Guanidinylation

Reagents

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

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

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

1H-Pyrazole-1-carboxamidine hydrochloride, Praxadine

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

402516-10G, $60.00

3,5-Dimethyl-1-pyrazolylformaminidium nitrate

Guanylating reagent for amines

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

S-alkylisothiouronium salts

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

(most active)

Convert Ornithine to Arginine

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

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.

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.

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

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

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

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

1/25/2014

293E

293T

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

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

picture: 1/29/2014

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

picture: 1/29/2014

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

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

2/6/14

2/17/14

Stability Test

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

HBTU

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate 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).