Photochemistry of three N-acetoacetyl amino acid methyl esters: Structure elucidation of the radiation products by gas chromatography/mass spectrometry

Article abstract of DOI:10.1002/(SICI)1096-9888(199812)33:12<1256::AID-JMS748>3.0.CO;2-Z

The photochemistry of three N-acetoacetyl α-amino acid (valine, tert-leucine, isoleucine) methyl esters was investigated. The products after UV irradiation in acetonitrile at 300 nm (direct excitation) were analyzed by gas chromatography coupled with chem

Full text of DOI:10.1002/(SICI)1096-9888(199812)33:12<1256::AID-JMS748>3.0.CO;2-Z

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 33, 1256È1260 (1998)
Photochemistry of Three N-Acetoacetyl Amino
Acid Methyl Esters: Structure Elucidation of the
Radiation Products by Gas Chromatography/Mass
Spectrometry
H. Budzikiewicz,* P. Dallakian, A. G. Griesbeck and H. Heckroth
Institut fur Organische Chemie der Universitat zu Koln, Greinstrasse 4, D-50939 Cologne, Germany
The photochemistry of three N-acetoacetyl a-amino acid (valine, tert-leucine, isoleucine) methyl esters was investi-
gated. The products after UV irradiation in acetonitrile at 300 nm (direct excitation) were analyzed by gas chro-
matography coupled with chemical ionization mass spectrometry. In all cases a complex product mixture was
found as the result of the np* excitation of the substrates. The major reaction paths were Norrish type I reactions
and hydrogen atom abstractions with concomitant radical cleavage and radical recombination steps. ( 1998 John
Wiley & Sons, Ltd.
KEYWORDS: photochemistry; a-amino acid methyl esters; gas chromatography/chemical ionization mass spectrometry
aqueous 1.75 M sodium carbonate solution, 10 mmol of
INTRODUCTION
diketene were added over a period of 30 min. After stir-
ring at room temperature for 16 h, the solvent was
evaporated under reduced pressure and the residue was
extracted with 100 ml of CH Cl . After treatment with
In a long-term research project, we are investigating the
photochemical behavior of N- and C-activated amino
acids and peptide model substrates.1,2 The background
of these studies is the understanding of energy and elec-
tron transfer processes in oligopeptides. In order to cor-
relate short- and long-distance processes with the
properties of the excited part of molecule, we have been
systematically investigating model compounds with
restricted reactive sites. The N-acetoacetyl a-amino acid
methyl esters 1aÈc used in this study were expected to
exhibit preferentially triplet nn* carbonyl photochemis-
try. The preferred reaction paths for these chromo-
phores are Norrish type I (a-CÈC homolysis) and
Norrish type II reactions (c-hydrogen atom
abstraction). The latter process was not feasible for sub-
strates 1 because of the strong NcH bond. We investi-
gated the complex product mixtures from direct
photolysis in acetonitrile using gas chromatography/
mass spectrometry (GC/MS).
2
2
80 ml of water, the organic phase was dried over
MgSO , Ðltered and evaporated under reduced pres-
4
sure. Compounds 1aÈc were obtained as a yellow oil.
Irradiation procedure
A solution (9.3 mmol l~1) of 1aÈc in acetonitrile in a
Pyrex vessel purged with a constant stream of dry nitro-
gen was irradiated for 15 h in a Rayonet RPR-208
photochemical reactor equipped with eight Ñuorescence
lamps (300 ^ 5 nm, ca. 800 W). The crude product mix-
tures were analyzed directly by GC/MS and by NMR
spectroscopy.
Instrumentation
All GC/CIMS measurements were performed with a
Finnigan Incos 500 quadrupole mass spectrometer
(Finnigan MAT, San Jose, CA, USA) coupled with a
Varian Model 3400 gas chromatograph (Varian Ana-
lytical Instruments, Sunnyvale, CA, USA) with a split/
splitless injector and an Optima-5 capillary column (25
m ] 0.25 mm i.d., 0.25 lm Ðlm thickness) (MachereyÈ
Nagel, Duren, Germany). The helium Ñow-rate was 1
ml min~1 (head pressure 55 kPa). Injection (1 ll) was
performed manually, with a split ratio of 10 : 1 and an
injection temperature of 250 ¡C. The oven temperature
program was initial temperature 60 ¡C, held for 1 min,
then increased at 10 ¡C min~1 to 250 ¡C, which was
held for 5 min. The transfer line temperature was
250 ¡C. The Incos 500 was equipped with ion sources
EXPERIMENTAL
Synthesis of substrates 1a–c
To a cooled solution (0 ¡C) of 10 mmol of the hydro-
chloride salt of the amino acid methyl ester in 5 ml of
* Correspondence to: H. Budzikiewicz, Institut fur Organische
Chemie der Universitat zu Koln, Greinstrasse 4, D-50939 Cologne,
Germany
Contract/grant sponsor: Deutsche Forschungsgemeinschaft.
Contract/grant sponsor: Fonds der Chemischen Industrie.
CCC 1076È5174/98/121256È05 $17.50
( 1998 John Wiley & Sons, Ltd.
Received 3 July 1998
Accepted 7 October 1998

N-ACETOACETYL AMINO ACID METHYL ESTERS
1257
Figure 1. Mass spectra of N-acetoacetylvaline methyl ester. Top,
EI (70 eV); bottom, CI(CH ). M ¼215.
Figure 2. CI(CH CN) mass spectra of N-acetoacetylvaline
methyl ester (top, M ¼215) and N-acetylvaline methyl ester
(bottom, M ¼173).
3
4
r
r
r
for CI and EI (70 eV). MS parameters were: ion source
set temperature 100 ¡C for CI and 180 ¡C for EI, elec-
tron multiplier voltage 1400 V (CI) and 1150 V (EI).
GC/MS data were acquired using Data General DG-20.
[CI(CH CN)]. EI, CI(CH ) and CI(CH CN) mass
3
4
3
spectra of N-acetylvaline methyl ester and N-
acetoacetylvaline methyl ester are compared in Figs 1
and 2. As can be seen, the EI mass spectra of these com-
pounds (e.g. N-acetoacetylvaline methyl ester, Fig. 1)
are characteristic, but the molecular ions cannot be rec-
ognized with certainty. Acetonitrile as CI reagent gas
has the advantage of not being corrosive towards metal
RESULTS
For these investigations we used electron ionization (EI)
and CI with methane [CI(CH )] and acetonitrile
surfaces (as, e.g., NH ). It has a high proton affinity
4
3
Figure 3. N-Acetoacetyl amino acid esters (1) and photochemically generated products 2–9. Amino acids: a, valine (R ¼iso-C H ); b,
tert-leucine (R ¼tert-C H ); c, isoleucine ÍR ¼CH(CH )CH CH Ë.
3
7
4
9
3
2
3
( 1998 John Wiley & Sons, Ltd.
J. Mass Spectrom. 33, 1256È1260 (1998)

1258
H. BUDZIKIEWICZ ET AL .
Table 1. CI(CH ) mass spectra of 1–9: m/z (relative intensity, % to base peak)
4
ÍMH ÉnCH CO˽
2
n ¼2
Compound (%)a
MH½ b
ÍMH ÉHCO CH ˽
2
ÍMH ÉCH OH˽
3
n ¼1
n ¼3
m/zh
3
1a (5.0)
2a (Ä0.3)
3a (68)
216c
156 (92)d
100
184 (42)
128 (30)
142 (67)
156 (38)
226 (8)
—
—
132 (53)
—
72 (23)
160 (97)
174
—
—
—
114 (78)
128 (45)
198 (10)
198 (5)
153 (78)
285 (8)
170 (38)
132 (32)
132 (32)e
216 (35)
216
—
72 (7)
72 (7)
4a (5.0)
5a (1.4)
6a (1.6)
7a (9.7)
8a (8.7)
9a (Ä0.3)
188
—
—
132 (53)
132 (24)
—
258
174 (66)
174 (8)
—
156 (22)
156 (11)
72 (3)
285 (20)
213
226 (3)
181 (39)
313 (14)
198 (24)
—
345 (95)
230
132 (11)f
—
—
—
214
132 (67)g
—
99 (46)
1b (7.1)
2b (1.5)
3b (65)
4b (3.0)
5b (1.7)
6b (2.2)
7b (11.0)
8b (8.0)
9b (0.5)
230
170 (82)
114
198 (37)
142 (67)
156 (58)
170 (37)
240 (10)
240 (2)
—
—
146 (31)
—
86 (18)
174 (90)
188
—
—
—
128 (73)
142 (43)
212 (8)
212 (3)
167
146 (9)
146 (10)e
230 (27)
230
—
86 (4)
86 (4)
170 (14)
170 (9)
86 (4)
228
202
—
—
146 (44)
146 (19)
—
272
188 (38)
188 (5)
—
272 (27)
227 (87)
373 (98)
244
195 (54)
341 (15)
212 (37)
—
313 (3)
184 (48)
146 (15)f
—
—
—
146 (44)g
—
99 (33)
1c (Ä0.3)
2c (3.4)
3c (65)
230
170 (62)
114 (80)
128 (51)
142 (35)
212 (9)
198 (24)
142 (32)
156 (35)
170 (17)
240 (6)
—
—
146 (49)
—
86 (50)
174
—
—
—
188
146 (13)
146 (18)e
230 (37)
230
—
86 (4)
86 (6)
4c (5.9)
5c (2.3)
6c (4.5)
7c (11.2)
8c (7.1)
9c ( Ä0.3)
202
—
—
146 (28)
146 (25)
—
272
188 (53)
188 (15)
—
170 (10)
170 (13)
86 (3)
272 (23)
227
212 (4)
240 (2)
167 (67)
313 (2)
195 (37)
341 (11)
212 (20)
—
373
146 (23)f
—
—
228 (69)
99 (96)
244 (78)
184 (18)
146 (11)g
—
a By GC/MS; normalized GC peak areas in % after the irradiation for 15 h in acetonitrile at 300 nm and 35 ¡C (the initial
concentration of 1 was 9.3 mmol lÉ1).
b Molecular masses were determined from ÍM ½H˽, ÍM ½C H ˽ and ÍM ½C H ˽ ions in the CI(CH ) spectra.
2
5
3
5
4
c The most abundant ion is given in bold type.
dm/z (relative intensity, %).
e ÍM ÉCH CHCO˽.
3
f ÍH NCH(R)COOCH ½H˽.
g ÍMH ÉCH CH COCHCO˽.
h Other characteristic peaks.
2
3
3
2
(PA) of 782 kJ mol~1, which lies between those of
methane (531 kJ mol~1) and isobutane (816 kJ
CI(CH ) mass spectra
4
mol~1).3h5 We successfully tested CI(CH CN) for some
3
The mass spectra obtained by GC/MS are presented in
Table 1. The molecular masses were determined from
the characteristic peaks of the protonated molecules
MH` and the ions [M ] C H ]` and [M ] C H ]`.
amino acid derivatives (e.g. N-acetoacetyl- and N-
acetylvaline methyl ester, Fig. 2). The protonated molec-
ular ions, cluster ions [MH ] CH CN]` and dimer
3
cluster ions [2M ] H]` were found to be the most
2 5
3 5
The ester function was readily identiÐed by the elimi-
abundant species. The essential absence of fragment
ions permits no further structure information to be
nation of CH OH, [MH [ 32]`, and HCO CH ,
3
2
3
[MH [ 60]`, from the protonated molecules.7,8 The
various substituents at the N-termini gave rise to ion a
(m/z 132 and 146 for valine and leucine derivatives,
respectively).
derived. In contrast, CI(CH ) produces protonated mol-
4
ecules, [M ] C H ]` and [M ] C H ]`, and also
2 5
3 5
characteristic fragment ions6 (Table 1).
The substrates 1aÈc and the photochemically gener-
ated products 2È9 detected in this GC/MS study are
shown in the Fig. 3. The main products under standard
irradiation conditions (the initial concentration of 1 was
9.3 mmol ml~1) were the Norrish type I cleavage pro-
ducts 3. The formation of the precursor radical
~CH CONHCH(R)COOCH is evident because of the
2
3
formation of the products 4, 7 and 8 due to competing
pathways. Substances 5 and 6 originate from radical
acetylation at the b- and d-positions, respectively (see
below).
The elimination of (CH CO) from the MH` ion is in
2
n
accordance with the N-termini of compounds 1, 3, 5,
and 6. The structural di†erences of isomers 5 and 6 are
readily recognized in the CI(CH ) spectra, while the EI
4
( 1998 John Wiley & Sons, Ltd.
J. Mass Spectrom. 33, 1256È1260 (1998)

N-ACETOACETYL AMINO ACID METHYL ESTERS
1259
underwent a multitude of secondary reactions. Hydro-
gen atom transfer to give the N-acetyl amino acid esters
3aÈc prevailed (ca. 70% in the original reaction
mixture). A less favorable path was coupling with an
acetonitrile radical to give the products 7aÈc. The com-
peting dimerization of the primarily formed precursor
radical ~CH CONHCH(R)COOCH led to the com-
2
3
pounds 8aÈc. For the substrates 1aÈc traces of the N-
formyl compounds 2aÈc were found as secondary
products of the alternative Norrish type I process a-
(OxCNH)ÈC bond cleavage; dashed line ii in Fig. 3).
The other minor products 4, 5, 6 and 9 derived from
complex coupling processes with a methyl or an acetyl
radical, respectively. Finally, it is remarkable that both
the alkyl substituent R and the ester group remained
intact even after prolonged irradiation. Comparison of
the photochemistry of N-acetoacetyl amino acid methyl
esters 1aÈc (at j \ 300 nm in acetonitrile) with that of
ex.
the N-acetyl amino acid methyl esters (at j \ 254 nm
ex.
in methanol) revealed that the latter compounds are
efficiently degraded by a-cleavage of the COOCH
3
group and, less efficiently, by a-cleavage of the alkyl
group (exempliÐed for the valine derivative in Table 28).
The analogous process was found to be characteristic
also in EI-induced fragmentation in both N-acetyl and
N-acetoacetyl amino acid methyl esters.
Figure 4. CI(CH ) mass spectra of two isomeric photochemically
generated products 5a (top) and 6a (bottom).
4
In contrast to EI, the characteristic fragmentations
under CI(CH ) conditions are the elimination of meth-
anol and of the protonated methoxycarbonyl group.
The most interesting feature is the elimination of one to
and CI(CH CN) spectra are similar (see, for example,
the spectra of 5a and 6a in Fig. 4).
4
3
three CH CO units from 1, 3, 5 and 6 (and accordingly
2
of C H COCH COÈH from 9). Loss of CH CO from
DISCUSSION
2 5
2
2
[M ] H]` of N-acetyl amino acids is well docu-
mented.9 For the degradation of the acetoacetyl group,
three mechanisms can be envisaged, as shown in Eqns
(1)È(3).
Process (1) corresponds to the loss of ketene from 3
and (2) represents an analogous H-transfer via a six-
membered transition state. Both result in the loss of the
entire acyl group, whereas (3) leads to the loss of
The predominant primary process for substrates 1a–c
after electronic excitation and intersystem crossing to
the triplet excited state was the cleavage of the a-(CxO)
ÈC bond with formation of an acyl radical and a
primary carbon radical (dashed line i in Fig. 3). This
clearly shows that the excitation energy is preferentially
localized in the ketone carbonyl group and not in the
amide carbonyl group. After homolysis, the radical pair
CH CO, and an enol is formed which would not be
expected to fragment by a further loss of CH CO. The
2
2
Table 2. Elimination processes of N-acetylvaline methyl ester under EI,
CI(CH ) and photodissociation (254 nm, in CH OH)
4
3
m/z (% to base peak
CI(CH )
Photochemical product,
molecular mass
Process
EI (70 eV)
4
M ÉCOOCH
114 (90)
114 (95–100)
115a
3
(a-cleavage)
M ÉCH O
142 (3)b
131 (9)
130 (3)c
142 (80; ÍMH ÉCH OH˽)
141c
—
3
M ÉCH CO
2
3
132 (40; ÍMH ÉCH CO˽)
2
M ÉC H
—
131
3
7
(a-cleavage)
a Main product after irradiation for 9 h in CH OH at 254 nm; the probable mecha-
nism is the formation of the precursor radical AcNHCH~CH(CH ) followed by
proton association.
3
3 2
b Takes place simultaneously with elimination of CH CO (m/z 99).
c Four minor products.
2
( 1998 John Wiley & Sons, Ltd.
J. Mass Spectrom. 33, 1256È1260 (1998)

1260
H. BUDZIKIEWICZ ET AL .
for 6 it may be less favorable for steric reasons. Mecha-
nism (3) explains the loss of the one and two CH CO
2
units (transfer of H from C-6 or C-4; see Fig. 4) from 5,
whereas from 6 only one CH CO will be lost. Hence the
2
assignment of the respective structures to the spectra is
reasonable.
CONCLUSION
The photochemically generated products from (alkyl-
substituted) N-acetoacetyl amino acid methyl esters
1aÈc after UV irradiation at 300 nm in acetonitrile were
observation that 1aÈc show a loss of (CH CO) but not
2
2
of CH CO suggests that mechanism (1) or (2) is oper-
2
ating. These considerations are useful for an explana-
investigated using GC/CI(CH )MS. Plausible mecha-
4
tion of the di†erences in the fragmentation patterns of 5
and 6. Compound 5 shows a pronounced loss of one,
nisms for the major reaction paths are described which
involve mainly the photochemistry of the ketone nn*
triplet. Norrish type I reactions are responsible for the
formation of cleavage and coupling products 2È9.
two and three CH CO molecules, whereas for 6 the loss
2
of (CH CO) is drastically reduced and that of one
2
2
CH CO dominates the spectrum. Mechanisms (1) and
(2) lead in either case to the elimination of (CH CO) ;
2
2
3
Acknowledgements
Financial support from the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie is gratefully acknowledged.
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( 1998 John Wiley & Sons, Ltd.
J. Mass Spectrom. 33, 1256È1260 (1998)