Hydrogen-Bonding Network Anchors the Cyclic Form of Sugar Arylhydrazones

Article abstract of DOI:10.1002/ejoc.201600462

The “classical” challenge, raised by Emil Fischer as to why one monosaccharide arylhydrazone adopts a cyclic structure but another an acyclic structure, is answered here. The present comprehensive analysis of hexose and hexosamine arylhydrazones, based on 2D NMR spectroscopy and theoretical modeling, has established that the chain of hydrogen bonds needed for conformational selection can only be completed for d-glucosamine derivatives. Thus, d-glucosamine 4-nitrophenylhydrazone exclusively adopts its cyclic form, but any configurational changes imply the formation of acyclic structures. In conclusion, three criteria dominate structure selection, namely 1) an amino function at the C-2 position, 2) the “all-equatorial” substitution mode of the pyranoid ring, and 3) an electron-withdrawing group on the arylhydrazone are all needed to get the cyclic form only.

Full text of DOI:10.1002/ejoc.201600462

DOI: 10.1002/ejoc.201600462
Full Paper
Glycochemistry
Hydrogen-Bonding Network Anchors the Cyclic Form of Sugar
Arylhydrazones
Viktória Goldschmidt Gőz,[a] István Pintér,[a] Antal Csámpai,[b] Imre Jákli,[c] Virág Zsoldos-
Mády,[c] and András Perczel*[a,c]
Abstract: The “classical” challenge, raised by Emil Fischer as to hydrazone exclusively adopts its cyclic form, but any configura-
why one monosaccharide arylhydrazone adopts a cyclic struc-
ture but another an acyclic structure, is answered here. The
present comprehensive analysis of hexose and hexosamine aryl-
hydrazones, based on 2D NMR spectroscopy and theoretical
modeling, has established that the chain of hydrogen bonds
needed for conformational selection can only be completed for
tional changes imply the formation of acyclic structures. In con-
clusion, three criteria dominate structure selection, namely
1) an amino function at the C-2 position, 2) the “all-equatorial”
substitution mode of the pyranoid ring, and 3) an electron-
withdrawing group on the arylhydrazone are all needed to get
the cyclic form only.
D
-glucosamine derivatives. Thus, D-glucosamine 4-nitrophenyl-
Introduction
have become key intermediates for the synthesis of various
heterocyclic compounds.[7,8] Hence, it is surprising that the
structural state of arylhydrazones in solution has never been
studied in detail.[9] Although Fischer had spelled out three iso-
The conformational properties of most natural products such
as carbohydrates are intimately and intrinsically linked to their
constitution and configuration. Determining, for example, the
mers of D-glucose phenylhydrazone in solution, namely α- and
configuration of an aldohexose (e.g., D-glucose) by NMR spec-
ꢀ-pyranosyl as well as the acyclic form isolated later,[10] their
conformational ratios were neither determined nor rationalized.
Several aldose arylhydrazones and their derivatives have been
studied by UV, IR, and NMR spectroscopy as well as by X-ray
diffraction.[11–14] Various synthetic approaches, particularly, the
formazan reaction, were devised to characterize the acyclic ald-
ose arylhydrazones in detail.[15] Interestingly, only those aryl-
troscopy is possible after resonance assignment by using suit-
able 2D experiments measuring three- and two-bond scalar
coupling constants,[1–3] by knowing the constitution of
D-gluc-
ose, and assuming that it has a pyranosyl form (>98 %). How-
ever, the same “tool kit” is practically useless if one cannot as-
sume that a single (or a very low number of) stereoisomer is
present in solution, as is the case with monosaccharide ox-
imes[4] or most monosaccharide arylhydrazones, which typically
contain a large amount of the acyclic form.
One of the oldest areas of glycoscience is the chemistry of
sugar arylhydrazones initiated by the classical Emil Fischer ex-
periment,[5] which is still an ongoing and challenging topic.
Over the years, important classes of acyclic and cyclic com-
pounds have been derived from arylhydrazones. Osazones, os-
ons, formazans as well as osotriazoles and tetrazolium salts[6]
hydrazones having a D-gluco configuration were found to adopt
a pyranoid ring. This structural feature was attributed to the
arrangement of all-equatorial hydroxy groups, as is the case
with D-glucose and its derivatives. Most computational data fo-
cused on establishing whether cyclic or acyclic forms of the
saccharide moieties are prevalent.[16] However, neither synthetic
nor theoretical studies were conducted to determine the con-
figurational and constitutional space and the driving force that
determines the 3D structures of monosaccharide arylhydraz-
ones.
[a] Laboratory of Structural Chemistry and Biology, Institute of Chemistry,
Eötvös University,
In this work we examined the structural properties of hexose
Pázmány Péter sétány 1/A, 1117 Budapest, Hungary
E-mail: perczel@chem.elte.hu
http://prot.chem.elte.hu/en/members/principal-investigator/
dr-andras-perczel
phenylhydrazones with different configurations (D-gluco, D-gal-
acto, -manno, and -talo). Furthermore, we expanded our re-
D
D
search to hexosamine phenylhydrazones and 4-nitrophenyl-
hydrazones as a new category of monosaccharide arylhydraz-
ones and we elucidated the influence of the amino group at
C-2 on the partition of the cyclic and acyclic forms. We focused
on establishing simple conditions with respect to monosaccha-
ride configuration and chemical composition, determining their
overall structural preference. The key aspect of this research
[b] Department of Inorganic Chemistry, Institute of Chemistry,
Eötvös University,
Pázmány Péter sétány 1/A, 1117 Budapest, Hungary
[c] MTA-ELTE Protein Modelling Research Group, Eötvös University,
Pázmány Péter sétány 1/A, 1117 Budapest, Hungary
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201600462.
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was to rationalize whether a cyclic (C form) or acyclic (A form) (Φ-Man 3, NO2-Φ-Man 4, Φ-Gal 5, NO2-Φ-Gal 6, NO2-Φ-Tal 7,
isomer or a conformational ensemble is in solution.
Φ-ManNH2 10+, NO2-Φ-ManNH2 11+), have typically been de-
scribed as ensembles of acyclic structures, showing no trace of
any cyclic isomer (Scheme 2). Moreover, both N-acetyl-D-glucos-
amine arylhydrazones (Φ-GlcNAc[17] 14, NO2-Φ-GlcNAc 15) are
Results and Discussion
presumed to exist as conformers of acyclic structures.
The present study comprises both C-2 and C-4 epimers of hex-
oses and hexosamines of phenylhydrazones and 4-nitrophenyl-
Owing to their tendency to undergo equilibrium cyclization,
phenylhydrazones of
D
-glucose (1),
D-galactosamine (Φ-GalNH2
hydrazones. At room temperature, arylhydrazones of D-glucose
12, NO2-Φ-GalNH2 13), and
D
-glucosamine (Φ-GlcNH2 8) can-
(Φ-Glc 1, NO2-Φ-Glc 2) present three different structural forms
in [D6]DMSO[5,14] (Scheme 1): Two cyclic and an acyclic form
were identified, with the acyclic isomer existing as an ensemble
of several conformers of comparable stability.
not be accessed as a single structure (Scheme 3). In high-resolu-
tion NMR spectroscopy in [D6]DMSO at room temperature, com-
pounds 1, 8, 12, and 13 show a conformational mixture of the
acyclic and both the cyclic forms, which have now been charac-
terized in detail.
In contrast, arylhydrazone derivatives with different relative
configurations, such as
D D D-talo
-manno-,[6] -galacto-,[11] or
Scheme 1. Formation of cyclic and acyclic isomers of
D-glucose phenylhydrazones 1 and 2. Reagents and conditions: i) phenylhydrazine·HCl, water,
NaOAc·3H2O or 4-nitrophenylhydrazine, MeOH, reflux.
Scheme 2. Formation of acyclic arylhydrazones 3–7, 10+, 11+, 14, and 15. Reagents and conditions: i) phenylhydrazine·HCl, water, NaOAc·3H2O; or 4-nitro-
phenylhydrazine, MeOH, reflux; ii) 4-nitrophenylhydrazine, MeOH, acetic acid, reflux; iii) 98 % phenylhydrazine, EtOH/water (3:1), acetic acid; or 4-nitrophenyl-
hydrazine, MeOH, reflux.
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Scheme 3. Formation of mixtures of cyclic and acyclic arylhydrazones 8+, 12+, and 13+ in [D6]DMSO solution detected by 1H and 13C NMR spectroscopy.
Reagents and conditions: i) 98 % phenylhydrazine, EtOH/water (3:1), acetic acid; ii) 4-nitrophenylhydrazine, MeOH, reflux.
In the complex structure analysis presented herein, the undergo ring-opening, as demonstrated by Takeda.[9] The iso-
hydrochloride salt of
D
-glucosamine 4-nitrophenylhydrazone mer ratio, as determined by 1H NMR spectroscopy, proved to
(NO2-Φ-GlcNH2, 9+) was used as the reference model as it is a be constant after 24, 72, and 168 h. The characteristic 1-H signal
monosaccharide derivative exclusively present as the ꢀ-pyran- (Figure 1) of the acyclic structure(s) appears between δ = 7 and
ose, 9+,C(ꢀ), in [D6]DMSO (Scheme 4). This unique conformer 8 ppm, surrounded by aromatic 1H resonances (Table 1). On the
was unambiguously proven by 1H and 13C NMR measurements. other hand, the very same 1-H chemical shift, if locked in a
With the experimental results we envisaged supplementing our pyranoid ring, is shifted upfield to δ = 3.5–5.0 ppm, presenting
structural analysis with comparative theoretical modeling stud- coupling constants characteristic of the alternative anomers
ies on a reasonable selection of possible pyranose isomers to (J1,2 ≈ 3–4 Hz for α and J1,2 ≈ 8–10 Hz for ꢀ). Similarly, 13C NMR
disclose some general rules on conformation selection. Note spectroscopy easily distinguishes the cyclic from the acyclic
that at this point no similar rule exists in the literature.
form(s) as the C-1 resonance for the former appears at around
δ = 85–95 ppm, whereas the resonance for the latter is at
1
around δ = 140 ppm. The assignments of H and 13C NMR sig-
nals were confirmed by 2D HSQC, COSY, and HMBC measure-
ments.
Scheme 4. Synthesis and the cyclic form of ꢀ-D-glucosaminyl-4-nitrophenyl-
hydrazine [9+,C(ꢀ)]. Reagents and conditions: i) 4-nitrophenylhydrazine,
MeOH, reflux.
For the detailed structure elucidation, state-of-the-art FTIR,
MS, and NMR analyses were performed. The IR spectra gave
solid evidence for the hydrazone structure (νNH at 3300–
3200 cm–1 and νC=N at 1630 cm–1). Interestingly, MS was not
suitable for distinguishing the isomeric forms as both the cyclic
(9) and acyclic (e.g., 10) isomers exhibit the same fragmentation
pattern (m/z = 298.2, 262.2, 208.1, 180.2, 162.1). On the other
hand, negative ionization mode MS was used to confirm that all
the isolated hexosamine arylhydrazones 8–13 are hydrochloride
salts.
Both 1D and 2D 1H and 13C NMR spectroscopy were used
to identify the structures and ratios of the isomers present in
[D6]DMSO solution at room temperature, the experimental con-
ditions under which cyclic aldose 4-bromophenylhydrazones
3
Figure 1. Characteristic 1H and 13C NMR chemical shifts and JH,H coupling
constants of cyclic and acyclic arylhydrazones used to determine their relative
ratio.
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Table 1. Characteristic 1H and 13C NMR spectroscopic data (δ and 3J) of the
acyclic (A) and cyclic isomers [C(α) and C(ꢀ)] of the arylhydrazones of hexoses
1–7, the hydrochloride salts 8+–13+, and the N-acetyl derivatives of hexos-
conformational ratios of all the hexosamines are independent
of the amino group protonation (Table 2). Owing to the com-
plexity of the NMR spectra, only the diagnostic and unambigu-
ously assigned resonances (e.g., 1-H, 2-H, 6-H or C-1, C-2, C-
6) were taken into account in the identification of the minor
components.
At this stage we had determined the configuration- and sub-
stituent-dependent conformational preference established for
both hexose and hexosamine arylhydrazones. To provide an
atomic explanation based on the relative thermodynamic stabil-
ity of the above conformation selection we carried out a series
of ab initio calculations.[18–20] For all the cationic species, opti-
mization gave two minima on the potential energy surface
(PES) representing the cyclic conformers C(ꢀ1) and C(ꢀ2), which
might coexist in different ratios depending on the relative con-
figuration of the carbohydrate fragment and on the substituent
pattern of the aromatic ring in the arylhydrazine residue
(Table 3). Similarly, two conformers of substantially different sta-
bility [C(ꢀ1) and C(ꢀ3)] were optimized for the free bases of the
amines 14 and 15 ([D6]DMSO, T = 298.7 K, c = 0.1–0.2
M).
[d]
Hexose/hexosamine
arylhydrazones
Isomer A[a] or C
and
% of α or ꢀ ano-
mer
δ [ppm][b]
3J1,2
[Hz]
1-H[c]
C-1[c]
Φ-Glc
1
2
A: 40[e]
C(α): 5
C(ꢀ): 55
A: 60
C(ꢀ): 40
A
A
A
A
A
7.13
4.9
141.3 6.4
92.7
91.2
3.4
9.1
3.71
7.36
3.79
7.16
7.36
7.27
7.45
7.41
7.20
4.10
4.12
NO2-Φ-Glc
147.2 6.4
91.2 8.5
Φ-Man
NO2-Φ-Man
Φ-Gal
NO2-Φ-Gal
NO2-Φ-Tal
Φ-GlcNH3
3
4
5
146.3 7.0
148.5 6.7
142.9 6.3
148.8 6.0
147.3 6.6
132.7 3.5
6
7
8+
A: 95
C(ꢀ): 5
C(ꢀ)
+
87.8
87.4
10.2
10.7
9.6
+
NO2-Φ-GlcNH3
9+
+
Φ-ManNH3
NO2-Φ-ManNH3
Φ-GalNH3
10+
11+
A
A
7.35
7.60
7.20
4.03
7.48
5.23
4.09
7.13
7.37
145.8 3.9
134.1 3.3
134.7 4.0
D
-glucose derivative 2 (Table 3).
+
12+ A: 93
C(ꢀ): 7
+
Table 3. Relative thermodynamic stability (ΔG) difference calculated for the
conformer pairs ꢀ2–ꢀ1 and ꢀ3–ꢀ1, respectively.
88.0
9.9
+
NO2-Φ-GalNH3
13+ A: 80
C(α): 5
140.7 3.5
Glucose or hexosamine arylhydra-
zones
Salt
Free base
89.5
87.9
3.5
10.4
C(ꢀ): 15
A
A
ΔG(ꢀ2–ꢀ1)[a]
ΔG(ꢀ3–ꢀ1)[a]
Φ-GlcNAc
NO2-Φ-GlcNAc
14
15
139.1 5.4
145.1 5.4
[kcal/mol]
[kcal/mol]
NO2-Φ-Glc
Φ-GlcNH2
NO2-Φ-GlcNH2
Φ-GalNH2
NO2-Φ-GalNH2
Φ-ManNH2
2
8
9
12
13
10
11
–
+3.40
+4.61
+3.87
+5.01
+5.38
+6.75
+3.24
[a] A = acyclic or open form, C = cyclic or pyranosyl form. [b] 1H chemical
shifts are referenced to [D6]DMSO. [c] The atoms are highlighted in Figure 1.
[d] 1H–1H vicinal coupling constants were measured to an accuracy of
0.3 Hz. [e] Ratio of different forms.
+1.50
+1.19
+0.55
+0.18
+0.44
+0.84
Thus, our results from the study of D-glucosamine 4-nitro-
NO2-Φ-ManNH2
phenylhydrazine (9+) show a strong but not yet explicitly re-
vealed intramolecular stabilizing interaction responsible for the
exclusive presence of a cyclic form (Table 1). Similarly, a lower
but still significant amount of cyclic forms can be determined
for five additional derivatives (1, 2, 8+, 12+, and 13+). To guess
the conformational propensities of the free derivatives, all the
hexosamine arylhydrazone salts were treated with 1.5 equiv. of
[a] The calculations were carried out at the B3LYP/6-311++G(2d,p) level of
theory using the IEFPCM solvent model (εDMSO = 46.7, T = 298.15 K).
For the arylhydrazones of
-glucose 1 and 2, the ratio[5,14] of
D
A and C is likely to be further modulated by the nature of the
substituent at the 4-position of the aryl ring of the hydrazone.
The two different ꢀ-pyranosyl forms, 2,C(ꢀ1) and 2,C(ꢀ3), were
found to be stabilized by a chain of hydrogen bonds of some-
what different architectures (Figure 2), spectacularly visualized
by the NBOs of selected interactions (e.g., overlap between the
n and σ* orbitals in the hydrogen bonds). In 2,C(ꢀ3), the repul-
sion between the O and α-N atoms highlighted by red and blue
lobes makes the overall conformer less stable than 2,C(ꢀ1) in
which the repulsive interaction is replaced by a hydrogen bond.
Even qualitative considerations regarding the structures of ꢀ1
and ꢀ3 might point to the importance of a sensitive balance
in the particular interactions that contribute differently to the
stability of a particular conformer.
The cyclic form of Φ-GlcNH2 is poorly populated (ca. 5 %),
whereas NO2-Φ-GlcNH2 is exclusively 100 % cyclic (Table 1). In
contrast, in the case of D-Glc derivatives, no such enhancement
is seen: Φ-Glc A/C ≈ 40:60 and NO2-Φ-Glc A/C ≈ 60:40. This
substituent-dependence could be explained by the presence of
an α-N···HO bond, involving a hydroxy group of elevated acidic
character compared with that of the ammonium group, rather
than the ꢀ-NH···Opyran bond, as reflected in the change in
1
triethylamine (TEA). H NMR measurements indicated that the
Table 2. Characteristic 1H and 13C NMR spectroscopic data (δ and 3J) of hexo-
samine arylhydrazones 8–13 in their free unprotonated forms ([D6]DMSO, T =
298 K, c = 0.1–0.2
M
+ 1.5 equiv. TEA).
[c]
Free hexosamine
arylhydrazones
Isomer A or C,
α or ꢀ anomer
δ [ppm][a]
1-H[b] C-1[b]
3J1,2
[Hz]
Φ-GlcNH2
8
A
C(α)
C(ꢀ)
A
A
A
7.17 137.5
4.3
3.1
9.1
8.2
4.8
4.5
10.0
3.55
10.2
5.07
3.71
6.96
7.52
7.22
4.32
7.46
4.06
91.0
91.6
NO2-Φ-GlcNH2
Φ-ManNH2
NO2-Φ-ManNH2
Φ-GalNH2
9[d]
10
11
12
139.4
134.1
139.7
96.8
140.7
88.0
C(ꢀ)
A
NO2-Φ-GalNH2
13
C(ꢀ)[e]
[a] 1H chemical shifts are referenced to [D6]DMSO. [b] The atoms are high-
lighted in Figure 1. [c] 1H–1H vicinal coupling constants are measured to an
accuracy of 0.3 Hz. [d] 3.7 equiv. of TEA was used to obtain the free base.
[e] Unlike the protonated form, the ꢀ-anomer dominates here.
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Table 4. Variation of hydrogen-bond lengths in the ꢀ1 pyranosyl conformers
of the different hexosamine arylhydrazones.
[a]
Glucose or hexosamine
arylhydrazones
α-N···HNH2/3
ꢀ-NH···O[a]
[Å]
[Å]
+
NO2-Φ-GlcNH3
9+
13+
8+
12+
9
13
8
12
1
2.644
2.638
2.622
2.617
2.798
2.800
2.786
2.776
2.517
2.605
2.690
2.692
2.752
2.751
2.607
2.548
2.708
2.679
2.736
2.653
+
NO2-Φ-GalNH3
+
Φ-GlcNH3
+
Φ-GalNH3
NO2-Φ-GlcNH2
NO2-Φ-GalNH2
Φ-GlcNH2
Φ-GalNH2
Φ-Glc
NO2-Φ-Glc
2
[a] The calculations were carried out at the B3LYP/6-311++G(2d,p) level of theory
by using the IEFPCM solvent model (εDMSO = 46.7, T = 298.15 K).
Figure 2. Two pyranosyl forms of D-glucose 4-nitrophenylhydrazone [2,C(ꢀ1)
and 2,C(ꢀ3)] with alternative hydrogen-bonding networks. Selected NBOs
overlap (n→σ*) illustrating the stabilization effect of the hydrogen bonds
highlighted by the yellow and blue lobes.
atomic distances induced by the nitro group (α-N···HO:
2.517 Å in Φ-Glc and 2.605 Å in NO2-Φ-Glc: Δd = +0.088 Å;
ꢀ-NH···Opyran = 2.736 Å in Φ-Glc and 2.653 Å in NO2-Φ-Glc:
Δd = –0.083 Å: Table 4). This view gains further support from
the fact that phenylhydrazine (pKb = 8.8) is a stronger base than
4-nitrophenylhydrazine (pKb = 10.3).[21]
The hydrogen-bond network stabilizing the cyclic form is
necessarily broken if either the C-2 or C-4 substituent is in the
axial position. Consequently, in [D6]DMSO, all the arylhydraz-
ones of the hexoses and hexosamines of D-manno configuration
are present as acyclic form(s) without detectable traces of any
C form for 3, 4, 10+, and 11+ in solution (Table 1). The de-
creased relative stability of the two C forms of the D-mannose
models can be attributed to an incomplete hydrogen-bonding
network in a hypothetical pyranosyl form, with unfavorable re-
pulsive interactions, as exemplified for the two low-energy con-
formers 11+,C(ꢀ1) and 11+,C(ꢀ2) by the NBO overlaps (red and
blue lobes in Figure 3). Note that in none of the optimized
Figure 3. Most stable, but still hypothetical, forms of protonated 2-amino-
2-deoxy-
undetectable by NMR spectroscopy in solution.
D
-mannopyranosyl-4-nitrophenylhydrazine (11+,C), as they both are
Figure 4. Unfavorable 1,3-diaxial interactions lower the population of the C form of the
otherwise well stabilized by a chain of hydrogen bonds. Characteristic overlaps of the cyclic form are depicted by NBO analysis.
D
-galactosamine 4-nitrophenylhydrazine salt (13+,C) to around 20 %,
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D
-mannose structures is α-NH involved in any hydrogen bond. cance of this interaction compared with the hydrogen bond
On the other hand, repulsion between the lone pairs of Opyran operative between the α-N and NH2 group is confirmed by the
and ꢀ-NH makes the overall molecular fold less favorable.
In spite of the nitro group, the C form of NO2-Φ-GalNH2 (13),
the C-4 epimer of D-GlcNH2, makes only a small contribution
(A/C ≈ 80:20, Table 1). However, this observation is perfectly in
line with the present NBO analysis, which reveals that the axial
fact that although α-N is more basic in 8 than in 9, which
strengthens this hydrogen bond in 8 relative to that in 9, it
makes a minor contribution to the overall stability of the pyran-
ose structure.
The elimination of the net positive charge by removing the
OH group, an integrated part of the chain of hydrogen bonds, “extra” proton from R-NH3+, an unintegrated element of the
is involved in an unfavorable 1,3-diaxial interaction with 2-H network, can further enhance the stability of the C form. In 9,
and the axial lone pair of Opyran (Figure 4, highlighted by red the hydrogen-bond chain locks all the flexible dihedral angles
and blue lobes).
of the sugar moiety in a preferred orientation, thereby maximiz-
ing the overlap between favorable NBOs (blue and yellow lobes
in Figure 6). However, the network is rather fragile as the rear-
rangement of the C(ꢀ1) to the C(ꢀ3) form initiates the loss of
one hydrogen bond and the development of a repulsive inter-
action between the lone pairs of the α-N and Opyran atoms (red
and blue lobes). The latter conformational shift is associated
with a considerable amount of destabilization of about 4–
5 kcal/mol. Thus, in accordance with the ab initio study, C(ꢀ3)
is just weakly populated (ca. 0.2 % based on the calculation) in
[D6]DMSO solution at room temperature (Table 3).
The presented results reveal that the configuration, the na-
ture of the C-2 substituent, and the type of arylhydrazine influ-
ence the ratio of the acyclic and cyclic forms of monosaccharide
arylhydrazones. Thus, if all the substituents are in equatorial
positions on the ꢀ-pyranoid ring carrying the 4-nitrophenyl-
hydrazinyl and the amino groups at the 1- and 2-positions, re-
spectively, this framework is expected to become the dominant
conformer! Accordingly, in [D6]DMSO solution the HCl salt of
NO2-Φ-GlcNH2, 9+, is present exclusively in the cyclic form, as
confirmed by the NMR spectroscopic data. The hydrogen bonds
in 9 form a chain around the pyranoid moiety, providing ex-
treme stability for the C form, the exclusively detected isomer
of this compound (Figure 5). The present comparative ab initio
molecular modeling study disclosed two alternative pyranosyl
structures, ꢀ1 and ꢀ2, both stabilized by similar hydrogen-bond-
ing networks. They may coexist in solution in a rapidly intercon-
verting mode of a balanced equilibrium characterized by the
ratio C(ꢀ1)/C(ꢀ2) of around 85:15, calculated by the simple Gibbs
equation using the ΔG values derived from the theoretical
modeling study (Table 3). It must be pointed out here that due
to their rapid interconversion, which takes place so rapidly rela-
tive to the NMR time scale, measurements can produce time-
averaged spectra of the mixture of the two conformers with
highly dominant contributions from C(ꢀ1), according to the de-
tectable chemical shifts and coupling constants, compared with
those originating from C(ꢀ2).
Figure 6.
D-Glucosaminyl-4-nitrophenylhydrazine (free base), for both con-
formers a complete chain of hydrogen bonds anchors the 3D structure. Nei-
ther C(ꢀ1) nor C(ꢀ3) have residual internal rotational freedom.
The exclusive cyclic conformational behavior of 9+ coupled
with favorable NMR spectral properties offers the possibility of
cross-validating experimental and ab initio determined NMR pa-
1
rameters. Both the H and 13C chemical shifts along with the
1
vicinal H–1H scalar coupling constants were calculated by ab
initio methods and compared with experimental values
(Table 5), which allowed the ratio of the C(ꢀ1) and C(ꢀ2) con-
formers in DMSO to be determined.
The small difference (1.19 kcal/mol, Table 3) between the
free-energy values of the C(ꢀ1) and C(ꢀ2) conformers of 9+
points to their comparable populations in solution. Thus, the
vicinal coupling constants measured in [D6]DMSO for the skele-
tal and OH proton pairs (Table 5) could be diagnostic and used
to scale/obtain populations of the C forms. The measured and
Figure 5. Two low-energy cyclic conformers of D-glucosaminyl-4-nitrophenyl-
hydrazine·HCl [9+,C(ꢀ1) and 9+,C(ꢀ2)] in equilibrium. The NBO analysis shows
hydrogen bonds for both conformers, as highlighted by yellow and blue
lobes. The OH protons not involved in the network are shown in red.
3
calculated values of the Jn-H,(n+1)-H coupling constants charac-
terizing the interaction of the nonacidic skeletal protons and
Furthermore, the higher acidity of the ꢀ-NH group in 9 short- the corresponding dihedral angles (154 4 and 175 2°) found
ens the hydrogen-bond length in ꢀ-NH···Opyran by 0.1 Å with in the measured and optimized structures, respectively, are in
respect to that of the parent Φ-GlcNH2 (8; Table 4). The signifi- good agreement.
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Table 5. Measured ([D6]DMSO, T = 298 K, c = 0.2
M
) and calculated [GIAO-B3LYP/6-311++G(2d,p)] 1H NMR chemical shifts (δ), vicinal 1H coupling constants (J), and
dihedral angles for 2-amino-2-deoxy-ꢀ-D
-glucopyranosyl-4-nitrophenylhydrazine (9+) and vicinal 1H coupling constants (J) used to calculate dihedral angles by using the
Karplus equation.[22]
Resonance
type
9+,C(ꢀ)
C(ꢀ1) conformer, major[e]
C(ꢀ2) conformer, minor[e]
Measured
δ [ppm][a] 3J [Hz][b]
Calculated dihedral
Calculated
Calculated
angle θ ≈ f(3J)[d]
δ [ppm][a] 3J [Hz][b]
Dihedral angle [°][c] δ [ppm][a] 3J [Hz][b]
Dihedral angle [°][c]
1-H[f]
4.12
3J1,2 = 9.7
1-H/2-H: 153
1-H/NH: 159
2-H/3-H: 153
–
–
–
4.52
3J1,2 = 8.1
1-H/2-H: 175
1-H/NH: 176
2-H/3-H: 174
3-H/4-H: 174
4-H/5-H: 176
5-H/6A-H: 179
5-H/6B-H: 60
4.25
3J1,2 = 8.1
3J1,NH = 10.3
3J2,3 = 9.0
3J3,4 = 6.9
3J4,5 = 7.7
3J5,6A = 7.8
1-H/2-H: 175
1-H/NH: 175
2-H/3-H: 174
3-H/4-H: 175
4-H/5-H: 179
5-H/6A-H: 172
5-H/6B-H: 69
3J1,NH = 10.7
3J2,3 = 9.7
m[g]
3J1,NH = 10.4
3J2,3 = 9.0
2-H
3-H
4-H
5-H
6A-H
6B-H
3-OH[g]
4-OH
6-OH
2.82
3.47
3.13
3.15
3.72
3.51
5.94
5.38
4.64
3.21
3.97
3.87
3.67
4.07
4.38
2.64
4.17
1.26
2.86
3.67
3.56
3.34
3.63
3.79
2.33
2.11
2.41
3J3,4 = 6.8
m
m
3J4,5 = 8.2
3J5,6A = 8.7
3J6A,6B = 7.7
3J5,6B = 4.5
3J3-OH,3 = 2.2
3J4-OH,4 = 0.2
3J6A,6B = 9.5
6A-H/6B-H: 151
3J5,6B = 3.0
3J3-OH,3 = 5.4[e] 3-OH/3-H: 38
2-H/3-H: 54
2-H/3-H: 76
3J3-OH,3 = 5.7
3J4-OH,4 = 4.8
2-H/3-H: 30
2-H/3-H: 38
3J4-OH,4 = 5.2
3J6-OH,6A = 8.6
3J6-OH,6B = 4.0
4-OH/4-H: 40
6-OH/6A-H: 13
6-OH/6B-H: 47
3J6-OH,6A = 2.9 6-OH/6A-H: 50
3J6-OH,6B = 2.3 6-OH/6B-H: 71
3J6-OH,6A = 0.2 6-OH/6A-H: 73
3J6-OH,6B = 1.3 6-OH/6B-H: 169
[a] 1H chemical shifts measured or calculated for the optimized structure 9+,C(ꢀ) referenced to [D6]DMSO or TMS. [b] Vicinal coupling constants measured to
3
3
an accuracy of 0.3 Hz or calculated for the optimized structure of 9+,C(ꢀ). [c] Dihedral angle optimized. [d] JH,H = 10.4cos 2θ – 1.5cos θ + 0.2, JH,OH = 5.76
3
– 2.05cos θ + 6.78cos 2θ, JH,NH = 12cos 2θ + 0.2. [e] Conformer ratio estimated by Gibbs equation using free-energy values determined from frequency
calculations. [f] 1-H: the proton at C-1, as shown in Figure 1. [g] J3-OH,3: vicinal coupling constant between the OH and H at C-3. [g] Multiplet and so J was
not determined.
However, because each modeling study presented here was
D-glucosamine derivative(s). Therefore, the C-2 epimers of 4-
carried out without using a highly demanding exact solvent nitrophenylhydrazone obtained by replacement by N nucleo-
model, with uncertain position and number of DMSO mol- philes at C-2 might be distinguished. We can now provide a
ecules, the apparent mismatch between the experimental and simple rule for the formation of the most probable molecular
3
calculated values of the Jn-OH,n-H coupling constants can be conformation occurring in solution at room temperature. Thus,
attributed to undefined solvent-induced intermolecular the relationship between the molecular conformations of hex-
hydrogen bonds involving OH protons that perturb the ideal ose and hexosamine arylhydrazones and their configurations
intramolecular hydrogen-bonding system and change the cal- can be estimated with a higher level of confidence and vice
culated dihedral angles. Nevertheless, the dihedral angles origi- versa.
nating from the measured coupling constants show acceptable
matches for 3-OH and 4-OH, which are integrated parts of the
hydrogen-bond chain in both conformers. On the other hand,
Conclusions
the more significant mismatch between the measured and cal-
The present work is the first detailed investigation of the struc-
tural criteria controlling the selection of cyclic or acyclic struc-
tures of the new category of compounds, hexosamine aryl-
3
3
culated vicinal coupling constants J6-OH,6A and J6-OH,6B can be
ascribed to the flexible nature of the terminal position of 6-OH,
which is the most easily accessible hydrogen-donor fragment
exposed to the acceptor [D6]DMSO molecules. This situation is
particularly characteristic for the dominant conformer C(ꢀ1), in
which the flanking proton of the 6-OH group is not involved in
any intramolecular hydrogen-bonding system.
hydrazones. The new 2-amino-2-deoxy-ꢀ-D-glucopyranosyl-4-
nitrophenylhydrazine has proved to be a unique example of
hexosamine arylhydrazones that exists exclusively in the cyclic
form. As expected, any axial substituent of the pyranoid ring, as
in the case of the D-galacto- or D-manno derivatives, diversifies
As all of these findings suggest that a crucial chain of
hydrogen bonds, capable of anchoring the arylhydrazine sub-
stituent in an optimal position, is extended to the OH groups,
it can be stated that theoretical modeling studies, carried out
at a higher level of theory, might provide a realistic picture of
the orientation of the OH groups in a pyranoid system. The
reasonable match between the results of these cross-checking
methods confirms that both the calculated and measured pa-
rameters are relevant in the structural analysis of pyranoses and
related molecular architectures.
the conformational distribution, thereby resulting in an ensemble
of mainly acyclic structures. Therefore, conditions favoring the
cyclic structure are 1) an amino group at the C-2 position, 2) the
“all-equatorial” substitution mode of the pyranoid ring, and 3) an
electron-withdrawing group (e.g., NO2) on the arylhydrazone
moiety. The consequence is the formation of the cyclic structure
stabilized by a complete chain of hydrogen bonds.
Experimental Section
General: All chemical reagents were purchased from Sigma–
Aldrich, Alfa Aesar, VWR, or Molar Chemicals. Melting points were
determined with a Boetius micro-melting-point apparatus. TLC was
performed on silica gel 60 F254, 230 mesh (E. Merck) and the spots
were detected by UV detection (254 nm) and destruction with 5 %
Outlook
Our results show that only the
tions of the arylhydrazones of hexosamines allow the formation
D-gluco and D-galacto configura-
of the pyranosyl form, but this structure is exclusive only for H2SO4 solution. Column chromatography was performed on Kiesel-
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gel 60 (0.040–0.063 nm, E. Merck). Optical rotations were deter-
mined with a Jasco P-2000 polarimeter at 589 nm. IR spectra were
recorded with an FTIR Bruker IFS 28 spectrophotometer. All NMR
experiments were performed at 298 K with a Bruker Avance DRX
500 MHz spectrometer equipped with a TXI probe with a z gradient
forming the optical rotation measurement. This work was sup-
ported by the Hungarian National Science Fund (OTKA) (grant
number NK101072).
Keywords: Conformation analysis · Hydrogen bonds ·
Carbohydrates · Hydrazones · Ab initio calculations
1
operating at 500.128 MHz for H and 125.757 MHz for 13C. Sample
concentrations ranged from 0.1 to 0.2
M. The NMR spectra were
recorded in [D6]DMSO using the solvent residual peak as the 1H
internal reference (δ = 2.5 ppm, [D6]DMSO). 2D NMR measurements
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550.
(1H–1H COSY, H–13C HSQC, and H–13C HMBC) were performed by
using standard Bruker[23] pulse programs. Spectra evaluation was
carried out with the TopSpin 3.2 software. Electrospray ionization
mass spectrometry (ESI-MS) was performed with a Bruker Daltonics
Esquire 3000+ mass spectrometer operating in continuous sample
injection mode at a flow rate of 10 μL/min. Samples were dissolved
in a mixture of acetonitrile/water (1:1, v/v) with NH4OAc buffer. Mass
spectrawererecordedinpositiveandnegativeionmodesintherange
m/z = 50–3000.
1
1
[4] V. Deulofeu, Adv. Carbohydr. Chem. 1949, 4, 119–151.
[5] E. Fischer, Ber. Dtsch. Chem. Ges. 1884, 17, 579–584.
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Tetrahedron: Asymmetry 1995, 6, 945–956.
All calculations were performed at the B3LYP/6-311++G(2d,p) level
of theory by using the IEFPCM solvent model as implemented in
the Gaussian 09[24] suite of programs. The NBO analyses were per-
formed on the previously DFT-optimized structures by using the
NBO 5.0/5.G program with Gaussian 09 using the same basis set
and solvent model. The optimized structures are available from the
authors.
[9] Y. Takeda, Carbohydr. Res. 1979, 77, 9–23.
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2563–2567.
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1997, 212, 191–192.
General Synthetic Procedures
[14] S. L. Zhdanov, A. A. Potehin, Zh. Org. Khim. 1979, 15, 1384–1392.
[15] G. Zemplén, L. Mester, Acta Chem. Acad. Sci. Hung. 1952, 2, 9–14.
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[17] A. Messmer, I. Pintér, V. Zsoldos-Mády, A. Neszmélyi, J. Hegedűs-Vajda,
Acta Chim. Hung. 1983, 113, 393–402.
[18] a) A. D. Becke, J. Chem. Phys. 1993, 98, 5648–5652; b) C. Lee, W. Yang,
R. G. Parr, Phys. Rev. B 1988, 37, 785–789; c) P. J. Stephens, F. J. Devlin,
C. F. Chahalowsky, M. J. Frisch, J. Phys. Chem. 1994, 98, 11623–11627.
[19] W. J. Hehre, L. Radom, P. v. R. Schleyer, J. A. Pople, Ab initio Molecular
Orbital Theory, Wiley, New York, 1986.
[20] NBO, v. 5.0: E. D. Glendening, J. K. Badenhoop, A. E. Reed, J. E. Carpenter,
J. A. Bohmann, C. M. Morales, F. Weinhold, Theoretical Chemistry Insti-
tute, University of Wisconsin, Madison, USA, 2001.
[21] Calculated values from the values of pKa: Advanced Chemistry Develop-
ment (ACD/Labs) Software V11.02, 1994–2016.
Synthesis of Hexose Phenylhydrazones Starting with Phenyl-
hydrazine Hydrochloride:[25]
D-Hexose (0.99 g, 5.5 mmol) was dis-
solved in hot water (1.4 mL), and a mixture of sodium acetate tri-
hydrate (0.99 g, 7.5 mmol) and phenylhydrazine hydrochloride
(0.99 g, 6.9 mmol) was dissolved in hot water (5 mL). The solutions
were cooled and then mixed, after 5–15 min the product precipi-
tated from solution. The precipitate was filtered and washed cold
water, ethanol, and diethyl ether.
Synthesis of Hexosamine Phenylhydrazones Starting with
Phenylhydrazine Solution: D-Hexosamine hydrochloride or D-gluc-
ose (0.9 mmol) was dissolved in 50 or 75 % EtOH (3 mL) and then
acetic acid (0.05 mL) and 97 % phenylhydrazine (0.14 mL, 1.4 mmol)
were added to the solution. The mixture was allowed to stand at
room temperature until the starting material had disappeared. The
solution was then concentrated. The residue was crystallized from
diethyl ether or tetrahydrofuran, filtered, and dried.
[22] R. R. Fraser, M. Kaufman, P. Morand, G. Govil, Can. J. Chem. 1969, 47,
403–409.
[23] Pulse Program Catalogue: I. 1D & 2D NMR Experiments, Bruker BioSpin
GmbH, 2006.
[24] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R.
Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Na-
katsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G.
Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hase-
gawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A.
Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Broth-
ers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K.
Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,
N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R.
Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G.
Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D.
Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaus-
sian 09, revision B.01, Gaussian, Inc., Wallingford, CT, 2010.
[25] L. Mester, A. Messmer, Phenylhydrazones, in: Methods in Carbohydrate
Chemistry, vol. 2 (Eds.: M. L. Whistler, M. L. Wolfrom), Academic Press,
New York, 1963, p. 117–141.
Synthesis of Hexose and Hexosamine 4-Nitrophenylhydrazones
Starting with 4-Nitrophenylhydrazine: 4-Nitrophenylhydrazine
(0.16 g, 1.1 mmol) was added dropwise to a solution of
D-hexose
or -hexosamine (1.1 mmol) in methanol/water (3/1.5 mL) and ace-
D
tic acid (0.05 mL). The mixture was heated at reflux for 1.5–3 h,
cooled, and the product filtered and washed with cold ethanol. If
the product did not precipitate from solution the reaction mixture
was concentrated. The residue was treated with tetrahydrofuran or
diethyl ether and the product was filtered and washed.
Supporting Information (see footnote on the first page of this
1
article): Analytical data, H and 13C NMR spectra and results of mo-
lecular modeling.
Acknowledgments
The authors express their gratitude to Anita Kapros for perform-
ing the MS measurement and to Szebasztián Szaniszló for per-
Received: April 13, 2016
Published Online: ■
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Glycochemistry
Cyclic or acyclic? Criteria determining
the cyclic form of sugar arylhydraz-
ones are listed: The buildup of a chain
of hydrogen bonds, the key element
of stability, was determined by high-
resolution NMR spectroscopy and QM
data.
V. G. Gőz, I. Pintér, A. Csámpai,
I. Jákli, V. Zsoldos-Mády,
A. Perczel* ............................................. 1–9
Hydrogen-Bonding Network An-
chors the Cyclic Form of Sugar Aryl-
hydrazones
DOI: 10.1002/ejoc.201600462
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