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resulted in altered splitting patterns for each methyl group in
and no shift was detected during the titration. Consequently,
we can exclude any hydration/dehydration processes that
occur at the C3 position (this position is readily hydrated in
aqueous solution upon deprotection). These findings are in
agreement with NMR studies that describe a high tendency of
monohydration of a-diketones in aqueous solutions of up to
70%;[13] however, we believe that the electron-withdrawing
effects of the adjacent alcohol also serve to increase the ratio
of monohydration of the diketone in DPD. These findings
allow us to assign the signals from species I to (4S)-3,3,4,5-
tetrahydroxypentan-2-one ((4S)-THP, structure E, Sche-
me 1a). The corresponding species C1 and C2 are in equilib-
rium with E and this can be detected in NOESY and ROESY
spectra (Figure S5 in the Supporting Information). However,
we are not able to fully exclude the linear diketone A and
both nonhydrated cyclic forms B1 and B2 (Scheme 1a), but we
postulate that they are of minor importance in aqueous
solutions. This is also in agreement with the calculated
equilibrium constant of the hydration of this molecule, which
favors hydration, as well as the equilibrium that was suggested
in another study.[7b,14]
Our structural analysis of DPD by NMR spectroscopy has
opened up an entirely new series of equilibrium structures
that were previously not thought to be important. However,
we wished to probe the chemical basis of these equilibrating
chemical partners further. Therefore, we synthesized two
derivatives as model systems for the cyclic and the linear
species of DPD (Figure 2). As our model for the cyclized form
we examined the CF3 analogue of DPD (2); interestingly, this
derivative is the most active agonist in V. harveyi that has been
described to date and is only present in the cyclic form,
hydrated at the C3 position.[9] In our analysis, the equilibrium
of 2 is highly pH-dependent (Figure 2a; see also Figure S6 in
the Supporting Information). Thus, the 1H NMR signal
changes for the C4 and C5 positions are very similar to the
cyclic isomers of natural DPD, and again show complete
hydration at C3, whereas the spectra of 2 and DPD parallel
each other and reemphasize that the equilibrium of the
hydrated species E with C1 and C2 is important.
We synthesized the methylated primary alcohol 5-MeO-
DPD (3) as our linear model compound, which is unable to
engage in any ring closing events. The titration of 3 in the
range of pH 1–7 indicates the presence of a single major
species over the entire pH range, as shown by 1H NMR
spectroscopy (Figure S7 in the Supporting Information). Only
one new minor species that makes up less than 5% of the
sample was detected, and the 13C NMR spectrum clearly
shows the presence of only one carbonyl moiety (Figure 2b).
This linear structure thus houses a single ketone, which
further strengthens our hypothesis that the linear form of
DPD is species E in aqueous solution (Scheme 1a). However,
seemingly in contradiction, diketone A has always been
implicated as the major linear species of DPD in solution,
largely because the addition of 1,2-phenylenediamine gives
the corresponding quinoxaline derivative.[3b,7a,b] To test this,
we treated 3 with an aqueous solution of 1,2-phenylenedi-
amine and achieved complete conversion to the correspond-
ing quinoxaline derivative (Figure S7 in the Supporting
Information). To reconcile this finding, we postulate that
the H NMR spectra (1JCH = 127.9–129.1 Hz and JCH = 5.0–
6.1 Hz). Nevertheless, the inclusion of these two isotopically
labeled compounds allowed us to focus on two carbon atoms
in the 13C NMR spectrum, C1 and C2, which undergo
significant changes (see above) based on hydration or
cyclization. Analysis of the labeled compounds indeed
revealed that the signals in the 13C NMR spectrum are of
better resolution than the proton signals, and we detected
three major (d = 20.0, 20.6, and 25.2 ppm) and four minor
signals for the methyl carbons at acidic pH values. Basic
titration resulted in a decrease in signal intensity of the major
signals and the appearance of new signals, which are clearly
separated from the original signals, at d = 23.1 ppm. However,
these new signals overlap with each other, which indicates
that the new species are all of similar structures. From the
13C NMR spectrum of the monolabeled compound 4 we can
surmise that eight or nine different DPD species are in
equilibrium at physiological pH (Figure S4 in the Supporting
Information). To quantify each labeled signal, we determined
the relaxation time for the 13C nuclei, a technique that is
rarely used in literature for small molecules, but is made
possible with the multiple 13C nuclei. Thus, the overlapping
signals at d = 23.1 ppm were quantified to be 1.5–2 times more
abundant at pH 7 than the three originally identified major
signals (d = 20.0, 20.5, and 25.2 ppm). Surprisingly, quantifi-
cation revealed that the ratio of the original signals is constant
over the whole pH range, with an excess of 4.3–4.6 times the
amount of both cyclic species relative to the linear species.
This clearly shows that the abundance of the original species
is reduced with higher pH, but that the linear/cyclic equilib-
rium is not influenced by the new species at physiological pH.
The additional 13C label that is incorporated into com-
pound 5 at position C2 is important because multiple ring-
closing equilibria are taking place at this position. Further-
more, hydration and dehydration processes could be followed
at this position, which again are readily detectable by
13C NMR spectroscopy (Figure 1b). Importantly, this com-
pound allows direct assignments of each methyl group to the
corresponding quaternary signal of the C2 position by using
the C–C coupling constants. We identified three major species
that we have labeled as species I, II, and III as seen in
Figure 1b (species II consists of both closed species).
1
2
The signal at d = 211 ppm corresponds to a linear species
of DPD that contains a carbonyl group and can be assigned to
1
the methyl group of species I at d = 25.2 ppm with JCC
=
42 Hz (Figure 1b). Furthermore, we assigned both cyclic
forms of DPD at d = 20.0 and 20.5 ppm to the quaternary
carbon signals at d = 104.0 and 104.5 ppm with a higher
coupling constant (1JCC = 47–48 Hz for species II). The ten-
dency of higher coupling constants for the more rigid cyclic
forms relative to the linear form is in accordance with
literature values.[12] Yet, the newly observed signals from
1
species III have an even higher coupling constant of JCC
=
51 Hz. These large values provide a glimpse that other cyclic
forms exist; however, these NMR spectra did not serve to
fully dissect their exact structure.
We detected only one major signal at d = 211 ppm that
corresponds to the C2 carbonyl group of the linear species
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Angew. Chem. Int. Ed. 2012, 51, 4204 –4208