1.4 Polyplexes as delivery systems
1.4.3 Sequence-defined oligomers
In previous chapters, many facts were stated, considering minor changes in nanocarrier structures that can significantly increase cell transfection. As in nature, precise and unique nucleotide or amino acid sequences build macromolecules such as nucleic acids or proteins which play a crucial role in organisms. The slightest change in their sequence or substitution of a cornerstone can totally change the function of protein or gene. This observation could be also exploited for design of polymeric carriers. Properties such as size, monodisperse molecular weight, exact monomer order, orientation, topology (linear, branched, comb, dendritic), number, and attachment sites of subunits can play crucial role in biological activity. To achieve this, classical solid-phase peptide synthesis can be used, developed by Robert Bruce in 1950s and 1960s, enabling precise and highly efficient assembly of different building blocks into sequence-defined oligomers. This precise synthetic strategy can provide answers to structure related activity relationships.(19) Various building block can be used, ranging from artificial polymeric components, artificial amino acids (Stp, Sph, Gttp), natural protected amino acids, to lipids.(42,46) So far, a lot of research has been done about using natural amino acids such as lysine, histidine, arginine or proline,based on that some promising delivery vehicles have been developed.(47,48)
13
Figure 2: Sequence-defined oligoaminoamide architectures: linear, two-arm, three-arm, four-arm, and comb
In studies of peptide-based transfection agents, some structure related activity was observed.
Natural amino acids such as lysine, arginine, or ornithine, both linear and branched, showed very efficient nucleic acid binding. Additionaly, lysine with two amino groups (α, ε) can introduce branching points and provide more branched oligoaminoamide-sequences.(40) Stability can be improved with incorporation of cysteine through covalent disulfide cross-linkage that can improve transfection up to 60-fold.(44) Furthermore, disulfides can be reduced in the reductive cytosol, which can facilitate payload release from the vehicles at the target site.(40) bis (acyl)-modified lysine tryptophan and tyrosine can improve the compacting of payload through hydrophobic interactions.(19,49,50) With histidine, based on its imidazole ring with pKa of ∼ 6, buffering can be increased because of protonation in acidifying endosomes and the transfection efficiency can be increased from 10- to 100-fold.(38,40,51) These building blocks, as mentioned before, can be assembled by solid-phase-assisted synthesis with common Fmoc strategy, and furthermore many Fmoc protected α-amino acids are commercially available on the market. In such a way, many linear, two-arm, three-arm, four-arm, and comb architectures can be synthesized. Four-arm oligomers showed that they are very effective pDNA-compacting carriers. Because of their large cationic backbone, the compaction is much better compared to two-arms oligomers.
However, two-arm oligomers showed better compaction for siRNA.(51) In addition, precise PEGylation and a targeting ligand can be attached.(19,40)
14 1.4.4 Functional delivery domains
As mentioned before, dissociation and aggregation of nanoparticles and blood cells, unspecific interactions with complement and other blood components can lead to acute toxicity. To avoid that, applying hydrophilic macromolecules can shield the surface of polyplexes, prevent undesired interactions, dissociation, aggregation and prolong blood circulation time. The predominantly used shielding agent is PEG, with some alternatives such as pHPMA, hydroxyethyl starch, hyaluronic acid available.(33) To improve targeting specificity, selectivity and efficiency, some targeting ligands, specific to certain tissues, can be attached, especially concering cancer tissues that have upregulated receptors on its surface. The main groups of targeting ligands are antibodies, antibody fragments, aptamers, glycoproteins, peptides, and small molecules.(38) So far, the following ligands were used as targeting domains: cMBP, GE11, cRGD, VEGFR, EGFR ligands, transferrin and folate to name a few.(30) To conjugate shielding or targeting domain to the carrier, click-chemistry can be applied. The DBCO motif enables post-functional copper free click-chemistry with azido-lysines, which were introduced in oligoaminoamides systems, via strain-promoting alkyne-azide cycloaddition. Moreover, this linker increases the distance of the two units for facilitating a crosslinking reaction with a second oligomer and faster disassembly in the reductive cytosol.(52,53)
15 2 AIMS OF THE THESIS
Delivery efficiency of non-viral carriers remains the main obstacle for successful delivery of nucleic acids as medical agents. While viral vectors show several magnitudes higher overall delivery efficiency, they exhibit high levels of immunogenicity. Hence, non-viral vehicles present a promising alternative to viral vectors due to their high-level of flexibility in structural design and many possibilities for further improvements. Recent developments in the solid-phase synthesis method allow us to assemble amino acids and similar agents quickly and easily into cationic oligomers. The latter are very efficient at complexing and stabilizing nucleic acids by forming sub-micrometer complexes called polyplexes that can protect them from degradation and facilitate delivery of nucleic acids into cells.
The following three aims were set for this master’s thesis:
First, two novel artificial amino acids were to be synthesized, based on PEI with repeating 1,2-diaminoethane motifs which is well known for its nucleic acid binding and endosomal buffering abilities. The first artificial amino acid Stp comprises four 1,2-diaminoethane motifs and the second Sph five 1,2-diaminoethane motifs. To connect the oligoamine segments in a more biodegradable manner and to enable the use of a building block in SPAS, succinic acid needs to be coupled on one terminal primary amine of four and five 1,2-diaminoethane sequences, respectively.
Second, a small library of eleven well-defined four-arm oligoaminoamides with different architectures and functionalities had to be generated via the SPAS approach to identify structure related activity relationships of DNA delivery carriers. In addition to the synthesis of Stp and Sph artificial amino acids, a different combination of amino acid sequences, for which a positive effect for transfection was reported, had to be incorporated into the four-arm structures. Four-four-arm oligoaminoamides were synthesized in a way where a good comparison between Stp and Sph artificial amino acids could be observed.
Third, four-arm structured polyplexes had to be formed with pDNA in three N/P ratios, and biophysical parameters had to be evaluated, such as pDNA complexation and binding ability, along with polyplexes’ size, and zeta potential.
16 3 MATERIALS AND METHODS
3.1 Materials
Table 1: Solvents used for experimental procedures
Solvents CAS-No. Supplier
Acetone6 67-64-1 LMU chemical supply center; Faculty of chemistry and pharmacy
Acetonitrile1 75-05-8 VWR Int. (Darmstadt, Germany) Chloroform6 67-66-3 VWR Int. (Darmstadt, Germany)
Chloroform-d2 865-49-6 Euriso-Top (Saint-Aubin Cedex, France) DCM anhydrous3 75-09-2 Acros organics (Geel, Belgium)
DCM3 75-09-2 Fisher Scientific (Schwerte, Germany) Deuterium oxide2 7789-20-0 Euriso-Top (Saint-Aubin Cedex, France) DMF4 68-12-2 Iris Biotech (Marktredewitz, Germany) Ethanol absolute3 64-17-5 Vwr Int. (Darmstadt, Germany)
Ether6 - LMU chemical supply center; Faculty of chemistry and pharmacy
Ethyl acetate5 141-78-6 Staub & Co. (Nürnberg, Germany) Methanol3 67-56-1 Fisher Scientific (Schwerte, Germany) Millipore Water8 - In-house purification, using fully automatic
Evoqua Ultra Clear GP TWF DI/EDI Water Purification System
MTBE7 1634-04-4 Brenntag (Mülheim/Ruhr, Germany) n-butanol6 71-36-3 Sigma-Aldrich (Munich, Germany) n-heptane6 142-82-5 Grüssing (Filsum, Germany)
n-hexane6 110-54-3 Grüssing GmbH (Filsum, Germany) NMP4 872-50-4 Iris Biotech (Marktredewitz, Germany) THF4 109-99-9 Fisher Scientific (Schwerte, Germany)
1 HPLC grade; 2 1H-NMR grade (> 99.9 %); 3 analytical grade; 4 peptide grade; 5 purum; 6 purissimum; 7 synthesis grade; 8 purified, deionized
Table 2: Chemicals and reagents used for experimental procedures Chemicals and reagents CAS-No. Supplier
1 M HCl 7647-01-0 VWR Chemicals BDH® (Insmaming, Germany) 2,5-Dihydroxybenzoic
acid 490-79-9 Sigma-Aldrich (Munich, Germany) 2-chlorotritylchloride
resin 42074-68-0 Iris Biotech (Marktredewitz, Germany)
2-Hydroxy-5-methoxybenzoic acid 2612-02-4 Sigma-Aldrich (Munich, Germany) Acetic acid 64-19-7 Sigma-Aldrich (Munich, Germany)
17 Chemicals and reagents CAS-No. Supplier
Acetic anhydride 108-24-7 Sigma-Aldrich (Munich, Germany) Agarose 9012-36-6 Sigma-Aldrich (Munich, Germany) Boc anhydride 24424-99-5 Sigma-Aldrich (Munich, Germany) Boric acid 10043-35-3 Sigma-Aldrich (Munich, Germany) Bromophenol blue 115-39-9 Sigma-Aldrich (Munich, Germany) CaCl2 anhydrous 10043-52-4 Grüssing GmbH (Filsum, Germany Celite 61790-53-2 Sigma-Aldrich (Munich, Germany) Concentrated HCL - LMU chemical supply center; Faculty of
chemistry and pharmacy D-(+)-Glucose
monohydrate 14431-43-7 Merck Millipore (Darmstadt, Germany)
DBU 6674-22-2 Sigma-Aldrich (Munich, Germany)
DIPEA 7087-68-5 Sigma-Aldrich (Munich, Germany)
EDT 540-63-6 Sigma-Aldrich (Munich, Germany)
EDTA disodium salt
dihydrate 6381-92-6 Sigma-Aldrich (Munich, Germany) Ethyl trifluoroacetate 383-63-1 Sigma-Aldrich (Munich, Germany) Fmoc-Ala-Wang resin 04-12-2043 Novabiochem® (Darmstadt, Germany) Fmoc-Gly-OH 29022-11-5 Iris Biotech (Marktredewitz, Germany) Fmoc-L-Arg(Pbf)-OH 154445-77-9 Iris Biotech (Marktredewitz, Germany) Fmoc-L-Cys(Trt)-OH 103213-32-7 Iris Biotech (Marktredewitz, Germany) Fmoc-L-His(Trt)-OH 109425-51-6 Iris Biotech (Marktredewitz, Germany) Fmoc-L-Lys(Boc)-OH 71989-26-9 Iris Biotech (Marktredewitz, Germany)
Fmoc-L-Lys(Fmoc)-OH 78081-87-5 Iris Biotech (Marktredewitz, Germany) Fmoc-L-Lys(N3)-OH 159610-89-6 Iris Biotech (Marktredewitz, Germany) Fmoc-L-Trp(Boc)-OH 143824-78-6 Iris Biotech (Marktredewitz, Germany) Fmoc-L-Tyr(tBu)-OH 71989-38-3 Iris Biotech (Marktredewitz, Germany) Fmoc-OSu 82911-69-1 Iris Biotech (Marktredewitz, Germany) GelRed™ - Biotum (Biotium, Hayward, CA, USA) Glycerin 56-81-5 Sigma-Aldrich (Munich, Germany) HBTU 94790-37-1 Multisyntech GmbH (Witten, Germany) HEPES 7365-45-9 Biomol (Hamburg, Germany)
HOBt 123333-53-9 Sigma-Aldrich (Munich, Germany)
KCN 151-50-8 Sigma-Aldrich (Munich, Germany)
Liquid nitrogen - LMU chemical supply center; Faculty of chemistry and pharmacy
Na2SO4 anhydrous 7757-82-6 ORG Laborchemie GmbH (Bunde, Germany) NaHCO3 144-55-8 LMU chemical supply center; Faculty of
chemistry and pharmacy
18 Chemicals and reagents CAS-No. Supplier
NaOH 1310-73-2 ORG Laborchemie GmbH (Bunde, Germany) Ninhydrin 485-47-2 Sigma-Aldrich (Munich, Germany) Nitrogen - LMU chemical supply center; Faculty of
chemistry and pharmacy
pCMV-luc - Plasmid Factory GmbH (Bielefeld, Germany) PEHA 4067-16-7 Sigma-Aldrich (Munich, Germany)
Phenol 108-95-2 Sigma-Aldrich (Munich, Germany) Pipperidine 110-89-4 Iris Biotech (Marktredewitz, Germany) PyBOP 437-1 Multisyntech GmbH (Witten, Germany) Pyridine 110-86-1 Acros organics (Geel, Belgium)
Sephadex® G-10 9050-68-4 GE Healthcare (Freiburg, Germany) Silica gel 7631-86-9 Acros organics (Geel, Belgium) Succinic anhydride 108-30-5 Sigma-Aldrich (Munich, Germany)
TEA 121-44-8 Grüssing GmbH (Filsum, Germany)
TEPA x 5 HCl 4961-41-5 Sigma-Aldrich (Munich, Germany)
TFA 76-05-1 Acros organics (Geel, Belgium)
TIS 6485-79-6 Sigma-Aldrich (Munich, Germany)
tri-Sodium citrate
dihydrate 6132-04-3 Sigma-Aldrich (Munich, Germany) Triton™ X-100 9002-93-1 Sigma-Aldrich (Munich, Germany) Trizma® Base 77-86-1 Sigma-Aldrich (Munich, Germany)
Table 3: Buffers and matrix used for experimental procedures
Buffer Composition
Electrophoresis loading buffer
6 mL of glycerin; 1.2 mL of 0.5 M EDTA; 2.8 mL of Millipore water; 0.02 g of bromophenol blue
HBG 20 mM HEPES buffer with 5 % (w/v) glucose; pH 7.4
Super-DHB (10 mg/mL)
9:1 (w/w) mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid in TA30 (AcN/Millipore water (3:7) with 0.1 % (v/v) TFA)
TBE buffer Trizma base 10.8 g; boric acid 5.5 g; EDTA 0.75 g; and 1 L of Millipore water
TLC staining solution 0.4 g of ninhydrin dissolved in 200 mL of
100:4.5:0.5 (v/v/v) n-butanol/Millipore water/acetic acid
19
Buffer Composition
Trisodium citrate buffer
(0.1 M; pH 5.5) 0.1 M of trisodium citrate dihydrate, adjusted to pH 5.5
Table 4: Materials used for experimental procedures
Materials Supplier
1.5 mL Eppendorf tube Brand (Wertheim, Geramny) 15- and 50-mL disposable centrifuge
test tube Lab Logistcs Group GmbH (Meckenheim,
Germany) 2- and 10-mL polypropylene syringe
microreactors with
polytetrafluoroethylene filters Multisyntech GmbH (Witten, Germany) 5 mL Eppendorf tube Eppendorf AG (Hamburg, Germany) Alu-laboratory-pop-up foil 230 x
270 LMU chemical supply center; Faculty of
chemistry and pharmacy
Boiling chips SARSTEDT AG & Co. KG (Nümbrecht, Germany)
Disposable UV-Cuvette micro;
70 μL BRAND GMBH + CO KG (Wertheim
Germany)
Folded capillary zeta cell Malvern Panalytical (Worcestershire, UK)
Dry ice LMU chemical supply center; Faculty of
chemistry and pharmacy
Filter paper (150 mm) Lab logistics (Meckenheim, Germany)
Ice LMU chemical supply center; Faculty of
chemistry and pharmacy
100 Sterican® 0.90 x 70 mm B. Braun Melsungen AG (Melsungen, Germany)
Oil bath LMU chemical supply center; Faculty of chemistry and pharmacy
Pipette tips (10 mL) Eppendorf (Hamburg, Germany) Pipette tips (10 μL,
200 μL, 1000 μL) Brand (Wertheim, Geramny)
TLC Silica gel 60 F254 Merck Millipore (Darmstadt, Germany)
20 3.2 Methods
3.2.1 Synthesis of the Stp building block 3.2.1.1 bis-tfa-TEPA(Boc3)
F
Figure 3: Reaction scheme of bis-tfa-TEPA(Boc3) synthesis
118.9 g of TEPA x 5 HCl were weighed into the 2 L round-bottom flask which was fastened on the ring stand with utility clamps and a magnetic stir bar was put inside. TEPA x 5 HCl was dissolved in 130 mL of DCM and 370 mL of MeOH and stirred at 1000 rpm on the magnetic stirrer. 223 mL of TEA, which was added to transform TEPA x 5 HCl into the free form, was slowly added to the solution that was cooled down to 0 °C in a dewer with an ice bath. 100 g of TFAEt was dissolved in 250 mL of DCM in a 500 mL beaker. The dropping funnel was fastened on the ring stand with utility clamps and installed with a round-bottom flask. (See Figure 7: Dropping apparatus). The TFAEt solution was poured into the dropping funnel and added dropwise (1 drop per 3 seconds) to the reaction mixture which was then stirred overnight at RT and 1000 rpm. The dropping funnel was closed with a chlorcalcium tube. 234.79 g of Boc anhydride was weighed into the beaker and dissolved in 200 mL of DCM. Additional 179 mL of TEA was added to the reaction mixture that was stirred at 1000 rpm. The solution with Boc anhydride was poured into the dropping funnel and added dropwise (1 drop per 3 seconds) to the reaction mixture under stirring at RT and 1000 rpm. After the addition, solution was stirred overnight. Organic solvents were evaporated using a rotary evaporator (Büchi Rotavapor R-114, Büchi Waterbath B-480) first at 800 mbar and then the pressure slowly dropped to 388 mbar. The water bath had a temperature of 50 °C. The solution was concentrated to 800 mL and the organic phase was washed, using a separating funnel, two times with 250 mL of 5 % NaHCO3 solution (w/w) (50 g of NaHCO3 were dissolved in 950 g of Millipore water) and three times with Millipore
21
water. The organic phase was collected into the Erlenmeyer tube, diluted with 500 mL of DCM and dried with Na2SO4
anhydrous (The drying of the solution was terminated when the portions of Na2SO4 anhydrous were floating freely in the solvent without clotting after addition and shaking). The suspension was filtered using a Büchner funnel with a filter paper inside and a side-arm flask with a tube leading to a vacuum pump and subsequently transferred into a 2 L round-bottom flask. The organic solvent was evaporated first, by using first rotary evaporator at 840 mbar and 60 °C and second, by a vacuum line (<10 mbar) until a yellowish waxy solid substance was obtained.
A reflux apparatus was assembled, consisting of a 2 L round-bottom flask with bis-tfa-TEPA(Boc3) inside put into an oil bath, installed with the reflux condenser, and fastened on a ring
stand with utility clamps. The oil bath was heated to 80 °C and a magnetic stirrer was set to 600 rpm. The magnetic stir bar was put inside the round-bottom flask and subsequently DCM was added portion wise until everything was dissolved (150 mL). In the next step n-hexane was added portion wise until clouding on the surface of the solution could be discerned.
After the occurrence of clouding, heating was stopped, the mixture was let to slowly cool down to RT and it was put into the fridge at 7 °C overnight. Next day, clean crystals could be seen which were filtrated using Büchner funnel with four filter papers inside and side-arm flask with a tube leading to a vacuum pump and washed with n-hexane. The final product was at the end dried using vacuum line overnight (<10 mbar), analyzed by 1H-NMR and ESI-MS (See 1H-NMR, ESI-MS), and stored in the fridge at 7 °C.(54)
bis-tfa-TEPA(Boc3) TEPA(Boc3)
Figure 5: Reaction scheme of TEPA(Boc3) synthesis
Figure 4: Reflux apparatus
22
20 g bis-tfa-TEPA(Boc3) was dissolved in 175 mL of absolute ethanol in a 1 L round-bottom flask and 195 mL of a 3 M aqueous sodium hydroxide solution (120 g of NaOH were dissolved in 1 L of Millipore water) was subsequently slowly added approximately within 30 min during the stirring with the magnetic stir bar on the magnetic stirrer set to 500 rpm.
The resulting mixture was stirred for 20 h overnight. After 20 h the ethanol had evaporated as far as it was possible using a rotary evaporator (Büchi Rotavapor R-114, Büchi Waterbath B-480). The pressure was dropped to 120 mbar and the water bath had the temperature of 60 °C. The mixture was filled into a separating funnel and the water phase was extracted in a fume hood four times with 100 mL of DCM. Afterwards, the organic phase was dried over Na2SO4 anhydrous and shaken for 5 min until the solution was dry (See bis-tfa-TEPA(Boc3)). The suspension was filtered using Büchner funnel with a filter paper inside and a side-arm flask with a tube leading to a vacuum pump and subsequently transferred into a tared 1 L round-bottom flask. The organic solvent was evaporated first by the rotary evaporator at the pressure of 800 mbar and water bath heated up to 40 °C and subsequently by a vacuum line at the pressure smaller than 10 mbar until a highly viscous compound was yielded. The final product was solidified, stored in the fridge at 7 °C and analyzed by
1H-NMR and ESI-MS (See 1H-NMR, ESI-MS).(54) 3.2.1.3 Fmoc-Stp(Boc3)-OH
Figure 6: Reaction scheme of Fmoc-Stp(Boc3)-OH synthesis
23
The mass of isolated TEPA(Boc3) was determinated by weighing the round-bottom flask with TEPA(Boc3) and subtracting the tare. TEPA(Boc3) was dissolved in 50 mL of THF in a round-bottom flask, a magnetic stir bar was added, and the mixture was stirred on the magnetic stirrer at 600 rpm in the fume hood until TEPA(Boc3) was completely dissolved. The round-bottom flask was fastened onto the ring stand with utility clamps, placed into a dewer and installed with a dropping funnel. Underneath was a magnetic stirrer set to 600 rpm. Acetone and dry ice were filled into the dewer and the temperature of -75 °C was maintained by adding dry ice from time to time (At the optimal temperature, there were no bubbles in the bath and also some solid dry ice could be observed at the bottom.). The first additions of dry ice were added very carefully because at the
beginning, a lot of gas had been generated, causing a lot of bubbling and splashing. At the end, the dewer and round-bottom flask were covered with aluminum foil. 1.25 eq. of Succinic anhydride according to the yield of TEPA(Boc3) was weighed into a beaker and dissolved in 400 mL of THF. Half of it was poured into the dropping funnel and with the other half following later on. The dropping funnel was closed with a chlorcalcium tube. The solution was slowly added dropwise to the reaction mixture, approximately 1 drop per 5 seconds, and additional dry ice was added to maintain the right temperature. When the addition was completed, the reaction mixture was stirred at 600 rpm and -75 °C for one hour and then for one additional hour at RT.(54)
3 eq. of DIPEA according to the yield of TEPA(Boc3) was slowly added to the reaction mixture under stirring at 600 rpm and the batch was cooled down to 0 °C by putting it into the ice bath (dewer with ice). 1.5 eq. of Fmoc-OSu according to the yield of TEPA(Boc3) was weighed into the beaker and dissolved in 60 mL of acetonitrile and 30 mL of THF. A round-bottom flask was installed with a dropping funnel. When the reaction mixture cooled down, the Fmoc-OSu solution was poured into the dropping funnel and the flask was washed with 30 mL of THF. The solution was subsequently added dropwise (1 drop per 3 seconds) into the reaction mixture that was stirred over the night at 600 rpm. The next day, the solution was concentrated to approximately 50 mL using a rotary evaporator at the pressure of
Figure 7: Dropping apparatus
24
320 mbar and a water bath at the temperature of 60 °C. The concentrate was poured into the separating funnel with 100 mL of DCM inside. The organic phase was washed five times with 100 mL of trisodium citrate buffer (0.1 M; pH 5.5). After that, the organic phase was dried over Na2SO4 anhydrous under slight shaking (For more details see TEPA(Boc3)). The solution was filtered directly into the 1 L round-bottom flask using a filter paper and a filter funnel. Into the round-bottom flask was subsequently added an adequate amount of celite (around 32 g) to obtain a very dense consistence. The organic solvent was evaporated using first a rotary evaporator (800 mbar at 40 °C) and second a vacuum line (<10 mbar).(54) 3.2.1.4 Purification of Fmoc-Stp(Boc3)-OH by a dry column vacuum chromatography A separating funnel and a sintered glass Büchner
funnel were installed with a side-arm adapter, connected to a vacuum pump, and fastened on a ring stand with utility clamps. The sintered glass Büchner funnel was gradually filled with a silica gel and every new layer of silica gel, which was approximately 5 mm high, was first pressed without and then with applied vacuum to produce a compact silica gel bed that was approximately 10 cm high. On the top of the silica gel bed a filter paper was laid. The column was then poured with 800 mL of n-heptane for two times under applied vacuum. The crude Fmoc-Stp(Boc3)-OH was pulverized using a mortar and a pestle and added evenly on the filter paper of the column and the
second filter paper was added on the top. 100 mL fractions of solvents were added with a measuring cylinder as shown in the table below, the vacuum was applied, and eluates were collected into the boiling tubes.(54)
Table 5: Solvent composition for dry column vacuum chromatography
Fraction 1 2 3 4 5 6 7 8 9 10 … N
25
A solvent gradient of n-heptane-ethyl acetate was used to remove Fmoc byproducts and ethyl acetate-methanol to elute our final product. Ethyl acetate was purified before use (See Ethyl
A solvent gradient of n-heptane-ethyl acetate was used to remove Fmoc byproducts and ethyl acetate-methanol to elute our final product. Ethyl acetate was purified before use (See Ethyl