H.-G. Schmarr et al.
H
H
O
O
compound (later assigned as 3b) according to Gerritsma et al.16
(Figure 1, route C). GC and GC-MS data of the compound
obtained from route A are consistent with those of the reference
compound SBMP 4b from Sigma-Aldrich (Table S-1). Consider-
ing earlier results,2,13 1,2-dihydro-3-sec-butyl-1-methyl-2-pyrazi-
none (or 3-sec-butyl-1-methyl-1H-pyrazin-2-one) 3b was
considered a possible product obtained from route C. Direct
comparison of the 1H-NMR spectra of commercial SBMP 4b and
its deuterated isotopologue d3-4b from route A and of 3b and
its deuterated isotopologue d3-3b showed disappearance of the
methyl-singlets at 3.96 and 3.49 ppm, respectively (full spectral
listings are in the experimental supplementary information
section). This chemical shift difference of almost 0.5 ppm and a
remarkable upfield shift of the aromatic protons by about 1 ppm
for 4b clearly demonstrate the success of incorporating the
CH3-groups in different positions in compounds 4b and 3b.
The pattern of the other signals of d3-4b is comparable and in
accordance with published data.11,12,20 In the 13C-NMR spectra
of compound 3b and of commercial MP 4b (Figure S-2), the
signal of the heteroatom bound methyl group is shifted about
16 ppm from 37.3 ppm in 3b to 53.3 ppm in 4b. The positions of
this methyl groups were unambiguously assigned by hetero-
nuclear correlation NMR (1H-13C- and 1H-15N-HMBC) experiments
(Figures S-3 and S-4). This way, the 15N chemical shifts were
estimated for both nitrogen atoms in 3b with ꢀ217 and
ꢀ54 ppm. This is comparable with literature values for the
1H-pyrazin-2-one with ꢀ198 and ꢀ36 ppm.21 It could be shown
(Figure S-3) that the introduction of the methyl group (3.5 ppm)
is at the nitrogen in the amide position (ꢀ217 ppm), whereas
the CH hydrogen (3.3 ppm) of the sec-butyl group shows a
coupling to the nitrogen in 4 position (ꢀ54 ppm) as expected.
This demonstrates that the synthesis pathway suggested by
Gerritsma et al. (route C) lead to the 3-alkyl-1-methyl-1H-pyrazin-
2-ones 3, and not to the desired MPs 4. Such finding underlines
the significance of simple retention index information for
characterization of compounds amenable to GC, as spectral
data can be similar.
H2N
H2N
O
R
+
H
N
N
OH
R
O
R
N
Cl
R
POCl3
N
1
N
1*
N
2
CH2N2
B)
C)
A)
2 :1
NaH,
CH3I
NaOCH3,
CH3OH
N
O
R
N
O
R
N
4
N
3
1a - 4a: R = CH(CH3)2
1b - 4b: R = CH(CH3)CH2CH3
1c - 4c: R = CH2CH(CH3)2
Figure 1. Routes to synthesize MPs as described in the literature.
following the approach of stable isotope dilution analysis (SIDA)
for quantification.17
In the work described here, we synthesized the desired
deuterated 2-[2H3]methoxy-3-alkylpyrazines d3-4a–c for wine
aroma analysis. Considering the published methods described
for MP synthesis, we first followed the method described by
Gerritsma et al.16 (Figure 1, route C) as it seemed to be the most
convenient approach. However, the products obtained showed
different gas chromatographic (GC) properties compared with
reference MPs 4a–c. This paper clarifies some of the previously
published results and summarizes the chromatographic and
spectroscopic characterization of compounds obtained by the
method described by Gerritsma et al.16 and MPs, respectively
their deuterated isotopologues.
Results and discussion
Furthermore, a closer investigation of the mass spectra of
In our hands, the deuterated derivatives obtained after the deuterated 3b (3c) and 4b (4c) revealed a loss of 15 and 18
approach of Gerritsma et al.16 (Figure 1, route C) were not mass units (representing a methyl or a [2H3]methyl group,
the designated MPs d3-4a–c, as they showed considerably respectively) which can also be used for differentiation of the
higher retention indices after GC analysis, than those for two structures. Gerritsma et al.16 described the loss of either
the commercial non-deuterated MP reference substances. We fragments from the molecular ion (M = 169) on the example of
obtained substances, which could later be assigned to the what they believed to be d3-4c. In their mass spectral
structures of d3-3a–c. Incorporation of deuterium into an interpretation, this methyl group (or [2H3]methyl group) loss
organic molecule leads to a somewhat lowered retention index could have come either from the methoxy position or the alkyl
in gas liquid chromatography, despite its higher absolute side chain, as they had found fragment ions at both m/z = 151
molecular mass. This effect is thus called inverse isotope effect18 and 154 in the case of the deuterated compound. In our hands,
and has been attributed to the different binding-length of C–H such fragmentation was only observed for compounds d3-3b
and C–D as summarized by Matucha et al.19 The unexpected and and c (Figure S-5) and not for d3-4b and c (Table S-1).
drastically higher retention indices of more than 200 (on a 5% Interestingly, in a thesis conducted at the same institution as
phenylmethylpolysiloxane stationary phase) or 600 (on
a
Gerritsma’s earlier work,22 Chen11 observed this discrepancy in
polyethylene glycol stationary phase) units for d3-3a–c and mass spectra from compounds synthesized according to route A
4a–c were an indication for a differing chemical nature of the (modified according to Masuda et al.10) as well. Unfortunately,
substances. Unfortunately, Gerritsma and co-workers provided no clear statement had been given that the earlier described
only MS and NMR data, but MS data acquisition had obviously synthesis obviously leads to wrong compounds 3 rather than
been achieved without prior GC separation. A full listing of the desired MPs 4. This is the more surprising, as the same
retention and MS data can be found in the experimental group recently published (as co-authors) a work on quantitative
supplementary section (Table S-1).
analysis of MPs in juice and wine using SIDA.12 The original
To verify synthesis procedures, deuterated sec-butyl pyrazine synthetic route of Karmas and Spoerri7 (A) was followed to
derivative was synthesized according to the originally described obtain deuterated MPs d3-4. However, commenting their
pathway (Figure 1, route A; d3-4b) as well as the non-deuterated earlier work16 had not been an option. With regard to the
J. Label Compd. Radiopharm 2011, 54 438–440
Copyright r 2011 John Wiley & Sons, Ltd.
Schmarr
