Mass Spectra of a-Amino Acid Oxazolidinones? Remy Liardon and Ursula Ott-Kuhn Research Department, Nest16 Products Technical Assistance Co. Ltd., P.O. Box 88, CH-1814 La Tour-de-Peilz, Switzerland
Petr Husek Research Institute of Endocrinology, Narodni trida 8, 11694 Prag I, Czechoslovakia
2-Bis-(chlorodifluoromethyl)-4-substituted-1,3-oxazolidin-5-ones, a new type of a -amino acid derivative for gas chromatographicseparation, have been studied by low resolution mass spectrometry. These derivatives are obtained by reacting a-amino acids with dichlorotetrafluoroacetone. Their structure has been established or confirmed for most protein amino acids and several non-protein a -amino acids. The mechanismsresponsible for the mass spectral pattern have been rationalized. An interesting feature of this derivatization procedure is that it distinguishes aspartic and glutamic acid from the respective amides. The structure of asparagine and glutamine derivatives has been established. A survey of oxazolidinone mass spectra has provided a list of diagnostically useful ions. Each amino acid can be identified by one or two of its most abundant fragments.
INTRODUCTION
The application of gas chromatography (GC) to the analysis of amino acids has been the object of many investigations. Various procedures have been proposed to convert these compounds into suitable volatile derivatives. A comprehensive review on this topic has been presented by Husek and Macek.' The most widely used derivatives are the N-perfluoroacyl-0- alkyl esters. This is essentially the result of the remarkable work of Gehrke et ~ 1 . Several ~ ' ~ authors have later proposed various modifications of the original Some years ago a new derivatization method, based on the specific reaction of 1,3-dichlorotetrafluoroacetone DCTFA) with a-amino acids, was proposed by H ~ s e k . ~ This * procedure is characterized by much milder conditions than are generally applied for the derivatization of amino acids. The reactions are carried out in slightly acidic media and the temperature never exceeds 70 "C.The first application of the procedure was the determination of tyrosine and thyroid hormones, triiodothyronine and thyroxin, in human blood. More recently it was shown that the same method could be applied for the determination of aspartic and glutamic acids and their amides. An important advantage of the procedure was that asparagine and glutamine did not hydrolyse. They could be determined simultaneously with aspartic and glutamic acids, in the same run." Further developments extended the method to all aamino acids.12 As an example of biomedical application it was tested on the determination of free amino acid in human serum and urine. Complete aminograms were obtained with 50 to 100 ~a mp1es.l~ t Abbreviations: DCTFA = dichlorotetrafluoroacetone; HFBA =
\
heptafluorobutyric anhydride; IBFC = isobutylchloroformate; DAPA = a,@-diaminopropionic acid; DABA = LI, y-diaminobutyric acid.
The principle of the derivatization is illustrated in Scheme 1. At first DCTFA reacts with a-amino acids and converts them into 2-bis-(chlorodifluoromethyl)-4substituted- 1,3-oxazolidinones. In subsequent steps the remaining polar groups are modified with heptafluorobutyric anhydride (HFBA), methanol and isobutylchloroformate (IBFC) in the same reaction vessel. The resulting derivatives present good gas chromatographic properties. Subsequently in the text they will be referred to simply as amino acid oxazolidinones. Samples for derivatization are prepared in the same way as holds for N-perfluoroacyl-0-ethyl esters. The formation of oxazolidinone does not involve any special preconditioning of the sample. 0 II
0
II
R-CH-C-OH
I
-+
NH2
+
ClF2C \ CLF&
/
R-CH-C-OH
';"? I ClFZC-C-OH
I
c=o
CFzCl
--+
R
7
q
0
HNx
CIF,C
CF,CI
Scheme 1. Reaction mechanism for the formation of a-amino acid oxazolidinone.
At present the determination of all protein a-amino acids as oxazolidinone is achieved by means of two G C columns. While they could all be separated in a single run, Arg, His, Trp and Cys would come out with a poor yield. Special analytical conditions had to be found for this group. Typical chromatograms of the two G C runs are shown in Figs. 1 and 2. In the development of this derivatization procedure, mass spectrometry was used extensively to determine or confirm the structure of the derivatives. The mass spectra of 26 amino acid oxazolidinones have been measured and analysed. The results of this work are presented here.
CCC-0306-042X/79/0006-0381$05.50 @ Heyden & Son Ltd, 1979
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 381
R. LIARDON, U. OTT-KUHN AND P. HUSEK
The mass spectra were obtained on a HewlettPackard 5992 combined gas chromatograph mass spectrometer. The samples were chromatographed on two different columns: (A) 10 m x 0.3 mm i.d. glass capillary coated with OV-17; (B) 1.2 m x 2 mm i.d. glass column containing 3% SE-30 on Chromosorb W 45/60. Operational temperatures were: injection port, 200 "C; column A, 70 to 220"C, programmed at 8"Cmin-'; column B, 150 to 260 "C, programmed at 8 "Cmin-'. Helium was used as carrier gas. The capillary column A was interfaced with the mass spectrometer by means of a restrictor. A 0.8 ml min-' constant flow was admitted into the ion source, while the remainder of the column effluent was vented out. For column B a membrane separator replaced the restrictor.
-
i
RESULTS AND DISCUSSION Figure 1. Amino acid standard mixture analysed on column A and nitrogen specific detector. For conditions, see text.
EXPERIMENTAL Amino acids, as a single compound or in standard mixtures, were converted into oxazolidinone derivatives according to the procedure of Husek."'"
To our knowledge, substituted 1,3-oxazolidin-5-ones have not been the object of any previous study by mass spectrometry. Therefore, the identification of amino acid oxazolidinones necessitated first the elucidation of the fragmentation mechanism of this type of compound. From the mass spectra of those for which there was no structural ambiguity, we could extrapolate to the other amino acid derivatives and confirm or establish their structure. The results of this study are detailed below. Basic fragmentation pattern From a study of the mass spectra of all protein a-amino acid oxazolidinones, we could establish the existence of a common fragmentation pattern which is determined by the structure of the oxazolidinone ring. These reactions are detailed in Scheme 2. Two competitive processes occur with the fragmentation of the molecular ion. The first consists of loss of a chlorine atom, immediately followed by the elimination of CO giving ion b. The second process is the loss of CFzCI. Alternatively, the positive charge remains on the oxazolidinone moiety c, or is carried away by the CFzCl fragment (m/z85).The two reaction pathways join together with the formation of d. The relative abundances of the ions formed in the above processes vary considerably from one amino acid to the other. Depending on the nature of R, other reactions can take place and compete successfully with the basic fragmentation processes. These additional reactions are discussed in the following sections.
Aliphatic amino acids
5 Time (rnin)
10
Figure 2. Amino acid standard mixture analysed on column B. For conditions. see text.
382 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
Figures 3 to 9 present the mass spectra for the oxazolidinone derivatives of several aliphatic amino acids, including norleucine and a-amino caprylic acid. The fragmentation mechanism of these compounds consists primarily of the reaction described in the previous section. Further decomposition of d also takes place, except for the derivatives of glycine and alanine. It involves the transfer of one or two hydrogens from the aliphatic chain. This process i s illustrated in Scheme 3. @ Heyden & Son Ltd, 1979
a-AMINO ACID OXAZOLIDINONES R-CH
100.
1 .1
u.
255 L
200
HN,O
]1
226 237
1I
I1 I,
250 m /z
300
350
400
450
Figure 7. Mass spectrum of isoleucine oxazolidinone (mol. wt 31 1).
-"Y CF,CI
+CFZCl m / z 85
I001
c=o I kFJI
69 1
d
C
Scheme 2. Basic fragmentation reactions of amino acid oxazolidinone molecular ion. m/z
Figure 8. Mass spectrum of norleucine oxazolidinone (rnol. wt 311).
I
,
,
I
,
250
, .
, , , ,
300
350
m*u'I.
il -
m/z
Figure 3. Mass spectrum of glycine oxazolidinone (mol. wt 255).
I30 142
n
*.ILL,.
50
100
170 L.
150
254 L 276 4
1
200
250
300
350
400
450
Figure 9. Mass spectrum of a-amino caprylic acid oxazolidinone (mol. wt 339).
m/z
Figure 4. Mass spectrum of alanine oxazolidinone (rnol. wt 269).
170 I 184
100,
When a hydrogen is present in the y-position of the aliphatic chain, a McLafferty type rearrangement takes place, initiating the fragmentation sequence shown in Scheme 4. This process is particularly important for isoleucine and valine which possess five or six yhydrogens respectively. The relative abundance of fragments d to i for the various aliphatic amino acids are reported in Table 1, showing how the size and the structure of the sidechain affect the fragmentation pattern. Serine and threonine
m/z
Figure 5. Mass spectrum of valine oxazolidinone (rnol. wt 297).
,
69
100.
I
198
m/z
Figure 6. Mass spectrum of leucine oxazolidinone (mol. wt 31 1).
Figures 10 and 11 present the mass spectra for serine and threonine HFB-oxazolidinones. The presence of the HFB group in these molecules is readily recognized by the appearance of m / z 169 [C3F7]' in their mass spectrum. The HFB carbonyl induces the fragmentation described in Scheme 5 . The transposition of the ahydrogen via a McLafferty rearrangement leads to the elimination of heptafluorobutyric acid and the formation of ion k.This species is identical to the molecular ion of an &$-unsaturated aliphatic amino acid oxazolidinone. Its fragmentation follows the basic pattern R-CH=CH
0 I1 R'-CH-CH=NH-C-CFzCI
+?-
u
d
0 +
c*
I1
1'
f f
R'-CH=CH-NH2-C-CF2CI
\
OH + I HzN=C-CFzCI e, m / z 130
Scheme 3. Decomposition pathways for fragment ion d.
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BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 383
R. LIARDON, U. OTT-KUHN AND P. HUSEK
Table 1. Relative abundance of aliphatic amino acid oxazolidinone fragment ions
\
,S=O;
Fragmenta
R
d
Glycine
H
Alanine Valine Norleucine Leucine lsoleucine a-Amino caprylic acid
CH3 (CH3).nCH CH3(CH2I4 CH&H2CH(CHJCH2 (CH3(CH2)2CH(CH3)
a
e
10010092 19 73 18 86 24 42 9
f
g
-
-
h
i
I
- - -
62 15 100 100 - 7 100 2 16 55 30 100
I
HNXo
6 25 11 8
45 15 100
1
R'-CH
0
II
R,--cH~\$
II
6 15
k
HNXo
C CH3(CH&
l
I
HNYo
HN+
See Schemes 2-4.
J
\ R'-CH
II
R'
C
k H OH
I HN
I
-
+
X0
d
\I
1"
H?' I
C=O
I
I
HNXo g,
Scheme 5. Main fragmentation pathways for 0-HFB serine and threonine oxazolidinone molecular ion.
m l z 255
-CF2CI
i
detailed above, resulting in the formation of n, with 1 and rn as alternate intermediates. II HN
+
-
Aromatic amino acids
c=o
HN I
n
CF2CI
I CF,CI
I
h, m / z 170
i, m / z 142
Scheme 4. McLafferty rearrangement and decomposition of aliphatic amino acid oxazolidinone molecular ion.
The mass spectra of phenylalanine, tyrosine and tryptophan are presented in Figs. 12 to 14. For these compounds, the fragmentation consists almost only of scission of the 0 C-C bond (Scheme 6), which may be accompanied by the transfer of a hydrogen. The aromatic or heterocyclic ring and the a-nitrogen occur to make this bond particularly labile. The positive charge is carried away by the phenyl or indole moiety. In tyrosine and tryptophan, the resulting ions undergo further fragmentation, as shown below.
I54
I00
n m/z
Figure 10. Mass spectrum of 0-HFB-serine oxazolidinone (mol. wt 481 1.
100
w
150
200
250 m /z
300
350
400
450
Figure 11. Mass spectrum of 0-HFB-threonine oxazolidinone (mol. wt 495).
384 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
200
250 mb
345
300
u '
69 ~
100
150
350
400
450
Figure 12. Mass spectrum of phenylalanine oxazolidinone (mot. wt 345).
K c
50
282
232
I , ..I., ,,
50
50
,,,,
100
~
,
l~
150
,
,
200
250 m/z
2~
1
300
344
,
350
444
494 ~
450
500
Figure 13. Mass spectrum of 0-HFB-tyrosine oxazolidinone (mot. wt 557).
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a-AMlNO ACID OXAZOLIDINONES
1’
07’
0
[email protected]
+C
HNXo
J7!-OGCHz
C3F7-0 m / z 275
m / z 303
c=o
c=o
I
COCZFS
\
I
C3F7
C3F7 m / r 326
m / z 276
QC’+ m / z 129
Sch me 6. Fragmentation reactions fo phenylalanine, 0-HFB-tyrosine and N-HFB-tryptophan oxazolidinone molecular ions.
When a simplified procedure is applied for the determination of selected amino acids, tryptophan oxazolidinone may be analysed in the non-acylated form.I4 The corresponding mass spectrum is shown in Fig. 15. It consists essentially of ion m / z 130 which results from the cleavage of the @-bond.
A mechanism explaining the formation of the fragment corresponding to mass peak m / z 81 is proposed in Scheme 8. a,w- Diamino acids
The general structure of the HFB-oxazolidinone derivatives of a,@-diamino acids is shown below.
Aspartic and glutamic acid The addition of methanol to DCTFA in the second step of the derivatization procedure leads to the formation of bis-(difluorochloromethyl) methoxymethanol. In the presence of HFBA, this compound reacts with the wcarboxyl group of aspartic and Iutamic acid oxazolidinones to form complex esters.’ The mass spectra of these derivatives are presented in Figs. 16 and 17. It can be seen that most fragments originate from scission of the bonds which are activated by the ester group or the a-nitrogen (Scheme 7). These processes may also involve the transposition of one or two hydrogens.
0
I1
c,F,c-NH-~cH~)~-,-----~~
H N X o
L IM
1001
6
;E!
r5B
50
50
I
100
150
200
250
300
- . ,
350
.
400 u14 ,
,
450
,
326
I
1
467
450
517
500
Figure 15. Mass spectrum of non-acylated tryptophan oxazolidinone (mol. wt 384).
580
550
600
650
700
750
800
850
rn /z Figure 14. Mass spectrum of Nind-HFB-tryptophan oxazolidinone (mol. wt 580).
@ Heyden & Son Ltd, 1979
50
100
150
200
250 m /z
300
400
450
500
Figure 16. Mass spectrum of aspartic w-[bis-(chlorodifluoromethy1)methoxy methyl] ester oxazolidinone (mol. wt 525).
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 385
R. LIARDON, U. OTT-KUHN AND P. HUSEK
-z
168
1001
-$
I
a%&
' 8
55
100
I
1
50
a%=
= S0
454
50
100
I50
200
250 m /z
300
350
450
500 m/z
Figure 17. Mass spectrum of glutamic acid w-IbisfchlorodifluoromethyI)methoxy methyl] ester oxazolidinone (mol. wt 539).
Figure 19. Mass spectrum of N6-HFB-a,S-diaminobutyric acid oxazolidinone (mol. wt 494).
266
1-+
:296
I
69 1
-
CFzCI 2IZ:JIZ
50
HNXo
100
200
150
250 m /z
300
,~ 350
-
389 405 423
400
.
450
Figure 20. Mass spectrum of N"-HFB-ornithine oxazolidinone (mol. wt 508).
1''
:)I0
CF,CI j 0 jZB2 I ill! CH,-O-C~O~C+CH,-CH, 1 .. CF,CI'
780
I..
213:326
5 :
HNXo
226 169
Scheme 7. Breakdown pattern for the molecular ion of aspartic and glutamic acid o-[bis-(chlorodifluoromethy1)methoxymethyllester oxazolidinone.
196
I
,+.I
50
100
150
200
250 m /z
___ 400 450 419
,
300
350
Figure 21. Mass spectrum of N"-HFB-lysine oxazolidinone (mol. wt 522).
CF,CI 0 11 I CH3-0-C-0-CI CFzCl
l'+ -CI -P
CFzCl 0 I It CH3-0-C-0-C\I + CF2
effect of the two nitrogens on the same bond. Fragment g is observed only in the spectrum of this same compound. This supports the assumption that its formation involves the transposition of the amide hydrogen onto the oxazolidinone carbonyl via a 6-membered transition
...
Scheme 8. Reaction pathway for fragment ion mlz81 in the mass spectra of aspartic and glutamic acid derivatives.
Figures 18 to 21 present the mass spectra of four compounds belonging to this series: a,B-diaminopropionic acid (DAPA) ( n = l), a,y-diaminobutyric acid (DABA) ( n = 2), ornithine ( n = 3) and lysine ( n = 4). It can be seen that the fragmentation pattern of these compounds depends considerably on the length of the aliphatic chain. An attempt to rationalize these reactions is detailed in Schemes 9 to 11. The structure of DAPA oxazolidinone favours the formation of ion m l z 226 by combining the activation
0 H 0 II I P + I1 C3F~C-NH-CH-~=NH-C-CFzCl
0
I1
-+ C3F7C-NH-CH=CH
d
1''
m / z 238
TI: ^^^ m / z 227
HNXo Mol. wt 480
1 C,F,CO . ,
\
&6
0
1
\
It
+
C3F,C-NH=CHZ m / z 226
OH C,F,C=NH2 I +
m / z 214
50
100
150
200
250 m /I
300
350
400
450
Figure 18. Mass spectrum of NY-HFB-n,y-diaminopropionicacid oxazolidinone (mol. wt 480).
386 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
g,m/z 255
m / z 170
Scheme 9. Breakdown pattern for NY-HFB-a,y-diamino pro. pionic acid oxazolidinone molecular ion.
0Heyden & Son Ltd, 1979
a-AMINO ACID OXAZOLIDINONES 0
0
II
II
+
C~F$-NH-CH~-CH~-CH=NH-C-CFZCI
0
1'
I1
+ C~FTC-NH-C~H~
d
m l z 252
T 0
01"
c,F,c-NH-cH,-cH,-,-+ II
HNXo
m / z 226
m/r214
Mol. wt 494
1
\
CHz=CH-CH
II
Cn,-Ln
CHz=CH-CH
II
1
HhX" m/z 218
m l r 168
Scheme 10. Breakdown pattern for N'-HFB-a,S-diaminobutyric acid oxazolidinone molecular ion.
HNXo Mol.wt 508,522
\ 0
I1
C~F~C-NH-[CH~]Z,~-CH-CH
1+1
H
+NH
I I
c=o
d
CFZCI
1
0,
m l z 266,280
C-CFzCl
mechanism does not take place in the fragmentation of the other members of that series. It may be only the consequence of subtle variations in the thermodynamics of the competing reactions. The fragmentation of ornithine and lysine derivatives is detailed in Scheme 11. The main process involves first the formation of d according to the reaction shown in Scheme 1. It is followed by the elimination of difluorochloroacetamide, resulting in 0. In the fragmentation of DAPA and DABA derivatives, the same reaction is of only minor importance. Its predominance in the decomposition of ornithine and lysine has been interpreted as an indication that these molecules present a particularly favourable configuration. This is the case if the reaction mechanism involves the formation of a 5- or 6-membered ring, as shown below. A very similar reaction has been reported in the fragmentation of NTFA(HFB)-O-alkyl esters of ornithine and 1 y ~ i n e . l ~ Proline and hydroxyproline
II
0 Scheme 11. Fragmentation pathway for N"-HFB-ornithine and lysine oxazolidinone molecular ion.
state. The absence of ion g from the spectra of the higher homologues indicates that aliphatic hydrogens are less readily transferred. The main fragmentation pathway of the molecular ion of D AB A-oxazolidinone consists of the transposition of a hydrogen onto the HFB carbonyl via a McLafferty rearrangement (Scheme 10). Further decomposition of the oxazolidinone moiety results in the formation of ions m / z 218 and m / z 168. It is not quite clear why this @ Heyden & Son Ltd, 1979
Figures 22 and 23 present the mass spectra of proline and hydroxyproline derivatives. The mechanism of fragmentation of proline oxazolidinone follows 182
295 .
50
100
1
300
350
400
4 50
Figure 22. Mass spectrum of proline oxazolidinone (mol. wt 295).
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 387
R. LIARDON, U. OTT-KUHN AND P. HUSEK
CIF,C-C,
".'\r "+
F k O
0
m / z 230
I
c=o
+o
I
CF,C1
MoLwt 507
m / z 180
CIF,C m / z 208
Scheme 12. Fragmentation pathways for 0-HFB-hydroxyproline oxazolidinone molecular ion.
precisely the basic pattern described above. It results in a very stable fragment ion d at m / z 182. For hydroxyproline the first reaction appears to be a McLafferty rearrangement induced by the HFB group. The resulting fragment can be considered formally as the molecular ion of an unsaturated proline oxazolidinone. Its further decomposition again follows the basic fragmentation pattern of the oxazolidinone ring (Scheme 12). Sulfur-containing amino acids
The mass spectra of methionine and methylcysteine oxazolidinone are presented in Figs. 24 and 25. They are dominated by mass peak m / z 61, which results from the &cleavage induced by the sulfur atom (Scheme 13).The predominance of this mechanism is more pronounced for methylcysteine, in which both sulfur and nitrogen atoms activate the same &bond.
Arginine
Figure 27 presents the mass spectrum of arginine HFBoxazolidinone. A complete understanding of the fragmentation pattern, shown by this spectrum, could not be achieved. The reaction mechanisms, proposed in Scheme 15, explain only the formation of some of the observed fragment ions. Prominent ions like m / z 215 and 180, as well as most of the high mass fragments, could not be rationalized. We suspect them to belong to the mass spectrum of a second compound eluting at exactly the same retention time as the expected arginine derivative. However, this could not be confirmed under our experimental conditions.
n
216
50
K
200
250
329
300
350
400
450
Figure 24. Mass spectrum of methionine oxazolidinone (mol. wt
61
m/z
150
I00
244 282284
329).
Scheme 13. Main fragmentation reaction for methylcysteine and methionine oxazolidinone molecular ion.
Figure 26 presents the mass spectrum for cystine oxazolidinone. It shows a very complex fragmentation pattern. The formation of most primary fragments is readily explicable, as detailed in Scheme 14. On the other hand, it has not yet been possible to rationalize the reaction pathways leading to the lower mass ions. In particular, no satisfactory explanation has been found for the base peak at m / z 107.
!
0s
3j
50/&
,,,, ,
I ,
68 85
,
,,
me
I,
,
230
,
,
, ~ ,, ,
,
,
200
252
250 m /z
315 ~
_A.
300
-
1 -
350
I
400
-~
450
Figure 25. Mass spectrum of methylcysteine oxazolidinone (mol. wt 315).
g;
294
394
422
d--,
50
332
.-+w. --
I80
1.. 007
a 5n
---I50
100
150
200
250 m/z
300
350
400
450
5
O
L
n
h
202 , 220 , 253
154''~
50
100
150
200
250
300
333
-L Ah.-300 350 400 450
m /z
Figure 23. Mass spectrum of 0-HFB-hydroxyproline oxazolidinone (mol. wt 507).
388 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
Figure 26. Mass spectrum of cystine oxazolidinone (mol. wt 600).
0Heyden & Son Ltd, 1979
a-AMINO ACID OXAZOLIDINONES HS-S-CH
,-?"
3'm/r 2 4 8 - y m/z 220 169'e2
m / z 433 HNXo
215 296
T 1
m / z 300
m/z
Figure 27. Mass spectrum of N,N'-HFB-arginine oxazolidinone (mol. wt 746).
HNXo m / r 268
-z -
Scheme 14. Partial breakdown pattern for cystine oxazolidinone
57
100 I
Eg
#< 5 0 j l L
molecular ion.
n 0
Histidine
I20
1
b
222 Z5O
&A L T ~
150
100
,I -L 200
- -
Petr Husek Research Institute of Endocrinology, Narodni trida 8, 11694 Prag I, Czechoslovakia
2-Bis-(chlorodifluoromethyl)-4-substituted-1,3-oxazolidin-5-ones, a new type of a -amino acid derivative for gas chromatographicseparation, have been studied by low resolution mass spectrometry. These derivatives are obtained by reacting a-amino acids with dichlorotetrafluoroacetone. Their structure has been established or confirmed for most protein amino acids and several non-protein a -amino acids. The mechanismsresponsible for the mass spectral pattern have been rationalized. An interesting feature of this derivatization procedure is that it distinguishes aspartic and glutamic acid from the respective amides. The structure of asparagine and glutamine derivatives has been established. A survey of oxazolidinone mass spectra has provided a list of diagnostically useful ions. Each amino acid can be identified by one or two of its most abundant fragments.
INTRODUCTION
The application of gas chromatography (GC) to the analysis of amino acids has been the object of many investigations. Various procedures have been proposed to convert these compounds into suitable volatile derivatives. A comprehensive review on this topic has been presented by Husek and Macek.' The most widely used derivatives are the N-perfluoroacyl-0- alkyl esters. This is essentially the result of the remarkable work of Gehrke et ~ 1 . Several ~ ' ~ authors have later proposed various modifications of the original Some years ago a new derivatization method, based on the specific reaction of 1,3-dichlorotetrafluoroacetone DCTFA) with a-amino acids, was proposed by H ~ s e k . ~ This * procedure is characterized by much milder conditions than are generally applied for the derivatization of amino acids. The reactions are carried out in slightly acidic media and the temperature never exceeds 70 "C.The first application of the procedure was the determination of tyrosine and thyroid hormones, triiodothyronine and thyroxin, in human blood. More recently it was shown that the same method could be applied for the determination of aspartic and glutamic acids and their amides. An important advantage of the procedure was that asparagine and glutamine did not hydrolyse. They could be determined simultaneously with aspartic and glutamic acids, in the same run." Further developments extended the method to all aamino acids.12 As an example of biomedical application it was tested on the determination of free amino acid in human serum and urine. Complete aminograms were obtained with 50 to 100 ~a mp1es.l~ t Abbreviations: DCTFA = dichlorotetrafluoroacetone; HFBA =
\
heptafluorobutyric anhydride; IBFC = isobutylchloroformate; DAPA = a,@-diaminopropionic acid; DABA = LI, y-diaminobutyric acid.
The principle of the derivatization is illustrated in Scheme 1. At first DCTFA reacts with a-amino acids and converts them into 2-bis-(chlorodifluoromethyl)-4substituted- 1,3-oxazolidinones. In subsequent steps the remaining polar groups are modified with heptafluorobutyric anhydride (HFBA), methanol and isobutylchloroformate (IBFC) in the same reaction vessel. The resulting derivatives present good gas chromatographic properties. Subsequently in the text they will be referred to simply as amino acid oxazolidinones. Samples for derivatization are prepared in the same way as holds for N-perfluoroacyl-0-ethyl esters. The formation of oxazolidinone does not involve any special preconditioning of the sample. 0 II
0
II
R-CH-C-OH
I
-+
NH2
+
ClF2C \ CLF&
/
R-CH-C-OH
';"? I ClFZC-C-OH
I
c=o
CFzCl
--+
R
7
q
0
HNx
CIF,C
CF,CI
Scheme 1. Reaction mechanism for the formation of a-amino acid oxazolidinone.
At present the determination of all protein a-amino acids as oxazolidinone is achieved by means of two G C columns. While they could all be separated in a single run, Arg, His, Trp and Cys would come out with a poor yield. Special analytical conditions had to be found for this group. Typical chromatograms of the two G C runs are shown in Figs. 1 and 2. In the development of this derivatization procedure, mass spectrometry was used extensively to determine or confirm the structure of the derivatives. The mass spectra of 26 amino acid oxazolidinones have been measured and analysed. The results of this work are presented here.
CCC-0306-042X/79/0006-0381$05.50 @ Heyden & Son Ltd, 1979
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 381
R. LIARDON, U. OTT-KUHN AND P. HUSEK
The mass spectra were obtained on a HewlettPackard 5992 combined gas chromatograph mass spectrometer. The samples were chromatographed on two different columns: (A) 10 m x 0.3 mm i.d. glass capillary coated with OV-17; (B) 1.2 m x 2 mm i.d. glass column containing 3% SE-30 on Chromosorb W 45/60. Operational temperatures were: injection port, 200 "C; column A, 70 to 220"C, programmed at 8"Cmin-'; column B, 150 to 260 "C, programmed at 8 "Cmin-'. Helium was used as carrier gas. The capillary column A was interfaced with the mass spectrometer by means of a restrictor. A 0.8 ml min-' constant flow was admitted into the ion source, while the remainder of the column effluent was vented out. For column B a membrane separator replaced the restrictor.
-
i
RESULTS AND DISCUSSION Figure 1. Amino acid standard mixture analysed on column A and nitrogen specific detector. For conditions, see text.
EXPERIMENTAL Amino acids, as a single compound or in standard mixtures, were converted into oxazolidinone derivatives according to the procedure of Husek."'"
To our knowledge, substituted 1,3-oxazolidin-5-ones have not been the object of any previous study by mass spectrometry. Therefore, the identification of amino acid oxazolidinones necessitated first the elucidation of the fragmentation mechanism of this type of compound. From the mass spectra of those for which there was no structural ambiguity, we could extrapolate to the other amino acid derivatives and confirm or establish their structure. The results of this study are detailed below. Basic fragmentation pattern From a study of the mass spectra of all protein a-amino acid oxazolidinones, we could establish the existence of a common fragmentation pattern which is determined by the structure of the oxazolidinone ring. These reactions are detailed in Scheme 2. Two competitive processes occur with the fragmentation of the molecular ion. The first consists of loss of a chlorine atom, immediately followed by the elimination of CO giving ion b. The second process is the loss of CFzCI. Alternatively, the positive charge remains on the oxazolidinone moiety c, or is carried away by the CFzCl fragment (m/z85).The two reaction pathways join together with the formation of d. The relative abundances of the ions formed in the above processes vary considerably from one amino acid to the other. Depending on the nature of R, other reactions can take place and compete successfully with the basic fragmentation processes. These additional reactions are discussed in the following sections.
Aliphatic amino acids
5 Time (rnin)
10
Figure 2. Amino acid standard mixture analysed on column B. For conditions. see text.
382 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
Figures 3 to 9 present the mass spectra for the oxazolidinone derivatives of several aliphatic amino acids, including norleucine and a-amino caprylic acid. The fragmentation mechanism of these compounds consists primarily of the reaction described in the previous section. Further decomposition of d also takes place, except for the derivatives of glycine and alanine. It involves the transfer of one or two hydrogens from the aliphatic chain. This process i s illustrated in Scheme 3. @ Heyden & Son Ltd, 1979
a-AMINO ACID OXAZOLIDINONES R-CH
100.
1 .1
u.
255 L
200
HN,O
]1
226 237
1I
I1 I,
250 m /z
300
350
400
450
Figure 7. Mass spectrum of isoleucine oxazolidinone (mol. wt 31 1).
-"Y CF,CI
+CFZCl m / z 85
I001
c=o I kFJI
69 1
d
C
Scheme 2. Basic fragmentation reactions of amino acid oxazolidinone molecular ion. m/z
Figure 8. Mass spectrum of norleucine oxazolidinone (rnol. wt 311).
I
,
,
I
,
250
, .
, , , ,
300
350
m*u'I.
il -
m/z
Figure 3. Mass spectrum of glycine oxazolidinone (mol. wt 255).
I30 142
n
*.ILL,.
50
100
170 L.
150
254 L 276 4
1
200
250
300
350
400
450
Figure 9. Mass spectrum of a-amino caprylic acid oxazolidinone (mol. wt 339).
m/z
Figure 4. Mass spectrum of alanine oxazolidinone (rnol. wt 269).
170 I 184
100,
When a hydrogen is present in the y-position of the aliphatic chain, a McLafferty type rearrangement takes place, initiating the fragmentation sequence shown in Scheme 4. This process is particularly important for isoleucine and valine which possess five or six yhydrogens respectively. The relative abundance of fragments d to i for the various aliphatic amino acids are reported in Table 1, showing how the size and the structure of the sidechain affect the fragmentation pattern. Serine and threonine
m/z
Figure 5. Mass spectrum of valine oxazolidinone (rnol. wt 297).
,
69
100.
I
198
m/z
Figure 6. Mass spectrum of leucine oxazolidinone (mol. wt 31 1).
Figures 10 and 11 present the mass spectra for serine and threonine HFB-oxazolidinones. The presence of the HFB group in these molecules is readily recognized by the appearance of m / z 169 [C3F7]' in their mass spectrum. The HFB carbonyl induces the fragmentation described in Scheme 5 . The transposition of the ahydrogen via a McLafferty rearrangement leads to the elimination of heptafluorobutyric acid and the formation of ion k.This species is identical to the molecular ion of an &$-unsaturated aliphatic amino acid oxazolidinone. Its fragmentation follows the basic pattern R-CH=CH
0 I1 R'-CH-CH=NH-C-CFzCI
+?-
u
d
0 +
c*
I1
1'
f f
R'-CH=CH-NH2-C-CF2CI
\
OH + I HzN=C-CFzCI e, m / z 130
Scheme 3. Decomposition pathways for fragment ion d.
@ Heyden & Son Ltd, 1979
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 383
R. LIARDON, U. OTT-KUHN AND P. HUSEK
Table 1. Relative abundance of aliphatic amino acid oxazolidinone fragment ions
\
,S=O;
Fragmenta
R
d
Glycine
H
Alanine Valine Norleucine Leucine lsoleucine a-Amino caprylic acid
CH3 (CH3).nCH CH3(CH2I4 CH&H2CH(CHJCH2 (CH3(CH2)2CH(CH3)
a
e
10010092 19 73 18 86 24 42 9
f
g
-
-
h
i
I
- - -
62 15 100 100 - 7 100 2 16 55 30 100
I
HNXo
6 25 11 8
45 15 100
1
R'-CH
0
II
R,--cH~\$
II
6 15
k
HNXo
C CH3(CH&
l
I
HNYo
HN+
See Schemes 2-4.
J
\ R'-CH
II
R'
C
k H OH
I HN
I
-
+
X0
d
\I
1"
H?' I
C=O
I
I
HNXo g,
Scheme 5. Main fragmentation pathways for 0-HFB serine and threonine oxazolidinone molecular ion.
m l z 255
-CF2CI
i
detailed above, resulting in the formation of n, with 1 and rn as alternate intermediates. II HN
+
-
Aromatic amino acids
c=o
HN I
n
CF2CI
I CF,CI
I
h, m / z 170
i, m / z 142
Scheme 4. McLafferty rearrangement and decomposition of aliphatic amino acid oxazolidinone molecular ion.
The mass spectra of phenylalanine, tyrosine and tryptophan are presented in Figs. 12 to 14. For these compounds, the fragmentation consists almost only of scission of the 0 C-C bond (Scheme 6), which may be accompanied by the transfer of a hydrogen. The aromatic or heterocyclic ring and the a-nitrogen occur to make this bond particularly labile. The positive charge is carried away by the phenyl or indole moiety. In tyrosine and tryptophan, the resulting ions undergo further fragmentation, as shown below.
I54
I00
n m/z
Figure 10. Mass spectrum of 0-HFB-serine oxazolidinone (mol. wt 481 1.
100
w
150
200
250 m /z
300
350
400
450
Figure 11. Mass spectrum of 0-HFB-threonine oxazolidinone (mol. wt 495).
384 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
200
250 mb
345
300
u '
69 ~
100
150
350
400
450
Figure 12. Mass spectrum of phenylalanine oxazolidinone (mot. wt 345).
K c
50
282
232
I , ..I., ,,
50
50
,,,,
100
~
,
l~
150
,
,
200
250 m/z
2~
1
300
344
,
350
444
494 ~
450
500
Figure 13. Mass spectrum of 0-HFB-tyrosine oxazolidinone (mot. wt 557).
@ Heyden & Son Ltd, 1979
a-AMlNO ACID OXAZOLIDINONES
1’
07’
0
[email protected]
+C
HNXo
J7!-OGCHz
C3F7-0 m / z 275
m / z 303
c=o
c=o
I
COCZFS
\
I
C3F7
C3F7 m / r 326
m / z 276
QC’+ m / z 129
Sch me 6. Fragmentation reactions fo phenylalanine, 0-HFB-tyrosine and N-HFB-tryptophan oxazolidinone molecular ions.
When a simplified procedure is applied for the determination of selected amino acids, tryptophan oxazolidinone may be analysed in the non-acylated form.I4 The corresponding mass spectrum is shown in Fig. 15. It consists essentially of ion m / z 130 which results from the cleavage of the @-bond.
A mechanism explaining the formation of the fragment corresponding to mass peak m / z 81 is proposed in Scheme 8. a,w- Diamino acids
The general structure of the HFB-oxazolidinone derivatives of a,@-diamino acids is shown below.
Aspartic and glutamic acid The addition of methanol to DCTFA in the second step of the derivatization procedure leads to the formation of bis-(difluorochloromethyl) methoxymethanol. In the presence of HFBA, this compound reacts with the wcarboxyl group of aspartic and Iutamic acid oxazolidinones to form complex esters.’ The mass spectra of these derivatives are presented in Figs. 16 and 17. It can be seen that most fragments originate from scission of the bonds which are activated by the ester group or the a-nitrogen (Scheme 7). These processes may also involve the transposition of one or two hydrogens.
0
I1
c,F,c-NH-~cH~)~-,-----~~
H N X o
L IM
1001
6
;E!
r5B
50
50
I
100
150
200
250
300
- . ,
350
.
400 u14 ,
,
450
,
326
I
1
467
450
517
500
Figure 15. Mass spectrum of non-acylated tryptophan oxazolidinone (mol. wt 384).
580
550
600
650
700
750
800
850
rn /z Figure 14. Mass spectrum of Nind-HFB-tryptophan oxazolidinone (mol. wt 580).
@ Heyden & Son Ltd, 1979
50
100
150
200
250 m /z
300
400
450
500
Figure 16. Mass spectrum of aspartic w-[bis-(chlorodifluoromethy1)methoxy methyl] ester oxazolidinone (mol. wt 525).
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 385
R. LIARDON, U. OTT-KUHN AND P. HUSEK
-z
168
1001
-$
I
a%&
' 8
55
100
I
1
50
a%=
= S0
454
50
100
I50
200
250 m /z
300
350
450
500 m/z
Figure 17. Mass spectrum of glutamic acid w-IbisfchlorodifluoromethyI)methoxy methyl] ester oxazolidinone (mol. wt 539).
Figure 19. Mass spectrum of N6-HFB-a,S-diaminobutyric acid oxazolidinone (mol. wt 494).
266
1-+
:296
I
69 1
-
CFzCI 2IZ:JIZ
50
HNXo
100
200
150
250 m /z
300
,~ 350
-
389 405 423
400
.
450
Figure 20. Mass spectrum of N"-HFB-ornithine oxazolidinone (mol. wt 508).
1''
:)I0
CF,CI j 0 jZB2 I ill! CH,-O-C~O~C+CH,-CH, 1 .. CF,CI'
780
I..
213:326
5 :
HNXo
226 169
Scheme 7. Breakdown pattern for the molecular ion of aspartic and glutamic acid o-[bis-(chlorodifluoromethy1)methoxymethyllester oxazolidinone.
196
I
,+.I
50
100
150
200
250 m /z
___ 400 450 419
,
300
350
Figure 21. Mass spectrum of N"-HFB-lysine oxazolidinone (mol. wt 522).
CF,CI 0 11 I CH3-0-C-0-CI CFzCl
l'+ -CI -P
CFzCl 0 I It CH3-0-C-0-C\I + CF2
effect of the two nitrogens on the same bond. Fragment g is observed only in the spectrum of this same compound. This supports the assumption that its formation involves the transposition of the amide hydrogen onto the oxazolidinone carbonyl via a 6-membered transition
...
Scheme 8. Reaction pathway for fragment ion mlz81 in the mass spectra of aspartic and glutamic acid derivatives.
Figures 18 to 21 present the mass spectra of four compounds belonging to this series: a,B-diaminopropionic acid (DAPA) ( n = l), a,y-diaminobutyric acid (DABA) ( n = 2), ornithine ( n = 3) and lysine ( n = 4). It can be seen that the fragmentation pattern of these compounds depends considerably on the length of the aliphatic chain. An attempt to rationalize these reactions is detailed in Schemes 9 to 11. The structure of DAPA oxazolidinone favours the formation of ion m l z 226 by combining the activation
0 H 0 II I P + I1 C3F~C-NH-CH-~=NH-C-CFzCl
0
I1
-+ C3F7C-NH-CH=CH
d
1''
m / z 238
TI: ^^^ m / z 227
HNXo Mol. wt 480
1 C,F,CO . ,
\
&6
0
1
\
It
+
C3F,C-NH=CHZ m / z 226
OH C,F,C=NH2 I +
m / z 214
50
100
150
200
250 m /I
300
350
400
450
Figure 18. Mass spectrum of NY-HFB-n,y-diaminopropionicacid oxazolidinone (mol. wt 480).
386 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
g,m/z 255
m / z 170
Scheme 9. Breakdown pattern for NY-HFB-a,y-diamino pro. pionic acid oxazolidinone molecular ion.
0Heyden & Son Ltd, 1979
a-AMINO ACID OXAZOLIDINONES 0
0
II
II
+
C~F$-NH-CH~-CH~-CH=NH-C-CFZCI
0
1'
I1
+ C~FTC-NH-C~H~
d
m l z 252
T 0
01"
c,F,c-NH-cH,-cH,-,-+ II
HNXo
m / z 226
m/r214
Mol. wt 494
1
\
CHz=CH-CH
II
Cn,-Ln
CHz=CH-CH
II
1
HhX" m/z 218
m l r 168
Scheme 10. Breakdown pattern for N'-HFB-a,S-diaminobutyric acid oxazolidinone molecular ion.
HNXo Mol.wt 508,522
\ 0
I1
C~F~C-NH-[CH~]Z,~-CH-CH
1+1
H
+NH
I I
c=o
d
CFZCI
1
0,
m l z 266,280
C-CFzCl
mechanism does not take place in the fragmentation of the other members of that series. It may be only the consequence of subtle variations in the thermodynamics of the competing reactions. The fragmentation of ornithine and lysine derivatives is detailed in Scheme 11. The main process involves first the formation of d according to the reaction shown in Scheme 1. It is followed by the elimination of difluorochloroacetamide, resulting in 0. In the fragmentation of DAPA and DABA derivatives, the same reaction is of only minor importance. Its predominance in the decomposition of ornithine and lysine has been interpreted as an indication that these molecules present a particularly favourable configuration. This is the case if the reaction mechanism involves the formation of a 5- or 6-membered ring, as shown below. A very similar reaction has been reported in the fragmentation of NTFA(HFB)-O-alkyl esters of ornithine and 1 y ~ i n e . l ~ Proline and hydroxyproline
II
0 Scheme 11. Fragmentation pathway for N"-HFB-ornithine and lysine oxazolidinone molecular ion.
state. The absence of ion g from the spectra of the higher homologues indicates that aliphatic hydrogens are less readily transferred. The main fragmentation pathway of the molecular ion of D AB A-oxazolidinone consists of the transposition of a hydrogen onto the HFB carbonyl via a McLafferty rearrangement (Scheme 10). Further decomposition of the oxazolidinone moiety results in the formation of ions m / z 218 and m / z 168. It is not quite clear why this @ Heyden & Son Ltd, 1979
Figures 22 and 23 present the mass spectra of proline and hydroxyproline derivatives. The mechanism of fragmentation of proline oxazolidinone follows 182
295 .
50
100
1
300
350
400
4 50
Figure 22. Mass spectrum of proline oxazolidinone (mol. wt 295).
BIOMEDICAL MASS SPECTROMETRY, VOL 6, NO. 9, 1979 387
R. LIARDON, U. OTT-KUHN AND P. HUSEK
CIF,C-C,
".'\r "+
F k O
0
m / z 230
I
c=o
+o
I
CF,C1
MoLwt 507
m / z 180
CIF,C m / z 208
Scheme 12. Fragmentation pathways for 0-HFB-hydroxyproline oxazolidinone molecular ion.
precisely the basic pattern described above. It results in a very stable fragment ion d at m / z 182. For hydroxyproline the first reaction appears to be a McLafferty rearrangement induced by the HFB group. The resulting fragment can be considered formally as the molecular ion of an unsaturated proline oxazolidinone. Its further decomposition again follows the basic fragmentation pattern of the oxazolidinone ring (Scheme 12). Sulfur-containing amino acids
The mass spectra of methionine and methylcysteine oxazolidinone are presented in Figs. 24 and 25. They are dominated by mass peak m / z 61, which results from the &cleavage induced by the sulfur atom (Scheme 13).The predominance of this mechanism is more pronounced for methylcysteine, in which both sulfur and nitrogen atoms activate the same &bond.
Arginine
Figure 27 presents the mass spectrum of arginine HFBoxazolidinone. A complete understanding of the fragmentation pattern, shown by this spectrum, could not be achieved. The reaction mechanisms, proposed in Scheme 15, explain only the formation of some of the observed fragment ions. Prominent ions like m / z 215 and 180, as well as most of the high mass fragments, could not be rationalized. We suspect them to belong to the mass spectrum of a second compound eluting at exactly the same retention time as the expected arginine derivative. However, this could not be confirmed under our experimental conditions.
n
216
50
K
200
250
329
300
350
400
450
Figure 24. Mass spectrum of methionine oxazolidinone (mol. wt
61
m/z
150
I00
244 282284
329).
Scheme 13. Main fragmentation reaction for methylcysteine and methionine oxazolidinone molecular ion.
Figure 26 presents the mass spectrum for cystine oxazolidinone. It shows a very complex fragmentation pattern. The formation of most primary fragments is readily explicable, as detailed in Scheme 14. On the other hand, it has not yet been possible to rationalize the reaction pathways leading to the lower mass ions. In particular, no satisfactory explanation has been found for the base peak at m / z 107.
!
0s
3j
50/&
,,,, ,
I ,
68 85
,
,,
me
I,
,
230
,
,
, ~ ,, ,
,
,
200
252
250 m /z
315 ~
_A.
300
-
1 -
350
I
400
-~
450
Figure 25. Mass spectrum of methylcysteine oxazolidinone (mol. wt 315).
g;
294
394
422
d--,
50
332
.-+w. --
I80
1.. 007
a 5n
---I50
100
150
200
250 m/z
300
350
400
450
5
O
L
n
h
202 , 220 , 253
154''~
50
100
150
200
250
300
333
-L Ah.-300 350 400 450
m /z
Figure 23. Mass spectrum of 0-HFB-hydroxyproline oxazolidinone (mol. wt 507).
388 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 9, 1979
Figure 26. Mass spectrum of cystine oxazolidinone (mol. wt 600).
0Heyden & Son Ltd, 1979
a-AMINO ACID OXAZOLIDINONES HS-S-CH
,-?"
3'm/r 2 4 8 - y m/z 220 169'e2
m / z 433 HNXo
215 296
T 1
m / z 300
m/z
Figure 27. Mass spectrum of N,N'-HFB-arginine oxazolidinone (mol. wt 746).
HNXo m / r 268
-z -
Scheme 14. Partial breakdown pattern for cystine oxazolidinone
57
100 I
Eg
#< 5 0 j l L
molecular ion.
n 0
Histidine
I20
1
b
222 Z5O
&A L T ~
150
100
,I -L 200
- -
