Hydrolytic degradation of N,N′-ethylenedimaleimide: Crystal structures of key intermediates and proposed mechanisms

Article abstract of DOI:10.1016/j.molstruc.2016.07.015

Maleimide groups are used extensively in bioconjugation reactions, but limited mechanistic studies are available regarding their hydrolysis reactions. In this paper, five single-crystal structures related with the reaction of four-step hydrolytic degradation of N,N′-ethylenedimaleimide have been investigated. On the basis of experimental results, the reaction mechanisms without or with water catalysis are proposed, which could provide some enlightenment into the study of similar hydrolytic degradations.

Full text of DOI:10.1016/j.molstruc.2016.07.015

Accepted Manuscript
Hydrolytic degradation of N,N'-ethylenedimaleimide: Crystal structures of key
intermediates and proposed mechanisms
Xue-Jie Tan, Shuang-Shuang Cheng, Yan Shi, Dian-Xiang Xing, Yun Liu, Hui Li,
Wen-Quan Feng, Jian-Bo Yang
PII:
S0022-2860(16)30686-X
10.1016/j.molstruc.2016.07.015
MOLSTR 22718
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 1 March 2016
Revised Date: 3 July 2016
Accepted Date: 8 July 2016
Please cite this article as: X.-J. Tan, S.-S. Cheng, Y. Shi, D.-X. Xing, Y. Liu, H. Li, W.-Q. Feng, J.-B.
Yang, Hydrolytic degradation of N,N'-ethylenedimaleimide: Crystal structures of key intermediates and
proposed mechanisms, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.07.015.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Five single-crystal structures related with the hydrolytic degradation of N,N'-ethylenedimaleimide
have been investigated and the mechanisms without or with water catalysis have been proposed.

ACCEPTED MANUSCRIPT
Hydrolytic degradation of N,N'-ethylenedimaleimide: crystal
structures of key intermediates and proposed mechanisms
Xue-Jie Tan
, Shuang-Shuang Cheng, Yan Shi, Dian-Xiang Xing, Yun Liu, Hui Li, Wen-Quan Feng, Jian-Bo Yang
School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan, Shandong Province,
250353, P. R. China.
Abstract
Maleimide groups are used extensively in bioconjugation reactions, but limited
mechanistic studies are available regarding their hydrolysis reactions. In this paper, five
single-crystal structures related with the reaction of four-step hydrolytic degradation of N,
N’-ethylenedimaleimide have been investigated. On the basis of experimental results, the reaction
mechanisms without or with water catalysis are proposed, which could provide some
enlightenment into the study of similar hydrolytic degradations.
1. Introduction
Maleimides are an important class of substrates for biological, pharmacological and chemical
applications.[1 and the references therein] The most important aspects are its ability to react
through Michael addition with nucleophilic functional groups, especially amine and thiols, as well
as its propensity to undergo Diels–Alder cycloadditions, which have led to its use for site-specific
biomolecule ligation and modification. [2 and the references therein].On August 19th, 2011,
brentuximab vedotin (Adcetris) received FDA approval for relapsed Hodgkin lymphoma and
anaplastic large cell lymphoma,[3] On February 22nd, 2013, another antibody-drug conjugate
(ADC) ado-trastuzumab emtansine (Kadcyla) also received FDA approval for patients with
HER2+ metastatic breast cancer.[4] Both of these therapeutics share common structural features
found in most ADCs: a thiosuccinimide linkage, formed through the reaction of thiol and alkyl
maleimide functionalities[5]. However, it has become apparent that thiosuccinimide formation is
reversible, so in the bloodstream, the variable stability of ADCs formed with traditional N-alkyl
maleimides usually leads to loss of drug and the released drug may show toxicities [6]. The
thiol–maleimide adduct undergo two competing reactions: hydrolysis of the thiosuccinimide ring
and cleavage of the thioether bond. The former can greatly retard the latter and help to develop a
stable formulation for ADC product [6(b), 7].
As is known, the hydrolysis of N-alkylmaleimide ring has two steps: the first hydrolysis
product is N-alkylmaleamic acid, which can be further hydrolyzed into maleic acid with
elimination of N-alkylamine. According to the results of Dahlgren [8], Kirby [9] and Matsui [10],
the secondary hydrolysis reaction of the maleimide moiety hardly occurs under their experimental
conditions (in aqueous solution over the temperature range 10-50 ℃ below pH 9). But they did
not explain the reason and the mechanisms of the continual hydrolysis processes are still unclear
now. More importantly, the crystal structures of the products in the two-step hydrolysis have
* Corresponding author. E-mail: tanxuejie@163.com, Tel.: +86 531 89631208; fax: +86 531 89631207.
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hardly been explored.
In this paper, a symmetrical bismaleimide (N, N’-ethylenedimaleimide, 2, Scheme 1) was
chosen as the model to explain how these hydrolytic degradations occur. In order to have a better
understanding of the degradation mechanism, we have isolated nearly all key intermediates and
studied their single crystal structures.
2. Experimental
2.1. Materials and measurements
All chemicals were purchased from Aladdin-reagent Chemicals and were used without
further purification. Elemental (C, H, N) analyses were carried out with a Perkin–Elmer 2400
1
microanalyzer. H NMR spectra were run on a Bruker Avance 400 MHz instruments. The
chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane, SiMe4 (δ = 0
ppm), referenced to the chemical shifts of residual solvent peak [deuterated dimethyl sulfoxide
(DMSO-d6)]. Melting points were determined on a WRS-2A electrothermal digital melting point
apparatus (Shanghai precision & scientific instrument Co., Ltd, China).
2.2. Synthesis and crystallization
Synthesis of unsaturated analogue of demethyl-cantharidin (UDMC) follows methods in the
literature [11]. Condensation of ethanediamine (EDA) with UDMC in anhydrous toluene gave the
crude product of 1, which was filtered, washed and then recrystallized in acetonitrile. 2 was
prepared by heating 1 over 120 in DMF for 12 h, employing a reverse Diels-Alder reaction. The
solvent was evaporated under reduced pressure to afford 2, which was recrystallized in
acetonitrile. Then a solution of 2 in ethanol/H2O was refluxed for 24 h. The solvent was
evaporated under reduced pressure to yield white residue. The residue was dissolved in turn with
dichloromethane, methanol and water. The solvents were subsequently removed by evaporation. 3,
4 and 6 were found in dichloromethane, methanol and water, respectively. The general reactions
are shown in Scheme 1:
Scheme 1. the reaction sequence and hydrolysis products in this paper. Systematic names for five
products:
1,
2,
4,
2,2'-(1,2-ethanediyl)bis(3a,4,7,7a-te-trahydro-4,7-epoxy-1,3-bishydroisoindole-1,3-dione);
N,N'-ethylenedimaleimide;
3,
4-(N-maleimidoethyl)
amino-4-oxobutenoic
acid;
N,N'-ethylenediamino-bis(4-oxobutenoic acid); 6, Ethylenediammonium bis(hydrogen maleate).
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Compound 5 is predicted but not obtained experimentally.
The physico-chemical characterization results are listed below:
1 Elemental analysis: found (calc. for C18H16N2O6): C, 60.72 (60.67%); H, 4.65 (4.53%); N,
7.98 (7.86%); HRMS (ESI): m/z calcd for C18H16N2O6+H+: 357.1087 [M+H+]; found: 357.1093;
1
M.p.184.4-184.9℃. HNMR (DMSO): δ (ppm) 6.510(s, 4H, olefinic protons), 5.072(s, 4H,
methine protons linked to bridge O, O-CH-), 3.528(s, 4H, methylene protons, -CH2-), 2.850(s, 4H,
methine protons, -CH-). 13C NMR (DMSO): δ (ppm) 175.99(carbonyl carbons), 136.38(olefinic
carbons), 80.19(methine carbons linked to bridge O, O-CH-), 46.99(methine carbons, -CH-),
35.10(methylene carbons, -CH2-). FT-TR(cm-1,KBr): 3098(w, ν C=C-H), 3032(w, ν C=C-H),
2993(w, ν C-H), 2957(w, ν C-H), 1770(s, ν C=O), 1720(vs, ν C=O), 1398(vs, ν C-N), 1165(s, ν
C-O-C); UV/Vis (CH3CN) λmax/nm (ε/L·mol-1·cm-1): 206.0(2.3×105).
2 Elemental analysis: found (calc. for C10H8N2O4): C, 54.52 (54.55%); H, 3.69 (3.66%); N,
12.79 (12.72%); M.p.188.7 -190.1℃. 1HNMR (DMSO): δ (ppm) 6.911(s, 4H, olefinic protons),
3.726 (s, 4H, methylene protons, -CH2-).
3 Elemental analysis: found (calc. for C10H10N2O5): C, 50.48 (50.42%); H, 4.31 (4.23%); N,
1
11.79 (11.76%); M.p.149-151℃. HNMR (DMSO): δ (ppm) 6.815(s, 2H, olefinic protons in
maleimide cycle), 6.522 (d, J=12.4 Hz, 1H, olefinic protons in maleamic acid), 6.094 (d, J=12.4
Hz, 1H, olefinic protons in maleamic acid), 3.704(m, 2H, methylene protons, -CH2-), 3.375(m, 2H,
methylene protons, -CH2-). 13C NMR (DMSO): δ (ppm) 172.22(C=O in maleimide cycle),
171.72(C=O in maleamic acid), 167.42(C=O in maleamic acid), 138.20(C=C in maleamic acid),
136.29(C=C in maleimide cycle), 132.39(C=C in maleamic acid), 51.68(-CH2- linked to
maleimide N), 37.32(-CH2- linked to –NH-).
4 Elemental analysis: found (calc. for C10H12N2O6): C, 46.92 (46.88%); H, 4.70 (4.72%); N,
10.97 (10.93%); M.p.182-184℃(lit.[12] 197℃). 1HNMR (DMSO): δ (ppm) 6.526 (d, J=12.4 Hz,
2H, olefinic protons in maleamic acid), 6.090 (d, J=12.4 Hz, 2H, olefinic protons in maleamic
acid), 3.402(s, 4H, methylene protons, -CH2-).
6 Elemental analysis: found (calc. for C10H16N2O8): C, 41.17 (41.10%); H, 5.56 (5.52%); N,
+
9.63 (9.59%); M.p.133-135℃. 1HNMR (DMSO): δ (ppm) 7.671(s, 6H, -NH3 ), 6.020 (s, 4H,
olefinic protons in maleic acid), 3.921(s, 4H, methylene protons, -CH2-). 13C NMR (DMSO): δ
(ppm) 167.16(C=O), 136.09(C=C), 41.00(-CH2-).
2.3. X-Ray Crystallographic Analysis
The X-ray diffraction measurements were made on a Bruker APEX II CCD area
detector diffractometer at 293/298K for compounds
1 to 6 (Mo Ka radiation, graphite
monochromator, λ = 0.71073 Å). The structures were solved by SHELXL-97. The
absorption correction was done using the SADABS program.[13] Software packages APEX
II (data collection), SAINT (cell refinement and data reduction), SHELXTL (data reduction,
molecular graphics and publication material), DIAMOND (simplifying crystal packing
diagram) were also used.[14
,
15, 16] All non-hydrogen atoms were refined with anisotropic
displacement parameters. In
1, hydrogen atoms were added to the structure model on
calculated positions but in the rest four crystal structures, the positions of all hydrogen atoms
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(except H9 in
3) were experimentally determined in electron density maps and refined
without any constraints.
3. Results and Discussion
3.1. Single-crystal X-ray crystallography
Crystal data, data collection and structure refinement details of the five compounds are
summarized in Table 1.
Table 1 Crystallographic data and structure refinements summary for four products
Compounds
1
2
3
4
6
Chemical
formula
Mr
C18H16N2O6
C10H8N2O4
220.18
C10H10N2O5
238.20
C10H12N2O6
256.22
C5H8NO4
146.12
356.33
block/colorless
triclinic
P -1
Crystal habit
Crystal system
Space group
a /Å
block/colorless block/colorless block/colorless block/colorless
monoclinic
P 21/n
monoclinic
P 21/c
monoclinic
C 2/c
triclinic
P -1
8.239(4)
10.295(5)
10.351(5)
96.549(5)
107.849(6)
103.068(6)
798.0 (6)
2
6.1101(4)
11.6530(7)
7.1284(5)
90.00
6.046(4)
24.896(15)
7.529(5)
90.00
7.2617(7)
7.3552(5)
21.1448(17)
90.00
5.731(3)
6.552(3)
8.794(4)
92.605(7)
96.025(6)
105.321(6)
315.8(3)
2
b /Å
c /Å
α /°
β /°
103.940(7)
90.00
101.202(9)
90.00
92.161(7)
90.00
γ /°
V /Å3
492.60(6)
2
1111.7(12)
4
1128.57(16)
4
Z
Dcalc. /g·cm–3
ꢀ /mm–1
T /K
1.485
1.423
1.508
1.537
1.483
0.113
0.117
0.116
0.126
0.134
298
293
298
293
293
372
F(000)
Rint
228
496
536
154
0.0544
0.0243
0.0452
0.1251/1170
1.030
0.0542
0.0497
0.1088/2544
0.917
0.0400
0.0642
0.1754/1304
1.045
0.0191
0.0476
0.1266/1374
1.029
0.0633
R1 [I>2σ(I)]
wR2/reflections
S
0.1719/2769
1.025
3. 1. 1. Crystal structure of 1
As an unsaturated norcantharimide (UNCI) dimer, 1 has appeared before, [17] but no detailed
structure information has been reported. Monomeric and packing structures of 1 are depicted in
Fig. 1. As can be seen from the figure, the polycyclic imide skeleton has the exo-conformation,
which is more stable than the endo- structure and inevitably becomes the overwhelmingly major
products under thermodynamic control. [18, 11(c)] As for the –CH2-CH2- linker, 1 prefers
antigauche mode. Though the asymmetric unit contains one complete dimer, the structure adopts
“approximately” C2 point-group symmetry and the C2 axis nearly parallel to [4 7 -6] direction
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through the center of C9-C10 bond (perpendicular to the bond). “Approximately” means the two
nearly identical halves have some differences (Table S1, All “S” numbered tables and figures are
in Supporting Information). For example, the double bond length of C5=C6 is 1.300(3) Å, but that
for C15=C16 is 1.304(3) Å.
Fig. 1. (a) Atom numbered molecular structure of 1 with displacement ellipsoids for non-H atoms drawn at the
30% probability level at 298 K. (b) Two infinite 1D chains of 1 formed through four kinds of intermolecular
C–H···O H-bonds (shown orange, green, red and blue dotted lines respectively, color figure available online), view
along the [1 1 1] direction. The sloping chain extends along the [-4, 3, -7] direction and the horizontal one extends
along the c axis. (c) 2D structure formed by intermolecular C–H···O H-bonds, view along the [1 1 1] direction.
The right half is illustrated by the simplified structures, i.e. polycyclic imide skeleton except N is simplified by its
center gravity (red balls at each end of the chains) and all H atoms have been omitted for clarity. (d) 3D structure
formed by the packing of single-sheet architecture, view along the [1 0 1] direction. The upper two and lower two
sheets are illustrated by the simplified dimers. The different objects in this figure are not drawn to scale.
In the packing structure of 1, no valuable π-π stacking interactions can be found and the
dominant force is hydrogen bonding. The geometries of hydrogen bonds are listed in Table S2. As
can be seen, four kinds of intermolecular C–H…O hydrogen bonds are present and they adopt C2
point-group symmetry too. Hydrogen bonds C4—H4···O5(x, y, z+1) and C11—H11···O3(x, y, z-1) are
symmetry related (both involve carbonyl O), and both of them can link the dimer units into the
same 1D chains, spread along the crystallographic c axis (Fig. 1b). Similarly, hydrogen bonds
C9—H9A···O1 (–x+1, -y+2, -z+2) and C10—H9B···O4 (–x, -y+2, -z+1) are symmetry related (both involve
bridge O), but they must work together to link the dimer units into 1D chains, which extend along
the [-4, 3, -7] direction (Fig. 1b). If all the four H-bonds are considered, two-dimensional
supramolecular layers with rhombic meshes are formed (Fig. 1c), which are aligned parallel to the
ac-plane of the crystal (Fig. 1d). Packing of these layers in the crystal is stabilized only by van der
Waals forces.
3. 1. 2. Crystal structure of 2
Compound 2 is a well known bismaleimide and its spectra and physical data have been
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reported in the literatures [19], however its single-crystal structure has not been unambiguously
determined till now.
Dimer 2 is not as complicated as 1 in symmetry. The structure of 2 adopts Ci point group
symmetry with an inversion center between the center two C atoms (C5-C5A). The asymmetric
unit contains only one half-molecule, so the two halves are really identical. As can be seen from
Fig. 2(a), dimer 2 maintains the same antigauche conformation as that in dimer 1.
Fig. 2. (a) Atom numbered molecular structure of 2 with displacement ellipsoids for non-H atoms drawn at the
30% probability level at 293 K. Symmetry code of A: −x+1, −y, −z. (b) Two kinds of π-π interactions, shown
with red and blue dotted lines, respectively. The red one corresponds to Cg1→Cg1 #1, #1: -x, 1-y, -z, d=3.5519(11)
Å. The blue dotted lines correspond to Cg1→Cg1 #2, #2: 1/2+x, 1/2-y, 1/2+z, d=5.1775(12) Å. (c)
Two-dimensional structure formed by the second kind of π-π interactions (shown blue dotted lines), view
perpendicular to the spreading plane, i.e. the crystallographic (2 0 3) plane. The right half is illustrated by the
simplified structures, i.e. imide ring except N is simplified by its center gravity (red balls at each end of the chains)
and all H atoms have been omitted for clarity. (d) Three-dimensional structure formed by two kinds of π-π
interactions, view along the b axis. The sloping chain extending along the c axis is formed by the first kind of π-π
interactions and the horizontal layers spreading along the (2 0 3) plane are formed by the second kind of π-π
interactions. Only five layers are present and their adjacent layers have been omitted for clarity. The upper two and
lower two layers are illustrated by the simplified structures.
Contrary to that in dimer 1, no classical H-bonds were found and only π-π stacking
interactions occur in the packing structure of 2. The geometries of two kinds of π, π –interactions
are listed in Table S4 and the schematic illustration is shown in Fig. 2(b). The first kind of π, π
–interactions (red dotted line) link dimer 2 into 1D chains stretching along the c axis (Fig. 2(d)),
while the second kind of π, π –interactions (blue dotted lines) link dimer 2 into 2D layers parallel
to the crystallographic (2 0 3) plane (Fig. 2(c)). The latter planes are further pillared by the former
chains to generate 3D networks (Fig. 2(d)).
3. 1. 3. Crystal structure of 3
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Compound 3 is new both in molecule structure and in crystal structure. It’s the result of
partly hydrolysis of dimer 2, i.e. only one maleimide ring in dimer 2 is converted into
4-oxobutenoic acid. The O–H distances are 1.09 (3) Å and 1.39 (3) Å, respectively, and it appears
that the O, H, O atoms are nearly collinear (178°) (Fig. 3(a)). This intra-molecular hydrogen bond
O2···H1—O3 (O···O 2.472 (2) Å) gives a 7-membered ring with an average r.m.s. deviation from
planarity of 0.0764 Å. The 5-membered maleimide ring has a relatively better coplanarity with an
average r.m.s. deviation only 0.0039 Å. The dihedral angle between the two planes is 54.5°. As for
the -C5-C6- single bond linker between these two planes, 3 prefers gauche conformation. As
mentioned above, both dimers 1 and 2 adopt antigauche mode. In fact, compound 4 also
crystallizes in antigauche mode. Then why only 3 prefers gauche conformation? Results from
DFT calculations about different conformations of 2, 3 and 4 prove that their gauche modes are
more stable than corresponding antigauche modes (Fig. S9 and Table S6). But the energy
separations are very small, for example, the energy difference between the gauche
and antigauche conformations of 3 is around 0.45 kcal/mol in gas phase and only 0.20 kcal/mol in
solution. So the mode which facilitates intermolecular hydrogen bonding and π-π stacking in the
condensed phase will become the end conformation, though the end conformation may have
slightly higher energy. That’s the case in 2 and 4.
Fig. 3. (a) Atom numbered molecular structure of 3 with displacement ellipsoids for non-H atoms drawn at the
30% probability level at 298 K. (b) Three types of 1D chains formed through three kinds of intermolecular
H-bonds (N1—H4···O1i, i: x+1, −y+1/2, z+1/2; C2—H2···O4ii, ii: x, −y+1/2, z−1/2; C8—H9···O3iii, iii: x+1, y,
z+1, shown blue, red and green dotted lines respectively), view along the b axis. The horizontal chain extends
along the [4 0 7] direction and the sloping two chains extend along the c axis and the [5 0 3] direction. (c) One
interesting 1D bowl-like zigzag chain, view perpendicular to the extending direction, i.e. c axis. (d) End-on view
of the aforementioned 1D bowl-like zigzag chain, view along the extending direction. (e) The 2D layer structure
formed by H-bonds further stack into 3D network through π-π interactions (orange dotted lines between two
molecules above the arrow). Among the five superimposed end-on-view chains illustrating the 2D layer, the upper
one and the lower one are simplified into bowls. At last, all these bowl-like zigzag chains are simplified into bowls
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in the 3D network.
In compound 3, there are three kinds of intermolecular hydrogen bonds (Table S7), which
join molecules into three kinds of infinite chains running in the [5 0 3], [4 0 7] and c directions,
respectively (Fig. 3(b)). All these chains are parallel to the ac plane, so their crosslinking leads to
the formation of layers lying on the ac plane. These layers are overlapped in the b-direction to
form 3D network. It’s worthy to note that the zigzag chains along the c direction exhibit an
unusual bowl shape (Figs. 3(c) and 3(d)). So an interesting packing pattern (Fig. 3(e)) is obtained
due to the involvement of intermolecular hydrogen bonding and the occurrence of the π-π stacking
interactions (Table S8)
.
3. 1. 4. Crystal structure of 4
Like 2, dimer 4 (with Ci point group symmetry) resides on a crystallographically imposed
inversion center and the asymmetric unit contains only one half-molecule. As shown in Fig. 4(a),
the central ethylene group exhibits an antigauche conformation and the symmetry-related two
4-oxobutenoic acid groups have linear O-H···O hydrogen bonds (178°).
Fig. 4. (a) Atom numbered molecular structure of 4 with displacement ellipsoids for non-H atoms drawn at the
30% probability level at 293 K. Symmetry code of A: −x+2, −y+2, −z+1. (b) Three kinds of intermolecular
H-bonds (N1—H4···O1i, N1—H4···O2i, C5—H6···O2i, i: x, y+1, z, shown with blue, red and orange dotted lines
respectively) link the dimers into 1D chains extending along the b axis. (c) 2D layer network formed by the fourth
kind of intermolecular H-bonds (C2—H2···O1ii, ii: −x+3/2, y+1/2, −z+1/2, shown green dotted lines), lying the
crystallographic (17 0 -17) plane, view along the a axis. (d) 3D structure formed by the packing of 2D layers,
view along the b axis.
Four types of intermolecular N−H···O and C−H···O hydrogen bonds are found in the
packing structure (Table S10). Three of them link the dimers into 1D chains extending along the b
axis (Fig. 4(b)) and the other one link adjacent dimers into 2D layers parallel to the
crystallographic (17 0 -17) plane (Fig. 4(c)). No hydrogen bonds or π-π interactions can be found
between these 2D hydrogen-bonded networks. So the 2D layers stack along the direction of the
a-axis into 3D networks mainly through Van der Waals forces (Fig. 4(d)).
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3. 1. 5. Crystal structure of 6
Though the X-ray crystal structure of 6 was known [20], its H-bonding pattern was not
described clearly in the original report.
Molecular and packing structures of 6 are depicted in Fig. 5. Relevant bond distances and
angles are given in Table S11, and the geometries of hydrogen bonds are listed in Table S12.
X-ray structural analysis reveals that the asymmetric unit comprises of a deprotonated maleate
anion, and one half protonated ethylenediammonium dication located in an inversion center (Fig.
5(a)). Hydrogen transfer from maleate acid to ethylenediamine was observed after the hydrolysis
of dimer 4, yielding three oppositely charged spheres. Our DFT calculations prove that the proton
transfer is inevitable in implicit solvent model of CPCM, i.e. no stable structure including neutral
fragments (maleate acid and ethylenediamine) can be obtained in ethanol solution. They can
coexist only in gas phase, with around 39.8 kcal/mol higher energy than that of ionic fragments
(two deprotonated maleate anions and one protonated ethylenediammonium dication), and the
former will always be optimized into the latter in ethanol solution. These ionic fragments were
held together by electrostatic interactions as well as hydrogen bond (N1—H6···O4). Linear
O-H···O hydrogen bonds still occur in the maleate anion, but less linear with the angle only 173°
instead of aforementioned 178° in 3 and 4.
Fig. 5. (a) Atom numbered molecular structure of 6 with displacement ellipsoids for non-H atoms drawn at the
30% probability level at 293 K. Symmetry code of A: −x+2, −y+2, −z+1. (b) Three kinds of intermolecular
H-bonds (N1—H7···O3i, N1—H7···O4i, C2—H2···O2i, i: x−1, y, z, shown with blue, red and yellow dotted lines
respectively) link the dimers into 1D chains extending along the a axis. (c) Three kinds of 1D chains extending
along different directions: the horizontal chain along the b axis (link by N1—H8···O4ii, ii: −x+1, −y+1, −z), the
sloping chain along the [-11 5 -29] direction (link by N1—H8···O1iii, iii: x−1, y, z−1) and the end-on view of the
aforementioned 1D chain along the a axis. (d) The aforementioned three kinds of chains weave with each other
leading to the formation of 3D structure, view along the a axis.
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In the packing structure of 6, there are five kinds of intermolecular N−H···O and C−H···O
hydrogen bonds (Table S12), each of which can link the compound into 1D chains. Three types of
hydrogen bonds involving the molecules with the symmetry code “x−1, y, z” coexist in the same
kind of 1D chains extending along the a axis (Fig. 5(b)), which look like chairs from the point of
end-on view, i.e. along the a axis (Figs. 5(c) and 5(d)). The other two types of intermolecular
hydrogen bonds join molecules into two kinds of 1D chains running in the [-11 5 -29] and b
directions, respectively (Fig. 5(c)). At last, these 1D chains with different orientations weave with
each other leading to the formation of 3D network with microporous structure.
In summary, dimer 1 possesses axial symmetry and dimers 2, 4, 6 possess central symmetry,
they all exhibit antigauche conformation, while 3 prefers slightly more stable gauche mode. All
five compounds crystallize in the centrosymmetric space group and the dominating forces are
hydrogen bonding as well as π-π stacking interactions, which link the molecules into interesting
1D, 2D and 3D networks.
3.2. Proposed Mechanisms of Hydrolysis
The hydrolytic degradation pathways of 2 without or with water catalysis were proposed
based on the identified crystal structures of these degradation products and shown in Scheme 2. It
can be seen that all degradation reactions induced by water occur in the C–N bonds, which are
broken step by step until the elimination of the last O=C–N bond. Nevertheless, product 5 is hard
to be obtained experimentally, which means the activation energy of the last step may be the
lowest one. The chemical elucidation of the proposed hydrolytic degradation pathways need to be
proved further in future study.
Scheme 2. Proposed mechanisms without or with water catalysis of continual four-step hydrolytic degradation
reaction of N, N’-ethylenedimaleimide (dimer 2).
In Mechanism-1, TS structures form tetragonal rings due to the attack of one water molecule,
while the TS structures in Mechanism-2 produce stable hexagonal rings in the presence of two
water molecules. So it can be deduced that the activation energies in Mechanism-2 should be
lower than that in Mechanism-1, i.e. one water molecule plays role of a catalyst. Obviously
such speculation needs to be investigated further.
4. Conclusions
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Five single-crystal structures related with the same reaction have been investigated. 1, 2, 4, 6
are dimers linked by –CH2-CH2- group and only 3 has non-symmetry related skeleton, which is
the product of partly hydrolysis of dimer 2. Though all four dimers exhibit antigauche
conformations, gauche modes are in fact more stable than corresponding antigauche modes, which
have been proved by DFT calculations as well as the crystal structure of 3. It is hydrogen bonding
and π-π stacking interactions in the condensed phases that drive these dimers into antigauche
modes. Still because of hydrogen bonding and π-π stacking interactions, the molecules form
different kinds of packing structures, which have been investigated and graphically represented.
Among these five crystal structures, the most interesting one is that of 3, in which one kind of
intermolecular hydrogen bonds link molecules into bowl shape zigzag chains and these “bowls”
are further overlapped into 2D and 3D networks by other kinds of intermolecular hydrogen
bonding and π-π stacking interactions.
It should be mentioned that what set these molecules apart are not only their crystal structures
but also their experimental evidence in the investigation of hydrolytic degradation mechanism.
Two reaction pathways have been proposed. Mechanism-1 involves only one H2O molecule,
which acts as reactant; Mechanism-2 involves two H2O molecules, which act as reactant as well
as catalyst. The detailed chemistry needs to be investigated further.
5. Acknowledgments
The authors are thankful to the Science & Technology Development Projects of Shandong
Province in China (No. 2011GGX10706 and 2012GGX10702) and the National Natural Science
Foundation of PR China (NSFC, No. 21103100) for financial support. Generous support in
computing resources from the National Supercomputer Center in Jinan, China, is greatly
appreciated.
Supplementary Material
Supplementary Information available: Tables S1 – S26, Figures S1 - S9 mentioned in the text.
Crystallographic information files of five compounds. CCDC < 1446298, 1454849, 1454850, 1454851, and
1454852> contains the supplementary crystallographic data for < 1, 2, 3, 4 and 6 >. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif ,
or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
References and notes
[1] V. Ondruš, L. Fišera, V. Bradac, ARKIVOC, v (2001) 60-67.
[2] V. Rao, S. Navath, M. Kottur, J. R. McElhanon, D. V. McGrath, Tetrahedron Lett. 54 (2013) 5011–5013.
[3] P. D. Senter, E. L. Sievers, Nat. Biotechnol. 30 (2012) 631–637.
[4] P. F. Peddi, S. A. Hurvitz, Future Oncol. 9 (2013) 319–326.
[5] R. P. Lyon, J. R. Setter, T. D. Bovee, S. O. Doronina, J. H. Hunter, M. E Anderson, C. L. Balasubramanian, S.
M. Duniho, C. I. Leiske, F. Li, P. D. Senter, Nat. Biotechnol. 32 (2014) 1059-1062.
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[6] (a) S. D. Fontaine, R. Reid, L. Robinson, G. W. Ashley, D. V. Santi, Bioconjugate Chem. 26 (2015) 145–152; (b)
S. C. Alley, D. R. Benjamin, S. C. Jeffrey, N. M. Okeley, D. L. Meyer, R. J. Sanderson, P. D. Senter,
Bioconjug. Chem. 19 (2008) 759–765; (c) B. Q. Shen, K. Xu, L. Liu, H. Raab, S. Bhakta, M. Kenrick, K. L.
Parsons-Reponte, J. Tien, S. F. Yu, E. Mai, D. Li, J. Tibbitts, J. Baudys, O. M. Saad, S. J. Scales, P. J.
McDonald, P. E. Hass, C. Eigenbrot, T. Nguyen, W. A. Solis, R. N. Fuji, K. M. Flagella, D. Patel, S. D.
Spencer, L. A. Khawli, A. Ebens, W. L. Wong, R. Vandlen, S. Kaur, M. X. Sliwkowski, R. H. Scheller, P.
Polakis, J. R. Junutula, Nat. Biotechnol. 30 (2012) 184–189; (d) V. L. Chudasama, F. S. Stark, J. M.
Harrold, J. Tibbitts, S. R. Girish, M. Gupta, N. Frey, D. E. Mager, Clin. Pharmacol. Ther. 92 (2012) 520–527.
[7] (a) C. P. Ryan, M. E. B. Smith, F. F. Schumacher, D. Grohmann, D. Papaioannou, G. Waksman, F. Werner, J.
R. Baker, S. Caddick, Chem. Commun. (Camb.) 47 (2011) 5452–5454; (b) P. Knight, Biochem. J. 179 (1979)
191–197.
[8] G. Dahlgren, N. L. Simmerman, J . Phys. Chem. 69 (1965) 3626-3630.
[9] A. J. Kirby, P. W. Lancaster, J. Chem. Soc., Perkin Trans. 2 (1972) 1206-1214;
[10] S. Matsui, H. Aida, J. Chem. Soc., Perkin Trans. 2 12 (1978) 1277-1280.
[11] (a) S. Baggio, A. Barriola, P. K. de Perazzo, J. Chem. Soc., Perkin Trans. 2 (1972) 934-937; (b) B. M.
Ramirez, G. D. de Delgado, W. O. Velasquez, G. M. Perdomo, Z. Kristallogr.-New Crystal Structures, 213
(1998) 197-198; (c) Y. W. Goh, B. R. Pool, J. M. White, J .Org. Chem. 73 (2008) 151-156.
[12] M. Viswanathan, Asian J. Chem. 17 (2005) 2611-2615.
[13] G. M. Sheldrick, SADABS, Bruker AXS Inc., Madison, Wisconsin, USA, 2004.
[14] G. M. Sheldrick, Acta Crystallogr, Sect. A, 64 (2008) 112-122.
[15] Bruker APEX II (version 2.0-1), SAINT (version 7.23A), XPREP (version 2005/2), SHELXTL (version 6.1).
Bruker AXS Inc., Madison, Wisconsin, USA, 2005.
[16] K. Brandenburg, H. Putz, DIAMOND. Crystal Impact GbR, Bonn, Germany, 2005.
[17] (a) L. P. Deng, Z. Yong, W. F. Tao, J. F. Shen, W. Wang, J. Heterocyclic Chem. 48 (2011) 158-161; (b) V. Rao,
S. Navath, M. Kottur, J. R. McElhanon, D. V. McGrath, Tetrahedron Lett, 54 (2013) 5011-5013.
[18] (a) M. W. Lee, W. C. Herndon, J. Org. Chem. 43 (1978) 518-518; (b) P. X. Iten, A. A. Hofmann, C. H.
Eugster, Helv. Chim. Acta 62 (1979) 2202-2210 and litt. therein.
[19] (a) Y. H. Kim, W. E. Stites, 47 (2008), 8804–8814; (b) D. Habibi, O. Marvi, ARKIVOC, xiii (2006) 8-15; (c)
R. Tona, R. Häner, Mol. BioSyst., 1 (2005) 93-98; (d) A. A. Kumar, K. Dinakaran, M. Alagar, J. Appl. Polym.
Sci., 89 (2003) 3808-3817; (e) I. M. Brown, T. C. Sandreczki, Macromolecules, 23 (1990) 4918–4924; (f) P.
Kovacic, R. W. Hein, J. Am. Chem. Soc. 81 (1959) 1187–1190.
[20] J. C. Barnes, T. J. R. Weakley, Acta Cryst. C53 (1997) IUC9700018.
Keywords
:
N,N'-ethylenedimaleimide, crystal structure, key intermediates, hydrolysis mechanism
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Highlights
1) Five single-crystal structures have been investigated; molecular conformations and weak
interactions have been analyzed.
2) The hydrolytic degradation mechanisms of N,N'-ethylenedimaleimide without or with water
catalysis have been proposed.