Intramolecular Cyclization Assistance for Fast Degradation
of Ornithine-Based Poly(ester amide)s
Caroline de Gracia Lux, Jason Olejniczak, Nadezda Fomina, Mathieu Lessard-Viger, and Adah
Almutairi*
Skaggs School of Pharmacy and Pharmaceutical Sciences, Departments of NanoEngineering and of
Materials Science and Engineering, University of California, San Diego, La Jolla, California 92093
Table of contents:
1. Synthesis of 4a-Boc, 4b-Boc, monomers and polymers 1-Boc and 2-Boc (S2-S5)
2. Photolysis and dimer degradation studies (S5-S7)
3. Procedures for GPC and 1H NMR degradation of polymers 1-Boc and 2-Boc (S7-S8)
4. Degradation of 1-Boc followed by MALDI-TOF (S9)
5. Nanoparticles formulation (S9)
6. TEM images of nanoparticles 1 (S10-S12)
S1
1. Synthesis.
Scheme S1. Synthesis of Polymers 1-Boc and 2-Boc.
Compound 4a-Boc. Fmoc-Orn(Boc)OH (0.3 g, 0.66 mmol), hexanediol (0.026 g, 0.22 mmol)
and DMAP (0.03 g, 0.2 mmol) were dissolved in 6 mL of DCM and DMF (5/1) under nitrogen
atmosphere. A solution of DCC (0.150 g, 0.726 mmol) in 1 mL DCM was added to the reaction
mixture dropwise. The reaction mixture was stirred overnight at room temperature. The solvents
were removed under reduced pressure; the residue was dissolved in ethyl acetate and washed
with 5% NaHCO3, water and brine. The organic phase was dried over Na2SO4 and concentrated
under reduced pressure. The product was purified by flash-chromatography on silica gel using a
linear gradient of hexane/ethyl acetate (100%/0% - 0%/100%). Yield: 0.186 g (84%). 1H NMR
(600 MHz, DMSO): 7.88 (d, 4H, J=7.2 Hz), 7.75 (d, 2H, J=7.8 Hz), 7.70 (dd, 4H, J=7.2 Hz,
J=3.6 Hz), 7.41 (t, 4H, J=7.2 Hz), 7.32 (t, 4H, J=7.2 Hz), 6.78 (t, 2H, J=5.4 Hz), 4.30 – 4.20 (m,
6H), 4.03 – 3.95 (m, 6H), 2.93-2.87 (m, 4H), 1.69 – 1.40 (m, 12H), 1.36 (s, 18H), 1.28-1.20 (m,
4H) ppm.
13
C NMR (150 MHz, DMSO): 172.4, 156.1, 155.6, 143.8, 143.7, 140.7, 127.6, 127.1, 125.2,
120.1, 77.4, 65.7, 64.3, 53.8, 46.6, 33.4, 28.2, 28.0, 26.1, 24.9.
HRMS: composition: C56H70N4O12Na; theor. mass: 1013.4882; measured mass: 1013.4878.
S2
Compound 4b-Boc. Fmoc-Lys(Boc)OH (2.1 g, 4.48 mmol), hexanediol (0.176 g, 1.49 mmol)
and DMAP (0.272 g, 13 %) were dissolved in 20 mL of DCM under nitrogen atmosphere. A
solution of DCC (1.017 g, 4.92 mmol) in 5 mL DCM was added to the reaction mixture
dropwise. The reaction mixture was stirred overnight at room temperature. The solvents were
removed under reduced pressure; the residue was dissolved in ethyl acetate and washed with 5%
NaHCO3, water and brine. The organic phase was dried over Na2SO4 and concentrated under
reduced pressure. The product was purified by flash-chromatography on silica gel using a
gradient of hexane/ethyl acetate (100%/0% - 0%/100%). Yield: 1.44 g (95%)
1
H NMR (400 MHz, DMSO):7.88 (d, J = 7.4 Hz, 4H), 7.77 – 7.65 (m, 6H), 7.41 (t, J = 7.4 Hz,
4H), 7.32 (t, J = 7.4 Hz, 4H), 6.77 (s, 2H), 4.34 – 4.17 (m, 6H), 4.05 – 3.91 (m, 6H), 2.88 (s, 4H),
1.99 (d, J = 0.8 Hz, 2H), 1.71 – 1.22 (m, 40H) ppm.
Monomer 1-Boc. 4a-Boc (0.15 g, 0.15 mmol) was dissolved in 4 mL of DMF and 0.2 mL of
piperidine was added. The reaction mixture was stirred at room temperature for 1h. The solvents
were removed under reduced pressure. The residue was partitioned between hexane and water,
aqueous fraction was washed with hexane 3 times. Water was removed by lyophilization. Yield:
0.048g (58.5%).
HRMS: composition: C26H50N4O8Na; theor. mass: 569.3521; measured mass: 569.3520.
1
H NMR (600 MHz, DMSO): 6.77 (t, 2H, J=5.4 Hz), 4.05-3.95 (m, 4H), 3.28 – 3.24 (m, 6H),
2.88 (dd, 4H, J=12 Hz, J=6 Hz), 1.58 – 1.31 (m, 34 H) ppm.
13
C NMR (150 MHz, DMSO): 176.0, 155.7, 77.4, 63.9, 53.9, 43.7, 32.0, 28.4, 28.1, 26.0, 25.0
ppm.
Monomer 2-Boc. 4b-Boc (0.10 g, 0.10 mmol) was dissolved in 16 mL of DMF and 0.42 mL of
piperidine were added. The reaction mixture was stirred at room temperature for 1h. The solvents
S3
were removed under reduced pressure. The residue was partitioned between hexane and water,
the aqueous fraction was washed with hexane 3 times. Water was removed by lyophilization.
Yield: 0.020g (35.5%).
1
H NMR (400 MHz, DMSO): 6.76 (s, 2H), 4.03-3.90 (m, 4H), 3.33 – 3.26 (m, 6H), 2.87 (d, J =
6.4 Hz, 4H), 1.68 – 1.17 (m, 38H) ppm.
13
C NMR (150 MHz, DMSO): 175.8, 155.6, 77.3, 63.8, 53.9, 34.3, 29.3, 28.3, 28.1, 25.0, 22.5
ppm.
Polymer 1-Boc. Monomer 1-Boc (0.21 g, 0.39 mmol) was dissolved in 2 mL DCM under
nitrogen atmosphere and pyridine (0.19 mL, 2.3 mmol) was added. Adipoyl chloride (0.057 mL,
0.39 mmol) was added to the reaction mixture dropwise (white precipitate formed upon
addition). The reaction was stirred at room temperature overnight. The polymer was isolated by
precipitation in 10 mL of hexane.The low molecular weight fraction was removed by preparative
GPC chromatography on LH-20 resin with DMF as eluent. Yield: 0.06 g (34%). Molecular
weight (relative to polystyrene standards): Mw = 36 000 Da (PDI 1.4).
1
H NMR (600 MHz, DMSO): 8.10 (d, J=7.2 Hz, 2H), 6.77 (t, J=5.4 Hz, 2H), 4.17 (dd, J=13.8
Hz, J=8.4 Hz, 2H), 4.04 – 3.97 (m, 4H), 2.89 (dd, J=12.6 Hz, J=6.0 Hz, 4H), 2.10 (s, 4H), 1.65 –
1.24 (m, 42 H) ppm.
13
C NMR (100 MHz, CDCl3): 65.4, 52.2, 40.1, 35.8, 29.4, 28.6, 28.4, 26.5, 25.5, 25.0 ppm.
Polymer 2-Boc. Monomer 2-Boc (0.13 g, 0.22 mmol) was dissolved in 0.4 mL DCM under
nitrogen atmosphere and pyridine (0.11 mL, 1.33 mmol) was added. Adipoyl chloride (0.033
mL, 0.22 mmol) was added to the reaction mixture dropwise (white precipitate formed upon
addition). The reaction was stirred at room temperature overnight. The polymer was isolated by
precipitation in 20 mL of methanol. The low molecular weight fraction was removed by
S4
preparative GPC chromatography on LH-20 resin with DMF as eluent. Yield: 0.05 g (33%).
Molecular weight (relative to polystyrene standards): Mw = 31 000 Da (PDI 1.8).
1
H NMR (400 MHz, DMSO): 8.11 (m, 2H), 6.76 (m, 2H), 4.19-4.10 (m, 2H), 4.08-3.95 (m, 4H),
2.87 (d, J = 6.4 Hz, 4H), 2.11 (s, 4H), 1.68 – 1.17 (m, 46H) ppm.
2. Photolysis and dimer degradation studies. A 0.1 mg/mL solution of compound 4a
or 4b in acetonitrile/phosphate buffer pH 7.4 or 6.0 (1/2) was placed into a quartz semi-micro
cuvette and irradiated at 350 nm for 15 min. The solution was incubated at 37˚C and aliquots
were removed periodically and injected into HPLC-MS to determine the presence of the
intermediates by integrating the peaks of the single ions, m/z = 791 (for IM 1, [M+H], z = 1),
m/z = 455 (for IM 2, [M+H], z= 1), m/z = 355 (for Fmoc-Orn, [M+H], z=1), and m/z = 359 (for
Fmoc-lactam, [M+H+Na], z=1) respectively (Figure S1). The lactam derivative (Fmoc lactam)
was formed from ornithine-based dimers 4a but not from lysine based dimers 4b upon irradiation
at pH 7.4 (Figure S2).
S5
S6
Figure S1. Dimer 4a degradation followed by HPLC-MS after 15 min UV irradiation (blue) at
pH 7.4 (a) and 6.0 (b). As a control, a non-irradiated solution of 4a was injected as well for both
pH values (red).
Figure S2. Dimer 4b degradation followed by HPLC-MS after 15 min UV irradiation (blue) at
pH 7.4. As a control, non-irradiated solution of 4b was injected (red).
3. Procedures for GPC and 1H NMR degradation of 1-Boc, 2-Boc
a) GPC: In a typical procedure, 10 mg of polymer (1-Boc or 2-Boc) were treated with a mixture
of TFA and DCM (1 mL, 1/1) for 30 min. The solvents were removed and the deprotected
polymer was dissolved in 0.2 M phosphate buffer (pH 7.4 or 6.0) and purified on PL-10
desalting column. The polymer solution was incubated at 37˚C and aliquots were removed
S7
periodically and injected into an aqueous GPC system calibrated with PEO standards (0.2 M
phosphate buffer (pH 7.4 or 6.0) /acetonitrile (8/2) as eluent).
As a control, a solution of 1-Boc in 0.2 M phosphate buffer (pH 7.4 or 6.0) /acetonitrile (3.5/6.5)
was incubated at 37˚C and aliquots were removed periodically. The solvents were removed at
reduced pressure; the polymer samples were dissolved in DMF and injected into an organic GPC
system calibrated with polystyrene standards (DMF with 0.01% LiBr as eluent) (Figure S3a-b).
Figure S3. Stability of 1-Boc at pH 7.4 (a) and pH 6.0 (b).
b) NMR: In a typical procedure, 10 mg of polymer (1-Boc and 2-Boc) were treated with a
mixture of TFA and DCM (1 mL, 1/1) for 30 min. After removal of solvents the deprotected
polymer was dissolved in water and purified on PL-10 desalting column. The solution was
lyophilized and the purified polymer was dissolved in a deuterated 0.2 M phosphate buffer (pH
7.4). The polymer solution was incubated at 37˚C and 1H NMR (500 MHz) spectra were taken
periodically to estimate the degree of polymer degradation as the percent of the cleaved ester
bonds.
S8
4.
Degradation of 1-Boc followed by MALDI-TOF. MALDI-TOF spectrum of the
degraded 1-NH2 was taken after 21 h of incubation in PBS 1 x (pH 7.4) at 37˚C using transindoleacrylic acid as a matrix. The ion source voltages were set to 19 kV and 16.35 kV. Linear
detector voltage was set to 2.675 kV. The spectra were produced in a positive ion mode and
averaged over 20 shots.
Figure S4. Lactam by-products observed by MALDI after deprotection of polymer 1-Boc
followed by 21 h incubation in PBS at pH 7.4.
5. Nanoparticles formulation
Polymer 1 (20 mg) was dissolved in a mixture of 0.4 mL of DMF and 0.3 mL of CHCl3
containing 0.25 mg of iron oxide nanoparticles (50 µL, 10 nm, 5mg/mL in toluene). The mixture
was electrosprayed at 20 kV (Gamma High Voltage, ES30) at a flow rate of 0.5 mL/hr (KD
Scientific) using a 25 gauge needle for 30 minutes. Samples were collected onto microscope
glass slides on an aluminum plate collector at a distance of 20 cm.
S9
6. TEM images of nanoparticles 1
The particles 1 obtained by sonication (~2 mg/slide) were removed from their glass slide
substrate by sonication in water (50 mL), washed with water and finally dispersed in 5 mL dilute
triethylamine/water solution (pH 8.0). Particle solution was irradiated at 350 nm for 5 or 15 min
and incubated at 37 °C for one to four days. The same procedure was followed but on nonirradiated particles. Finally, 5µL of each solution was placed on copper grids (400 mesh, with a
carbon film on, purchased from Electron Microscopy Sciences (Hatfield, PA).
Figure S5. TEM images of nanoparticles 1 after 24 h incubation at 37°C in dilute triethylamine/
water solution (pH 8.0) without irradiation.
S10
Figure S6. TEM images of nanoparticles 1 upon 5 min irradiation followed by 24 h incubation at
37°C in dilute triethylamine/ water solution (pH 8.0).
Figure S7. TEM images of nanoparticles 1 upon 15 min irradiation followed by 24 h incubation
at 37°C in dilute triethylamine/ water solution (pH 8.0).
S11
Figure S8. TEM images of nanoparticles 1 upon 15 min irradiation followed by 4 days
incubation at 37°C in in dilute triethylamine/ water solution (pH 8.0).
Figure S9. TEM images of nanoparticles 1 after 4 days incubation at 37°C in dilute
triethylamine/ water solution (pH 8.0) without irradiation.
S12