Synthesis of Symmetrical and Unsymmetrical ... - ACS Publications

Bioconjugate Chem. 2007, 18, 579−584

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Synthesis of Symmetrical and Unsymmetrical PAMAM Dendrimers by Fusion between Azide- and Alkyne-Functionalized PAMAM Dendrons Jae Wook Lee,†,* Jung Hwan Kim,† Hee Joo Kim,† Seung Choul Han,† Ji Hyeon Kim,‡ Won Suk Shin,§ and Sung-Ho Jin§,* Department of Chemistry, Dong-A University, Hadan-2-dong, Busan 604-714, Korea, Department of Chemical & Biochemical Engineering, Dongguk University, Seoul 100-715, Korea, and Department of Chemistry Education & Center for Plastic Information System, Pusan National University, Busan 609-735, Korea . Received August 17, 2006; Revised Manuscript Received January 25, 2007

A new convergent synthetic method for the synthesis of PAMAM dendrimers has been developed. The fusion between propargyl-functionalized PAMAM dendrons and azido-functionalized PAMAM dendrons via the Cu(I)-catalyzed Huisgen [2 + 3] dipolar cycloaddition reaction (click chemistry) of an alkyne and an azide leads to the formation of symmetric PAMAM dendrimers in high yields. Furthermore, the coupling reactions between the different generation dendrons afford the size-differentiated unsymmetrical PAMAM dendrimers.

INTRODUCTION Dendrimers, which are prepared by repetition of a given set of reactions using either divergent or convergent strategies, are highly branched and regular macromolecules with well-defined structures and have served as functional objects in nanotechnology and nanoscience (1, 2). Recent advances in nanomedicine showed that dendrimers are a novel class of nanoscale carriers that can be multifunctionally used for targeting, imaging, and treatment of biological systems (3). Especially PAMAM dendrimers, which are nanoscopic spherical macromolecules composed of polyamidoamino units with repeating dendritic branching, have been extensively studied in many fields such as drug, drug and gene delivery, and in magnetic resonance imaging. The interior cavities of PAMAMs are often used to encapsulate hydrophobic or hydrophilic drugs (4, 5). In order to better control the release rate, imaging, and targeting properties of drugs, the terminal groups of PAMAMs have been functionalized with various moieties, such as drugs, biospecific ligands, and fluorescent tags (6, 7). These dendrimer-based nanodevices hold great promise for various biomedical applications. In the future, more expanded applications of PAMAM dendrimers rely on efficient and practical synthetic procedures. The early synthetic efforts in PAMAM dendrimer synthesis applied the divergent synthesis procedure building the dendrimers from the core by an iterative synthetic procedure (8). The convergent approach of dendrimer synthesis introduced by Fre´chet and co-workers revolutionized the synthetic approaches to develop monodisperse dendrimers (9, 10). The convergent methodology installs the core in the final step, enabling the incorporation of various functionalities. It provides greater structural control than the divergent approach due to its relatively low number of coupling reactions at each growth step. The ability to prepare well-defined (un)symmetrical dendrimers is the most attractive feature of the convergent synthesis method. Therefore, the synthesis of PAMAM dendrimers via the convergent approach is a significant challenge. However, an * Corresponding authors. E-mail: [email protected]; E-mail: shjin@ pusan.ac.kr. † Dong-A University. ‡ Dongguk University. § Pusan National University.

example in the convergent synthesis of PAMAM dendrimers by the amide coupling between carboxylic acid and amine is reported (11). In continuation of our efforts in the convergent synthesis of dendrimers, we here report the efficient synthesis of symmetric and unsymmetric PAMAM dendrimers by 1,3dipolar cycloaddition of azide- and alkyne-functionalized PAMAM dendrons. The reactions employed in the synthesis of dendrimers should be high yielding without any side reactions. Well-known processes, such as Michael reaction, Williamson ether synthesis, amidations, and reductions have been used extensively in the synthesis of dendrimers (10). The Cu(I)-catalyzed Huisgen [2 + 3] dipolar cycloaddition reaction between an organic azide and an alkyne (12, 13), the best example of click chemistry (14, 15), is clearly a breakthrough in the synthesis of dendrimers (16, 17). The reaction is characterized by mild and simple reaction conditions, reliable 1,4-regiospecific 1,2,3-triazole formations, and tolerance toward water as well as a wide range of functionalities (18). Although many reports have been found in the synthesis of the triazole-mediated dendritic materials using click chemistry (19-26), there are a few reports that demonstrate the synthesis of the PAMAM dendrimer in spite of its importance for new material design (27-29).

EXPERIMENTAL PROCEDURES General. 1H NMR spectra were recorded on a 300 or 500 MHz NMR spectrometer using the residual proton resonance of the solvent as the internal standard. Chemical shifts are reported in parts per million (ppm). When peak multiplicities are given, the following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; d of d, doublet of a doublet; m, multiplet; br, broad. 13C NMR spectra were proton decoupled and recorded on a 75 or 125 MHz NMR spectrometer using the carbon signal of deuterated solvent as the internal standard. FAB and MALDI mass spectra were obtained from Korea Basic Science Institute (KBSI) in Daegu or Daejeon and POSTECH. Flash chromatography was performed with 37-75 µm silica gel. Analytical thin layer chromatography was performed on silica plates with F254 indicator, and the visualization was accomplished by UV lamp or using an iodine chamber. Polydispersity (PDI) of dendrimers was determined by gel permeation chromatography (GPC) analysis relative to polystyrene calibration (Agilent 1100 series GPC, Plgel 5µm

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MIXED-C, refractive index detector) in THF solution. All chemicals were obtained from commercial sources and used as received, unless otherwise mentioned. THF was distilled over Na/Ph2CO ketyl. General Procedure for the Preparation of Dendrimers 3-Gmn from Propargyl-PAMAM Dendrons 1-Dm and Azido-PAMAM Dendrons 2-Dn. A mixture of propargyldendrons 1-Dm (0.10 mmol) and azido-dendrons 2-Dn (0.11 mmol) in THF-H2O (4:1, 1 mL) in the presence of 5 mol % CuSO4‚5H2O with 10 mol % sodium ascorbate was stirred at room temperature for ∼4 h. The reaction mixture was poured into brine (20 mL), and the resulting solution was extracted with EtOAc (20 mL × 4). The combined organic phase was dried with sodium sulfate, concentrated, and purified by column chromatography (EtOAc/methanol system) to afford the desired product. 3-G11. 95% yield; IR 2955, 2840, 1734, 1437, 1258, 1199, 1174, 1044 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.02 (quintet, J ) 6.6 Hz, 2H), 2.39 (t, J ) 6.7 Hz, 6H), 2.47 (t, J ) 7.0 Hz, 4H), 2.71 (t, J ) 6.8 Hz, 4H), 2.77 (t, J ) 7.0 Hz, 4H), 3.62 (s, 6H), 3.64 (s, 6H), 3.78 (s, 2H), 4.31 (t, J ) 7.0 Hz, 2H), 7.55 (s, 1H); 13C NMR (75 MHz, CDCl3): δ ) 173.3, 173.2, 144.6, 123.4, 52.0, 51.9, 50.8, 49.5, 49.3, 48.9, 48.2, 33.0, 32.8, 28.5; MS (FAB): m/z ) 500 [M+ + H]; HRMS (FAB) calcd for C22H37N5O8: 499.2642. found: 500.2837 [M+ + H]. PDI ) 1.01. 3-G22. 97% yield; IR 3314, 2952, 2828, 1734, 1663, 1533, 1437, 1357, 1257, 1198, 1176, 1046 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.02 (m, 2H), 2.34 (m, 4H), 2.41-2.44 (m, 30H), 2.54-2.55(m, 8H), 2.73-2.77 (m, 16H), 3.28 (t, J ) 4.7 Hz, 8H), 3.66 (s, 24H), 3.84 (s, 2H), 4.36 (t, J ) 6.4 Hz, 2H), 6.97 (br s, 2H), 7.15 (br s, 2H), 7.67 (s, 1H); 13C NMR (75 MHz, CDCl3): δ ) 173.4, 172.7, 172.4, 143.8, 123.5, 53.3, 52.0, 50.2, 50.0, 49.6, 49.6, 48.2, 47.8, 37.5, 37.5, 34.2, 33.9, 33.4, 33.0, 28.4; MS (FAB): m/z ) 1301 [M+ + H]; HRMS (FAB) calcd for C58H101N13O20: 1299.7286. found: 1300.7364 [M+ + H]. PDI ) 1.02. 3-G33. 94% yield; IR 3294, 2954, 2918, 2848, 1737, 1658, 1649, 1542, 1437, 1257, 1200, 1177, 1046 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.02 (m, 2H), 2.34-2.36 (m, 10H), 2.42 (t, J ) 6.5 Hz, 48H), 2.53 (t, J ) 5.7 Hz, 24H), 2.57 (m, 8H), 2.74 (t, J ) 6.6 Hz, 32H), 2.79 (t, J ) 6.3 Hz, 16H), 3.263.27 (m, 24H), 3.65 (s, 48H), 3.82 (s, 2H), 4.35 (t, J ) 6.4 Hz, 2H), 7.07-7.09 (m, 8H), 7.59 (br, 2H), 7.73 (s, 1H), 7.77 (br, 2H); 13C NMR (125 MHz, CDCl3): δ ) 173.3, 173.2, 144.8, 123.3, 52.0, 51.9, 50.8, 49.5, 49.3, 49.0, 48.2, 33.1, 32.8, 28.5; MS (MALDI): m/z calcd for C130H229N29O44: 2900.6573. found: 2901.6875 [M+ + H]. 3-G31. 92% yield; IR 3303, 2952, 2827, 1735, 1664, 1651, 1539, 1436, 1257, 1198, 1176, 1043 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.04 (m, 2H), 2.36 (t, J ) 6.5 Hz, 6H), 2.42 (t, J ) 6.6 Hz, 24H), 2.53 (t, J ) 5.8 Hz, 12H), 2.58 (t, J ) 5.5 Hz, 4H), 2.74 (t, J ) 6.6 Hz, 20H), 2.78-2.82 (m, 12H), 3.28 (t, J ) 5.4 Hz, 12H), 3.66 (s, 24H), 3.67 (s, 6H), 3.84 (s, 2H), 4.33 (t, J ) 6.9 Hz, 2H), 7.04 (br s, 4H), 7.66 (s, 1H), 7.73 (br s, 2H); 13C NMR (75 MHz, CDCl3): δ ) 172.9, 172.8, 172.13, 172.06, 143.0, 123.1, 52.7, 52.3, 51.5, 50.2, 49.7, 49.0, 48.9, 47.7, 47.0, 37.2, 37.0, 33.6, 33.3, 32.5, 32.2, 27.9; MS (FAB): m/z ) 1701 [M+ + H]; HRMS (FAB) calcd for C76H133N17O26: 1699.9608. found: 1700.9686 [M+ + H]. PDI ) 1.03. 3-G32. 92% yield; IR 3310, 2952, 2827, 1735, 1664, 1651, 1537, 1437, 1257, 1197, 1177, 1045 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.02 (m, 2H), 2.33-2.36 (m, 2H), 2.41-2.43 (m, 36H), 2.53, (m, 12H), 2.59 (m, 4H), 2.73-2.75 (m, 24H), 2.81 (m, 16H), 3.26-3.27 (m, 16H), 3.66 (s, 36H), 3.83 (s, 2H), 4.35 (t, J ) 6.9 Hz, 2H), 6.98 (br s, 2H), 7.05 (br s, 4H), 7.66

(s, 1H), 7.74 (br s, 2H); 13C NMR (75 MHz, CDCl3): δ ) 172.9, 172.1, 143.2, 123.0, 52.7, 52.3, 51.5, 49.7, 49.4, 49.0, 47.7, 47.1, 37.2, 376.0, 33.7, 33.5, 33.3, 32.5, 32.1, 27.9; MS (FAB): m/z ) 2101 [M+ + H]; HRMS (FAB) calcd for C94H165N21O32: 2100.1930. found: 2101.2008 [M+ + H]. PDI ) 1.03. 3-G41. 95% yield; IR 3295, 2952, 2829, 1737, 1649, 1539, 1436, 1257, 1198, 1176, 1045 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.03 (m, 2H), 2.35 (m, 18H), 2.42 (m, 48H), 2.53 (m, 24H), 2.57 (m, 4H), 2.74 (m, 36H), 2.79 (m, 28H), 3.27 (m, 28H), 3.65 (s, 54H), 3.83 (s, 2H), 4.33 (t, J ) 6.9 Hz, 2H), 7.05 (br s, 8H), 7.63 (br s, 4H), 7.69 (s, 1H), 7.83 (br s, 2H); 13C NMR (125 MHz, CDCl ): δ ) 173.4, 173.2, 173.1, 172.8, 3 172.7, 143.7, 123.8, 53.3, 52.9, 52.5, 52.0, 50.9, 50.5, 50.3, 49.7, 49.5, 48.3, 47.6, 37.9, 37.6, 37.2, 34.2, 33.9, 33.1, 32.8, 32.4, 30.1, 28.6; MS (MALDI): calcd for C148H261N33O50: 3300.8895. found: 3323.7861 [M+ + Na]. 3-G42. 88% yield; IR 3290, 2952, 2828, 1734, 1648, 1545, 1436, 1257, 1199, 1177, 1043 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.05 (m, 2H), 2.34 (m, 18H), 2.41 (m, 60H), 2.52 (m, 28H), 2.73 (m, 72H), 3.26 (m, 32H), 3.64 (s, 60H), 3.79 (s, 2H), 4.34 (m, 2H), 7.01 (br s, 2H), 7.07 (br s, 8H), 7.63 (br s, 4H), 7.71 (s, 1H), 7.83 (br s, 2H); 13C NMR (125 MHz, CDCl3): δ ) 173.43, 173.39, 172.9, 172.8, 143.0, 124.4, 53.7, 53.3, 52.9, 52.0, 51.9, 50.5, 50.2, 49.7, 48.4, 47.4, 37.9, 37.6, 34.8, 34.2, 33.1, 32.9, 30.1, 28.6; MS (FAB): m/z ) 3702.2 [M+ + H]; MS (MALDI): calcd for C166H293N37O56: 3701.1701. found: 3702.2282 [M+ + H]. 3-G43. 76% yield; IR 3286, 2952, 2827, 1734, 1651, 1539, 1437, 1257, 1195, 1177, 1043 cm-1; 1H NMR (500 MHz, CDCl3): δ ) 2.01 (m, 2H), 2.35 (m, 28H + 6), 2.41-2.44 (m, 56H), 2.53-2.57 (m, 40H), 2.73-2.76 (m, 56H), 2.80 (m, 32H), 3.27 (m, 40H), 3.66 (s, 72H), 3.80 (s, 2H), 4.36 (m, 2H), 7.07 (br s, 12H), 7.59 (br s, 2H), 7.65 (br s, 4H), 7.73 (s, 1H), 7.84 (br s, 2H); 13C NMR (125 MHz, CDCl3): δ ) 173.4, 172.9, 172.82, 172.75, 143.2, 124.2, 53.3, 52.9, 52.0, 50.5, 50.3, 49.7, 46.0, 37.9, 37.6, 34.5, 34.2, 33.1, 32.7, 30.0, 28.5; MS (MALDI): calcdforC202H357N45O68: 4501.5861.found: 4524.6025 [M+ + Na].

RESULTS AND DISCUSSION In the convergent synthesis of the dendrimers, two feasible methods are the fusion of two dendrons and the stitch of multifunctional group with dendrons. We have developed the stitching method for the synthesis of symmetric dendrimers using click chemistry between an alkyne and an azide (17, 21, 22, 26). At this time, we are fascinated to utilize the fusion method of two dendrons for the synthesis of symmetric and unsymmetric PAMAM dendrimers. The synthetic strategy for the PAMAM dendrimers 3-Gmn utilized a fusion of the propargyl-functionalized PAMAM dendrons 1-Dm and the azido-functionalized PAMAM dendrons 2-Dn (Figure 1). The propargyl-functionalized PAMAM dendrons 1-Dm (m ) 1-4: generation of dendron) and azido-functionalized PAMAM dendrons 2-Dn (n ) 1-3: generation of dendron) are synthesized by the divergent approach using propargylamine and azidopropylamine as a focal point, respectively (27, 28). This methodology involves typical stepwise and iterative two-step reaction sequences, consisting of amidation of methyl ester groups with a large excess of ethylenediamine and Michael addition of primary amines with methyl acrylate to produce methyl ester terminal groups. Stitching together different dendrons effectively and efficiently without any ensuing problems in isolation and purification is a major challenge. The clue was provided by click chemistry due to the high yields and lack of byproducts. Building dendrimers via the fusion approach of dendrons

Technical Notes

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Figure 1. Structures of dendrons 1-Dm and 2-Dn and synthetic strategy for PAMAM dendrimers 3-Gmn.

together allows the synthesis of symmetric and unsymmetrical dendrimers. To efficiently connect the propargyl focal point PAMAM dendrons 1-Dm with azide-PAMAM dendrons 2-Dn for the synthesis of PAMAM dendrimer, the click condition using Cu(I) species was selected as synthetic method. The substrate-specificity in the copper-catalyzed cycloaddition reaction between an azide and an alkyne was

found to influence at the reaction time. The click reaction of alkyne-PAMAM dendrons 1-Dm, conducted from the conditions of 5 mol % of CuSO4‚5H2O with 10 mol % of sodium ascorbate, was completed at room temperature within a short time which may potentially be explained by anchimeric assistance due to the amine moiety presented in dendrons (27, 30).

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Figure 4. GPC diagrams of dendrimers 3-G11 and 3-G22 obtained from THF eluent. Figure 2. 1H NMR spectra of PAMAM dendrimers (a) 3-G11, (b) 3-G22, and (c) 3-G33.

Figure 3. IR spectra of PAMAM dendrimers (a) 3-G11, (b) 3-G22, and (c) 3-G33.

Thus, the reaction of alkyne-dendron 1-D1 and azidodendrons 2-D1 in 5 mol % CuSO4‚5H2O with 10 mol % sodium ascorbate in a 4:1 solvent ratio of THF to H2O for 1.5 h at room temperature afforded the desired product 3-G11 in 98% yield. The generation of product and disappearance of the dendrons were monitored by TLC runs of the reaction mixture. Given the success in the synthesis of first generation dendrimer 3-G11, we expanded this reaction to get higher generation dendrimers. The reactions of 1-D2 with 2-D2 and of 1-D3 with 2-D3 afforded the PAMAM dendrimers 3-G22 and 3-G33 in yields of 97% and 94%, respectively, after 2.5 and 4 h. For completion of the reaction between two dendrons, the higher generation dendrons take slightly longer time than the lower generation dendrons which can be imagined by the simple steric hindrance of dendrons. This result showed that the formation of triazole can be regarded as a new connector to construct the symmetric PAMAM dendrimers from dendrons. The symmetric PAMAM dendrimers were purified by column chromatography, and the structures were confirmed by 1H and 13C NMR spectroscopy, IR spectroscopy, and FAB or MALDI mass spectra. From the 1H NMR spectra (CDCl3), peaks of the methylene protons adjacent to the carbon of triazole, the triazole proton, and the methylene protons adjacent to the nitrogen of triazole in dendrimers 3-Gmn were found at 3.78, 7.55, and 4.31 ppm for 3-G11, 3.84, 7.67, and 4.36 ppm for 3-G22, 3.82, 7.73, and 4.35 ppm for 3-G33, respectively (Figure 2). As the dendrimer generation increased, peaks of the triazole proton shifted gradually to downfield which may be influenced by the dendritic effect (31). The IR spectra also shows the disappearance of acetylene peak at ∼3277 cm-1 and the azide peak at ∼2096 cm-1 in the final dendrimers (Figure 3) while the 1H NMR

Figure 5. 1H NMR spectra of PAMAM dendrimers (a) 3-G31, (b) 3-G32, (c) 3-G41, and (d) 3-G42.

Figure 6. IR spectra of PAMAM dendrimers (a) 3-G41, (b) 3-G42, and (c) 3-G43.

revealed no alkyne peak at around δ 2.46 ppm. Their mass spectra were exhibited very good correlation with the calculated molecular masses. Analysis of the dendrimers by gel-permeation chromatography (GPC) from THF eluent shows very low polydispersity values, PDI ) 1.01 and 1.02 for 3-G11 and 3-G22, respectively (Figure 4). Unfortunately, the PDI value by GPC analysis of 3-G33 could not be obtained due to their poor solubility and aggregation property in THF. To probe the viability of our approach, we next turned our attention toward the construction of unsymmetrical PAMAM dendrimers. Unsymmetric dendrimers were assembled through the click reactions between different generations of dendrons.

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Technical Notes

(Figure 8). Unfortunately, the PDI value by GPC analysis of other dendrimers also could not be obtained due to their poor solubility and/or aggregation properties in THF.

CONCLUSION

Figure 7. FAB mass spectrum of dendrimer 3-G32.

We have demonstrated for the first time that click reaction between the propargyl-functionalized PAMAM dendrons and the azido-functionalized PAMAM dendrons lead to the formation of symmetric PAMAM dendrimers in high yields. Furthermore, the coupling reactions between the different generation dendrons afforded the size-differentiated unsymmetrical PAMAM dendrimers. This method may then provide an insight into designing various (un)symmetrical dendrimers such as amphiphilic dendrimers. We are currently working toward synthesis of various functional dendrimers using this strategy for various applications.

ACKNOWLEDGMENT This research was supported by the University IT Research Center (ITRC) Project of the Ministry of Information and Communication (J.W.L) and by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10600000157-06J0000-15710) (S.H.J). We also thank the Korean Ministry of Education (BK 21 Program) for graduate studentships. Figure 8. GPC diagrams of dendrimers 3-G31 and 3-G32 obtained from THF eluent.

We used 1-D3 and 1-D4 dendrons as higher generation dendrons for developing unsymmetric dendrimers. The reaction of third generation alkyne-focal dendron 1-D3 with 2-D1 and 2-D2 in the presence of 5 mol % CuSO4‚5H2O with 10 mol % sodium ascorbate in a 4:1 solvent ratio of THF to H2O afforded the unsymmetrical PAMAM dendrimers 3-G31 and 3-G32 in yields of 92% and 91%, respectively, after 2 h. The reaction of fourth generation alkyne-focal dendron 1-D4 with 2-D1, 2-D2, and 2-D3 provided the unsymmetrical PAMAM dendrimers 3-G41, 3-G42, and 3-G43 in yields of 95%, 88%, and 76%, respectively, after 2, 2.5, and 4 h. The products were separated by column chromatography. The low yield of 3-G43, in the absence of any side product(s) as observed by TLC, could be due to significant retention of the polar dendrimer in silica column. To improve the isolated yield of dendrimers, the membrane dialysis could be applied. Therefore, the results showed that the formation of triazole between alkyne-dendrons and azidedendrons are found to be an efficient connector to construct various unsymmetric dendrimers and may be applied for the synthesis of functional materials. We are currently investigating the synthesis of functional unsymmetric dendrimers using the dendrons peripherally modified with various substituents. The structures of all unsymmetric PAMAM dendrimers were also confirmed by 1H and 13C NMR spectroscopy and mass spectra. From the 1H NMR spectra (CDCl3), we observed that the chemical shifts of the methylene protons adjacent to the carbon of triazole, the triazole proton, and the methylene protons adjacent to the nitrogen of triazole in unsymmetric dendrimers were similar to those in the corresponding dendron(s) of symmetric dendrimers (Figure 5). IR data also confirmed that neither alkyne (∼3277 cm-1) nor azide (2096 cm-1) residues remain in the final dendrimer (Figure 6). Their mass spectra were exhibited very good correlation with the calculated molecular masses (Figure 7). Analysis of the dendrimers by gelpermeation chromatography (GPC) from THF eluent shows very low polydispersity value, PDI ) 1.03 for both 3-G31 and 3-G32

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