In one aspect, the disclosure provides a conjugate comprising an insulin molecule having an A-chain and a B-chain; an affinity ligand covalently bound to the A-chain; and a monovalent glucose binding agent covalently bound to the B-chain, wherein the affinity ligand competes with glucose for non-covalent binding with the monovalent glucose binding agent. In the absence of glucose, the monovalent glucose binding agent binds the affinity ligand to produce a "closed" inactive form of the insulin molecule. When free glucose is added, it competes with the affinity ligand for binding with the monovalent glucose binding agent to produce an "open" active form of the insulin molecule. The monovalent glucose binding agent and affinity ligand are covalently bound to the insulin molecule. The disclosure also provides methods of using these conjugates and methods of making these conjugates. In another aspect, the disclosure provides exemplary conjugates. The disclosure also provides alternative conjugates that are not necessarily activated by glucose.