Molecular recognition of isomeric protonated amino acid esters monitored by ESI-mass spectrometry

Article abstract of DOI:10.3762/bjoc.10.78

Two new 9,9'-spirobifluorene-derived crown ethers were prepared and used to recognise constitutionally isomeric amino acid derivatives. The performance of the receptors was evaluated by ESI-mass spectrometry using the isomer labelled guest method (ILGM).

Full text of DOI:10.3762/bjoc.10.78

Molecular recognition of isomeric protonated amino
acid esters monitored by ESI-mass spectrometry
Andrea Liesenfeld and Arne Lützen*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 825–831.
University of Bonn, Kekulé-Institute of Organic Chemistry and
Biochemistry, Gerhard-Domagk-Str.1, D-53121 Bonn, Germany
doi:10.3762/bjoc.10.78
Received: 13 December 2013
Accepted: 21 March 2014
Published: 09 April 2014
Email:
Arne Lützen* - arne.luetzen@uni-bonn.de
*
Corresponding author
This article is part of the Thematic Series "Chemical templates".
Guest Editor: S. Höger
Keywords:
amino acids; isomer labelled guest method (ILGM); mass
spectrometry; molecular recognition; 9,9’-spirobifluorenes; template
© 2014 Liesenfeld and Lützen; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Two new 9,9’-spirobifluorene-derived crown ethers were prepared and used to recognise constitutionally isomeric amino acid
derivatives. The performance of the receptors was evaluated by ESI-mass spectrometry using the isomer labelled guest method
(
ILGM). This method revealed the preferred binding of L-norleucine and L-leucine compared to L-isoleucine for both receptors.
Furthermore, non-covalent isotope effects demonstrate the relevance of dispersive interactions for the overall binding event. These
effects also provide hints for the relative spatial orientation of the guest molecules within the host–guest complex, and thereby
prove the importance of the spirobifluorene moiety for the observed binding of the protonated amino acid esters.
Introduction
The separation of constitutionally isomeric amino acids is of
practical interest. This is particularly true for leucine (Leu),
isoleucine (Ile), and norleucine (Nle), especially since the first
two are both proteinogenic amino acids (Figure 1). However, it
is a great challenge to separate molecules that have the same
molecular mass and do not differ significantly in structure.
Figure 1: L-Norleucine, L-isoleucine, and L-leucine.
Hence, the chemical and physical properties are very similar,
and the isomers leucine, isoleucine, and norleucine are difficult
to separate employing commonly used analytical methods like Therefore, we thought to look for a supramolecular approach to
crystallization, enzymatic separation methods, or modified thin- achieve amino acid recognition by an artificial host [6-11] with
layer chromatography [1-5].
regard to isomer separation upon isomer-selective molecular
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recognition by a concave template acting as a host, thereby that prevents substrates of a certain shape to be accommodated
avoiding the necessity to establish or break additional covalent in the concave binding site of the templates. Since the 9,9’-
chemical bonds. Hence, we have prepared two new templates spirobifluorene moiety provides such a rigid concave, non-polar
based on a 9,9’-spirobifluorene core and tested them with scaffold that has been demonstrated to be a valuable part of
regard to their ability to recognize the three isomeric amino some receptors [12,13], we decided to employ this motif. There-
acids in form of their protonated methyl esters. We decided to fore, we designed the two compounds 1 and 2 shown in
use ESI-mass spectrometry for these tests since this technique is Figure 2 that differ only in the bridging element between the
fast to perform, consumes only trace amounts of material, and crown ether group and the spirobifluorene.
can be used to explore competitive experiments that are diffi-
cult to perform using UV–vis or NMR spectroscopy.
The synthesis of 1 and 2 started from 1-bromo-4-methoxyben-
zene which was transferred into 2-bromo-4’-methoxybiphenyl
Results and Discussion
Design and synthesis of the concave
templates
(
3) in 92% yield via lithiation with t-BuLi, transmetallation with
zinc(II) bromide, and subsequent Negishi cross-coupling reac-
tion with 1-bromo-2-iodobenzene [14,15]. 3 was then trans-
Ammonium ions exhibit strong binding affinity towards crown formed into the corresponding Grignard reagent which was
ether moieties. Hence, we decided to use this motif to achieve reacted with 9-fluorenone to afford tertiary alcohol 4 in 55%
binding of the leucine isomers as their protonated ester deriva- yield. Adopting a protocol of Tour et al. [16] led to 2-methoxy-
tives. This provides the major part of the overall binding 9,9'-spirobifluorene (5) in 95% yield via acidic condensation of
energy. However, to distinguish the three isomers the receptors 4. Next, the methoxy group was cleaved quantitatively by reac-
need to provide further elements that either provide additional tion with boron tribromide, followed by hydrolysis to afford
binding sites for the non-polar parts of the substrates, e.g., via phenol 6 which was finally transferred into the corresponding
attractive dispersive interactions, or provide steric hindrance triflate 7 in 64% yield (Scheme 1).
Figure 2: Concave templates 1 and 2.
Scheme 1: Syntheses of the 2-(9,9’-spirobifluorene-2-yl)trifluoromethansulfonate (7).
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Triflate 7 was then subjected to a Sonogashira cross-coupling anyway. Thus, the host–guest complexes would be charged and
reaction and a Suzuki cross-coupling reaction followed by treat- supposedly easy to detect by mass spectrometry if they can be
ment with boron tribromide to obtain the ethynylated and separated from the counter-ions.
arylated alcohols 8 in 95% yield and 9 in 87% yield over both
steps, respectively. Finally, deprotonation by sodium hydride Nevertheless, it still sounds kind of paradox to use mass spec-
and reaction with tosylated 18-crown-6 derivative 10 [17,18] trometry to study isomeric complexes due to their identical
derived from commercially available (1,4,7,10,13,16-hexaoxa- mass/charge ratio. This problem can be circumvented by the use
cyclooctadecan-2-yl)methanol afforded the desired target com- of isotopically labelled substrates in the sense of an isomer
pounds 1 and 2 in moderate yields (Scheme 2).
labelled guest method (ILGM) (Figure 3) which is closely
related to the enantiomer labelled guest method (ELGM) intro-
duced by Sawada [22].
Molecular recognition studies
With our crown ether derivatives 1 and 2 in hands we studied
their recognition behaviour towards the L-leucine isomers. Here, a competitive recognition experiment using a non-labelled
Usually, spectroscopic techniques like NMR or UV–vis spec- substrate and a mass-labelled quasi-isomer is performed to
troscopy are used for this purpose. However, mass spectrom- reveal the relative affinity of a receptor towards the different
etry has become a major analytical tool in supramolecular isomers. In this way, the mass spectrometric analysis easily
chemistry in recent years [19-21] and seemed to be perfectly allows direct identification of the individual host–guest
suited in this case, since we were planning to recognise the complexes. This is usually more complicated with other tech-
amino acid derivatives in form of their protonated alkyl esters niques such as, e.g., NMR spectroscopy because it is more diffi-
Scheme 2: Synthesis of the two receptors 1 and 2.
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Beilstein J. Org. Chem. 2014, 10, 825–831.
Figure 3: Schematic presentation of the isomer labelled guest method (ILGM).
cult to assign the signals to the individual host–guest complexes allow an individual detection but also small enough not to cause
and the analysis might be additionally hindered or even be any problems due to mass discrimination phenomena.
impossible due to severe signal overlapping.
It is important to note that these measurements are obviously
In our case, we used the methyl groups of the ester function as not biased by, e.g., different solvation energies of the structural
the mass label by employing either the normal protiated methyl isomers which could cause different ESI response factors
group or a trideuteromethyl group as the labelled one. To test because we did not observe different intensity ratios when we
the relative affinity of a receptor towards two isomeric changed the overall concentration of our samples. Another
substrates we prepared solutions that contain 1:1:1 mixtures of important point that has to be mentioned here, however, is that
the receptor, a non-labelled guest, and an isotopically-labelled intensity differences of the complexes might also be the result
guest. These solutions were analysed by ESI-mass spectrom- of differences in the tendency to dissociate under the conditions
etry (Figure 4). The intensity ratios of the signals of the of the ESI–MS experiment. Unfortunately, the low mass of the
host–guest complexes can then be used to conclude which guest leucine derivatives investigated here did not allow to study this
is bound stronger since the mass difference is large enough to phenomenon directly because the FTICR spectrometer we used
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Beilstein J. Org. Chem. 2014, 10, 825–831.
L-isoleucine, and L-leucine that were calculated according to
Equation 1.
(1)
Table 1: ILGM ratios for the recognition of the three pairs of L-leucine
isomers by templates 1 and 2.
pair of isomers
1
2
Figure 4: ESI-mass spectrum (positive mode) of a 1:1:1 mixture of 1,
protonated L-leucine methyl ester (LeuOMe), and protonated
L-isoleucine trideuteromethyl ester (IleOMe-d3) in a 1:1 mixture of
CH2Cl2/MeOH (m/z = 792.4 LeuOMe–1, m/z = 795.5 IleOMe-d3–1).
L-NleOMe/L-IleOMe
L-IleOMe/L-LeuOMe
L-NleOMe/L-LeuOMe
1.5
0.7
1.0
1.2
0.8
0.9
is tuned in a way that it does not allow detection of such low
molecular mass ions with the necessary accuracy. Hence, we
optimized our ESI conditions using larger and well-detectable These ratios translate into the following relative affinities of our
protonated amino acid esters like protonated phenylalanine receptors:
benzyl ester to make sure that the conditions are mild enough
not to cause dissociation.
Receptor 1: L-NleOMe ≈ L-LeuOMe > L-IleOMe
Having made sure that the method is in principle suitable to Receptor 2: L-LeuOMe ≥ L-NleOMe > L-IleOMe
study the recognition of protonated amino acid derivatives in a
competitive fashion there is still one more factor that has to be Interestingly, isoleucine methyl ester is the worst guest in both
taken into account: non-covalent isotope effects [23] might also cases although the effect is not large enough yet to think about
cause significant differences of the signals’ intensities. There- an application for the separation of the isomers on a larger scale
fore, every experiment has to be repeated with the different via supramolecular transport, for instance. However, these
order of isotope labelling and in addition one should measure results are still promising with regard to the development of a
the same isomer in both forms – non-labelled and labelled – in template that allows the efficient separation of isoleucine and
order to quantify this effect (Figure 5).
leucine which is obviously the most interesting challenge. The
non-covalent isotope effects clearly demonstrate that the ester
group of the amino acid derivative must interact with our
templates, and therefore, these effects tell us something about
the relative spatial orientation of the template and the substrate
within the host–guest complexes (Figure 6).
In fact, the deuterated guests were always bound less good than
the protiated guests. This can be rationalized by assuming
attractive dispersive interaction to play a significant role during
the recognition event. The deuterated group is on average
slightly smaller than the protiated group [14-26] which causes a
smaller van der Waals volume of the deuterated group [27-32].
In a first approximation, however, the dispersive interactions
get stronger with an increasing size of the volume and conse-
quently the contact area, and hence, the deuterated compounds
ability to interact via those interactions is weaker [33-35]. This
Figure 5: ESI-mass spectrum (positive mode) of a 1:1:1 mixture of 1,
protonated L-leucine methyl ester (LeuOMe), and protonated L-leucine
trideuteromethyl ester (LeuOMe-d3) in a 1:1 mixture of CH2Cl2/MeOH
(m/z = 792.4 LeuOMe–1, m/z = 795.5 LeuOMe-d3–1).
Table 1 lists the results of the ILGM measurements with regard observation perfectly agrees with the conclusion that the ester
to the affinity of templates 1 and 2 towards L-norleucine, group gets into close contact with the non-polar parts of the
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Beilstein J. Org. Chem. 2014, 10, 825–831.
Supporting Information
Experimental data of all new compounds and of the ESI
mass spectrometric experiments.
Supporting Information File 1
Experimental data, ESI-mass spectrometric experiments.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-78-S1.pdf]
Acknowledgements
Financial support from the DFG (SFB 624) is gratefully
acknowledged.
Figure 6: Two different motifs for the binding of substrates to the
templates.
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