Synthesis of dendronized PAMAM grafted ROMP polymers

Abstract Oxanorbornene cored 0.5, 1.5 and 2.5 generation ester terminated PAMAM dendronized monomers with 2 and 6 carbon-containing alkyl spacer in the middle of oxanorbornene core and dendronized branching were synthesized. Monomers were polymerized through the ring-opening metathesis polymerization (ROMP) technique by using 2nd and 3rd generation Grubbs catalysts to get dendronized ROMP polymers. It was found that Grubbs 3rd generation catalyst works well for 0.5 generation dendronized ROMP polymer whereas Grubbs 2nd generation catalyst works for the higher generation. We also found the linker effect during polymerization as six-carbon linker monomers polymerize with high yield along with higher generation. Finally, dendronized ROMP-based PAMAM encapsulated zero valent Cu nanoparticle was synthesized by using a reducing agent, NaBH4. The catalytic activity of this nanoparticle was further investigated from the reduction of 4-nitrophenol to 4-aminophenol. Graphical Abstract


Introduction
Poly(amidoamine) (PAMAM) dendrimer was the first established dendrimer among the dendrimers family that has been synthesized, characterized, commercialized [1,2] and well-studied for gene delivery, [3] battery technology, [4] antibacterial agents, [5,6] catalysis, [7,8] catalytic application [9,10] and magnetic imaging. [11,12] PAMAM dendrimers are composed of repeating poly(amidoamine) units [1] which are highly biocompatible represented by cytotoxicity tests on PAMAM dendrimers. [13] But higher generation perfect dendrimer synthesis is not easy without structural deviation [14] due to incomplete conversion of generation, structural defects, [15] steric hindrance of huge numbers of surface functional groups and changing of self-assembly from cylindrical to spherical shape. [16] To overcome these difficulties, the interest in graft polymer with dendronized branching has grown rapidly. Dendronized graft polymers [17] are in which different generations of dendronized branched are joined to the linear backbone of polymers as a pendant group. [18] The synthesis of dendronized graft polymer is a tough and tricky task, particularly when a higher generation bulky branch is connected to the backbone and surface modification is targeted through functionalization. [19][20][21] Mainly three routes are used to synthesize dendronized polymers-(i) graft-from method, (ii) graft-to method and (iii) grafting-through method. [22] Grafting-from technique of dendronized block polymers begins with the synthesis of the main backbone of polymer containing multi-initiator (predetermined number) sites on the surface which are subsequentially used to initiate further block polymerizations. Based on polymerization strategies taken in two steps, initiating groups can be introduced directly or protectively after the first-step polymerization. [23,24] In the grafting-from synthesis method, repeating unit composition and chain length can be controlled as the backbone is synthesized first.
Nevertheless, the initiating surface-active groups and backbone initiate significant steric effects which sometimes are directed to insufficient initiation in the process of polymerization. [25,26] In the grafting-through synthesis technique or macromonomer method, linear backbone and dendronized branches are synthesized individually. This technique consists of the reaction of terminal functional polymers and several functional groups containing an initial polymer backbone. Due to the individual development of the side chain and polymer backbone, the chain length of polymer can control properly through monomer composition by selecting suitable polymerization techniques. As the branches are synthesized through a proper polymerization strategy in the beginning, their structures can be controlled as well as those in the grafting-onto approach and well-defined polymers can be synthesized.
Dendrimer encapsulated metal nanoparticles (DENs) have drawn enormous interest among researchers because of their particular chemical and physical characteristic. DENs [27] are different from their bulk counterparts [28] and for DENs synthesis dendrimers are used as a template due to having a well-defined uniform structure, [29,30] encapsulation capacity, [31,32] and access control of small molecules selectively through high branching. [29,33] DENs have received huge interest in the field of sensors, [34] optoelectronics, [35] biochemistry, catalysis [36] and among all applications, catalysts are extensively applicable in energy production, energy conversion, environmental remediation, pollution abatement, and specialty chemical production. [37][38][39] Copper(II)-PAMAM nanocomposite was first studied as dendrimer intramolecular complexes between PAMAM dendrimer and metal as DENs. [40] These Cu 2þ PAMAM complexes can be simply determined by color changes, UV-Vis spectra and EPR spectra. In water solution, Cu 2þ ions with hexahydrate show a weak absorption band at 810 nm stemming from d-d transition in the absence of PAMAMs. [41,42] Cu 2þ ions form complex easily with PAMAM and d-d transition shifts to 605 nm. Reduction of Cu 2þ dendrimer complex done by a reducing agent, that is, NaBH 4 . After the addition of excess NaBH 4 into the dendrimer, Cu 2þ solution immediately changes color of the solution from blue to golden brown due to the formation of dendrimer encapsulating copper nanoparticles. Through PAMAM dendrimer encapsulated Cu nanoparticle uses are in biomedicine and biomedical imaging but the catalytic application is mostly focused among researchers. [43][44][45] Oxanorbornene polymerization using ruthenium-based 2nd and 3rd generation Grubbs catalyst [46] is a potential technique for high resistance of ruthenium toward various functional groups and the living character of ring-opening metathesis polymerization (ROMP). [47,48] Among all other uncontrollable radical polymerization of vinyl group, [49][50][51] ROMP [52][53][54] of dendronized macromolecule is the best technique for controlled polymerization. It is known that the exo-isomer of oxanorbornene gave a proper polymer backbone, however, endo-isomer of oxanorbornene is highly shielded alkene from the catalyst and did not polymerize even after extended reaction time. [55] Again, the linker between the polymerizable core and the pendent dendronized branch plays an important role to overcome steric congestion around olefin bond of the core which is the polymerizable unit. Jung and coworkers [56] have synthesized Newkome types of dendronized ROMP by using various spacer lengths and found that doubling the length of the linker improves the rate of polymerization. Moreover, the fourth generation dendronized pendant ROMP without a spacer and with a biphenyl spacer showed a uniform conformation, whereas the polymer with the flexible ethylene spacer exhibited a much more semi-flexible chain. [57] Higher generation (G5) showed the extended rod-like conformation but lower generation smaller dendron exhibited the most semi-flexible chain. These findings represent that length and conformation of linkers and the generation of the pendant dendronized branch have a significant effect on conformation and rate of ROMP polymerization. [58] Schluter and coworkers did not find any effect of linker on polymerization time, yield or molar mass of radical polymer of dendronized poly(methacrylate)s. [59] On the other hand, Kim et al [18] reported that without any linker between norbornene derivative and G4 ester dendron did not polymerize because of the huge steric hindrance. But when they applied ethylene and rigid biphenyl linker complete conversion was observed at 50 C and even rigid biphenyl linker possesses better polydispersity. A similar effect was reported that one carbon spacer polymerize 50% whereas ten carbons spacer polymerize 98% conversion. [56] ROMP is a unique type polymer due to having double bond in the backbone that can functionalized to get special properties. Though many works have done on dendrimer templated Cu nanoparticles preparation [60] and dendronized ROMP [61,62] but there is no remarkable work is found on PAMAM dendronized branched ROMP polymer templated Cu nanoparticle synthesis to be used as a catalyst for the reduction in presence of NaBH 4 . Here, reported dendronized monomers consist of PAMAM dendron branching like Tomalia's dendrimers that were built by the use of [1!2]-C-branched monomers. [1,44] Since neither azides nor alkynes or amine is compatible with Grubbs catalyst for polymerization, [63] we designed ester terminated half-generation dendron with 2 and 6 carbon spacer between pendent dendron and polymerizable oxanorbornene via ROMP. In total six polymers have been synthesized through ROMP by Grubbs 2nd and 3rd generation catalysts. Among them, the highest generation polymer is used to prepare ROMP-PAMAM encapsulated Cu nanoparticles to see catalytic activity by reducing 4-nitrophenol to 4-aminophenol which is monitored UV-Vis spectroscopy. Figure 1 shows the synthesized monomers in the study.

Instrumentation
FT-IR spectrometer (model: Nicolet iS10), produced by Thermo Fisher Scientific, USA, was used for FT-IR spectra in a wave number range 4000-650 cm À1 . 1 H and 13 C NMR spectra were recorded on a Bruker 500 MHz spectrometer at 25 C using TMS as the internal standard. Molecular weights and polydispersity index (PDI) of polymers were measured by gel permeation chromatography (GPC) using tetrahydrofuran (THF) relative to polystyrene standards. Ultravioletvisible (UV-vis) absorption spectra were recorded in 1-cm quartz cells with Cary 60 UV-vis spectrometer (Agilent, USA).

Synthesis of compound 2
3.02 g (49.44 mmol) ethanolamine was taken in 15 mL methanol solvent in a 250 mL flask. 15.80 mL (174.23 mmol) methyl acrylate was added to ethanolamine solution drop wise and kept the reaction mixture with continuous stirring for 4 days at ambient temperature. [66,67] Then, unreacted reagents were removed by rotary evaporator at 60 C with high vacuum. The product was found as a yellowish oily dense liquid with nearly 95% yield and the structure of the product was elucidated by FT-IR and 1 H NMR in

Synthesis of monomer M1
3.00 g (44.07 mmol) of 3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (1) was dissolved in 55 mL dry THF at 60 C under inert condition and then cooled the solution at ambient temperature. [68] 8.50 g of 0.5 generation dendronized compound (2) was dissolved in 10 mL dry THF and added to compound (1) solution by a syringe. 4.70 g triphenylphosphine (PPh 3 ) was dissolved in 10 mL dry THF and added to the reaction mixture through a syringe. The reaction flask was kept in an ice bath to make 0 C and 3.90 mL diisopropylazodicarboxylate (DIAD) was added to the cool reaction mixture dropwise by a syringe for half an hour. Finally, the reaction flask was covered by aluminum foil and kept stirring for 48 h at ambient temperature. After completion of the reaction, solvent from product mixture was removed by rotary evaporator and precipitated in cold petroleum ether through product dropping in cold petroleum ether of a 250 mL beaker. Synthesized product was purified by column chromatography against silica with ethyl acetate and hexane mixture 1:6 (v/v). Yellowish solid product percentage of yield is 75%. FT

Synthesis of compound 3
2.55 g (6.71 mmol) of 0.5 generation dendronized monomer M1 was dissolved by 50 mL methanol in a flask and ethylenediamine methanol solution (8.05 g, 134.16 mmol EDA in 20 mL MeOH) was added to the 0.5 generation dendronized monomer solution with continuous stirring to get 1.0 generation compound 3. [69] The stirring was continued at ordinary temperature for 4 days and then excess reagents were removed by the rotary evaporator at 60 C bath temperature. Though all methanol was removed by rotary evaporator, ethylenediamine still might remain in the product which was removed by the addition of 50 mL n-butanol by making an isotropic mixture of lower boiling point that was removed by rotary evaporator at high vacuum. Finally, biotech grade membrane of 0.1-0.5 kDa MWCO cellulose ester tube was used for 6 h to get rid of ethylenediamine.

Synthesis of monomer M2
2.50 g (29.14 mmol) of methyl acrylate in methanol (40 mL) was added to 2.12 g (4.85 mmol) of amine terminated 1.0 dendronized compound 3. The magnetic stirring of the reaction was continued at ambient temperature for 72 h and then unreacted reagents were removed by a rotary evaporator. Synthesized 1.5 generation dendronized monomer M2 was found as yellowish jelly like solid with very good yield (>92%).

Synthesis of compound 5
Amine terminated six-carbon linker core compound 5 was synthesized by following a published article. [70] 2.73 g, (16.5 mmol) Diels-Alder adduct (4) and 11.5 g (98.7 mmol) 1,6-diaminohexane were heated together at 80 C for 2h. Then the reaction was cooled at room temperature and diluted by 25 mL of dichloromethane (DCM). The organic layer was separated by a funnel using water (25 mL Â 5) and then brine (25 mL Â 3). The organic layer was dried over MgSO 4 and concentrated by vacuum in a rotary evaporator to get pure product. Final product was found as yellowish solid with yield of 51%. FT

Synthesis of monomer M3
2.07 g (24.09 mmol) methyl acrylate was taken in methanol (20 mL) solvent and followed by adding to amine terminated core compound 5 (2.01 g, 8.03 mmol). [69,71] The reagent mixture was continued to stir at ambient temperature for 48 h and unreacted reagents were evaporated by a rotary evaporator and finally synthesized 0.5 generation dendronized monomer M3 was yellowish powder.

Synthesis of compound 6
Six carbon linker 1.0 generation dendronized compound 6 was synthesized by amidation reaction followed by a standard method. [69] 2.51 g, (5.75 mmol) of monomer M3 was dissolved in 100 mL methanol and 6.90 g (115.01 mmol) ethylenediamine was also dissolved in 50 mL methanol in two different flasks. Both solutions were mixed with continuous stirring by a magnetic rotator to get full generation monomer. The reaction was continued at ambient temperature for 72 h and solvent was removed by rotary evaporator at 60 C bath temperature. Though all methanol was removed by rotary evaporator, ethylenediamine still might remain in the product which was removed by the addition of 50 mL n-butanol by making an isotropic mixture of the lower boiling point that was removed by rotary evaporator at high vacuum. Finally, biotech grade membrane of 0.

Synthesis of monomer M4
3.49 g (40.55 mmol) of methyl acrylate was dissolved in 20 mL methanol which was added to a methanol (20 mL) solution of amine terminated 1.0 dendronized compound 6 (3.33 g, 6.75 mmol). The mixture of both solutions was stirred at room temperature for 4 days and a rotary evaporator was used to remove excess reagents under vacuum. Finally synthesized 1.5 generation dendronized monomer was yellowish gel with percentage of yield of 92%. FT

Synthesis of compound 7
4.02 g (4.80 mmol) of 1.5 generation dendronized monomer M4 was dissolved in 100 mL methanol in a 250 mL round bottom flask. 10 equivalent weight of ethylenediamine (11.54 g, 192.35 mmol) for each ester terminal of M4 was also dissolved in 50 mL methanol. Ethylenediamine methanol solution was added to M4 monomer solution with continuous stirring by magnetic starrier to get full generation compound 7. [69] The reaction was continued at room temperature for 6 days and a rotary evaporator was used to remove excess reagents under vacuum. Though all methanol was removed by rotary evaporator, ethylenediamine still might remain in the product which was removed by the addition of 50 mL n-butanol by making an isotropic mixture of lower boiling point that was removed by rotary evaporator at high vacuum. Finally, biotech grade membrane of 0.1-0.5 kDa MWCO cellulose ester tube was used for 12 h to get rid of ethylenediamine.

Polymerization procedure
For polymerization, 0.2 g of M1 and M3 monomers were dissolved in 2 mL dry dichloromethane into two separate vials individually and 0.2 g of M2, M4 and M5 monomers were dissolved by trifluoroethanol in three separate sealed tubes. 0.0088 g Grubbs 2 nd generation catalyst was weighted in two separate vials and 0.0088 g Grubbs 3rd generation catalyst was weighted in three separate vials individually for targeting molecular weight of each polymer 20,000 g/mole. Catalyst of each vial was dissolved in 0.5 mL dry dichloromethane. Grubbs 3rd generation catalyst solutions were added to M1 and M3 monomer solution vials at a time under nitrogen condition and kept vigorous stirring for five days at room temperature. Whereas Grubbs 2nd generation catalyst solutions were added to M2, M4 and M5 monomers solution of the sealed tube at a time and kept vigorous stirring over five days at 80 C temperature under inert atmosphere. Polymerizations were terminated by adding ethylvinyl ether (30% solution in DCM) and continued stirring more half an hour. Polymers were precipitated by adding diethyl ether and washed several times by centrifuging over diethyl ether. Diethylether was decanted and precipitated solid polymer was dried by nitrogen flow and stored in a dark place.

Preparation of dendronized ROMP encapsulated copper nanoparticle
ROMP based PAMAM encapsulate Cu was synthesized according to appropriate procedure. [45] 2 mg/mL (1.22 Â 10 À9 mM) of six-carbon linker 2.5 generation dendronized ROMP (P5) stock solution was prepared in distilled water. Stock solution of CuSO 4 Á5H 2 O (14 mg/mL, 4.91 Â 10 À8 mM) and NaBH 4 (7.6 mg/mL, 2.01 Â 10 À7 mM) were also prepared in distilled water. 500 mL P5 stock solution was taken in 10 mL volumetric flask and then distilled water was added up to the mark and then it was taken in a vial for mixing magnetic stirring. After that 21 mL copper sulfate stock solution was added to the vial. The solution was stirred for 20 min to form Cu 2þ complex with tertiary amine group of the polymer and the solution color was turned to light blue. After then, 18 mL NaBH 4 stock solution was added to the Cu 2þ complex solution and stirred for almost 2 min to convert Cu 2þ ions to zerovalent Cu atoms to get ROMP-based PAMAM encapsulated Cu nanoparticle (CN1). We also performed for the synthesis of nanoparticles CN2 and CN3 which were prepared with excess NaBH 4 (2 x eq.) and excess CuSO 4 (4Â eq.) by following the same procedure.

Synthesis of two carbon linker monomers (M1 and M2)
Two different oxanorbornene cored ester terminated PAMAM dendronized monomers with two carbon linker in between oxanorbornene core and dendronized branching were synthesized. All steps of the monomers synthesis route with two-carbon linker are shown in Scheme 1. Pure exo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (1) was synthesized through Diels-Alder cycloaddition reaction from furan and maleimide. [72] The half-generation ester terminated dendron was synthesized from ethanolamine [66,67] through Michael addition reaction. FT-IR spectrum gave a broad spectrum at 3456 cm À1 for -OH and C ¼ O peak of ester spectra at 1720 cm À1 ( Figure S1). The disappearance of vinyl peaks (6.5 ppm) of methyl acrylate and the appearance of methoxy peaks of 6H at 3.69 ppm confirmed the successful conversion of 0.5 generation dendron ( Figure S2). The synthesized dendron (2) was incorporated toexo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (1) through Mitsunobu reaction to get monomer M1. After purifying the compounds over column chromatography, monomer M1 was characterized via NMR. In the 1 H NMR spectrum, the peak at 6.51 ppm indicates the olefin protons of oxanorbornene, 3.65 ppm represents methoxy protons of monomer M1 ( Figure S4) whereas in the 13 C NMR spectrum, 13 C peaks at 136.6 ppm for C ¼ C of olefin, 172.6 ppm representing ester carbonyl (C ¼ O) ( Figure S5). Theoretical molar mass of M1 is 380.39 g/mol which was found 381.16 g/mol for the compound ([C 18 H 24 N 2 O 7 ]þH) þ through mass spectroscopy analysis ( Figure S6). 1.0 generation dendronized compound 3 was synthesized by the reaction of ethylenediamine and M1. The progress of full generation dendronized compound formation was monitored through FT-IR by disappearance of ester peak at 1732 cm À1 and newly formed amide peaks at 1643 cm À1 and 1549 cm À1 ( Figure S7). Similarly, the disappearance of methoxy peak at 3.65 ppm and appearance proton peak at O ¼ CNH-at 8.32 ppm in 1 H NMR spectra confirmed successful conversion of 1.0 generation dendronized compound 3 ( Figure S8). Finally, two carbon linker monomer M2 was synthesized by the reaction of methylacrylate with 1.0 generation compound 3. Newly appeared ester peak at 1733 cm À1 in FT-IR indicates 1.5 generation dendronized monomer M2 ( Figure S9). In 1 H NMR methoxy peak at 3.65 ppm confirmed formation of the product ( Figure S10).

Synthesis of six-carbon linker monomers (M3, M4 and M5)
Three different oxanorbornene cored ester terminated PAMAM dendronized monomers with six carbon linker in between oxanorbornene core and dendronized branching were synthesized. All steps of monomers M3, M4 and M5 synthesis routes with six carbon linker are shown in Scheme 2.
Exo-isomer of oxanorbornene (compound 4) was prepared through Diels-Alder cycloaddition reaction from furan and maleic anhydride in THF. [72] Olefinic protons of oxanorbornene were observed at 6.59 ppm as a singlet in 1 H NMR ( Figure S12). Olefinic carbon and carbonyl carbon Scheme 1. Synthesis of two carbon linker dendronized monomers.
peaks of 13 C NMR are at 137.3 and 171.8 ppm, respectively, which reconfirmed the exo-stereoisomer structure of the starting product ( Figure S13). The core was synthesized by reacting 1,6-diaminohexane and oxanorbornene through the imide route. Olefinic protons and hexyl protons are at 6.53 ppm and 1.25-1.75 ppm, respectively, at 1 H NMR spectra ( Figure S15). However, olefinic carbons and hexyl carbons are at 136.5 ppm and 26.0-41.5 ppm, respectively, indicating the product ( Figure S16). After then methyl acrylate was reacted with core compound 5 to get monomer M3 through Michael addition reaction. Disappearance of vinyl peaks of methyl acrylate and appearance of methoxy peaks of six protons at 3.69 ppm confirmed the successful conversion of 0.5 generation dendronized monomer ( Figure  S18). After then 1.0 generation dendrimer was synthesized by reacting with ethylenediamine. The progress of 1.0 generation dendronized compound formation was monitored through FT-IR by disappearance of ester peak at 1733 cm À1  and newly formed amide peaks at 1603 cm À1 and 1445 cm À1 ( Figure S21). Similarly, in 1 H NMR peak, disappearance of methoxy peak of 3.69 ppm and appearance of amide HNC ¼ O peak at 7.46 ppm confirmed the successful conversion of 1.0 generation. Compound 6 was found as a mixture of both exo and endo isomer together with the molar ratio of 1.00:0.57 confirmed by two peaks at 6.55 and 6.53 ppm in 1 H NMR in Figure S22. Carbon NMR of C ¼ C double bond of exo and endo isomer was reported at 138.0 and 134.6 in the literature. [73] We tried to separate both isomers from each other by column chromatography but failed as retention time was very close. The generation was extended to 1.5 generation by reacting with methyl acrylate with dendronized compound 6. In FT-IR, the appearance of ester peak at 1733 cm À1 along with the previous amide peaks at 1643 cm À1 and 1549 cm À1 indicates 1.5 generation dendrimer ( Figure S23). In 1 H NMR, the peak at 6.55 ppm indicates olefin protons, 3.58 ppm corresponds to methoxy protons of ester. The monomer M4 contains 30% endo isomer with exo-isomer that was observed in NMR olefin protons peak at 6.53 ppm and 5.59 ppm in Figure S24. We also tried to separate the isomers by column chromatography but failed as retention time both exo and endo monomers are very close. 2.0 generation dendronized monomer was synthesized by reaction of ethylenediamine with monomer M4. The progress of 2.0 generation dendronized compound formation were monitored through FT-IR by disappearance of ester carbonyl peak at 1733 cm À1 and newly formed amide peaks at 1632 cm À1 and 1552 cm À1 ( Figure S25). Similarly, disappearance of methoxy peak at 3.59 ppm from 1 H NMR peak and the appearance of amide (O ¼ CNH) peak at 7.90 ppm confirmed the successful conversion of 2.0 generation dendronized compound 7 ( Figure S26). Finally, 2.5 generation dendronized monomer M5 was synthesized by adding methyl acrylate with 2.0 generation dendronized compound 7. The successful synthesis of the monomer and ester to amide conversion from 0.5 generation to full generation was confirmed by FT-IR through the appearance of ester peak at 1731 cm À1 along with previous amide I and amide II peaks at 1643 cm À1 and 1543 cm À1 . In 1 H NMR of M5 have a methoxy peak at 3.61 ppm along with other dendronized branch peaks at 2.00-3.55 ppm and olefin protons peak at 6.55 and 6.49 ppm representing at least 20% endo product with endo monomer M5 ( Figure S28). Conversion of all generations was monitored by FT-IR is shown in Figure 2.
For the purification of full generation dendronized compounds, firstly ethylenediamine was removed under a high vacuum by a rotary evaporator maintaining water bath temperature of 65 C. Then, the remaining trace amount of ethylenediamine was removed by making azeotropic mixture through 50 mL of n-butanol with methanol solution of ethylenediamine and repeating the process for three times. Finally, the product was further dialyzed with liquid-phase polymer retention (LPR) method with 50% water-methanol solution for 48 h by using dialysis membrane of desired molecular weight cutoff size (MWCO) 0.5, 1.0 kDa depending on molecular weight of the product. The product was found highly pure form with percentage of yield in between the range of 90-93%.

Synthesis of polymers
For polymer synthesis, we used Grubbs 2nd generation and Grubbs 3rd generation catalyst in various solvents such as trifluoroethanol, DCM and THF. Half generation dendron grafted monomers M1 and M3 were not polymerized by Grubbs 3rd generation catalyst in trifluoroethanol at two hours reaction time at room temperature. But monomer M1 and monomer M3 were polymerized in dry DCM by Grubbs 3rd generation catalyst at room temperature over five days to obtain polymers P1 and P3, respectively. Both monomers were also polymerized by Grubbs 2nd generation under the same condition, solvent and reaction time.
Monomers M2, M4 and M5 were tried to polymerize by Grubbs 3rd generation catalyst in trifluoroethanol at 60 C for five days and THF at room temperature for 24 h but monomers were not polymerized. Finally, monomers M2 and M5 were polymerized in trifluoroethanol in sealed tube at 80 C over five days and found polymers P2 and P5, respectively, but unfortunately, we were not able to polymerize monomer M4 (Figure 3) to obtain P4.
One of the reasons for unsuccessful P4 is the presence of high endo stereoisomer (30%) present in monomer M4. In contrast monomer M5 also contain 20% endo isomer but it is polymerized with lower yield. [18,55,56] In Figure 4, monomer M3 remains near about 5% endo isomer as an impurity but it is polymerized with very high yield. It could be concluded that monomer with lower generation dendronized group can polymerize easily (M1 & M3) than higher generation dendronized group (M5). [56] Moreover, the monomer containing six carbon chain linker polymerize more easily than the monomer containing two carbon linker. [18,56] Rumetal carbine complex formation in the initiation step and bulky dendronized side groups inhibiting effect during carbene intermediate formation might be the reason behind the findings. [58] However, it was previously demonstrated that self-assemble and self-organization of dendritic monomer can provide a supramolecular reactor for the polymerizable group concentration and yields higher conversion. [74] Overall, it seems that aggregate size, packing efficiency of each monomer and monomers containing a flexible spacer affect the polymerization process. [51,74] Polymerization was confirmed by the total disappearance of olefine peaks at 6.3-6.6 ppm and new peak appears at 4.0-5.0 ppm from the double bond of the polymer backbone. [75] 1 H NMR spectra of the polymer of six-carbon linker 0.5 generation monomer M3 is given in Figure 4. The manifestation of vinylic hydrogen newly appeared by the ROMP pathway resulted in both cis-and trans-double bond formation and the integration of these two signals in 1 H NMR indicates the relative cis/trans ratio in the polymer backbone. The stereoselectivity of cis/trans-isomer of the synthesized polymers was calculated by the ratio of 1 H NMR signals integration of cis and trans proton at 5.6-6.2 ppm. The observed integration of 1 H NMR signal of proton represents that trans proton peak is much greater than the cis proton peak of each polymer. [76] For example, polymer P1 showed higher trans configurations than cis configurations with trans/cis ratio 1.75. The ratio of trans/cis isomer for synthesized polymers is shown in Table 1. The reason is that the trans configuration is more favorable than cis configurations that are formed by the double bonds (C ¼ C) of the backbones in the polynorbornene. [55,77,78] Moreover, average trans content in ROMP is significantly dependent on monomer concentration and it increases in the diluted monomer. [78] 1 H NMR end group analysis technique was used for the determination of the number average molecular weight (M n ) of the purified polymer by comparing with the assumed Figure 3. Structure of synthesized dendronized polymers. molecular weight. [79] Degree of polymerization is calculated from the integration of the olefin (-CH ¼ CHcis/trans-) peaks compared with the styrene end group of the Grubbs catalyst in the 1 H NMR spectra of the polymer. The original carbene moiety connected phenyl end group of the catalyst is visible at catalyst approximately 7.2-7.5 6 ppm and other branching peaks remained the same. Integration for phenyl group is taken as 1and therefore for each proton integration is 0.2. Integration of the (-CH ¼ CHcis,trans ) peaks compare to the Grubbs catalyst belonging styrene end group is 20.07 for two protons and 10.03 for each proton. Hence the number of repeating units is 50.175. Molecular weight of polymer was calculated by multiplying the molecular weight of the monomer and degree of polymerization ¼19,086 g/mole. Our targeted molecular weight was 20,000 which is very close to our calculated molecular weight ( Table 1).
The molecular weight (M n ) of the synthesized polymer has also been determined by GPC. However bimodal distribution of the polymers was observed and with a > 1.4 proves the uncontrolled fashion of polymerization. We performed different polymerization solvent and reaction conditions most probably solvent chelation and backbiting or cross-metathesis formation after very long reaction times though the polymerization result in higher . It should also point out that the self-assemble and strong aggregation of dendritic monomers might also slow down to migrate from their assembly to the Ru-carbene intermediate complex.
The catalyst efficiency on molecular weight was investigated. [80] It is well known that the initiation efficiency of Grubbs catalyst 3rd generation is 10 times higher than Grubbs 2nd generation catalyst. [81] So, it is expected that Grubbs 3rd generation catalyst control the polymerization by initiation and propagation efficiencies and form narrow distribution. [82] Increasing the tertiary amine concentration enhances the complexation with Ru-center after exchange the pyridine-ligand complex present in Grubbs 3rd generation. [83,84] However, we did not see dramatic effect on the polymerization of P3. Interestingly increasing the higher generation dendronized branching, P5, Grubbs 2nd generation catalyst works better.  The catalyst efficiency on the polymerization kinetic was also investigated. The monomer M1 possess the highest polymer efficiency was also investigated with Grubbs 2nd generation (G2) and the Grubbs 3rd generation (G3) catalysts for monitoring the total monomer (M1) conversion to P1 polymer by ROMP reaction. 1 H NMR spectroscopy technique (CDCl 3 ) was applied using n-tetradecane as internal standard. [85] Figure 5 illustrates that disappearance of the monomer H a proton at 6.44 ppm. Then appearing of new polymer signal H a (trans) proton signal at 6.02 ppm and H b (cis) proton signal at 5.72 ppm.
According to NMR result, 86.33% of the monomer was converted to the polymer by G2 catalyst within 1 min where trans and cis were found equal (trans50%-cis 50%) in the polymer backbone. The polymerization raised to 99.89% conversion after 5 min and determined trans and cis were 60% and 40% respectively. Finally, 100% conversion was reached at 15 min with 50% trans and 50% cis during efficiency monitoring of G2 catalyst. However, during the efficiency test of G3 upon monomer M1, polymerization has been completed (100%) within 1 min and their percentage of stereoisomers was found as trans 57%-cis 43%.
Although G2 catalyst was shown to have tremendous activity in a large number of ROMP reactions, it generally provides polymers with high molecular weight, which is uncontrolled, and broad polydispersities. [81,86] It is reported that the initiation capacity of G3 catalyst is much faster than G2 catalyst. [46] Because G3 catalyst has strongly ligated to the N-heterocyclic carbenes with weakly coordinating pyridine and exhibited fast initiation kinetics due to the unstable nature of the pyridine ligands. [87] Here, the same conclusion was observed at least for that monomer M1 according to the literature.

The catalytic activity of ROMP-based PAMAM polymer encapsulated Cu nanoparticles
In a typical example, P5 encapsulated Cu nanoparticles were prepared to investigate the catalytic activity of the complex. Initially, P5 solution was stirred for 20 min with CuSO 4 solution to form PAMAM Cu 2þ complex which turned to ROMP based PAMAM encapsulated Cu nanoparticle. Two different P5 encapsulated Cu nanoparticles were prepared (N1 and N2) and details were given in supporting info Table S1. At the beginning, first the calibration curve of 4-nitrophenol was drawn using UV-vis spectrometer ( Figure S35). The formation of colored complexes was also observed by eyes. (Figure  6) Cu-P5 complex was blue but after the addition of NaBH 4 the color of the solution turns to immediately to yellow and absorption decreased due to the formation of the nanoparticles. [41,88] Zeta potential of Cu 2þ with polymer P5 and Cu0 has been measured and found þ2 mV and À26 mV respectfully ( Figure S36) that represents the successful conversion of Cu 2þ polymer to Cu0. The method and reaction conditions used in 4-nitrophenol reduction are given in Table 2.
The actual absorption of 4-nitrophenolate ion and 4-aminophenol is 400 nm and 310 nm, respectively. [89] In presence of 2.44 Â 10 À12 m MP5 encapsulated Cu nanoparticles catalyst (CN1), resulted in gradual decrease of absorption k 400 nm and increase the absorption band 310 nm which means that 4-nitrophenol is gradually reducing to 4-aminophenol in 90 min after addition of NaBH 4 À . On the other hand, increasing the concentration of Cu-P5 complex 100 fold resulted in a six time faster reduction. Thus, it was observed that reduction time for 4-nitrophenol is proportionally related to amount of P5-Cu complex usage. Cu amounts in the polymer complex were also determined by atomic absorption spectrometry (AAS) to speculate about the catalytic activity of the complex. Cu amounts for the complex that show slower reduction time was found to be 0.4832 ppm and faster reduction time was 25 ppm. After the addition of a catalyst, disappearance of 4-nitrophenol absorption peak at 400 nm was observed and 15 min the peak was completely disappeared for the highest catalyst concentration (Figure 7). Characteristic band for the 4-aminophenol at k 310 nm did not increase efficiently and the reason might be the complexation of 4-aminophenol with the polymer complex.

Conclusion
In total four different dendronized ROMP type polymers were synthesized and characterized. It could be concluded that monomer with small generation containing six carbon chain linker can polymerize more easily than monomer containing two carbon linker. Moreover, exo-isomer of oxanorbornene polymerizes rather than endo isomer of oxanorbornene containing dendrimer. The polydispersity of the polymer was found to be 1.28-2.04. Unsuccessful polymerization was observed for the 1.5 generation dendritic monomer, P4, possessing six carbon linker. Kinetics studies for monitoring the total monomer (M1) conversion to P1 polymer was established using Grubbs G3 and G2 catalyst. Fast initiation and conversion efficiency was observed for G3. ROMP-PAMAM encapsulated Cu nanoparticle was synthesized for 2.5 generation dendronized polymer, P5, and monitored by UV-vis spectrometer. The catalytic activity of the particle with NaBH 4 for reducing 4-nitrophenol to 4aminophenol was observed and reduction performance was significantly dependent on the catalyst concentration.

Funding
Financial support by the Turkish Govt. Scholarship (No. 13BD110201), is gratefully acknowledged.   Figure 7. UV-vis spectra for the reduction of 4-nitrophenolate ion to 4-amino phenol by different concentration of catalyst, recipe CN1 and CN2.