Synthesis of PHP-TREN

PHP-TREN co-polymer synthesis

8/6/14

monomer	TREN-1	TREN-2	TREN-3
DBP	184 mg	184 mg	184 mg
DAP	38 mg	38 mg	0
TREN	61 mg	30 mg	61 mg
MeOH	150 ul	150 ul	150 ul
	Form solid rx product after adding TREN	yellowish liquid	form clear crystal, it sticks on the wall, gel-like polymer, not soluble in water
	DNA binding using Sybe-Gold: good	DNA binding using Sybe-Gold: good	DNA binding using Sybe-Gold: MeOH soluble, ok, water phase is gel-like suspension, not binding to DNA

 react at 50 oC for 24 hours

DAP is required for  polymer synthesis

Purify polymer by adding isopropanol to ppt TREN-1 and TREN2. Add water to dissolve pellet

Transfection

O(ornithine)+DBP+AG(agmatine); see page 000726 for synthesis

Before Isopropanol Purification

O(ornithine)+DBP+AG(agmatine)	O+CBA+AG	TREN-1, cells are dead	TREN-2, cells are dead	TREN-3, MeOH	TREN-3, H2O
O+DBP+AG, 1:10 dilution	O+CBA+AG, 1:10 dilution	TREN-1, 1:10 dilution	TREN-2, 1:10 dilution	TREN-3, 1:10 dilution	TREN-3, 1:10 dilution
GuP-1	GuP-2	GuP-3	Stability-1		GFP

Picture of 8-10-14

After Purification

QC: 8-15-14, 1:10	QC: 8-15-14-P, 1:10	TREN-1-P, 1:10, 90% cells are dead	TREN-1-P, 1:30	TREN-2-P, 1:10	TREN-2-P, 1:30
8-15-14, 1:30	8-15-14-P, 1:30	L3K	L2K	TurboFect	ViaFect
8-15-14, 1:50	8-15-14-P, 1:50	GuP-1

Picture of 8-10-14

8/11/14

8/12/14

Synthesis of PHP-TREN

8/16/14

Mix A: 184 mg DBP, 38 mg DAP, 75 ul MeOH

Divide into 3 vials

Mix B: 184 mg DBP, 75 mg DAP, 75 ul MeOH

Divide into 3 vials

Reactions

	A1	A2	A3	B1	B2	B3
DBP	61 mg	61 mg	61 mg	61 mg	61 mg	61 mg
DAP	12.5 mg	12.5 mg	12.5 mg	25 mg	25 mg	25 mg
TREN	10 mg in 75 ul MeOH	20 mg in 75 ul MeOH	30 mg in 75 ul MeOH	10 mg in 75 ul MeOH	20 mg in 75 ul MeOH	30 mg in 75 ul MeOH
Total MeOH	150 ul	150 ul	150 ul	150 ul	150 ul	150 ul
Appearance	yellowish oil	white ppt + yellowish oil	white ppt + yellowish oil	yellow/white gel, white gel is not soluble in H2O	white polymer	white polymer

50 oC react o/n (24 hours)

Reaction Products

Add 1 ml isopropanol to ppt. Dissolve pellet in 500 ul water

dilute in 10 mM HEPES, pH 7.0: 1:30 and 1:10 dilutions

8/18/14 Transfection

use 3 ul 1:10 and 1:30 diluted polymer, 1 ug DNA, 24 well plate

1:10 A1	1:10 A2	1:10 A3	1:10 B1	1:10 B2	1:10 B3
1:30 A1	1:30 A2	1:30 A3	1:30 B1	1:30 B2	1:30 B3
8/6/14 TREN1 1:10	8/6/14 TREN1 1:30	8/6/14 TREN2 1:10	8/6/14 TREN2 1:30	ProteinOne liposome #1 DOTAP-DOPE	ProteinOne liposome #2 DOTAP-DOPE-PEG
L2K

8/19/14

8/20/14

8/22/14

A1, 1:10, 2 ug DNA	A2, 1:10, 2 ug DNA	A3, 1:10, 2 ug DNA	B1, 1:10, 2 ug DNA	B2, 1:10, 2 ug DNA	B3, 1:10, 2 ug DNA
L2K, 2ugDNA

8/19/14

8/20/14

8/22/14

Transfection of Diluted PHP-TREN

8/22/2014:

Transfection, 1ug DNA, 293T

Dilution	1:30	1:50	1:75	1:30	1:50	1:75
	A1	A1	A1	A2	A2	A2
	A3	A3	A3	B1	B1	B1
	B2	B2	B2	B3	B3	B3
	TREN1	TREN1	TREN1	TREN2	TREN2	TREN2

Conclusion: A2 and A3 are the best

Picture 8/23/2014

8/24/14

8/25/14

8/26/14

8/27/14

Scale up synthesis of PHP-TREN

Synthesis

9/6/14

369 mg DAP

1.8 g DBP

+ 1 ml MeOH to dissolve

+ 950 mg TREN (should be 885 mg, 6.5 mmole,  but added too much) + 2.0 ml MeOH

Mix and react at 50 oC o/n, 24 hours

+ 30 mg cystamine --> 60 0C react 3 hours

+ 7 ml isopropanol, mix, spin 5 min-->brownish oil-like bottom phase, remove top phase (isopropanol)

+ 5ml H2O--> 7.5 ml total vol, take out 10 ul for transfection assay

Add 200 mg pyrazole-1-carboxyamidine hydrochloride, react at 25 oC for 2 hours---> take 10 ul for transfection assay

Add 10 ml isopropanol, spin--remove top phase (isopropano).

Transfection

9/8/14

1:30, 1:50, 1:75, 1:100 dilution

3 ul diluted + 1 ug DNA

PHP-TREN, 1:30	GU-PHP-TREN, 1:30	PHP-DSP, 1:30	PHP-TREN, A2, 1:50	L3K
PHP-TREN, 1:50	GU-PHP-TREN, 1:50	PHP-DSP, 1:50	PHP-TREN, A3, 1:50	L2K
PHP-TREN, 1:75	GU-PHP-TREN, 1:75	PHP-DSP, 1:75	PHP-TREN, TREN1, 1:100
PHP-TREN, 1:100	GU-PHP-TREN, 1:100	PHP-DSP, 1:100	PHP-TREN, B1, 1:50

9/9/2014 morning

9/9/2014 afternoon

9/10/14

9/11/14

9/12/14

9/13/14

9/13/14 afternon

9/14/14

9/14/14

GU-PHP_DSP,1:30	GU-PHP-TREN, B1, 1:30	GU-PHP-TREN, A3, 1:30	PHP-TREN, B1, 1:30	L3K	GU-PHP-TDSP, 1:30
GU-PHP_DSP, 1:50	GU-PHP-TREN, B1,   1:50	GU-PHP-TREN, A3,  1:50	PHP-TREN, B1, 1:50	L2K	GU-PHP-TDSP, 1:50
GU-PHP_DSP, 1:75	GU-PHP-TREN, B1, 1:75	GU-PHP-TREN, A3, 1:75	PHP-TREN, B1, 1:75	GU-PHP-TREN, A3, 1:30	GU-PHP-TREN, B1, 1:30
GU-PHP_DSP, 1:100	GU-PHP-TREN, B1, 1:100	GU-PHP-TREN, A3, 1:100	Mix of Gu_PHP-DSP and Gu_PHP-TREN, A3, at 3:1 ratio, 1:30	GU-PHP-TREN, A3, 1:50	GU-PHP-TREN, B1, 1:50

PHP-TREN-B1 Large Scale

Synthesis

10/25/14

1.5 g DAP + 3.6 g DBP + 1.5 ml MeOH, dissolve, feel heat

+ (200 ul TREN + 0.5 ml MeOH)

mix 50 oC react o/n

add 1 ml MeOH--> 60 oC for 3 hours

Purification

10/26/14

+ H2O to 10 ml, + 40 ml isopropanol, spin, repeat once

dissolve in H2O and adjust to 10 ml-->P

Derivatization

P1	PHP-TREN-B1-Cho	25 ul P + 2.5 ul 0.5 mg/ul Cho-Chl
P2	PHP-TREN-B1-PEI	25 ul P + 2.5 ul 0.4 mg/ml PEI + 3.5 ul DSP
P3	PHP-TREN-B1--PEI-Cho	25 ul P + 2.5 ul PEI-Cho + 3.5 ul DSP

Transfection

10/27/14

R: PHP-TREN-A3-Cho-Gu (oil-like)

RP: purify R using isopropanol

Q: PHP-TREN-B1-Cho-Gu, 10-9-14, white pellet

QP: purified Q using isopropanol

Result

10/29/14

10/30/14

cell dead RP, P2, P3

10/31/14

11/1/14