Editing Guanidinylation (section)

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

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

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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> 

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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> 

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[[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>