Discovery of new mutually orthogonal bioorthogonal cycloaddition pairs through computational screening

The sydnone-dibenzocyclooctyne and norbornene-tetrazine cycloadditions are both bioorthogonal and mutually orthogonal, used for simultaneous labeling of two targets.


Small molecule reaction kinetics
The reaction rates between diaryltetrazine 13 and 5-norbornene-2-endo-acetic acid 12 were measured under pseudo-first order reaction conditions by using 10-80 fold excess 5-norbornene-2-endo-acetic acid 12 in water/methanol mixtures. Stock solutions of diaryltetrazine 13 were prepared in 9/1 water/methanol (0.1 mM) and 5-norbornene-2-endo-acetic acid 12 in methanol (1, 3, 5 and 8 mM). Equal volumes (0.5 mL each) of stock solutions, resulting final concentration 0.05 mM of tetrazine and 0.5, 1.5, 2.5, 4 mM of norbornene were mixed by maintaining MeOH:H 2 O (55:45). The exponential decay in UV absorbance of tetrazine 13 at 320 nm was measured over time. The pseudo-first order reaction kinetics for DIBAC 10 and benzyl azide were measured by following the exponential decay in UV absorbance of DIBAC 10 at 310 nm upon reaction with 20-100 fold excess benzyl azide in methanol-water (55:45). Stock solutions, 0.05 mM of DIBAC 10 in methanol and 1, 2, 3, 4 and 5 mM of benzyl azide in 9/1 water/methanol were prepared. The pseudo-first order reaction kinetics for BARAC 6 and benzyl azide were measured by following the exponential decay in UV absorbance of BARAC 6 at 306 nm upon reaction with 20-100 fold excess benzyl azide in acetonitrile-water (1:1). Spectra were recorded on a Shimadzu UV-Vis-NIR spectrometer. All the data were recorded at 23 º C using spectral band-width (SBW) = 1.0 nm, path length = 1.0 cm with increment in data point collection at 2 seconds. The observed rate constants k′ were determined by fitting each data set to a single-exponential equation. The k′ values were then plotted against the concentration of norbornene 12 or benzyl azide and subjected to a linear fit to yield a plot with slope k 2 , the second order rate constant. Each kinetic experiment was performed in triplicate and the three k 2 values were averaged. All data was processed on Origin8 software program. Due to direct overlap in UV absorbance of DIBAC or BARAC with N-phenyl sydnone, the rate constants were calculated from competition experiments between N-phenyl sydnone and benzyl azide with DIBAC or BARAC via NMR spectroscopy. On the basis of the product ratios of NMR competition experiments and the determined rate constants for the benzyl azide cycloadditions of DIBAC or BARAC, the rate constants for the N-phenyl sydnone cycloadditions of DIBAC or BARAC were obtained.

Cross-reactivity kinetics
The cross reactivity of DIBAC 10 with diaryltetrazine 13 and 5-norbornene-2-endo-acetic acid 12 with sydnone 1 was monitored via NMR spectroscopy at room temperature over a period of 24 h. No reaction was observed between either reaction pair after 24 h.

Preparation of protein conjugation
Bovine serum albumin (BSA) and Ovalbumin from chicken egg white (OVA) conjugates were prepared by treating the appropriate protein with the succinimidyl esters of either DIBAC (DIBAC-NHS) (CP-2033, Conju-Probe, San Diego, CA) or 5-norbornene-2-endo-acetic acid (Nor-NHS) (Sigma-Aldrich) using standard coupling conditions. In short, BSA and OVA (0.5 mL of a 20 mg/mL solution in PBS pH 7.4) were combined with DIBAC-NHS and Nor-NHS (100 µL of 20 mM solution in DMSO) respectively. The reaction mixture was incubated at 37 °C with shaking for 3 h then allowed to stand at rt for 12 h. Column purification was performed to remove excess DIBAC-NHS and Nor-NHS from the protein-conjugates. The reaction mixture was loaded onto a Zeba™ Spin Desalting Column, pre-equilibrated with 10 mL PBS, and the eluted fractions were collected.

Synthesis of sydnone derivatives
Sydnones 1 and 15 were synthesized according to literature procedure. 6 The 1 H and 13 C NMR spectroscopic data were consistent with previously reported values. (1) (15)

Synthesis of BARAC
Synthesis of BARAC 6 was carried out following the literature reported protocol. 8 All the intermediates were characterized and in agreement with the previously reported data. The final product was purified by semi-preparative HPLC (30% to 75% CH 3 CN in water over 35 min) and the 1 H and 13 C NMR spectroscopic data were identical to that previously reported in the literature. 8 Figure S1. HPLC trace of purified BARAC 6 using a gradient of 30% to 75% acetonitrile in water over 35 min.
DMF was removed under reduced pressure and the dye-conjugates were purified by semipreparative reverse phase HPLC (10% to 85% CH 3 CN in water over 35 minutes, 3mL/min flow rate). The identity and purity of the conjugates were confirmed by analytical HPLC and HRMS.   Figure S5. Determination of the second-order rate constant k 2 of diaryltetrazine 13 and 5norbornene-2-endo-acetic acid 12 via the same method as described in Figure S4. The calculated rate constant was 1.05 ± 0.04 M -1 s -1 .

General Procedure for reaction rate determination by NMR competition experiments:
To a solution of benzyl azide (100 µmol) and phenyl sydnone 1 (100 µmol) in solvent, DIBAC 10 (10 µmol) or BARAC 6 (10 µmol) was added. After 4 hrs of shaking, the solvents were evaporated and crude material was dissolved in CD 3 CN. A 1 H NMR spectrum was recorded to determine the ratio of the cycloadducts derived from benzyl azide and phenyl sydnone. DIBAC 10-benzyl azide cycloaddition was carried out in CD 3 OD:D 2 O (55:45) and BARAC-benzyl azide cycloaddition was conducted in CD 3 CN:D 2 O (1:1). Competition spectrum is shown in Figure S12. S37 Figure S14. The 13 C NMR spectra of cycloadducts 7 and 11.