Selective Iridium-Catalyzed Reductive Amination Inside Living Cells

Abstract

Given that amino groups are ubiquitous in bioactive molecules, abiotic routes to incorporate them into cellular species offer new opportunities to study and manipulate living systems. In the present work, we report the first biocompatible method to prepare 1°, 2°, or 3° amines selectively starting from an aldehyde and nitrogen precursor through iridium-catalyzed reductive amination. To prevent overalkylation, we developed a nontoxic self-immolative agent comprising 4-(1-aminoethyl)phenol that can condense with carbonyl groups and undergo 1,6-elimination upon reduction to the desired 1° amines. The use of an electron-poor half-sandwich Ir catalyst favored the formation of amine over alcohol products. To synthesize 2° or 3° amines, the aldehydes were combined with the appropriate 1° or 2° amine, respectively, under our standard reaction conditions. Our method is sufficiently mild to perform on proteins, as demonstrated by the conversion of aldehyde-containing allysine residues in bovine serum albumin to lysine. Importantly, we showed that Ir-catalyzed reductive amination could be applied inside living cells, such as by generating the alkaloid phenethylamine or calcium-reducing drug cinacalcet to elicit different biological responses. The amines formed via intracellular reductive amination were quantified by high performance liquid chromatography, revealing that turnover numbers of up to ∼20 were achieved. This work is expected to enable greater versatility and precision in transforming a wide range of aldehyde-containing entities within living environments, further expanding our biosynthetic chemistry toolbox.

Figure 1.

Structures of Ir complexes used in this study (A) and comparison of products obtained from the reaction of 1a with Ir and HCOONH₄ (B). The reaction products were quantified by GC using pentamethylbenzene as an internal standard. Products not detected are marked with an asterisk (*) above the corresponding column. Yield % = [(moles of product formed × reaction stoichiometry/moles of starting aldehyde) × 100].

Figure 2.

Evaluation of nitrogen-precursors as possible self-immolative donors (A) and comparison of products obtained from the reaction of 1a with Ir, HCOONa, and 10 (B). In Part B, no secondary (4a) and tertiary (5a) products were obtained. The reaction products were derivatized by treatment with ethyl chloroformate and then quantified by gas chromatography using an internal standard. Products not detected are marked with an asterisk (*) above the corresponding column. Yield % = [(moles of product formed × reaction stoichiometry/moles of starting aldehyde) × 100].

Figure 3.

Reductive amination of various aldehydes to the corresponding primary amines using Ir6, HCOONa, and 10. The reaction products were derivatized by treatment with ethyl chloroformate and then quantified by gas chromatography using an internal standard. Unless indicated (e.g., 2i, 2k), no alcohol products were observed. Yield % = [(moles of product formed × reaction stoichiometry/moles of starting aldehyde) × 100]. See Table S5 for more details.

Figure 4.

Proposed pathway for the conversion of 1a to 3a (A), kinetic studies of reductive amination and amine cleavage (B), and effects of pH and H₂O₂ on the amine cleavage efficiency (C). The kinetic studies were performed in DMSO/PBS (5:95) at 37 °C and the products were analyzed by HPLC (reductive amination) or GC (amine cleavage). [A_o] = concentration of starting material, [B] = concentration of product.

Figure 5.

Synthesis of secondary (A and B) and tertiary (C) amines using Ir6, HCOONa, and an appropriate nitrogen-containing starting material. The reaction yields were quantified using GC with pentamethylbenzene as an internal standard. Cinacalcet (part B) was obtained from the reaction of aldehyde 1p (1 mM), (R)-41 (2 mM), Ir6 (40 μM, 4 mol%), and HCOONa (10 mM) in DMSO/PBS (5:95) at 37 °C for 24 h. Yield % = [(moles of product formed × reaction stoichiometry/moles of starting aldehyde) × 100]. See Tables S10–S12 for more details.

Figure 6.

Oxidation of the lysine side chains in bovine serum albumin (BSA) to allysine (BSA^Ald), followed by reductive amination using Ir6 in the presence of 10 and HCOONa to produce BSA^RA. The bottom plots show the carbonyl density (left) and lysine content (right) in the protein samples after various treatments. The data were analyzed using one-way ANOVA and are presented as the mean ± standard deviation (n = 4 per group). The p-values are indicated as follows: ns = not significant (p > 0.05), * = p < 0.05, ** = p < 0.01, and *** = p < 0.001, **** = p < 0.0001.

Figure 7.

A) Diagram showing treatment of NIH-3T3 mouse fibroblast cells with aldehyde 1c, followed by Ir-catalyzed reduction to the corresponding alcohol 2c or amine-containing natural compound 3c. At the conclusion of the experiment, the cells were lysed and the contents were derivatized with Fmoc-Cl prior to product analysis. B) Cell viability data obtained from carrying out reductive amination of 1c inside NIH-3T3 cells. C) HPLC analysis of products generated inside cells after treatment with 1c, 10, Ir1 or Ir6, and HCOONa. The turnover numbers (TONs) were calculated by dividing the product concentration by the intracellular iridium concentration.

Figure 8.

A) Diagram showing treatment of SH-SY5Y human neuroblastoma cells with aldehyde 1p and amine (R)-41, followed by Ir-catalyzed reduction to the corresponding drug molecule cinacalcet (Cin). B) Cell viability data obtained from carrying out reductive amination of 1p using an Ir catalyst and (R)-41 inside SH-SY5Y cells. C) HPLC analysis of products generated inside cells after treatment with 1p, (R)-41, Ir1 or Ir6, and HCOONa. The turnover numbers (TONs) were calculated by dividing the product concentration by the intracellular iridium concentration. The identity of the peak marked with an asterisk (*) is currently unknown.

Scheme 1.

Possible routes for reductive amination using Ir catalysts with HCOONH₄ (A) or HCOONa and a self-immolative nitrogen donor (B). In Part B, no secondary or tertiary amines were observed. R = alkyl or aryl, X′ = self-immolative group.

Chart 1.

Reductive amination of aldehydes to amines using conventional (A) and transfer hydrogenation-based methods (B). We have applied our method in living cells to synthesize phenethylamine and cinacalcet (C). R = alkyl or aryl; R′, R′′ = H, alkyl, or aryl; X′ = self-immolative group; Ir_A = Cp* iridium catalyst with bipyridine ligand, Ir_B = Cp* iridium catalyst with picolinamidate ligand.