Characterization of D-glucaric acid using NMR, X-ray crystal structure, and MM3 molecular modeling analyses

Article abstract of DOI:10.1016/j.carres.2011.08.016

D-Glucaric acid was characterized in solution by comparing NMR spectra from the isotopically unlabeled molecule with those from d-glucaric acid labeled with deuterium or carbon-13 atoms. The NMR studies provided unequivocal assignments for all carbon atoms and non-hydroxyl protons of the molecule. The crystal structure of d-glucaric acid was obtained by X-ray diffraction techniques and the structure was a close match to the low energy conformation generated from a Monte-Carlo-based searching protocol employing the mm3 molecular mechanics program. The molecule adopts a bent structure in both the crystalline and computationally generated lowest-energy structure, a conformation that is devoid of destabilizing eclipsed 1,3-hydroxyl interactions.

Full text of DOI:10.1016/j.carres.2011.08.016

Carbohydrate Research 346 (2011) 2551–2557
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Characterization of D-glucaric acid using NMR, X-ray crystal structure,
and MM3 molecular modeling analyses
Travis T. Denton a, , Kenneth I. Hardcastle b, Michael K. Dowd c, Donald E. Kiely a,
⇑
a Shafizadeh Rocky Mountain Center for Wood and Carbohydrate Chemistry, The University of Montana, Missoula, MT 59812, USA
b Emory University Crystallography Laboratory, Department of Chemistry, Emory University, Atlanta, GA 30322, USA
c USDA Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70126, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 March 2011
Received in revised form 15 August 2011
Accepted 17 August 2011
Available online 31 August 2011
D
-Glucaric acid was characterized in solution by comparing NMR spectra from the isotopically unlabeled
molecule with those from -glucaric acid labeled with deuterium or carbon-13 atoms. The NMR studies
provided unequivocal assignments for all carbon atoms and non-hydroxyl protons of the molecule. The
crystal structure of -glucaric acid was obtained by X-ray diffraction techniques and the structure was
D
D
a close match to the low energy conformation generated from a Monte-Carlo-based searching protocol
employing the MM3 molecular mechanics program. The molecule adopts a bent structure in both the crys-
talline and computationally generated lowest-energy structure, a conformation that is devoid of destabi-
lizing eclipsed 1,3-hydroxyl interactions.
Keywords:
D-Glucaric acid
Characterization
NMR
Ó 2011 Elsevier Ltd. All rights reserved.
X-ray
Molecular mechanics
1. Introduction
to provide us with some conformational information on D-glucaric
acid in aqueous solution employing 1H NMR, in the solid state via
an X-ray crystal structure analysis and computationally, employing
a new MM3 molecular mechanics protocol.13 An added incentive for
this study stemmed from a forecast in a US Department of Energy
publication pointing to the significant potential commercial value
D
-Glucaric acid (2) is a naturally occurring aldaric acid that is
found in small amounts in a variety of vegetables and fruits1,2
and is touted for its dietary value, particularly as a cancer preven-
tative agent.1
D
-Glucaric acid was first reported by Sohst and
Tollens in 18883 by the nitric acid oxidation of
D-glucose (1) and
of D-glucaric acid as an important renewable chemical building
isolated as its monopotassium salt (3, Fig. 1). Although nitric acid
has been widely employed over the past century as a general agent
for the oxidation of aldoses to their corresponding aldaric acids,
other oxidative processes have also been used for these conver-
block derived from widely available D
-glucose.14
2. Results and discussion
sions.4–6 A crystalline form of
D-glucaric acid was generated by
2.1. NMR studies on
D
-glucaric acid (2)
-glucaric acid were carried out in order to
Rehorst by treating the silver salt with hydrochloric acid,7 whereas
Hirasaka et al. converted the monopotassium salt (3) to 2 using a
cation exchange resin.8
The NMR studies on
D
unequivocally assign the proton and carbon chemical shifts of the
molecule and compare the results with those from earlier re-
ports.15–17 The NMR studies described here differ from the previ-
ously reported studies in that NMR data were obtained directly
D
-Glucaric acid has been of particular interest to us as a diacid
monomer for condensation polymerizations with diamines to give
the corresponding poly( -glucaramides)
-glucaramides).9–12 Poly(
are conformationally complex structures given that the repeating
asymmetric -glucaryl unit has four chiral carbons. To gain addi-
tional insight into possible conformations the -glucaryl group
D
D
from samples of crystalline D-glucaric acid dissolved in D2O rather
D
than in salt form or where acyclic 2 was in equilibrium with its
acid/lactone and dilactone forms. In addition, results from three
different isotopically labeled samples of 2 were used for verifica-
tion of specific chemical shift assignments. 1H NMR and 13C NMR
chemical shifts and coupling constants for 2 (D2O, Table 1) were
obtained from spectra recorded at 600 MHz and 150 MHz,
respectively. Spectral assignments for 2 were made from detailed
analyses of HOMO and HETERO nuclear, two dimensional NMR
spectra (COSY and HSQC, respectively—Supplementary data).
D
might adopt in these polyamides, we undertook a study designed
⇑
Corresponding author. Tel.: +1 406 543 6126; fax: +1 406 543 2304.
E-mail addresses: travis.denton@mail.ewu.edu (T.T. Denton), don@rivertop.com,
donald.kiely@umontana.edu (D.E. Kiely).
Present address: Department of Chemistry and Biochemistry, Eastern Washington
University, Cheney, WA 99004, USA.
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.08.016

2552
T. T. Denton et al. / Carbohydrate Research 346 (2011) 2551–2557
O
OH
OH
3
1
OH
5
KOH
HO
O
6
4
2
OH
O
OH
4
OH
O
OH
OH
1
O
3
O K
5
HO
HO
2
HO
OH
6
2
OH
O
OH
OH
H
1
3
Figure 1. General oxidative conversion of
D-glucose (1) to D-glucaric acid (2), isolated as monopotassium D-glucarate (3).
applying conformational nomenclature previously established18
to the inner three C2–C3, C3–C4, and C4–C5 backbone bonds. The
Table 1
NMR assignments for 2
crystal structures of a number of D-glucaric acid derivatives have
Hydrogen No.
2
3
4
5
been reported, some of which adopt varying bent conformations
with others being extended. Included among the former deriva-
Chemical shift (ppm)
Vicinal 1H–1H coupling
J value (Hz)
Carbon No.
Chemical shift (ppm)
4.48
4.14
3.96
4.36
J2,3
3.00
J3,4
5.87
J4,5
5.14
6
tives are potassium sodium
-glucarate,20 and diammonium
methyl
-glucaramide19 and dipotassium
formationally extended. A conformational characteristic found
with crystalline -glucaric acid is the absence of hydroxyl groups
D
-glucarate hydrate,19 monopotassium
-glucarate,21 whereas N,N0-di-
-glucarate19 are con-
1
2
3
4
5
D
D
176.74
72.47
72.43
74.12
72.26
176.43
D
D
D
eclipsed in 1,3-parallel interactions, which is in contrast to what
was observed with extended structures noted above. Extended
Supporting 1H and 13C NMR spectral information was garnered
from spectra of 2-2H, 1-13C and 6-13C isotopically labeled samples
of 2 (2a, 2b, and 2c, respectively, Fig. 3) generated by the nitric acid
N,N0-dimethyl
D
-glucaramide19 represents a previously reported
conformational model for the glucaryl repeating unit in poly-D-glu-
oxidation of correspondingly labeled
The 1H NMR spectra of unlabeled
-[2-2H]- (2a), -[1-13C]- (2b), and
shown in Figure 2. The spectrum of
D
-glucose.
caramides that includes the diamido functionality of typical gluca-
ric acid-based polyamides.
D
-glucaric acid (2) and labeled
D
D
D
-[6-13C]-glucaric acid (2c) are
Crystalline D-glucaric acid (2) is extensively hydrogen bonded
D
-[2-2H]-glucaric acid (2a) is
as shown in Figure 5. All six hydroxyl groups donate to intermolec-
ular hydrogen bonds with the O4 and O7 hydroxyl groups each
donating into two interactions. Except for the terminal hydroxyl
oxygen atoms (i.e., O2 and O7), all the molecule’s oxygen atoms
accept hydrogen bonds. Two oxygen atoms, the O1 carbonyl oxy-
gen atom and the O3 hydroxyl oxygen atom accept hydrogen
bonds from two donating hydroxyl groups. The hydrogen bonds
form a single cooperative network, but do not form either closed
rings or infinite chain structures. No intramolecular hydrogen
bonds exist.
missing the doublet at d 4.48 ppm (H2) seen in that of unlabeled
2 and shows the signal at d 4.14 ppm (H3) as a doublet in place
of the quartet of unlabeled 2. These spectral changes confirm the
assignment of the H-2 proton chemical shift (4.48 ppm) and clearly
show the J3,4 coupling (5.87 Hz) from the H3 doublet at 4.14 ppm.
The1H NMR spectra of -[1-13C]- and [6-13C]-glucaric acid, 2b and
D
2c, in addition to supporting the proton chemical shift assignments
for 2, also have some notable spectral differences. The spectrum of
2b displays the H2 signal (4.48 ppm) as a doublet of doublets, con-
sistent with the added 13C1–C2–1H2 two bond coupling
(J = 5.03 Hz), and the H-3 signal (4.14 ppm) as a complex multiplet,
resulting from the additional 13C1–C2–C3–1H3 vicinal coupling
(J = 1.76 Hz). The H4 and H5 signals of 2c are also more complex
than those of 2 due to the additional 13C6–C5–C4–H4 vicinal cou-
pling (J = 4.11 Hz) and 13C6–C5–H5 two bond coupling
(J = 4.12 Hz), respectively. The 13C spectra of 2b and 2c were partic-
ularly valuable in that each was distinguished by a single enhanced
downfield signal, allowing for direct chemical shift assignment of
C1 and C6 in 2 at 176.74 ppm and 176.43 ppm, respectively.
The chemical shifts and 1H–1H coupling constant values re-
ported here update those values previously reported.15–17 The
assignments for 2 given in Ref. 16 were also aided from spectral
Employing the method of Dowd et al.,13 a Metropolis Monte-
Carlo search routine was coupled to the MM3 molecular mechanics
program22–24 and a 20,000-step search of the
D-glucaric acid con-
formational space was run. The search was conducted at a dielec-
tric constant of 3.5 to reduce the strength of intra-molecular
hydrogen bonds, and a shaking routine was used to keep the search
from becoming trapped in a single low-energy region.13
The conformational search found 3310 distinct conformers.
Twenty-one conformers were found within 1 kcal/mol of the global
energy minimum (Table 2), the group falling into four conforma-
tional families, 3G+4G+, 2Gꢀ4G+, 2Gꢀ4Gꢀ, and 4G+. No fully extended
conformations were observed among the lowest-energy structures.
The lowest energy conformer had backbone torsion angles of
178.5° (C1–C2–C3–C4), 61.3° (C2–C3–C4–C5), and 58.5° (C3–C4–
data acquired with D
-[2-2H]-glucaric acid, but details were not re-
ported. A recent report17 gives comparable but slightly different 1H
chemical and 13C shift values for 2. However, in that report, spectra
were recorded of an equilibrium mixture of 2 as its open chain dia-
cid, lactone/acid and dilactone forms in DCl/D2O and not as a single
component in D2O, as reported here.
C5–C6), comparable to those in crystalline D-glucaric acid of
169.17° (C1–C2–C3–C4), 57.84° (C2–C3–C4–C5), and 67.93° (C3–
C4–C5–C6), respectively. This backbone shape, 3G+4G+, accounted
for 57% of the conformers found with 1 kcal/mol of the computed
structures (Table 2). These angles produce a ‘bent’ shape that
avoids sterically unfavorable 1,3-oxygen–oxygen interactions.25
Although the backbone conformation of the global MM3 mini-
2.2. Comparison of the X-ray crystal structure and
computationally generated structures of
D
-glucaric acid
mum (Fig. 6) closely matched that of the D-glucaric acid crystal
structure (Fig. 4), differences in the structures were apparent. In
the modeled structure, the terminal C1–C2 and C5–C6 carboxyl
moieties and the hydroxyl groups had torsional orientations that
The structure of
D-glucaric acid in the crystal state was deter-
mined to have a sickle-like (bent) 2G+3G+ conformation (Fig. 4),

T. T. Denton et al. / Carbohydrate Research 346 (2011) 2551–2557
2553
D-Glucaric Acid (2)
H2
H5
H4
H3
4.55
4.50
4.45
4.40
4.35
4.30
4.25
4.20
4.15
4.10
4.05
4.00
3.95
3.90
3.85 3.80
2
D
-[2- H]-Glucaric Acid (2a)
H2
H5
H3
H4
4.55
4.50
4.45
4.40
4.35
4.30
4.25
4.20
4.15
4.10
4.05
4.00
3.95
3.90
3.85 3.80
13
D
-[1- C]-Glucaric Acid (2b)
H4
H5
H3
H2
4.55
4.50
4.45
4.40
4.35
4.30
4.25
4.20
4.15
4.10
4.05
4.00
3.95
3.90
3.85 3.80
13
D
-[6- C]-Glucaric Acid (2c)
H2
H5
H4
H3
4.55
4.50
4.45
4.40
4.35
4.30
4.25
4.20
4.15
4.10
4.05
4.00
3.95
3.90
3.85 3.80
Chemical Shift (ppm)
Figure 2. 1H NMR spectra (600 MHz) of
D-glucaric acid (2) and isotopically labeled D-glucaric acids (2a–2c).
differed from the crystal form. As an elevated dielectric constant
was the only consideration applied to mimic the condensed phase
during the modeling, the hydroxyl and carboxyl groups would not
be influenced by intramolecular and environmental effects while
the crystal structure would be sensitive to intermolecular hydro-
gen bonding and packing effects. Hence, differences in hydroxyl

2554
T. T. Denton et al. / Carbohydrate Research 346 (2011) 2551–2557
O
O
HO
H
HO
H
H
HO
H
HO
2H
H
R
R
HO
HO
O
O
H
OH
HO
H
OH
HO
2. R=OH
3. R=O
2a, R=OH
3a, R=O
K
K
O
O
HO
H
HO
H
H
HO
H
HO
H
H
R
R
13C
13C
O
HO
HO
O
H
OH
HO
H
OH
HO
2b, R=OH
3b, R=O
2c, R=OH
3c, R=O
K
K
Figure 3.
D-Glucaric acid (2), isotopically labeled
D-glucaric acid (2a–2c), monopotassium
D-glucarate (3) and monopotassium salts (3a–3c) of 2a–2c.
and carboxyl orientations between the solid-state and modeled
conformers were not surprising.
Hydrogen–hydrogen coupling constants were calculated for
each MM3 conformer with the Karplus equations of Haasnoot
et al.26 Population-averaged coupling constants for the three
internal backbone bonds were in the 3.4–4.6 Hz range and were
within range of the experimental values (Table 1). Calculated
coupling constants averaged over the whole population were: J2,3
=
Table 2
Distribution of MM3 generated conformers within 1 kcal/mol of the lowest-energy
structure
C-1ꢁ ꢁ ꢁC-4 angle
C2ꢁ ꢁ ꢁC5 angle
C3ꢁ ꢁ ꢁC6 angle
Conformer
designation
Number
found
Figure 4. X-ray crystal structure of
D-glucaric acid (2).
180
ꢀ60
ꢀ60
180
60
180
180
180
60
ꢀ60
60
3G+4G+
2Gꢀ4Gꢀ
2Gꢀ4G+
4G+
12
5
3
60
1
Figure 5. The unit cell of crystalline
bonding system associated with the crystal structure.
D-glucaric acid (2) showing the hydrogen
Figure 6. The lowest-energy MM3 conformer (3G+4G+) of
D-glucaric acid (2).

T. T. Denton et al. / Carbohydrate Research 346 (2011) 2551–2557
2555
4.63 Hz (3.00 Hz), J3,4 = 3.69 Hz (5.87 Hz), J4,5 = 3.4 Hz (5.14 Hz).
The experimental values are in parenthesis. Because many con-
formers had relatively low energies, the calculated values resulted
from the contribution of several forms and not from a single dom-
inant backbone orientation. Hence, the modeling does not support
the existence of an ‘average’ solution structure but rather a mix-
ture of many structures. In addition, calculated coupling constants
for the lowest-energy conformer, which had a backbone structure
concentrated. The syrupy residue was seeded with a crystal from
above and stored at 4 °C for 12 h. The crystals were removed from
the flask, washed, quickly, with 97% ethanol and the fine solid and
ethanol were removed with a pipette to afford X-ray quality,
crystalline
D
-glucaric acid: mp 119.3 °C (lit.8 117–118 °C, lit.7
125–126 °C); ½a 2D0
ꢃ
+5.7 (c 0.064, D2O, 5 min.) [lit.8 +6.1 (c 1.0,
H2O, 5 min), lit.7 +6.9 (c 1.0, H2O); 1H NMR (D2O): d 4.48 (d, 1H,
H2, J2,3 = 3.00 Hz), 4.36 (d, 1H, H5, J4,5 = 5.14 Hz), 4.14 (dd, 1H,
in good agreement with that of crystalline
D
-glucaric acid, were
H3, J
2,3 = 3.00 Hz, J3,4 = 5.87 Hz), 4.00 (m, 1H, H4). 13C NMR
markedly different (J2,3 = 0.56 Hz, J3,4 = 9.87 Hz, and J4,5 = 2.65 Hz)
from the experimental results. This indicates that the aqueous
(150 MHz, D2O): d 176.74 (C1), 176.43 (C6), 74.12 (C4), 72.47
~
(C2), 72.43 (C3), 72.26 (C5); IR (KBr):
m
3492–2917 (s, O–H stretch),
and crystalline conformations of
D
-glucaric acid must be quite dif-
1729 (s, C@O stretch), 1692 (s, C@O stretch) cmꢀ1
.
ferent. Jeffrey has made a similar observation regarding alditols.27
3.1.3. Conformational modeling
The 1996 version of MM322–24 was used for modeling preferred
-glucaric acid conformations. Default parameters were used with
3. Experimental
D
the full-matrix optimization method. A dielectric constant of 3.5
was chosen for this work, as dielectric constants between 3.0 and
4.0 have been used in prior MM3 studies of carbohydrates and have
3.1. General methods
been generally found to yield preferable results.19,28 Because
D-
All solvents and ion exchange resins were purchased from com-
mercial sources and used without further purification.
glucose and
-[6-13C]-glucose were purchased from Cambridge
Isotope laboratories, Inc., Andover, MA and
-[2-2H]-glucose was
D
-[1-13C]-
glucaric acid has 11 torsion angles that define its conformation, a
full staggered search of the confomational space would require
311 or greater than 177,000 optimizations. To reduce the computer
D
D
purchased from Omicron Biochemicals, Inc., South Bend, IN. Solu-
tions were concentrated in vacuo using a rotary evaporator with
a vacuum of 15–20 mbar and bath temperature of 40 °C. Melting
points were determined using a differential scanning calorimeter
(Jade DSC, Perkin–Elmer, Shelton, CT) scanning from 5 °C to
250 °C at 10 °C/min and are uncorrected. 1H NMR and 13C NMR
spectra were recorded at room temperature and 600 MHz and
150 MHz, respectively, on a Varian Unity spectrometer. Chemical
shifts were reported in parts per million (ppm, d) using tert-butyl
alcohol [1.203 ppm (1H), 30.695 ppm (13C)] in D2O as an internal
standard. Two-dimensional HOMO and HETERO nuclear spectra
were obtained using standard macros in the Varian Unity software.
All NMR spectra were transformed and processed with ACD/Spec-
Manager software using the standard 1D NMR and 2D NMR
macros. Infrared spectra were recorded on a Thermo Nicolet 633
FT-IR spectrophotometer as KBr pellets. Optical rotations were
measured at room temperature at the sodium D-line using a
Perkin–Elmer 241 polarimeter and 1-dm tubes.
cost, the MM
Metropolis-Monte-Carlo search routine to allow for a quicker more
focused search of the low-energy regions of the molecule.13
3 program was embedded within a Unix-based
A
‘shaking’ routine was included in the search to prevent the search
from being trapped within a region of low energy. A 20,000-step
search found essentially all of the low-energy MM3 forms, as re-
peated searches produced essentially the same set of lowest energy
forms. In addition, a routine was written to calculate the Karplus
hydrogen–hydrogen coupling constants from the Haasnoot et al.
model.26 Transition-state structures were excluded from the con-
former population by checking for imaginary vibrational frequency
for each structure as it was found.
3.1.4. Monopotassium
To a mixture of
-[2-2H]-glucose (536 mg, 2.96 mmol) and
D
-[2-2H]-glucarate (3a)
D
sodium nitrite (ca. 5 mg) at room temperature was added concen-
trated nitric acid (1 mL) and the solution was stirred for 5 min
when an exothermic, brown/green gas generating reaction
occurred. After an additional 5 min, when gas evolution had sub-
sided, the solution was warmed to 60 °C, stirred for 2 h, and then
concentrated under reduced pressure. The residue was dissolved
in water, the solution cooled to 0 °C, the pH of the solution
adjusted to 11 with aqueous potassium hydroxide solution (45%)
and maintained above pH 10 for 1 h by addition of potassium
hydroxide solution as needed. The solution was acidified to a pH
of about 3.5 with concentrated hydrochloric acid to precipitate
3.1.1. D-Glucaric acid (2)
To a slurry of Dowex 50WX8-100 H+ form ion-exchange resin
(20.8 mL, 43.7 mmol H+) in deionized water (40 mL) was added
solid monopotassium
D-glucarate (1, 10 g, 40.3 mmol) and the
resultant slurry was stirred at room temperature for exactly
10 min. The resin was removed by gravity filtration and washed
with water (10 mL). The combined filtrate was frozen (dry ice/2-
propanol) and the water was removed by lyophilization to afford
a mixture of small crystals and an amorphous, off-white solid.
Approximately 2 mL of the amorphous solid was removed from
the flask, dissolved in a minimum amount of boiling water. The
solution was cooled to room temperature, seeded with a few crys-
tals from the flask, cooled to 4 °C and held overnight. The crystals
formed were removed from the vial and triturated with acetone
(10 mL). The acetone was carefully removed with a pipette and
the crystalline solid was again washed with acetone (2 ꢂ 10 mL).
monopotassium D
-[2-2H]-glucarate (3a). The slurry was cooled to
0 °C, stirred for 30 min and the solid formed was isolated by filtra-
tion. The solid was then washed with a minimal amount of ice
water to remove colored material, washed with acetone, and then
dried at room temperature under vacuum to give monopotassium
D
-[2-2H]-glucarate (3a, 239 mg, 23% yield) as a white powder. The
powder was used in the next step without further purification.
3.1.5.
D
-[2-2H]-Glucaric acid (2a)
The D-glucaric acid crystals obtained (398 mg) were not suitable
for X-ray structure determination, as they appeared fractured
and slightly opaque, but were useful as seed crystals.
A solution of 3a (40 mg) in D2O (2 mL) was treated with Dowex
50WX8-100 H+ form ion-exchange resin (ca. 2 mL, previously
washed three times with D2O) for 5 min, the solid materials were
removed by filtration and the 1H NMR spectra of resulting
D-
[2-2H]-glucaric acid (2a) was recorded: 1H NMR (D2O): d 4.35 (d,
1H, H5, J4,5 = 4.99 Hz), 4.13 (d, 1H, H3, J3,4 = 5.87 Hz), 3.96 (dd,
1H, H4, J4,5 = 4.99 Hz, J3,4 = 5.87 Hz). 13C NMR (150 MHz, D2O): d
176.72 (C1), 176.42 (C6), 74.07 (C4), 72.36 (C3), 72.27 (C5).
3.1.2. X-ray quality, crystalline D-glucaric acid (2)
Monopotassium
D-glucarate (1) was treated with Dowex
50WX8-100 H+ form ion-exchange resin as above and following
removal of the resin by filtration, the combined filtrate was

2556
T. T. Denton et al. / Carbohydrate Research 346 (2011) 2551–2557
3.1.6. Monopotassium
D
-[1-13C]-glucarate (3b)
3.1.10. Collection of X-ray diffraction data and solution of the
crystal structure for -glucaric acid, 2
D
-[1-13C]-Glucose (250 mg, 1.38 mmol) was oxidized according
D
to the procedure for 3a to afford 3b as a white powder (143 mg,
42% yield). The powder was used in the next step without further
purification.
A suitable crystal of 2 was coated with Paratone N oil, sus-
pended in a small fiber loop and placed in a cooled nitrogen gas
stream at 100 K on a Bruker D8 SMART 1000 CCD sealed tube diffrac-
tometer with graphic monochromated Cu K
tion. Data were measured by means of a combination of
a
(1.54178 Å) radia-
and
3.1.7. D
-[1-13C]-Glucaric acid (2b)
u
x
scans with 10-s frame exposures and 0.3° frame widths. Data col-
lection, indexing and initial cell refinements were carried out with
SMART29 software. Frame integration and final cell refinements were
A solution of 3b (143 mg) in D2O (1 mL) was protonated accord-
ing to the procedure for 3a and the NMR spectra of 2b was re-
corded: 1H NMR (D2O):
d 4.45 (d, 1H, H2, J2,3 = 3.0 Hz,
30
done with SAINT software. The final cell parameters were deter-
JC1,H2 = 5.03 Hz), 4.34 (d, 1H, H5, J4,5 = 4.96 Hz), 4.14 (ddd, 1H, H3,
J2,3 = 3.23 Hz, J3,4 = 5.00 Hz, JC1,H3 = 1.76 Hz), 4.96 (d, 1H, H-4,
J4,5 = 4.99 Hz, J3,4 = 5.59 Hz). 13C NMR (150 MHz, D2O): d 176.83
(C1), 176.54 (C6), 74.12 (C4), 72.75 and 72.36 (C2, d,
JC1,C2 = 58.34 Hz), 72.41 (C3), 72.36 (C5).
mined from least-squares refinement of 4070 reflections. The
structure was solved by direct methods and difference Fourier
techniques (SHELXTL, V5.10).31 Hydrogen atoms were found in differ-
ence maps and were refined isotropically. Scattering factors and
anomalous dispersion corrections are taken from the International
Tables for X-ray Crystallography.32 Structure solution, refinement,
graphics, and publication materials were generated with SHELXTL
V5.10 software. Crystal data and structure refinement for D-gluca-
ric acid (2) are shown in Table 3.
3.1.8. Monopotassium
D
-[6-13C]-glucarate (3c)
-[6-13C]-glucose (250, 1.38 mmol) was oxidized following the
,
D
procedure for
D
-[2-2H]-glucose to afford 3c as a white powder
(153 mg, 42% yield). The powder was used in the next step without
further purification.
Acknowledgments
3.1.9. D
-[6-13C]-Glucaric acid (2c)
Partial support for this research was from the US Department of
Agriculture (USDA-CSRESS), Grant Nos. 2005-34463-11561 and
2006-34463-16886 and is gratefully acknowledged.
A solution of 3c (20 mg) in D2O (2 mL) was protonated accord-
ing to the procedure for 3a and the NMR spectra of 2c recorded: 1H
NMR (D2O): d 4.47 (d, 1H, H2, J2,3 = 3.27 Hz), 4.35 (dd, 1H, H5,
J4,5 = 4.90 Hz, JH5,C6 = 4.12 Hz), 4.13 (dd, 1H, H3, J2,3 = 3.33 Hz,
J3,4 = 5.87 Hz), 3.96 (ddd, 1H, H4, J4,5 = 4.99 Hz, J3,4 = 5.59 Hz, H4,
JH4,C6 = 4.11 Hz). 13C NMR (150 MHz, D2O): d 176.74 (C1), 176.45
(C6), 74.09 (C4), 72.50 (C2), 72.47 and 72.08 (C5, d,
JC5,C6 = 59.47 Hz), 72.49 (C3).
Supplementary data
Details of X-ray crystallographic data for compound 2 are in-
cluded as supplementary data. Tables of atomic coordinates and
equivalent isotropic displacement parameters, bond lengths and
angles, anisotropic displacement parameters, hydrogen coordi-
nates, torsion angles, and5 hydrogen bonds of 2 have been included
as supplementary data.
Complete crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic Data Centre,
CCDC No. 798054. Copies of this information may be obtained free
of charge from the Director, Cambridge Crystallographic Centre, 12
Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1223 336033,
email: deposit@ccdc.cam.ac.uk or via: http://www.ccdc.cam.ac.uk.
COSY and HSQC spectra of 2 are also included.
Table 3
Crystal data and structure refinement for D-glucaric acid (2)
Identification code
D
-Glucaric acid
Empirical formula
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
C6H10O8
173(2) K
1.54178 Å
Monoclinic
P21
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.08.016.
a = 6.7589(4) Å
b = 8.6385(6) Å
c = 7.2420(4) Å
421.89(5) Å3
2
a
= 90°
b = 93.832(2)°
= 90°
c
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
h range for data collection 6.12–66.11°
Index ranges
References
1.654 Mg/m3
1.412 mmꢀ1
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0.36 mm ꢂ 0.33 mm ꢂ 0.21 mm
ꢀ7 6 h 6 7, ꢀ8 6 k 6 8,
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