JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2003; 16: 148–156
Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.621
Synthesis, characterization and immunochemical
evaluation of cephalosporin antigenic determinants
Francisco Sánchez-Sancho1†, Ezequiel Perez-Inestrosa1*, Rafael Suau1, Marı́a I. Montañez1,
Cristobalina Mayorga2, Maria J. Torres2, Antonino Romano3 and Miguel Blanca4
1
Department of Organic Chemistry. Faculty of Science. University of Malaga, Malaga, Spain
Research Unit for Allergic Diseases, Carlos Haya Hospital, Malaga, Spain
3
Department of Internal Medicine and Geriatrics, UCSC-Allergy Unit, C.I. Columbus and IRCCS Oasi Maria S.S. Troina, Italy
4
Allergic Service, Hospital Universitario La Paz, Madrid, Spain
2
Lack of knowledge of the exact chemical structure of cephalosporin antigenic determinants has hindered
clinical interpretation of adverse reactions to these drugs and delayed understanding of the mechanisms
involved in the specific recognition and binding of IgE molecules to these antigenic determinants. We
further resolve the relationship between structure and activity of proposed antigenic chemicals, including
the rational design and synthesis of these haptenic structures. Comparative RAST inhibition studies of the
synthesized molecules revealed that they were recognized by IgE antibodies induced by cephalosporin
antibiotics. Thus, these data indicate that recognition is mainly directed to the acyl side chain and to the blactam fragment that remains linked to the carrier protein in the cephalosporin conjugation course.
Copyright # 2003 John Wiley & Sons, Ltd.
Keywords: immediate reactions; lactams; IgE; antibiotics; molecular recognition; cephalosporins; immunochemistry
Received 31 October 2002; revised 3 March 2003; accepted 4 March 2003
INTRODUCTION
b-Lactam antibiotics, mainly penicillins and cephalosporins
(Fig. 1), are the drugs that most frequently cause adverse
reactions by specific immunologic mechanisms (Dewdney,
1977; Weiss and Adkinson, 1988). The great use of these
antibiotics, as well as the wide diversity of their chemical
structures, has led to the appearance of immunologic
responses that recognize each of these structures as
different, with clinical relevance in the induction of allergic
reactions (Blanca, 1995).
Of all the b-lactams, penicillins are the best studied so far
(Weiss and Adkinson, 1988; Blanca et al., 1994), probably
due to their greater prescription and consumption (Batchelor
et al., 1965; Levine and Ovary, 1961). Since penicillins have
very similar chemical structures, differing only in the nature
of the side-chain group (Fig. 1), benzylpenicillin has
traditionally been considered as the reference model for
the study of allergy to b-lactams. The ‘major’ antigenic
determinant is benzylpenicilloyl (Fig. 1, R=PhCH2), formed
by the nucleophilic opening of the b-lactam ring by the
amino group of proteins found in either plasma or cell
membranes (Dewdney, 1977; Batchelor et al., 1965; Moreno
*Correspondence to: E. Perez-Inestrosa, Department of Organic Chemistry,
Faculty of Science, University of Malaga, E-29071 Malaga, Spain.
E-mail: [email protected]
†Current address: Pharma Mar, S.A. Madrid, Spain.
Contract/grant sponsor: FEDER; contract/grant number: 1FD97-0516.
Abbreviations used: AMP, ampicillin; AX, amoxycillin; MDM, minor
determinant mixture; PG, penicillin G; PLL, poly-L-lysine; PPL, penicilloylpolylysine; RAST, radioallergosorbent test.
Copyright # 2003 John Wiley & Sons, Ltd.
et al., 1995), resulting in a covalent binding of the antibiotic
to the carrier protein to yield the hapten-carrier conjugate.
Nevertheless, a number of studies have shown that
betalactam structures with different side chains contribute
to the specific response, and it has become clear that these
new conjugates formed with these penicillins have also to be
considered for immunologic tests (Moreno et al., 1995).
Although cephalosporins have an increasing role in the
induction of allergic reactions (Romano et al., 2000), they
have barely been investigated and the determinants involved
in the immunological responses are largely unknown.
Despite the structural similarities between penicillins and
cephalosporins, they differ considerably in the products
obtained from the nucleophilic opening of the b-lactam ring.
The equivalent ‘major’ antigenic determinant for cephalosporins would be the cephalosporoyl (Batchelor et al.,
1966). However, this compound is unstable and suffers an
extensive fragmentation process (Fig. 1).
The immunologic behavior of these antibiotics is
determined by their intrinsic chemical reactivity, which is
related with the capacity of the b-lactam carbonyl group to
act as the acylating agent to form the corresponding
antigenic determinants with protein amino groups (Batchelor et al., 1966; Kaiser and Kukolja, 1972). The reactivity
of this group is higher in penicillins, due to the strain in the
chemical structure caused by the cis fusion between the two
heterocycles of four and five atoms. In cephalosporins the
heterocycles are of four and six atoms, which results in less
strain in the b-lactam ring (Lowe, 1979). Thus, in penicillins
the nucleophilic ring opening takes place more quickly and
effectively. Furthermore, the conjugate obtained in this way,
the ‘major’ antigenic determinant, is stable enough to be
CEPHALOSPORIN ANTIGENIC DETERMINANTS
149
Figure 1. Structures of some frequently used penicillins and cephalosporins and their nucleophilic ring
opening. Whereas the penicilloyl derivative is a stable and chemically well-characterized structure, the
cephalosporoyl analog evolves to further uncharacterized end products.
isolated and characterized by common methods. However,
in cephalosporins, the lower reactivity of the b-lactam ring
slows the reaction. Nevertheless, the existence of a good
leaving group at position 3' of the dihydrothiazine ring,
which occurs in most clinically important cephalosporins,
enhances the reactivity of the b-lactam carbonyl via the
associated elimination of R2. In fact, the rate of opening of
the b-lactam ring and the antibacterial activity both increase
as the ability of the group at 3' to act as a leaving group
increases (Kaiser and Koklja, 1972; Holden, 1984).
Experimental and theoretical studies have been interpreted
in terms of b-lactam ring opening non-concerted with the
departure of the leaving group R2 (Faraci and Pratt, 1984).
Nevertheless, no intermediate metabolite corresponding to
cephalosporoyl has yet been isolated and fully characterized. The conjugate obtained this way is unstable and
Copyright # 2003 John Wiley & Sons, Ltd.
undergoes a process of multiple fragmentation in the
dihydrothiazine portion, which leads to a great number of
degradation products, more so if the great variety of
chemical structures for cephalosporins is taken into account
(Fig. 1). This complicates the isolation and characterization
of the haptenic structures and the possible antigenic
determinants formed.
Several studies have attempted to elucidate the possible
chemical structures obtained after the reaction of different
cephalosporins with ammonia, amino acids and simple
amino compounds, and under hydrolysis conditions on the
basis of UV (Hamilton-Miller et al., 1970a) and NMR
spectra (Grabowski et al., 1985; Vilanova et al., 1993;
Llinás et al., 1998), as well as isolation by HPLC analysis
(Vilanova et al., 1993; Llinás et al., 1998). Thus, structures
such as polymers, piperazinediones, penaldates and penaJ. Mol. Recognit. 2003; 16: 148–156
150
F. SÁNCHEZ-SANCHO ET AL.
maldates have been proposed (Dewdney, 1977; HamiltonMiller et al., 1970a; Grabowski et al., 1985; Vilanova et al.,
1993; Llinás et al., 1998; Manhas and Bose, 1971).
However, the assignments are somewhat tentative and
ammonolysis (Grabowski et al., 1985) in liquid ammonia at
50 °C has characterized the intermediate of cephamycin.
Initially the cephalosporin undergoes b-lactam cleavage and
has a sufficient lifetime for carbon-13 spectral characterization.
Direct observation of cephalosporoate intermediates in
aqueous solutions has been also reported (Pratt and Faraci,
1986). The combination of absorption and NMR spectral
evidence shows that, in those cases investigated, 3'
elimination from cephalosporins is not concerted. These
cephalosporoates are chemical compounds whose stability
and lability depend on the nature of the R2 substituents. The
authors conclude that, with better leaving groups than those
examined, a concerted elimination might occur. As a result,
it is claimed that comparison of the hydroxide-catalyzed
elimination rate constant suggests that the intermediate
cephalosporoate would not be seen during alkaline hydrolysis of cephalosporins with good 3' leaving groups.
The production of monoclonal antibodies has shown that
cephalosporins can generate unique structures capable of
inducing a specific immunologic response without crossreacting with the classical structures (Nagakura et al.,
1990). Consequently, allergic reactions to cephalosporins
may occur by sensitization to determinants similar to those
of penicillins or to specific cephalosporin haptens (Blanca et
al., 1994; Pham and Baldo, 1996; Baldo A, 1999). On the
other hand, there is evidence that the C-7 substitution (R1
acyl chain) plays a dominant role in determining the
specificity of immunologic reactions between individual
cephalosporins and between penicillins and cephalosporins
to a greater degree than that observed for penicillins
(Manhas and Bose, 1971).
EXPERIMENTAL
Chemical synthesis
General. Melting points were determined on a Gallenkamp
instrument and are given uncorrected. MS (EI) were
recorded on an HP-MS 5988A spectrometer operating at
70 eV. HRMS were recorded on an AutoSpecE, CACTI,
University of Vigo (Spain). NMR spectra were recorded on
a Bruker WP-200 SY instrument at 200 MHz for 1H and
50.3 MHz for 13C. Chemical shifts are given relative to the
residual signal of solvents, dH 7.24 ppm and dC 77.0 ppm for
deuteriochloroform. NOE experiments were recorded on a
Bruker (500 MHz), University of Santiago (Spain). Optical
rotations were determined by using a Perkin-Elmer 241
digital polarimeter. Elemental analyses were carried out in
the Microanalytical Laboratory, SCAI, University of
Malaga. TLC analyses were performed on silica gel 60 F
256 plates and column chromatography was carried out on
silica gel 60 (70–230 mesh). Organic solutions were dried
with MgSO4 and concentrated in vacuo.
Compounds 2 (Paruszewski et al., 1996) and 3 (Wagatsuma et al., 1973) were prepared according to the sequence
Boc-Ala-OH → Boc-Ala-nBu(2) → Ala-nBu(3) by proceCopyright # 2003 John Wiley & Sons, Ltd.
dures common for the preparation of peptides and showed
spectroscopic data according to that reported in the
literature.
The following compounds were prepared according to
literature procedures: 4a (Bucourt et al., 1978); 4c (Boger et
al., 1997) and 4f (Kuisle et al., 1999). 4b, 4d and all other
reagents were purchased from Aldrich, Sigma or Fluka.
Procedure for the synthesis of compounds 5a–f. Solutions
of the corresponding acids 4a–f were sequentially treated
with isobutylchloroformate and the amine, 3. After workup, the corresponding pure compounds 5a–f were obtained.
(2S)-N1-butyl-2-({(methoxyimino)[2-(trytilamino)-1,3thiazol-4-yl]acetyl}amino)propanamide (5a): white solid
(635 mg, 60% yield); m.p. = 96–100 °C; [a]D = 37.0 (c = 1
in CHCl3). Analysis calculated for C32H35N5O3S—C,
67.46; H, 6.19; N, 12.39; S, 5.63; found—C, 67.54; H,
6.08; N, 12.19; S, 5.34.
(2S)-N1-butyl-2-[2-(thienylacetyl)amino]propanamide
(5b): white solid (225 mg, 40% yield); recrystallized with
CHCl3-hexane; m.p. = 154–155 °C; [a]D = 43.7 (c = 1,
MeOH). Analysis calculated for C13H20N2O2S—C, 58.18;
H, 7.51; N, 10.44; S, 11.95; found—C, 58.03; H, 7.49; N,
10.19; S, 11.84. HRMS (EI) calculated for C13H20N2O2S—
268.1245; found—268.1245.
(2S)-2-{[(2R)-(tert-butoxycarbonylamino)(4-hydroxyphenyl)acetyl]amino}-N1-butylpropanamide (5c): white solid (243 mg, 55% yield); m.p. = 172–174 °C; [a]D = 111.8
(c = 1.7, CHCl3). Analysis calculated for C20H31N3O5—C,
61.05; H, 7.94; N, 10.68; found—C, 59.94; H, 8.06; N,
10.25. (2S)-2-{[(2R)-(tert-butoxycarbonylamino)(phenyl)
acetyl]amino}-N1-butylpropanamide (5d): white solid
(310 mg, 80% yield); recrystallized with CH2Cl2-Et2O;
m.p. = 194–196 °C; [a]D = 105.9 (c = 1, CHCl3). Analysis
calculated for C20H31N3O4—C, 63.64; H, 8.28; N, 11.13;
found—C, 63.31; H, 8.48; N, 11.01.
(2S)-N1-butyl-2-{[(2R)-phenyl(tetrahydro-2H-pyran-2-yloxy)acetyl]amino}propanamide (5e): thick oil (219 mg,
57% yield). Analysis calculated for C20H30N2O4—C,
66.27; H, 8.34; N, 7.73; found—C, 66.13; H, 8.43; N, 7.68.
(2S)-N1-butyl-2-{[2-furyl(oxo)acetyl]amino}propanamide (5f): yellow solid (760 mg, 40% yield); m.p. = 84–86
°C; [a]D = 82.1 (c = 1, CHCl3). Analysis calculated for
C13H18N2O4—C, 58.63; H, 6.81; N, 10.52; found—C,
58.63; H, 6.91; N, 10.51.
Preparation of compounds 1a–f. (2S)-2-{[(2-amino-1,3thiazol-4-yl)(methoxyimino)acetyl]amino}-N1-butylpropanamide (1a): compound 5a was treated with a 50% aqueous
formic acid solution for 30 min at 70 °C. After work-up,
pure 1a (71%) was obtained: white solid; m.p. = 83–86 °C;
[a]D = 46.9 (c = 1, MeOH). 1H NMR (200 MHz, CD3OD):
= 7.05 (s, 1H), 4.51 (q, 1H, J = 7.3 Hz), 4.05 (s, 3H), 3.31
(m, 2H), 1.70–1.30 (m, 4H), 1.48 (d, 3H, J = 7.3 Hz), 1.01 (t,
3H, J = 7.3 Hz). 13C NMR (50 MHz, CD3OD): = 174.4,
171.5, 165.0, 150.3, 143.6, 111.9, 63.2, 50.7, 40.3, 32.6,
21.0, 17.8, 14.2. MS (EI) m/z (%): 327 (7) [M]‡, 296 (3),
227 (92), 197 (52), 156 (80), 125 (100), 83 (23). Analysis
calculated for C13H21N5O3S—C, 47.69; H, 6.47; N, 21.39;
S, 9.79; found— C, 47.84; H, 6.12; N, 21.05; S, 9.62. HRMS
(EI) calculated for C13H21N5O3S—327.1365; found—
327.1361.
J. Mol. Recognit. 2003; 16: 148–156
CEPHALOSPORIN ANTIGENIC DETERMINANTS
Hydrolysis of tert-butoxycarbonyl group. General procedure for the synthesis of compounds 1c and 1d: an ethyl
acetate solution of the corresponding 5c and 5d was treated
with 3 M aqueous HCl solution, at room temperature.
Quantitative yield, white solids.
(2S)-2-{[(2R)-amino(4-hydroxyphenyl)acetyl]amino}N1-butylpropanamide, hydrochloride (1c): m.p. = 182–184
°C; [a]D= 85.0 (c = 0.2, CH3OH). 1H NMR (200 MHz,
D2O): = 7.35 and 6.96 (AA'BB', 4H), 5.08 (s, 1H,), 4.24 (b
q, 1H, J = 7.3 Hz), 3.20 (b t, 1H, J = 6.7 Hz), 1.54–1.13 (m,
4H), 1.25 (d, 3H, J = 7.3 Hz), 0.87 (t, 3H, J = 7.3 Hz). 13C
NMR (50 MHz, D2O): = 175.4, 169.6, 158.2, 130.8, 124.6,
117.3, 57.0, 51.2, 40.1, 31.4, 20.3, 17.5, 13.9. Analysis
calculated for C15H24ClN3O3—C, 54.62; H, 7.33; N, 12.74;
found—C, 54.43; H, 7.01; N, 12.51.
(2S)-2-{[(2R)-amino(phenyl)acetyl]amino}-N1-butylpropanamide, hydrochloride (1d): m.p. = 196–198 °C;
[a]D = 50.0 (c = 0.5, CH3OH). 1H NMR (200 MHz,
D2O): = 7.51 (s, 5H), 5.13 (s, 1H), 4.26 (q, 1H,
J = 7.3 Hz), 3.21 (t, 2H, J = 7.3 Hz), 1.59–1.19 (m, 4H),
1.26 (d, 3H, J = 7.3 Hz), 0.89 (t, 3H, J = 7.3 Hz). 13C NMR
(50 MHz, D2O): = 175.4, 169.4, 132.8, 131.4, 130.7,
128.9, 57.5, 51.2, 40.2, 31.4, 20.3, 17.5, 13.9. Analysis
calculated for C15H24ClN3O2—C, 57.41, H, 7.71; N, 13.39;
found— C, 57.03; H, 7.75; N, 13.26.
(2S)-N1-Butyl-2-{[(2R)-hydroxy(phenyl)acetyl]amino}
propanamide (1e): compound 5e was treated with p-TsOH
in MeOH at room temperature (80% yield); m.p. = 138–140
°C; [a]D = 94.1 (c = 1, CHCl3). 1H NMR (200 MHz, D2O):
= 7.32 (m, 5H), 7.05 (b d, 1H, J = 6.7 Hz), 6.18 (b t, 1H,
J = 4.9 Hz), 5.02 (d, 1H, J = 3.5 Hz), 4.41 (dq, 1H,
J = 6.7 Hz, J = 7.3 Hz), 3.68 (d, 1H, J = 3.5 Hz), 3.05
(distorted q, 1H, J = 6.7 Hz), 1.40–1.07 (m, 4H), 1.31 (d,
3H, J = 7.3 Hz), 0.81 (t, 3H, J = 7.3 Hz). 13C NMR
(50 MHz, CDCl3): = 172.3, 171.8, 139.4, 128.7, 128.5,
126.7, 74.3, 48.7, 39.2, 31.3, 19.8, 18.1, 13.6. MS (EI) m/z
(%): 278 (<1) [M]‡, 235 (1), 206 (3), 179 (21), 171 (100),
129 (54), 118 (67), 107 (35), 79 (42). Analysis calculated for
C15H22N2O3—C, 64.73, H, 7.97; N, 10.06; found—C,
64.28; H, 8.04; N, 10.11. HRMS (EI) calculated for
C15H22N2O3—278.1630; found—278.1634.
(2S)-N1-Butyl-2-{[2-furyl(syn-metoxyimino)acetyl]amino} propanamide (1f): compound 5f was treated with
methoxylamine hydrochloride and Na2CO3 in MeOH:H2O
(1:3) at pH 7. The mixture was refluxed for 22 h. The crude
mixture of syn/anti isomers (relative proportion 6:1) was
flash chromatographied (CHCl3/MeOH 9.8:0.2) to afford
the pure syn-1f (22% yield): m.p. = 102–105 °C;
[a]D = 48.3 (c = 1, CHCl3). 1H NMR (200 MHz, D2O):
= 7.49 (d, 1H, J = 1.8 Hz), 6.79 (b s, 1H), 6.78 (d, 1H,
J = 3.7 Hz), 6.45 (dd, 1H, J = 3.7 Hz, J = 1.8 Hz), 6.17 (b s,
1H), 4.58 (q, 1H, J = 7.3 Hz), 4.04 (s, 3H), 3.25 (distorted q,
2H, J = 6.7 Hz), 1.45 (d, 3H, J = 6.7 Hz), 1.5–1.2 (m, 4H),
0.90 (t, 3H, J = 7.3 Hz). 13C NMR (50 MHz, CDCl3):
= 171.1, 160.4 and 145.7, 144.7, 144.0, 113.9, 111.8, 63.5,
49.2, 39.4, 31.5, 19.9, 19.2, 13.7. MS (EI) m/z (%): 295 (5),
195 (100), 165 (99), 152 (41), 124 (98), 94 (74), 93 (90).
HRMS (EI) calculated for C14H21N3O4—295.1532;
found—295.1536.
Copyright # 2003 John Wiley & Sons, Ltd.
151
Immunochemical studies
Subjects. Sera from four patients who suffered an
anaphylactic shock after the intake of ceftriaxone (sera 1
and 2) or cefuroxime (sera 3 and 4) were used for the
immunochemical studies. They were three females and one
male, with ages ranging from 21 to 47 years. In all cases the
blood was collected between 2 and 6 months after the
reaction. The patients were evaluated using skin tests to
different cephalosporin compounds, including the culprit
drug, and in vitro specific cephalosporin IgE antibodies
were determined by radioallergosorbent test (RAST).
Skin tests. Prick and intradermal tests were carried out
using penicilloylpolylysine (PPL) (Allergopharma Merck,
Reinbeck, Germany), minor determinant mixture (MDM)
(Allergopharma), penicillin G (PG) (Normon S.A., Madrid,
Spain), amoxycillin (AX; SB SmithKline Beecham, Madrid,
Spain) (20 mg/ml) and ampicillin (AMP; Normon, Madrid,
Spain) (20 mg/ml) as described (Romano et al., 2000). The
final concentrations were 5 10 5 mmol/L for PPL,
2 10 2 mmol/L for MDM, 10,000 IU/mL for PG, and
20 mg/mL for AMP and AX (the last three were diluted in
0.9% NaCl). They were also performed with different
cephalosporins, using cefuroxime (Curoxim, Glaxo, Madrid, Spain), ceftazidime (Glazidim, Glaxo), cefotaxime
(Zariviz, Hoechst Roussel, Milan, Italy), ceftriaxone
(Rocefin, Roche, Basel, Switzerland) cephalothin (Keflin,
Lilly, Sesto Fiorentino, Italy) and cefamandole (Mandokef,
Lilly) at concentrations of 2 mg/ml in 0.9% NaCl. All of the
above reagents were initially tested on volar forearm skin by
the prick method and reactions were considered positive
when a wheal >3 mm in diameter was present 20 min later.
When prick tests were negative, 0.01 ml of the reagent
solution was injected intradermally in volar forearm skin.
Readings were made 20 min after injections. Results were
considered positive when there was an increase in the wheal
of >3 mm. Positive controls for prick and intradermal tests
were made with histamine (at 10 and 1 mg/mL, respectively). Normal saline was used as a negative control. The
concentration used for cephalosporins had proved to be nonirritant in a control group of 40 healthy subjects.
Radioimmunoassay for IgE determination. These were
made by RAST to PG, AX, AMP and to different
cephalosporins (ceftazidime, ceftriaxone, cefuroxime, cefotaxime, cefadroxile, cephalexine) conjugated to poly-Llysine (PLL) (Sigma) as previously described (Blanca et al.,
1992). Blood samples were obtained when patients were
evaluated and sera were kept at 20 °C until assayed.
Samples were considered positive if they were higher than
2.5% of label uptake, which was the mean ‡2 SD of the
negative control group, with total IgE ranging from 8 to 1
300 kU/l (Blanca et al., 1992).
Study of the immunochemical recognition. In order to
determine the capacity of the different synthetic structures
to be recognized by specific IgE antibodies, competitive
inhibition immunoassays were carried out with sera from
the four patients with immediate allergic reactions to
cephalosporin (ceftriaxone and cefuroxime) as described
(Moreno et al., 1995). This was undertaken using in the fluid
J. Mol. Recognit. 2003; 16: 148–156
152
F. SÁNCHEZ-SANCHO ET AL.
Figure 2. Proposed aminolysis pathway of cephalosporins and retrosynthetic analysis for proposed haptenic
structure 1 derived after b-lactam opening by amine nucleophiles.
phase the sera from the patients incubated with 10-fold
concentrations (from 150 to 15 mM) of the five compounds
synthesized, 1a–f. Only two concentrations were used in
order to save sera, because many determinations were
required. Based on previous studies these concentrations,
although high, were known not to induce non-specific
binding (Moreno et al., 1995). After 18 h, disks sensitized
with ceftriaxone for sera 1 and 2 and cefuroxime for sera 3
and 4, conjugated to PLL, were added. Results were
expressed as percentage inhibition with respect to the noninhibited serum.
RESULTS AND DISCUSSION
Synthesis
Previous work by others on the conjugation of different
cephalosporins with simple amino compounds (HamilstonMiller et al., 1970b) and our preliminary observations by
NMR techniques employing n-butylamine as the nucleophile, point towards the formation of the cephalosporoyl
conjugate at the first moment of the reaction. This
compound suffers extensive fragmentation of the dihydrothiazine portion as soon as it is formed, leading to a
complex mixture of compounds difficult to isolate and
analyze. Nevertheless, as can be inferred by NMR data, the
acyl side chain would appear to remain virtually unaffected,
giving rise to a conjugate such as compound 1 (Fig. 2), with
a penaldate-like structure.
Obviously, the carbon atom from which the G group
derives, in the final compound 1, is originally like a
carbonyl group and its nature is a priori speculative, since in
the physiological media it can undergo different side
reactions and become oxidized to a diverse extent. This
particular characteristic of 1 should certainly modulate the
extent of recognition by IgE antibodies, but in order to
Copyright # 2003 John Wiley & Sons, Ltd.
explore the inherent nature of this biological process, we
decided initially to establish the basis of the structural
requirements. Furthermore, the possibility of using different
aminoacids would increase the versatility of this synthetic
strategy to produce diverse molecules, which would help
further understand the nature of the recognition process.
Due to the difficulties encountered in the isolation of pure
compounds from the complex mixtures obtained in the
conjugation process, we decided to synthesize conjugates
with compound 1 structure in which G is a methyl group.
These would allow us to have access to essentially pure
compounds with defined chemical structures and to evaluate
their ability to recognize IgE antibodies to cephalosporin. A
retrosynthetic analysis for the synthesis of these compounds
is shown in Fig. 2. They were devised so as to be derived
from the corresponding acyl side chain, plus an alpha
aminoacid and butylamine, which are readily available
starting materials.
The synthetic route is depicted in Fig. 3. We chose Lalanine as the model aminoacid (G = CH3, Fig. 2) to
evaluate the extent of the recognition process by the
antibody. The synthetic scheme was essentially the same for
all the compounds obtained, differing only in the nature of
the protecting groups employed in each acyl side chain (4a–
f) and therefore in the final deprotection step.
Compound 2 was obtained in good yield by reaction of NBOC-L-alanine with n-butylamine via previous carboxyl
group activation as a mixed anhydride. This compound was
subsequently deprotected under acidic conditions to obtain
compound 3, which is conveniently functionalized and
ready for amide formation with acyl side chains 4a–f to
obtain 5a–f. The last step of the synthetic scheme was
carried out under the same conditions employed for the
synthesis of compound 2 and took place in good to moderate
yields for 1a–e. All the compounds obtained, as well as the
intermediates in their syntheses, were conveniently analyzed by spectroscopic and analytical techniques, all the
J. Mol. Recognit. 2003; 16: 148–156
CEPHALOSPORIN ANTIGENIC DETERMINANTS
153
Figure 3. Synthetic route for the products 1a±f. Compound 3 was obtained as the base structure, to which the
libraries of acyl side chains were coupled by amide linkage.
Table 1. Results of skin tests to different cephalosporins and penicillin derivatives, including the culprit
cephalosporin
Penicillin derivatives
Pat
1
2
3
4
Drug involved
PPL
MDM
Cephalosporin derivatives
PG
AX
AMP
‡
‡
‡
Ceftriaxone
Ceftriaxone
Cefuroxime
Cefuroxime
CL
CM
CZ
CT
‡
‡
‡
CU
‡
‡
CX
‡
‡
Penicillin derivatives: PPL, benzylpenicilloyl determinant; MDM, minor determinant mixture; PG, penicillin G; AX, amoxicillin; AMP,
ampicillin.
Cephalosporin derivatives: CL, cephalothin; CM, cefamandole; CZ, ceftazidime; CT, ceftriaxone; CU, cefuroxime; CX, cefotaxime.
Table 2. Results of skin tests and speci®c IgE antibody RAST to different cephalosporins and penicillins, including
the culprit cephalosporin
Penicillin
Pat
1
2
3
4
Cephalosporin
Drug involved
PG
AX
AMP
CDX
CLX
CZ
CT
CU
CX
Ceftriaxone
Ceftriaxone
Cefuroxime
Cefuroxime
0.1
0.71
0.06
0.2
0
0.04
0.11
0
0
0.11
0.13
0.4
0.18
0.02
0.12
0.03
0.41
0.19
0.31
0.09
0.14
0.31
0.01
0.67
4.53
17.7
0.16
1.99
0.09
1.86
9.43
20.8
0.46
40.7
0.89
13.9
Penicillins: PG, penicillin G; AX, amoxicillin; AMP, ampicillin.
Cephalosporins: CDX, cefadroxil; CLX, cephalexine; CZ, ceftazidime; CT, ceftriaxone; CU, cefuroxime; CX, cefotaxime.
Copyright # 2003 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2003; 16: 148–156
154
F. SÁNCHEZ-SANCHO ET AL.
Figure 4. RAST inhibition studies with sera from four patients allergic to cephalosporins. (A) RAST inhibition
with sera from the patients allergic to ceftriaxone at two concentrations (150 and 15 mM) of different
compounds. In both sera (serum 1 and 2) the maximum inhibition was seen with 1a, the synthesized
compound having the same acyl side chain as ceftriaxone. (B) RAST inhibition with sera from the patients
allergic to cefuroxime at two concentrations (150 and 15 mM) of different compounds. For these patients,
compounds with chemically similar acyl side chains show comparable inhibition levels, with the compound
having the same side chain as cefuroxime producing the highest inhibition (1f).
data being in agreement with the proposed structures. The
synthesis of 1f (syn configuration) required, in the final step,
a dehydrating condensation between the keto-carbonyl
group of the a-furanacetic moiety in 4f and methoxyamine.
This yielded the two syn/anti isomers in the carbon nitrogen
double bond and further exhaustive chromatographic
separations were necessary to separate the two isomers,
Copyright # 2003 John Wiley & Sons, Ltd.
the relative syn/anti configuration of which was determined
by NOE experiments.
Immunochemical studies
The recognition of the different structures synthesized was
J. Mol. Recognit. 2003; 16: 148–156
CEPHALOSPORIN ANTIGENIC DETERMINANTS
made by competitive inhibition studies of RAST immunoassay using human sera from the four patients with
allergic reactions to cephalosporins. The patients were
diagnosed by skin test (Table 1) and RAST studies (Table
2), which demonstrated IgE antibodies that specifically
recognized the cephalosporin involved in the immediate
reaction and, in some cases, cross-reactivity to other
cephalosporins and/or penicillins.
The RAST inhibition studies provided more precise
information concerning the specific recognition by the IgE
antibodies of the synthetic compounds, because their
structure and concentration are well characterized. This
method was undertaken with serum from each of the four
patients included. The results with serum 1 [Fig. 4(A)],
specific to ceftriaxone, showed that the optimal recognition
was made with the synthetic structure 1a, which included
the same side chain structure as ceftriaxone, and also to the
other structures in decreasing order 1e, 1b, 1d, with no
recognition of 1c. Serum 2 [Fig. 4(A)] showed a strong
binding with structure 1a, which had the same structure as
the side chain of several cephalosporins, i.e. ceftriaxone,
which induced the reaction, cefotaxime and ceftazidime.
These results were confirmed by skin test and, to a certain
extent, RAST. The inhibition with serum 3 [Fig. 4(B)],
specific to cefuroxime, showed that optimal recognition was
obtained with the structure containing its corresponding side
chain 1f, followed by 1a and 1b. The results obtained with
serum 4 [Fig. 4(B)] showed a specific recognition of the
structure 1f, containing the side chain of cefuroxime, which
was the culprit cephalosporin in the reaction. There was also
a specific binding with the structure 1a, which had a
chemical similarity to 1f. The IgE antibodies present in this
serum showed no specific binding with the other structures
assayed. These results indicated that differences existed in
the behavior of specific IgE antibodies of serum from the
four patients studied. In sera 2, 3 and 4 the recognition was
more specific to the acyl side chain structure, whereas in
serum 1, besides the side chain, the butyl-alanyl moiety also
seemed to contribute to the recognition process.
These results reveal that structures 1a–f, representing a
fragment of the proposed cephalosporoyl intermediate, are
suitably recognized by IgE antibodies directed to cephalosporins, and that specificities are related to the acyl side
chain as well as to the b-lactam fragment, which remains
linked to the carrier protein in the conjugation process.
In contrast to the great number of studies related to the
chemistry of the synthesis and reactivity of penicillins, in
which the chemical structures are unmistakably established,
the chemistry of cephalosporins lacks this conclusive
accuracy, mainly in reactivity, despite which the same
chemical repercussions have been assumed for cephalosporins. This supposition has been reflected in the
immunological and clinical postulates, the negative consequences of which may well be very important.
Despite the lack of certainty about the chemical structure
of the antigenic determinant responsible for allergic reactions to cephalosporins, several studies (Pham and Baldo,
1996; Baldo, 1999; Harle and Baldo, 1990) have reported
immunological results, although the conclusions have to be
taken with a certain degree of speculation. Because the
haptenic determinant of cephalosporins produced by their
degradation is largely unknown, more definite studies
Copyright # 2003 John Wiley & Sons, Ltd.
155
require each free drug to be used as the skin test agent to
detect antibodies reactive to these antibiotics (Baldo, 1999;
Weiss and Adkinson, 1988). Besides the requirement for
specific immunoassays to detect IgE antibodies to individual
cephalosporins, and the need to define the allergenic
determinants in these drugs, other potentially helpful data
on a number of fundamental and applied aspects of
cephalosporin immunochemistry are absent. These include
allergic cross-reactivity with other b-lactams, the contribution of side-chain structures to allergenicity, and the extent
of heterogeneity of cephalosporin allergenic determinants.
Consequently, many questions on cross-reactivities between
cephalosporins, and between cephalosporins and penicillins,
cannot currently be answered with confidence. This raises
difficulties in the selection of antibiotics for some penicillinand/or cephalosporin-allergic subjects.
The antigen binding activity is found in the variable
region of antibodies, but there are structural constraints
conditioning its ability to bind specifically to one or more
closely related small molecules. Thus, synthesis of 1a–f has
provided, for the first time, structural information concerning
the chemical implications in the recognition requirements,
and has allowed us to evaluate the extent of the heterogeneity
indicated by the evident and fine differences in recognition
of different sera. We propose this synthetic methodology as
the right course for the systematic study of the chemical
implications in these recognition processes in order to define
the allergenic determinants of cephalosporins.
CONCLUSION
A series of six aN-acyl-L-alanylbutanamides was prepared
in four steps from N-BOC-L-alanine and the appropriate
acids. These chemically well-defined structures comprised
the entire acyl side chain and the aminoacidic residue
included in the b-lactam moiety of the cephalosporins
studied, and were linked as amide functions to an aliphatic
(n-butyric) chain. They were used to study the molecular
basis of the recognition of different cephalosporins by IgE
antibodies from subjects allergic to ceftriaxone and
cefuroxime. Based on the results obtained in RAST
inhibition studies, which were well correlated with both
skin test and RAST results, fine structural recognition was
detected between IgE antibodies in different sera and
compounds 1a–f. Thus, these synthesized compounds
incorporate the appropriate epitope recognizable by IgE
antibodies and provide the basis to understand the pathway
of cephalosporin conjugation to carrier proteins, to determine the structural requirements needed to be recognized by
IgE antibodies and, finally, to obtain an efficient in vitro test
to detect these antibodies. Chemical structure modifications
are now under way in our laboratories to enhance the
specificity and sensitivity of the recognition process.
Acknowledgements
F.S.-S. is grateful for a fellowship linked to this project. The authors are
also grateful to Professor D. Dominguez for recording the NOE
experiments. The authors thank Ian Johnstone for help with the English
language.
J. Mol. Recognit. 2003; 16: 148–156
156
F. SÁNCHEZ-SANCHO ET AL.
REFERENCES
Baldo BA. 1999. Penicillins and cephalosporins as allergensÐ
structural aspects of recognition and cross-reaction. Clin.
Exp. Allergy 29: 744±749.
Batchelor FR, Dewdney JM, Gazzard D. 1965. Penicillin allergy:
the formation of the penicilloyl determinant. Nature 206: 362±
364.
Batchelor FR, Dewdney JM, Weston RD, Wheeler AW. 1966. The
immunogenicity of cephalosporins derivatives and their
cross-reaction with penicillin. Immunology 10: 21±33.
Blanca M. 1995. Allergic reactions to penicillins. A changing
world? Allergy 50: 777±782.
Blanca M, Mayorga C, Perez E, Suau R, Juarez C, Vega JM,
Carmona MJ, Perez-Estrada M, Garcia J. 1992. Determination
of IgE antibodies to the benzyl penicilloyl determinant. A
comparison between poly-L-lysine and human serum albumin as carriers. J. Immunol. Meth. 153: 99±105.
Blanca M, Vega JM, Garcia J, Miranda A, Carmona MJ, JuaÂrez C.
1994. New aspects of allergic reactions to beta-lactamsÐ
crossreactions and unique speci®cities. Clin. Exp. Allergy 24:
407±415.
Boger DL, Borzilleri RM, Nukui S, Beresis RT. 1997. Synthesis of
the vancomycin CD and DE ring systems. J. Org. Chem. 62:
4721±.
Bucourt R, Heymes R, Lutz A, PeÂnasse L, Perronnet J. 1978.
Cephalosporines a chaines amino-2 thiazolyl-4 acetyles.
In¯uence de la presence et de la con®guration d'un grupe
axyimino sur l'activite antibacterienne. Tetrahedron 34:
2233±2243.
Dewdney JM. 1977. Antigens. In Immunology of the Antibiotics,
Sela M (ed). Academic Press: New York; 73±245.
Faraci WS, Pratt RF. 1984. Elimination of a good leaving group
from the 3'-position of a cephalosporin need not be concerted
with b-lactam ring opening: TEM-2 b-lactamase-catalyzed
hydrolysis of pyridine-2-azo-4'-(N',N'-dimethylaniline) cephalosporin (PADAC) and of cephaloridine. J. Am. Chem. Soc.
106: 1489±1490.
Grabowski EJJ, Douglas AW, Smith GB. 1985. Ammonolysis of
cephalosporins: 13C NMR characterization of the intermediates from b-lactam ring cleavage prior to loss of the 3'-group.
J. Am. Chem. Soc. 107: 267±268.
Hamilton-Miller JMT, Newton GGF, Abraham EP. 1970a. Products of aminolysis and enzymic hydrolysis of the cephalosporins. Biochem. J. 116: 371±384.
Hamilton-Miller JMT, Richards E, Abraham EP. 1970b. Changes
in proton magnetic resonance spectra during aminolysis and
enzymic hydrolysis of cephalosporins. Biochem. J. 116: 385.
Harle DG, Baldo BA. 1990. Drugs as allergens: an immunoassay
for detecting IgE antibodies to cephalosporins. Int. Arch.
Allergy Appl. Immunol. 92: 439±444.
Holden KG. 1984. Cephalosporins. In Comprehensive Heterocyclic Chemistry, Vol. 7, Katritzky AR (ed). Pergamon Press:
Oxford; 285±339.
Kaiser GV, Kukolja S. 1972. Modi®cation of the b-lactam ring. In
Cephalosporins and Penicillins. Chemistry and Biology,
Copyright # 2003 John Wiley & Sons, Ltd.
Flynn EH (ed). Academic Press: New York; 74±133.
Kuisle O, QuinÄoa E, Riguera R. 1999. A general methodology for
automated solid-phase synthesis of depsides and depsipeptidesÐpreparation of a valinomycin analog. J. Org. Chem.
64: 8063±8075.
Levine BB, Ovary Z. 1961. Studies on the mechanism of the
formation of the penicillin antigen III. The N-(D-a-benzylpenicilloyl) group as an antigenic determinant responsible for
hypersensitivity to penicillin G. J. Exp. Med. 114: 875±904.
LlinaÂs A, Vilanova B, Frau J, MunÄoz F, Donoso J, Page MI. 1998.
Chemical-reactivity of penicillins and cephalosporinsÐintramolecular involvement of the acyl-amido side-chain. J. Org.
Chem. 63: 9052±9060.
Lowe G. 1979. b-Lactam antibiotics. In Comprehensive Organic
Chemistry, Vol. 5, Barton D, Ollis D (eds). Pergamon Press:
Oxford; 289±320.
Manhas MS, Bose AK. 1971. b-Lactams: Natural and Synthetic.
Part 1, Bose AK (ed). Wiley-Interscience: New York.
Moreno F, Blanca M, Mayorga C, Terrados S, Moya MC, PeÂrez E,
Suau R, Vega JM, GarcõÂa J, Miranda A, Carmona MJ. 1995.
Studies of the speci®cities of IgE antibodies found in sera
from subjects with allergic reactions to penicillins. Int. Arch.
Allergy Immunol. 108: 74±81.
Nagakura N, Shimizu T, Masuzawa T, Yanagihara Y. 1990. Anticephalexin monoclonal antibodies and their cross-reactivities to cephems and penams. Int. Arch. Allergy Appl.
Immunol. 93: 126±132.
Paruszewski R, Rostai®nska-Suchar G, Strupinska M, Jaworski P,
Stables JP. 1996. Synthesis and anticonvulsant activity of
some amino acid derivatives. Part 1: alanine derivatives.
Pharmazie 51: 145±148.
Pham NH, Baldo BA. 1996. b-Lactam drug allergens: ®ne
structural recognition patterns of cephalosporin-reactive IgE
antibodies. J. Mol. Recognit. 9: 287±296.
Pratt RF, Faraci WS. 1986. Direct observation by 1H NMR of
cephalosporoate intermediates in aqueous solution during
the hydrazinolysis and b-lactamase-catalyzed hydrolysis of
cephalosporins with 3' leaving groups: kinetics and equilibria
of the 3' elimination reaction. J. Am. Chem. Soc. 108: 5328±
5333.
Romano A, Mayorga C, Torres MJ, Artesani MC, Suau R, Sanchez
F, Perez E, Venuti A, Blanca M. 2000. Immediate allergic
reactions to cephalosporins: cross-reactivity and selective
response. J. Allergy Clin. Immunol. 106: 1177±1183.
Vilanova B, MunÄoz F, Donoso J, Blanco FG. 1993. HPLC and 1H
NMR studies of alkaline-hydrolysis of some 7-(oxyiminoacyl)cephalosporins. Helv. Chim. Acta 76: 2789±2802.
Wagatsuma M, Terashima S, Yamada SI. 1973. Amino acids and
peptides. VI. Novel peptide bond formation catalyzed by
metal ions. IV. Formation of optically active amino acid
amides and peptide amides. Chem. Pharm. Bull. 21: 422±427.
Weiss M, Adkinson NF. 1988. Immediate hypersensitivity reactions to penicillin and related antibiotics. Clin. Allergy 18:
515±540.
J. Mol. Recognit. 2003; 16: 148–156