Synthesis of Fluorogenic Arylureas and Amides and Their Interaction with Amines: A Competition between Turn-on Fluorescence and Organic Radicals on the Way to a Smart Label for Fish Freshness

We describe the synthesis of fluorogenic arylureas and amides and their interaction with primary or secondary amines under air and light in organic-aqueous mixtures to give rise to a new class of persistent organic radicals, described on the basis of their electron paramagnetic resonance (EPR), as well as UV–vis, fluorescence, NMR, and quantum mechanics calculations, and their prospective use as multi-signal reporters in a smart label for fish freshness.

S25 Figure S34. EPR spectrum of the interaction of 4 with pyrrolidine. In blue is given the fit described in text (see Figure  S27). Experimental details: 100 l of a 10 −2 M solution of 4 in DMSO, 100 l of water and 100 l of DMSO were allowed to mix for 1 h. Then, 100 l of a 10 −2 M solution of pyrrolidine in DMSO were added. After shaking, 200 l of the mixture were pipetted into a flat cell. The present spectrum was recorded 10 min after the beginning of the experiment. Data collection: modulation amplitude 0.02 mT, time constant 40.96 ms, conversion time 327.68 ms, gain 6.32 10 4 , power 20 mW, the spectrum is averaged from 3 scans and microwave frequency 9.7727 GHz. Figure S35. EPR spectrum of the interaction of 5 with pyrrolidine. In blue is given the fit described in text (see Figure  S27). Experimental details: 100 l of a 10 −2 M solution of 5 in DMSO, 100 l of water and 100 l of DMSO were allowed to mix for 1 h. Then, 100 l of a 10 −2 M solution of pyrrolidine in DMSO were added. After shaking, 200 l of the mixture were pipetted into a flat cell. The present spectrum was recorded 11 min after the beginning of the experiment. Data collection: modulation amplitude 0.02 mT, time constant 40.96 ms, conversion time 327.68 ms, gain 6.32 10 4 , power 20 mW, the spectrum is averaged from 3 scans and microwave frequency 9.7716 GHz.

Procedure
The experimental procedure consisted of placing the solution of 1 in the cuvette, adding with a microsyringe a certain number of equivalents of pyrrolidine, shaking the cuvette and recording the absorption spectrum obtained in each case for every 1:pyrrolidine ratio. As a result of the addition of pyrrolidine, the colour paled from orange to yellow.

Titration with pyrrolidine in 3:1:1 (v/v) DMSO/water/acetone
The absorption band centred at 390 nm decreases meanwhile another absorption band centred at 317 nm increases. There is an isosbestic point at 340 nm that disappears for excesses larger than 1 equiv of pyrrolidine.

Titration with pyrrolidine in DMSO
The absorption band centred at 405 nm decreases meanwhile another absorption band centred at 305 nm increases. There is an isosbestic point at 336 nm that disappears for excesses larger than 1 equiv of pyrrolidine.

S28
Fluorescence measurements: The detection of amines by the probe 1 can be followed by fluorescence measurements considering a two steps process, because the kinetic effect observed is very sharp. It consists of an initial extremely fast increase in the fluorescence followed by a very slow process of fluorescence intensity decay. The first step is related with the formation of a complex between the probe and the analyte and can be studied kinetically by using ultrafast techniques such as stopped flow or by fluorescent titrations measuring different ratios 1:pyrrolidine immediately after the addition of the amine (t= 0). On the other hand, the second step corresponds to the decay of the complex, which can be studied by recording the fluorescence decay over time under pseudo-first order conditions (big excess of pyrrolidine): 1 + Pyrrolidine ↔ Complex → (Decomp.) Pyrrolidine was selected as an example of secondary amine. The mechanisms was extrapolated to many other amines, such as the biogenic amines. Therefore, the results were compared between pyrrolidine, histamine, putrescine and cadaverine. Then, real samples of fish were studied upon decomposition and the possibility of using as an intelligent label was studied.

b) Titration with pyrrolidine in DMSO:
The emission band centred at 507 nm increases with the concentration of pyrrolidine. λ exc = 336 nm.   Plotting the rate constants calculated versus pyrrolidine concentration allowed us to obtain the equilibrium constant: k Pyr k k obs v   Figure S41. Representation of the rate constant observed versus pyrrolidine concentration.
According to these results and after validating the regression by using least median of squares, a mathematical tool to eliminate outliers, an equilibrium constant of 147.16 ± 49.14 M -1 was achieved for the decomposition process taking place after the 1:Pyrrolidine complex formation. Quantum yields was measured for 1 in the presence of pyrrrolidine, used as reference, and histamine, putrescine and cadaverine. 1 was dissolved in DMSO, the reference was quinine sulfate in H 2 SO 4 0.05M. The process was repeated three times and the results combined (after normalizing to 25 days result). The combination gives an exponential increase in the amines produced by the fish at 25 ºC. This increase starts to be noticeable after 3-5 days, since that point the presence of biogenic amines is clear. Apart from the solution it was tested by the same way in gel, by this process, rotten fish is heated and the gas extracted to a vial that contains the gel sample: Figure S44.

Figure S45
Standard orientation Description of the molecular orbitals SOMO: Figure S46: Single Occupied Molecular Orbital (SOMO) has mainly -antibonding character located over the C=N bond of the protonated nitrogen and over the 1-indene carbon atom and the aryl group, as well as -bonding over the aryl group bonded to the five members ring. The molecular orbital has slight contributions of -bonding interactions between hydrogen atoms and the former nitrile nitrogen atom and on carbon atom of the five membered ring. SOMO+1: Figure S47: SOMO+1 is located on the arm of the molecule not affected by the formation of the radical. It has mainly antibonding character located over the aryl fragment near to the nitrile groups, and -antibonding character in the nitrile groups. Nevertheless, this orbital has slight -bonding character in the C-C interactions of the nitrile groups and the 1-indene carbon atom with the aryl fragment.
SOMO-1: Figure S48: SOMO-1 is a -bonding molecular orbital delocalized over the phenyl rings directly bonded to the ether oxygen atom and with slight -antibonding interactions with a p orbital of this oxygen atom and with p orbitals of the urea nitrogen atoms.
-antibonding -antibonding -bonding S37 SOMO-2: Figure S49: SOMO-2 is a -bonding molecular orbital delocalized over the biphenyl rings of the arm not affected by the radical. There are also small contributions of p orbitals of the nitrile nitrogen atoms, the 1-indene exocyclic carbon atom, the oxygen atom and the nitrogen atom of the urea directly bonded to the biphenyl fragment.
SOMO-3: Figure S50: SOMO-3 is a -bonding molecular orbital extended over the phenyl rings bonded to the ether oxygen. There is also participation of a p orbital of the oxygen ether. The molecular orbital has different participation of orbitals of atoms of each arm of the ether. In the arm not affected by the radical there is participation of the nitrogen atom of the urea fragment directly bonded to the phenyl ring of the ether and a p orbital of the oxygen atom of this fragment urea. In the arm affected by the radical, there is participation of p orbitals of both nitrogen atoms of the fragment urea and a -bonding participation of the phenyl ring directly bonded to the urea fragment.