Fig. 3. Reaction efficiencies for APN, chemical shift changes, and spin-lattice relaxation times of APN probe candidates.

(A) A series of probe candidates and their conversion rates during enzymatic reactions with APN. Conditions: 3.0 mM compound in 100 mM phosphate buffer (pH 7.4), hAPN (9.7 nM), 37°C, 40 min, n = 3. Wavy lines indicate bonds that are cleaved by APN. (B) Magnetic parameters of the ¹³C atoms in probe candidates, highlighted as blue or red circles in (A). Δδ, change in ¹³C chemical shift between probe and product generated by hydrolysis by APN. Measurement conditions, 100 mM each of the probe and product in phosphate buffer [90 mM (pH 7.4)] containing 10% D₂O, 37°C. T₁, spin-lattice relaxation time. Measurement conditions, 500 mM compound in phosphate buffer [90 mM (pH 7.4)] containing 10% D₂O, 37°C, 9.4 T. (C) Time course of Ala-NH₂ (5) and Ala-Gly-NMe₂ (11) conversion by APN. Conditions, 3.0 mM compound in phosphate buffer [100 mM (pH 7.4)], hAPN (9.7 nM), 37°C, n = 3. (D) T₁ values and contributions of R₁^DD of Ala-[1-¹³C]Gly-NMe₂ and Ala-[1-¹³C]Gly-d₂-NMe₂. Measurement conditions, 10 mM compound in D₂O (In D₂O), in phosphate buffer [90 mM (pH 7.4)] containing 10% D₂O (In PB), in DPBS containing 10% D₂O (In DPBS), or in human serum containing 10% D₂O (In serum), 37°C, 9.4 T; 3 T (HP), ca. 1.4 mM compound in DPBS containing 3.4 mM glycerol, 32 μM OX063, and 227 μM EDTA, 37 °C, 3.0 T under a hyperpolarized state. R₁^DD, inverse of T₁ relaxation time by dipole-dipole interaction; R₁^tot, the total value of the inverse of T₁ relaxation times. HP, hyperpolarized state. Error bars indicate the SD. n.d. indicates that the product (Ala) was not detected.