General synthetic remarks and materials
Moisture-free conditions were employed only where indicated. Reagents and solvents were purchased from TCI America, ChemPep, Alfa Aesar, BDH, and Sigma-Aldrich, anhydrous whenever possible, and were used as received unless otherwise indicated. FluoroFlash fluorous solid-phase extraction cartridges were purchased from Fluorous Technologies Incorporated.
Design of alkylating agents
A generalized structure of Nimzyme-amenable substrate analogs as prepared in this manuscript is shown in Fig. 1c. A perfluoroalkanoyl moiety imparts the required fluorophilic character to allow for on-chip purification, and an arginine moiety ensures high ionization efficiency in NIMS . Avoiding reliance on the substrate moiety to impart ionizability also allows for direct comparison of NIMS peak heights to determine relative abundance of chemical species. A tri(ethylene glycol) linker was installed to improve enzyme–substrate accessibility and enhance the probe's solubility in water.
Nucleophilic substitution chemistry was chosen to couple substrates of interest to the probe's invariant portion because many biologically relevant substrates possess nucleophilic functional groups. Hence, we incorporated a p-toluenesulfonate ester (tosylate) leaving group into the invariant portion of the Nimzyme probe, resulting in alkylating agents 1 and 2 (Fig. 1d).
Synthesis of alkylating agents
A synthetic route to 1 and 2 was devised, which starts from readily available starting materials. A synthetic scheme, detailed synthetic protocols, and spectral data of all intermediates are reported in the Electronic Supplementary Material (Scheme S1 and S2 and Figs. S1–S11). Briefly, 2-(2-(2-aminoethoxy)ethoxy)ethanol—obtained by means of a Gabriel synthesis from 2-(2-(2-chloroethoxy)ethoxy)ethanol—was coupled to Fmoc-Arg(Pbf)-OH using conventional solution-phase peptide synthesis methodology. The Fmoc group was removed with diethylamine and the resulting amine was acylated with perfluorooctanoyl chloride under Schotten–Baumann conditions. The resulting alcohol was tosylated to afford 1 in 26 % yield with respect to Fmoc-Arg(Pbf)-OH (four steps). Lastly, the Pbf protecting group was removed with 90:10 TFA/MeOH to afford 2.
The alkylation and deprotection reactions were monitored by NIMS of 0.1 μL of the reaction mixture dissolved in 10 μL methanol. Fluorous solid-phase extraction (F-SPE) was performed on FluoroFlash 2 g cartridges as follows: The cartridges were preconditioned with 1 mL N,N-dimethylformamide (DMF) and 7 mL 80:20 MeOH/H2O, loaded with the crude alkylation reaction mixture, washed with 7 mL 80:20 MeOH/H2O, and eluted with 10 mL MeOH. Each F-SPE cartridge was used only once. Possible product isomerism was ruled out by LC/MS (see ESM). Because the quantities prepared here are too small to be accurately weighed, their yields were determined relative to a known concentration of Nz-OMe (12 in the ESM) by mixing them in a 1:1 ratio and determining the ratio of NIMS peak intensities. 12 was chosen as an internal standard because its mass does not overlap with any of the reagents, products, or possible side products and can reasonably be assumed to ionize similarly to 3 through 11. For amine nucleophiles (products 5, 6, and 11), using the hydrochloride form led to the formation of what was presumed to be “Nz(Pbf)-Cl” (NIMS calc'd for [M+H]+ 972.2; found 972.1; characteristic 3:1 M+H:M+H+2 ratio). Hence, we either purchased or generated the free base form of these substrates. Representative reactions using alkylating agents 1 and 2 are described in detail below. All others are described in the ESM; ‘Nz’ = 2-(2-(N
α-perfluorooctanoylargininamidoethoxy)ethoxy)ethyl (structural formula shown in Fig. 2a)
9—Ibuprofen Nz ester
Into a borosilicate test tube with stir bar, 5.7 mg ibuprofen sodium salt (25 μmol, 50 eq), 10 μL 50 mM Nz-OTs (2) in DMF, and 490 μL DMF were added. The mixture was stirred at 70 °C for 3 h. After cooling to room temperature (RT), 5 mL 1 M aqueous NaOH was added, the resulting solution was extracted with 3 × 5 mL chloroform, and each of the extracts was in turn washed with another 5 mL 1 M aqueous NaOH. A few crystals of NH3Cl were added to the chloroform extracts, which were filtered and evaporated in vacuo to yield a colorless residue which was taken up in MeOH 78 % yield.
Into a borosilicate test tube with stir bar, 50 μL 100 mM Nz(Pbf)-OTs (1) in DMF, 200 μL more DMF, and 53.1 mg (250 μmol, 50 eq) (1R,2R)-2-amino-1-(4-nitrophenyl)-1,3-propanediol were added. The mixture was stirred at 110 °C for 2 h, cooled to RT, and F-SPE purified. The eluent was evaporated in vacuo, redissolved in 2 mL 90:10 TFA/MeOH, and left to stir at RT for 12 h. The TFA/MeOH was evaporated under a gentle stream of nitrogen, leaving a white residue which was taken up in MeOH 45 % yield.
NIMS surface fabrication
The production of NIMS chips has been described elsewhere . Briefly, a silicon wafer is cleaned thoroughly with methanol, followed by anodic etching with 25 % hydrofluoric acid (w/v) in ethanol in a custom-made Teflon etching chamber using a current of 2.4 A for 15 min. Next, the chips are coated by adding the perfluorinated initiator liquid bis(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tetramethyl-disiloxane for 20 min. Excess initiator is blown off with nitrogen.
Enzymatic activity assay
Overnight cultures of Escherichia coli DH1 harboring either pBbB8k-GFP or pBbB8k-CAT (the construction of these is described in the ESM) were diluted 1:10 in LB with 50 μg mL−1 kanamycin, and grown for 1 h at 37 °C, after which they were induced with 0.2 % (w/v) arabinose. After 2 h of growth, 0.5 mL of the culture was centrifuged at ∼12,000×g for 1 min, the pellet was resuspended in 0.25 mL aqueous 50 mM sodium phosphate (pH 7.5), and sonicated for 30 s. Acetyl-CoA was added to a final concentration of 2 mM and 11 to a final concentration of 0.5 mM. The mixture was mixed and incubated at room temperature for 5 min and subsequently quenched with an equal volume of methanol. A 0.3-μL droplet of the mixture was spotted onto a NIMS chip and the excess liquid removed 5 s later by touching it with a Kimwipe (Kimberly-Clark). The spotted area was washed with 2 × 1 μL deionized water. The NIMS chip was taped to a modified standard matrix-assisted laser desorption/ionization (MALDI) plate, which was then loaded into an Applied Biosystems 4800 MALDI time of flight (TOF)/TOF mass spectrometer. Agilent ESI-L Low Concentration Tuning Mix was spotted nearby on the NIMS chip to allow for mass calibration of the instrument. Spectra were acquired in manual mode and positive polarity.