Synthesis of Dendronized Poly(l-Glutamate) via Azide-Alkyne Click Chemistry

Poly(l-glutamate) (PGlu) was modified with a second-generation dendron to obtain the dendronized polyglutamate, P(Glu-D). Synthesized P(Glu-D) exhibited a degree of polymerization (DPn) of 46 and a 43% degree of dendronization. Perfect agreement was found between the P(Glu-D) expected structure and the results of nuclear magnetic resonance spectroscopy (NMR) and size-exclusion chromatography coupled to a multi-angle light-scattering detector (SEC-MALS) analysis. The PGlu precursor was modified by coupling with a bifunctional building block (N3-Pr-NH2) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) coupling reagent. The second-generation polyamide dendron was prepared by a stepwise procedure involving the coupling of propargylamine to the l-lysine carboxyl group, followed by attaching the protected 2,2-bis(methylol)propionic acid (bis-MPA) building block to the l-lysine amino groups. The hydroxyl groups of the resulting second-generation dendron were quantitatively deprotected under mild acidic conditions. The deprotected dendron with an acetylene focal group was coupled to the pendant azide groups of the modified linear copolypeptide, P(Glu-N3), in a Cu(I) catalyzed azide-alkyne cycloaddition reaction to form a 1,4-disubstituted triazole. The dendronization reaction proceeded quantitatively in 48 hours in aqueous medium as confirmed by 1H NMR and Fourier transform infrared spectroscopy (FT-IR) spectroscopy.


Synthesis of Bis-MPA-Acetonide
Bis-MPA (16.5 g, 120 mmol) and PTSA monohydrate (0.23 g, 1.2 mmol) were suspended in acetone (90 mL). 2,2-Dimetoxypropane (22 mL, 180 mmol) was added to this mixture and stirred at room temperature for 3 h. Afterwards, 2 M ammonium in ethanol (0.6 mL, 1.2 mmol) was added to the clear reaction mixture, which was stirred for five more minutes. The reaction mixture was evaporated under reduced pressure and the solid residue was dissolved in dichloromethane (150 mL). Organic phase was washed with deionized water (3 × 20 mL), dried over Na2SO4, and evaporated under reduced pressure. Yield: 17.8 g (83%).

Synthesis of γ-Benzyl-L-Glutamate NCA (BGlu NCA)
A 250 mL flame-dried round-bottom flask was charged with BGlu (6.2 g, 26 mmol) and triphosgene (3.7 g, 13 mmol) under argon. Then, dry THF (130 mL) was added and the reaction mixture was stirred at 55 °C for 90 min. The clear reaction mixture was concentrated under vacuum followed by precipitation in hexane. The product was crystallized from THF/hexane three times. Yield: 6.1 g (89%).

Synthesis of Poly(γ-Benzyl-L-Glutamate) (PBGlu)
BGlu NCA (5.0 g, 19 mmol) was dissolved in dry DMF (62 mL) under argon in an ice-bath [1]. A solution of hexylamine (0.38 mmol) in dry DMF (1 mL) was added and the reaction mixture was stirred in an ice bath for two days. Then, the reaction mixture was precipitated into cold deionized water. The product was collected by centrifugation, washed with water several times, and freeze-dried to obtain white powdery material. Yield

Synthesis of Poly(L-Glutamate) (PGlu)
PBGlu (1.0 g, 4.6 mmol benzyl ester groups) was dissolved in TFA (11 mL) under argon and anisole (2.75 mL, 23 mmol) was added to the mixture. The mixture was chilled in an ice bath and then MSA (10.2 mL, 158 mmol) was slowly added. The reaction mixture was stirred in ice bath for 20 min followed by stirring for 30 min at room temperature. Afterwards, the crude product was precipitated using ice-cold diethyl ether and collected by centrifugation. The product was dissolved in saturated NaHCO3, dialyzed (CE, 100-500 Da) against deionized water and finally freeze-dried to obtain PGlu as sodium salt. Yield: 0.6 g (86 %). SEC-MALS: Mn: 6.8 Da, Mw: 7.1 Da, ĐM: 1.04. MALDI-TOF MS: Mapex: 5012 Da.

Synthesis of 3-Azidopropane-1-Amine
3-Bromopropane-1-amine hydrobromide (4.38 g, 20 mmol) was dissolved in deionized water (67 mL) and then NaN3 (3.90 g, 60 mmol) was added. The reaction mixture was stirred at 80 °C for 20 h and, afterwards, it was concentrated under reduced pressure to one-third of the volume. The mixture was chilled in the ice bath and diethyl ether (50 mL) was added. Next, NaOH (2.40 g, 60 mmol) was slowly added while stirring. Under the clear organic phase an emulsion was formed which turned clear after addition of a small amount of brine (2 mL). The organic phase was separated and the water phase was washed with diethyl ether (3 × 20 mL). The combined organic phase was dried over Na2SO4 and diethyl ether was evaporated under reduced pressure. Yield: 1.5 g (75%).

Synthesis of Dendronized Poly(L-Glutamate) (P(Glu-D))
Dendron D (82 mg, 0.2 mmol) was dissolved in deionized water (8 mL) and P(Glu-N3) (67 mg, 0.16 mmol) was added. CuSO4 × 5H2O (6 mg, 0.02 mmol) and sodium-(+)-L-ascorbate (15 mg, 0.07 mmol) were added to the reaction mixture, which was then stirred at room temperature for two days.   Explanation to Figure S2: In the gHSQCad spectrum (2S, C) the integral values of the four CH3 signals 15 and 15ʹ are, on one hand, positive (red color) while, on the other hand, the lysine CH2 signalsʹ 6, 7 and 8 integral values are negative (blue color). Signals 6, 7, and 8 were assigned using 2D NMR spectra by the following procedure: doublet b (7.63 ppm), assigned to the amide proton signal in position Nα correlates with proton 5 (4.34 ppm) in the COSY spectrum ( Figure S2D). Proton signal 5 also correlates with the signal at 1.62 ppm (signal 6). According to the gHSQCad spectrum the proton signal at 1.62 ppm (signal 6) is correlated to the carbon signal at 32.0 ppm. The signal of proton 6 (1.62 ppm) is, according to the COSY spectrum, correlated to the proton signal at 1.25 ppm, which was, thus, assigned to position 7. The gHSQCad spectrum shows correlation between proton signal at 1.25 ppm and carbon signal at 22.3 ppm. The remaining CH2 signal in the gHSQCad spectrum was assigned to the CH2 group in position 8 and the assignation was confirmed by COSY, where correlation between signals 8 and 9 was observed.
Next, the carbon signal at 27.8 ppm was assigned to a CH2 carbon according to the gHSQCad spectrum and it was observed that this carbon signal correlates to the proton signal at 3.87 ppm. COSY spectrum shows correlation between the amide proton a signal and the proton signal at 3.87 ppm which was, therefore, assigned to the position 3. Furthermore, the proton signal 3 also correlates with the signal at 3.10 ppm, which was thus assigned to the acetylene proton 1. In the gHSQCad spectrum an anomaly was observed for the signal 1. For this signal, assigned to the acetylene CH group, the integral value was expected to be positive (red color), however, the spectrum shows a negative integral value. The reason for this anomaly lies in a very high one-bond C-H coupling constant value for the acetylene group (~250 Hz) [2]. The gHSQCad experiment was set for the coupling constant of 150 Hz, which agrees sufficiently with most C-H systems.   Figure S4. Structures of the species as assigned from the PBGlu MALDI-TOF mass spectrum ( Figure S3D).