Synthesis, characterization and structural aspects of new haptens for PAHs

Article abstract of DOI:10.1016/j.molstruc.2010.12.023

Two new haptens for PAHs, 4-(naphthalen-2-yl)-4-oxobutanoic acid (I) and 4-(anthracen-1-yl)-4-oxobutanoic acid (II), were synthesized and confirmed by elemental analysis, IR, and ¹H NMR. Single crystal X-ray structure showed that compound I crystallizes in the triclinic crystal system (space group = P-1). A classic hydrogen bonded dimer is formed by the carboxylic acid group from two molecules. The R22 (8) graph-set motif links the molecules into pairs around inversion centers in the unit cell. The O-H?O hydrogen-bonded interactions involving these pairs are very strong and stabilize molecular packing of these dimers into a unique assembly. Compound II crystallizes in the Monoclinic crystal system (space group = P21/c). Similarly to compound I, a classic hydrogen bonded dimer is formed by the carboxylic acid group from two molecules. The R22 (8) graph-set motif links the molecules into pairs around inversion centers in the unit cell. Besides, an intramolecular hydrogen bonding causes the formation of a planar six-membered ring, which is also coplanar with the adjacent anthracene ring. The geometries of the corresponding parts of the haptens are almost the same as those of naphthalene and anthracene. Ab initio Hartree-Fock computational calculation provided the supports that the size, shape (geometry) and electronic properties at the corresponding parts of the haptens did not change significantly, compared to those of naphthalene and anthracene. The haptens were coupled with bovine serum albumin (BSA) to make antigents. The coupling ratio of I-BSA was 1:20, and II-BSA 1:37, respectively. These results showed that the new haptens could be used to induce specific antibodies for PAHs.

Full text of DOI:10.1016/j.molstruc.2010.12.023

Journal of Molecular Structure 987 (2011) 206–213
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, characterization and structural aspects of new haptens for PAHs
b,
Yi Li a, Mingming Yang b, Yuanyuan Liu b, Rongqing Wei a, Xiaoning Liu a, , Fangshi Li
⇑
⇑
a College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, PR China
b College of Science, Nanjing University of Technology, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new haptens for PAHs, 4-(naphthalen-2-yl)-4-oxobutanoic acid (I) and 4-(anthracen-1-yl)-4-oxob-
utanoic acid (II), were synthesized and confirmed by elemental analysis, IR, and 1H NMR. Single crystal
X-ray structure showed that compound I crystallizes in the triclinic crystal system (space group = P-1).
A classic hydrogen bonded dimer is formed by the carboxylic acid group from two molecules. The R22
(8) graph-set motif links the molecules into pairs around inversion centers in the unit cell. The O–
HÁ Á ÁO hydrogen-bonded interactions involving these pairs are very strong and stabilize molecular pack-
ing of these dimers into a unique assembly. Compound II crystallizes in the Monoclinic crystal system
(space group = P21/c). Similarly to compound I, a classic hydrogen bonded dimer is formed by the carbox-
ylic acid group from two molecules. The R22 (8) graph-set motif links the molecules into pairs around
inversion centers in the unit cell. Besides, an intramolecular hydrogen bonding causes the formation of
a planar six-membered ring, which is also coplanar with the adjacent anthracene ring. The geometries
of the corresponding parts of the haptens are almost the same as those of naphthalene and anthracene.
Ab initio Hartree–Fock computational calculation provided the supports that the size, shape (geometry)
and electronic properties at the corresponding parts of the haptens did not change significantly, com-
pared to those of naphthalene and anthracene. The haptens were coupled with bovine serum albumin
(BSA) to make antigents. The coupling ratio of I-BSA was 1:20, and II-BSA 1:37, respectively. These results
showed that the new haptens could be used to induce specific antibodies for PAHs.
Received 2 November 2010
Received in revised form 13 December 2010
Accepted 13 December 2010
Available online 21 December 2010
Keywords:
Hapten of PAHs
4-(Naphthalen-2-yl)-4-oxobutanoic acid
4-(Anthracen-1-yl)-4-oxobutanoic acid
Crystal structure
Hartree–Fock calculation
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
cules that are unable to induce an immune response. Therefore,
the target molecule is derivatized into a hapten, by introducing a
Polycyclic aromatic hydrocarbons (PAHs) are a large group of
organic compounds containing two or more fused aromatic rings
constituted of carbon and hydrogen atoms. PAHs have received
considerable attention because of their rich chemistry [1–5], phys-
ical properties [6], technological and industrial applications [1],
aromaticity [7–15], and role as a major class of environmental pol-
lutants and carcinogens [16–18].
Conventional analytical methods for PAHs, such as gas chroma-
tography (GC) and high-performance liquid chromatography
(HPLC), are time-consuming and cumbersome [19–21]. Immuno-
chemical methods, such as enzyme-linked immunosorbent assays
(ELISAs) and immunosensors, are fast, selective, and inexpensive
analytical methods that complement traditional chromatographic
analytical procedures in the environmental analysis of PAHs [22–
27].
functional group, in order to link it to a carrier protein, for example,
bovine serum albumin (BSA). The immunoconjugate thus formed is
immunogenic and a mammal can be immunized to produce spe-
cific antibodies to the antigen. The key step in developing an
immunoassay for PAHs is the design and preparation of optimum
haptens for immunogens and competitors. The performance of
the antibody in immunoassays is greatly affected by the property
of the hapten.
There have been some haptens for PAHs reported [26,28,24],
however, the antibody specificity resulting from the newly de-
signed hapten is often unpredictable, and this knowledge comes
only after time-consuming and laborious animal experiments.
It would be more attractive to be able to predict the specificity
of the antibodies obtained with a given hapten. As antigen–anti-
body recognition is based on the steric criteria and the interactions
resulting from the electronic properties of the molecules, molecu-
lar modeling may be helpful as it allows the determination of vol-
umes and charges of the compounds [29].
Immunochemical methods are based on the specific interaction
of antibodies with antigens. PAHs are low-molecular-mass mole-
To have a better understanding of such hapten molecules from
the molecular and sub-molecular level, we report here the synthe-
sis of two novel haptens for PAHs (Fig. 1, compounds I and II). The
⇑
Corresponding authors. Tel./fax: +86 25 83587347 (X. Liu), tel./fax: +86 25
83587450 (F. Li).
E-mail addresses: xiaoningliu@163.com (X. Liu), fangshi.li@njut.edu.cn (F. Li).
0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2010.12.023

Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
207
Fig. 1. Scheme of the synthesis of compounds I and II.
crystal structure and the isolated state conformation of the haptens
were studied by X-ray crystallography and ab initio calculation.
And the electronic structures of the haptens and the conjugates
were discussed.
1680, 1624, 1433, 1403, 1367, 1254, 1230, 1170, 933, 812; 1H
NMR (300 MHz, CDCl3) d 8.51 (s, 1H, Ar–H), 8.06–7.54 (m, 6H,
Ar–H), 3.48 (t, J = 6.7 Hz, 2H, CH2), 2.89 (t, J = 6.7 Hz, 2H, CH2).
2.3. Synthesis of 4-(anthracen-1-yl)-4-oxobutanoic acid (II)
2. Experimental
Hapten (II) was synthesized according to the method reported
in the literature [31] with some modification. A suspension of
anthracene (7.14 g, 0. 04 mol) and succinic anhydride (4.0 g,
0.04 mol) in CH2C12 (100 mL) was cooled in an ice bath, and
AlC13 (5.5 g, 0.041 mol) was added in four portions over 20 min
with vigorous mechanical stirring. The deep-red mixture was stir-
red at 0–5 °C for another 60 min and stored in the refrigerator
overnight. Ice and dilute HCl were added with stirring to decom-
pose the complex. The mixture was digested on a boiling water
bath for 2 h to evaporate the organic solvent. The mixture was
cooled and decanted. The solid was separated and washed thor-
oughly with H2O. The solid was digested with dilute K2CO3 solu-
tion on a boiling water bath for 30 min. The remaining solid was
collected by filtration. To the filtrate, EtOAc (100 mL) was added,
followed by the dropwise addition of concentrated HCl with stir-
ring. The layers were separated and the aqueous layer was ex-
tracted with EtOAc. The combined organic extracts were dried
(Na2SO4) and evaporated under reduced pressure. The oily residue
was treated with a small amount of benzene. The yellow precipi-
tate was filtered, air-dried, and recrystallized from EtOAc. m.p.
177–179 °C; yield: 3.1 g, 55%. Anal. calcd. for C18H14O3: C 77.68, H
2.1. Materials and instruments
All chemicals were reagent grade and were used without fur-
ther purification.
1H NMR spectra were recorded on a Bruker AV-500 spectrome-
ter at 500 MHz or a Bruker AV-300 spectrometer at 300 MHz using
CDCl3 or DMSO-d6 as the solvent, with tetramethylsilane as the
internal standard. Chemical shifts (d) are given in parts per million
(ppm), and coupling constants (J) are given in Hertz (Hz). IR spectra
were recorded in KBr disk using a Nicolet 380 FT-IR spectropho-
tometer and only diagnostic absorbances (kmax) are reported. Ele-
mental analyses were performed with a Flash EA-1112 elemental
analyzer. Melting points were measured on an X-4 microscope
electrothermal apparatus and were uncorrected. X-ray crystallo-
graphic analyses were performed on a Nonius CAD4 single-crystal
diffractometer using graphite-monochromated Mo K
(k = 0.71073) Å.
a radiation
2.2. Synthesis of 4-(naphthalen-2-yl)-4-oxobutanoic acid (I)
5.07; found: C 77.56, H 5.10. IR (KBr, m
, cmÀ1): 3042, 2921, 1710,
Hapten (I) was synthesized according to the method reported in
the literature [30] with some modification. A suspension of naph-
thalene (3.84 g, 0.03 mol) and succinic anhydride (3.0 g, 0.03 mol)
in PhNO2 (60 mL) was cooled in an ice bath, and AlC13 (8.5 g,
0.065 mol) was added in four portions over 20 min with vigorous
mechanical stirring. The mixture was stirred at 0–5 °C for 10 h,
and then at 25 °C for 24 h. Ice and dilute HCl were added with stir-
ring. The mixture was decanted. The brown solid was separated
and washed thoroughly with H2O and Hexane. The solid was tritu-
rated with a small amount of benzene and air-dried. m.p. 157–
159 °C; Yield: 2.8 g, 41%. Anal. calcd. for C14H12O3: C 73.67, H
1671, 1615, 1537, 1408, 1349, 1254, 1197, 1169, 1081, 958, 903,
877, 732. 1H NMR (500 MHz, DMSO-d6) d 12.20 (s, 1H, OH), 9.22
(s, 1H, Ar–H), 8.68 (s, 1H, Ar–H), 8.33–8.08 (m, 4H, Ar–H), 7.62–
7.56 (m, 3H, Ar–H), 3.40 (t, J = 6.5 Hz, 2H, CH2), 2.72 (t, J = 6.5 Hz,
2H, CH2).
2.4. Preparation of Hapten-protein conjugates
The hapten-protein conjugates were prepared by the slightly
modified NHS ester method [32]. The coupling ratio of I-BSA was
1:20, and II-BSA 1:37, respectively.
5.30; found C 73.52, H 5.32. IR (KBr, m
, cmÀ1): 3059, 2931, 1714,

208
Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
2.5. X-ray structure determination of the haptens
and bond angles are listed in Table 4. The molecular structures of I
and II are shown in Figs. 2 and 4, respectively.
Colorless crystals of I suitable for X-ray analysis were grown
from concentrated solution of methanol, and yellow crystals of II
from acetonitrile.
2.6. Quantum chemical calculations
A crystal was put on a glass fiber. The diffraction data were col-
lected on a Nonius CAD4 single-crystal diffractometer equipped
The first task for the computational work was to determine the
optimized geometry of the compounds I and II. The spatial coordi-
nate positions of the compounds I and II, as obtained from the
X-ray structural analysis, were used as the initial coordinates for
their theoretical calculations. The quantum mechanical calculations
were carried out using the Gaussian 03 program using the Hartree–
Fock (HF) method with a 6-31G (d) basis set level [34,35]. The
geometrical, electronic, and energy parameters were extracted from
the Gaussian files based on the optimized structures.
with a graphite-monochromated Mo K
a radiation (k = 0.71073 Å)
by using a /2h scan mode at 298 K. The crystal structure was
x
solved by the direct method and refined by the full-matrix least-
squares procedure on F2 using SHELXL-97 program [33]. Positions
of hydrogen atoms were located by geometrical calculation (x, y,
z and Uiso fixed to 1.2 times Uiso of the atom they are bound to).
Crystal and experimental data for I and II are listed in Table 1.
The atomic coordinates of non-hydrogen atoms and their thermal
parameters of I and II are listed in Tables 2 and 3. The bond lengths
3. Results and discussion
3.1. Hapten design
Table 1
X-ray crystal data and structure refinement of I and II.
For any kind of immunochemical method, the specific antibody
is the key reagent. To produce antibody against a small molecule
like a PAH, the latter must be conjugated with a carrier, usually
some protein such as albumin [36,37]. Unfortunately, PAHs have
no functional group for direct conjugation with proteins. Therefore,
a derivative (hapten) of PAH must be synthesized. According the
criteria of structure design for haptens [38–40], that the hapten
should be a near perfect mimic of the target structure in size, shape
(geometry), and electronic properties, we designed two new hap-
tens for PAHs (Fig. 1). Hapten I has a carboxyl group with four
length of spacer arm at 2-position of naphthalene, and II at 1-posi-
tion of anthracene.
Chemical formula
I
II
Formula weight
CCDC number
Temperature (K)
Wavelength (Å)
Crystal system, Space
group
a (Å)
b (Å)
c (Å)
C
14H12O3
C18H14O3
798276
293(2)
0.71073
Monoclinic, P21/c (No.
14)
18.021(4)
5.2290(10)
14.788(3)
90.00
90.51(3)
90.00
1393.4(5), 4
1.327
0.090
584
759778
293(2)
0.71073
Triclinic, P-1 (No. 2)
5.6250(11)
7.6220(15)
13.537(3)
99.36(3)
97.39(3)
99.14(3)
558.23(19), 2
1.358
a
(°)
b (°)
c
(°)
V (Å3), Z
Density (calc.) (g cmÀ3
)
Abs. coeff. (mmÀ1
F(0 0 0)
)
0.095
240
3.2. Single crystal X-ray crystallography of the haptens
h Ranges (data
collection)
Index ranges
1.54–25.27
1.13–25.29
Single crystal X-ray diffraction study has shown that compound
I crystallizes in the Triclinic crystal system (space group = P-1). A
packing diagram of I in the unit cell is shown in Fig. 3. A classic
hydrogen bonded dimer is formed by the carboxylic acid group
from two molecules (Table 5). The R22 (8) graph-set motif [41] links
the molecules into pairs around inversion centers in the unit cell.
The O–HÁ Á ÁO hydrogen-bonded interactions involving these pairs
are very strong and stabilize molecular packing of these dimers
0 6 h 6 6
À9 6 k 6 9
À16 6 l 6 16
2248
0.013
2026
À21 6 h 6 0
0 6 k 6 6
À17 6 l 6 17
2622
0.091
2538
Full-matrix least-
squares
1.002
Reflections collected
Rint
Independent reflections
Refinement method on F2 Full-matrix least-
squares
1.004
Goodness-of-fit on F2
Final R indices [I > 2
R indices (all data)
r(I)]
0.0488
0.0632
0.144/À0.272
0.0674
0.1641
0.145/À0.133
Table 3
Residual (e ÅÀ3
)
Atomic coordinates of non-hydrogen atoms (II) and their thermal parameters.
Atom
x
y
z
U (eq)
Table 2
Atomic coordinates of non-hydrogen atoms (I) and their thermal parameters.
C1
O1
C2
O2
C3
O3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
0.26224(17)
0.19395(15)
0.29759(18)
0.01361(15)
0.35175(18)
0.05722(14)
0.3879(2)
0.4410(2)
0.4607(2)
0.4295(2)
0.37356(19)
0.3386(2)
0.8180(7)
0.4585(6)
0.8428(7)
0.4799(6)
1.0295(7)
0.2233(5)
1.0498(8)
1.2291(9)
1.3985(9)
1.3825(8)
1.1960(7)
1.1699(8)
0.9862(7)
0.9636(8)
0.7865(9)
0.6244(7)
0.6312(6)
0.4597(7)
0.2713(7)
0.1240(8)
0.2924(8)
0.2685(2)
0.13925(16)
0.1850(2)
0.11187(18)
0.1704(2)
0.00616(19)
0.0856(2)
0.0713(3)
0.1431(4)
0.2239(3)
0.2422(3)
0.3248(2)
0.3408(2)
0.4260(2)
0.4420(2)
0.3722(2)
0.2870(2)
0.2176(2)
0.2411(2)
0.1594(3)
0.0904(3)
0.0525(9)
0.0970(10)
0.0613(10)
0.0986(10)
0.0595(10)
0.0943(10)
0.0780(12)
0.0899(14)
0.0898(14)
0.0811(12)
0.0606(10)
0.0680(11)
0.0572(10)
0.0778(12)
0.0837(13)
0.0660(11)
0.0504(9)
Atom
x
y
z
U (eq)
O1
O2
O3
C1
C2
C3
C4
C5
C6
C7
0.6995(3)
0.9663(2)
0.2354(2)
0.7661(3)
0.5705(3)
0.6424(3)
0.4342(3)
0.4727(3)
0.2826(3)
0.3038(3)
0.1079(3)
0.1326(4)
0.3532(4)
0.5450(3)
0.5261(3)
0.7203(3)
0.6957(3)
0.8657(2)
0.9652(2)
0.6741(2)
0.8856(2)
0.8013(2)
0.8189(2)
0.7365(2)
0.7352(2)
0.6536(2)
0.6510(2)
0.5685(3)
0.5714(3)
0.6558(3)
0.7353(2)
0.7353(2)
0.8173(2)
0.8166(2)
1.46691(9)
1.37329(9)
1.12317(9)
1.38016(12)
1.29244(12)
1.19024(12)
1.10492(13)
0.99783(12)
0.92175(12)
0.81878(12)
0.73941(13)
0.64160(14)
0.61678(14)
0.69075(13)
0.79394(12)
0.87373(13)
0.97201(13)
0.0765(5)
0.0642(5)
0.0701(5)
0.0470(5)
0.0473(5)
0.0435(4)
0.0428(4)
0.0384(4)
0.0395(4)
0.0391(4)
0.0504(5)
0.0600(5)
0.0600(6)
0.0512(5)
0.0396(4)
0.0469(5)
0.0465(5)
0.28497(19)
0.2506(2)
0.1975(2)
C8
C9
0.1747(2)
C10
C11
C12
C13
C14
0.20430(18)
0.17310(17)
0.11276(18)
0.08697(19)
0.0501(2)
0.0572(10)
0.0637(10)
0.0763(12)
0.0717(11)

Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
209
Table 4
Theoretical and X-ray crystallography data of I and II.
I
II
Parameters
X-ray
Calculated
Parameters
X-ray
Calculated
O1–C1
O2–C1
O3–C4
C1–C2
C2–C3
C3–C4
C4–C5
C5–C6
C5–C14
C6–C7
C7–C12
C7–C8
1.302(2)
1.214(2)
1.216(2)
1.492(2)
1.511(2)
1.511(2)
1.492(2)
1.374(2)
1.420(2)
1.412(2)
1.419(2)
1.421(2)
1.352(3)
1.411(3)
1.360(3)
1.414(2)
1.418(2)
1.356(2)
1.3299
1.1891
1.197
C1–C2
1.400(4)
1.441(4)
1.458(4)
1.221(3)
1.399(4)
1.223(4)
1.423(4)
1.426(4)
1.305(4)
1.356(5)
1.425(5)
1.328(5)
1.430(5)
1.385(5)
1.384(5)
1.414(4)
1.354(5)
1.396(5)
1.372(4)
1.472(4)
1.510(4)
1.503(4)
1.499(5)
1.3907
1.4299
1.4505
1.1959
1.3914
1.1867
1.4354
1.4209
1.3317
1.348
C1–C10
C1–C14
O1–C15
C2–C3
O2–C18
C3–C4
C3–C8
O3–C18
C4–C5
C5–C6
C6–C7
C7–C8
C8–C9
C9–C10
C10–C11
C11–C12
C12–C13
C13–C14
C14–C15
C15–C16
C16–C17
C17–C18
1.5055
1.5216
1.5176
1.4997
1.3654
1.4218
1.4149
1.4091
1.4216
1.3573
1.4176
1.3582
1.4202
1.4194
1.3575
1.4322
1.3478
1.435
C8–C9
C9–C10
C10–C11
C11–C12
C12–C13
C13–C14
1.3883
1.3885
1.4344
1.3453
1.4271
1.3546
1.5058
1.5252
1.5273
1.5102
O2–C1–O1
O2–C1–C2
O1–C1–C2
C1–C2–C3
122.66(15)
124.72(16)
112.62(15)
114.19(15)
111.61(14)
120.04(15)
120.41(15)
119.54(14)
119.17(15)
118.20(15)
122.62(15)
121.34(15)
119.00(15)
121.90(15)
119.10(15)
120.42(17)
120.56(18)
120.61(17)
120.51(17)
122.59(15)
118.80(15)
118.62(15)
121.19(15)
120.67(15)
122.2805
125.9762
111.7434
112.0937
112.1655
120.7656
120.6854
118.549
119.2261
118.0797
122.6942
121.3972
119.0342
121.7855
119.1803
120.6154
120.1566
120.5423
120.5537
122.2415
118.9517
118.8068
120.9283
120.6074
C4–C3–C2
O3–C4–C5
O3–C4–C3
C5–C4–C3
C6–C5–C14
C6–C5–C4
C14–C5–C4
C5–C6–C7
C6–C7–C12
C6–C7–C8
C2–C1–C10
C2–C1–C14
C10–C1–C14
C3–C2–C1
C2–C3–C4
C2–C3–C8
C4–C3–C8
C5–C4–C3
C4–C5–C6
C7–C6–C5
118.0(3)
124.1(3)
117.9(3)
121.7(4)
121.0(4)
119.9(3)
119.1(4)
121.2(4)
119.0(4)
121.8(4)
121.2(4)
118.1(4)
124.1(4)
117.7(4)
122.9(4)
121.5(4)
119.4(3)
119.1(4)
121.9(4)
119.4(4)
123.3(4)
118.4(3)
118.4(3)
123.1(3)
123.1(3)
116.3(3)
120.6(3)
111.6(3)
112.2(3)
121.8(4)
122.0(4)
116.1(4)
117.8507
124.1966
117.9041
122.1844
121.8723
119.5772
118.5502
120.8095
120.5773
120.4241
120.7333
118.6163
122.4741
118.9043
121.8089
120.5586
119.9184
119.5102
120.8871
119.9011
121.9999
119.7144
114.866
C12–C7–C8
C9–C8–C7
C6–C7–C8
C9–C8–C3
C9–C8–C7
C3–C8–C7
C8–C9–C10
C11–C10–C9
C10–C11–C12
C11–C12–C13
C11–C12–C7
C13–C12–C7
C14–C13–C12
C13–C14–C5
C10–C9–C8
C9–C10–C11
C9–C10–C1
C11–C10–C1
C12–C11–C10
C11–C12–C13
C14–C13–C12
C13–C14–C1
C13–C14–C15
C1–C14–C15
O1–C15–C14
O1–C15–C16
C14–C15–C16
C17–C16–C15
C18–C17–C16
O2–C18–O3
O2–C18–C17
O3–C18–C17
125.3693
120.0166
119.2785
120.5029
111.817
116.0185
122.2231
124.1248
113.6265
Fig. 2. (Left): X-ray structure with atoms numbering of I. Ellipsoids are drawn at 50% probability. (Right): The theoretical optimized geometric structure of I.

210
Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
Fig. 3. Packing diagram of I in the unit cell. Hydrogen bonds are shown as dashed lines.
Fig. 4. (Left): X-ray structure with atoms numbering of II. Ellipsoids are drawn at 50% probability. (Right): The theoretical optimized geometric structure of II.
Table 5
Table 6
Hydrogen bonding parameters (Å, °) of I.
Hydrogen bonding parameters (Å, °) of II.
D–HÁ Á ÁA
d(D–H)
d(HÁ Á ÁA)
d(DÁ Á ÁA)
<D–HÁ Á ÁA
D–HÁ Á ÁA
d(D–H)
d(HÁ Á ÁA)
d(DÁ Á ÁA)
<D–HÁ Á ÁA
O1–H1A–O2 #1
0.8200
1.8600
2.6743(19)
174.00
O3–H3A–O2 #1
C2–H2A–O1
0.8200
0.9300
1.8400
2.1700
2.654(4)
2.822(5)
173.00
126.00
Symmetry transformations used to generate equivalent atoms #1 2 À x, 2 À y, 3 À z.
Symmetry transformations used to generate equivalent atoms: #1 Àx, 1 À y, Àz.
into a unique assembly. The naphthalene ring parts are plane (the
dihedral angle of C9–C8–C7–C6 is 1.27°) and form parallel layers
with a distance of 2.870 Å between the dimers. The dihedral angle
of the naphthalene ring and the R22 (8) graph-set motif is 3.52°.
Compound II crystallizes in the Monoclinic crystal system
(space group = P21/c). A packing diagram of II in the unit cell is
shown in Fig. 5. Similarly to compound I, a classic hydrogen
bonded dimer is formed by the carboxylic acid group from two
molecules (Table 6). The R22 (8) graph-set motif links the molecules
into pairs around inversion centers in the unit cell. Besides, The
intramolecular C2–HÁ Á ÁO1 hydrogen bonding causes the formation
of a planar six-membered ring, which is also coplanar with the
adjacent anthracene ring (Fig. 5 and Table 6). The anthracene ring
parts are plane (the dihedral angle of C5–C4–C3–C2 is 0.62° and
C3–C2–C1–C14 is 1.45°) and form parallel layers with a distance
of 2.484 Å between the dimers. The dihedral angle of the anthra-
cene ring and the R22 (8) graph-set motif is 80.08°.
According to the X-ray data in Table 4, the bond lengths and an-
gles of the corresponding parts of I and II are almost same with
those of naphthalene and anthracene [12]. The biggest difference
between I and naphthalene is 0.0099 Å at (C7–C12), while between
II and anthracene is 0.0311 Å at (C12–C13), respectively.
3.3. Quantum chemical calculations
3.3.1. Molecular geometry of the haptens
For comparison with the X-ray study, the optimized structures
of I and II by HF calculations are shown in Figs. 2 and 4,
respectively.
Fig. 5. Packing diagram of II in the unit cell. Hydrogen bonds are shown as dashed.

Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
211
Fig. 6. The frontier molecular orbitals (HOMO and LUMO) for I and II by using HF.
Fig. 7. The electrostatic potential isosurfaces of I and II. The blue areas indicate positive potential and the red areas indicate negative potential.
It is interesting that the orientation of the O1 atom in the theo-
retically computed molecule and in the molecule calculated from
diffraction data is absolutely different (Fig. 4). The reason perhaps
is that the calculation was based on an isolated gaseous molecule,
while the X-ray diffraction on solid-state molecules. Crystal
particles present periodically ordered in the micro space. It is the
order that fix the orientation of the O1 atom in crystal state.
The optimized structure parameters are also listed in Table 4.
Because of the hydrogen bonds (Figs. 3 and 5, Tables 5 and 6),
the theoretical values of the bond lengths and angles related to

212
Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
Fig. 8. The charge distribution of I-BSA and II-BSA.
the carboxyl groups are some different from their crystal data.
Whereas other parameters computed are almost same with the
values obtained from X-ray crystallographic data.
4. Conclusion
Two new haptens for PAHs were synthesized. Hapten I has a
carboxyl group with four length of spacer arm at 2-position of
naphthalene, and II at 1-position of anthracene. Single crystal
X-ray structure showed that a classic O–HÁ Á ÁO hydrogen bonded
dimmer is formed by the carboxylic acid group for both of the hap-
tens. The geometries of the corresponding parts of the haptens are
almost the same as naphthalene and anthracene. The HF computa-
tional calculations provided the supports that the size, shape
(geometry) and electronic properties at the corresponding parts
of the haptens did not change significantly, compared to naphtha-
lene and anthracene. These results showed that the new haptens
could be used to induce antibodies for PAHs.
3.3.2. Frontier molecular orbital and energies of the haptens
The frontier-orbital energies of a compound can play an impor-
tant role in biological activity [42]. EHOMO is a rough measure of the
electron-donating ability of a compound and, normally, increasing
its value can increase the biological activity, while the ELUMO acts in
reverse. It is clear from the graphical representation of the HOMO
and LUMO for I and II (Fig. 6), that the electron cloud of HOMO is
mainly focused on the naphthalene and anthracene rings. These
parts were most likely the important portions of the epitopes for
antibody recognition.
Supplementary material
3.3.3. Electrostatic potential isosurface map of the haptens
From the electrostatic potential isosurface map of a compound,
we can get which region is electron rich region, which is the lack
e-region, so we can know which atoms susceptible to nucleophile
attack, which attacked by electrophilic reagents.
Fig. 7 is the electrostatic potential isosurface maps of I and II.
The blue areas indicate positive potential and the red areas indi-
cate negative potential. For the corresponding parts of naphthalene
and anthracen, the positive electrostatic potential is more evenly
distributed at the edge of the rings. The negative electrostatic po-
tential is distributed in both sides of the ring plane. Compared to
those of naphthalene and anthracen [43], the electrostatic poten-
tial isosurface of the corresponding parts of I and II do not change
significantly.
Crystallographic data for the structural analysis of the synthe-
sized compounds have been deposited at the Cambridge Crystallo-
graphic Data Center, 12 Union Road, Cambridge, CB2 1EZ, UK, and
are available free of charge from the Director on request quoting
the deposition number CCDC 759778 and 798276 (fax: C44 1223
336033, e-mail: deposit@ccdc.cam.ac.uk).
Acknowledgement
This work was supported by the National High-tech R&D Pro-
gram of China (No. 2007AA02Z200 and 2007AA06A402).
References
3.3.4. Charge distribution of the conjugates
[1] R.G. Harvey, Polycyclic Aromatic Hydrocarbons, John Wiley, New York, 1997.
[2] P. Garrigues, M. Lamotte, Polycyclic Aromatic Compounds: Synthesis,
Properties, Analytical Measurements, Occurrence and Biological Effects,
Gordon and Breach Science Publishers, Amsterdam, 1993.
[3] J.R. Dias, Handbook of Polycyclic Hydrocarbons: Part A, Benzenoid
Hydrocarbons (Physical Sciences Data, No. 30), Elsevier, New York, 1987.
[4] J.C. Fetzer, Large (C P 24) Polycyclic Aromatic Hydrocarbons: Chemistry and
Analysis, John Wiley, New York, 2000.
[5] M.L. Lee, M.V. Novotny, K.D. Bartle, Analytical Chemistry of Polycyclic Aromatic
Compounds, Academic Press, New York, 1981.
[6] R.A. Hites, W.J. Simonsick Jr., Calculated Molecular Properties of Polycyclic
Aromatic Hydrocarbons, Elsevier, New York, 1987.
The immunoconjugate region determines the priority of the rec-
ognition sites of the antibodies. The more the similarity between
the corresponding region of a compound and the specific region
of an immunoconjugate, the higher the affinity of the resulting
antibodies for this analyte.
The charge distributions of naphthalene, anthracen, and the
conjugates are shown in Fig. 8 for comparison. The biggest change
of the charge distribution at the corresponding parts of I-BSA and
II-BSA is at the carbon atom connected to the carbonyl group. The
changes are from (À0.2092) e to (À0.1300) e for I-BSA, and from
(À0.1810) to (À0.0682) for II-BSA, respectively. The changes are
not significant.
[7] P.V.R. Schleyer, Chem. Rev. 101 (2001) 1115.
[8] G. Portella, J. Poater, J.M. Bofill, P. Alemany, M. Sola, J. Org. Chem. 70 (2005)
2509.
[9] J. Poater, X. Fradera, M. Duran, M. Sola, Chem. Eur. J. 9 (2003) 400.

Y. Li et al. / Journal of Molecular Structure 987 (2011) 206–213
213
[10] K.B. Wiberg, J. Org. Chem. 62 (1997) 5720.
[28] C.R. Spier, E.S. Bromage, T.M. Harris, M.A. Unger, S.L. Kaattari, Anal. Biochem.
387 (2009) 287.
[29] N. Delaunay-Bertoncini, V. Pichon, M.C. Hennion, J. Chromatogr. A 999 (2003)
3.
[30] A.V. Wiznycia, J. Desper, C.J. Levy, Chem. Commun. 37 (2005) 4693.
[31] M.N. Inbasekaran, D.T. Witiak, J. Med. Chem. 23 (1980) 278.
[32] C.J. Wu, F.S. Li, J. Agro-Environ. Sci. 26 (2007) 1080 (Journal written in Chinese.
CAN: 150:391462).
[33] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112.
[34] Y. Li, J.Y. Xia, Li, F.S. Li, Comput. Appl. Chem. 26 (2009) 1503 (Journal written in
Chinese. CAN: 152:557814).
[11] S.T. Howard, T.M. Krygowski, Can. J. Chem. 75 (1997) 1174.
[12] C.H. Suresh, S.R. Gadre, J. Org. Chem. 64 (1999) 2505.
[13] P.J. Garratt, Aromaticity, Wiley, New York, 1986.
[14] E. Clar, The Aromatic Sextet, John Wiley and Sons Ltd., London, 1972.
[15] G.M. Badger, Aromatic Character and Aromaticity, Cambridge University Press,
Cambridge, 1969.
[16] A.H. Neilson, PAHs and Related Compounds: Biology, Springer, New York,
1998.
[17] A. Luch, The Carcinogenic Effects of Polycyclic Aromatic Hydrocarbons,
Imperial College Press, London, 2005.
[18] D.M. Jerina, H. Yagi, R.E. Lehr, D.R. Thakker, M. Schaefer-Ridder, J.M. Karle,
W.W.A.W. Levin, R.L. Chang, A.H. Cohney, Polycyclic Hydrocarbons and Cancer,
Academic Press, New York, 1978.
[19] D.L. Poster, M.M. Schantz, L.C. Sander, S.A. Wise, Anal. Bioanal. Chem. 386
(2006) 859.
[35] D.Y. Wu, Y. Li, F.S. Li, Comput. Appl. Chem. 26 (2009) 329 (Journal written in
Chinese. CAN: 151:562151).
[36] M.P. Marco, S. Gee, B.D. Hammock, Trends Anal. Chem. 14 (1995) 341.
[37] C.P. Price, D.J. Newman, Principles and Practice of Immunoassay, Grove’s
Dictionaries, 1991.
[20] S. Moret, L.S. Conte, J. Chromatogr. A 882 (2000) 245.
[21] J. de Boer, R.J. Law, J. Chromatogr. A 1000 (2003) 223.
[22] P.M. Kramer, Anal. Chim. Acta 376 (1998) 3.
[23] D. Barcelo, A. Oubina, J.S. Salau, S. Perez, Anal. Chim. Acta 376 (1998) 49.
[24] K.A. Fahnrich, M. Pravda, G.G. Guilbault, Anal. Lett. 35 (2002) 1269.
[25] A. Szekacs, H.M. Le, D. Knopp, R. Niessner, Anal. Chim. Acta 399 (1999) 127.
[26] K. Li, R.L. Chen, B.T. Zhao, M. Liu, A.E. Karu, V.A. Roberts, Q.X. Li, Anal. Chem. 71
(1999) 302.
[38] F.S. Li, J.Y. Xia, Y. Li, Agro-Environ. Sci. 27 (2008) 1697 (Journal written in
Chinese. CAN: 152:34630).
[39] F.S. Li, F. Sun, X.J. Liu, H.H. Cui, Agric. Sci. China 6 (2007) 964.
[40] M.H. Goodrow, B.D. Hammock, Anal. Chim. Acta 376 (1998) 83.
[41] J. Bernstein, R.E. Davis, L. Shimoni, N.L. Chang, Agnew. Chem. Int. Ed. 34 (1995)
1555.
[42] S.W. Yao, V.H.C. Lopes, F. Fernandez, X. Garcia-Mera, M. Morales, J.E.
Rodriguez-Borges, M.N.D.S. Cordeiro, Bioorg. Med. Chem. 11 (2003) 4999.
[43] J.W. Zou, B. Zhang, G.X. Hu, Z.C. Shang, Q.S. Yu, Acta Chim. Sinica 62 (2004) 241
(Journal written in Chinese. CAN: 140:186519).
[27] Y.F. Zhang, Q.M. Zhang, Z.X. Gao, S.G. Dai, J. Agro-Environ. Sci. 25 (2006) 537
(Journal written in Chinese. CAN: 148:162781).