Propargylamine

A controlled pore glass support (CPG) derivatized with 2′-O-TBDMS-G, 2′-O-methyl-G, deoxycytosine (dC) or deoxythymidine (dT), 5′,N-protected 2′-O-methylribo- (A, C, G or U), 2′-O-TBDMS-ribo (A, C, G or U) and deoxyribo (dA, dC, dG or dT,) phosphoramidites, 1-O-dimethoxytrityl-hexyl-disulfide,1′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Thiol-Modifier C6 S–S) were purchased from Glen Research Inc (Sterling, VA, USA). (Pyrene-1-yl-methyl)amine hydrochloride, N,N-diisopropylethylamine (DIPEA), trileucine, α-tocopherol, propargylamine, hexamethylenediamine, were purchased from Sigma-Aldrich (St. Louis, MO, USA), N,N′-disuccinimidyl carbonate (DSC), cholesterol chloroformate, folic acid, oleylamine were purchased from Acros Organics (Geel, Belgium), Cy3-azide, 10 mM Cu(II)-TBTA Stock in 55% DMSO, ascorbic acid were purchased from Lumiprobe (Russia), dodecamethylenediamine was from Fluka (St. Louis, MO, USA), 12-amino-1-dodecanol was from TCI Chemicals (India), and sodium dodecaborate Na₂[B₁₂H₁₂] was from AviaBor (Dzerzhinsk, Russia). N-(2-hydroxyethyl)phenazinium chloride was obtained in ICBFM SB RAS as described in previous work [44 (link)]. All solvents (THF, DMSO, CH₃CN (various vendors)) were dried by 3 Å molecular sieves or by distillation and stored over CaH₂. Kieselgel F254 thin-layer chromatography plates were purchased from Merck (Kenilworth, NJ, USA).

Solvents were purchased from Iris Biotech, Marktredwitz, Germany (DMF, DCM) or Sigma-Aldrich, Burlington, MA, USA (MeOH, Et2O, THF). THF was distilled before use. Other solvents were used without further purification. Fmoc-protected aminoacids ((Boc-Lys(Fmoc)-OH, Boc-D-Lys(Fmoc)-OH and Fmoc-Har(Pbf)-OH), TFA and TIS (triisopropylsilane) were purchased from Iris Biotech. Coupling reagents as well as preloaded resin Fmoc-Arg(Pbf) with 0.55 mmol/g loading were obtained from Activotec, Cambridge, UK. Propargylamine, 2-Methyl-3-butyn-2-amine and N-(9H-fluoren-9-ylmethoxycarbonyloxy) succinimide (Fmoc-OSu) were purchased from Sigma-Aldrich. Imidazole-1-sulfonyl azide hydrochloride was provided by Trimen Chemicals, Łódź, Poland.
All researched compounds 1L, 1D, 2L, 2D, 3L, 3D, 4L and 4D were obtained manually using solid-phase peptide synthesis according to standard procedures employed for the Fmoc strategy [37 (link)]. All presented general procedures are described for typical synthesis at 0.1 mmol scale.

Purified L-α-phosphatidylcholine (eggPC) and L-α-phosphatidyl-DL-glycerol (eggPG) were purchased from Avanti Lipids Polar Inc. (Alabaster, AL, United States). Biobeads SM-2 (20–50 mesh size) were obtained from Bio-Rad Laboratories Inc. (Hercules, CA, United States). Triton X-100, sodium cyanoborohydride, and propargylamine were purchased from Sigma-Aldrich (Steinheim, Germany). Hyaluronic acid sodium salt (M_r = 10 kDa, sHA) was procured from LifeCore Biomedial (Chaska, MN, United States). HS–(CH)₁₁–EG₃–OH (EG₃OH) and HS–(CH)₁₁–EG₆–N₃ (EG₆N₃) were obtained from Prochimia (Sopot, Poland). Alkylated RGD peptide (sequence: GRGDSP) was purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). Dialysis tube (MWCO: 3,500 Da) was purchased from VWR (Radnor, PA, United States). Integrin α_IIbβ₃ was extracted from outdated human blood platelets obtained by the Red Cross Germany, according to the protocol from Müller et al. with modifications (Müller et al., 1993 (link); Hu et al., 2000 (link)).

The synthesis of the arborescent PBG substrates, either randomly or chain end-functionalized with carboxylic acid groups, was described earlier [16 (link)]. To obtain alkyne-functionalized arborescent PBG substrates, propargylamine was coupled with the carboxylic acid moieties using N,N′-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt). For example, chain end carboxylic acid-functionalized G1PBG (0.202 g, 6.64 × 10⁻⁵ mol CO₂H, 1 eq.) was placed in a 10-mL round bottomed flask with 4 mL of dry DMF. DIC (52 μL, 3.32 × 10⁻⁴ mol, 5 eq.) and HOBt (45 mg, 3.32 × 10⁻⁴ mol, 5 eq.) were then added to the reaction, followed by propargylamine (8 μL, 1.33 × 10⁻⁴ mol, 2 eq., 98%, Sigma-Aldrich, Etobicoke, ON, Canada). The reaction was stirred overnight under N₂ at RT. The crude product was purified by preparative SEC in DMF, to ensure the complete removal of excess propargylamine. The purified product was concentrated to 4–6 mL, precipitated in methanol, centrifuged, and dried overnight under vacuum. Yield: 0.153 g (76%). SEC (DMF): M_n = 280,000 g/mol, M_w/M_n = 1.05 (MALLS). ¹H NMR (300 MHz, d₆-DMSO) δ: 8.2–7.8 (b, 1H), 7.28–7.20 (s, 5H), 5.03–4.89 (s, 2H), 4.35–3.80 (b, 1H), 3.80–3.70 (b, 2H), 3.10–2.90 (b, 1H), 2.33–1.70 (b, 4H), 1.35–1.10 (b, 10H), 0.80–0.70 (b, 3H).

All chemicals were used as provided without further purification unless noted otherwise. Tetrahydrofuran, triethylamine, oxalyl chloride, propargyl amine, sodium ascorbate, neocuproine, hexa (ethylene glycol) dithiol, glycerol ethoxylate, PEG 600 diol and hexafluorobenzene were purchased from Sigma Aldrich. Deuterobenzene, deuterated dimethyl sulfoxide and deuterochloroform were purchased from Cambridge Isotope Laboratories (CIL). Copper(ii) acetate was obtained from Acros Organics and 2,2-dimethoxy-2-phenylacetophenone (DMPA) from TCI. TBTA was synthesized following a previously published procedure.⁴³ Perfluorotripropylamine (FC 3283), methoxyperfluorobutane (HFE 7100), 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE 7500) and Krytox FSH were obtained from Miller-Stephenson. Acetonitrile (MeCN) was obtained from EMD and hexafluoroisopropanol (HFIP) from Alfa-Aesar. Thiourea was purchased from Chem-Impex International and ethanol, methanol, sodium hydroxide and hydrochloric acid were obtained from Macron Fine Chemicals.

All solvents were purchased from local commercial suppliers: DMF, DCM, MeOH, and Et₂O (puriss) were used without further purification; THF (puriss) was freshly distilled before the use according to the standard protocols. Fmoc protected amino acids and coupling reagents were purchased from Iris Biotech GmbH. Wang resin with preloaded Fmoc-Arg(Pbf) with a capacity of 0.57 mmol/g was obtained from Activotec (Comberton, Cambridge, UK). Propargylamine was purchased from Sigma-Aldrich and Fmoc-OSu was obtained from Merck. HPLC grade solvents can and MeOH were purchased from Merck.
All compounds 1–23 were obtained manually using mixed solid phase peptide synthesis and solution phase according to standard coupling procedures for Fmoc/tBu strategy. The quantity of Wang polystyrene resin was fixed to carry out the synthesis in a scale of 0.15 mmol.