1. Analytical application of alkylation for the study of amino acids
  Nino G. Todua, Kirill Tretyakov, Anzor Mikaia
National Institute of Standards and Technology, USA
INTRODUCTION
Alkylation is widely applied to amino acids and their condensation products of natural origin prior to their analysis by mass spectrometry. However systematic studies of behavior of alkylated amino acids under mass spectrometry conditions have not been carried out. EI spectra and GC retention index values of partially and completely alkyl substituted amino acids have been acquired in the present study for the NIST/NIH/EPA mass spectral library. Fragmentation pathways are evaluated and diagnostically important ions are identified.
Mono- di- and trimethyl-L-threonines demonstrate competing fragmentation processes. Loss of carbmethoxy radical and ethylene oxide as a result of hydrogen rearrangement is characteristic for all three. However carbmethoxy elimination with the formation of ions a takes over with the increase of Nitrogen atom basicity . The latter determines stability of resulting imminium cations:
Scheme 1. Formation of nitrilium cations in the case of L-Threonine, N,O-dimethyl-, methyl ester, and 2-methyl-L-Phenylalanine, methyl ester
and its N-methyl derivative.
EXPERIMENTAL
Chemicals: Commercial Glycine, L-Alanine, beta-Alanine, L-Phenylalanine, L-Valine, L-Leucine, L-Isoleucine, L-Threonine, L-Serine, L-Threonine, L-Methionine, L-Lysine, L-Histidine, L-Tyrosine, L-Proline, and L-Tryptophan were objects of the study. Alkylation of these amino acids has been accomplished with the use of commercial derivatization reagents: (a) alkyl (methyl, trideuteromethyl, ethyl, n.-propyl, isopropyl, 1,1-dimethyl-propyl, n.-butyl, sec.-butyl, tert.-butyl, 2-methylbutyl, n.-pentyl, isopentyl and neopentyl) iodides in presence of NaOH or NaH, and (b) .25M trimethylsulfonium hydroxide/methanol.
Instrumentation: EI mass spectra were acquired on GC-MS instruments with quadrupole analyzers.
Micro-synthesis: Procedure 1. (1) Alkyl iodide in a ratio 1:5 was gradually added to a suspension of 1 mmole of amino acid and powdered NaOH in 30 microliter of acetonitrile at 5oC. The mixture was stirred for 15 minutes at 25oC, and then analyzed by GCMS.
Procedure M Trimethylsulfonium hydroxide/methanol solution was gradually added to 1 mmole of “dry” amino acid at 5oC and stirred for 15 minutes at 25oC.
RESULTS AND DISCUSSION
Fragmentations of methylated prolines and tyrosines proceed according the general dissociation rules or organic compounds under EI conditions, no surprises have been observed. In Tyrosine case most peaks in the spectra correspond to imminium cations and just few ions include aryl part of a molecule (Fig 5a), and spectra of prolines and prolinamides are rich of ions containing proline heterocycle (Fig 5b,c).
Methylated α-amino acids under electron ionization undergo simple cleavages of C(2)-N , C(2)-C(1) and C(2)-C(3) bonds. These processes may be accompanied by hydrogen rearrangements. The simplest representative of this class L-glycines show peaks of molecular ions and dimethylmethyleneminium cations (Fig. 1a,b). For L- valine derivative peak of [M-C3H7]+ appears of significant intensity along with a base peak of an ion [M-COOCH3]+ ; these two peaks can be used for molecular weight determination for the corresponding permethylated amide (Fig. 1c,d).
Table 1. Characteristic ions in the spectra of methyl ester of S-methyl-L-cystein (I) and its N-methyl- (II) and N,N-dimethyl-analogs (III).
Ion I II
III
M+.
149(11)
163(3)
177(4)
[M-CH3]+
132(13) -
[M-COOCH3]+
90(88)
104(49)
118(39)
[M-CH3SCH2]+
88(100)
102(100)
116(100)
[M-CH3SCH2-HCOOCH3]+
28(12)
42(32)
56(15)
[M-COOCH3-CH3S]+
43(27)
57(23)
71(24)
[M-COOCH3-CH3SH]+
42(19)
56(10)
70(5)
[CH3N≡CH]+ , c
42(11)
Figure 3. L-Threonine, O-methyl, methyl ester (a); L-Threonine, N,O-dimethyl-, methyl ester (b);
L-Threonine, N,N,O-trimethyl-, methyl ester (c), L-Threonine, N-methyl-O-trideuteromethyl-, trideuteromethyl ester (d).
Diagnostically important ions for N-alkylated amino acids appear to be nitrilium cations (c, CH3N+≡CH, m/z 42). They are a result of a complex fragmentation processes of molecular ions of amino acids containing N-methyl group, and include methylamino and C(2) atoms: this ion remains intact in the spectrum of N-methyl-O,O’-di(trideuteromethyl) amino acid (Fig 3 d) and formation of homologous ion [CH3N≡CCH3] + at m/z 56 is observed in the spectrum of 2-methyl-amino acid (Fig. 4b). Elimination one methyl group precedes the formation of c ions in the case of N,N-dimethyl-derivatives: in the spectra of N-methyl-N-trideuteromethyl-L-threonines two c ions have been observed with the composition of [CH3N+≡CH] at m/z 42 and [CD3N+≡CH] at m/z 45.
Formation of methyl- and dimethylnitrilium cations in the case of N-methyl-analogs of L-Threonine and L-phenylalanine are presented in Scheme 1. Activation of C(2)-C(3) bond by phenyl and amino groups simplifies ion c formation (Scheme 1).
Figure 1. Glycine, N,N-dimethyl-, methyl ester (a); Glycinamide, N,N,N(2),N(2)-tetramethyl- (b);
L-Valine, N,N-dimethyl-, methyl ester (c); Valinamide, N,N,N(2),N(2)-tetramethyl- (d).
Spectra of isomeric permethyl-L-leucine and permethyl-L-isoleucine contain diagnostically important fragmentation pathways similar to alkanes and the resulting ions are good enough for their differentiation (Fig. 2)
Figure 5. L-Tyrosine, N,N,O-trimethyl, ethyl ester (a); L-Prolinamide, N-(1)-tert.-butoxycarbonyl-, N,N-dimethyl- (b);
L-Proline, N-methyl-, methyl ester (c).
Methylated cysteins exhibit fragmentation patterns similar to other amino acid derivatives (Table 1) by undergoing cleavages of C(2)-C(1) and C(2)-C(3) bonds. The resulting ions may eliminate methylthio radical and methylthiol molecule at a less extent.
CONCLUSIONS
General and specific fragmentation processes of alkylated amino acids are determined and diagnostically important ions for structure elucidation are identified.
Composition and the origin of alkylnitrilium cations are determined.
Figure 2. L-Leucine, N,N-dimethyl-, methyl ester (a); L-Isoleucine, N,N-dimethyl-, methyl ester (b).
Figure 4. L-Phenylalanine, 2-methyl-, methyl ester (a); L-Phenylalanine, 2-methyl-N-methyl-, methyl ester (b).