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Research Article

Received: 27 February 2008 Revised: 9 April 2008 Accepted: 10 April 2008 Published online in Wiley Interscience: 9 May 2008

(www.interscience.com) DOI 10.1002/mrc.2246

15
N– 1H and 15N– 13C couplings in 15N-enriched
dihydroxamic acids
Jan Schramla∗ and Petr Ciglerb
15 N-enriched dihydroxamic acids (HONHCO(CH ) CONHOH, n
2 n = 0, 1, and 2) were prepared and their spectra NMR (1 H, 13 C, 15 N)
recorded in dimethyl sulfoxide (DMSO) solutions with the aim of determining 15 N coupling constants (15 N– 1 H and 15 N– 13 C).
The results supplement chemical shifts published earlier and yield additional support to the structural conclusions derived
from other NMR parameters. Notably, no trace of hydroximic structures could be found in the 15 N NMR spectra of these acids.
The values of 15 N– 13 C coupling constants backed by theoretical calculations support the assignments made earlier for all
of the major conformers and for the minor conformer of succinohydroxamic acid. The enrichment revealed that the minor
component of malonodihydroxamic acid solution previously considered to be the ZE conformer is in fact the monohydroxamic
acid (HOOC–CH2 –CO–NH–OH). Copyright  c 2008 John Wiley & Sons, Ltd.

Keywords: 15 N NMR; 13 C NMR; 1 H NMR; coupling constants; J(15 N– 13 C); J(15 N– 1 H); dihydroxamic acids; E/Z conformers; quantum
chemistry

Introduction Z and E isomers of acetohydroxamic acid (102.2 and 100.0 Hz


in dimethyl sulfoxide(DMSO)-d6 , respectively) and determined
Several years ago, we succeeded in determining the structures of also 1 J(15 N– 13 C) and 2 J(15 N– 13 C) in the Z isomer (12 and 12 Hz,
dihydroxamic acids (HONHCO(CH2 )n CONHOH, with n = 0, 1, 2, 3, sign not determined). Similar absolute values of these couplings
4, and 6) in solution.[1] Using model data from the literature[2,3] and were found in benzohydroxamic acid 1 J(15 N– 1 H) = 102.2 Hz,
experimentally determined 1 H, 13 C, and 15 N chemical shifts (all in 1 J(15 N– 13 C) = 13.7 Hz, and 2 J(15 N– 13 C) = 11.2 Hz but in
natural abundance), we were able to conclude that the hydroxamic its disilylderivatives, which have Z-benzohydroximic structure,
groups assume Z-Z and Z-E combinations of conformers, with the 1 J(15 N– 13 C) coupling was estimated to be <0.6 Hz while
the former prevailing. One exception is oxalodihydroxamic acid, 2 J(15 N– 13 C) = 10.6 Hz, and 3 J(15 N– 13 C) = 3 Hz.[8]
which assumes only the Z-Z conformer in solution as in the
solid state.[4,5] In describing the structure of hydroxamic groups,
we follow the conventions described in the literature.[1 – 3,6] The Experimental
symbols E and Z denote the configuration or conformation
around the C–N bond irrespective of the degree of double bond Preparation of dihydroxamic acids
character. In the E conformation the dihedral angle O C–N–O
The dihydroxamic acids were prepared using a modification of
(α) is 180◦ ± 30◦ (Scheme 1), while in the Z conformation α is
the procedure reported by Hurd and Botteron.[9] To 0.50 mmol
0◦ ±30◦ . The arrangement around the N–O bond is specified by the
of dicarboxylic acid ethylester, 1.2 ml of 1.5 M hydroxylamine
dihedral angle H–N–O–H (β). In relation to the molecular carbon
solution (98% 15 N enriched, Chemotrade, Leipzig) in dry MeOH
skeleton, the orientation of the hydroxamic group is described
(1.5 mmol) was added in one portion under an argon atmosphere.
by the dihedral angle γ , which refers to either the dihedral
The reaction mixture was stirred and cooled to 0 ◦ C. A solution of
angle O C–C O (in oxalodihydroxamic acid) or to the angle
sodium methoxide (56 mg; 0.55 mol) in 1 ml of dry methanol was
O C–C–C (in higher homologues). Conventional descriptors (ap,
added dropwise, and the reaction mixture was stirred overnight
sp, ac, and sc) are sometimes used to specify the values of these
at room temperature. In the case of the oxalic and malonic
dihedral angles.[7]
acid derivatives, the resulting white disodium dihydroxamates
Some of these dihydroxamic acids have proven to have remark-
were filtered and washed with 0.5 ml of dry methanol. The free
able enzyme inhibition activity (to be published elsewhere). In
dihydroxamic acids were liberated from aqueous solutions of
the course of the inhibition studies it became necessary to pre-
pare acids enriched in the 15 N isotope. The enriched compounds
provide the opportunity to measure additional NMR parameters

(such as 15 N coupling constants) and to independently test the Correspondence to: Jan Schraml, Institute of Chemical Process Fundamentals
of the ASCR, v.v.i., Rozvojová 135, 165 02 Prague 6, Czech Republic.
previous structural conclusions. This is particularly valuable in the
E-mail: schraml@icpf.cas.cz
case of oxalodihydroxamic acid, which has very few measurable
NMR parameters. The results of these measurements and back- a Institute of Chemical Process Fundamentals of the ASCR, v. v. i., Rozvojová 135,
up theoretical calculations are presented here. Unfortunately, 16502 Prague 6, Czech Republic
only very few data are available for comparison of these cou- b Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and
plings in hydroxamic structures with unique structural flexibility. Biochemistry of the ASCR, v. v. i., Flemingovo n. 2, 166 10 Prague 6, Czech
748

Brown et al.[2] determined and assigned 1 J(15 N– 1 H) couplings in Republic

Magn. Reson. Chem. 2008, 46, 748–755 Copyright 


c 2008 John Wiley & Sons, Ltd.
15 N couplings in dihydroxamic acids

O OH O (O or C)
O OH H H
N α β γ
R H C N C
Z conformer
H R C N
O H
N α: O=C-N-O β: H-N-O-H γ: O=C-C=O or O=C-C-C
R OH

E conformer dihedral angles α, β, and γ

Scheme 1. Confomers of hydroxamic acids and their dihedral angles (shown with negative values of α, β, and γ ).

dihydroxamates on a Dowex (Lewatit CNP 80) column (weakly time of 1.0 s, and relaxation delays of 10–60 s. The inverse-
acidic catex, H+ form). Lyophilization of these eluates provided gated broadband 1 H decoupling mode was used to suppress
the final dihydroxamic acids in good yields (80–90%). The the negative NOE; in some cases monoresonance spectra were
disodium succinodihydroxamate was partially soluble in methanol; also measured.
thus, the reaction mixture was diluted with water and directly For the 2D correlation experiments (gHMBC and gHSQC) each
loaded onto the column. The eluate was lyophilized, and the of the 1 H and 13 C NMR spectral widths was broken into two
sticky residue was triturated with acetone to yield a fine nonoverlapping regions (1 H: 1.5–4.0 and 8.5–11.0 ppm and 13 C:
powder of succinodihydroxamic acid. Before measurements, the 24–30 and 166–178 ppm). The FIDs were recorded without carbon
dihydroxamic acids were dried in vacuo over P2 O5 for at least 1 day. decoupling with a long acquisition time of up to 2 s in order
The purity and identity of the acids were tested by NMR spec- to produce sufficiently accurate values of coupling constants.
troscopy. A noticeable impurity was found in succinodihydroxamic Typically, 512 increments were used and the data were zero-filled
acid, and it was identified by MS (FAB and EI) as 1-hydroxy- to 2K. Various estimates of 13 C– 1 H coupling constants were used
2,5-pyrrolidinedione with the following NMR parameters: 13 C: for optimization of polarization transfer delays.
δ(C O) = 172.94, 1 J(15 N– 13 C) = 12.5 Hz, δ(CH2) = 25.41,
2 J(15 N–C– 13 C) = 9.9 Hz; 1 H: δ(CH2) = 2.58; 15 N: δ = −166.9.
Calculations
This by-product was also observed in our earlier work.[1] The minor
component in the solution of succinodihydroxamic acid consti- The geometry of all compounds was optimized with the DFT
tuted about 8% of the major one. Owing to low concentration and B3LYP method and the atomic basis set 6-31G∗∗ without any
lack of structural symmetry, it was not possible to determine all association or solvent effects included. The shielding constants
the possible NMR parameters for the minor component. were calculated using the gauge-independent atomic orbital
(GIAO) method,[11] indirect NMR spin–spin coupling constants
were calculated using the coupled perturbed DFT method[12 – 14]
NMR measurements with the B3LYP functional. Total coupling constants between
nuclei A and B separated by n bonds n J(A,B) were calculated
The NMR spectra of the acids were measured in dry DMSO-d6 ; the as a sum of the diamagnetic spin orbit, paramagnetic spin
concentration of the compounds varied between 5–10 mg/ml. orbit, Fermi–contact, and spin–dipolar terms.[15,16] The atomic
The lines of the solvent were used as references (δ(1 H) = 2.500; basis used for the NMR calculation was the basis usually called
δ(13 C) = 39.70). 15 N chemical shifts were referenced to external IgloIII.[17] All calculations were made with the G03 program
neat nitromethane (δ(15 N) = 0.00) in a capillary; no susceptibility package.[18] A referee suggested that the 15 N coupling constant,
correction was applied. calculated using Dunning’s correlation consistent basis set[19]
The 1 H, 13 C, and 15 N NMR spectra were measured on a Varian augmented with diffuse functions (aug-cc-pVDZ) would reproduce
INOVA 500 spectrometer (operating at 499.871, 125.695, and these couplings better as found in substituted pyrroles.[20] The
50.667 MHz, respectively). In all cases, including the gHMBC[10] values of large 1 J(15 N– 1 H) couplings calculated in this base
and gHSQC[10] pulse sequences, the standard software (vnmr indeed fit experimental values better by a few percent (in
6.1C) was used. The spectra were recorded at 25 ◦ C using either low-energy conformers) but the agreement in other (smaller)
an indirect detection probe (1 H and 2D spectra) or a broad- couplings involving 15 N and 1 H nuclei worsened considerably
band switchable probe. Both were 5 mm probes equipped with (e.g. 1 J(15 N– 13 C) and 2 J(13 C–N– 1 H) by about 100 and 20%,
z-gradient coils. The 1 H NMR spectra were measured with an respectively).
acquisition time of 4 s, relaxation delay was 2–5 s in a spectral
width of 8 kHz, no FID weighting was applied, and data were
zero-filled to 128 K. Selectively, 15 N decoupled 1 H NMR spectra Results and Discussion
(1 H{15 N}) were recorded under the same conditions using 15 N
Molecular geometry optimization
coherent decoupling with a decoupling field of approximately
100 Hz. The 13 C NMR spectra were measured using a spectral Since the results of 15 N NMR measurements of the enriched
width of 16 000 Hz. WALTZ decoupling was applied both during compounds did not reveal the presence of hydroximic structures
acquisition (2 s) and relaxation delay (2–5 s). Zero-filling to 256 K (with δ(15 N) around −80 ppm[21] ), the computational efforts
and 0.5 Hz line broadening were used in data processing. 15 N FIDs were directed only to hydroxamic structures. The calculated
749

were acquired with a spectral width of 30 kHz, an acquisition structures are denoted here by the capital bold letters O, M,

Magn. Reson. Chem. 2008, 46, 748–755 Copyright 


c 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc
J. Schraml and P. Cigler

Table 1. NMR parameters calculated for the three most stable conformers of oxalodihydroxamic acid compared to the experimentally determined
valuesa

Conformer O-1-ZZ O-2-ZEb O-3-EE Experimentalc

Relative energy [kJ/mole] 0.00 7.04 58.94


Dihedral angles α −6.4 −0.1 −179.2 154.2 –
[◦ ]d
β 169.2 179.9 179.9 −110.3 –
γ 174.8 179.9 179.9 −72.9 –
Chemical shifts δ(13 C O) 162.71 166.59 159.26 182.51 156.91
[ppm]
δ(15 N–H) −225.42 −221.02 −220.76 −231.37 −212.59
δ(1 H–N) 9.37 9.69 7.28 6.93 11.462
δ(1 H–O) 7.05 7.03 11.28 4.26 9.150
RMSDe 8.31 6.16f 18.73 –
Coupling 1 J(15 N– 1 H) −112.1 −115.8 −106.4 −86.0 −106.3
constants [Hz]
1 J(15 N– 13 C) −19.1 −21.6 −19.3 −4.7 −17.1
2 J(15 N–C– 13 C) −11.9 −9.6 −11.1 −9.2 12.0
2 J(13 C–N– 1 H) +10.1 +10.5 +6.0 +1.4 11
RMSDg 3.58 3.85f 14.90 –
a The spin–spin coupling constants J are in Hz; the chemical shifts are given as ppm (δ scale).
b The first column refers to the parameters for the Z part and the second column to the E part. The 2 J(15 N–C– 13 C) couplings are listed according to
the position of the 15 N nucleus.
c The signs of measured couplings are indicated only when experimentally determined.
d
For definitions of dihedral angles see Scheme 1.
e Root mean square deviation between calculated and experimental chemical shifts, in ppm.
f Average.
g Root mean square deviation between calculated and experimental coupling constants, in Hz.

or S (standing for oxalo-, malono-, or succinodihydroxamic acid differences are small and that they were calculated for molecules
structures, respectively) followed through by a conformer number in the gas phase without considering any association. The most
(starting with 1 for the most stable structure) and by a letter stable structure, M-1-EE, has EE geometry and is stabilized
combination indicating the basic conformation of the hydroxamic by two intramolecular hydrogen bonds. The two hydroxamic
groups (E or Z). For example, S-2-ZZ indicates the second most groups are almost magnetically equivalent. The next stable EE
stable conformer of succinodihydroxamic acid, which has both structure (M-4-EE) is 3.65 kJ/mole higher in energy, it is no longer
hydroxamic groups in the Z conformation. symmetrical, and the hydrogen bond involves only one hydroxyl
In the case of oxalodihydroxamic acid, geometry optimization proton. None of the low-energy structures with ZZ geometry
starting from initial estimates close to idealized combinations of experiences any intramolecular hydrogen bonding, and the two
hydroxamic groups in E and Z conformations without symmetry hydroxamic groups are distinctly nonequivalent.
restrictions produced stable geometries for all three possible Succinodihydroxamic acid was treated similarly to malonodi-
combinations (ZZ, ZE, and EE). The most stable ZZ conformer hydroxamic acid. All three possible combinations of hydroxamic
(denoted as O-1-ZZ) has a structure that is slightly different from group conformations are among the four lowest energy con-
the usual idealized picture; it is almost planar (Table 1). The ZE formers listed in Table 3, and none of them is stabilized by an
conformer (O-2-ZE), which is higher in energy by 7.04 kJ/mole, is intramolecular hydrogen bond. In two conformers, S-1-ZZ and
planar, stabilized by one hydrogen bond between the hydroxyl of S-4-EE, the hydroxamic groups are equivalent. The two nonequiv-
the E group and the carbonyl oxygen of the Z group. Finally, the alent groups in the S-2-ZZ conformer have some calculated NMR
least stable EE conformer (O-3-EE) is nonplanar. parameters that are very close; they also differ very little from
In the case of malonodihydroxamic acid, the results of geometry those calculated for the S-1-ZZ conformer.
optimization depend strongly on the initial guess. In order not to The two hydroxamic groups of succinodihydroxamic acid are
miss the true energy minimum, we tried all structures usually isolated, and so the calculated energy order of conformers agrees
considered for a hydroxamic group (i.e. E and Z, ap and sp for with that assumed previously[1] in assigning the structure of this
dihedral angle β) for each of the two groups independently as acid on the basis of data reported for monohydroxamic acids[2]
well as the orientation of each with respect to the carbon skeleton and glutarodihydroxamic acid.[3]
(ap, sp, sc, and ac for dihedral angle γ ). Optimization starting However, while the energy differences between the ZZ and
from any EZ combination led either to an EE or a ZZ combination. ZE conformers of succinodihydroxamic acid are small, the
Optimization of ZZ structures led to dihedral angles β close to difference between those of oxalodihydroxamic acid is large,
160◦ , while in the E structures the angles are between 115◦ and clearly favoring the O-1-ZZ conformer. Malonodihydroxamic acid
140◦ . The relative energies and geometry parameters for the five appears anomalous in this respect, not only due to the absence of
most stable structures are given in Table 2 together with the a ZE conformer among the calculated stable conformers but also
750

calculated NMR parameters. It should be noted that the energy due to the fact that the M-2-ZZ, M-3-ZZ, and M-4-EE conformers

www.interscience.wiley.com/journal/mrc Copyright 
c 2008 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2008, 46, 748–755
15 N couplings in dihydroxamic acids

Table 2. NMR parameters calculated for the five most stable conformers of malonodihydroxamic acida

Confomer M-1-EE M-2-ZZ M-3-ZZ M-4-EE M-5-ZZ

Relative energy [kJ/mole] 0.00 2.91 3.10 3.65 5.08


Dihedral angles α 176.6 176.2 8.5 11.3 10.0 −11.3 −163.1 −165.1 −9.8 −7.6
[◦ ]b
β 116.3 115.6 −156.5 −152.7 −161.2 153.0 111.0 135.4 160.7 162.9
γ −92.2 −92.1 −47.3 −135.4 45.9 138.0 −74.9 −109.8 −46.4 −163.3
Chemical shift δ(13 C O) 181.25 181.18 176.85 173.52 176.11 173.25 188.73 180.79 176.47 168.75
[ppm]
δ(15 N–H) −228.73 −228.62 −227.59 −224.48 −227.01 −224.51 −229.16 −229.91 −225.63 −223.69
δ(1 H–N) 7.41 7.41 7.70 9.97 7.66 10.11 7.18 6.92 10.20 7.68
δ(1 H–O) 7.76 7.76 7.48 7.31 7.48 7.34 5.02 8.76 7.56 7.58
δ(1 H–C) 3.99 3.99 3.54 2.79 3.99 3.99 3.93 4.00 3.32 2.82
δ(13 CH2 ) 44.25 42.33 41.87 42.46 40.93
Coupling 1 J(15 N– 1 H) −107.6 −107.5 −102.3 −100.8 −101.2 −102.9 −96.9 −99.4 −109.4 −102.5
constants [Hz]
1 15
J( N– 13 C) −16.6 −16.6 −11.5 −9.6 −9.8 −10.7 −10.5 −11.2 −14.0 −11.5
2 J(15 N–C– 13 C) −7.7 −7.7 −10.0 −8.4 −8.4 −10.7 −7.4 −8.4 −9.1 −10.8
3 J(15 N–C–C– 1 H) −1.6 +0.2 −0.4 −1.2 −1.0 −2.6 −2.3 +0.2 −0.9 −2.9
+0.2 −1.6 −0.8 −3.4 −3.4 −1.3 +0.2 −1.8 −1.1 −1.4
3 J(15 N–C–C– 13 C) −0.3 −0.3 −3.3 +0.6 +0.7 −1.1 +0.1 +0.1 +0.9 −1.0
2 J(13 C–N– 1 H) +9.2 +9.2 +9.6 +6.6 +6.7 +9.7 +4.7 +5.9 +9.7 +9.6
2 13
J( C–C– 1 H) −6.4 −5.0 −7.9 −7.3 −7.3 −6.6 −7.1 −6.8 −7.7 −5.7
−5.00 −6.46 −3.0 −5.9 −6.10 −3.32 −4.4 −5.71 −6.66 −3.00
1 J(13 C– 1 H) +137.15 +128.39 +127.26 +130.00 +127.11
+137.09 +130.2 +130.23 +136.39 +128.80
a The spin–spin coupling constants J are given in Hz; the chemical shifts are in δ scale (ppm units). The data in each column represent one of the

hydroxamic groups; E and Z groups can be distinguished by their dihedral angles α.


b For the definitions of dihedral angles see Scheme 1.

have similar energies (2.9–3.7 kJ/mole) relative to the M-1-EE are further split into not fully resolved doublets by a long-range
conformer. coupling 4 J(15 N–C–C–N– 1 H) of 1.2 Hz). The line at δ = 9.150 can
The reported relative energies of conformers should be therefore be indirectly assigned to the H–O proton. Analogously,
considered with caution. A number of recent computa- the doublet (J = 106.3 Hz) observed in 15 N NMR spectra measured
tional studies of much simpler formohydroxamic[7,22 – 24] and without 1 H decoupling collapses into a singlet (δ = −212.59)
acetohydroxamic[23,25] acids reached conclusions that varied with when proton decoupled. The 13 C NMR spectrum exhibited four
the computational method and basis set employed and, natu- lines (in a 1 : 1 : 1 : 1 ratio) due to 15 N couplings (17.1 and 12.0 Hz)
rally, are affected by hydration or ionization. To the best of our to two magnetically nonequivalent 15 N nuclei. A gHMBC 1 H– 13 C
knowledge, among the dihydroxamic acids only glutarodihydrox- correlation experiment (Fig. 1) established that the larger of the
amic acid has been similarly treated[3] with the result that the two splittings is due to the same nitrogen nucleus as the one-bond
15
ZZ conformer is 15.1 kJ/mole less stable than the EE conformer. N– 1 H coupling (106.3 Hz), and it follows from the positive tilt of
Hydration by one molecule of water reduces this difference to the reduced coupling pattern (due to passive 15 N couplings) that
4.5 kJ/mole, but addition of another water molecule renders the the two one-bond couplings (15 N– 13 C and 15 N– 1 H) have the same
dihydrate of the ZZ conformer 5.2 kJ/mole more stable than that sign. This finding is in agreement with the literature,[26] which lists
of EE conformer. Unfortunately, forcing the studied molecules into both signs as negative. The experimental results are summarized
the ZZ conformation by hydration is not applicable to these studies in Table 1 and compared with the results of calculations for the
as it would effectively decouple the most interesting 1 J(15 N– 1 H) three most stable conformers (in the gas phase). On the basis of
coupling by proton exchange. comparison with other data, we deduced[1] that the conformer
present in solution has a ZZ arrangement. The present calculations
show that the EE conformer is energetically unfavorable, and its
NMR spectra and calculated parameters
calculated NMR parameters differ most from those observed. The
In agreement with our previous study, all the measured (1 H, 13 C, ZE conformer (planar), with two nonequivalent groups, is unlikely
and 15 N) NMR spectra of oxalodihydroxamic acid confirm the to contribute significantly to possible exchange averaging of the
presence of one compound only, and all chemical shifts agree NMR parameters. Despite its stabilization by one hydrogen bond,
within experimental error with those reported earlier.[1] Doublet it is still less favored than the ZZ conformer by 7.04 kJ/mole.
splitting (106.3 Hz) of the proton line at δ = 11.462 was proven to The spectra of malonodihydroxamic acid indicate the presence
be due to 15 N coupling by heteronuclear decoupling experiments, of two compounds in a ratio of approximately 1 : 12; previously,
thus confirming the previous assignment of this 1 H chemical shift the major component was assigned to the ZZ conformer and
751

to the H–N proton[1] (In very dry solutions, the doublet lines the minor to the ZE conformer of malonodihydroxamic acid.[1]

Magn. Reson. Chem. 2008, 46, 748–755 Copyright 


c 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc
J. Schraml and P. Cigler

Table 3. NMR parameters calculated for the four most stable conformers of succinodihydroxamic acid compared to experimentally determined
valuesa

Confomer S-1-ZZ S-2-ZZ S-3-ZE S-4-EE Experimentalb

Relative energy [kJ/mole] 0.00 0.64 1.95 7.17 Major Minorc


Dihedral angles α 12.70 −10.1 −12.5 −13.0 158.0 157.5 –
[◦ ]d
β 152.60 151.0 153.1 150.6 −111.1 112.6 –
γ −41.50 40.5 −43.4 −39.3 −1.7 5.6 –
C–C–C–C 180.0 177.7 167.0 164.9 –
Chemical shifts δ(13 C O) 181.53 181.53 181.32 183.71 192.09 192.94 168.33 168.88/174.92
[ppm]
δ(15 N–H) −227.22 −227.87 −227.15 −227.21 −227.43 −227.14 −215.97 −215.97/−214.52
δ(1 H–N) 7.48 7.51 7.49 7.37 6.91 6.88 10.435 10.441/9.788
δ(1 H–O) 7.55 7.59 7.56 7.60 4.46 4.48 8.703 8.712/9.150
δ(1 H–C) 2.76 2.72 2.69 3.38 2.06 3.03 2.178 2.178/2.178
2.19 2.34 2.12 2.88 2.76 3.02
δ(13 CH2 ) 33.34 33.46 32.99 31.49 33.53 30.97 28.00 26.66/26.92
RMSDe 7.56 7.58f 11.17g,h 6.70g,h 11.36 – –
Coupling 1 J(15 N– 1 H) −95.5 −96.6 −95.9 −93.6 −88.2 −87.8 −102.4 102.3/100.4
constants [Hz]
1 J(15 N– 13 C) −7.2 −7.8 −7.4 −6.3 −5.8 −5.2 −12.1 12/15
2 15
J( N–C– 13 C) −11.4 −11.4 −11.4 −10.9 −8.2 −8.0 11.8 11/5
3 J(15 N–C–C– 1 H) −1.7 −0.6 −1.6 −1.8 +0.1 +0.1 1
−3.1 −0.1 −3.2 −3.1 0.0 +0.1
2 J(13 C–N– 1 H) +7.4 +7.9 +7.5 +6.9 +2.1 +1.9 10.6
1 J(13 C– 1 H) +129.3 +128.5 +129.7 +126.0 +127.3 +127.7 129.2
+125.1 +126.7 +124.7 +126.9 +127.0 +125.2
RMSDi 4.06 3.59f 11.05gj 8.85gj 7.53 – –
a
The spin–spin coupling constants J are given in Hz; the chemical shifts are given in ppm. The couplings are listed according to the position of 15 N
nucleus.
b The sign of measured couplings is indicated only when experimentally determined. The estimated error is ±1 for the last digit indicated; the error

varies with the method used for determination.


c Some 13 C NMR lines are broad, leading to values of lower accuracy. Assignment of the chemical shifts to the two hydroxamic groups follows that

reported earlier.[1] The first value is assumed to be due to the Z conformer and the second to the E conformer; assignment of the coupling constants
follows from that of the chemical shifts.
d
For the definitions of dihedral angles see Scheme 1.
e Root mean square deviation between experimental and calculated chemical shifts, in ppm.
f RMSD for the average.
g RMSD for the minor product.
h RMSD of the average relative to the major conformer 9.61.
I Root mean square deviation between experimental and calculated coupling constants, in Hz.
j RMSD of the average relative to the major conformer 5.88.

The chemical shifts of the major component measured here same sign in malonodihydroxamic acid. However, no three-bond
are consistently somewhat smaller (13 C by 0.3–0.5 ppm, 1 H by 3 J(15 N–C–C– 13 C) coupling was observed. According to the root
0.04 ppm) than observed previously. The same differences are mean square deviations (RMSD), the values calculated for the M-5-
observed in the chemical shifts of the minor component. The ZZ conformer fit the chemical shifts best; the fit is worst for the two
15 N isotopic enrichment leads to a triplet splitting of the 1 H NMR EE conformers. Similarly, the fits of the coupling constants of the ZZ
line of the CH2 group. Such splitting (J = 1.5 Hz) is observed on conformers are somewhat better than those for EE conformers, but
the line of the major component only. The corresponding line the differences do not provide sufficient guidance for conformer
of the minor component is split only into a doublet, indicating assignment.
that the minor component is not the dihydroxamic but rather The spectra of succinodihydroxamic acid indicate the presence
the monohydroxamic acid and that the previous assignment was of two isomers in addition to the side product described in the
erroneous. Since the two hydroxamic groups are equivalent in Experimental Part. The chemical shifts of the major component
the measured NMR spectra of malonodihydroxamic acid, Table 4 agree well with the values reported earlier (13 C chemical shifts
compares the experimental results for the major conformer with are 0.03–0.13 ppm smaller and 1 H shifts are 0.01–0.04 ppm larger
computational estimates calculated as the arithmetic means than the literature values) and identified as the ZZ conformer.[1]
of the values obtained for the corresponding nuclei in the Again, the tilt in the gHMBC (1 H– 13 C) correlation confirms that
1 15
two groups (Table 2) of a given conformer. An experiment J( N– 1 H) and 1 J(15 N– 13 C) have the same sign, which is according
similar to that depicted in Fig. 1 indicates that the two one- to the literature,[26] negative. Heteronuclear gHSQC correlation
752

bond couplings, 1 J(15 N– 13 C) and 1 J(15 N– 1 H), also have the revealed that the 3 J(1 H–C–C– 1 H) coupling is 7.8 Hz (sign not

www.interscience.wiley.com/journal/mrc Copyright 
c 2008 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2008, 46, 748–755
15 N couplings in dihydroxamic acids

Figure 1. A gHMBC 2D 13 C– 1 H correlation spectrum of oxalodihydroxamic acid, showing the correlation between NH protons and C O carbons. The
spectrum on the top is from an independently measured 1D 13 C NMR spectrum. As indicated, the large splittings along the F2 and F1 axes are due to
1 15
J( N– 1 H) and 1 J(15 N– 13 C) couplings, respectively. The smaller splitting along the F1 axis is due to 2 J(15 N–C– 13 C) coupling with the remote 15 N nucleus,
the spin state of which is independent of the status of the directly attached nitrogen. The positive tilt indicates that the two one-bond couplings have
the same sign; the sign of the two-bond coupling remains undetermined.

Table 4. Malondihydroxamic acid. Experimental NMR parameters compared to the average calculated valuesa

Conformer Parameter M-1-EE M-2-ZZ M-3-ZZ M-4-EE M-5-ZZ Exptl.

Chemical shifts δ(13 C O) 181.21 175.18 174.68 184.76 172.61 163.52


[ppm]
δ(15 N–H) −228.68 −226.04 −225.76 −229.53 −224.66 −213.71
δ(1 H–N) 7.41 8.83 8.88 7.05 8.94 10.494
δ(1 H–O) 7.76 7.39 7.41 6.89 7.57 8.897
δ(1 H–C) 3.99 3.17 3.99 3.97 3.07 2.751
δ(13 CH2 ) 44.25 42.33 41.87 42.46 40.93 38.39
RMSDb 9.90 7.21 6.97 11.10 6.00 –
Coupling 1 J(15 N– 1 H) −107.57 −101.54 −102.06 −98.20 −105.96 −103.3
constants [Hz]
1
J(15 N– 13 C) −16.62 −10.58 −10.26 −10.87 −12.77 −13.5
2 J(15 N–C– 13 C) −7.71 −9.20 −9.55 −7.88 −9.94 11.6
3 J(15 N–C–C– 1 H) −0.71 −1.43 −2.09 −0.93 −1.56 −1.5
3 J(15 N–C–C– 13 C) −0.27 −1.40 −0.23 +0.09 −0.06 1.0
2 J(13 C–N– 1 H) +9.18 +8.12 +8.21 +5.30 +9.63 11.6
2
J(13 C–C– 1 H) −5.72 −6.02 −5.85 −5.99 −5.76 6.4
1 J(13 C– 1 H) +137.12 +129.30 +128.75 +133.20 +127.96 129.8
RMSDc 3.61 1.94 1.94 3.53 1.54 –
a The spin–spin coupling constants J are in Hz; the chemical shifts are in ppm units. The sign of measured couplings is indicated only when
experimentally determined.
b Root mean square deviation of calculated chemical shifts from the respective experimental values, in ppm.
c
Root mean square deviation of calculated coupling constants from the respective experimental values, in Hz.

determined) and that the 2 J(15 N–C– 13 C) and 3 J(15 N–C–C– 1 H) ZZ conformers agree best with the experimental results; the
couplings also have the same sign, although the value of the latter difference between the two is insignificant. The predicted NMR
coupling could not be determined with sufficient accuracy. While parameters for the two less stable conformers, S-3-ZE (average)
3 J(15 N–C–C– 1 H) couplings of both signs are found, 2 J(15 N–C– 13 C) and S-4-EE, differ slightly more from the experimental values.
couplings are generally assumed to be positive.[26] Hence, we However, the increase in the RMSD is almost entirely due the
assigned a positive sign to the 3 J(15 N–C–C– 1 H) couplings. The 13
C chemical shifts (in all cases the calculated shifts exceed those
753

values of NMR parameters calculated for the S-1-ZZ and S-2- found experimentally).

Magn. Reson. Chem. 2008, 46, 748–755 Copyright 


c 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc
J. Schraml and P. Cigler

The chemical shifts of the minor product (approx. 10% of of the NH proton is larger than that of the OH proton. The
the major one) agree with those reported earlier and assigned theoretical calculations on the level used describe the major
as the ZE conformer.[1] In this case, the lines are somewhat trends in the chemical shifts of 1 H, 13 C, and 15 N nuclei and their
broad, which did not allow precise determination of the coupling coupling constants, but they are not yet accurate enough to allow
constants. In Table 3 the experimental parameters are compared unmistakable conformer assignment.
with those calculated for the S-3-ZE conformer. The previously
published assignment of the 13 C NMR lines[1] agrees with the
Acknowledgements
order predicted by the calculations; assignment of the coupling
constant follows from the assigned chemical shifts. The agreement This work was supported in part by a grant from the Ministry
between calculated and experimentally determined values is less of Education (MSMT) of the Czech Republic within the Program
satisfactory than in the major product. 1M6138896301 ‘Research Centre for New Antivirals and Antineo-
Considering that the calculated values include the chemical plastics’ to P.C.
shifts of three different nuclei (with 13 C chemical shifts covering The authors gratefully acknowledge the financial support
a range of 140 ppm) and coupling constants between these provided by the Grant Agency of the Academy of Sciences (grant
nuclei over one and two bonds, the overall fit is remarkably no. IAA400720706) and by the Czech Science Foundation (grant
good. In detail, it still leaves much to be desired. The calculated no. 203/06/0738).
13
C chemical shifts are larger than those found experimentally
by 4–12 ppm; the largest deviations (12 ppm) occur for the
carbonyl carbons of malono- and succionodihydroxamic acids.
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