESI head was 4 kV, the heater capillary temperature 250 ° C, the nitrogen sheath gas flow 20 and the sweep gas flow 5 units. Building blocks were dissolved in chloroform and analyzed in positive and negative mode.

3.2.9.3 MALDI-TOF MS

One μL of Super-DHB matrix was applied on a MTP AnchorChip (Bruker Daltonics, Bremen, Germany) and subsequently 1.5 μL of the sample solution (0.5 mg/mL in Millipore water) were added to the matrix spot under rapid pipetting. After the matrix dried and crystalized, samples were analyzed in positive or negative reflector mode using an Autoflex II mass spectrometer (Bruker Daltonics, Bremen, Germany).

38 4 RESULTS AND DISCUSSION

4.1 Synthesis of the Stp and Sph building blocks To improve the buffering capacity in

the lower endosomal pH, compaction of nucleic acid at a neutral pH, and subsequent transfection of nanocarriers, two building blocks were synthesized.

They were designed in a way that

enabled their use in SPAS using common Fmoc strategy and sequence-defined oligoaminoamides with precise topology and so that site-specific functionalization could be realized.⁽⁵⁴⁾ The blocks originate from PEI, which is very toxic, but if we break it into smaller segments and connect them in a more biodegradable and biocompatible manner, we can attain a very efficient transfection agent.⁽⁵⁷⁾ TEPA and PEHA are four and five sequences of a repeating diaminoethane motif, respectively and represent smaller, less toxic PEI sequences. Furthermore, coupling PEHA and TEPA with succinic anhydride resulted in reduced toxicity.⁽⁴³⁾ The purpose of succinic acid at one terminal primary amine, is to connect one oligoamine segment to the other and make the oligoaminoamide more biodegradable.⁽⁵⁴⁾ TEPA is a commercially available compound, whereas PEHA may be purchased, but is available only as a mixture of isomers. As recent studies reported that linear PEI are more efficient transfection agents, PEHA was purified, so that a linear isomer was obtained.⁽⁴¹⁾ The linear PEHA isomer was distilled using high-vacuum distillation at a pressure of 1.5 mbar. Fraction with linear PEHA isomer collected at 190–195 °C was precipitated with concentrated HCl what resulted in a 24.1 % yield. In the past, PEHA was purified using Kugelrohr distillation method which is not as efficient as high-vacuum distillation. The latter provides more efficient rectification and therefore cleaner fractions can be obtained.⁽⁵⁸⁾ From here on, Stp and Sph building blocks were synthesized with the same steps, except for different amounts of reagents due to one additional amino group. The Sph building block was synthesized for first time according with the improved protocol, where the volumes of needed solvents for dissolving are smaller and some unnecessary extraction steps are skipped. This could be also the reason for lower yield at the end, still these changes should theoretically not affect drastically affect the final yield.

Figure 12: Artificial aminoacid, Stp and Sph

Figure 13: Reaction scheme of Sph synthesis

The primary amines were firstly transiently protected with TFAEt as trifluoroacetamides to allow site-specific Boc- protection of the secondary amines by Boc anhydride in a one-pot-reaction. Furthermore, the Boc group served as protection due to unwanted side-reactions with secondary amines when using SPAS. TFAEt preferentially reacts with primary amines due to their stronger nucleophilicity in comparison to secondary amines. Only 2.1 equivalents were added dropwise into the cooled mixture to minimize the side-reaction with secondary amines. The intermediate products bis-tfa-TEPA(Boc3)/bis-tfa-PEHA(Boc4) were separated from the remaining TFAEt, Boc anhydride and other related structures through extraction with 5 % NaHCO3 solution and water. At the end, purification occurred via crystallization to remove some smaller 1,2-diaminoethane sequences that could still be present, due to an impure starting component. Structures were confirmed with ¹H-NMR and ESI-MS. At this step, pentaboc- and tetraboc-adduct, respectively, could occur. However, the spectra showed this type of adduct appearing only in traces. The yield for bis-tfa-TEPA(Boc3) was 58.98 % and for bis-tfa-PEHA(Boc4) 54.62 %. The reason for a lower yield of Sph might lie in crystallization. In the next step, the trifluoroacetamide protection group was removed with alkaline hydrolysis using a 3 M NaOH solution, and the organic compound was extracted with DCM to remove sodium salt of TFA. Structures were confirmed with ¹H-NMR and ESI-MS. This step resulted in a very high yield of 96.7 % and 92.39 % for TEPA(Boc3) and PEHA(Boc4), respectively. In the last step, primary amines were coupled with succinic anhydride and Fmoc-OSu and the final Fmoc-Stp(Boc3)-OH and Fmoc-Sph(Boc4)-OH were obtained. To selectively couple succinic acid only to one primary amine, the mixture was cooled down to -75 °C using dry ice and only 1.25 equivalents of

succinic anhydride was added dropwise. The reaction is preferentially directed to the one primary amine because after one primary amine is coupled, the second one becomes less nucleophilic, which leads the

reaction to new molecule with no reacted primary amines.

Subsequently, the last primary amine was coupled with a reactive ester, Fmoc-OSu, in the presence of DIPEA, which serves as proton scavenger, making the reaction very efficient. The Fmoc group is base labile group and is introduced to prevent random polymerization.

Subsequently, the organic phase was washed five times with a trisodium citrate buffer (0.1 M; pH 5.5) to remove unreacted primary

amines which are protonated and therefore hydrophilic. Still, other impurities are present, such as “double Fmoc” (Fmoc on both primary amines, Fmoc-PEHA(Boc)4-Fmoc), or not reacted Fmoc, or succinic acid on both primary amines, or not reacted TEPA(Boc3) and PEHA(Boc4), respectively. To remove these byproducts, Stp and Sph were isolated using DCVC (See Figure 8: Dry column vacuum chromatography apparatus). Silica gel was used as a stationary phase. A solvent gradient of n-heptane-ethyl acetate was used to remove Fmoc byproducts, occurring around the 30^th fraction, and ethyl acetate-methanol to elute our final product. To determine, if Fmoc byproducts were separated from our product, each fraction was analyzed by TLC using silica gel aluminum plates for fluorescence quenching at 254 nm and solvent mixture of 7:3 CHCl3/MeOH. Fmoc byproducts are more lipophilic and have a Rf > 0.9 whereas the final product has a lower Rf of 0.6. The Stp building block separated from Fmoc byproducts very efficiently, whereas Sph did not. At the fraction, where the strongest fluorescence quenching of our product was detected, a strong spot of Fmoc byproducts also appeared. Consequently, these fractions were collected separately.

Isolated Stp and Sph were analyzed by ¹H-NMR and ESI-MS. Spectra of Stp and Sph that had no traces of Fmoc byproducts on TLC plate were as expected, whereas a ¹H-NMR Figure 14: Example of DCVC purification TLC. At the bottom, individual fractions are presented.

Fluorescence quenching spots with Rf values around 0.9 are caused by Fmoc by-products and product Fmoc-Stp(Boc3)-OH has an Rf of 0.6.

spectrum of another Sph batch had shown an additional spot on the TLC plate at a Rf around 0.9 where Fmoc byproducts can be observed. The spot displayed an additional Fmoc group, meaning that not all molecules reacted with the succinic anhydride, instead coupling into an additional Fmoc group, or have possibly couped to the succinic anhydride. However, the beside and Sph peak, the ESI-MS spectrum had, an additional intensive pick at 1099.55 g/mol, which corresponds to the molar mass of a “double Fmoc” adduct, and there was no sign of an adduct that had an additional Fmoc group coupled to succinic acid. To check if this byproduct interferes with further steps of our synthesis, where it should theoretically not, the [G-Sph-G-Sph-G-Sph-G] sequence was synthesized for both batches of Sph using an automatic synthesizer. The sequence mass was measured by MALDI-TOF MS, and the results showed that the mass of the clean batch was as expected 1186.93 g/mol, whereas the batch containing Fmoc byproducts was 893.57 g/mol. According to their mass, Fmoc-PEHA(Boc4)-Fmoc (“double Fmoc”) could interfere with our synthesis. Double Fmoc cannot couple with free amines, meaning that the Sph block could be omitted from the sequence, and that the mass of the dummy structure comprising the impure Sph, is lower for exactly one Sph building block. The reason why double Fmoc appeared later and not during

In document UNIVERZA V LJUBLJANI FAKULTETA ZA FARMACIJO URBAN MATE MAGISTRSKO DELO ENOVITI MAGISTRSKI ŠTUDIJ FARMACIJE Ljubljana, 2021 (Strani 49-0)