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1 Supporting Information for:
2 Sulfated quaternary amine lipids: a new class of inverse charge
3 zwitterlipids
4
5 Vincent J. Venditto,a Aaron Dolor,a Aditya Kohli,b Stefan Salentinig,c Ben J. Boydc and Francis C. Szoka, Jr.a,b
6
7 aDepartment of Bioengineering and Therapeutic Sciences, School of Pharmacy, University of California
8 San Francisco, San Francisco, CA 94143. bThe UC Berkeley-UCSF Graduate Program in Bioengineering,
9 University of California Berkeley, Berkeley, CA 94720. cDrug Delivery, Disposition and Dynamics,
10 Monash Insititute of Pharmaceutical Sciences, Monash University, Victoria 3052, Australia.
11
12 This work was supported by NIH EB003008. VJ Venditto was funded by a grant from the National Institutes of
13 Allergy and Infectious diseases of the National Institutes of Health under Award Number F32AI095062. A
14 Dolor was funded by a fellowship from the National Science Foundation Graduate Research Fellowship
15 Program. The content is solely the responsibility of the authors and does not necessarily represent the official
16 views of the National Institutes of Health or the National Science foundation.
17
18 Address correspondence and reprint requests to Dr. Francis C. Szoka, Jr., Departments of Bioengineering and
19 Therapeutic Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California, San
2 Experimental Section page3 1. Materials………………………………………………………………………………………34 2. General synthetic scheme……………………………………………………………………..35 3. Chemical characterization…………………………………………………………………….46 4. Elemental analysis…………………………………………………………………………….67 5. Differential scanning calorimetry……………………………………………………………..68 6. Transmission electron microscopy……………………………………………………………79 7. Small angle X-ray scattering measurements…………………………………………………..7
1011 Tables and Figures.12 Table S1: Elemental Analysis of AS lipids………………………………………………………..813 Figure S1: BrPrOSO3-DIPEA (1) 1H……………………………………………………………...914 Figure S2: BrPrOSO3-DIPEA (1) 13C……………………………………………………………1015 Figure S3: DOAS (3a) 1H………………………………………………………………………..1116 Figure S4: DOAS (3a) 13C……………………………………………………………………….1217 Figure S5: DSAS 1H……………………………………………………………………………..1318 Figure S6: DSAS 13C…………………………………………………………………………….1419 Figure S7: DPAS 1H……………………………………………………………………………..1520 Figure S8: DPAS 13C…………………………………………………………………………….1621 Figure S9: DMAS (3a) 1H………………………………………………………………………..1722 Figure S10: DMAS (3a) 13C………………………………………………………………..……1823 Figure S11: DLAS 1H……………………………………………………………………………1924 Figure S12: DLAS 13C…………………………………………………………………………...2025 Figure S13: DCAS 1H…………………………………………………………………………....2126 Figure S14: DCAS 13C…………………………………………………………………………...2227 Figure S15: SAXS scattering profile for DOAS with increasing temperature…………………..2328 Figure S16: DOAS scattering profiles with increasing temperatures……………………………2329 Figure S17: Change in lamellar spacing with temperature for DOAS…...……………………...2430 Figure S18: DOAS data and fit for thickness pair distance distribution fuction at 20 oC……….2431 Figure S19: pt(r) calculated from DOAS SAXS data at 20 oC…………………………………..2532 Figure S20: DOAS data and fit for thickness pair distance distribution function at 80 oC……...2533 Figure S21: pt(r) calculated from DOAS SAXS data at 20 oC…………………………………..2634 Figure S22: Electron density within the bilayer at 20 oC and 80 oC……………………………..2635 Figure S23: SAXS scattering profile for DMAS with increasing temperature……………...…..2736 Figure S24: DMAS scattering profiles with increasing temperatures…………………………...2737 Figure S25: Change in particle size by DLS with increasing temperature…….………………...2838 Figure S26: Determination of polydispersity by DLS with increasing temperature…………….2939 Figure S27: Differential scanning calorimetry of DMAS in the presence of kosmotropic 40 salts as compared to DMPC……………………………………………………..304142434445
2
1 Experimental Section
2 1. Materials.
3 NMR measurements were performed on a Bruker (Billerica, MA) 300MHz Avance system and
4 analyzed using TopSpin software. Chemical shifts are expressed as parts per million using
5 tetramethylsilane or CDCl3 solvent peaks as internal standards. MALDI-TOF measurements were
6 performed on a Bruker Daltonics MicroFlex LT system (Billerica, MA). High Performance flash
7 chromatography (HPFC) was carried out using a Grace Reveleris Flash System (Columbia, MD) with
8 prepacked silica gel columns. Elemental analysis was performed by the Microanalytical Laboratory at
9 the University of California Berkeley using an ICP Optima 7000 DV instrument. Zeta potential and size
10 measurements were carried out using a Nano-ZS Dynamic Light Scattering Instrument from Malvern
11 (Westborough, MA). Differential Scanning Calorimetry (DSC) measurements were obtained using a
12 high temperature MC-DSC 4100 calorimeter from Calorimetry Sciences Corp. (Lindonk, UT).
13 Fluorescence measurements were made on a FLUOstar plate reader from BMG Labtech (Durham, NC)
14 with excitation at 485 nm and emission at 518 nm. TEM images were obtained using an FEI Tecnai 12
15 transmission electron microscope at the University of California Berkeley Robert D. Ogg Electron
16 Microscope Laboratory or the University of California, San Francisco Molecular Electron Microscopy
17 Lab.
18
19 2. General Synthetic Scheme.
20 Lipids were prepared in a two-step synthesis (Scheme 1) starting with the acylation of 3-
21 (dimethylamino)-1,2-propanediol as previously reported (Kohli, 2012). Synthesis of 1-bromo-3-
22 propanesulfate (1) was performed by stirring 1 mmol of 1-bromo-3-propanol at 0.2 M in DCM as 4
23 mmol sulfurtrioxide-pyridine complex (45%) and 1 mmol diisopropyl ethylamine was added. The
3
1 reaction was then heated to 40 °C overnight under nitrogen. The reaction was concentrated and taken up
2 in DCM to afford a solid, which was removed by filtration and the filtrate purified by silica gel flash
3 chromatography (0-10% methanol in DCM). The product eluted as the 1-bromo-3-propanesulfate –
4 diisopropyl ethylamine salt in a 1:1 ratio as determined by NMR.
5 The diacyl tertiary amine lipid (2a-f) (1 mmol) was then quaternized with 1-bromo-3-
6 propanesulfate (1) (3.5 mmol) and 2 mmol diisopropyl ethylamine in dimethylformamide at 0.15 M. The
7 reactions were heated to 60 °C overnight under nitrogen. A precipitate formed in the reactions with
1 Figure S15: SAXS scattering profile for DOAS (3a) with increasing temperature (background subtracted).
2
1
10
100
1000
10000
0.00 0.05 0.10 0.15 0.20 0.2520
30
4050
6070
Inte
nsity
q (Å-1)
Tem
perature (°C)
Scattering profiles for DOCS with increasing temperature (background subtracted)
3
4 Figure S16: DOAS (3a) scattering profile with increasing temperature.
5
DOCS scattering profiles with increasing temperature
q (Å-1)
0.05 0.10 0.15 0.20 0.25
Tem
pera
ture
(°C
)
30
40
50
60
700.1 1 10 100
23
1 Figure S17: Change in lamellar spacing with temperature for DOAS (3a).
2
Change in lamellar spacing with temperature for DOCS
Temperature
10 20 30 40 50 60 70 80
Latti
ce S
paci
ng (Å
)
43.5
44.0
44.5
45.0
45.5
46.0
46.5
3
4 Figure S18: DOAS (3a) SAXS data and fit for the thickness pair distance distribution function at 20 oC. 5 Structure factor for the lamellar phase (modified Caille Theory) number of bilayer: 200, bilayer spacing: 46.0 6 Å, Caille parameter: 0.14 Å-1.7
89
24
1 Figure S19: pt(r) calculated from SAXS data in Figure S18.
2
3
4 Figure S20: DOAS (3a) SAXS data and fit for the thickness pair distance distribution function at 80 oC. 5 Structure factor for the lamellar phase (modified Caille Theory) number of bilayer: 76.3, bilayer spacing: 43.8 6 Å, Caille parameter: 0.14 Å-1.
7
89
25
1 Figure S21: pt(r) calculated from SAXS data in Figure S20.
234 Figure S22: Electron density within the bilayer thickness calculated via deconvolution of the pt(r) functions.
56789
10111213
26
1 Figure S23: SAXS scattering profile for DMAS (3d) with increasing temperature.
2
1e-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
0.050.10
0.150.20
0.2520
30
40
50
60
70
80
Inte
nsity
q (Å -1)
Tem
pera
ture
(°C)
Scattering profiles for DMCS with increasing temperature (background subtracted)
3456 Figure S24: DMAS (3a) scattering profiles with increasing temperature.
7
DMCS scattering profiles with increasing temperature
q (Å-1)
0.05 0.10 0.15 0.20 0.25
Tem
pera
ture
(°C
)
30
40
50
60
700.1 1 10 100
27
1 Figure S25: Determination of particle size by dynamic light scattering with increasing temperature. The high 2 polydispersity index of these measurements are shown in Figure S26.3
4
Particle size dependence with temperature
Temperature in DLS (°C)
20 30 40 50 60 70 80
Zave
rage
radi
us (n
m)
200
250
300
350
400
450
5001000
2000
3000
4000
5000
DOCSDMCS
Temperature in DLS (°C)
30 40 50
Zave
rage
radi
us (n
m)
200
250
300
350
Cou
nts
220000
240000
260000
280000
300000
320000
340000
DOCS ZaveDOCS Counts
5
6
28
1 Figure S26: Determination of polydispersity by dynamic light scattering with increasing temperature for 2 DMAS.
3
Polydispersity of DMCS dispersion with increasing temperature
Temperature in DLS (°C)
10 20 30 40 50 60 70 80 90
Pol
ydis
pers
ity In
dex
0.2
0.4
0.6
0.8
1.0
4
5
6 Figure S27: Differential scanning calorimetry of DMAS in the presence of kosmotropic salts as compared to 7 DMPC.
8
DMAS
40 60 80
150 mM NaCl1M NaCl1M NaI1M NaClO41M NH4Cl1M (NH4)2SO41M Na2SO4