A Kinetic Photometric Assay for the Quantification of the Open-Chain Content of Aldoses

Article abstract of DOI:10.1002/ejoc.202001641

Aldoses exist predominantly in the cyclic hemiacetal form, which is in equilibrium with the open-chain aldehyde form. The small aldehyde content hampers reactivity when chemistry addresses the carbonyl moiety. This low concentration of the available aldeh

Full text of DOI:10.1002/ejoc.202001641

Communications
doi.org/10.1002/ejoc.202001641
Very Important Paper
A Kinetic Photometric Assay for the Quantification of the
Open-Chain Content of Aldoses
Hubert Kalaus+,[a] Alexander Reichetseder+,[b] Verena Scheibelreiter,[a] Florian Rudroff,[a]
Christian Stanetty,*[a] and Marko D. Mihovilovic[a]
ring forms, which are generally dominating by several orders of
magnitude with typical open-chain contents (OCC) of signifi-
cantly below 1%.
Aldoses exist predominantly in the cyclic hemiacetal form,
which is in equilibrium with the open-chain aldehyde form. The
small aldehyde content hampers reactivity when chemistry
addresses the carbonyl moiety. This low concentration of the
available aldehyde is generally difficult to ascertain. Herein, we
demonstrate a new kinetic determination of the (minute) open-
chain content (OCC) of aldoses. This kinetic approach exploits
the aldehyde-selectivity of 2-aminobenzamidoxime (ABAO),
which furnishes a strongly UV-active adduct. Simple formation
curves can be measured in a photometer or plate reader for
high-throughput screening. Under pseudo-first order kinetics,
these curves correlate with a prediction model yielding the
relative OCC. The OCCs of all parent aldoses (pentoses and
hexoses) were determined referencing against the two tetroses
with exceptionally high OCCs and were in very good agreement
with literature data. Additionally, the assay was extended
towards higher-carbon sugars with unknown OCC and also
applied to rationalise a lack of reactivity observed in a recent
synthetic investigation.
Nonetheless, utilising the aldehyde moiety represents a
powerful and low-threshold opportunity for the more general
organic chemist to exploit carbohydrates as a renewable source
of chirality in synthetic strategies. Established methodologies
include Wittig-type olefinations,[2] addition of organometallics
(Mg, In, Zn)[3] or carbanions (Kiliani-Fischer), and Carbonyl-
Umpolung as principle examples[4] (Figure 1, top). Despite its
intrinsic reactivity, the aldoses’ aldehyde moiety is underutilised
in both the field of carbohydrate chemistry and beyond.[5]
We are convinced that the low OCC, particularly of the more
popular monosaccharides, together with the difficulty to accu-
rately determine the degree of availability (OCC), is an additional
challenge to such studies. It prevents a separate discussion/
analysis of the actual chemistry of interest from the influence of
this pre-equilibrium when comparing different carbohydrate
species. We have observed examples for the dominating influence
of the OCC in our own work with the indium mediated
acyloxyallylation
(IMA)[3d,6]
as
well
as
NHC-catalysed
Carbohydrates are the predominant class of biomolecules
formed by Nature, fulfilling a multitude of purposes in all living
organisms: polysaccharides serve as structural components of
cells, oligosaccharides play a major role in the recognition
process of biomolecules, and monosaccharides and lower
oligosaccharides are essential energy sources within metabolic
cycles.[1] Chemically, each monosaccharide comprises a carbonyl
group (ketone or aldehyde) and a varying number of alkyl
hydroxyl groups. Owing to these structural features, the open-
chain form of carbohydrates is in equilibrium with hemiacetal-
dehomologation.[7] Also, in other reports, similar differences can be
deduced although rather implicitly reported.[5c,8]
We believe that the lack of a straightforward OCC determi-
nation is the reason why this important phenomenon is often
not fully addressed, thereby representing a burden for accurate
[a] H. Kalaus,+ V. Scheibelreiter, Prof. Dr. F. Rudroff, Dr. C. Stanetty,
Prof. Dr. M. D. Mihovilovic
Institute of Applied Synthetic Chemistry
TU Wien
Getreidemarkt 9, 1060 Vienna, Austria
E-mail: christian.stanetty@tuwien.ac.at
https://info.ias.tuwien.ac.at/bsc/stanetty-lab/
[b] A. Reichetseder+
Department of Pharmaceutical Chemistry
University of Vienna
Althanstraße 14, 1090 Vienna, Austria
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202001641
Figure 1. The open-chain content (OCC) of carbohydrates as a relevant
feature for reactions addressing their aldehyde moiety. The previous gold
standard of OCC determination and our new operationally simple UV-based
approach.
© 2021 The Authors. European Journal of Organic Chemistry published by
Wiley-VCH GmbH. This is an open access article under the terms of the
Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
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prediction and planning of synthetic outcome, creating a bias
against the use of carbohydrates for this purpose.
monosaccharide concentration was adjusted to 4 mM to ensure
a sufficient reaction rate to also enable the investigation of
monosaccharides with very low OCC. A tenfold excess of ABAO
was applied to ensure pseudo-first order kinetic condition,
required for facile analysis. Another prerequisite for the
approach is that the adduct formation and not the pre-
equilibrium is the rate-determining step; this was proven
experimentally to be clearly the case even with the slowest
investigated monosaccharide, glucose 1 (see ESI). The structure
of the postulated adducts, as well as their complete formation,
was confirmed by NMR (see ESI for selected examples of the
processed adducts).
The reaction progress is monitored by determination of the
UV-signal at 405 nm. The assay can be performed in a photo-
meter cuvette or in multi-well plates. Under pseudo-first order
conditions, the absorption curves follow a shape fitting the
mathematical formula, equation 1:
To date, the quantification of the minor component open-
chain form, which is often present below 0.1%, is challenging,
being reflected in largely fluctuating values reported for the
few carbohydrates for which such data exists at all.[9] The
currently available methods include circular dichroism,[10]
a
kinetic NMR-based assay,[11] or the quantification by 13C-NMR of
1-[13C]-labelled monosaccharides by the group of Serianni. The
latter approach is providing the most convincing data but relies
on the preparation of the monosaccharide of interest in its
isotopically labelled form, representing a major limitation for
broad and facile application[12] (Figure 1, middle). A simpler to
use approach to quantify the availability of certain aldoses as
aldehyde species is required.
Herein, we report an efficient, facile, and reliable way to
determine OCC values based on a UV-based kinetic assay utilising
the recently developed aldehyde-selective reagent, 2-amino-
benzamidoxime 2 (ABAO, Figure 1, bottom) from the Derda
group.[13] We hypothesised that by shifting the equilibrium from
closed to open forms, carbohydrates could be fully converted to
ABAO-adducts and that this formation can be easily followed
due to the strong UV-absorption (Figure 2a) of the ABAO
adducts. We further assumed that the derived rate of formation
could be correlated to the OCC. Hence, carbohydrates with a
higher OCC exhibit faster adduct formation as is exemplified in
Figure 2b with idose, arabinose, and galactose with decreasing
known OCC. Herein, we show the validity of this approach,
applying the assay to determine the OCC of all standard aldoses,
validated them against the literature, and finally present its
application in two recent synthetic challenges.
Abs ¼ Absmax � ð1 À eÀ a�t
Þ
(1)
Under the given assumptions and based on mathematical
derivation which is found in the ESI, the exponent α reflects
equation 2:
a ¼ K � k2 � ½ABAO�
(2)
and was obtained from software supported fitting or alter-
natively least-square fitting of the logarithmic plot of (1–Abs/
Absmax) against time. Herein, K is the equilibrium constant
between all open-chain species (Figure 2a) and k2 the rate
constant of adduct formation. With known and constant ABAO
concentration, the mathematical product K·k2 is deduced,
interlinking the existing pre-equilibrium (of interest) with the
adduct formation (Figure 2b).
The experimental setup of the assay is simple. Aqueous
solutions of the respective aldoses are pipetted into a buffered
solution (pH 4.5) of the ABAO component. Generally, the
Thus, with known k2, absolute values of K (and OCC) can be
deduced and the other way around. Therefore, at least one
reference carbohydrate with a reliable OCC value is required.
Among the parent aldoses, the two tetroses erythrose 7 and
threose 8 stand out with their extremely high OCCs of ~10%.
Therefore, these were selected as potential reference points,
1
and OCCs were conveniently quantified by H-NMR (ESI). The
assessment of erythrose 7 and threose 8 in the ABAO assay led
to the unexpected finding that despite comparable OCCs,
significantly differing slopes, reflecting different k2 values, were
found. Additionally, different absorption maxima were observed
(see Figure 3a) for the curves. This ambivalent behaviour
between 2,3-erythro and 2,3-threo configured monosaccharides
was consistently observed within the pentoses (Figure 3b) and
hexoses (Figure 3c and Figure 3d) in the course of the study.
The differences in the absorption maxima are attributed to
different ratios of the two epimers formed during adduct
formation within the two families, under the assumption of
different UV-absorption coefficients of those epimers. This
interpretation is supported by the performed NMR-measure-
ments (ESI). As for the different slopes of the curves of
formation, we concluded that the erythro configuration of the
2O,3O-diol significantly favours product formation over the
Figure 2. The kinetic aldehyde-selective ABAO assay enables quantification
of open-chain contents (OCC) of aldoses. (a) The overall reaction leading to
the UV-active adduct that is detected. (b) The principal concept for the
correlation between observed reaction rate and OCC.
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Figure 3. UV-Curves of ABAO-adduct formation for all parent aldoses revealing the presence of two families in respect to their relative 2,3-stereochemistry
exhibiting differences in absorption maxima and rate constants. (a) From the tetroses, erythrose 7 and threose 8, two different rate constants were derived,
that were applied for the two families, respectively. (b)–(d) The absorption curves of pentoses and hexoses (fast and slow adduct formation reflecting their
OCC values) are depicted exhibiting a clear difference in absorption maxima for the erythro and threo families again.
2O,3O-threo-configuration. Therefore, as a refinement of our
initial hypothesis, we decided to determine separate k2, erythro
and k2, threo values for the 2,3-erythro and the 2,3-threo-family,
respectively (see ESI for a visual representation of those families
in their Fischer projection).
These k2,erythro-, and k2,threo-values (8.89 L·molÀ 1 ·sÀ 1 and
3.76 L·molÀ 1 ·sÀ 1, respectively) were then used for the OCC
determination of the other parent aldoses, based on fitting of
their curves of formation and utilisation of equation 2 and
equation 4.
,
In the case of the tetroses, with the reliable OCC values
from the H-NMR spectra, and the equilibrium constant K can
be calculated via equation 3, the opposite calculation is
possible after transformation to equation 4.
On a technical note, due to their high OCCs and
1
consequently, very fast conversion at 4 mM, experiments with 7
and 8 were performed at 0.4 mM to generate better analysable
data. We confirmed, that the OCC values obtained by the assay
are robust to changes in concentration of sugar (0.4 and 4 mM)
and ABAO (4 to 40 mM). This was demonstrated for ribose,
spanning sugar/ABAO ratios between 1:1 and 100:1 with only
minor changes in OCC (see ESI).
OCC
1 À OCC
K ¼
(3)
K
The results of the OCC values of all parent aldoses up to the
hexoses are summarised in Table 2 (entry 1–14), clustered by
chain length, and sorted by the determined OCC-values (blue
for erytho- and red for threo-configuration). The obtained
numbers were compared to the literature values obtained from
OCC ¼
(4)
1 þ K
With known K for the two tetroses, based on the fitting of
the curves of formation to deliver α and by using equation 2,
k2-values specific for each of the two families were determined
(Table 1).
the
previous
NMR
studies
with
1-[13C]-labelled
monosaccharides[12] displaying a high degree of consistency.
The comparison of the absolute values provides good to
excellent matches in all cases. Noteworthy, OCCs spanning
several orders of magnitude can be obtained with the new
method. Further, our assay requires only a few minutes of
sample preparation time and relies on a simple photometer
compared to the requirement of the synthesis of labelled
materials and long measuring times on sophisticated NMR
equipment.
Table 1. Stepwise deduction of specific rate konstants k2 for the erythro-
and threo-families from NMR derived OCC values
Name
OCC
[%]
K
α·103
[sÀ 1
k2
]
[L·molÀ 1 ·sÀ 1
]
l-erythrose 7
d-threose 8
12.5[a]
11.7[a]
0.143
0.133
5.08
2.00
8.89
3.76
With the successful validation of our assay against the
reported OCCs of the full standard hexose tree, we set out to
[a] Determined as points of reference using 1H-NMR (600 MHz) at 4 mM
concentration.
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Scheme 1. ABAO-based OCC measurements elucidating the lack of reactivity of reluctant substrate 19 in the indium mediated acyloxyallylation.
apply it to two particular questions from a recent study on the
indium-mediated acyloxyallylation (IMA) of aldoses, which is
based on the organometallic addition to the aldehyde moiety.
This powerful two-carbon-homologation reaction (see
Scheme 1 for representative examples) requires a fast consump-
tion of carbohydrate-based starting material to be successful
due to a competing decomposition of the organometallic
reagent under the Barbier conditions used.[3d]
This, in our view, represents a prototypic complication when
carbohydrates are targeted to react in their (minority) open-
chain form. Nonetheless, we have successfully utilised IMA for
the preparation of l-glycero-d-manno-heptose 17 at scale
amongst more current applications.[6] In such an ongoing study,
we required further elongation of heptose 17 as well as its
homologous d-erythro-l-manno-octose 18[3c] (see Figure 4).
However, in these IMAs, we observed reduced reactivity
compared to their shorter-chain analogues. We thus used our
assay to determine the OCC of heptose 17 and octose 18 in
order to gain deeper insights into these observed differences.
In turn, we compared their OCCs with the ones of their
shorter analogues d-lyxose 9 and d-mannose 15 (see Figure 4
and ESI), which share the same relative stereochemistry –
(2O,3O,4O)-lyxo in the carbohydrate core. As shown in Figure 4,
the pentose lyxose 9 has a roughly 3.5 times higher OCC than
its hexose counterpart mannose 15, which correlates well with
their performances in the IMA reaction.[3c] Further formal chain
elongation (17, 18) led only to an insignificant change in OCC.
Thereby the new assay proved its value to quickly confirm or
rule out any effect of the OCC on the observed performance in
more complex transformations.
Table 2. OCC values determined by the ABAO assay and compared to
literature values (blue for erythro- and red for threo-configuration).
Entry Name
K·k2 ·103
OCC ABAO
[%][a]
OCC NMR
[%] lit.[9,12]
[L·molÀ 1 ·sÀ 1
]
1
2
3
l-erythrose 7
d-threose 8
d-arabinose 5 4.78
1270
500
12.5[b]
11.7[b]
0.13
12–12.5
10.6–12
0.11
4
5
6
7
8
9
10
11
12
13
14
d-lyxose 9
9.78
8.21
2.74
26.8
5.46
7.77
6.50
2.04
0.11
0.097
0.13
0.071
d-ribose 10
d-xylose 11
l-idose 4
d-altrose 14
l-gulose 12
d-talose 13
d-galactose 6
d-mannose 15 2.88
d-allose 16
d-glucose 1
0.092
0.072
0.71
0.79
0.14
0.093
0.082
0.081
0.052
0.026
0.0095
0.010
0.087
0.073
0.054
0.032
0.012
0.0097
1.03
0.37
[a] measured in triplicates with coefficients of variation below 5% as
shown with idose as an example (see ESI). The k2-values used for the
determination of the OCCs were selected based on the carbohydrate
family with k2, erythro =8.89 L·molÀ 1 ·sÀ 1 and k2, threo =3.76 L·molÀ 1 ·sÀ 1. [b]
Determined as points of reference using 1H-NMR (600 MHz) at 4 mM
concentration (ESI).
Figure 4. Effect of formal terminal chain elongation onto the OCC of
homologous monosaccharides as determined by the ABAO assay.
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The second application of the ABAO-assay addressed a
OCC is a limitation in their reactions. Thereby, it will help lift a
barrier and a bias against using this underutilised compound
class in syntheses towards complex organic matter. In this light,
our lab is looking deeper into mechanistic details of this
powerful interaction, and investigations with a broader scope of
carbohydrates and ABAO-derivatives are also ongoing.
more complex question, also related to another aspect of our
IMA study. In the systematic investigation of the IMA of
reducing sugars, we intensively studied unprotected erythrose
7 as well as 4-O-formyl-2,3-O-isopropylidene erythrose 20. Upon
installation of the protecting group, the diastereodivergent
product 22 can be achieved compared to 21 derived from 7.[3d]
However, compound 20 was only ever introduced as a
surrogate for its simpler variant 2,3-O-isopropylidene-erythrose Acknowledgements
19, which surprisingly and in marked contrast to 7 and 20 could
not be converted. Using the new assay, we set out to
corroborate our speculations that OCC is responsible with
discrete data. Thus, all three compounds of interest were
measured with the ABAO-assay: While 7 and 20 exhibited fast
ABAO-adduct formation, resulting in completion in minutes, 19
was very slowly converted over the course of several hours,
even at higher concentration (see time-courses in the ESI).
Different factors can now be deduced. For erythrose 7, the
combination of a high OCC of 12.5% and its rate constant in
the ABAO-adduct formation of 8.89 L·molÀ 1 ·sÀ 1 (vide supra)
confirms the reason for the fast conversion. The 4-O-formyl-2,3-
O-isopropylidene-erythrose 20 showed overall comparable
ABAO-adduct formation. However, it exhibits an OCC of 100%
(as proven by NMR), reflected in a ~20-fold lower rate constant
of 0.44 L·molÀ 1 ·sÀ 1 (see ESI). This low rate of formation is
attributed to the sterically demanding isopropylidene group in
close proximity to the carbonyl centre, thus impeding its attack.
In analogy to above, we used the rate constant of 20 as a
reference for 19 and could thereby deduce the OCC of the
protected lactol 19 from its fitted ABAO-curve. This resulted in a
low OCC value of only 0.5%, thus ~20 fold lower than in the
parent erythrose 7.
We thank H. Dienstbier and A. Ressmann for technical support.
The Austrian Science Fund FWF (Grant P29138) is gratefully
acknowledged for financial support. HK is currently funded by the
federal state of Lower Austria (BioSet project).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: Aldehydes · Analytical methods · Carbohydrates ·
Kinetics · UV/Vis spectroscopy
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Manuscript received: December 18, 2020
Revised manuscript received: February 15, 2021
Accepted manuscript online: March 17, 2021
As for carbohydrates, we are convinced that this new tool
will provide a valuable test for chemists to rapidly identify if the
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