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Draft

Improved Large Scale Synthesis and Characterization of

Small and Medium Generation PAMAM-dendrimers

Journal: Canadian Journal of Chemistry

Manuscript ID cjc-2017-0108.R2

Manuscript Type: Article

Date Submitted by the Author: 11-Apr-2017

Complete List of Authors: Ficker, Mario; University of Copenhagen, Dept. of Chemistry Paolucci, Valentina; University of Copenhagen, Dept. of Chemistry Christensen, Jørn; University of Copenhagen, Dept. of Chemistry;

Is the invited manuscript for consideration in a Special

Issue?: Dendimers

Keyword: PAMAM, dendrimer synthesis, up-scaling, polymer characterization

https://mc06.manuscriptcentral.com/cjc-pubs

Canadian Journal of Chemistry

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Improved Large Scale Synthesis and Characterization of Small and 1

Medium Generation PAMAM-dendrimers 2

3

Mario Ficker, Valentina Paolucci, Jørn B. Christensen* 4

Department of Chemistry, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg, DK-1871 5

Denmark 6

E-mail: [email protected] 7

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Abstract 1

Dendrimers are promising polymers for biomedical applications; however, most dendrimer formulations 2

have failed to move from laboratory science to up-scaled products for pre-clinical testing or GMP 3

production. This publications reports on an improved large scale PAMAM dendrimer synthesis that is 4

suitable to manufacture large amounts of highly pure and monodisperse dendrimers of generations G0 to G5. 5

Furthermore, an extended analytical guideline how to characterize PAMAM dendrimers with NMR, HPLC, 6

SEC-MALS, ESI, MALDI, UV-Vis, Fluorescence and IR spectroscopy is provided. 7

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Key words 10

PAMAM, dendrimer synthesis, up-scaling, polymer characterization 11

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Graphical Abstract 1

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Introduction 1

Dendrimers represent a highly interesting polymer class in the scientific community due to their 2

well-established properties in biomedical application.1-2 However, they have not fulfilled all the great 3

expectations that have fueled so much of the work done over the years.3 Many promising dendrimers 4

formulations have been developed such as transfection agents,4-5 anti-cancer drugs,6-7 and imaging agents.8-10 5

Nevertheless, the only dendrimer to our knowledge that has achieved clinical approval is lysine dendrimer 6

based Vivagel® by Starpharma.11 This raises the question, how Starpharma has achieved to push this 7

dendrimer family to the market and regarding the reasons behind the poor clinical translation of other 8

dendrimer families. In our opinion, from the biological point of view many dendrimer formulations have 9

proven to work, however, the main challenge relies on preserving purity and mono-dispersity when 10

dendrimers are up-scaled and manufactured for preclinical and clinical testing.2 Thanks to commercial GMP 11

partners and investors on board, Starpharma had the unique chance to preserve the properties found in 12

laboratory scale dendrimer systems through upscaling, pre-clinical and clinical testing. However, due to the 13

commercial character of Starpharma’s business model, none of the synthetic achievements how to GMP 14

manufacture and up-scale lysine dendrimers are publically available. 15

We currently encountered a similar challenge, being involved in a large scale EU-project in 16

nanomedicine,12 we had to synthesize large amounts of poly-amido-amine (PAMAM) dendrimers for a pre-17

clinical study with the ultimate goal to transfer the dendrimer synthesis into a GMP production. After a 18

careful analysis of the requirements with respect to amounts, purity, batch to batch reproducibility and 19

availability; we decided to synthesize the required PAMAM-dendrimers in-house. This allowed us to control 20

every step of the synthesis and achieve excellent quality of the final products. 21

In this paper, we describe our findings with respect to improved reaction conditions and purification 22

of half- and full generation PAMAM-dendrimers in order to overcome the critical limitations of this potential 23

class of polymers. In addition, we report on a detailed guidance of methods and techniques which can be 24

applied to fully characterize these dendritic polymers. 25

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Experimental 1

General 2

Standard laboratory glassware and plastic apparatus was used. Unless otherwise stated, all starting 3

materials were obtained from Sigma Aldrich and used as received. Solvents were HPLC grade and used as 4

received. Thin-layer chromatography was carried out using silica plates on aluminum (Merck, Silica 60-F254, 5

0.2 mm layer thickness) with detection under UV light and, if required treatment with 1 % solution of 6

ninhydrin in ethanol. UV/Vis spectra were recorded on a Perkin Elmer apparatus using 1 cm quartz cuvettes. 7

1H-NMR and 13C-NMR spectra were performed on a 300 MHz NMR (Bruker) apparatus (300 MHz 1H-8

NMR, 75 MHz 13C-NMR) or on a 500 MHz NMR (Bruker) apparatus (500 MHz 1H-NMR, 125 MHz 13C-9

NMR). Chemical shifts are reported in parts per million (ppm) downfield of TMS (tetramethylsilane) using 10

the resonance of the deuterated solvent as internal standard. Proton couplings are described as s (singlet), d 11

(doublet), t (triplet), q (quartet), br (broad) and m (multiplet), coupling constants are reported in Hertz. 12

A Jasco V-650 spectrophotometer (Jasco, Japan) and a Jasco Model FP-6200 spectrofluorometer 13

(Jasco, Japan) were used to collect absorption and emission spectra, respectively. All the spectra were 14

measured using 1 cm path quartz cuvettes and Milli-Q as solvent. The concentration of all PAMAM 15

dendrimers solutions was 0.4 mM. 16

A size-exclusion chromatography system (HPLC: Dionex Ultimate® 3000, Column: TSKgel® 17

GMPWXL HPLC Column) coupled with a multiangle light scattering (miniDAWN TREOS-AQUEOUS) 18

and refractive index (RI Detector Refractomax 521) detectors were used to acquire the RI and LS signals. 19

The number and weight average molecular mass values were obtained using ASTRA software. The SEC-20

MALS-RI system and ASTRA software were purchased from Wyatt technology Europe (Dernbach, 21

Germany), while the column was from Sigma Aldrich (Denmark). Citrate buffer (pH 2.9) was used as 22

solvent to collect SEC-MALS chromatograms. A 50 µL volume of sample solution prefiltered with 0.2 µm 23

Acrodisc ® Syringe Filter (Supor ® Membrane) was injected into the system with a flow rate of 0.5 mL/min. 24

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Batch measurements were performed using five solutions prepared by dilution in PBS (pH 7.4) within 1

concentration range 0.1 – 1.5 mg/mL. The solutions were prefiltered with 0.2 µm Acrodisc ® Syringe Filter 2

(Supor ® Membrane) and manually injected into the MALS detector until a plateux was reached. 3

MALDI was measured on a Bruker SolarisX XR instrument in positive mode with a SA matrix. 4

Sinaptic acid saturated in ethanol was pre-spotted (0.5 µL) on a MALDI plate, this was followed by spotting 5

approximately 1 mg of dendrimer dissolved in a mixture of water/MeCN/TFA (39.9/60/0.1) saturated with 6

synaptic acid. 7

For HPLC-MS analysis a Dionex Ultimate 300 PLC connected to an ESI-MS (MSQ Plus Mass 8

Spectrometer, Dionex) was used, measurements were performed on a Phenomenex Kinetex 5µm C4 100 Å 9

column (50 x 2.1 mm), the column was thermostated to 42 °C. As eluent system water and acetonitrile 10

(containing 0.1 % (v/v) TFA) was used, starting with 5 % acetonitrile until 0.5 min, then gradient from 5 % 11

to 35 % for 0.7 min, then gradient 35 % to 100 % over 3 min, finally plateau of 100 % acetonitrile. The flow 12

rate was set to 0.5 mL/min (300 bar). Absorption was followed using 215 nm as wavelength. The injection 13

volume was 2 µl, containing approximately 5 mg/mL dissolved in 5 % acetonitrile and 95 % water. 14

IR spectra were recorded on a Bruker FT-IR instrument using the attenuated total reflectance (ATR) 15

sampling technique, and the measurements were carried out on a thin film of each sample obtained by 16

evaporation from a solution of methanol. 17

18

Synthesis of Poly(amidoamine) (PAMAM) Dendrimers 19

20

Core: DAB-PAMAM-(CO2Me)4 [1] 21

Under nitrogen atmosphere, methyl acrylate (166 g, 1.93 mol) dissolved in methanol (100 mL) was 22

cooled to 0 °C, using an ice bath, and 1,4-Diaminobutane (15.15 g, 0.172 mol) dissolved in methanol (75 23

mL) was added dropwise to the acrylate solution over 1 h. The ice bath was kept for another 3 h and the 24

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reaction was then stirred overnight at room temperature and full conversion was checked by TLC. The 1

excess methyl acrylate and methanol were removed using a rotary evaporator. The final product was 2

obtained as colorless oil with a yield of 97% (71.55 g, 0.167 mol). 3

MW: 432.5 - 1H-NMR: (500 MHz, CDCl3) [ppm]: δ = 1.33-1.45 (m, 4 H); 2.35-2.41 (m, 4 H); 2.43 4

(t, 8 H,3J=7.2 Hz); 2.75 (t, 8 H,3J=7.2 Hz); 3.66 (s, 12 H) - 13C-NMR: (125 MHz, CDCl3) [ppm]: δ = 24.83; 5

32.51; 49.20; 51.49; 53.61; 173.05 - MS: MALDI-TOF: m/ z calc. = 433.247 [M+H]+; m/ z found = 433.254 6

[M+H]+. 7

G0: DAB-PAMAM-(NH2)4 [2] 8

DAB-PAMAM-Core (71.55 g, 0.167 mol) was dissolved in methanol (850 mL). Ethylene diamine (524 9

g, 8.7 mol, 13 eq. per surface group) was dissolved in methanol (110 mL) and cooled to 0 °C, using an ice 10

bath. The dendrimer solution was added to this solution dropwise over 1 h under nitrogen atmosphere. The 11

ice bath was kept for another 3 h and the reaction was then stirred for 4 days at room temperature. Workup 12

was done by removing as much methanol and EDA as possibly by applying vacuum. After that, azeotropic 13

distillation with a mixture of methanol/toluene 1/9 was performed until all EDA had been removed in 14

vacuum. The excess toluene was removed by azeotropic distillation with methanol. The final compound was 15

obtained as colorless oil in a quantitative yield (91.5 g, 0.167 mol). 16

MW: 544.7 - 1H-NMR: (500 MHz, MeOD-d4) [ppm]: δ = 1.30-1.36 (m, 4 H); 2.26 (t, 8 H,3J=6.8 Hz); 17

2.34-2.43 (m, 4 H); 2.62 (t, 8 H,3J=6.4 Hz); 2.65 (t, 8 H,3J=6.8 Hz); 3.14 (t, 8 H,3J=6.4 Hz) - 13C-NMR: 18

(125 MHz, MeOD-d4) [ppm]: δ = 25.84; 34.59; 42.07; 43.05; 50.86; 54.45; 175.32 - MS: MALDI-TOF: m/ z 19

calc. = 567.425 [M+Na]+; m/ z found = 567.425 [M+Na]+; SEC-MALS: Elution time = 19.55 min - UV/Vis: 20

ε215nm = 3560 M-1cm-1, ε280nm = 30 M-1cm-1- Fluorescence:

λexc = 360 nm, λem,max = 470 nm - FT-IR: ��[cm-21

1]= 3263 (m, νstrech(N-H Amide); 3076 (w, νstrech(C-H); 2934 (m, νstrech(C-H); 2862 (m, νstrech(C-H); 1629 (s, 22

νstrech(C=O Amide); 1545 (s, νbending(N-H Amide); 1463 (s); 1432 (s); 1314 (s); 1232 (m); 1191 (s); 1116 (m); 23

1027 (w); 945 (w); 818 (m). 24

General Synthesis of DAB-PAMAM Dendrimer of Generation G 0.5 to G5 25

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The PAMAM dendrimer synthesis consists of a repeating series of two reactions, first a branching of 1

the amine unit to two branches using a Michael addition of two methyl acrylate molecules per amine, this 2

leads to the half generation dendrimers G0.5; G1.5; G2.5; G3.5 and G4.5. This step is always followed by an 3

“activation” step where ethylene diamine (EDA) forms an amide, substituting the outer methyl esters, which 4

yields to new primary amines as the outer dendrimer layer (full generation dendrimers G1; G2; G3; G4 and 5

G5). 6

General Synthesis of Half Generation Dendrimers: 7

Methyl acrylate (3 eq. per dendrimer amine surface group) was dissolved in methanol (typical same 8

volume as the methyl acrylate) and cooled with an ice bath to 0 °C. Under nitrogen atmosphere, DAB-9

PAMAM-Gn (1 eq.) dissolved in methanol (10 w/w %) was added dropwise over 1 h. The ice bath was kept 10

for another 3 h and the reaction was afterwards stirred for two days at room temperature. Full conversion was 11

checked by KAISER test (1 % ninhydrin in ethanol) for remaining amines. The solvent and excess methyl 12

acrylate was removed on a rotary evaporator, followed by high vacuum. This gave the half generation 13

dendrimer in form of a slight yellowish oil. 14

15

General Synthesis of Full Generation Dendrimers: 16

DAB-PAMAM-Half-Generation (1 eq.) was dissolved in methanol (10 w/w %). Ethylenediamine (25 17

eq. per ester surface group) was dissolved in methanol (typical 25% of the ethylenediamine volume) and 18

cooled to 0 °C, using an ice bath. The dendrimer solution was added to this solution dropwise over 1 h under 19

nitrogen atmosphere. The ice bath was kept for another 3 h and the reaction was afterwards stirred for 4 days 20

at room temperature. Workup was done by removing as much methanol and EDA as possibly by applying 21

vacuum. After that, azeotropic distillation with a mixture of methanol/toluene 1/9 was performed until all 22

EDA was removed. The excess toluene was removed by azeotropic distillation with methanol. Dendrimers of 23

generations G2-5 were further purified by dialysis against first water or methanol. The full generation 24

dendrimer was typically obtained in form of white foam. 25

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G0.5: PAMAM(CO2Me)8 [3] 1

Reaction Scale: 20.5 mmol - MW: 1233.5 - 1H-NMR: (500 MHz, MeOD-d4) [ppm]: δ = 1.46-1.54 (m, 2

4 H); 2.40 (t, 8 H,3J=6.7 Hz); 2.49 (t, 16 H,3J=6.7 Hz); 2.50-2.55 (m, 4 H); 2.58 (t, 8 H,3J=6.4 Hz); 2.79 (t, 3

24 H,3J=6.7 Hz); 3.28 (t, 8 H,3J=6.4 Hz); 3.69 (s, 24 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 4

25.82; 33.61; 34.45; 38.46; 50.52; 50.81; 52.21; 53.80; 54.39; 174.75; 174.77 - MS: MALDI-TOF: m/ z 5

calc. = 1233.719 [M+H]+; m/ z found = 1233.719 [M+H]+- Yield: 25.1g, 20.3 mmol, 99%. 6

G1: PAMAM(NH2)8 [4] 7

Reaction Scale: 12.0 mmol - MW: 1457.9 - 1H-NMR: (500 MHz, MeOD-d4) [ppm]: δ = 1.46-1.53 8

(m, 4 H); 2.39 (t, 24 H,3J=6.7 Hz); 2.48-2.53 (m, 4 H); 2.60 (t, 8 H,3J=6.7 Hz); 2.75 (t, 16 H,3J=6.3 Hz); 2.82 9

(t, 24 H,3J=6.8 Hz); 3.27 (t, 24 H,3J=6.3 Hz) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 25.81; 34.37; 10

34.83; 38.60; 42.07; 43.07; 50.83; 51.19; 53.55; 54.39; 174.85, 175.20 - MS: ESI-MS: m/ z calc. = 1458.059 11

[M+H]+; m/ z found = 1458.071 [M+H]+; SEC-MALS: Elution time = 19.05 min - UV/Vis: ε215nm = 7930 M-12

1cm-1, ε280nm= 90 M-1cm-1 - Fluorescence: λexc = 360 nm, λem,max = 430 nm - FT-IR: ��[cm-1]= 3284 (m, 13

νstrech(N-H Amide); 3072 (w, νstrech(C-H); 2936 (m, νstrech(C-H); 2863 (m, νstrech(C-H); 1639 (s, νstrech(C=O 14

Amide); 1552 (s, νbending(N-H Amide); 1463 (s); 1360 (s); 1232 (m); 1198 (s); 1154 (m); 1127 (m); 1041 (w); 15

952 (w) - Yield: 16.7 g, 11.4 mmol, 95%. 16

G1.5: PAMAM(CO2Me)16 [5] 17


(m, 4 H); 2.41 (t, 24 H,3J=6.8 Hz); 2.49 (t, 32 H,3J=6.7 Hz); 2.49-2.53 (m, 4 H); 2.58 (t, 16 H,3J=6.5 Hz); 19

2.63 (t, 16 H,3J=6.5 Hz); 2.79 (t, 32 H,3J=6.7 Hz); 2.85 (t, 24 H,3J=6.8 Hz); 3.28 (t, 24 H,3J=6.5 Hz); 3.69 (s, 20

48 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 25.84; 33.62; 34.44; 34.78; 38.53; 38.65; 50.53; 50.85; 21

51.10; 52.24; 53.53; 53.83; 54.34; 174.68, 174.71; 174.77 - MS: MALDI-TOF: m/ z calc. = 1418.329 22

[M+2H]2+; m/ z found = 1418.331 [M+2H]2+- Yield: 32.5 g, 11.4 mmol, 99%. 23

G2: PAMAM(NH2)16 [6] 24

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4 H); 2.27 (t, 56 H,3J=6.9 Hz); 2.37-2.43 (m, 4 H); 2.48 (t, 24 H,3J=6.6 Hz); 2.63 (t, 32 H,3J=6.3 Hz); 2.70 (t, 2

56 H,3J=6.9 Hz); 3.15 (t, 56 H,3J=6.3 Hz) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 25.86; 34.47; 3

34.84; 36.63; 38.67; 39.98; 42.10; 43.09; 50.86; 51.18; 51.32; 53.56; 54.43; 174.75; 175.17 - MS: ESI-MS: 4

m/ z calc. = 1642.669 [M+2H]2+; m/ z found = 1642.713 [M+2H]2+; SEC-MALS: Elution time= 18.45 min, 5

Mw= 2,6 ± 0,3 kDa - UV/Vis: ε215nm = 8490 M-1cm-1, ε280nm= 190 M-1cm-1 - Fluorescence: λexc = 360 nm, 6

λem,max = 410 nm - FT-IR: ��[cm-1]= 3280 (m, νstrech(N-H Amide); 3076 (w, νstrech(C-H); 2935 (m, νstrech(C-H); 7

2822 (m, νstrech(C-H); 1633 (s, νstrech(C=O Amide); 1543 (s, νbending(N-H Amide); 1461 (s); 1434 (s); 1357 (s); 8

1248 (m); 1198 (s); 1151 (m); 1030 (w); 949 (w) - Yield: 12.08 g, 3.7 mmol, 62 %. 9

G2.5: PAMAM(CO2Me)32 [7] 10


4 H); 2.41 (t, 56 H,3J=6.7 Hz); 2.49 (t, 64 H,3J=6.7 Hz); 2.50-2.54 (m, 4 H); 2.58 (t, 32 H,3J=6.4 Hz); 2.61-12

2.67 (m, 24 H); 2.79 (t, 64 H, 3J=6.7 Hz); 2.85 (t, 56 H, 3J=6.8 Hz); 3.28 (t, 56 H,3J=6.4 Hz); 3.69 (s, 96 H) - 13

13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 25.87; 33.63; 34.47; 34.09; 34.89; 38.54; 38.68; 39.88; 50.55; 14

51.09; 51.17; 51.24; 52.26; 53.55; 53.84; 54.51; 174.6; 174.75 - Yield: 21.5 g, 3.6 mmol, 95%. 15

G3: PAMAM(NH2)32 [8] 16

Reaction Scale: 3.54 mmol - MW: 6936.9 - 1H-NMR:(500 MHz, MeOD-d4) [ppm]: δ = 1.32-1.43 (m, 17

4 H); 2.27 (t, 120 H,3J=6.4 Hz); 2.37-2.43 (m, 4 H); 2.48-2.55(m, 60 H); 2.62-2.65 (m, 64 H); 2.65-2.73 (m, 18

120 H); 3.14-3.19 (m, 120 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 25.94; 34.84; 36.62; 38.67; 19

40.00; 42.07; 42.99; 51.18, 53.56; 54.71; 174.74; 175.18 - MS: ESI: m/ z calc. = 1388.2 [M+5H]5+; m/ z 20

found = 1388.3 [M+5H]5+ SEC-MALS: Elution time = 18.10 min, Mw = 6,7 ± 2,1 kDa - UV/Vis: 21

ε215nm = 8820 M-1cm-1, ε280nm= 320 M-1cm-1 - Fluorescence: λexc = 360 nm, λem,max = - 410 nm - FT-IR: 22

��[cm-1]= 3273 (m, νstrech(N-H Amide); 3081 (w, νstrech(C-H); 2938 (m, νstrech(C-H); 2828 (m, νstrech(C-H); 1630 23

(s, νstrech(C=O Amide); 1545 (s, νbending(N-H Amide); 1462 (s); 1435 (s); 1356 (s); 1248 (m); 1199 (s); 1152 24

(m); 1027 (w); 950 (w) - Yield: 19.6 g, 2.83 mmol, 80%. 25

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G3.5: PAMAM(CO2Me)64 [9] 1


(m, 4 H); 2.38 (t, 120 H,3J=6.8 Hz); 2.47 (t, 128 H,3J=6.8 Hz); 2.53-2.65 (m, 120 H,); 2.74-2.88 (m, 248 H); 3

3.26 (t, 120 H,3J=6.8 Hz); 3.67 (s, 192 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 33.61; 34.77; 4

38.31; 38.64; 51.07; 52.24; 53.54; 53.82; 174.57; 174.67- Yield: 16.4 g, 1.32 mmol, 97%. 5

G4: PAMAM(NH2)64 [10] 6

Reaction Scale: 1.31 mmol - MW: 14242.5 - 1H-NMR:(500 MHz, MeOD-d4) [ppm]: δ = 1.40-1.51 7

(m, 4 H); 2.37 (t, 240 H,3J=6.4 Hz); 2.53-2.65(m, 128 H); 2.69-2.78 (m, 120 H); 2.79-2.91 (m, 248 H); 3.22-8

3.31 (m, 248 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 34.85; 38.66; 42.10; 43.04; 51.19, 53.57; 9

174.69; 175.12 - MS: SEC-MALS: Elution time = 17.85 min, Mw = 15,4 ± 1,3 kDa - UV/Vis: ε215nm = 8790 10

M-1cm-1, ε280nm= 330 M-1cm-1 - Fluorescence:

λexc = 360 nm, λem,max = 420 nm - FT-IR: ��[cm-1]= 3276 (m, 11

νstrech(N-H Amide); 3068 (w, νstrech(C-H); 2938 (m, νstrech(C-H); 2830 (m, νstrech(C-H); 1633 (s, νstrech(C=O 12

Amide); 1544 (s, νbending(N-H Amide); 1462 (s); 1433 (s); 1319 (s); 1198 (s); 1153 (m); 1036 (w) - Yield: 13

13.1 g, 0.92 mmol, 70%. 14

G4.5: PAMAM(CO2Me)128 [11] 15


(m, 4 H); 2.39 (t, 248 H,3J=6.8 Hz); 2.47 (t, 256 H,3J=6.8 Hz); 2.54-2.69 (m, 248 H,); 2.74-2.92 (m, 496 H); 17

3.22-3.331 (m, 248 H); 3.67 (s, 384 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 33.64; 34.78; 38.54; 18

38.67; 50.55; 51.09; 52.29; 53.57; 53.85; 174.59; 174.70 - Yield: 11.9 g, 0.47 mmol, 99%. 19

G5: PAMAM(NH2)128 [12] 20

Reaction Scale: 0.46 mmol - MW: 28853.1 - 1H-NMR:(500 MHz, MeOD-d4) [ppm]: δ = 1.42-1.52 (m, 21

4 H); 2.37 (t, 504 H,3J=6.4 Hz); 2.54-2.65(m, 248 H); 2.71-2.75 (m, 256 H); 2.76-2.94 (m, 504 H); 3.24-3.33 22

(m, 504 H) - 13C-NMR: (125 MHz, MeOD-d4) [ppm]: δ = 34.85; 38.66; 42.10; 43.04; 51.19, 53.57; 174.69; 23

175.12 - MS: SEC-MALS: Elution time = 17.45 min, Mw = 29,0 ± 5,2 - UV/Vis: ε215nm = 10330 M-1cm-1, 24

ε280nm= 4680 M-1cm-1 - Fluorescence: λexc = 360 nm, λem,max = 420nm - FT-IR: ��[cm-1]= 3275 (m, νstrech(N-H 25

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Amide); 3076 (w, νstrech(C-H); 2935 (m, νstrech(C-H); 2830 (m, νstrech(C-H); 1631 (s, νstrech(C=O Amide); 1545 1

(s, νbending(N-H Amide); 1463 (s); 1321 (s); 1200 (s); 1153 (m); 1036 (w) - Yield: 8.3 g, 0.28 mmol, 61%. 2

3

Results 4

Dendrimer Synthesis 5

PAMAM-dendrimers were invented by Donald Tomalia at Dow Chemicals and they were also one of 6

the first dendrimer families to become commercially available, which have made them the most extensively 7

investigated family of dendrimers.13 The divergent synthesis of PAMAM-dendrimers is deceptively simple; 8

basically only involving two different reactions that are repeated until the desired size dendrimer has been 9

reached (Scheme 1). The first step is branching of the primary amine unit to two branches using a Michael 10

addition of two methyl acrylate molecules per amine. This is followed by an “activation” step where ethylene 11

diamine (EDA) substituted the outer methyl esters to an amide, which leads to a new layer of primary amines 12

on the dendrimer surface (Figure 1).14-16 13

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1

2 3

Scheme 1: Synthesis of PAMAM dendrimers by alternating reactions with methyl acrylate and ethylene 4

diamine to build up the typical shell-like dendrimer structure. 5

6

The synthesis of PAMAM dendrimers appears easy and straight forward, but some mistakes and errors 7

need to be avoided to easily manufacture high amounts of good quality PAMAM dendrimers. 8

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1

2

Figure 1: Typical defects in PAMAM dendrimers due to intra-branch cyclization (A), incomplete amidation 3

(B), remaining EDA which can build up trailing dendrimers (C), and retro MICHAEL addition under heat 4

influence (D). 5

6

As depicted in Figure 1; there are four main types of side reactions that influence the purity of PAMAM-7

dendrimers synthesized by the divergent method; A: formation of cyclic amides, which prevent the growth of 8

the affected dendritic branch in the upcoming reaction steps – B) partial surface functionalization, which can 9

lead to missing dendrimer branches and also oligomers due to the reaction with neighboring dendrimers – C) 10

trailing generations, which result from residual 1,2-ethylenediamine/methyl acrylate, that have not been 11

properly removed by dendrimer purification – D) defect structures formed by retro-Michael addition due to 12

overheating the dendrimer during removal of solvents, reagents or drying. 13

Purification of type A, B and D damaged molecules by chromatography is not realistic on large scale 14

synthesis because most of these defected structures are very similar in size and properties to the main product 15

requiring preparative HPLC, which makes the best option to optimize the reaction conditions as much as 16

possible to minimize the level of impurities. Trailing dendrimer generations (defect type C) are the only 17

mistakes than can realistically be removed by methods like size exclusion chromatography or dialysis, since 18

they usually have a weight and size difference to the desired dendrimer by a factor of two. 19

In order to avoid most of the above mentioned mistakes, we report on an accurate description of large 20

scale PAMAM dendrimers synthesis providing valuable details on reaction and purification conditions. 21

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The PAMAM dendrimer synthesis starts from a diamine core. Ethylene diamine was historically used 1

as dendrimer core.13-14, 17 However, in our experience EDA-core dendrimers are prone to cyclisation reactions 2

in the first dendrimer steps, where a seven membered ring system is formed. In order to overcome this 3

obstacle, we recommend to use 1,4-diamino butane (DAB) or even longer carbon chains, where cyclisation 4

would lead to the kinetically unflavored formation of nine (in case of DAB) or even larger membered ring 5

systems (Figure 2). This phenomena seems also to be the reason wherefore most commercial dendrimers are 6

nowadays offered with 1,4-diamino butane core. The dendrimer core can still be considered a small 7

molecule, which has the advantage that the reaction can be monitored by TLC and silica column 8

chromatography is an option if impurities are detected. Nonetheless, in our experience if high grade 1,4-9

diaminobutane is used and methyl acrylate is freshly distilled prior to the reaction chromatographic 10

purification is not necessary. 11

12

Figure 2: EDA dendrimer cores can promote cyclization reactions in the first synthesis step, which can 13

be overcome by using larger diamines that correspond to unfavored ring sizes. 14

As a rule of thumb we used three equivalents of methyl acrylate per amine on the dendrimer surface. In 15

case of the dendrimer core, this resulted in six equivalents of methyl acrylate to form four new dendrimer 16

branches. If one looks carefully at the two dendrimer reaction steps, it appears that the amines of EDA can 17

perform a clean amidation of the dendrimer surface ester, while in the methyl acrylate reaction, only the 18

Michael receptor reacts and the methyl esters of methyl acrylate do not react with the core amines. This is a 19

potential problem in the dendrimer synthesis and it needs to be addressed carefully. 20

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The Michael addition is thermodynamically and kinetically favored compared to the competitive 1

amidation side reaction. In order to overcome this theoretical problem and to promote specifically the 2

Michael type reaction, we ran the reaction at low temperatures (0 °C). The dendrimer core, dissolved in 3

methanol, was added to a reaction mixture of methyl acrylate (six equivalents, freshly distilled) in methanol. 4

The reaction flask was cooled during the whole addition with a water/ice bath. The addition rate was 5

dropwise, which resulted in an addition time of at least one hour, for larger scale reactions multiple hour 6

addition times were applied. In this way the added diamine core experienced a high excess of methyl acrylate 7

and oligomer formation was limited, as demonstrated in the analytical section. After complete addition, the 8

reaction was cooled at 0 °C for another three hours; this was followed by stirring for two days at ambient 9

temperature (22 °C) to assure a quantitative reaction of all dendrimer surface groups. The reaction time could 10

be shortened when the reaction was monitored by TLC (Ninhydrine Kaiser18 test for amines). Workup was 11

achieved by removal of all volatiles (methanol and methyl acrylate) on a rotary evaporator. To avoid retro 12

Michael reaction (error type D), the water bath temperature of the evaporator did not exceed 30 °C. This was 13

followed by drying the material in a high vacuum system (less than 0.1 mbar) for 24-48 hours. The complete 14

removal of methyl acrylate from the dendrimer core was investigated using NMR and HPLC. If any residues 15

of methyl acrylate were found, we applied further vacuum. 16

The methyl ester terminated dendrimer core could afterwards be reacted with EDA to gain the full 17

generation amine terminated dendrimer (G0 with four amine surface groups). Theoretically, also longer 18

diamines like 1,4-diaminobutane can be used in the dendrimer branch growth, however, these molecules are 19

difficult to remove in the purification step, since their boiling points are higher than in the case of EDA. In 20

order to avoid intramolecular cyclization reactions and oligomer formation, we used a high excess of EDA, 21

typically 25-30 eq. per dendrimer methyl ester. EDA was always freshly distilled prior to the reaction to 22

avoid colored oxidation products that are usually found in older EDA. The dendrimer core was dissolved in 23

methanol and added to a solution of EDA in methanol. Again, we applied cooling to the reaction mixture 24

during the addition. This was necessary due to the high mixing energy that is released by EDA and methanol 25

and to keep the exothermic reaction at a low temperature to avoid retro-Michael reactions. The amidation 26

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reaction was quite slow compared to the previous Michael reaction. We typically used four days of stirring to 1

ensure full amidation with a sufficient safety time buffer. The work-up of the amidation step was found to be 2

the most critical step in the dendrimer synthesis, since residual EDA frequently leads to trailing generation 3

dendrimers where the remaining EDA basically forms a new dendrimer core in the consecutive methyl 4

acrylate reaction.19 The work-up was performed on a rotary evaporator connected to a strong vacuum pump 5

(less than 1 mbar) to remove all methanol and most EDA. In our experience, even long periods of vacuum 6

were not sufficient to remove all EDA. It was thus already suggested by Tomalia et al. to perform azeotropic 7

distillations with a 9:1 mixture of toluene and methanol.14-15 This can be done by dissolving the dendrimer in 8

this mixture and removing all volatiles on a rotary evaporator. This procedure was repeated at least ten times 9

to assure that all EDA is evaporated. In order to remove toluene residues from the dendrimer, a similar 10

azeoptropic distillation was established with pure methanol (app. five times). It has to be noted that during 11

this extensive procedure the water bath temperature of the rotary evaporator is kept to a maximum of 30 °C 12

to avoid retro Michael reactions. 13

The main tool for building up higher generation dendrimers was to carefully apply these two repeating 14

steps to the following cascade reactions. Key parameters were the fresh distillation of the EDA and methyl 15

acrylate starting materials and the careful removal of the corresponding excess amounts after the reaction. 16

The dendrimers were always added to a cooled solution of the EDA/methyl acrylate and high excess rates of 17

starting materials and long reaction times are essential to get full functionalization. If defects like trailing 18

dendrimers generations (most common impurity) were detected, we applied size exclusion chromatography 19

for low generation dendrimers like G1 and G2. Here, we made good experience with Sephadex G25, 20

visualization on TLC plates was achieved by ninhydrin18. For higher generation dendrimers (G2-G5) we 21

used dialysis to remove excess EDA and trailing generation dendrimers. A suitable dialysis cut off size had 22

to be chosen that is well below the nominal weight of the dendrimer (typically 1 or 2 kDa), since in our 23

experience the dendrimers are very flexible molecules that easily escape even membranes with half the 24

nominal weight cut off limit. Dialysis can be performed against methanol, however, we experienced smaller 25

losses with water since protonated dendrimers are more rigid20 and less likely to escape the membrane. The 26

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dendrimer/water solution can be lyophilized without difficulty. Applying these conditions and guidelines, we 1

achieved to synthesize large amounts of the dendrimer generations G0 to G5. Detailed procedures are listed 2

in the materials and methods section. 3

4

Dendrimer Characterization 5

PAMAM dendrimers are hyper-branched polymers that share properties with smaller molecules, like their 6

defined molecular structure; however, they have also size and shape properties reminiscent to proteins or 7

natural polymers like dextrans.21 With this section, we give an overview of the characterization of the 8

synthesized dendrimers that was used to characterize the dendrimers and to investigate their purity. Detailed 9

analytical protocols are given in the materials and method section, which can be used as a guideline for 10

dendrimer characterization, furthermore all analytical data is depicted in the corresponding section or 11

supporting information, which can be used as standard for comparison. 12

13

NMR Characterization 14

NMR is the most common chemical technique to evaluate the structure and purity of molecules.22 15

Compared to other macromolecules and proteins, dendrimers have a quite low number of peaks with well-16

defined structures in the 1H-NMR and 13C-NMR. This shows impressively the high symmetry within the 17

dendrimer and the mono-dispersity within the synthesized dendrimer batches. 18

Amine terminated PAMAM dendrimers were well known from the literature and the synthesized 19

compounds could be evaluated by comparing the 1H and 13C NMR data to the published data.14-15, 23-24 Figure 20

3 and Figure 4 depict a NMR-assignment of a synthesized G4-PAMAM dendrimer with 64 amine groups, 21

which was achieved using COSY, HSQC and HMBC. Full assignments and spectra for all dendrimer 22

generations are found in the supporting information. No impurities like EDA and methyl acrylate were 23

detected, and the defined peak structures indicates high quality mono-disperse dendrimer batches. The 24

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depicted NMR spectra demonstrates that with the described reaction and work-up conditions, where the 1

temperature was always kept below 30 °C, dendrimers are highly symmetric with no visible defects. This is 2

in strong contrast to NMR and other characterization data published on PAMAM synthesis using reaction 3

temperatures of 50 °C to 55 °C,25-26 where undefined NMR 1H-NMR signals and fragmentation peaks are 4

visible. 5

6

7

Figure 3: 1H-NMR assignment of a G4-PAMAM dendrimer with 64 amine surface groups, measured in 8

deuterated methanol. 9

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1

2

Figure 4: H-NMR assignment (top) and 13C-NMR assignment (bottom) of a G4-PAMAM dendrimer with 64 3

amine surface groups, measured in deuterated methanol. The inset shows the dendrimer carbonyl peaks in 4

the high ppm region. 5

6

HPLC Analysis and Mass Spectrometry 7

Fortunately, the rapid development in biochemistry promoted the introduction of innovative MS 8

techniques like electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI) and multi 9

angular light scattering (MALS), which were capable of measuring large structures like proteins and 10

dendrimers.27-29 Usually, these techniques are used in combination with chromatographic techniques like 11

HPLC or size exclusion chromatography to detect and separate impurities and defects in the dendrimer 12

structure.19 We used HPLC and SEC to study the purity of the synthesized dendrimers and we established 13

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reliable, ESI-MS, MALS and MALDI-TOF methods that can be used to obtain molecular weight 1

information. 2

HPLC is an excellent tool to assure dendrimer purity.30 It can detect small molecule impurities, like 3

methyl acrylate, EDA and other remaining molecules from the dendrimer synthesis, and it can as well 4

distinguish between dendrimer dimers and tailing generations. Ideally, an HPLC chromatogram is observed 5

with only a single dendrimer peak. A sharp peak indicates a well-defined mono-disperse product. Small 6

peaks in front or after the dendrimer peak indicate dendrimer oligomers (dimer, trimer etc.) or tailing 7

generations of dendrimers (resulting from insufficient EDA removal in the dendrimer synthesis). Mullen et 8

al. gave an excellent overview of dendrimer HPLC and common defects, impurities in commercially 9

available dendrimers, resulting in multiple peaks of dimers, trimers and trailing generations of dendrimers.19 10

11

Figure 5: HPLC chromatogram of a G5 amine dendrimer with a C-4 column. Eluent system: water / 12

acetonitrile (containing 0.1 % (v/v) TFA) was used, starting with 5 % acetonitrile until 0.5 min, then gradient 13

from 5 % to 35 % for 0.7 min, then gradient 35 % to 100 % over 3 min, finally plateau of 100 % acetonitrile. 14

15

HPLC analysis showed that the synthesized dendrimers were of high quality with no small molecule 16

impurities, furthermore, no oligomers, and tailing generation dendrimers could be detected in the 17

chromatogram. A very sharp peak structure was observed (Figure 5). Due to their strongly positively charged 18

polar surface amine dendrimers were run on a C4-column with TFA as counter ion and detection of the 19

dendrimer was accomplished using a 215 nm absorption detector which is sensitive to the amides in the 20

0.0 2.0 4.0 6.0 8.0 10.0 12.0 15.0

-200

500

1,000

1,600kso #936 S5 UV_VIS_1mAU

min

WVL:215 nm

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dendrimer backbone. Quantification is an option using the extinction coefficients and absorption properties 1

that are discussed in the following section. 2

3

Figure 6: Normalized SEC-MALS chromatograms of PAMAM dendrimers acquired in pH 2.9 citrate buffer 4

(G0 brown, G1 green, G2 red, G3 grey, G4 black and G5 blue) . 5

6

Size exclusion chromatography system coupled with LS and RI detectors represents an additional powerful 7

tool to investigate dendrimer purity and estimate molecular weight of PAMAM dendrimers,31 although 8

particular attention is required in the choice of buffer used as eluent. Citrate buffer (pH 2.9) allowed us to 9

obtain well resolved LS chromatograms for all generation PAMAM dendrimers as shown in Figure 6. Each 10

generation showed only one highly symmetric peak that reflects the excellent mono-dispersity (Table 1) and 11

purity of the sample. 12

13

14

15

16

time (min)

5.0 10.0 15.0 20.0 25.0

Relative Scale

0.0

0.5

1.0LS

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Table 1: Polydispersity of the synthesized G2-G5 PAMAM dendrimers measured with SEC-MALS. The 1

values for the small dendrimer generations G0 and G2 are not reported since the light scattering signals had 2

no significant intensity to calculate reliable values 3

Sample Polydispersity Mw/Mn

G2-PAMAM 1.04 ± 0.02

G3-PAMAM 1.02 ± 0.01

G4-PAMAM 1.04 ± 0.00

G5-PAMAM 1.08 ± 0.02

4

However, all MW calculated with ASTRA software presented higher values than the theoretical ones. This 5

was attributed to the interaction of citrate counter-ions with positively charged amines present in the 6

dendrimer when it was dissolved in acidic conditions. This result suggested that citrate buffer is not an ideal 7

solvent to determine precise molecular weight values. PBS buffer (pH 7.4) was then used to dissolve 8

PAMAM dendrimers and determine molar mass value. In this case, another type of problem was encountered 9

when a separation method such as size exclusion column chromatography was used. A distortion of the 10

chromatogram was observed as shown in the supporting information. This result was attributed to the near 11

absence of counter-ion shell surrounding the dendrimers that led to column interactions. It was, thus, 12

concluded that batch measurements of unfractionated samples in PBS buffer represented the best option to 13

estimate weight-average molecular weight Mw (Figure 7) and the experimental values obtained with ASTRA 14

software are shown in Table 2. The low generation dendrimers (G0 and G1) are not reported since the light 15

scattering signals by the low molecular weight dendrimers was not sufficient to calculate reliable values. 16

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1

Figure 7: MALS batch measurement of unfractionated G5 PAMAM dendrimer sample. A weight average 2

molecular weight of Mw = 29.0 ± 5.2 kDa was calculated, which is in good accordance with the theoretical 3

value of Mw = 28.9 kDa. 4

5

ESI is an excellent soft-ionization technique with little fragmentation (compared to FAB or EI). 6

However, the large amount of possibly pronated tertiary amine groups in the dendrimers and thus their broad 7

distribution of mass signal, represent a challenge. Sometimes dendrimers can be observed flying with over 8

20 H+ attached to the molecule. Finding and assigning the right peak is, thus, complicated. Furthermore, 9

using ESI, dendrimers can “fly” with entrapped guest molecules like H2O, which makes the peak 10

interpretation even more complicated. Nevertheless, it was possible to record ESI masses for all of the 11

dendrimers (Table 1). ESI was found a very reliable tool to investigate low generation dendrimer, which in 12

general have less protonation sites and a smaller capacity for entrapped solvent molecules. 13

14

15

time (min)

5.0 10.0 15.0 20.0 25.0 30.0

Relative Scale

0.0

0.5

1.0 LS

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Table 2: Overview of the MS characterization of the synthesized PAMAM dendrimers with MALD, 1

MALDI and ESI. 2

Sample Theoretical

molecular

weight (g/mol)

MALS

Weight-average

molecular

weight (kDa)

MALDI

(m/z)

ESI

(m/z)

G0-PAMAM

544,7

Low scattering

signal

545.4 [M+H]+

545.4 [M+H]+

G1-PAMAM

1457,9

Low scattering

signal

1458.1 [M+H]+

1458.1 [M+H]+

G2-PAMAM

3284,3

Mw= 2.6 ± 0.3

3285.3

[M+H]+

1642.7 [M+2H]+2

≙ 3283.4 Da

G3-PAMAM

6937,0

Mw= 6.7 ± 2.1

6958,2 [M+Na]+

1157.2 [M+6H]+6

≙ 6937.0 Da

G4-PAMAM

14242,4

Mw = 15.4 ± 1.3

11.5 – 15 kDa

broad

1577.6 [M+9H]+9

≙ 14189.4 Da

G5-PAMAM

28853,1

Mw = 29.0 ± 5.2

22-28 kDa

broad

1684.5 [M+17H]+17

≙ 28636.5 Da

3

MALDI is another soft ionization technique used for large biomolecules. The advantage of MALDI is 4

the dominance for single charged molecules;32 this makes the result easier to interpret than in the case of ESI, 5

which usually has many different charged species. A negative aspect of MALDI is the strong laser power 6

that is needed to bring higher generations of dendrimers to “fly”, this can lead to a serious degree of 7

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fragmentation of the dendrimer. An example is shown in Figure 8. A G2 PAMAM dendrimer with low laser 1

power gives the expected [M+H]+ signal (25 % laser power), while a G5 PAMAM dendrimer same sample 2

with higher laser power (80 %) led to fragmentation and a broad signal distribution. Unfortunately, in case of 3

the higher dendrimers (G3-5) a very high laser power (70-100 %) is needed to extract the dendrimer from the 4

matrix, this induces fragmentation and results in very broad peak shapes. This fragmentation makes it 5

difficult to detect failures in the dendrimer structure, since it cannot distinguish between structure failures or 6

laser induced fragmentation. 7

8

Figure 8: MALDI-TOF of a G2-PAMAM dendrimer with expected molecular mass peaks and of a G5-9

PAMAM dendrimer with a fragmentation pattern starting from the molecular mass of 28.8 kDa downwards. 10

11

In comparison, we found that MALDI and ESI are excellent tools to investigate precise molecular weight for 12

low and medium generations of dendrimer,33-35 for examples G0 to G3. For G4 and higher generations of 13

dendrimers we experienced that MALS is a reliable tool to get mass information. 14

15

16

17

18

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1

Uv-Vis and Fluorescence Spectroscopy 2

150 200 250 300 350 400 450 500 550 600 650

0

2000

4000

6000

8000

10000

12000

360 400 440 480 520 560 600 640 680

0

10

20

30

40

50

Extinctio

n C

oeffic

ient (M

-1cm

-1)

Wavelength (nm)

G0-NH2

G1-NH2

G2-NH2

G3-NH2

G4-NH2

G5-NH2

Inte

nsity (

cp

s)

Wavelength (nm)

G0-NH2

G1-NH2

G2-NH2

G3-NH2

G4-NH2

G5-NH2

3 4

Figure 9: Plot of calculated extinction coefficient spectra (left) and emission spectra (right, λexc = 360 nm) of 5

PAMAM dendrimers acquired in MilliQ water (concentration 0.4 mM). 6

7

High molar absorptivity was detected in the near ultraviolet region of the spectrum. All generations of 8

PAMAM dendrimers investigated in this work showed a strong absorption band around 215 nm (Figure 9), 9

which arises from n� π* transitions and it is typical for amide bonds. This represents an advantageous 10

property for the dendritic polymer since traditional HPLC separations are carried out using 11

spectrophotometric detection in this region of the spectrum. A smaller absorption band was detected around 12

280 nm that seemed to arise from symmetry forbidden amide transitions, which could be promoted by 13

increasing internal rigidity in higher dendrimer generation as this band increases with dendrimer size. 14

Fluorescence emission was collected using λexc = 360 nm and the resulted luminescence spectra are depicted 15

in Figure 9 (right). All the dendrimers showed a broad fluorescence band around 440 nm, which increases as 16

the generation grows. In a previous work, Lee et al.36 proposed that oxidation of OH-terminated PAMAM 17

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dendrimers was responsible for the blue luminescence observed in such dendritic polymer. Contrarily, a 1

more recent study carried out by Wang demonstrated that fluorescent chemical species was related to the 2

tertiary amines of the dendritic backbone.37 To our knowledge, origin of fluorescence in PAMAM 3

dendrimers remains a controversial property, since they do not exhibit traditional chromophore in their 4

chemical structure. Even though it has not been fully understood, luminescence arising from PAMAM 5

dendrimers provides an important tool, as an alternative to classical fluorophore labeling,38-39 to characterize 6

and apply them in cellular tracing studies.40 7

8

FT-IR spectroscopy 9

The FT-IR spectra of the PAMAM dendrimers showed a characteristic, fingerprint-like pattern (Figure 10). 10

However, a dendrimer generation dependency on the spectra was not found. Strong and characteristic bands 11

are formed by C-N stretching vibration (1545 cm-1) and C=O stretching vibrations (1630 cm-1) from the 12

amide units. Broad bands around 2900 cm-1 and 3300 cm-1 are attributed to C-H stretching vibrations and N-13

H stretching vibrations respectively. The FT-IR data was in good accordance with previous published data 14

on PAMAM dendrimers.41 15

16

Figure 10: FT-IR spectra of a G5 PAMAM dendrimer in absorption mode. 17

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1

Conclusion 2

We report on a robust large scale synthesis of PAMAM dendrimers, which improved previous procedures 3

with easily adoptable methods like distillation of EDA and methyl acrylate and temperature settings during 4

reaction and work-up that are always below 30 °C to avoid retro-Michael additions. Solvent conditions and 5

reaction equivalents of EDA and methyl acrylate are reported that led to the synthesis of highly pure 6

PAMAM dendrimers avoiding cyclization reactions, oligomer formation and trailing generation of 7

dendrimers. Applying these strategies, we managed to synthesize large amounts, such as approximately 20 g 8

of G4 and even higher amounts of low generation dendrimers. The dendrimers synthesized according to 9

these procedures where of high purity and mono-dispersity. The applied analytical techniques, such as NMR, 10

HPLC, SEC-MALS, ESI, MALDI, UV-Vis, fluorescence and IR investigated the dendrimers from multiple 11

aspects. The detailed analytical methods reported in this work can be used as guideline on how to 12

characterize dendrimers and to compare spectroscopic data. 13

14

15

Supplementary Material 16

Supplementary material is available with the article through the journal Web site. 17

18

19

20

21

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Acknowledgment 1

Financial support by the European Union’s Seventh Framework Programme (FP7-NMP-2012-Large-6) 2

under the grant agreement No 310337 of the CosmoPHOS-nano Large-Scale Project is gratefully 3

acknowledged. 4

5

6

7

8

9

10

References 11

12

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