Synthesis of CBA-polylysine

Synthesis of Reducible linear L-Lysine (L-lysine-modified copolymers (LLC)) by Michael Addition

L-ornithine

Mix 730.6 mg L-lysine HCl (4 mmoles) + 1060 mg CBA (4 mmoles)

Add 160 mg NaOH (4 mmoles) to neutralize the l-lysine acid

Add 10 ml MeOH/H2O (80:20 v/v)

React at 45 oC for 2 days, such as oil bath, (do not change temperature and time, otherwise yield will be low

Dialyse vs water MWCO 2000 for 2 days

Freeze dry

Small scale: 

36 mg L-polylysine + 53 mg CBA

8 mg NaOH

0.5 ml MeOH (80:20 v/v)

Conjugate of LLC to another polymer or amine

Add 6 ml ddh2O to 100 mg of redicible LLC (0.033 mmoles)

add EDC (192 to 960 mg), 5 to 20 molar excess of EDC

Add N-boc ethylenediamine (60 to 300 mg)

40 oC react for 4 hours, dark, N2

Dialyse MWCO 2000 for 2 days

transfer to sterilzed tube

Freeze-Dry for 2 days

Small scale: For 5 mg of LLC, add 12 mg N-boc ethylenediamine (20 molar excess), 38 mg EDC

MW: 3.6 kDa, Mn: 3.6 kDa, number of repeat unit: 6~8, MW = 436.64 D a for each repeating unit

Synthesis of reducible linear L-lysine-modified copolymers (LLC) without ethylenediamine

Figure imgf000015_0001

The lysine monomers are activated by neutralization of the acid form of the amino acid with sodium hydroxide (NaOH). The activated lysine is then reacted with CBA in a Michael addition reaction. Briefly, 730.6 mg (4 moles) L-lysine HCl and 1060 mg (4 moles) CBA are weighed and transferred to a 50 mL round bottom flask. Afterwards, 160 mg (4 moles) of NaOH is used to neutralize the HCl present in the L-lysine starting material. Then, 10 mL of methanol/water (MeOH/H2O) mixture of 80/20 volume ratio are added to the flask, which is then stirred in an oil bath at 45 "C to dissolve the reagents. The reaction is then stirred in the dark under a nitrogen atmosphere for 2 days (Fig. 1, Intermediate 1). The resulting solution is then dissolved in ultrapure water and the reaction solution is purified via dialysis (MWCO 2000) against water for 2 days to remove low molecular weight polymeric biproducts and remaining traces of the starting materials. The dialyzed solution is then transferred to a sterile conical tube and lyophilized for 2 days.

The resulting polymers are characterized with proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), and matrix assisted laser desorption ionization-time of flight (MALDI-TOF). The molecular weights of the various polymers are determined with size exclusion chromatography (SEC) and mass spectrometry with MALDI ionization. The mobile phase for SEC interfaced with an evaporative light scattering detector (ELSD) maintained at 40 °C is ammonium acetate buffer (0.15 M) of pH 4.4 with methanol (80/20) pumped at a rate of 1 mL/min. A polyethylene glycol standard kit is used to construct the calibration curves. The matrix for MALDI-TOF is saturated alpha-cyano-4- hydroxycinnamic acid dissolved in 50/50 acetonitrile/water with 0.1% TFA with a polymer concentration of 1 mg/ml. An equal volume (8 μL) of the polymer and the matrix prepared are spotted and air-dried on the plate.

Conjugation of N-boc ethylenediamine to synthesized reducible LLC

To sequester the free hydroxyl groups in intermediate 1 , which can reduce complexation with pDNA, N-boc ethylenediamine is selectively conjugated along the polymer backbone. In addition, to optimize the conjugation of N-boc ethylenediamine, 5 different copolymers are prepared by varying the amounts of EDC and N-boc. Briefly, 100 mg each of reducible LLC (0.033 mmoles) are weighed and transferred to five different round bottom flasks to which 6 ml of deionized water is added to each flask to fully dissolve the polymers with stirring. To the dissolved polymers 5, 10, 15, 20, and 25 molar excess of EDC and N-boc ethylenediamine (0.165, 0.33, 0.495, 0.66 and 0.825 mmoles respectively) are then added to each of the respective reaction mixtures, which is stirred in an oil bath at 40 0C in the dark under a nitrogen atmosphere for 4 hrs (Fig. 1, Intermediate 2). After this time, the reaction solutions are transferred to separate dialysis bags (MWCO 2000) and dialyzed against deionized water for 2 days to purify the copolymers from the starting materials. The dialyzed polymers are then transferred to sterile conical tubes and dried on a lyophilizer for 2 days. The resulting polymers are characterized with 1H NMR, GPC, and MALDI-TOF.

Deprotection of N-boc-ethylenediamine conjugated reducible LLC

The acid-labile N-boc amine protection group present on the terminal end of the conjugated ethylenediamine is removed with TFA/H2O mixture (75/25 v/v). Briefly, 100 mg (0.033 mmoles) of reducible LLC is added to a round bottom flask to which 10 mL of deionized H2O is added. Afterwards, 30 ml of TFA is added. The dissolved copolymers are then stirred for 30 min at room temperature (Fig. 1, final product). The final polymers are then purified by dialysis (MWCO 2000) against deionized water for 2 days to remove the free N-boc groups and TFA. The dialyzed polymers are then transferred to a sterile conical tube and dried on a lyophilizer for 2 days. The resulting polymers are characterized with 1H NMR, GPC, and MALDI-TOF. The amount of primary amines in intermediate 1, intermediate 2, and the final product is quantified with a ninhydrin assay at 570 nm. The assay is conducted at 100 0C for 15 min. Glycine is used to construct the standard curves.

Chemical structure determination LLC copolymers are synthesized through Michael addition using EDC as the coupling agent and the chemical structures are determined with 1H NMR (Fig. 1) (25). After synthesis and purification, chemical structure is determined with 1H NMR. The 1H NMR spectrum (300 MHz, D2O) of intermediate 1 showed the following peaks: δ 1.231 (-NH-CH- CH2-CH2-CH2-CH2-NH-), δ 1.434 ( NH-CH-CH2-CH2-CHrCH2-NH-), δ 1.581 ( NH-CH-CH2- CH2-CH2-CH2-NH-), δ 2.957 (-NH-CH-CH2-CH2-CH2-CH2-NH-), δ 3.178 (-NH-CH-CH2-CH2- CH2-CH2-NH-), δ 2.776 (-S-CH2-CH2-NH-), δ 3.436 (-S-CH2-CH2-NH-), δ 2.479 (-NH-CH2-CH2- CO-), δ 2.379 (-NH-CH2-CH2-CO-).

The 1H NMR spectrum (300 MHz, D2O) of intermediate 2 showed the following peaks: δ 1.114 (-NH-CH-CH2-CH2-CH2-CH2-NH-), δ 1.518 (-NH-CH-CH2-CH2-CH2-CH2-NH-), δ 1.719 (-NH-CH-CH2-CH2-CH2-CH2-NH-), δ 2.957 (-NH-CH-CH2-CH2-CH2-CH2-NH-, -S-CH2- CH2-NH-), δ 3.183 (-NH-CH-CH2-CH2-CH2-CH2-NH-, -NH-CH2-CH2-NH-), δ 2.508 (-NH-CH2- CH2-CO-), δ 3.431 (-S-CH2-CH2-NH-), δ 1.331 (terminal N-boc group).

Finally, the 1H NMR spectrum (300 MHz, D2O) of the final product showed the following peaks: δ 1.103 (-NH-CH-CH2-CH2-CH2-CH2-NH-), δ 1.565 (-NH-CH-CH2-CH2-CH2- CH2-NH-), δ 1.814 (-NH-CH-CH2-CH2-CH2-CH2-NH-), δ 2.81 1 ( NH-CH-CH2-CH2-CH2-CH2- NH-, -S-CH2-CH2-NH-, -NH-CH2-CH2-NH2), δ 3.178 (-NH-CH-CH2-CH2-CH2-CH2-NH-, -NH- CH2-CH2-NH2), δ 2.562 (-NH-CH2-CH2-CO-), δ 3.456 (-S-CH2-CH2-NH-) (Fig. 2). The number of conjugated ethylenediamine per polymer chain for the final polymer using 5, 10, 15, 20 and 25 excess molar ratio of EDC and N-boc-ethylenediamine is 1.95, 3.33, 3.84, 7.51 and 2.3 respectively (Table 1). The reducible LLC copolymers with 20 excess molar ratio of EDC and N-boc-ethylenediamine showed the maximum amount of ethylenediamine conjugation as determined with 1H NMR and it is then designated the optimum reducible LLC and selected for use in subsequent assays.

TABLE l

Figure imgf000017_0001

Molecular weight determination

The molecular weight of the synthesized LLC copolymers is determined using SEC and MALDI-TOF. The number average molecular weight (Mn) of intermediate 1, intermediate 2 and final product as determined by SEC are 3489, 3118, and 3601 Da with a polydispersity index (PDI) of 1.08, 1.10 and 1.06 respectively (Table 2). The number average molecular weight (Mn) of the LLC copolymers as determined by MALDI-TOF supported the calculated SEC data with molecular weights of 3538, 31 18 and 3468 Da lor intermediate 1, intermediate 2 and the final product respectively. The PDI of each is found to be 1.097, 1.10 and 1.09 respectively (Table 2). For the final product this corresponds to 6-8 repeating units (MW = 436.64 Da for each repeating unit). The ninhydrin assay confirmed that the final polymer had more primary amines (1.44XlO"4 ± 3.5xlO"5) compared to intermediate 1 (1.27x10' 4 ± 3.5xlO"5) and intermediate 2 (1.03XlO"4 ± 3.2xlO"5).

The degree of EDA conjugation to the copolymer backbone as determined with 1H NMR, was found to be 25, 42, 48, 94 and 29 % for the 5, 10, 15, 20, and 25 molar excess of N-boc EDA respectively. Thus, the data showed that only a maximum of 94 % conjugation of EDA to the polymer chains was possible, which was probably due to steric hindrance of approaching nearby EDA molecules at higher molar concentrations that limit further conjugation to the polymers. The reducible LLCs with 94% conjugated EDA was then used in subsequent assays as the final LLCs. PLL was able to effectively condense pDNA from an N/P ratio of 1/1 as evident by the absence of free pDNA from wells of the gel (Fig. 3, lanes 2 - 1 1) compared to LLCs where effective condensation occurred from an N/P ratio of 5/1 (Fig. 3, lanes 3 - 11). This could be due to the high MW PLL (~20900 Da) chains that would be more effective at condensing pDNA at this N/P ratio compared to low the MW LLC (~ 3200 Da) chains. However, both reducible LLCs and PLL were able to completely condense pDNA from an N/P ratio of 25/1 as apparent from the total absence of fluorescence in the wells (Fig. 3, lanes 7 - 11).