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
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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
40
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 30th 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.
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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 the first composition of solvents, might be due to encapsulation into the Sph building blocks, which eluted with the new mixture of solvents, consequently releasing “double Fmoc”, and producing a batch with impurities. Nevertheless, the yield was still high enough to synthesize all the structures. The final Stp and Sph step resulted in a 49.6 % and 46.2 % yield, respectively. If we combine all reactions the overall yield, was 28.29 % and 5.48 % for Stp and Sph, respectively. The peak of Sph can be seen at 955.54 g/mol, whereas the intensive peak appears at 1099.55 g/mol.
O
Figure 15: Stp and Sph building block for SPAS