Experiments in this work were performed in D2O to extend the lifetime of hyperpolarized spin order, and to produce a spin system with just two proton nuclei in the product molecule ([3,O-D2]malate). Application in biological systems means working in the presence of H2O, which reduces the fumarate singlet-state lifetime. The proton 𝑇 S was measured in a protonated phosphate buffer solution to be 8 s (see Supporting Information), which presents a challenge for applications of this particular molecular system; the hyperpolarized fumarate should be prepared in D2O, and only mixed with H2O at the point of delivery to minimize signal losses. As an alternative, it has been shown that other molecules can support proton singlet states that are relatively long-lived in protonated solvents . We note that the 8 s 𝑇 S was measured on [1-13 C]fumarate, and fluctuating dipolar coupling to the nearby C spin is an additional source of relaxation that will not be present in the unlabelled molecules used for singlet-contrast imaging. The conversion to malate occurring in a protonated solvent also leads to the formation of fully protonated malate, a three-spin system. In order to convert the initial parahydrogen-derived three-spin order into observable in-phase magnetization, the pulse sequence evolution (𝜏) delays need to be modified, and an overall lower transfer efficiency can be expected. We discuss the three-spin case further in the Supporting Information and provide the optimal theoretical 𝜏 delays.