On the detection of both carbonyl and hydroxyl oxygens in amino acid derivatives: A 17O NMR reinvestigation

Article abstract of DOI:10.1016/j.tetlet.2003.12.162

The hypothesis and the conclusions of previous ¹⁷O NMR studies on the detection of both oxygens of the carboxylic group of Boc-[ ¹⁷O]Tyr(2,6-diClBzl)-OH in DMSO-d₆ solution (Tetrahedron Lett. 2000, 41, 8651) are reconsidered. The appearance of two discrete resonances at 340 and 175ppm of this protected amino acid is not now attributed: (a) to the reduction of the intramolecular conformational exchange rate, due to the effect of intramolecular hydrogen bonding of the hydroxy part of the carboxyl with the carbonyl oxygen of the Boc-group, and (b) to the effect of solvent viscosity, suggested in the mentioned study. The cause of this phenomenon is now attributed to a strong hydrogen bonding of the polar proton acceptor solvent DMSO with the carboxy group, which effectively reduces the proton exchange rate, thus becoming slow on the ¹⁷O NMR time scale.

Full text of DOI:10.1016/j.tetlet.2003.12.162

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2243–2245
On the detection of both carbonyl and hydroxyl oxygens in
17
amino acid derivatives: a O NMR reinvestigation
Vassiliki Theodorou,a Anastassios N. Troganisb and Ioannis P. Gerothanassisa,*
aDepartment of Chemistry, University of Ioannina, GR-45 110 Ioannina, Greece
bDepartment of Biological Applications andTechnologies, University of Ioannina, GR-45 110 Ioannina, Greece
Received 6 October 2003; revised 11 December 2003; accepted 18 December 2003
Abstract—The hypothesis and the conclusions of previous 17O NMR studies on the detection of both oxygens of the carboxylic
group of Boc–[17O]Tyr(2,6-diClBzl)–OH in DMSO-d6 solution (V. Tsikaris et al., Tetrahedron Lett. 2000, 41, 8651) are reconsidered.
The appearance of two discrete resonances at 340and 175 ppm of this protected amino acid is not now attributed: (a) to the
reduction of the intramolecular conformational exchange rate, due to the effect of intramolecular hydrogen bonding of the hydroxy
part of the carboxyl with the carbonyl oxygen of the Boc-group, and (b) to the effect of solvent viscosity, suggested in the mentioned
study. The cause of this phenomenon is now attributed to a strong hydrogen bonding of the polar proton acceptor solvent DMSO
with the carboxy group, which effectively reduces the proton exchange rate, thus becoming slow on the 17O NMR time scale.
Ó 2004 Elsevier Ltd. All rights reserved.
17O NMR spectroscopy provides a powerful and sensi-
tive tool for studying intra- and intermolecular hydro-
gen bonding effects both in solution and in the solid
state.1–4 Since the oxygen atom is one of the most
important atoms constituting hydrogen-bonding struc-
tures, 17O NMR might provide novel and complemen-
tary information not readily available from other
methods.
complexes consisting of one carboxylic acid molecule
and two solvent molecules13–16 (Scheme 1B) in protic
solvents, such as H2O and MeOH. These phenomena
are considered to be very fast processes on the NMR
time scale and an average 17O resonance was usually
observed at 250–260 ppm.
Recently, Tsikaris et al.17 claimed that the two reso-
nances at 340.3 and 175 ppm observed for the carboxylic
oxygens of the labelled amino acid Boc–[17O]Tyr(2,6-
diClBzl)–OH (Scheme 2Aa) in DMSO-d6 solution,
should be attributed to two phenomena, which strongly
reduce the intramolecular conformational exchange
rate: firstly, to a strong intramolecular hydrogen bond
interaction between the carboxy group (COOH) and the
In carboxylic acids, only one resonance is observed in
the 17O NMR spectra for both C@O and OH oxy-
gens,1–9 due to the intermolecular proton transfer
through a cyclic dimeric form (Scheme 1A) in the solid
state,10;11 a cyclic dimer form or higher linear polymeric
forms in apolar media,11;12 or within hydrogen-bonded
H
O
O
O
O
O
H
O
O
H
O
R
C
R
C
C
R
R
C
H
H
CH3
CH3
H
H
S
H
O
O
O
(A)
(B)
(C)
Scheme 1.
Keywords: 17O NMR; 17O-labelled amino acid derivatives; DMSO; Hydrogen bonding.
* Corresponding author. Tel.: +30-2-6510-98389; fax: +30-2-6510-98682; e-mail: igeroth@cc.uoi.gr
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.12.162

2244
V. Theodorou et al. / Tetrahedron Letters 45 (2004) 2243–2245
CH COOR
CH2
(A)
(CH3)3C
O CO NH
(B)
COOR
N
COCH3
CI
CI
O
CH2
(a) R = H
(b) R = CH3
Scheme 2.
carbonyl part (C@O) of the Boc group, stabilising a
c-turn like structure and, secondly, to the effect of
DMSO viscosity. The purpose of this communication is
to critically evaluate the conclusions of Tsikaris et al.17
and to provide a coherent comment on the observation
of both carbonyl and hydroxyl oxygens in amino acid
derivatives.
In their report,17 Tsikaris and co-workers presented a
series of experiments in order to support their hypoth-
esis:
(i) The 17O NMR spectrum of HCl[17O]Tyr(2,6-diC-
lBzl)–OH in DMSO-d6 has only one resonance at
251.8 ppm, which was interpreted with the hypothe-
sis that a c-turn structure cannot exist. However, no
details were provided on the preparation of the salt.
In any case either with aqueous HCl or with HCl
gas, fast proton transfer of the carboxylic group
would be facilitated and one resonance would be
observed.
(ii) The 17O NMR spectrum of Boc–[17O]Tyr(2,6-diC-
lBzl)–OH in CDCl3, revealed a single resonance at
247.5 ppm. It was claimed that the viscosity of the
solvent strongly affects the intramolecular confor-
mational exchange rate, being rapid in the case of
chloroform. This hypothesis is erroneous for two
reasons. Firstly, the differences in viscosity between
DMSO (1.788 mPa s at 30 °C)18 and CHCl3
(0.512 mPa s at 30 °C)19 solutions would not be ex-
pected to reduce an intramolecular conformational
exchange of the ns time scale to an ms time scale
or ever longer. Secondly, in nonpolar solvents, such
as chloroform, a very fast intermolecular proton
transfer occurs through the cyclic dimeric or linear
polymeric forms of a carboxylic acid.
Figure 1. 17O NMR spectra at 54.4 MHz of Ac-[17O]ProOH
(C ¼ 40mM) (A),
[
17O]PhCOOH (C ¼ 30mM) (B), and
[
17O]PhCOOH (C ¼ 100 mM) (C) in DMSO-d6, containing 0.4 M of
water. NMR spectra were recorded on a Bruker AMX-400 spec-
trometer at 40 °C. 800 000 scans were accumulated with a repetition
rate of 20s ꢀ1 and an exponential line broadening of 200 Hz was
imposed on the accumulated data before Fourier transformation.
Two resonances at 320and 180ppm were observed for
the two oxygens (C@O, OH) of Ac–[17O]Pro–OH in
DMSO-d6 (Fig. 1A). DMSO is an important dipolar
aprotic solvent with a high dielectric constant, which
behaves as a strong hydrogen bond acceptor with
proton donor substrates, such as COOH and OH
groups.23;24 Dimethyl sulfoxide also interacts with other
acids and electrophiles25 enabling ionisation or other
processes. As the concentration of the acceptor is in-
creased, the cyclic dimer of the carboxylic acid (Scheme
1A) is suppressed effectively, in favour of the complex
with solvent molecules (Scheme 1C). DMSO, therefore,
disrupts the potential hydrogen bonds of the cyclic
dimers, the S@O oxygen interacting strongly with the
COOH, and reduces the intermolecular proton transfer,
resulting in a clear differentiation of the two oxygens.
The chemical shifts of the two resonances are in good
agreement with those observed for the two oxygens
(C@O, –OCH3) of the two methyl esters Ac–[17O]Pro–
OMe (335 and 130ppm) and Boc–[ 17O]Tyr-(2,6-
diClBzl)–OMe (342 and 135 ppm) in CDCl3 (40 °C). On
the contrary, the 17O NMR of Ac–[17O]Pro–OH in
CDCl3 indicates only one resonance at 255 ppm due to
In order to further support our hypothesis, we per-
formed some complementary experiments with
Ac–[17O]Pro–OH, which is one of the best characterised
models of c-turn structures,9;20 Ac–[17O]Pro–OMe21,
Boc–[17O]Tyr(2,6-diClBzl)–OMe21 (Scheme 2) and
[17O]PhCOOH.22 The samples were prepared by dis-
solving the correct amount of each compound in 600 lL
commercial DMSO-d6, which, according to proton
NMR spectra, contained 0.4 M of H2O.

V. Theodorou et al. / Tetrahedron Letters 45 (2004) 2243–2245
2245
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diazomethane. The [17O]amino acids were obtained by
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presence of [17O]H2O (10% atom 17O).
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acidic [17O]H2O (10% atom 17O).
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broad resonances for the two oxygens of the carboxyl in
DMSO-d6 at 40 °C (Fig. 1B). Since this carboxylic acid
cannot form a c-turn structure, the presence of two
resonances must be attributed to the formation of new
intermolecular hydrogen bonds with solvent molecules,
the PhCOOHÆDMSO complex (Scheme 1C), with dis-
ruption of the cyclic dimers (Scheme 1A). Further
confirmation that the two resonances are really con-
nected by proton exchange can be obtained from vari-
able concentration experiments. According to Meschede
and Limbach26 the pseudo-first order proton exchange
rate constant kobs observed by NMR (in the slow
exchange regime) is given by kobs ¼ K2kC, where K2 is
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acid, and k the true exchange rate in the dimer. There-
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(Fig. 1C) results in an averaged single resonance
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the dimers (Scheme 1A). IR spectra of PhCOOH, both
in DMSO and chloroform, are consistent with the 17O
NMR results.
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