A Facile Method to Quantify Synthetic Peptide Concentrations on Biomaterials

While it is well understood that peptides can greatly improve cell–material interactions, it is often challenging to determine the concentration of the peptide which decorates a material. Herein, we describe a straightforward method using readily, synthetically accessible Fmoc peptides and commercially available reagents to measure the concentration of peptides on nanoparticles, surfaces, and hydrogels. To achieve this, the Fmoc protecting group from immobilized peptides is removed under optimized basic conditions. The dibenzofulvene released can be quantified by HPLC or UV–vis spectroscopy, enabling a direct experimental measurement of the concentration of the peptide. We show that we can measure the concentration of a BMP-2 peptide mimic on a hydrogel to determine the concentration required to stimulate osteogenesis of human mesenchymal stem cells. We envision that this methodology will enable a more thorough understanding of the concentration of synthetic peptides decorated on many biomaterials (e.g., nanoparticles, surfaces, hydrogels) to improve deconvolution of the interactions at the cell–material interface.


Fmoc-Lys(methacrylamide) synthesis
To a 50 mL round bottom flask was added Fmoc-Lys(Boc) (5 g, 10.7 mmol, 1 eq) and a magnetic stirrer bar.Then, a 20 mL mixture containing 1:1 (v/v) trifluoroacetic acid (TFA)/dichloromethane (DCM) and 1 mL of deionized (DI) water was added.The reaction was allowed to stir at room temperature for 2 hours.The TFA/DCM mixture was then removed via rotary evaporation before the crude residue was resuspended in DCM and removed via rotary evaporation twice.The crude, Fmoc-Lys was then used in the next step without purification.
Fmoc-Lys-COOH was suspended in anhydrous DCM (20 mL).The flask was cooled in a sodium chloride ice bath, then N,N-diisopropylethylamine (3.72 mL, 21.4 mmol, 2 eq) was added dropwise.A small aliquot of the reaction mixture was removed with a Pasteur pipette, mixed with water, and the pH tested.If the pH was below pH 8, an additional 1 mL of N,N-diisopropylethylamine was added.This was repeated until pH was greater than 8.The flask was then sealed under nitrogen, then methacryloyl chloride (1.25 mL, 12.8 mmol, 1.25 eq) (was added dropwise.The reaction flask was covered with aluminium foil to protect the reaction from light and allowed to stir overnight and warm slowly to room temperature. The reaction mixture was then diluted with DI water (20 mL) and transferred to a separating funnel.The organic layer was washed with DI water (2  20 mL) and brine (20 mL), then dried with anhydrous sodium sulphate, filtered and concentrated via rotary evaporation.The crude mixture was then diluted into HPLC grade 30% acetonitrile in water (v/v) containing 0.1% (v/v) formic acid before being purified using reverse phase preparative high performance liquid chromatography (prep-HPLC) using the gradient below.Eluted fractions were collected based on UV absorbance measurements at 220 and 254 nm with pure Fmoc-Lys(methacrylamide) fractions confirmed using liquid chromatography/mass spectroscopy (LC-MS).

First-amino acid loading to the resin
To a 12 mL syringe fitted with a polypropylene frit is added 2-chlorotrityl chloride resin (0.5 g, 1.14 mmol/g loading).The resin is washed with dichloromethane (3  5 mL), then allowed to swell in dichloromethane (5 mL) on an orbital shaker (200 rpm) for 15 minutes.
Meanwhile, a solution containing Fmoc-Cys(Trt)-OH (3 eq) in 1:1 (v/v) N,N-dimethylformamide/dichloromethane is prepared, followed by the addition of S5 N,N-diisopropylethylamine (6 eq).The dichloromethane solution is expelled from the resin, then the Fmoc-Cys(Trt)-OH mixture is added to the resin.The resin is shaken on an orbital shaker for 16 hours at room temperature, then the solution is expelled.The resin is washed with dichloromethane (5  5 mL), then a mixture containing 9:1:0.5 (v/v) dichloromethane/methanol/N,N-diisopropylethyl amine is added to the resin, which is shaken on an orbital shaker (200 rpm) for 30 minutes.The solution is expelled, then the resin is wash with dichloromethane (3  5 mL) and N,N-dimethylformamide (3  5 mL).

Fmoc deprotections
To the resin is added 20% piperidine in N,N-dimethylformamide (5 mL), which is then shaken on an orbital shaker (200 rpm) for 1 minute then expelled.This step is repeated, but for 15 minutes.The resin is then washed with N,N-dimethylformamide (5  5 mL).

Fmoc-amino acid couplings
A mixture containing the next protected Fmoc amino acid to be coupled (3 e) is prepared by dissolving in a 0.5 M HATU in N,N-dimethylformamide solution (3 eq).Then, N,N-diisopropylethylamine (6 eq) is added to this mixture, which is stirred or sonicated to dissolve the Fmoc-amino acid.This mixture is then added to the resin, which is shaken on a orbital shaker (200 rpm) for 30 minutes.The solution is expelled, then the resin washed with N,N-dimethylformamide (5  5 mL).A Kaiser test is performed to determine if the reaction has gone to completion.If a positive Kaiser test result is obtained (i.e., blue-purple coloured solution or resin), this coupling step is repeated.

Figure S4 .
Figure S4.Characterisation of products from the Fmoc-deprotection.The product from the Fmoc deprotect of Fmoc-Arg(Pbf)-OH was purified via preparative HPLC. a The UV-Vis spectra of the isolated Fmoc deprotection product shows good agreement against a DBF standard and b an identical retention time to the DBF standard.
round bottom flask (50 mL), rinsed with trifluoroacetic acid (3  2 mL) and concentrated using rotary evaporation.Diethyl ether (45 mL) is added to the concentrated peptide to give a precipitate.The diethyl ether is decanted, and the precipitate then dissolved in 25% acetonitrile in HPLC grade water containing 0.1% (v/v) formic acid.Purification of the peptide was performed using reverse phase preparative HPLC (Shimadzu Prominence 1,2-ethanedithiol, 2.5% triisopropylsilane, 2.5% milliQ H 2 O and 92.5% trifluoroacetic acid (v/v) is added to the resin.The resin is shaken on the orbital shaker (200 rpm) for 3 hours.The solution is then S6 expelled into a