A Novel Amino Lipid Series for mRNA Delivery: Improved ... · A novel amino lipid series for mRNA...

YMTHE, Volume 26

Supplemental Information

A Novel Amino Lipid Series for mRNA Delivery:

Improved Endosomal Escape and Sustained

Pharmacology and Safety in Non-human Primates

Staci Sabnis, E. Sathyajith Kumarasinghe, Timothy Salerno, Cosmin Mihai, TatianaKetova, Joseph J. Senn, Andy Lynn, Alex Bulychev, Iain McFadyen, Joyce Chan, ÖrnAlmarsson, Matthew G. Stanton, and Kerry E. Benenato

A novel amino lipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates

Staci Sabnis1, E. Sathyajith Kumarasinghe1, Timothy Salerno1, Cosmin Mihai1, Tatiana Ketova1, Joseph J. Senn1, Andy Lynn1, Alex Bulychev1, Iain McFadyen1, Joyce Chan1, Örn Almarsson1, Matthew G. Stanton2, Kerry E. Benenato1*

1Moderna Therapeutics, 200 Technology Square, Cambridge, MA 02139, USA 2Current address: Generation Bio, 215 First Street, Suite 150, Cambridge, MA 02142, USA

Correspondence should be addressed to: Kerry E. Benenato, 200 Technology Square, Cambridge, MA 02139, USA; email: [email protected]

Supplemental Information

Figure S1.

Whole body and ex vivo luciferase imaging of CD-1 mice. CD-1 mice, 0.5 mg/kg dose luciferase mRNA, lipid 1-based LNP, i.v. bolus, 6 h.

mailto:[email protected]

liver spleen lung

Table S1.

Lipid purity. Lipid stability in ethanol over 96 h at 25 °C and 37 °C.

Lipid Purity (%)

Lipid t = 0 t = 96 h, 25 °C t = 96 h, 37 °C

1 95.17 95.15 96.47

2 97.59 98.64 97.82

3 91.81 93.17 93.86

4 93.80 93.21 94.29

5 94.37 94.71 95.01

6 97.017 97.67 98.10

7 92.08 92.07 91.90

8 95.58 96.23 96.13

9 98.64 97.38 95.72

Figure S2. hEPO expression in Sprague Dawley rats. hEPO serum concentrations, Sprague Dawley rats, i.v. bolus, 1 mg/kg hEPO mRNA, n =3, mean ± std. dev., p < 0.05 relative to MC3 AUC.

0 2 0 4 00

1 ×1 0 7

2 ×1 0 7

3 ×1 0 7

T im e (h )

hE

PO

se

rum

co

nc

en

tra

tio

n(p

g/m

L)

5

8

9

M C 3

Table S2.

Pharmacokinetic Parameters. Sprague Dawley rats (n = 3 per time point), 0.2 mg/kg dose hEPO mRNA, mean ± std. dev.

Tissue Lipid HL (hr) Tmax (hr)

Cmax (ng/mL) AUCall (hr*ng/mL)

Mean SE Mean SE

Plasma MC3 8.4 0.5 28,900 2350 69,100 84.2

5 1.2 0.5 44,200 3520 61,800 6520

8 2.3 0.5 37,500 2540 35,400 1470

Liver MC3 52.5* 24 33,000 2940 1,410,000 114,000

5 5.8 1 6050 91.3 16,900 1930

8 6.9 1 17,900 608 89,200 5780

Spleen MC3 33.3* 8 21,200 5380 787,000 61,000

5 10.6 4 3770 847 39,400 2750

8 15.2 4 20,400 4170 259,000 7050

* = 2 terminal datapoints estimation

PK analysis of the individual plasma and tissue concentration data was performed using a non-validated program (Phoenix WinNonlin®, Version 7.0 (Pharsight Corp., Mountain View, CA)). Kinetic parameters were estimated using a noncompartmental model (Plasma (200-202), Uniform weighting, extravascular dosing with sparse sampling). AUC was calculated using the linear trapezoidal rule.

All derived parameters are reported to 3 significant figures with the exception of time to peak concentration (Tmax) and halflife (HL), which are reported to one decimal place. No further statistical analyses were performed on the plasma concentration data or derived pharmacokinetic parameters.

Figure S3.

Tissue Distribution in CD-1 mice. Comparison of tissue distribution of MC3 and lipid 5 after three i.v. bolus doses, CD-1 mice (n = 3 per timepoint), 0.05 mg/kg mRNA, mean ± SEM.

L ive r

S p lee n

P lasm

a

K idn e y

H e a r t

L u n g

L ive r

S p lee n

P lasm

a

K idn e y

H e a r t

L u n g

0

1 0 0 0 0 0

2 0 0 0 0 0

3 0 0 0 0 0

4 0 0 0 0 0

AU

C0

-24

hr

(hr*

ng

/mL

)

D a y 1

D a y 8

D a y 1 5

M C 3 5

Table S3.

Metabolite profiling and characterization data for Lipid 5.a

a + Detected, ND Not Detected

Component Retention time (min)

Experimental m/z

Theoretical m/z

Mass error (ppm)

Mass shift

Elemental composition

Proposed biotransformation

0 min

30 min

60 min

90 min

C1 10.65 584.5257 584.5254 + 0.5 -126.1405 C35H69NO5 Ester hydrolysis on the 17 carbon chain ND + + +

5 12.49 710.6662 710.6662 0.0 0.0 C44H87NO5 - + + + +

Figure S4.

Human IgG expression in NHP. Cynomolgus monkey, 0.1 mg/kg human IgG mRNA, i.v., 60-min. infusion, n = 3, mean ± std. dev., p < 0.05 for lipid 5 relative to MC3 AUC.

0 .0

0 .5

1 .0

1 .5

2 .0

T im e (h )

IgG

se

rum

co

ne

ntr

ati

on

(mg

/mL

)

M C 3

5

0 2 4 7 11 14 18 21

Figure S5.

Lipid tissue distribution in NHP. Cynomolgus monkey, 0.2 mg/kg mRNA, 12 h, n = 2.

L ive r

S p lee n

B rain

H e a r t

L ymp h

Ma rr

o wL iv

e r

S p lee n

B rain

H e a r t

L ymp h

Ma rr

o w

1 0 0

1 0 0 0

1 0 0 0 0

1 0 0 0 0 0

1 0 0 0 0 0 0

ng

/g t

iss

ue

M C 3 5

Figure S6.

MC3 single-cell necrosis in rat liver and liver enzyme

Figure S7.

mRNA levels in NHP after repeat dose, n= 4, mean ± S.D.

-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

Predose 1 h 6 h 24 h Predose 1 h 6 h 24 h

Day 1 Day 29

seru

m m

RNA

conc

entr

atio

n

(ng/

mL)

0.1 mg/kg 0.3 mg/kg 1 mg/kg

Figure S8.

hEPO expression in ApoE KO and LDLr KO mice. 0.5 mg/kg hEPO mRNA, C57BL/6 WT mice, n = 5, mean ± S.D.

C 5 7B L /6 W

T

A p o E KO

C 5 7B L /6 W

T

A p o E KO

0

5 ×1 0 6

1 ×1 0 7

1 .5 ×1 0 7

hE

PO

se

rum

co

nc

en

tra

tio

n(p

g/m

L)

5

5

P B S

P B S

lip id 5 P B S

C 5 7B L /6 W

T

L D L r KO

C 5 7B L /6 W

T

L D L r KO

0

2 ×1 0 6

4 ×1 0 6

6 ×1 0 6

hE

PO

se

rum

co

nc

en

tra

tio

n(p

g/m

L)

5

5

P B S

P B S

lip id 5 P B S

Table S4. Lipid onset Tm measured by differential scanning calorimetry.

Lipid Onset Tm

(°C)

MC3 -20.7

Lipid 5 -33.4

Lipid 5 has a lower onset Tm than MC3 lipid as shown by DSC measurements.

Differential scanning calorimetry (DSC) data were recorded on a TA Instruments Q200 DSC. Tested 5 mg MC3 and lipid 5 lipids individually. Samples were equilibrated at 20 °C and then cooled to -80 °C, then heated up to 200 °C and back down to 20 °C. Ramp rate used was 5 °C/min and there were 5-minute isothermal holds at the end of each ramp.

Figure S9.

Quantifying the number of mRNA per LNP with single particle imaging microscopy.

IVT mRNA (mCherry) was fluorescently labeled at the poly-A tail using copper free click chemistry. 2’ Azido-ATP (Jena Bioscience) was incorporated at the 3’ end post in vitro transcription using yeast Poly(A) Polymerase, and DBCO conjugated Alexa 488 or Alexa 647 (Thermo Fisher) was covalently linked via a triazole moiety; the products were further purified using ethanol precipitation to remove unreacted fluorophores. The mRNA product was run on an Agilent Bioanalyzer micro-capillary electrophoresis system to check for conjugation efficiency (close to 100%). Labeled mRNA was co-formulated in Rhodamine labeled MC3 LNPs, and single particle imaging microscopy together with combinatorial analysis was used to evaluate the number of mRNA per LNP.

LNPs containing labeled mRNA were deposited on plasma treated glass coverslip-bottom dishes and imaged using Structured Illumination Microscopy (SIM) on a Zeiss Elyra S1 microscope using a 63× magnification (NA = 1.46) oil-immersion objective and 488, 561 and 647 nm laser excitation. Emission bandpass filters were 495–550, 570–620 nm, with a 655 nm long pass filter used for the 647 laser. The different channels were recorded sequentially,

within each channel the raw data contained 3 rotations, 5 phases and 0.1 μm spaced z stack images. The super-resolution images were then reconstructed from the raw data using ZEISS SIM processing software (representative section shown, A) and analyzed with Imaris image analysis software (Bitplane AG, Zurich, Switzerland). LNP objects were identified in the Rhodamine channel and classified as Alexa 488 mRNA (green) or Alexa 647 mRNA (red) positive using intensity thresholds derived from LNP samples incorporating unlabeled mRNA; several fields of view were analyzed per sample, and each field of view included in average 4000 LNP objects (C) The data was binned as function of Rhodamine intensity (LNP size) and the percentage of double positive (green and red) LNPs was calculated for each bin. Combinatorial composition analysis (B) was used to derive the number of mRNA per LNP from the percentage of double positive LNPs (D). The relative frequency of mRNA loading was plotted across the entire LNP population, and the population weighted average was calculated (E).

Figure S10.

Workflow for characterizing endosomal escape efficiency. HeLa cells were transfected with ATTO 647 labeled MC3 and lipid 5 LNPs encapsulating luciferase mRNA, and processed for single molecule FISH (smFISH, red) alongside cells electroporated with unformulated mRNA. Prior to smFISH hybridization, the cells were imaged for LNP uptake and the number of LNP per cell was evaluated by comparison with single LNP intensity (measured by direct deposition on glass substrates). mRNA molecules that egressed the endocytic organelles into the cytosol were identified by comparison with the electroporated sample, using object based image analysis.

Figure S11. Intensity-based classification of cytosolic vs. organelle trapped mRNA using single molecule FISH

Figure S12.

Co-staining with endosomal markers Lamp 1 and EEA1. HeLa cells were transfected with LNP encapsulated mRNA (FLuc) for 4h, then fixed in 4 % PFA, followed by permeabilization with 0.5 % Saponin in PBS, and blocking in with 0.1% Saponin/1% BSA/PBS); cells were co-incubated with primary antibodies Lamp1 (BD, Cat#555798) and EEA1 (Cell Signaling, Cat#3288) for 1h at RT diluted in blocking buffer at 1:2000 and 1:100 respectively. After washes and incubation with secondary antibody (1:1000 dilution, Invitrogen Cat#A11029, A11070) for 30 min, samples were fixed for 5 min in 4% PFA and processed for smFISH using custom designed probe against FLuc. Cells were stained with HCS Cell Mask (Invitrogen, Cat# H 32720) and Nuclear Stain (NucBlue Fixed Cells, Invitrogen, Cat#R37606) using manufacturer recommended protocol. Imaging was done on the Opera Phenix spinning disk confocal microscope using a 63x water immersion objective.

Cells transfected with LNPs show bimodal distribution of the smFISH signal with large aggregates colocalized with endocytic markers and single molecule objects. By contrast the electroporated sample shows homogenous distribution of single molecule objects that do not colocalize efficiently with endocytic markers.

Lipid Synthesis

All reagents were purchased from commercial vendors unless noted.

2-(Di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (1)

HON

Chemical Formula: C38H71NO

Molecular Weight: 557.99

To a solution of (6Z,9Z)-18-bromooctadeca-6,9-diene (4 g, 12.1 mmol) in CH3CN (26 mL) was added 2-aminoethan-1-ol (0.334 mL, 5.52 mmol), K2CO3 (3.36 g, 24.3 mmol), and KI (0.092 g, 0.552 mmol). The reaction was allowed to stir at 75 °C for 16 hours. The reaction mixture was cooled to room temperature, filtered, and the solids were washed with hexanes. The filtrate was extracted with hexanes, and the combined extracts were concentrated in vacuo. Purification by ISCO silica flash chromatography (0-15% MeOH in DCM) provided 2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (1.9 g, 62%).

UPLC/ELSD: RT = 4.20 min. HRMS (ESI): m/z calcd for C38H72NO+ (M+H), 558.561; found, 558.557. 1H NMR (300 MHz, CDCl3) δ: ppm 5.37 (m, 8H); 3.55 (t, 2H); 2.80 (m, 4H); 2.60 (t, 2H); 2.46 (m, 4H); 2.06 (m, 8H); 1.54-1.19 (m, 36H); 0.92 (m, 6H). 13C NMR (75 MHz, CDCl3): δ 130.20, 130.13, 127.99, 127.94, 58.31, 55.51, 53.85, 31.54, 29.68, 29.58, 29.57, 29.37, 29.31, 27.46, 27.24, 27.22, 27.17, 25.65, 22.59, 14.09. Dinonyl 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate (2)

Nonyl 8-bromooctanoate

O

OBr

Chemical Formula: C17H33BrO2 Molecular Weight: 349.35

To a solution of 8-bromooctanoic acid (5 g, 22 mmol) and nonan-1-ol (6.46 g, 45 mmol) in dichloromethane (100 mL) were added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4.3 g, 22 mmol) and DMAP (547 mg, 4.5 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction was diluted with dichloromethane and extracted with saturated NaHCO3 (aq.). The organic layer was separated and washed with brine, then dried over MgSO4, filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to obtain nonyl 8-bromooctanoate (6.1 g, 17 mmol, 77%). 1H NMR (300 MHz, CDCl3) δ: ppm 4.06 (t, 2H); 3.40 (t, 2H); 2.29 (t, 2H); 1.85 (m, 2H); 1.72-0.97 (m, 22H); 0.88 (m, 3H).

Dinonyl 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate (2)

HON

O

O

O

O

Chemical Formula: C36H71NO5


A solution of nonyl 8-bromooctanoate (0.200 g, 0.6 mmol) and 2-aminoethan-1-ol (0.016 g, 0.3 mmol) and N, N-diisopropylethylamine (0.074 g, 0.6 mmol) in THF/CH3CN (1:1) (3 mL) was allowed to stir at 63 °C for 72 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was extracted with ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried over Na2SO4 and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% MeOH in dichloromethane) to obtain dinonyl 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate (0.080 g, 0.13 mmol, 43%).

UPLC/ELSD: RT = 3.09 min. HRMS (ESI): m/z calcd for C36H72NO5 + (M+H), 598.541; found, 598.537.

1H NMR (300 MHz, CDCl3) δ: ppm 4.05 (br. m, 4H); 3.57 (br. m, 2H); 2.71-2.38 (br. m, 6H); 2.29 (m, 4H), 1.71-1.01 (br. m, 49H), 0.88 (m, 6H). 13C NMR (75 MHz, CDCl3): δ 173.93, 64.43, 58.29, 55.51, 53.82, 34.36, 31.86, 29.49, 29.26, 29.23, 29.14, 28.66, 27.26, 27.06, 25.93, 24.97, 22.67, 14.11. Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (3)

Nonyl 8-((2-hydroxyethyl)amino)octanoate

HN

O

OHO



A solution of nonyl 8-bromooctanoate (1.2 g, 3.4 mmol) and 2-aminoethan-1-ol (5 mL, 83 mmol) in ethanol (2 mL) was allowed to stir at 62 °C for 18 hours. The reaction mixture was concentrated under vacuum and the residue was extracted with ethyl acetate and water. The organic layer was separated and washed with water, brine and dried over Na2SO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain nonyl 8-((2-hydroxyethyl)amino)octanoate (0.295 g, 26%).

UPLC/ELSD: RT = 1.29 min. MS (ES): m/z calcd for C19H40NO5+ (M+H), 330.53; found, 330.42 1H NMR (300 MHz, CDCl3) δ: ppm 4.07 (t, 2H); 3.65 (t, 2H); 2.78 (t, 2H); 2.63 (t, 2H); 2.32-2.19 (m, 4H); 1.73-1.20 (m, 24H); 0.89 (m, 3H). Nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (3)

HON

OO



A solution of nonyl 8-((2-hydroxyethyl)amino)octanoate (0.150 g, 0.46 mmol), (6Z,9Z)-18-bromooctadeca-6,9-diene (0.165 g, 0.5 mmol) and N, N-diisopropylethylamine (0.065 g, 0.5 mmol) in ethanol (2 mL) was allowed to stir at reflux for 48 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% MeOH in dichloromethane) to obtain nonyl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (0.081 g, 0.14 mmol, 30%) as a HBr salt.

UPLC/ELSD: RT = 3.24 min. HRMS (ESI): m/z calcd for C37H72NO3+ (M+H), 578.551; found, 578.546.

1H NMR (300 MHz, CDCl3) δ: ppm 10.71 (br., 1H); 5.36 (br. m, 4H); 4.04 (m, 4H); 3.22-2.96 (br. m, 5H); 2.77 (m, 2H); 2.29 (m, 2H); 2.04 (br. m, 4H); 1.86 (br. m, 4H); 1.66-1.17 (br. m, 40H); 0.89 (m, 6H). 13C NMR (75 MHz, CDCl3): δ 173.93, 130.21, 130.13, 127.99, 127.94, 64.44, 53.82, 34.36, 31.87, 31.54, 29.68, 29.56, 29.49, 29.37, 29.30, 29.24, 29.14, 28.67, 27.43, 27.24, 25.94, 25.65, 24.97, 22.68, 22.59, 14.12, 14.09. Heptadecan-9-yl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (4)

2-(((9Z,12Z)-Octadeca-9,12-dien-1-yl)amino)ethan-1-ol

HONH

Chemical Formula: C20H39NO


A solution of (6Z,9Z)-18-bromooctadeca-6,9-diene (0.8 g, 2.4 mmol) and 2-aminoethan-1-ol (0.223 g, 3.1 mmol), K2CO3 (0.336 g, 2.4 mmol), KI (0.005 g, 0.3 mmol) in acetonitrile (8 mL) was allowed to stir at 66 °C for 18 hours followed by heating at reflux for 24 hours. The reaction mixture was concentrated under vacuum and the residue was extracted with ethyl acetate and water. The organic layer was separated and washed with water, brine and dried over Na2SO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-20% MeOH in dichloromethane) to obtain 2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (0.402 g, 54%).

UPLC/ELSD: RT = 1.79 min. MS (ES): m/z calcd for C20H40NO + (M+H), 310.55; found, 310.46 1H NMR (300 MHz, CDCl3) δ: ppm 5.39 (m, 4H); 3.67 (br. m; 2H); 2.82 (m, 4H); 2.66 (m, 2H); 2.27-1.97 (m, 6H); 1.53 (m, 2H); 1.33 (m, 16H); 0.92 (m, 3H).

Heptadecan-9-yl 8-bromooctanoate

O

OBr

Chemical Formula: C25H49BrO2


To a solution of 8-bromooctanoic acid (1.04 g, 4.6 mmol) and heptadecan-9-ol (1.5 g, 5.8 mmol) in dichloromethane (20 mL) were added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.1 g, 5.8 mmol), N,N-diisopropylethylamine (3.3 mL, 18.7 mmol) and DMAP (0.114 g, 0.9 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction was diluted with dichloromethane and extracted with saturated NaHCO3 (aq.). The organic layer was separated and washed with brine, then dried over MgSO4, filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to obtain heptadecan-9-yl 8-bromooctanoate (875 mg, 41%). 1H NMR (300 MHz, CDCl3) δ: ppm 4.89 (m, 1H); 3.42 (m, 2H); 2.31 (m, 2H); 1.89 (m, 2H); 1.73-1.18 (br. m, 36H); 0.88 (t, 6H). 13C NMR (75 MHz, CDCl3): δ 72.04, 37.51, 31.90, 29.73, 29.61, 29.29, 25.67, 22.68, 14.11. Heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate

HN

O

OHO



A solution of heptadecan-9-yl 8-bromooctanoate (3.8 g, 8.2 mmol) and 2-aminoethan-1-ol (15 mL, 248 mmol) in ethanol (3 mL) was allowed to stir at 62 °C for 18 hours. The reaction mixture was concentrated under vacuum and the residue was extracted with ethyl acetate and water. The organic layer was separated and washed with water, brine and dried over Na2SO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (3.1 g, 7 mmol).

UPLC/ELSD: RT = 2.67 min. MS (ES): m/z calcd for C27H56NO3+ (M+H), 442.68; found, 442.75 1H NMR (300 MHz, CDCl3) δ: ppm 4.89 (quint, 1H); 3.67 (t, 2H); 2.81 (t, 2H); 2.65 (t, 2H); 2.30 (t, 2H); 2.05 (br. m, 2H); 1.72-1.41 (br. m, 8H); 1.40-1.20 (br. m, 30H); 0.88 (t, 6H). 13C NMR (75 MHz, CDCl3): δ 173.55, 74.07, 60.40, 53.39, 51.15, 49.43, 34.62, 34.12, 31.83, 29.64, 29.50, 29.47, 29.21, 29.14, 29.09, 27.08, 25.29, 25.04, 22.63, 14.07. Heptadecan-9-yl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (4)

HON

OO



A solution of nonyl 2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (0.150 g, 0.48 mmol), heptadecan-9-yl 8-bromooctanoate (0.246 g, 0.50 mmol) and N, N-diisopropylethylamine (0.069 g, 0.50 mmol) in ethanol (2 mL) was allowed to stir at 65 °C for 18 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was partitioned between ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain heptadecan-9-yl 8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (0.138 g, 42%).


1H NMR (300 MHz, CDCl3) δ: ppm 5.37 (m, 4H); 4.86 (br. m, 1H); 3.53 (br. m; 2H); 2.78 (br. m, 2H); 2.58 (br. m, 2H); 2.45 (br. m, 4H); 2.28 (m, 2H); 2.05 (m, 4H); 1.68-1.15 (br. m, 57H); 0.89 (m, 9H). 13C NMR (75 MHz, CDCl3): δ 173.61, 130.19, 130.12, 127.98, 127.93, 74.11, 58.30, 55.49, 53.85, 34.70, 34.16, 31.88, 31.54, 29.68, 29.57, 29.55, 29.52, 29.36, 29.31, 29.25, 29.20, 27.46, 27.30, 27.24, 27.21, 27.16, 27.12, 25.64, 25.33, 25.13, 22.68, 22.59, 14.12. Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (5)

HON

O

O

O

O



A solution of nonyl 8-((2-hydroxyethyl)amino)octanoate (0.150 g, 0.42 mmol), heptadecan-9-yl 8-bromooctanoate (0.217 g, 0.47 mmol) and N, N-diisopropylethylamine (0.061 g, 0.47 mmol) in ethanol (2 mL) was allowed to stir at 65 °C for 18 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was partitioned between ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried and evaporated under vacuum. The residue was purified by silica gel chromatography [(0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (0.089 g, 30%).


1H NMR (300 MHz, CDCl3) δ: ppm 4.86 (m, 1H); 4.05 (t, 2H); 3.53 (br. m, 2H); 2.83-2.36 (br. m, 5H); 2.29 (m, 4H); 1.71-1.41 (br. m, 13H); 1.40-1.17 (br. m, 51H); 0.88 (m, 9H). 13C NMR (75 MHz, CDCl3): δ 173.81, 173.52, 74.03, 64.36, 58.31, 55.51, 53.82, 34.64, 34.30, 34.13, 31.84, 29.51, 29.48, 29.46, 29.23, 29.21, 29.15, 29.11, 28.64, 27.26, 27.23, 27.08, 25.91, 25.29, 25.09, 24.93, 22.64, 14.08. Di(heptadecan-9-yl) 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate (6)

N

HO

O

O

O

O



A solution of heptadecan-9-yl 8-bromooctanoate (0.280 g, 0.61 mmol) and 2-aminoethan-1-ol (0.015 g, 0.24 mmol) and N,N-diisopropylethylamine (0.078 g, 0.61 mmol) in EtOH (1 mL) was allowed to stir at 63 °C for 18 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was extracted with ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried over Na2SO4 and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain di(heptadecan-9-yl) 8,8'-((2-hydroxyethyl)azanediyl)dioctanoate (0.079 g, 42%).


1H NMR (300 MHz, CDCl3) δ: ppm 4.86 (m, 2H); 3.72-3.44 (br. m, 2H); 2.83- 2.34 (br. m, 5H); 2.28 (m, 4H); 1.69-1.39 (br. m, 16H); 1.39-1.16 (m, 62H); 0.88 (m, 12H). 13C NMR (75 MHz, CDCl3): δ 173.62, 74.12, 58.29, 55.52, 53.85, 34.70, 34.15, 31.87, 29.54, 29.51, 29.25, 29.20, 27.30, 27.06, 25.33, 25.13, 22.67, 14.11. Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (7)

Nonyl 8-((2-(dimethylamino)ethyl)amino)octanoate

NNH

O

O

Chemical Formula: C21H44N2O2


A solution of nonyl 8-bromooctanoate (0.35 g, 1.0 mmol) and N1,N1-dimethylethane-1,2-diamine (0.177 g, 2.0 mmol) in ethanol (0.5 mL) was allowed to stir at 65 °C for 18 hours. The reaction mixture was concentrated under vacuum and the residue was extracted with ethyl acetate and water. The organic layer was separated and washed with water, brine and dried over Na2SO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain nonyl 8-((2-(dimethylamino)ethyl)amino)octanoate (0.212 g, 59%).

UPLC/ELSD: RT = 1.09 min. MS (ES): m/z calcd for C21H45N2O2+ (M+H), 357.35; found, 357.56

1H NMR (300 MHz, CDCl3) δ: ppm 4.06 (p, 2H); 2.79-2.53 (m, 4H); 2,44 (s, 2H); 2.35-1.94 (m, 9H); 1.72-1.18 (m, 23H); 0.89 (m, 3H).

Heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (7)

NN

O

O

O

O

Chemical Formula: C46H92N2O4


A solution of nonyl 8-((2-(dimethylamino)ethyl)amino)octanoate (0.200 g, 0.56 mmol), heptadecan-9-yl 8-bromooctanoate (0.310 g, 0.67 mmol) and N,N-diisopropylethylamine (0.108 g, 0.84 mmol) in ethanol (0.5 mL) was allowed to stir at 65 °C for 18 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was partitioned between ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain heptadecan-9-yl 8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (0.047 g, 11%).

UPLC/ELSD: RT = 3.51 min. HRMS (ESI): m/z calcd for C46H93N2O4+ (M+H), 737.714; found, 737.708.

1H NMR (300 MHz, CDCl3) δ: ppm 4.84 (p, 1H); 4.04 (t, 2H); 2.95 (m, 2H); 2.78 (m, 6H); 2,44 (s, 6H); 2.28 (m, 4H); 1.70-1.41 (m, 14H); 1.41-1.14 (m, 48H); 0.87 (m, 9H). 13C NMR (75 MHz, CDCl3): δ 173.85,173.55, 74.19, 64.47, 54.82, 53.58, 50.02, 44.88, 34.58, 34.24, 34.12, 31.86, 29.52, 29.50, 29.48, 29.23, 29.03, 28.97, 28.92, 28.64, 26.95, 25.92, 25.32, 24.81, 22.66, 14.11. Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (8)

Undecyl 6-bromohexanoate

O

OBr



To a solution of 6-bromohexanoic acid (10 g, 51.3 mmol) and undecan-1-ol (17.7 g, 102.5 mmol) in dichloromethane (250 mL) were added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (9.8 g, 51.3 mmol), and DMAP (1.25 g, 10.3 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction was diluted with dichloromethane and extracted with saturated NaHCO3 (aq.). The organic layer was separated and washed with brine, dried over MgSO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-10% ethyl acetate in hexanes) to obtain undecyl 6-bromohexanoate (14.3 g, 80%). 1H NMR (300 MHz, CDCl3) δ: ppm 4.89 (m, 1H); 3.42 (m, 2H); 2.31 (m, 2H); 1.89 (m, 2H); 1.73-1.18 (br. m, 36H); 0.88 (t, 6H). 13C NMR (75 MHz, CDCl3): δ 173.48, 64.52, 34.09, 33.41, 32.41, 31.90, 29.60. 29.57, 29.52, 29.33, 29.25, 28.64, 27.66, 25.93, 24.13, 22.68, 14.11.

Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (8)

N

HO

O

O

O

O



A solution of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (0.150 g, 0.34 mmol), undecyl 6-bromohexanoate (0.142 g, 0.41 mmol) and N,N-diisopropylethylamine (0.053 g, 0.41 mmol) in ethanol (1 mL) was allowed to stir at 65 °C for 18 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was partitioned between ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (0.035 g, 14%).


1H NMR (300 MHz, CDCl3) δ: ppm 4.86 (m, 1H); 4.05 (t, 2H); 3.68-3.46 (br. m, 2H); 2.77-2.37 (br. m, 5H); 2.29 (m, 4H); 1.74-1.41 (m, 14H); 1.39-1.18 (m, 50H); 0.88 (m, 9H). 13C NMR (75 MHz, CDCl3): δ 173.74, 173.59, 74.10, 64.47, 58.31, 55.51, 53.81, 53.63, 34.68, 34.28, 34.15, 31.91, 31.86, 29.60, 29.58, 29.53, 29.51, 29.33, 29.26, 29.24, 29.19, 28.65, 27.29, 27.02, 26.94, 26.80, 25.93, 25.32, 25.12, 24.89, 22.68, 22.66, 14.11.

Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino)octanoate (9)

Nonyl 4-bromobutanoate

BrO

O



To a solution of 4-bromobutanoic acid (10 g, 59.8 mmol) and nonan-1-ol (17.3 g, 119.7 mmol) in dichloromethane (250 mL) were added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (11.5 g, 59.8 mmol), and DMAP (1.46 g, 11.9 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction was diluted with dichloromethane and extracted with saturated NaHCO3 (aq.). The organic layer was separated and washed with brine, dried over MgSO4, then filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-20% ethyl acetate in hexanes) to obtain nonyl 4-bromobutanoate (12.3 g, 70%). 1H NMR (300 MHz, CDCl3) δ: ppm 4.10 (t, 2H); 3.51 (t, 2H); 2.51 (t, 2H); 2.15 (m, 2H); 1.65 (m, 2H); 1.33 (m, 12H); 0.92 (m, 3H). 13C NMR (75 MHz, CDCl3): δ 172.58, 77.47, 77.04, 76.62, 64.78, 32.72, 32.50, 31.85, 29.46, 29.23, 29.22, 28.60, 27.80, 25.90, 22.66, 14.09.

Heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino)octanoate (9)

N

HO

O

O

O

O



A solution of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (0.150 g, 0.34 mmol), nonyl 4-bromobutanoate (0.200 g, 0.68 mmol) and N, N-diisopropylethylamine (0.117 µL, 0.68 mmol) in ethanol (1 mL) was allowed to stir at 65 °C for 18 hours. The reaction was cooled to room temperature and solvents were evaporated under vacuum. The residue was partitioned between ethyl acetate and saturated NaHCO3 (aq.). The organic layer was separated, dried and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain heptadecan-9-yl 8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino)octanoate (0.129 g, 58%).


1H NMR (300 MHz, CDCl3) δ: ppm 4.86 (p, 1H); 4.06 (t, 2H); 3.79 (br. m, 2H); 2.91-2.20 (m, 10H); 1.98-1.03 (m, 55H); 0.88 (m, 9H). 13C NMR (75 MHz, CDCl3): δ 173.62, 173.58, 74.10, 64.64, 58.48, 55.68, 53.69, 52.99, 34.68, 34.14, 32.04, 31.86, 29.53, 29.50, 29.48, 29.26, 29.24, 29.18, 28.63, 27.28, 26.90, 25.93, 25.32, 25.11, 22.66, 22.43, 14.10. Henicosan-11-yl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate (10) Henicosan-11-yl 6-bromohexanoate

Br

O

O

Chemical Formula: C27H53BrO2 Molecular Weight: 489.62

To a solution of 6-bromohexanoic acid (1.9 g, 9.6 mmol) and henicosan-11-ol (3.0 g, 9.6 mmol) in dichloromethane (20 mL) were added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.84 g, 9.6 mmol), and DMAP (0.23 g, 1.9 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction was diluted with dichloromethane and extracted with saturated NaHCO3 (aq.). The organic layer was separated and washed with brine, dried over MgSO4. The organic layer was filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-20% ethyl acetate in hexanes) to obtain henicosan-11-yl 6-bromohexanoate (3.9 g, 83%). 1H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H); 3.43 (t, 2H); 2.33 (t, 2H); 1.91 (m, 2H); 1.68 (m, 2H); 1.52 (m, 6H); 1.28 (m, 32H); 0.90 (m, 6H).

Henicosan-11-yl 6-((2-hydroxyethyl)amino)hexanoate

HONH

O

O

Chemical Formula: C29H59NO3 Molecular Weight: 469.80

A solution of henicosan-11-yl 6-bromohexanoate (2.0 g, 4.1 mmol) and 2-aminoethan-1-ol (5.0 g, 82 mmol) in ethanol (5 mL) was allowed to stir at 62 °C for 18 hours. The reaction mixture was concentrated in vacuum and the residue was extracted with ethyl acetate and water. The organic layer was separated and washed with water, brine and dried over Na2SO4. The organic layer was filtered and evaporated under vacuum. The residue was purified by silica gel chromatography (0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane) to obtain henicosan-11-yl 6-((2-hydroxyethyl)amino)hexanoate (1.4 g, 73%). UPLC/ELSD: RT = 2.87 min. MS (ES): m/z calcd for C29H60NO3

+ (M+H), 470.46; found, 470.45 1H NMR (300 MHz, CDCl3) δ: ppm 4.89 (p, 1H); 3.67 (t, 2H); 2.82 (t, 2H); 2.67 (t, 2H); 2.32 (t, 2H); 1.75-1.16 (m, 43H); 0.90 (m, 6H). Henicosan-11-yl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate (10)

HON

O

O

O

O

Chemical Formula: C46H91NO5 Molecular Weight: 738.24

A solution of henicosan-11-yl 6-((2-hydroxyethyl)amino)hexanoate (0.25 g, 0.53 mmol), undecyl 6-bromohexanoate (0.200 g, 0.56 mmol), KI (0.1 g, 0.59 mmol) and K2CO3 (0.29 g, 2.1 mmol) in a mixture of cyclopropyl methyl ether/acetonitrile (1:1, 4 mL) was allowed to stir at 80 °C for 18 hours. The reaction was cooled to room temperature, filtered and solvents were evaporated under vacuum. The residue was purified by silica gel chromatography (0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane) to obtain henicosan-11-yl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate (0.11 g, 28%). UPLC/ELSD: RT = 3.72min. HRMS (ESI): m/z calcd for C46H92NO5

+ (M+H), 738.697; found, 738.691. 1H NMR (300 MHz, CDCl3) δ: ppm 4.88 (m, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.60 (m, 2H); 2.48 (m, 4H); 2.32 (m, 4H); 1.72-1.41 (m, 15H); 1.28 (m, 52H); 0.90 (m, 9H). 13C NMR (75 MHz, CDCl3): δ 173.74, 173.44, 74.19, 64.47, 58.35, 55.52, 53.68, 53.63, 34.62, 34.28, 34.14, 31.91, 29.60, 29.56, 29.55, 29.34, 29.27, 28.66, 27.01, 26.95, 26.86, 25.94, 25.34, 25.05, 24.89, 22.69, 14.12.

8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid (11)

tert-Butyl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

NO

O

O

O

HO



To a solution of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (0.24 g, 0.54 mmol) and tert-butyl 8-bromooctanoate (0.17 g, 0.6 mmol) in CPME and acetonitrile (1:1, 6 mL) were added K2CO3 (0.3 g, 2.17 mmol) and KI (0.1 g, 0.6 mmol). The reaction was heated at 81 °C for 18 hours. The reaction was cooled to room temperature and the solvents evaporated under vacuum. The residue was dissolved in ethyl acetate and extracted with water. The organic layer was separated, dried with Na2SO4, filtered and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain tert-butyl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate (0.32 g, 83%).

UPLC/ELSD: RT = 3.59 min. MS (ES): m/z calcd for C39H78NO5+ (M+H), 640.59; found, 640.53

1H NMR (300 MHz, CDCl3) δ: ppm 4.88 (m, 1H); 3.57 (br. m, 2H); 2.74-2.37 (br. m, 6H); 2.34-2.16 (m, 4H); 1.72-1.19 (m, 58H); 0.90 (m, 6H).

8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid (11)

NO

O

OH

O

HO



To a solution of tert-butyl 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate (0.11 g, 0.17 mmol) in DCM was added trifluoroacetic acid (0.06 mL, 0.69 mmol) with stirring at room temperature. Upon completion of the reaction the volatiles were evaporated under vacuum. The residue was dissolved in ethyl acetate and extracted with water. The organic layer was separated, dried with Na2SO4, filtered and evaporated under vacuum. The residue was purified by silica gel chromatography [0-100% (mixture of 1% NH4OH, 20% MeOH in dichloromethane) in dichloromethane] to obtain 8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoic acid (0.015 g, 15%).

UPLC/ELSD: RT = 2.72 min. MS (ES): m/z calcd for C35H70NO5+ (M+H), 584.95; found, 584.85

1H NMR (300 MHz, CDCl3) δ: ppm 4.87 (p, 1H); 3.98 (m, 2H); 3.25-3.02 (br. m, 6H); 2.33 (m, 4H); 1.82-1.45 (br. m, 12H); 1.44-1.18 (br. m, 36H); 0.92 (m, 6H).

Hydrolytic Stability of Lipids

Lipids were diluted to 1 mg/mL with Deacon Lab ethanol and vortexed. Lipids purity was tested at t = 0 and t = 96 h under various conditions. Three sets of samples were prepared: 1) for t = 0 RT, 2) for t = 96 h RT, 3) for t = 96 h 37 °C with shaking. Set three was placed on a thermo-shaker at 37 °C and 500 rpm for 96 hours before analysis. 5 ml of sample was injected onto a Thermo Vanquish CAD UPLC with a Thermo Vanquish Accucore C18 100 x 2.1 mm column with a 40-min method. Column chamber was heated to 70 °C and CAD power was set to 1.20 (Buffer A: 0.1 % TFA water, Buffer B: 0.1 % TFA MeOH). Flow rate was 0.5 mL/min and the gradient used was 0-1 min. (10-15 % B), 1-15 min. (10-75 % B), 17-37 min. (75-100 % B), 39-39.5 (100-10% B). Data was analyzed using Thermo Chromelon software and % purity was determined relative to % area of the parent peak.

A Novel Amino Lipid Series for mRNA Delivery: Improved ... · A novel amino lipid series for mRNA...

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