With these cyclopropene glutamate derivatives in hand
With these cyclopropene-glutamate derivatives in hand, we tested their ability to ligate with popular bioorthogonal reagents (Fig 3). First, we tested their ability to ligate with a tetrazine (3,6-Di-2-pyridyl-1,2,4,5-tetrazine) via an inverse electron demand Diels-Alder reaction by monitoring the characteristic tetrazine absorbance peak at 520 nm. The ligation rate (second order rate constant, k2) between Boc- and ester-protected 2 with is 0.0001 M−1s−1 in MeCN (Fig. S6 in the ESI) which improves by 20-fold (k2 = 0.002 M−1s−1 1:1 PBS/MeOH) for cyclopropene-amino pi3 kinase 4 (Fig. S7 in the ESI) possibly due to both a relief in steric strain and diminished electron-withdrawing ability of carboxylate relative to the ethyl ester. Such an effect of cyclopropene-C3 substituent’s electron-withdrawing nature by inductive effect is known , . Next, we subjected the sterically strained (at C1) cyclopropene 9 to tetrazine ligation; however, we could not detect any measurable ligation between cyclopropenes 9 (or 11) (Fig. S8–S9 in the ESI). This suggests that inserting methylene units between the cyclopropene-C1 and substituents at C1 improves the tetrazine ligation.
On the other hand, photoclick chemistry presents an alternative to utilize strained cyclopropenes such as 11 for bioorthogonal applications . For this, we synthesized the 2-(4-methoxyphenyl)-5-phenyl-2H-tetrazole and assayed its light-dependent ligation with cyclopropene 11 using an HPLC/MS assay (Fig. S10–12 in the ESI). We observed the expected ligation product (Rt ∼ 18 mins), as a mixture of diastereomers, between the cyclopropene-glutamate 11 and the tetrazole in the first 15 mins, which reached saturation at around 120 mins. We also observed the TFA (cyclopropene 11 was used as a TFA salt) and tetrazole ligation product (Rt = 21 mins). Such carboxylic acid initialized tetrazole ligation are known and has been utilized for protein ligation , , .
To conclude, we have synthesized the second generation of cyclopropene-analogs of the amino acid neurotransmitter glutamate. They are stable in solution, on concentration, and in the presence of high concentrations of the biological nucleophile l-glutathione. Compared to the first generation of cyclopropene-glutamate, inserting a methylene spacer or substituting the α-proton with a methyl group significantly improves the stability of cyclopropene-glutamates. This synthetic adjustment also makes the corresponding starting material alkynes stable to silica-gel; thereby, reducing the ten steps required for the first-generation cyclopropene-glutamate synthesis to either four or six steps for the second-generation. Overall, these improvements combined, allowed us to obtain the cyclopropene-glutamate 4 and 11 in ∼ 0.5 g scale quantities which is >100-fold higher than the first-generation. Lastly, it opens the possibility of exploring additional substituents at the α-position, e.g., fluorine, trifluoromethyl, to study their biological properties. Future efforts in the lab are dedicated to obtaining isolated diastereomers of the cyclopropene-glutamates by generating enantiomerically pure alkyne 8 , , resolving diastereomers at cyclopropene-C3 using a chiral reagent  or testing the efficacy of chiral rhodium catalyst  for mono-substituted (at C3) cyclopropenes.
Acknowledgments We thank Dr. Bela Ruzsicska, Director of the analytical instrumentation laboratory, Stony Brook University, and the Stony Brook University Institute for Chemical Biology and Drug Discovery for maintaining and acquiring high-resolution mass spectrometry data. We thank Stony Brook University NMR coordinators Francis Picart, Dr. James Marecek and Dr. Fang Liu for maintaining the NMR facility. We thank Prof. Nicole S. Sampson for access to the plate-reader for conducting kinetic experiments, and graduate student Wang Yao in the lab of Prof. Ming-Yu Ngai for carrying out chiral HPLC measurements. Lastly, we thank John A. Mannone for assistance with this work. This work was supported by a grant to S.T.L. from the National Science Foundation 1451366.