Paul.xu at 22:55, 13 August 2014

2014-08-13T22:55:49Z

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== Reagents ==

=== 1H-1,2,4-Triazole-1-carboxamidine hydrochloride ===

Guanylating agent using water and alcohols as solvents, high yields, mild conditions 

[[Image:1H-1,2,4-Triazole-1-carboxamidine hydrochloride.png]] 

http://www.sigmaaldrich.com/catalog/product/aldrich/76082, 76082-25G, $62.70 

 

=== 1H-Pyrazole-1-carboxamidine hydrochloride, Praxadine ===

[[Image:1H-Pyrazole-1-carboxamidine hydrochloride .png]] 

http://www.sigmaaldrich.com/catalog/product/aldrich/402516 

402516-10G, $60.00 

 

 

=== 3,5-Dimethyl-1-pyrazolylformaminidium nitrate ===

Guanylating reagent for amines 

[[Image:3,5-Dimethyl-1-pyrazolylformaminidium nitrate .png]] 

http://www.sigmaaldrich.com/catalog/product/aldrich/d182257, D182257-25G, $150.00 

 

=== S-alkylisothiouronium salts ===

'''1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea '''[http://www.sigmaaldrich.com/catalog/product/aldrich/439908 www.sigmaaldrich.com/catalog/product/aldrich/439908] 

(most active)

[[Image:1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea.png]]

 

== Convert Ornithine to Arginine ==

[[Image:Ornithine to arginine.png]] 

 

 

== Grafting polymers with Fmoc-Arg(Pbf)-OH ==

Novabiochem or Sigma http://www.sigmaaldrich.com/catalog/product/aldrich/47349, 47349-5G $77.50, 47349-25G, $258.50 

[[Image:Fmoc-Arg(Pbf)-OH .png]] 

 

Amino acid coupling to the PAMAM was per- formed in anhydrous DMF for 4 h at room temper- ature with 4 equivalents of HOBt, HBTU, Fmoc- Arg(pbf)-OH and 7.3 equiv. of DIPEA, respectively. The product was precipitated in ethyl ether and washed with excess ether. The Fmoc groups of Fmoc-Arg(pbf)-coupled dendrimer was removed by adding 30% piperidine in DMF (v/v). After 1 h of deprotection reaction, the mixture was precipitated in ethyl ether and washed with excess ether. The reagent (95:2.5:2.5, trifluoroacetic acid/triisopropylsilane/ H 2 O, v/v) was used for deprotection of pbf groups of arginine (6 h at room temperature) and the final product was precipitated in ethyl ether and washed with excess ether. The product was solubilized in water and dialyzed against pure water at 4 8C for overnight. Then, the product was collected after freeze-drying yielding white powder. The overall synthesis scheme of PAMAM-Lys was the same as described above. Fmoc-Lys(Boc)-OH was used instead and 90% TFA was used finally to deprotect BOC groups (1 h at room temperature). The yields for the products were usually over 99%. 

 

Briefly, to a solution of 4.0 equiv of HOBt, 4.0 equiv of HBTU, 4.0 equiv of Fmoc-Arg(pbf)-OH, and 8.0 equiv of DIPEA (equivalent to one primary amine terminal of the amine- terminated dendrimer) in 5.0 mL of anhydrous DMF was added a solution of 50 mg of amine-terminated TEA-core PAMAM dendrimer in 5.0 mL of DMF. The reaction solution was stirred at 25 °C for 2 days. The resulting product was then precipitated with methanol/diethyl ether (1.0 mL/20 mL) at 4 °C for 12 h (three times). The residual precipitate was dissolved in 3.0 mL of DMF and mixed with a solution of 3.0 mL of 30% piperidine in DMF (v/v), then stirred at 25 °C for 2 h to deprotect the Fmoc groups. The resulting product was then precipitated with methanol/diethyl ether (1.0 mL/20 mL) at 4 °C for 12 h (three times). A solution of 7.6 mL of trifluoroacetic acid (TFA), 2.0 mL of triisopropylsilane (TIS), and 2.0 mL of water (95:2.5:2.5, v/v/v) was used to remove the pbf groups at 25 °C for 1 h. The resulting crude product was purified by the dialysis in water (800−1000 mL) (dialysis protocol: at the beginning, the water was changed every 1 h three times, then every 2−3 h three times). The dialyzed product was lyophilized to yield the corresponding arginine- terminated dendrimer. 

 

[[Image:Arginine grafted dendrimer.png|650px|Arginine grafted dendrimer.png]]

PAMAM G4 was allowed to react with 4 equivalents of Fmoc-Abu-OH (GABA), HOBt, HBTU, and 8 equivalents of DIPEA in anhydrous DMSO for 16 h. After the first reaction, PAMAM G4-GABA-Fmoc was precipitated using cold ether and dried using N 2 gas. Next, 30% (v/v, DMSO) piperidine was used to deprotect the Fmoc groups for 1 h in the dark. Next, PAMAM G4-GABA-NH 2 was further reacted with 4 equi- valents of Fmoc-Arg(pbf)-OH, HOBt, HBTU, and 8 equivalents of DIPEA in anhydrous DMSO for 16 h. The result- ing PAMAM G4-GABA-Arg(pbf)-Fmoc was precipitated, dried, and deprotected in the same manner described above. To deprotect the pbf groups on Arg units, the product was dissolved in deprotection solution (TFA:TIS:H 2 O, 95%: 2.5%:2.5%) and reacted for 6 h. After the deprotection reaction, the final products were precipitated and dried. To separate the final product from the un-reacted molecules, the final product was dialyzed against distilled water using a dialysis membrane (MWCO3500, Spectra/por ® , Spectrum Laboratories, CA, USA) for 2 days. After dialysis, the final product was lyophilized and collected in a microcentrifuge tube. The synthesis scheme is shown in Scheme 1. For struc- ture identification, PAMAM G4 derivatives were analyzed using 400 MHz 1 H NMR spectroscopy 

== Guanidinylation of polymers with 1-H-pyrazole-1-carboxamidine (P) ==

=== GU of PEI25 and PEI 2K: ===

Dissolve PEI (0.010 mol amine residues/L) in 50 mM potassium carbonate aqueous solution. Add pyrazole-1-carboxamidine (5 eq. per amine residue).

Stire  for 20 h

Dialysis (M.W.C.O = 1,000 Da.) against distilled water .

The conversion yield   12% and 19% for PEI 25 kDa and PEI 2 kDa, respectively. 

 

 

[[Image:1H-Pyrazole-1-carboxamidine hydrochloride PEI reaction.jpg]]

0.75 g 1,3DAP + 1.8g 1,3,DBP, 1.5 ml MeOH react 50 oC 1hr, add 10 mg cystamine, react 36 hours at 50 oc 

dissolve in 10 ml H2O 

wash 3 times with 40 ml EtOH, spin down, dssolve in 5 ml H2O, final vol is 5 ml 

+ 0.75 ml PEI/Pyrozale 

+120 mg CBA 

40 oC for 3 hours 

Final is 6 ml liquid 

divide into 2 X3 ml: 

'''Lot 011014P''': add 50 mg 1-H-pyrazole-1-carboxamidine (P) to 3 ml stock 

'''Lot 011014''': did not add 1-H-pyrazole-1-carboxamidine (P) 

'''Conclusion''': Guanidinylation improve transfection efficiency.

293E is more sensitive than 293T, after a few days most 293T cells are dead, while 293T are still alive 

=== Results ===

293E, 1 ug DNA in 50 ul DMEM + 3 ul diluted TR in 50 ul DMEM

{| width="573" cellspacing="1" cellpadding="1" border="1"
|-
| 011014P, 1:25 dilution 
| 011014P, 1:50 dilution 
| 011014P, 1:75 dilution 
| 011014P, 1:100 dilution 
|-
| 011014, 1:25 dilution 
| 011014, 1:50 dilution 
| 011014, 1:75 dilution 
| 011014, 1:100 dilution 
|-
| 12/31/13, 1:25 dilution 
| 12/31/13, 1:50 dilution 
| Promega ViaFect 
| Lipofectamine 2000 
|}

 

[[Image:Tr qc 1 24 14 2.JPG]] 

293T, 1 ug DNA in 50 ul DMEM + 3 ul diluted TR in 50 ul DMEM

{| width="573" cellspacing="1" cellpadding="1" border="1"
|-
| 011014P, 1:25 dilution 
| 011014P, 1:50 dilution 
| 011014P, 1:75 dilution 
| 011014P, 1:100 dilution 
|-
| 011014, 1:25 dilution 
| 011014, 1:50 dilution 
| 011014, 1:75 dilution 
| 011014, 1:100 dilution 
|-
| 12/31/13, 1:25 dilution 
| 12/31/13, 1:50 dilution 
| Promega ViaFect 
| Lipofectamine 2000 
|}

[[Image:TR QC 1 24 14 1.JPG]]

1/25/2014

293E 

[[Image:2014 1 25 TR QC 1.JPG]]

293T 

[[Image:2014 1 25 TR QC 2.JPG]]

 

== Post Guanidinylation ==

1/24/2014:

Transfection: 293T, DNA: 0.5 ug, DMEM: 25 ul, diluted polymer TR: 1.5 ul 

1/26/2014: take picture under blue light 

{| width="693" cellspacing="1" cellpadding="1" border="1"
|-
|
1 ml of Lot 12/31/13 stock + '''10''' mg P, 1:'''50 '''dilution 

| 1 ml of Lot 12/31/13 stock + '''10''' mg P, 1:'''100''' dilution 
| 1 ml of Lot 12/31/13 stock + '''10''' mg P, 1:'''150''' dilution 
|
1 ml of Lot 12/31/13 stock + '''20''' mg P, 1:'''50''' dilution 

| 1 ml of Lot 12/31/13 stock + '''20''' mg P, 1:'''100''' dilution 
| 1 ml of Lot 12/31/13 stock + '''20''' mg P, 1:'''150''' dilution 
|-
|
1 ml of Lot 12/31/13 stock + '''30''' mg P, 1:'''50''' dilution 

| 1 ml of Lot 12/31/13 stock + '''30 '''mg P, 1:'''100''' dilution 
| 1 ml of Lot 12/31/13 stock + '''30 '''mg P, 1:'''150''' dilution 
|
1 ml of Lot 12/31/13 stock + '''40 '''mg P, 1:'''50''' dilution 

| 1 ml of Lot 12/31/13 stock + '''40 '''mg P, 1:'''100''' dilution 
| 1 ml of Lot 12/31/13 stock + '''40''' mg P, 1:'''150''' dilution 
|-
| 1 ml of Lot 2/8/13 stock + 40 mg P, 1:'''50''' dilution 
| 1 ml of Lot 2/8/13 stock + 40 mg P, 1:'''100''' dilution 
| 1 ml of Lot 1/13/13 stock + 40 mg P, 1:'''50''' dilution 
| 1 ml of Lot 1/13/13 stock + 40 mg P, 1:'''100''' dilution 
|
1 ml of Lot 2013-3-1 stock + 40 mg P, 1:'''50''' dilution

will discard 

|
1 ml of Lot 2013-3-1 stock + 40 mg P, 1:'''100''' dilution

will discard 

|-
| 1 ml of Lot '''011014P''' stock + 10 mg P, 1:'''50''' dilution 
| 1 ml of Lot '''011014P''' stock + 10 mg P, 1:'''150''' dilution 
| 1 ml of Lot '''011014P '''stock + 20 mg P, 1:'''50''' dilution 
| 1 ml of Lot '''011014P''' stock + 20 mg P, 1:'''150''' dilution 
| 1 ml of Lot '''011014P''' stock + 30 mg P, 1:'''50 '''dilution 
| 1 ml of Lot '''011014P '''stock + 30 mg P, 1:'''150''' dilution 
|}

[[Image:2014 1 26 gu 3.JPG]]

picture: 1/29/2014 

[[Image:Guanidinylation 1 29 14 1.JPG]] 

 

{| width="540" cellspacing="1" cellpadding="1" border="1"
|-
| 1 ml of Lot 011014 stock + '''10''' mg P, 1:'''50 '''dilution 
| 1 ml of Lot 011014 stock + '''10''' mg P, 1:'''100''' dilution 
| 1 ml of Lot 011014 stock + '''10''' mg P, 1:'''150''' dilution 
| 1 ml of Lot 011014 stock + '''20''' mg P, 1:'''50''' dilution 
| 1 ml of Lot 011014 stock + '''20''' mg P, 1:'''100''' dilution 
| 1 ml of Lot 011014 stock + '''20''' mg P, 1:'''150''' dilution 
|-
| 1 ml of Lot 011014 stock + '''30''' mg P, 1:'''50''' dilution 
| 1 ml of Lot 011014 stock + '''30 '''mg P, 1:'''100''' dilution 
| 1 ml of Lot 011014 stock + '''30 '''mg P, 1:'''150''' dilution 
| Lipofectamine 3000 
| 
| 
|}

[[Image:2014 1 26 gu 1.JPG]]

picture: 1/29/2014 

[[Image:Guanidinylation 1 29 14 2.JPG]]

To make working solution: 

1:50: add 20 ul to 1000 ul 10 mM Hepes-KOH buffer 

1:100: add 10 ul to 1000 ul 10 mM Hepes-KOH buffer 

 

== Comparing other transfection reagents ==

{| width="452" cellspacing="1" cellpadding="1" border="1"
|-
| 011014+30P 1:50, 3 ul, 1 ug DNA 
| L3K 
| L2K 
| Attractant 
|-
| TurboFect 
| Transit 2020 
| Dharmafect Duo 
| Fugene 9 
|-
| ExtremeGene HP 
| PEI MAX 
| LPI25K 
| BPEI25K 
|}

2/5/14 

[[Image:Transfection comp 2 5 2014.JPG]] 

2/6/14

[[Image:Transfection comp 2 6 2014.JPG]] 

2/17/14

[[Image:Transfection comp 2 17 14 2.JPG]] 

 

== Stability Test ==

[[Image:TR comp 2014 2 22 1.JPG]] 

[[Image:TR comp 2014 2 22 2.JPG]] 

{| width="567" cellspacing="1" cellpadding="1" border="1"
|-
| 2013-3-1 
| 011014P+30Gu (form precipitate after freezing-thawing a few times) 
| ViaFect 1.5 ul 
| ViaFect 3.0 ul 
| TurboFect 
| L3K 
|-
| L2K 
| Transit 2020 
| Atractene 
| DaharmaFect Duo 
| PureFectin 
| Transit-TKO 
|-
| 12/31/13, 10mg Gu, 1:50 
| 12/31/13, 20mg Gu, 1:50 
| 12/31/13, 30mg GU, 1:50 
| 12/31/13, 40mg Gu, 1:50 
| 2/8/13, 1:50 
| 1/31/13 1:50
|-
| 2013-3-1 1:50
| 011014P, 10mg Gu, 1:50
| 011014P, 20mg Gu, 1:50
| 011014P, 30mg Gu, 1:50
| 011014, 10mg Gu, 1:50
| 011014, 20mg Gu, 1:50
|-
| 011014, 30mg Gu, 1:50
| Transit 2020 
| 
| 
| 
| 
|}

 

== Guanidinylation using O-methylisourea hemisulfate ==

== HBTU ==

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate [http://www.sigmaaldrich.com/catalog/product/aldrich/12804?lang=en&region=US Sigma 12804-1G-F $31.90]

Recent studies show that crystal as well as solution structure of this coupling agent is a guanidinium N-oxide and not an uronium compound;1 Coupling reagent for peptide synthesis; advantages are: very low racemization, simple reaction conditions, very short reaction time, and high yields

In a solution of bPEI (30mg, dissolved in 5mL dry DMF) and TEA (6.07 𝜇L, 2.5 equivalents), HBTU (6.62mg, 0.17mmol for 10% substitution) was added and the reaction mixture was stirred for 12 h at ambient temperature. Then the reaction mixture was poured in a dialysis bag (12 kDa cut-off) and dialyzed against 1M HCl solution for 24 h followed by against 1M NaCl (pH 8.5) solution for 24 h and deionized water for 48 h with intermittent change of respective solutions/water. Subsequently, the dialyzed solution was lyophilized to obtain TP1 as white solid in ∼78% yield. Similarly, other modified polymers, namely, TP2, TP3, TP4, and TP5, were prepared by varying the amounts of HBTU in 75–83% yield. These modified polymers were then characterized by 1H-NMR. TP5: 1H-NMR (D2O) 𝛿 (ppm): 2.4–2.65 (m, −NCH2 −, PEI) 2.75 (br s, −NCH3, TMG).

[[Image:PEI gu HBTU reaction.jpg|650px|PEI gu HBTU reaction.jpg]]

[[Image:PEI gu HBTU.jpg|650px|PEI gu HBTU.jpg]]

[[Category:Transfection]] [[Category:Paul]]

Guanidinylation - Revision history

Paul.xu at 22:55, 13 August 2014