Selectivity and stability of N-terminal targeting protein modification chemistries

Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no ‘one size fits all’ approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field.


S15
A solution of compound 8 (0.44 g, 1.4 mmol, 0.8 equiv.) in acetone (1 mL) was added to a mixture of K2CO3 (0.34 g, 2.5 mmol, 1.5 equiv.) and compound 21 (0.40 g, 1.6 mmol, 1.0 equiv.) in acetone (4 mL, 0.3 м), and the reaction mixture was stirred at 60 °C for 17 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure and the residue obtained was dissolved in methanol (9.7 mL, 0.1 м) and stirred at room temperature for 1 h. The reaction mixture was then concentrated under reduced pressure, and saturated aqueous NaHCO3 solution (20 mL) was added. The aqueous was extracted with
After cooling to room temperature, the reaction mixture was diluted with CH2Cl2 (50 mL) and the organics washed with saturated aqueous NaHCO3 solution (2 × 20 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (50% S16 EtOAc:Petrol, Rf 0.50 in 66% EtOAc:Petrol), and pure fractions were concentrated under reduced pressure to afford a yellow oil (0.61 g), which was used immediately in the next step without characterisation.

4-(2-(2-(2-Methoxyethoxy)ethoxy)ethoxy)benzaldehyde (7)
A solution of compound 8 (280 mg, 880 µmol, 1.1 equiv.) in THF (0.5 mL) was added to a mixture of K2CO3 incomplete, and so a further portion of K2CO3 (0.28 g, 2.0 mmol, 2.4 equiv.) was added and the reaction S18 mixture was stirred at 66 °C for 48 h. The reaction mixture was allowed to cool to room temperature, then water (20 mL) was added and the aqueous was extracted with CH2Cl2 (3 × 20 mL). The combined organics were dried over MgSO4, filtered, and concentrated under reduced pressure. The brown oil obtained was purified by flash column chromatography (33-50% EtOAc:Petrol, Rf 0.18 in 33% EtOAc:Petrol), and pure fractions were concentrated under reduced pressure to afford the title compound (0.16 g, 0.6 mmol, 71%) as a pale-yellow oil with spectroscopic data in accordance with the literature. 10   that appear to be a slightly red/orange colour) and transferred into 5 mL 2×YT media containing ampicillin (100 µg/mL) and chloramphenicol (35 µg/mL). The inoculated media was incubated at 37 °C, 180 rpm overnight. 2 mL of the overnight culture was then taken, the cells were pelleted by centrifugation (2 min, 1000 S19 g), and 1.5 mL of the supernatant was removed. The cells were resuspended in the remaining 500 µL, and 50% glycerol (750 µL) was added, pipetting the solution several times to ensure mixing. The resulting solution was flash frozen using liquid N2 and the resultant glycerol stock stored at -80 °C.

Protein expression
Protein expression: The glycerol stocks described above were plated onto ampicillin-and chloramphenicolcontaining LB agar and grown at 37 °C overnight. A single colony was then selected and grown in 5 mL of 2×YT, containing the appropriate antibiotics, at 37 °C and 200 rpm for ~ 8 hrs. This culture was then transferred into 100 mL 2×YT, containing the appropriate antibiotics, and incubated overnight at 37 °C and 200 rpm. After this time, as a preliminary test to confirm that cells contained both the desired plasmids, a portion of the cells were pelleted via centrifugation, and were found to be red/pink in colour.
10 mL of the overnight culture was then used to inoculate 1000 mL of 2×YT, containing the appropriate antibiotics, in a non-baffled conical flask, and the flask was incubated at 37 °C and 180 rpm until OD600 ≈ 0.6.
The culture was then cooled to 16 °C and shaken at 150 rpm before addition of a solution of isopropyl ß-D-

Screening of reagents under differing conditions
Each of the reagents 1-7 were used to modify RNase A under the conditions outlined in the General Procedures A, C, D and E for 23 h. Compound

Conversion (%)
General Procedure A General Procedure C General Procedure D General Procedure E

Optimisation of 2-EBA concentration
To minimise off-target reactivity with 2-EBA 5, RNase A and myoglobin were modified according to General

Protein panel modification
For the modification of RNase A and myoglobin: reagents 1-7 were applied under the conditions outlined in General Procedure A. For compound 7, 5 equiv. of sodium cyanoborohydride in 5 µL of water was also added, and compound 7 was added as a more concentrated solution (45 µL, 11 mM) to keep the total volume constant.
For the modification of Clostripain LC: As described above, on a 19.5 µL scale.
For the modification of CjX183-D WT: As described above, on a 22.9 µL scale.
For the modification of CjX183-D R51K: As described above, on a 30.6 µL scale.

Stability to competitive dipeptide
Stability of protein conjugates upon addition of DiAla as a competitor S45 a). For the modification of RNase A with 100 equiv. reagent: RNase A was modified with compounds 1-7 according to General Procedure A, on a 200 μL scale. For compound 7, 5 equiv. of sodium cyanoborohydride in 10 µL of water was also added, and compound 7 was added as a more concentrated solution (90 µL, 11 mM) to keep the total volume constant. The reaction was then split into 3 aliquots (65 µL).
To these, were added a different concentration of L-alanyl-L-alanine (DiAla; 26