Editing Guanidinylation

== Reagents<br>  ==

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

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

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

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

<br> 

=== 1H-Pyrazole-1-carboxamidine hydrochloride, Praxadine <br>  ===

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

http://www.sigmaaldrich.com/catalog/product/aldrich/402516<br> 

402516-10G, $60.00<br> 

<br> 

<br> 

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

Guanylating reagent for amines<br> 

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

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

<br> 

=== S-alkylisothiouronium salts <br> ===

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

(most active)

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

<br>

== Convert Ornithine to Arginine<br>  ==

[[Image:Ornithine to arginine.png]]<br> 

<br> 

<br> 

== Grafting polymers with Fmoc-Arg(Pbf)-OH <br>  ==

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

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

<br> 

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

<br> 

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

<br> 

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

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

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

=== GU of PEI25 and PEI 2K:<br>  ===

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

Stire&nbsp; for 20 h 

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

The conversion yield &nbsp; 12% and 19% for PEI 25 kDa and PEI 2 kDa, respectively.<br> 

<br> 

<br> 

[[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<br> 

dissolve in 10 ml H2O<br> 

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

+ 0.75 ml PEI/Pyrozale<br> 

+120 mg CBA<br> 

40 oC for 3 hours<br> 

Final is 6 ml liquid<br> 

divide into 2 X3 ml:<br> 

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

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

'''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<br> 

=== Results<br>  ===

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<br> 
| 011014P, 1:50 dilution<br> 
| 011014P, 1:75 dilution<br> 
| 011014P, 1:100 dilution<br>
|-
| 011014, 1:25 dilution<br> 
| 011014, 1:50 dilution<br> 
| 011014, 1:75 dilution<br> 
| 011014, 1:100 dilution<br>
|-
| 12/31/13, 1:25 dilution<br> 
| 12/31/13, 1:50 dilution<br> 
| Promega ViaFect<br> 
| Lipofectamine 2000<br>
|}

<br> 

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

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<br> 
| 011014P, 1:50 dilution<br> 
| 011014P, 1:75 dilution<br> 
| 011014P, 1:100 dilution<br>
|-
| 011014, 1:25 dilution<br> 
| 011014, 1:50 dilution<br> 
| 011014, 1:75 dilution<br> 
| 011014, 1:100 dilution<br>
|-
| 12/31/13, 1:25 dilution<br> 
| 12/31/13, 1:50 dilution<br> 
| Promega ViaFect<br> 
| Lipofectamine 2000<br>
|}

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

1/25/2014 

293E<br> 

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

293T<br> 

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

<br> 

== Post Guanidinylation<br>  ==

1/24/2014: 

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

1/26/2014: take picture under blue light<br> 

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

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

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

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

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

will discard<br> 

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

will discard<br> 

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

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

picture: 1/29/2014<br> 

[[Image:Guanidinylation 1 29 14 1.JPG]]<br> 

<br> 

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

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

picture: 1/29/2014<br> 

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

To make working solution:<br> 

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

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

<br> 

== Comparing other transfection reagents<br>  ==

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

2/5/14<br> 

[[Image:Transfection comp 2 5 2014.JPG]]<br> 

2/6/14 

[[Image:Transfection comp 2 6 2014.JPG]]<br> 

2/17/14 

[[Image:Transfection comp 2 17 14 2.JPG]]<br> 

<br> 

== Stability Test<br>  ==

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

[[Image:TR comp 2014 2 22 2.JPG]]<br> 

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

<br> 

== Guanidinylation using&nbsp;O-methylisourea hemisulfate  ==

== HBTU  ==

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate&nbsp;<span style="line-height: 1.5em;">[http://www.sigmaaldrich.com/catalog/product/aldrich/12804?lang=en&region=US Sigma 12804-1G-F $31.90]</span> 

<span style="line-height: 1.5em;">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</span> 

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