Expected and unexpected products from reacting Sanger’s reagent with p-phenylenediamine

p-Phenylenediamine reacts with Sanger’s reagent in hot ethanol to give the expected mono- and di-substitution products, but in ethanol at room temperature, it gave exclusively 2-nitro-5-fluorophenyl-p-phenylenediamine, where a hydrogen atom is displaced by attack at an activated, unsubstituted position. The reactions of p-phenylenediamine and aniline with Sanger’s reagent were compared in the cheap, ‘green’ solvent ethanol. An unexpected product forms from reacting Sanger’s reagent with p-phenylenediamine


Introduction
Sanger's reagent, 2,4-Dinitrofluorobenzene (DNFB) 1, is used for the chromatographic detection and quantification of amino acids, peptides and proteins (Figure 1). 1 Its effectiveness is based on the reaction of the reagent with free alpha and epsilon amino groups to form stable, yellow dinitrophenyl derivatives. 1 It allows the terminal amino acid of a peptide chain to be determined. 1elective cleavage of the labelled peptide chain allowed the amino sequence of insulin to be determined by examining the overlap of peptide sequences. 1][4][5][6][7][8][9][10][11][12] (Figure 2).2,4-Dinitrofluorobenzene 1 and p-phenylenediamine 6 were interesting reagents to react together with a view to making isomers of trimer 7, that might expand the range of hair dyes, and to explore the reactivity of DNFB 1.
The main product 8, from a bis adduct formation reaction, was difficult to purify by chromatography owing to its poor solubility and pigmentary properties.This accounts for the low yield.A quantity of the half-coupled product 9 was also formed.A front-running compound 10 13 was also formed in variable but significant quantities and was characterised by its proton and carbon NMR spectra.This known compound forms here by the displacement of fluorine with ethanol in the hot solution.In hot isopropanol, the isopropyl analogue of compound 10 did not form, so the product was mainly compound 8 (89%) contaminated with a small amount of compound 9. Isopropanol is more hindered than ethanol so the formation of a substitution product from it and DNFB 1 is suppressed.
Compound 8 crystallises in the monoclinic space group P2 1 /n with half a molecule in the asymmetric unit with the complete molecule generated by crystallographic inversion symmetry (Figure 4).The dihedral angle between the central and pendant aromatic rings is 39.94 (7)° and the N2/ O1/O2 and N3/O3/O4 nitro groups are twisted from the latter ring by 4.84 (10) and 17.48 (7)°, respectively.An intramolecular N -H 16)°] and the same group also participates in a much weaker intermolecular N -H•••O interaction.
An experiment with p-phenylenediamine 6 and two equivalents of DNFB 1 at room temperature in EtOH in the presence of two equivalents of Et 3 N was conducted to minimise or avoid the reaction of EtOH with DNFB 1.An unexpected product 11 was formed in high yield (Figure 5).Compound 11 was not isolated previously from a column along with compounds 8-10 (Figure 4) but the column was complex in yellow colour and the failure to isolate compound 11 does not prove that none of it formed as a minor product.
Compound 11 crystallises in the monoclinic space group P2 1 /c with two molecules in the asymmetric unit (Figure 6).In the N1 molecule the dihedral angle between the aromatic rings is 54.29 (16)° and the equivalent angle in the N4 molecule is 51.22 (15)°.The nitro group is close to coplanar with its attached ring in both molecules [dihedral angles of 5.23 (7)  Only a mono coupled product formed so the second primary amine is less reactive.A fluorine atom was clearly present from the NMR spectra as it couples with hydrogen and carbon.The primary amine of p-phenylenediamine 6 has reacted at an unsubstituted position of DFNB 1 and  eliminated a nitro group as nitrous acid with Et 3 N.An addition-elimination mechanism is drawn in Figure 7.
This is an unusual way for the reaction proceed.Et 3 N is present when the reaction was done at a low and high temperature (Figure 7) so is probably not changing the reaction pathway.p-Phenylenediamine behaves like a stronger nucleophile, owing to repulsion between the nitrogen lone pairs, a higher energy HOMO, and can drive the alternative reaction pathway.The para nitro group is eliminated rather than the ortho nitro group which is more sterically crowded.Both the fluorine substituted site and the unsubstituted site are activated by two nitro groups.The product is still bright yellow so it would be detectable if any of it formed with an amino acid or peptide chain.
One possible explanation for the experimental outcome might be the strength of the carbon-fluorine bond which is very strong.Related addition-elimination reactions are also known for 1,3-dinitrobenzene 14,15 under oxidative conditions and the nitration of furan in acetic anhydride 16,17 which is the least aromatic of the π-excessive heterocycles.A nitro group is not eliminated though.The displacement of hydrogen, rather than a halogen, has previously been called vicarious nucleophilic substitution where the leaving group is attached to the nucleophile. 18,19FB 1 was reacted with aniline 14 to compare with the products formed from p-phenylenediamine 6 (Figure 8).The expected and known substitution product 15 was formed 20 in which the fluorine atom has been displaced.No other products were formed in significant amounts.The extra equivalent of aniline binds to the HF rather than using Et 3 N to do this.
The asymmetric unit of 15 (space group P2 1 /n) consists of one molecule (Figure 9) in which the dihedral angle between the aromatic rings is 50.32 (5)°, in agreement with the crystal structure of a sample of 15 which has been reported (Cambridge Structural Database reference code TAMHEX). 21It was prepared here under controlled, comparable conditions for a comparison with structure 11.

Conclusion
p-Phenylenediamine 6 reacts with DNFB 1 in hot ethanol to give the expected products 8 and 9, but in ethanol at room temperature, an unexpected product 11 forms.The expected products form by displacement of fluorine and the unexpected product forms by the displacement of hydrogen from an unsubstituted position.Both sites of attack are activated by two nitro groups.The fluorine is strongly electron withdrawing but the carbon-fluorine bond is very strong, which might influence the outcome.A mechanism is drawn which rationalises how the reaction can proceed.It is an addition-elimination mechanism in which the negatively charged complex must protonate first before the nitro group is eliminated.Either nitro group could eliminate depending upon where the intermediate anion protonates.The energy barrier leading to the more stable product is higher as it requires a higher temperature to form.The reactions of p-phenylenediamine 6 and  aniline 14 with DNFB 1 were compared and showed that aniline 14 gave the expected product.The product is still bright yellow so would act as a label if this reaction had occurred in the Sanger's method.However, preliminary studies have shown that amino acids react with DNFB 1 entirely as expected.Compound 11 is unknown by a Reaxy's search.

Experimental
Infrared spectra were recorded on a diamond-attenuated total reflection (ATR) Fourier transform (FTIR) spectrometer.Ultraviolet (UV) spectra were recorded using a Perkin Elmer Lambda 25 UV-Vis spectrometer with EtOH as the solvent.The term sh means shoulder. 1H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at 400 and 100.5 MHz, respectively, using a Varian 400 spectrometer.Chemical shifts, δ, are given in ppm and using a solvent peak as an internal reference.Coupling constants, J, are given in Hz.High-resolution mass spectra were obtained at the University of Wales, Swansea, using an Atmospheric Solids Analysis Probe (ASAP) (positive mode) instrument: Xevo G2-S ASAP.Melting points were determined on a Kofler hot-stage microscope.

Crystal structure determinations
The crystal structure of compound 8 (red prism, 0.38 × 0.10 × 0.05 mm, recrystallised from dichloromethane:light petroleum ether), compound 11 (red plate, 0.18 × 0.13 × 0.05 mm, recrystallised from dichloromethane:light petroleum ether) and compound 15 (orange rod 0.32 × 0.05 × 0.03 mm, recrystallized from dichloromethane:light petroleum ether) were established using intensity data collected on a Rigaku CCD diffractometer (Cu Kα radiation, λ = 1.54178Å) at 100 K.The structures were routinely solved by dual-space methods using SHELXT 26 and the structural models were  completed and optimised by refinement against |F| 2 with SHELXL-2018. 27For compounds 8 and 15, the N-bound hydrogen atoms were found in difference maps and their positions were freely The N-bound H atoms in compound 11 (N -H = 0.88 Å) and the C-bound H atoms in all structures were placed geometrically (C -H = 0.95 Å) and refined as riding atoms.The constraint U iso (H) = 1.2U eq (carrier) was applied in all cases.Full details of the structures and refinements are available in the deposited cifs.The crystal of compound 12 was found to be non-merohedrally twinned and data quality is poor, resulting in rather high R-factors, but the structure has been unambiguously determined.

Figure 1 .
Figure 1.The formation of a terminal amino acid and peptide chains labelled with Sanger's reagent 1.1 Compound 5 forms when a peptide bond along the chain hydrolyses.

Figure 6 .
Figure 6.The molecular structure of 11 showing 50% displacement ellipsoids.Hydrogen bonds are indicated by double-dashed lines.

Figure 7 .
Figure 7.A mechanism for the formation of unexpected product 11.

Figure
Figure A protocol for reacting amino acids with Sanger's reagent 1 under controlled comparable conditions, used in this work, followed by chromatography.1