The investigation of N-dinitrophenyl derivatives of amino acids by electron / chemical ionization using a particle beam interface 1

The EI and CI mass spectra of DNP-amino acids and oligopeptides give characteristic mass spectra when a particle beam interface is used for introduction. They differ from mass spectra obtained after direct insertion into the ion source: In the particle beam interface the major part of the molecules suffers degradation by contact with metal surfaces such as decarboxylation and reduction of the nitro groups. The final products are benzimidazole derivatives carrying in 2-position the residue of the respective amino acid. These products show characteristic fragmentation reactions which allow to identify isomeric amino acids. For DNP-diand oligopeptides an identification of the N-terminal amino acid is always possible, that of the C-terminus with restrictions.


Introduction
The labelling of N-terminal free amino groups of peptides as 2,4-dinitrophenyl (DNP) derivatives was introduced by Sanger [2].In contrast to other derivatives the DNP amino acids (free or as their methyl esters) have been investigated by mass spectrometry only scarcely, especially in combination with chromatographic techniques: Paper chromatography with subsequent isolation of the fractions and identification by EI [3], and liquid chromatography (LC) in combination with flow FAB [4] or using a particle beam (PB) separator and EI [5] were discussed.FAB [4] and CI [6] mass spectra allow the determination of the molecular masses only.In the EI spectra [M-• COOCH 3 ] + (for aliphatic amino acids usually responsible for the base peak, aromatic and functionalized amino acids show side chain characteristic fragments) and to some extent also [M-• R] + can be observed in addition to M +• of varying abundance [3,6,7].Interestingly, the EI spectra after PB separation differed drastically from those obtained with direct sample introduction [5], however, no explanation was offered.In the following we will show that the additional ions observed after PB separation are due to surface catalized reactions which offer additional structural information and allow to some extent to differentiate between DNP amino acids having the same molecular mass and thus being indistinguishible by the techniques mentioned before.

Instrumentation
MS25 RFA (EB geometry) mass spectrometer (electron energy 70 eV for EI, 150 eV for CI; source temperature 250 • C) with a particle beam interface (both Kratos Analytical, Manchester, GB) to the LC-9A HPLC (Shimadzu, Kyoto, Japan), column Nucleosil C18, 5 µm (Knauer, Berlin, D).Samples: 2 mg DNP amino acid dissolved in 1 ml of CH 3 OH, injected amounts 2-5 µl.HPLC solvent CH 3 OH/50 mM NH 4 OAc buffer 77 : 23 (v/v).MAT 900 ST (Finnigan MAT, Bremen, D) for CI, high resolution and linked scan measurements; sample amount 10-20 µl and 2-5 µl (CI), respectively.For CI (both positive and negative) the HPLC solvent (see above) entering the ion source was sufficient as ionizing gas.Addition of NH 3 or CH 4 gave no increase in sensitivity and did not change the appearance of the spectra.

Syntheses
N-(2-Nitrophenyl) amino acids.To 100 mg (0.76 mmol) of Leu, Ile or Nle dissolved in 10 ml of 0.1 M borate buffer (pH 9.2) 50 µl of 2-fluoronitrobenzene were added dropwise under stirring.The reaction mixture was stirred for 15 hrs, then brought to pH 1 with 1 N HCl.Solid material was removed by centrifugation, suspended in 2 ml of H 2 O and extracted twice with 2 ml of CH 2 Cl 2 each.The united organic phases were dried with MgSO 4 , CH 2 Cl 2 was distilled off i.v.Remainig traces of CH 2 Cl 2 were removed by dissolving the residue several times in CH 3 OH and distilling off the solvent i.v.The resulting orange-red oil was subjected to mass spectrometric investigation without further purification.

N-(2,4-Dinitrophenyl)-dipeptides.
To a solution of 0.13 mmol of the respective dipeptide in 5 ml of 0.1 M borate buffer (pH 9.2) 150 µl of 2,4-dinitrofluorobenzene were added and the mixture was stirred in the dark for 4 hrs at 60 • C. The organic phase was removed and from the residue solid material was separated by centrifugation.The remaining aqueous phase was brought to pH 1 with 1 N HCl.The resulting yellow precipitate was separated by centrifugation and dissolved in 3 ml of CH 2 Cl 2 .The solution was washed twice with 1 ml of H 2 O each, dried with MgSO 4 and brought to dryness i.v.
2-sec-butyl-benzimidazole was obtained from 1,2-diamino benzene and 2-methyl butyric acid following a literature procedure [9].The product was recrystallized several times from CH 3 OH/H 2 O until pure according to GC/MS.

Electron ionization (EI)
The EI spectrum of DNP-Leu (1) obtained with a direct inlet system (Fig. 1) shows the expected fragments: Loss of • COOH by α-cleavage leads to the main fragment (m/z 252) while the competing loss of • C 4 H 9 (m/z 240) is of minor importance ([M-• COOH] + and [M-• R] + will be mentioned in the following discussions only when there are peculiarities).Elimination of C 3 H 6 and of C 4 H 8 from m/z 252 (m/z 210 and 196) as demonstrated by linked scan measurements are the only other characteristic processes.From these ions m/z 252 appears with about 10% rel.int. in the EI spectrum of DNP-Leu after introduction of the sample via the particle beam (PB) separator (Fig. 2).Instead a series of predominant ions can be seen which recur in the mass spectra of other DNP-amino acids.They are the result of surface-catalized reactions [10], viz.decarboxylation and reduction.
Reactions of substrate molecules with substances adsorbed at the metal surfaces of the ion source (O 2 , H 2 O, solvents, CI reactand gases, etc.) have been studied in detail [11,12], especially the reduction of aromatic nitro groups, adsorbed solvents being the reducing agent [12][13][14].It was shown, i.a., [12] that the reactions do occur in the ion source whose temperature has essentially no effect, and that the   5) and the Nle-derivative (7b) is the much lower intensity of the loss of • C 3 H 7 as compared with that of C 3 H 6 for the latter (loss of an i-as compared with n-propyl radical).It is important to note that the PB-EI mass spectra of the three isomeric compounds can be well distinguished.As mentioned above, this is not possible for any other mass spectrometric technique (EI spectra show only differences in the abundances of some ions).In the same way as the isomeric leucins also Val and Nva can easily be distinguished by their PB-EI spectra (Fig. 6).The one of Nva corresponds to those of Leu and Nle, only the masses of the ions containing the alkyl group are shifted by 14 u (most notable is 8b, the lower homolog of 5 at m/z 175).For Val (9b) the alkyl group is too short for a McLafferty rearrangement, hence only loss of • CH 3 (m/z 160) and of • C 3 H 5 (m/z 134) is possible.The same picture offers DNP-α-aminobutyric acid: 10b loses • CH 3 (m/z 146) and • C 2 H 3 (m/z 134).The ion 10a (m/z 191) is of high abundance (Fig. 7).Accordingly, DNP-Ala loses H . from 11b (Fig. 8), and DNP-Gly shows no further loss from 12b (Fig. 9).Again, 12a shows appreciable abundance.Also DNP-Phe loses H • from 13b and from 13a; in the lower mass region C 7 H + 7 (m/z 91) has an abundance of 30% (Fig. 10).Due to the better charge stabilization as compared with the tropylium ion the indolyl-CH + 2 ion (m/z 129, which subsequently loses HCN, m/z 102) is     In the PB-EI spectrum of DNP-Arg (Fig. 14) 18a is weakly and 18b hardly recognizable.The main ions are derived from degradations of the guanidino group: The base peak m/z 60 comes from protonated guanidine, other abundant ions can be represented as 11b, 19b-21b (m/z 147, 190, 173 and 160 -the last one possibly rearranging to a diazepinium structure).The ion m/z 123 -observed in the spectra of several DNP amino acids -is probaly ionized 1,2,4-triaminobenzene.
The spectra of DNP-Gln (Fig. 15) and of DNP-Asn (Fig. 16) show the expected pattern.Free DNP-Glu forms an abundant [M-• COOH] + ion (m/z 268) which loses H 2 O (m/z 250) and subsequently CO (m/z 222) which suggests a lactam formation between the α-amino-and the γ-carboxyl group as DNP-Asp does not show the loss of CO (a ß-lactone would be improbable).24b (m/z 205) also loses H 2 O (m/z 187) and subsequently CO (m/z 159) (Fig. 17).DNP-Asp yields [M-• COOH] + (m/z 254) which in turn loses H 2 O (m/z 236) less readily than DNP-Glu.Also 25b is hardly recognizable.The main ion is the decarboxylation product of 25b (11, m/z 147) indicating again that a lactam formation is not possible (Fig. 18).DNP-Pro mainly gives 26 (m/z 203) and 27 (m/z 173), the analogs of 4 and 5. 26 is isobaric with 28 which by dehydrogenation of the pyrrolidine ring gives 29 (m/z 199, confirmed by exact mass measurements) (Fig. 19).Due to the possibility of H 2 O losses the spectrum (Fig. 20) of DNP-Hyp is more complex.The hydroxy derivatives of 26 (m/z 219) and 27 (30, m/z 189) can lose H 2 O (m/z 201 and 171).29 can be formed more easily than from DNP-Pro.The base peak occurs at m/z 145 (C 8 H 7 N 3 ) formed by the loss of CH 2 =CHOH from 30 possibly with subsequent rearrangement to aminochinoxaline (31) (note that an ion m/z 145 can also be observed in the spectrum of DNP-Pro, though of much lower abundance).The ion m/z 175 is the nitro analog of 31.
The DNP derivatives of several dipeptide methyl esters had been investigated by EI after direct introduction [15].The ion DNP-NH=CHR + is of medium to high abundance, but there are other important     ions whose genesis is more complex complicating the identification of samples of unknown composition.We, therefore, investigated the DNP derivatives of several dipeptides (Leu-Gly, Gly-Leu, Gly-Ile, Ala-Val, Val-Ala, Gly-Gly).In all cases the spectra correspond to those of the N-terminal DNP-amino acid (cf.Figs 6 and 21).Thus the N-terminal amino acid can be identified readily and even isomeric and isobaric amino acids can be distinguished.

Positive ion chemical ionization (PCI)
PB-PCI mass spectra of DNP amino acids show the same surface catalized degradations as discussed for the PB-EI spectra as exemplified for DNP-Leu and DNP-Val (Figs 22, 23; cf.Figs 2 and 6).The   24).In addition, an abundant ion resulting from the cleavage of the peptide bond (32, m/z 102) can be seen which confirms the identity of the C-terminal amino acid in cases where the [M + H] + ions cannot be recognized unambiguously (though generally they are more abundant than the M +• ions in the EI spectra).PCI spectra of higher DNP peptides allow only the identification of the N-terminus).

Negative ion chemical ionization (NCI)
The NCI spectra of are more complicated (see ions 33-36) than the PCI spectra and do not allow to distinguish between isomeric structures (Fig. 25).Especially the spectra of DNP-di-and higher peptides contain ions the formation of which is difficult to understand.The sensitivity and the reproducibility are worse than in the PCI mode.The NCI technique does not offer any advantages.

Conclusions
Only a minor portion of DNP-amino acids and DNP-oligopeptides reaches the ion source undecomposed and gives rise to the typical EI or CI spectra when a particle beam interface is used for connection with a liquid chromatograph.The major part suffers surface-catalized decomposition [13], especially decarboxylation and reduction of the nitro groups (for detailled discussions with respect of the various parameters of the PB interface and the ion source [11,12]) resulting finally in the formation of benzimidazole derivatives carrying in 2-position the residue of the respective amino acid.These processes offer a major advantage: CI spectra obtained with a direct inlet system show essentially [M + H] + (in the positive mode) or M −• (in the negative mode [6]) and in EI spectra loss of the complete side chain is observed.Characteristic fragmentation processes of the substituted benzimidazoles allow an identification of amino acids with isobaric (as Gln and Lys) or even isomeric residues (as, e.g., Leu, Ile and Nle).For DNP-di-and higher peptides the N-terminally substituted amino acids are clearly identifiable as they show the same degradation patterns as the DNP-amino acids.In the case of dipeptides the C-terminal amino acid can be identified in the PCI spectra by the abundant ions formed by the loss of the residue (with the restriction that isobaric and isomeric residues cannot be distinguished in this way).NCI spectra do not offer any advantages.

Fig. 4 .
Fig. 4. PB-EI-mass spectrum of DNP-Nle (M = 297 u).Strong evidence for the formation of 2-alkylbenzimidazole derivatives by the surface-catalized degradations offers a comparison of the PB-EI mass spectra of the N-(2-nitrophenyl)-derivatives of Leu, Ile and Nle with those of 2-n-, 2-i-and 2-sec-butylbenzimidazole (Fig. 5).Up to m/z 174 (corresponding to m/z 189 above) the mass spectra agree well by pairs (additional ions in the spectra of the amino acid derivatives are a weak M +• , m/z 252, and [M-• COOH] + , m/z 207).Fragmentation of the alkyl group by a McLafferty rearrangement yields the ions m/z 132 for Leu and Nle and m/z 146 for Ile.In the same way as the isomeric leucins also Val and Nva can easily be distinguished by their PB-EI spectra (Fig.6).The one of Nva corresponds to those of Leu and Nle, only the masses of the ions containing the alkyl group are shifted by 14 u (most notable is 8b, the lower homolog of 5 at m/z 175).For Val (9b) the alkyl group is too short for a McLafferty rearrangement, hence only loss of • CH 3 (m/z 160) and of • C 3 H 5 (m/z 134) is possible.The same picture offers DNP-α-aminobutyric acid: 10b loses • CH 3 (m/z 146) and • C 2 H 3 (m/z 134).The ion 10a (m/z 191) is of high abundance (Fig.7).Accordingly, DNP-Ala loses H . from 11b (Fig.8), and DNP-Gly shows no further loss from 12b (Fig.9).Again, 12a shows appreciable abundance.Also DNP-Phe loses H • from 13b and from 13a; in the lower mass region C 7 H + 7 (m/z 91) has an abundance of 30% (Fig.10).Due to the better charge stabilization as compared with the tropylium ion the indolyl-CH + 2 ion (m/z 129, which subsequently loses HCN, m/z 102) is