Synthesis of FX and preparation of FX@HP

1. CXCR4 antagonistic monomer X was synthesized using the same method as previously reported[1]. Synthesis of Tri-tert-butyl-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate A: Boc₂O (3.27 g, 15.0 mmol) in CH₂Cl₂ (75mL) solution was added dropwise for 2 h to a solution of cyclam (1.00 g, 5.0 mmol) in CH₂Cl₂ (200 mL) at 0 °C. The mixture was then warmed to room temperature and stirred overnight. After concentrated and purified by chromatography (AcOEt→10:1 AcOEt:CH₃OH), tri-Boc cyclam (A, 2.10 g) as a white set foam was obtained.

2. Synthesis of Tri-tert-butyl-11-(4-(chloromethyl)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate B: The mixture of α,α′-dichloro-p-xylene (7.44 g, 41.6 mmol), anhydrous K₂CO₃ (1.44 g, 10.4 mmol) and A (4.17 g, 8.33 mmol) dissolved in CH₃CN solution (150 mL) was refluxed for 6 h before filtering and washed with acetone. The filtrate was concentrated to give a white solid that was heated with 2:1 hexanes:AcOEt and concentrated to a small volume to allow the excess dichloroxylene to crystallize out. The filtrate was further purified by chromatography (2:1→1:1 hexanes:AcOEt) and the desired mono-cyclam product (compound B, 4.31 g) as a white set foam was obtained.

3. Synthesis of Tri-tert-butyl-11-(4-((3-hydroxypropylamino)methyl)benzyl)-1,4,8,11-tetraazacyclo tetradecane-1,4,8-tricarboxylate C: B (2.14 g, 3.35 mmol), anhydrous K₂CO₃ (0.69 g, 5.0 mmol) and 3-amino-1-propanol (2.6 mL, 2.55 g, 33.5 mmol) were added in CH₃CN (15 mL) and the mixture was stirred overnight. The formed solid was filtered and washed with AcOEt (50 mL). The filtrate was washed with water (2 × 10 mL), saturated NaCl (10 mL), dried (MgSO₄), and concentrated to give a viscous liquid, which was then purified by chromatography (10:1 CH₂Cl₂:CH₃OH, adding 0.5% NH₃ later) to give the amino alcohol as a white foam.

4. Synthesis of Tri-tert-butyl-11-(4-((tert-butoxycarbonyl(3-hydroxypropyl) amino)methyl)benzyl)-1,4,8,11-tetraazacyclotetra decane-1,4,8-tricarboxylate D: To a solution of C (2.16 g, 3.18 mmol) in CH₂Cl₂ (30 mL), Boc₂O (1.08 g, 4.77 mmol) in CH₂Cl₂ (5 mL) was added and the mixture was stirred overnight. It was concentrated to give a light yellow liquid and purified by chromatography (1:1 hexanes:AcOEt) to obtain the Boc-protected amino alcohol as a white set foam.

5. Synthesis of Tri-tert-butyl-11-(4-((tert-butoxycarbonyl(3-(1,3-dioxoisoindolin-2-yl)propyl)amino) methyl) benzyl)- 1,4,8,11-tetraazacyclo tetradecane-1,4,8-tricarboxylate E: A mixture of D (0.89 g, 1.14 mmol), PPh3 (0.61 g, 2.3 mmol), phthalimide (0.19 g, 1.3 mmol), and anhydrous THF (15 mL) was heated under N₂ protection until all the solid was dissolved, before it was cooled to 0 °C. DEAD (0.37 mL, 0.41 g, 2.3 mmol) was added dropwise and the mixture was warmed to room temperature, after which it was concentrated to give a white solid. This was heated with 2:1 hexanes:AcOEt until all the solid was dissolved and filtered through silica gel, collecting the fractions that contained product. These fractions where concentrated to a small volume and any solid that crystallized out was removed by filtration. The filtrate was further purified by chromatography (2:1 hexanes:AcOEt) to obtain compound E, 1.03 g.

6. Synthesis of Tri-tert-butyl-11-(4-(((3-aminopropyl) (tert-butoxycarbonyl) amino) methyl)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate (X): E (1.03 g, 1.1 mmol) and NH₂NH₂ (0.36 mL, 0.36 g, 11 mmol) were added in CH₃OH (20 mL), and the mixture was stirred overnight. It was concentrated to give a white solid that was mixed with CH₂Cl₂ and filtered. This was repeated until no more amine was extracted from the solid. The combined filtrates were concentrated and purified by chromatography (10:1 CH₂Cl₂:CH₃OH, adding 0.5% NH₃ latter) to give a white foam (compound X, 0.66 g). 

7. Synthesis of FX[2]: In a typical polymerization reaction, a Boc-protected CXCR4 antagonistic cyclam monomer X (76.3 mg, 0.1 mmol) and N,N’-cystaminebisacrylamide (CBA) (26 mg, 0.1 mmol) were dissolved in 0.7 ml of methanol/water (7/3 v/v), and the polymerization was carried out at 50°C for 14 days. Then, another 4 mg of cyclam was added, stirring for an additional 2 days. After isolating by evaporation, the protecting Boc groups were removed by stirring in trifluoroacetic acid/dichloromethane solution overnight. The product was further evaporated, dissolved in acidified water (pH 4), and dialyzed against acidified water (pH 4) using dialysis membrane with a molecular weight cutoff (MWCO) of 1 kDa. Lyophilization was used to obtain the final product RX. Then, RX (30 mg) was dissolved in 2 mL methanol and mixed with methanol solution of HFBA (4.1 mg, 0.01mmol). Triethylamine (1.7 μL, 0.02 mmol) was added and the reaction mixture was stirred at room temperature under nitrogen in dark for 48 h. The mixture was diluted into water (pH 4, HCl) and dialyzed (MWCO 1 kDa) against water for 2 days before final lyophilization to obtain FX.

8. Preparation of HP: Human serum albumin (HSA) (100 mg) was dissolved in 50 ml ultrapure water with constant stirring at 37 ^oC, and 100 μl of dithiothreitol (DTT) (10 mg/ml) was added. After 15 min, PTX (10 mg/ml, ethanol as solvent) was added for another 2 h reaction until light blue opalescence is observed. The HSA-PTX (HP) aqueous suspension was extensively dialyzed with membrane (MW: 8 K) to remove any remnant DTT before lyophilization. 

9. Preparation of FX@HP: Different amounts of FX were added into HSA-PTX aqueous suspension before mixed with a pipette and further sonicated with a sonicator for twice. The FX@HP aqueous suspension was further kept at 4^oC after ultrafiltration.

Thanks for your request for the detailed protocol. Below are the original research articles where this protocol was used:

References:

[1] Y. Wang, S.T. Hazeldine, J. Li, D. Oupicky, Development of functional poly(amido amine) CXCR4 antagonists with the ability to mobilize leukocytes and deliver nucleic acids, Advanced healthcare materials, 4 (2015) 729-738.

[2] Z. Li, Y. Wang, Y. Shen, C. Qian, D. Oupicky, M. Sun, Targeting pulmonary tumor microenvironment with CXCR4-inhibiting nanocomplex to enhance anti-PD-L1 immunotherapy, Sci Adv, 6 (2020) eaaz9240.