Synthesis of mercapto-(+)-methamphetamine haptens and their use for obtaining improved epitope density on (+)-methamphetamine conjugate vaccines

Article abstract of DOI:10.1021/jm2004943

This study reports the synthesis of the mercapto-hapten (S)-N-(2-(mercaptoethyl)-6-(3-(2-(methylamino)propyl)phenoxy)hexanamide [3, (+)-METH HSMO9] and its use to prepare METH-conjugated vaccines (MCV) from maleimide-activated proteins. MALDI-TOF mass spectrometry analysis of the MCV synthesized using 3 showed there was a high and controllable epitope density on two different carrier proteins. In addition, the MCV produced a substantially greater immunological response in mice than previous METH haptens, and a monoclonal antibody generated from this MCV in mice showed a very high affinity for (+)-METH (K_D = 6.8 nM). The efficient covalent coupling of (+)-METH HSMO9 to the activated carrier proteins suggests that this approach could be cost-effective for large-scale production of MCV. In addition, the general methods described for the synthesis of (+)-METH HSMO9 (3) and its use to synthesize MCV will be applicable for conjugated vaccines of small molecules and other substances of abuse such as morphine, nicotine, and cocaine.

Full text of DOI:10.1021/jm2004943

ARTICLE
pubs.acs.org/jmc
Synthesis of Mercapto-(+)-methamphetamine Haptens and Their Use
for Obtaining Improved Epitope Density on (+)-Methamphetamine
Conjugate Vaccines
F. Ivy Carroll,*,† Bruce E. Blough,† Ramakrishna R. Pidaparthi,† Philip Abraham,† Paul K. Gong,† Liu Deng,†
Xiaodong Huang,† Melinda Gunnell,‡ Jackson O. Lay, Jr.,§ Eric C. Peterson,‡ and S. Michael Owens‡
†Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park,
North Carolina 27709, United States
‡Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock,
Arkansas 72205, United States
§Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States
S Supporting Information
b
ABSTRACT: This study reports the synthesis of the mercapto-hapten (S)-N-(2-(mercaptoethyl)-6-(3-(2-
(methylamino)propyl)phenoxy)hexanamide [3, (+)-METH HSMO9] and its use to prepare METH-con-
jugated vaccines (MCV) from maleimide-activated proteins. MALDI-TOF mass spectrometry analysis of the
MCV synthesized using 3 showed there was a high and controllable epitope density on two different carrier
proteins. In addition, the MCV produced a substantially greater immunological response in mice than previous
METH haptens, and a monoclonal antibody generated from this MCV in mice showed a very high affinity for
(+)-METH (KD = 6.8 nM). The efficient covalent coupling of (+)-METH HSMO9 to the activated carrier
proteins suggests that this approach could be cost-effective for large-scale production of MCV. In addition, the general methods
described for the synthesis of (+)-METH HSMO9 (3) and its use to synthesize MCV will be applicable for conjugated vaccines of
small molecules and other substances of abuse such as morphine, nicotine, and cocaine.
ethamphetamine’s (METH) ease of illegal
(+)-Msynthesis, low cost, long duration of action,
and powerful rewarding effects lead to destructive individual and
societal health in the United States.1 Chronic METH use causes
addiction, neurological damage, and altered behavioral and
cognitive functions.2ꢀ5 Acutely, METH can produce renal and
liver failure, cardiac arrhythmias, heart attack, stroke, and
seizures.2,6,7 At present there are no specific pharmacotherapies
for treating these adverse medical effects.
Since METH’s psychostimulant and addictive properties are
mediated through sites of action in the central nervous system
(CNS), METH must first cross the bloodꢀbrain barrier to
produce its neurological effects. A novel treatment strategy is
to use high affinity anti-METH antibody medications as phar-
macokinetic antagonists to prevent or slow METH’s access to
these CNS sites of action.8ꢀ10 Anti-METH immunotherapy
includes high affinity, preformed anti-METH monoclonal anti-
bodies (mAb) for immediate protection, and anti-METH anti-
bodies generated over time through active immunization with
METH-conjugate vaccines (MCV).11 With both types of im-
munotherapy, preclinical studies suggest that the two major
determinants of therapeutic success are the affinity of the
antibodies for METH and the ability to maintain adequate levels
of anti-METH antibodies in the vascular circulation.8ꢀ10
The important process of creating high affinity anti-METH
antibodies starts with immunization of animals with a MCV
consisting of a METH-like hapten attached (conjugated) to an
antigenic carrier protein (e.g., albumin, keyhole limpet
hemocyanin) in a specific molecular orientation. The METH-
like hapten is composed of a METH backbone molecular
structure linked to a spacer group at a site that is distal to the
key molecular features to be recognized by the immune system.
The other end of the linker terminates in a functional group that
can readily form covalent attachments to the antigenic carrier
protein. The linker arm also serves to separate the hapten from
the carrier protein at an optimal distance to maximize affinity and
specificity for METH.12,13 The chemical bonds formed at both
ends of the linker and the length of this spacer arm (e.g., nine
carbon atoms) are critical to the quality (e.g., affinity and
specificity for METH) and quantity (i.e., amount of antibody)
of the immune response.
We previously described the synthesis of METH haptens 2a
[designated (+)-METH MO10] and 2b [designated (+)-METH
MO6] and their use for the development of highly refined mAb
medications for treating METH abuse.12 These METH haptens
proved extremely valuable for producing anti-METH mAb and
for understanding immune system structureꢀactivity require-
ments for control of the specificity and affinity of anti-METH
antibodies. However, the anti-METH serum antibody titers to
Received: April 21, 2011
Published: June 17, 2011
r
2011 American Chemical Society
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Scheme 1a
a Reagents: (a) THF BF3, THF, 0 °C; (b) HCO2NH4, 5% Pd/C, CH3OH, 50 °C; (c) BBr3, CH2Cl2, ꢀ78 °C; (d) (Boc)2O, CH2CI2, Et3N; (e)
3
Br(CH2)5CO2CH3, NaH, DMF; (f) LiOH, H2O, CH3OH, THF; (g) (C6H5)3CS(CH2)2NH2, EDCl, HOAt, DMF; (h) TFA, CH2Cl2; (i) Hg(OAc)2,
C2H5OH; (j) H2S, C2H5OH.
MCVs were relatively low, and the immune response was
inconsistent. These factors made it an unsatisfactory prototype
MCV for use in humans. We also knew from our longer term
studies of phencyclidine-like haptens that the quality and quan-
tity of immune response are greatly improved with greater
phencyclidine hapten epitope densities (i.e., 8ꢀ12 haptens per
albumin molecule).14
for the treatment of human METH addiction will require high
affinity antibodies with KD for METH in the range of e10 to 20
nM.9 Previous METH hapten structureꢀactivity studies also
showed that the best chance for achieving these high affinity
antibodies would be through immunization with MCV contain-
ing haptens with long linker chains connected to the meta
position on the aromatic ring of METH.8,9,12 In this report, we
present the design and synthesis of the METH hapten 3
[designated (+)-METH HSMO9], which has an 11-atom linker
chain, and report that conjugation of 3 to antigenic carrier
proteins leads to MCV with significantly higher hapten to carrier
protein ratios than 2a, lower excesses of hapten to carrier protein,
and improved immune responses.
Thus, we reasoned that the low immune response to these
previous MCV was primarily due to the low METH hapten
epitope density (i.e., moles of hapten per mole of antigenic
carrier protein) on the MCV. Hapten epitope density on the
carrier protein is important because if there are too few haptens
on the carrier protein, the immune system will generate low levels
of anti-METH antibodies or even no immune response. For
example, when we covalently bound (+)-METH MO10 haptens
to prototype ovalbumin (OVA) and bovine serum albumin
(BSA) carrier proteins using a carbodiimide procedure, we could
only achieve a maximum METH hapten epitope density of
approximately 5:1 with both OVA and BSA.12,13,15 This relatively
low epitope density from the MCV synthesis occurred even when
the starting hapten/carrier protein ratio was g60:1. Studies of
the anti-METH immunological response to these antigens and
antigens with <5:1 epitope densities in mice revealed that MCV
with an epitope density of 5:1 produced a modest to weak
immune response, and epitope densities of <5:1 failed to
stimulate an anti-METH immune response. These data sug-
gested that procedures used for conjugation of hapten to protein
carriers were both inefficient and inadequate for our needs.
We hypothesized that an improved hapten to carrier protein
conjugation synthesis procedure could lead to increased MCV
epitope densities that utilized lower excesses of hapten to carrier
protein in the MCV synthesis. On the basis of over a decade of
preclinical testing of active vaccines and monoclonal antibodies
produced from METH conjugate vaccines, we think immunotherapy
’ CHEMISTRY
The synthesis of the METH hapten 3 is shown in Scheme 1.
Reduction of the N-formyl compound of 412 using diborane in
tetrahydrofuran provided the N-methyl compound 5. Subjection
of 5 to transfer hydrogenation using ammonium formate and 5%
palladium on carbon catalyst in methanol yielded 6. O-Demethy-
lation of 6 using boron tribromide in methylene chloride afforded
the phenol 7. Treatment of 7 with di-tert-butyl dicarbonate in
methanol containing triethylamine gave the N-Boc-protected 8.
The sodium salt of the phenol 8 prepared by using sodium
hydride in dimethylformamide was alkylated with methyl 6-bro-
mohexanoate to give 9. Hydrolysis of 9 using lithium hydroxide
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in a waterꢀmethanolꢀtetrahydrofuran mixture afforded the acid
10. Coupling 10 with 2-(tritylmercapto)ethylamine using 1-eth-
yl-3-(3-diethylaminopropyl)carbodiimide (EDCI) and 1-hydro-
xy-7-azabenzotriazole (HOAt) in dimethylformamide followed
by removal of N-Boc with trifluoroacetic acid in methylene
chloride yielded 11. Compound 11 was detritylated by treatment
with mercuric acetate in ethanol to give the mercuric acetate
sulfhydryl derivative which was treated with hydrogen sulfide to
give the free thiol 3 isolated as the tartrate salt. The free thiol
must be handled in nonoxidative conditions to avoid dimer
formation.
(+)-METH HSMO9 was coupled to commercially available
maleimide-activated BSA 12 by combining a solution of 3 in
degassed conjugation buffer with a reconstituted solution of the
maleimide-activated protein in buffer as shown in Scheme 2. The
commercially available lyophilized maleimide-activated BSA
contains buffer, and the solution is reconstituted with distilled
water. A portion of the reconstituted maleimide-activated protein
was set aside as an analytical control. The optimum conditions
for haptens incorporation were determined by assessing various
molar ratios of haptens to protein. Ratios of 10:1, 15:1, 20:1, and
25:1 were evaluated by adding various amounts of (+)-METH
HSMO9 stock solution to a stock solution of maleimide activated
protein. The protein solutions became cloudy after addition of
the hapten, suggesting changes in solubility due to hapten
incorporation. The optimum conditions from this experiment
were used to scale up production of (+)-METH HSMO9
MCV13 for studies in mice to determine immunological
response.
(+)-METH HSMO9 was also coupled to commercially avail-
able maleimide-activated OVA in a similar fashion. Several molar
ratios of hapten to protein were explored including 20:1, 30:1,
40:1, and 50:1. As with BSA, protein solutions became cloudy
after hapten addition, suggesting incorporation.
’ ANALYTICAL CHARACTERIZATION
The extent of hapten incorporation was determined by
MALDI-TOF using procedures developed for high mass
analysis.16 Figure 1 shows a representative MALDI-TOF mass
spectrum of a maleimide activated OVA protein before (control)
and after conjugation with (+)-METH HSMO9. Before the
MALDI-TOF analysis was conducted, the salts from each protein
solution were removed by equilibrium dialysis with multiple
changes of distilled water. As little as 20 μL of the MCV was
needed for this procedure. Previous experiments showed that
multiple steps of dialysis-based desalting lowered the measured
m/z value as alkali ions (usually Na+) were replaced with protons
until the solution was essentially salt free. These salts could not
be adequately eliminated using a ZipTip procedure nor did it
improve upon the results obtained from repeated dialysis. On the
basis of these observations and the potential loss of sample using
ZipTips for purification, desalting by equilibrium dialysis alone
was judged to be the best approach.
Scheme 2a
The MALDI measured mass value of the maleimide-activated
OVA was about 47 268 ( 100 Da. A peak at m/z 23 572 showed
the doubly charged species. Because of the low resolution at the
high m/z values for the MCV, the center of mass (centroid) of
the roughly Gaussian shaped peaks (see Figure 1) represents the
a Reagents: (a) (i) distilled H2O; (ii) HSMO9 hapten (3), phosphate
saline buffer, room temp, 2 h.
Figure 1. MALDI TOF mass spectra of maleimide activated OVA protein (a) before and (b) after reaction with (+)-METH HSMO9 hapten (3).
Before the MALTI-TOF analysis was conducted, the salts from protein solutions were removed by equilibrium dialysis with multiple changes of distilled
water. The left axis of parts a and b is signal intensity in arbitrary units.
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overlapping masses of an ensemble of components rather than a
single molecular species.
After (+)-METH HSMO9 was desalted, hapten epitope
density on the MCV could be calculated by the mass difference
between the maleimide-activated OVA (before hapten conjuga-
tion, Figure 1a) and the (+)-METH HSMO9-OVA final product
(Figure 1b). In this example the measured mass of the final
product was 49 951 ( 100 Da. Because we used an extensive (but
simple) desalting step before MALDI-TOF analysis, the mass
difference of 2683 Da could be attributed exclusively to (+)-
METH HSMO9 hapten incorporation. Dividing the 2683 Da
mass by the hapten mass (339 Da) yielded an average hapten
epitope density of 7.9 moles of (+)-METH HSMO9 per mole of
maleimide-activated OVA in this experiment. The noninteger
value resulting from this calculation (and the increased peak
width) in Figure 1b compared to Figure 1a occurred because the
data in Figure 1b reflect a distribution (or family) of haptenated
species (e.g., 7ꢀ9 haptens per mole of protein) rather than
incorporation of a single specific integer value of hapten per
protein molecule.
Figure 2. Relationship between the molar ratio of METH hapten to
maleimide activated BSA at the start of the synthesis versus the final
METH hapten epitope density at the end of reaction. Epitope density of
BSA was determined by MALDI-TOF MS.
reported that the METH antibody binding function of mono-
clonal antibodies generated from haptens with short linkers
attached at the para position of the aromatic ring is significantly
inactivated in vivo in rats within 24 h of antibody treatment.
However, this does not happen with monoclonal antibodies
generated from active immunization of mice with longer chain
METH-like haptens coupled to linker groups at the meta
position of the aromatic ring.10 On the basis of these discoveries,
we designed (+)-METH HSMO9 with a relatively long spacer
group connected to the meta position on the aromatic ring. In
addition, the linker in the maleimide-activated BSA and OVA
(see structure 12) used in our studies has a cyclohexylmethylene
in the spacer arm that is reported to decrease the rate of
hydrolysis of the maleimide group compared to similar
reagents.21 The only other example of the use of a Michael
addition in METH vaccine development using METH is the very
recent report by Moreno et al.22 In this study the spacer arm of
the maleimide-activated protein had three methylene groups (see
structure 14).
’ RESULTS AND DISCUSSION
The relationship between the degree of hapten incorporation
(i.e., epitope density) and the immune response is not known for
most haptenated antigens because it is usually very difficult to
accurately measure, yet it is a critical factor in immune response
optimization. A recent trend in vaccine development is to couple
haptens to proteins via the Michael addition of a free thiol to a
maleimide-activated protein. These reactions are high yielding
and efficient. They have been primarily used to couple peptides
to large antigenic proteins where an efficient chemical coupling
reaction is beneficial to overcome any steric issues of linking two
relatively large moieties. For example, the Danishefsky laboratory
has applied this linking approach to the synthesis of pentavalent
and hexavalent antigenic constructs in which multiple carbohy-
drate molecules are attached at each member of a peptide chain,
then linked to keyhole limpet hemocyanin.17
Most clinical vaccines are based on neutralizing the disease
processes of proteins, peptides, or carbohydrates. Not until
recently have clinically useful vaccines against small pharmaco-
logical active molecules been extensively explored, and most of
the studies involved drugs of abuse such as cocaine and
nicotine.18 Conjugation of small molecules to large antigenic
proteins using carboxylic acid coupling reactions can effectively
generate very high affinity monoclonal antibodies in mice, as was
observed for our MCV.10,12 However, greater and more repro-
ducible immune responses will be needed for human active
vaccines.8
OVA and BSA are not suitable for human vaccines because
these proteins are part of the diet of many humans. They can,
however, be used in animal studies as a proof of principle and
more importantly are small enough (<100 000 Da) to be directly
analyzed by MALDI-TOF for determination of hapten incor-
poration. Moreover, maleimide-activated OVA and BSA are
commercially available making the need to activate native protein
unnecessary.
An important problem with the current generation of nicotine
and cocaine conjugate vaccines for treating addiction is the poor
and unsustained immune response over time in human subjects.
Indeed, only about one-third of human subjects mounted even a
modest immune response.19,20 Therefore, anti-addiction vac-
cines that produce immune responses in 100% of the subjects
should be an important criterion for all anti-addiction vaccines.
To achieve this goal, improvements in epitope density along with
other factors are needed.
Toward the goal of developing a better MCV, a study was
conducted on the use of the Michael addition approach with our
methamphetamine-based haptens, (+)-METH HSM09 using
OVA and BSA as antigenic proteins. In previous studies, we
Initial probe experiments suggested that haptens additions
were significantly greater than 5:1 for both BSA and OVA;
therefore, in order to optimize epitope densities using this
approach, a range of molar equivalents were studied. Haptens
to protein molar ratios of 10:1, 15:1, 20:1, and 25:1 were
evaluated for additions to maleimide activated BSA, using BSA
with 14ꢀ16 available functional maleimides (as reported by
Pierce). The number of available maleimide-active sites on each
carrier protein varies between commercial batches and was
provided by the vendor (ThermoFisher Scientific). The opti-
mum molar ratio for maximum hapten incorporation was 20:1,
which provided an epitope density of 10:1 (Figure 2). This
finding was double the epitope density of our methamphetamine
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haptens on BSA compared to our earlier efforts using the
carboxylic acid coupling methods.12 Importantly we found a
linear relationship between final epitope density (y-axis) and the
starting molar ratios of (+)-METH HSMO9 hapten/protein
(x-axis) when using hapten/protein ratios up to 20:1. Less than a
2-fold excess of hapten to the number of maleimide active sites
was needed to achieve maximum covalent coupling with (+)-
METH HSMO9 and BSA in this experiment. In a later scale-up
experiment using a new batch of commercial maleimide-acti-
vated BSA with 18 functionally active maleimide sites, we used a
molar ratio of 22:1 and found 12 (+)-METH HSMO9 haptens
per BSA. This was a 140% increase in hapten incorporation
compared to coupling (+)-METH MO10 to BSA using carbo-
diimide chemistry material.12 This MCV was used for immuniza-
tion experiments (see below). Both experiments with BSA
suggested that maximum incorporation of METH haptens
requires only an approximate 1.8- to 2.0-fold excess of hapten
to the number functional maleimide sites. This suggests that
fairly precise epitope densities could be easily achieved for
refined studies of the effect of epitope density on immune
response. Furthermore, this appeared to be a very efficient
synthesis process, which would likely be cost-effective for large-
scale production of MCV.
A similar set of experiments were conducted using maleimide-
activated OVA with 12 functional maleimides, using 20:1, 30:1,
40:1, and 50:1. Western blot analysis of the MCV (data not
shown) suggested significant hapten incorporation. Mass spec-
tral analysis of the 30:1 experiment showed a hapten incorpora-
tion rate of eight (+)-METH HSMO9 haptens per maleimide-
activated OVA. This is a 60% increase in efficiency of coupling
compared to the carbodiimide coupling of (+)-METH MO10 to
OVA (+)-METH MO10 haptens per OVA (results not
shown).12
high epitope densities (24ꢀ29 METH haptens per BSA) and
produced high levels of circulating antibodies (45ꢀ108 μg/mL
of antibody) in mice after immunization. However, the anti-(+)-
METH immune response after their third immunization de-
creased. The anti-METH polyclonal antibodies in the mice were
only in the range of moderate to poor affinity (82ꢀ169 nM).
When we used similar short chain linkers coupled at the para
position, we generated similar low affinity antibodies in mice.10
However, very high affinity (9ꢀ16 nM) anti-METH monoclonal
antibodies that were produced from active immunization of mice
with longer chain METH-like haptens were coupled to linker
groups at the meta position of the aromatic ring.8,9,12
’ CONCLUSIONS
(+)-METH HSMO9 proved to be significantly better than
previous METH haptens by several measures. The major ad-
vantages include the ability to achieve high and easy to control
epitope densities on at least two different carrier proteins and the
substantially greater immunological response to this MCV in
mice following active immunization. We also showed the MCV’s
broader utility by generating a very high affinity mAb with a KD
for (+)-METH of 6.8 nM and a KI for (+)-MDMA of 1.1 nM.
Very efficient covalent coupling to the carrier proteins suggests
this hapten could be cost-effective for large-scale production of a
MCV. This is the second report of the synthesis of a mercapto-
hapten from a drug of abuse and its use to prepare conjugates for
antigenic proteins. The general methods reported for metham-
phetamine will be applicable to other small molecules and
substances of abuse such as morphine, nicotine, and cocaine.
’ EXPERIMENTAL SECTION
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were
recorded on a 300 MHz (Bruker AVANCE 300) spectrometer. Chemi-
cal shift data for the proton resonances were reported in parts per million
(ppm) relative to internal standard. Optical rotations were measured on
an AutoPol III polarimeter, purchased from Rudolf Research. Elemental
analyses were performed by Atlantic Microlab (Norcross, GA). Purity of
compounds (>95%) was established by elemental analyses. Analytical
thin-layer chromatography (TLC) was carried out on plates precoated
with silica gel GHLF (250 μM thickness). TLC visualization was
accomplished with a UV lamp or in an iodine chamber. All moisture-
sensitive reactions were performed under a positive pressure of nitrogen
maintained by a direct line from a nitrogen source. Matrix-assisted laser
desorption ionization (MALDI) mass spectra were acquired using a
Bruker (Bilerica MA) Reflex III time-of-flight (TOF) mass spectrometer
(MS) in the linear, positive ion mode. Samples were prepared for
MALDI MS by using both equilibrium dialysis and a ZipTipC18
(Millipore, Billerica, MA) to remove salts prior to analysis. Anhydrous
solvents were purchased from Aldrich Chemical Co. CMA is a mixture of
80% chloroform, 18% methanol, and 2% concentrated ammonium
hydroxide. [3H]METH [(+)-[20,60-3H(n)]methamphetamine; 28.3
Ci/mmol; 0.521 mCi/mL] for radioimmunoassay was synthesized with
the radiolabel at the 2 and 6 positions of the aromatic ring by the
Research Triangle Institute (Research Triangle Park, NC) for the
National Institute on Drug Abuse.
The importance of increased epitope density was assessed in
two types of immunotherapeutic applications: active immuniza-
tion and production of monoclonal antibodies. For studies of
active immunization, mice that were immunized with the (+)-
METH HSMO9 BSA MCV (hapten epitope density of 12)
showed a substantial increase in their anti-METH immune
response after the second and subsequent MCV booster im-
munization. The second immunological boost is typically the
point at which the most substantial anti-METH IgG immune
response occurs in animals. This significant increase occurred in
90% of the mice, and continued booster injections maintained
these high anti-METH titers. By comparison, no more than 10%
of the mice responded with anti-METH antibodies when im-
munized with (+)-METH MO10 BSA (hapten epitope density
of 5). Weak immune responses and a low percentage of immune
responders have also been significant problems for early clinical
trials of active vaccines for nicotine and cocaine in humans.19,20
In a second series of immunological studies, we produced a
monoclonal antibody using mice immunized with HSM09 BSA
MCV. From one of these mice, we generated a very high affinity
mAb with (+)-METH KD of 6.8 nM and (+)-MDMA KI of 1.1
nM. Our previous highest affinity mAb against a meta position
hapten linker was 9 nM.13 This is the highest affinity mAb against
(+)-METH and (+)-MDMA that we have ever produced against
a METH conjugate vaccine.
(S)-N-(2-(Mercaptoethyl)-6-(3-(2-(methylamino)propyl)-
phenoxy)hexanamide (3) Tartrate. Compound 11 (0.630 g, 0.001
mol) was dissolved in ice-cold EtOH (12 mL). Mercuric acetate (0.775
g, 0.002 mol) was then added, and the mixture was slowly brought to
room temperature and stirred overnight. The residue resulting from
concentration of the solution was washed with diethyl ether (5 ꢁ
10 mL). After the ethereal layer was decanted, the resulting residue
In studies by Moreno et al.,22 a series of constrained and
unrestrained METH-like haptens were synthesized and then
linked to antigenic carrier proteins like BSA through a sulfhydryl
linker. The best three of these METH conjugate vaccines showed
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containing mercuric salts was suspended in EtOH (20 mL) and treated
with hydrogen sulfide gas for 2 h under moderate pressure. The black-
orange mercuric salt precipitate was removed using a syringe filter under
N2 atmosphere to isolate the free mercaptan into a round-bottom flask
containing (L)-tartaric acid (150 mg, 0.994 mmol). The mixture was
purged with positive flow of N2 gas to remove the solvent from the
mercaptan tartrate salts. This oily residue was redissolved in minimum
amount of degassed MeOH, filtered, and the solvent was removed
under a stream of N2 gas. Washing the oily substance with
(CH3)2CHOHꢀpentanes, followed by drying under high vacuum for
a few days, gave a white hygroscopic foam that was amenable for
transferring to a vial (under N2) to yield 0.565 g (87%) of 3 as a
hygroscopic solid. [R]20D +5.90 (c 0.290, MeOH). 1H NMR (300 MHz,
CDCl3) δ 7.18 (t, J = 8.0 Hz, 1H), 6.67ꢀ6.82 (m, 3H), 6.57 (t, J = 5.4
Hz, 1H), 3.93 (t, J = 6.3 Hz, 2H), 3.55 (q, J = 6.0 Hz, 2H), 2.76ꢀ2.92 (m,
3H), 2.71 (dd, J = 13.1, 7.0 Hz, 1H), 2.60 (dd, J = 13.1, 6.3 Hz, 1H), 2.40
(s, 3H), 2.07ꢀ2.33 (m, 4H), 1.64ꢀ1.87 (m, 4H), 1.44ꢀ1.60 (m, 2H),
1.07 (d, J = 6.1 Hz, 3H). 13C NMR (300 MHz, CDCl3) δ 130.9 (CH),
129.8 (CH), 121.9 (CH), 116.0 (CH), 112.5 (CH), 67.9 (CH2), 56.7,
43.7 (CH2), 38.8 (CH2), 38.2 (CH2), 36.8 (CH2), 34.2, 29.4 (CH2),
26.2 (CH2), 25.8 (CH2), 19.9. IR (CH2Cl2): 1666 cmꢀ1 (NCdO).
LCMS (ESI) m/z 339.9 (M + 1)+.
(S)-1-(3-Methoxyphenyl)-N-methyl)-N-((S)-1-phenylethyl)-
propan-2-amine (5). To an ice-cooled stirring solution of 4 (4.87 g,
0.016 mol) in THF (50 mL) was added BH3ꢀTHF solution (1.0 M,
57 mL, 0.057 mol) dropwise over 15 min. After the addition, the reaction
mixture was brought to room temperature and stirred overnight. The
reaction mixture was carefully quenched with MeOH to provide a
colorless oil. The oil was purified by flash chromatography (silica gel,
80.0 g Analogix column) using hexaneꢀEt2OꢀNEt3 (10:1:0.1) as
eluent to yield 4.56 g (94%) of 5 as a clear, colorless oil. This compound
was used in the next step without further purification.
dropwise over 15 min. After the addition, the mixture was brought to
room temperature and allowed to stir at 25 °C for 8 h. The reaction
mixture was carefully quenched with methanol at ice-cold condition and
concentrated under vacuum. Addition of methanol followed by con-
centration was carried out at least five more times to ensure that the
boron compounds were converted into its volatile. The resulting brown
oily residue was purified by flash chromatography (silica gel, 220 g ISCO
column) using CH2Cl2ꢀCMA (1:1) as the eluent to yield 7 (2.92 g,
73%) as a brown-colored oily substance. [R]20D +7.59 (c 1.305, MeOH).
1H NMR (300 MHz, CD3OD) δ 7.17 (dt, J = 8.3, 2.3 Hz, 1H, H-5),
6.57ꢀ6.86 (m, 3H, H-2, 4, 6), 3.39ꢀ3.54 (m, 1H, H-8), 3.07 (dd, J =
13.6, 5.3 Hz, 1H, H-7), 2.71 (s, 3H, H-10), 2.69 (dd, J = 13.6, 9.0 Hz, 1H,
H-7), 1.25 (d, J = 6.8 Hz, 3H, H-9). 13C NMR (300 MHz, CD3OD) δ
159.0 (C), 138.4 (C), 131.0 (CH), 121.4 (CH), 117.2 (CH), 115.3
(CH), 57.8 (CH), 40.2 (CH2), 31.0 (CH3), 15.8 (CH3).
(S)-tert-Butyl 1-(3-Hydroxyphenyl)propan-2-yl(methyl)-
carbamate (8). A stirred solution of 7 (2.75 g, 0.011 mol) in MeOH
(20 mL), triethylamine (6.17 g, 0.056 mol), and di-tert-butyl dicarbonate
(2.0 g, 0.009 mol) was heated at reflux for 30 min. The reaction mixture
was concentrated and the oil residue dissolved in EtOAc (100 mL). The
EtOAc solution was successively washed with H2O (50 mL), saturated
NaHCO3 (50 mL), and brine (50 mL), and the organics were dried
(MgSO4) and concentrated. The resulting oil was purified by column
chromatography (silica gel, 40.0 g ISCO column) using CMA (100%) as
eluent to provide 2.30 g (95%) of 8 as a brown oil. [R]20D +31.8 (c 1.650,
MeOH). 1H NMR (300 MHz, CDCl3) δ: major rotomer, 7.11 (t, J = 7.2
Hz, 1H), 6.50ꢀ6.85 (m, 3H), 4.31 (bs, 1H), 2.75 (s, 3H), 2.50ꢀ2.72 (m,
2H), 1.23ꢀ1.60 (m, 9H), 1.13 (bs, 3H); minor rotomer (diagnostic
peaks), 4.56 (bs, 1H). 13C NMR (500 MHz, CDCl3) δ 156.3 (C), 156.1
(C), 140.7 (C), 129.4 (CH), 120.9 (CH), 116.0 (CH), 113.4 (CH), 79.6
(C), 52.8, 40.3 (CH2), 28.4, 28.2, 27.5, 18.5; minor rotomer (diagnostic
peaks), 140.2 (C), 115.8 (C), 50.6, 27.9, 17.6; rotomer ratio, 1.3:1.0
(based on 1H NMR).
(S)-Methyl 6-(3-(2-(tert-Butoxycarbonyl(methyl)amino)-
propyl)phenoxy)hexanoate (9). A solution of 1.04 g (0.004 mol)
of phenol 8 in 5 mL of DMF was carefully added to a suspension of 0.267
g of 60% NaH (washed with hexanes in 5 mL of DMF) followed by the
addition of 0.920 g (0.004 mol) of methyl 6-bromohexanoate in 2 mL of
DMF. After 16 h of being stirred, the reaction mixture was quenched
with water and extracted with CH2Cl2 (3 ꢁ 75 mL). The combined
organic layers were washed with H2O (2 ꢁ 75 mL), dried (MgSO4), and
concentrated to yield an oil. The oil was purified by flash chromatog-
raphy using hexaneꢀEt2O (4:1) as the eluent to yield 0.798 g (66%) of 9
as an oil. [R]24D +32.5 (c 2.340, MeOH). 1H NMR (300 MHz, CDCl3)
δ: major rotomer, 7.14 (t, J = 7.9 Hz, 1H), 6.51ꢀ6.86 (m, 3H), 4.30 (bs,
1H), 3.91 (t, J = 6.4 Hz, 2H), 3.66 (s, 3H), 2.47ꢀ2.86 (m, 5H), 2.34 (t, J
= 7.5 Hz, 2H), 1.56ꢀ1.89 (m, 4H), 1.44ꢀ1.56 (m, 2H), 1.19ꢀ1.41 (m,
9H), 1.11 (bs, 3H); minor rotomer (diagnostic peaks), 4.40 (bs, 1H).
13C NMR (300 MHz, CDCl3) δ 174.0 (C), 159.1 (C), 155.5 (C), 140.6
(C), 129.2 (CH), 121.3 (CH), 115.3 (CH), 112.1 (CH), 79.1 (C), 67.5
(CH2), 52.6, 51.4, 40.6 (CH2), 34.0 (CH2), 29.0 (CH2), 28.3, 27.7, 25.7
(CH2), 24.7 (CH2), 18.3, 17.6. MS (EI) m/e 416.5 (MNa)+, 394.6
(MH)+, 338.6 (MH ꢀ OtBu)+, 294.2 (MH ꢀ C5H10O2)+; rotomer
ratio, 1.4:1.0 (major to minor, based on 1H NMR). Anal. (C22H35NO5)
C, H, N.
(S)-1-(3-Methoxyphenyl)-N-methylpropan-2-amine (6). To
a methanol solution (50 mL) of 5 (2.45 g, 0.0082 mol) was added
ammonium formate (2.70 g, 0.042 mol) and 5% Pd/C (0.245 g). The
solution was gently warmed at ∼50 °C for a period of 4 h. The cooled
reaction mixture was filtered through a pad of Celite under moderate
pressure. The Celite pad was washed with methanol (3 ꢁ 10 mL), and
the collected organics were concentrated to yield an oilysubstance, which
was dissolved in diethyl ether (100 mL) containing a small amount of
NEt3 (10 mol %). The mixture was vacuum filtered (to remove excess
formate salts), and the filtrate was concentrated to give an oil. The oil was
subjected to flash chromatography (silica gel, 40.0 g ISCO column) using
hexaneꢀEt2OꢀNEt3 (10:1:0.1) as the eluent to yield 1.37 g (93%) of 5
as a clear, colorless oil. [R]20 +5.83 (c 1.15, MeOH). 1H NMR (300
D
MHz, CDCl3) δ 7.21 (t, J = 7.9 Hz, 1H, H-5), 6.70ꢀ6.83 (m, 3H, H-2, 4,
6), 3.80 (s, 3H, H-11), 2.80 (hextet, J = 6.4 Hz, 1H, H-8), 2.65 (dd, J =
13.6, 6.4 Hz, 1H, H-7), 2.63 (dd, J = 13.6, 6.4 Hz, 1H, H-7), 2.39 (s, 3H,
H-10), 1.07 (d, J = 6.4 Hz, 3H, H-9). 13C NMR (300 MHz, CDCl3) δ
159.6 (C-3), 141.1 (C-1), 129.3 (C-5), 121.6 (C-6), 115.0 (C-2), 111.4
(C-4), 56.2 (C-8), 55.1 (C- 11), 43.5 (C-7), 33.9 (C-10), 10.7 (C-9).
Compound 6 was converted into its HCl salt by slow addition of HCl
(2.0 M in Et2O) to the ice-cold ethereal solution: mp 132ꢀ134 °C.
[R]20D +2.251 (c 1.435, MeOH). 1H NMR (300 MHz, CDCl3) δ 9.65
(bs, 2H), 7.22 (t, J = 7.5 Hz, 1H, H-5), 6.69ꢀ6.88 (m, 3H, H-2, 4, 6),
3.79 (s, 3H, H-11), 3.46 (dd, J = 12.8, 3.8 Hz, 1H, H-8), 3.28ꢀ3.41 (bm,
1H, H-8), 2.82 (dd, J = 12.8, 10.6 Hz, 1H, H-7), 2.72 (bs, 3H, H-10),
1.35 (d, J = 6.4 Hz, 3H, H-9). 13C NMR (500 MHz, CDCl3) δ 159.8 (C-
3), 137.5 (C-1), 129.8 (C-5), 121.5 (C-6), 114.8 (C-2), 112.6 (C-4),
57.0 (C-8), 55.2 (C-11), 39.4 (C-7), 30.1 (C-10), 15.5 (C-9). Anal.
(C11H18ClNO) C, H.
(S)-6-(3-(2-(tert-Butoxycarbonyl(methyl)amino)propyl)-
phenoxy)hexanoic Acid (10). A mixture of 9 (0.657 g, 0.0017 mol)
and LiOH H2O (0.258 g, 0.006 mol) in THFꢀMeOHꢀH2O (1:1:1,
3
10 mL each) was refluxed for 8 h. The reaction mixture was concentrated
and the resulting residue dissolved in CH2Cl2 and 3.0 N HCl (1:1,
75 mL). The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (3 ꢁ 25 mL). The combined organic layers were
washed with H2O (2 ꢁ 50 mL), dried (MgSO4), and concentrated to
yield an oil. The oil was used in the next step without any further
(S)-3-(2-(Methylamino)propyl)phenol Hydrobromide (7).
To a stirred solution of 6 (2.90 g, 0.016 mol) in CH2Cl2 (50 mL) at
ꢀ78 °C, boron tribromide solution (13.25 g, 0.053 mol) was added
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Journal of Medicinal Chemistry
ARTICLE
1
purification. [R]24 +33.57 (c 0.855, MeOH). H NMR (300 MHz,
vendor). A portion of the maleimide activated protein stock solution
(0.5 mL) was kept aside for later analysis as a control representing the
starting conditions prior to conjugation reactions. The remaining
activated protein solution (1.5 mL) was transferred into a sterile
cryogenic vial and added to the (+)-METH HSMO9 hapten stock
solution. The reaction mixture was purged with N2 and incubated for 2 h
under N2 at ambient temperature with gentle stirring. After the synthesis
was complete at 2 h, aliquots of the OVA-MCV and BSA-MCV reaction
mixture were sealed in vials and shipped under ice-cold conditions to
laboratories at the University of Arkansas at Fayetteville (Fayetteville,
AR) for MALDI-TOF determination of the (+)-METH HSMO9
epitope density and to the University of Arkansas for Medical Sciences
(Little Rock, AR) for immunological studies.
Mass Spectrometry Analysis. Mass spectra were obtained of (+)-
METH, (+)-METH MO10, and (+)-METH HSMO9 using a Bruker
Reflex III MALDI TOF mass spectrometer operated in the linear mode
with an acceleration voltage of approximately 25 kV. Pulsed ion extraction
with a delay of 700 ns was used. The instrument was calibrated using a
commercial reference material from the manufacturer (Bruker Protein
Standard II) for high mass (10ꢀ100 kDa) range calibration. Ion
suppression was used up to m/z 10 kDa to enhance the high mass ions.
While developing the methods for MALDI-TOF analysis of the MCV,
we realized that the protein solution salt content from buffers adversely
affected the method. To optimize analytical conditions, we compared
several methods for removal of interfering salts using the OVA-MCV. For
the experiment, we compared results from the MALDI-TOF analysis of
proteins without desalting (control), after dialysis against distilled H2O
(model HTD96b; HT Dialysis LLC, Gales Ferry, CT) and after desalting
on a ZipTipC18 (Millipore Corporation, Bedford, MA).
D
CDCl3) δ: major rotomer, 7.16 (t, J = 7.9 Hz, 1H), 6.61ꢀ6.83 (m, 3H),
4.33 (bs, 1H), 3.94 (t, J = 6.3 Hz, 2H), 2.54ꢀ2.83 (m, 5H), 2.39 (t, J =
7.3 Hz, 2H), 1.66ꢀ1.85 (m, 4H), 1.46ꢀ1.60 (m, 2H), 1.25ꢀ1.44 (m,
10H), 1.14 (bs, 3H); minor rotomer (diagnostic peaks), 4.53 (bs, 1H).
13C NMR (300 MHz, CDCl3) δ 178.4 (C), 159.0 (C), 155.6 (C), 140.6
(C), 129.2 (CH), 121.4 (CH), 115.4 (CH), 112.1 (CH), 79.3 (C), 67.5
(CH2), 52.7, 51.2, 40.6 (CH2), 33.8 (CH2), 31.6, 28.9 (CH2), 28.3, 27.8,
25.6 (CH2), 24.4 (CH2), 22.6, 20.6, 18.4, 17.6, 14.1. MS (EI) m/e 402.2
(MNa)+, 380.5 (M)+, 378.4 (M ꢀ H)+, 280.3 (MH ꢀ DeBoc)+; rotomer
ratio, 1.3:1.0 (based on 1HNMR). Anal. (C21H33NO5 0.75H2O) C, H, N.
3
(S)-6-(3-(2-Methylamino)propyl)phenoxy)-N-(2-(tritylthio)-
ethyl)hexanamide (11). To a solution of 10 (1.0 g, 0.0026 mol) and
2-(tritylmercapto)ethylamine (1.07 g, 0.003 mol) in 15 mL of DMF at
0 °C was added HOAt in DMF (0.5 M, 0.0065 mol), EDCI HCl (1.82 g,
3
0.0095 mol), and NEt3 (1.33 mL) under a N2 atmosphere. The reaction
mixture was slowly brought to room temperature and stirred for 24 h.
The residue obtained on concentration was quenched with saturated
NaHCO3 and extracted with CH2Cl2 (3 ꢁ 150 mL). The combined
organics were washed with brine, dried (MgSO4), and concentrated to
an oil which was purified by flash chromatography using EtOAcꢀhexane
(1:1) to give 1.52 g (85%) of an oil. [R]21D +13.9 (c 1.00, MeOH). 1H
NMR (300 MHz, CDCl3) δ: major rotomer, 7.41 (ad, J = 7.41 Hz, 6H),
7.28 (dt, J = 6.8, 1.0 Hz, 6H), 7.20 (dt, J = 7.3, 1.0 Hz, 3H), 7.14 (d, J =
7.9 Hz, 1H), 6.63ꢀ6.79 (m, 3H), 5.60 (bs, 1H), 4.32 (bs, 1H), 3.92 (t,
J = 6.4 Hz, 2H), 3.09 (q, J = 6.1 Hz, 2H), 2.55ꢀ2.79 (m, 5H), 2.33ꢀ2.48
(m, 2H), 2.12 (t, J = 7.6 Hz, 2H), 1.70ꢀ1.84 (m, 2H), 1.60ꢀ1.70 (m,
2H), 1.43ꢀ1.58 (m, 2H), 1.30ꢀ1.58 (m, 9H), 1.13 (bs, 3H); minor
rotomer (diagnostic peaks), 4.52 (bs, 1H). 13C NMR (300 MHz,
CDCl3) δ major rotomer: 172.7 (C), 159.0 (C), 144.7 (C), 137.8
(C), 129.5 (CH), 129.2 (CH), 128.0 (CH), 126.8 (CH), 121.3 (CH),
115.3 (CH), 112.2 (CH), 79.3 (C), 67.5 (CH2), 52.5, 40.6 (CH2), 38.1
(CH2), 36.6 (CH2), 32.1, 29.0 (CH2), 28.4 (CH2), 25.8 (CH2), 25.4
(CH2), 14.2; minor rotomer (diagnostic peaks), 155.5 (C), 140.6 (C),
79.1 (C), 66.8, 60.4, 33.7, 28.8, 25.5, 24.5, 21.0; rotomer ratio, 1.4:1.0
(based on 1H NMR).
Immune Response to (+)-METH HSMO9 BSA MCV in Mice.
Female BALB/C mice were housed in an animal care facility with a 12 h
light/dark cycle (6:00 a.m. to 6:00 p.m.) and an average 22 °C ambient
temperature. They received food and water ad libitum. Animal protocols
were in accordance with the Guide for the Care and Use of Laboratory
Animals, as adopted and promulgated by the National Institutes of
Health. They were approved by the Institutional Animal Care and Use
Committee of the University of Arkansas for Medical Sciences (Little
Rock, AR) prior to beginning the experiments.
The above oil was dissolved in CH2Cl2 (20 mL) at room temperature,
and trifluoroacetic acid (5.0 mL) was added dropwise over ∼5 min and
stirred for an additional 10 min. The oily residue obtained on concen-
tration was coevaporated with MeOH (3 ꢁ 10 mL) followed by
purification using flash chromatography with CH2Cl2ꢀCMA (1:1),
For studies of active immunization, female BALB/c mice (10 per
group; Charles River Laboratories, Wilmington, MA) were subcuta-
neously immunized with either 20 μg of (+)-METH HSMO9 BSA (with
a (+)-METH HSMO9 hapten epitope density of 12) or 20 μg of (+)-
METH MO10 BSA (with a (+)-METH MO10 hapten epitope density
of 5). The antigens were emulsified in Freund’s complete adjuvant
(EMD Chemicals, Gibbstown, NJ) near each front and hindquarter per
the adjuvant manufacturer’s instructions. This initial immunization was
followed by 20 μg booster immunizations at 3ꢀ6 week intervals with
Freund’s complete adjuvant. Immune serum was collected 10ꢀ14 days
after each boost, and serum antibody titers were determined by
examining binding to [3H]-(+)-METH using radioimmunoassay.15
Production of a Monoclonal Antibody against (+)-METH
HSMO9 BSA in Mice. For production of monoclonal antibodies,
female BALB/c mice (five per group) were immunized by the sub-
cutaneous and intraperitoneal routes with 20 μg of (+)-METH HSMO9
BSA. The (+)-METH HSMO9 hapten epitope density was 10 (see
Figure 2). This MCV was emulsified in Sigma Adjuvant System (Sigma
Chemical Co., St. Louis, MO) according to the manufacturer’s instruc-
tions. This initial immunization was followed by 20 μg booster im-
munizations at 3 weeks followed by two booster injections at two 6-week
intervals. Immune serum was collected 10ꢀ14 days after each boost, and
serum antibody titers were determined by examining binding to
[3H]-(+)-METH using radioimmunoassay.15 The generation, produc-
tion, and characterization of the monoclonal antibodies were as de-
scribed by Peterson et al.13
eluting to yield 1.06 g (81%) of 11 as an oil. [R]22 +0.561 (c 0.535,
D
MeOH). 1H NMR (300 MHz, CDCl3) δ 9.31 (bs, 2H), 7.41 (ad, J = 7.3
Hz, 6H), 7.28 (at, J = 7.3, 6H), 7.14ꢀ7.24 (m, 4H), 6.67ꢀ6.80 (m, 3H),
5.60 (t, J = 5.3 Hz, 1H), 3.91 (t, J = 6.3 Hz, 2H), 3.31 (bs, 1H), 3.20 (dd, J
= 13.2, 4.4 Hz, 1H), 3.08 (q, J = 6.1 Hz, 2H), 2.58ꢀ2.75 (m, 5H), 2.41 (t,
J = 6.3 Hz, 2H), 1.71ꢀ1.85 (m, 2H), 1.58ꢀ1.71 (m, 2H), 1.42ꢀ1.54 (m,
2H), 1.26 (d, J = 6.4 Hz, 3H). 13C NMR (300 MHz, CDCl3) δ 159.5
(C), 144.6 (C), 137.8 (C), 129.9 (CH), 129.5 (CH), 128.0 (CH), 126.8
(CH), 121.3 (CH), 115.5 (CH), 113.3 (CH), 67.6 (CH2), 56.7, 50.8,
39.5 (CH2), 38.1 (CH2), 36.5 (CH2), 32.0 (CH2), 30.2, 28.9 (CH2),
25.7 (CH2), 25.3 (CH2), 15.5. MS (EI) m/e 581.9 (MH)+, 307.7 (M ꢀ
C21H20S)+.
Conjugation of Maleimide Activated OVA and Maleimide
Activated BSA to (+)-METH HSMO9 Hapten. Representative
conditions for synthesis of MCV using maleimide activated OVA or
maleimide activated BSA are as follows. An amount of 2 mg of
maleimide-activated OVA or maleimide activated BSA (ThermoFisher
Scientific) was reconstituted in 1.0 mL of distilled H2O for a 2 mg/mL
solution, which contains 44.4 μmol or 27.8 μmol of protein, respectively.
At the same time, (+)-METH HSMO9 hapten (3) stock solutions
(11.38 or 20.8 mM, respectively) were prepared by dissolving (+)-
METH HSMO9 hapten (22 mg or 42.0 μmol for OVA; 7 mg or 11.64
μmol for BSA) in degassed reaction buffer (2.0 mL, supplied by the
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Journal of Medicinal Chemistry
ARTICLE
’ ASSOCIATED CONTENT
(9) Gentry, W. B.; Ruedi-Bettschen, D.; Owens, S. M. Anti-(+)-
methamphetamine monoclonal antibody antagonists designed to pre-
vent the progression of human diseases of addiction. Clin. Pharmacol.
Ther. 2010, 88, 390–393.
S
Supporting Information. Elemental analysis results of
b
compounds 3, 6, 9, and 10. This material is available free of
(10) Laurenzana, E. M.; Hendrickson, H. P.; Carpenter, D.; Peter-
son, E. C.; Gentry, W. B.; West, M.; Che, Y.; Carroll, F. I.; Owens, S. M.
Functional and biological determinants affecting the duration of action
and efficacy of anti-(+)-methamphetamine monoclonal antibodies in
rats. Vaccine 2009, 27, 7011–7020.
(11) Byrnes-Blake, K. A.; Carroll, F. I.; Abraham, P.; Owens, S. M.
Generation of anti-(+)methamphetamine antibodies is not impeded by
(+)methamphetamine administration during active immunization of
rats. Int. Immunopharmacol. 2001, 1, 329–338.
(12) Carroll, F. I.; Abraham, P.; Gong, P. K.; Pidaparthi, R. R.;
Blough, B. E.; Che, Y.; Hampton, A.; Gunnell, M.; Lay, J. O., Jr.;
Peterson, E. C.; Owens, S. M. The synthesis of haptens and their use for
the development of monoclonal antibodies for treating methampheta-
mine abuse. J. Med. Chem. 2009, 52, 7301–7309.
(13) Peterson, E. C.; Gunnell, M.; Che, Y.; Goforth, R. L.; Carroll,
F. I.; Henry, R.; Liu, H.; Owens, S. M. Using hapten design to discover
therapeutic monoclonal antibodies for treating methamphetamine
abuse. J. Pharmacol. Exp. Ther. 2007, 322, 30–39.
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 919 541-6679. Fax: 919 541-8868. E-mail: fic@rti.org.
’ ACKNOWLEDGMENT
This research was supported by the National Institute on Drug
Abuse under Projects U01 DA23900, DA05477, and DA11560
and by P20 RR15569 from the National Center for Research
Resources (NCRR), a component of the National Institutes of
Health (NIH).
’ ABBREVIATIONS USED
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(+)-AMP, (+)-amphetamine; (ꢀ)-AMP, (ꢀ)-amphetamine; BSA,
bovine serum albumin; c-BSA, cationized bovine serum albumin;
CNS, central nervous system; DAWN, Drug Abuse Warning
Network; ED, emergency department; EDCI, 1-ethyl-3-(3-di-
methylaminopropyl)carbodiimide; ELISA, enzyme-linked im-
munosorbent assay; HOAT, 1-hydroxy-7-azabinatriazole; mAb,
monoclonal antibody (both singular and plural); MALDI, ma-
trix-assisted laser desorption/ionization; (+)-METH HSMO9,
mercapto-hapten (S)-N-(2-(mercaptoethyl)-6-(3-(2-(methylamino)-
propyl)phenoxy)hexanamide; MCV, METH-conjugate vaccine;
(+)-MDMA, (+)-3,4-methylenedioxymethamphetamine; (ꢀ)-
MDMA, (ꢀ)-3,4-methylenedioxymethamphetamine; (()-MDMA,
(()-3,4-methylenedioxymethamphetamine; (+)-METH, (+)-
methamphetamine; (ꢀ)-METH, (ꢀ)-methamphetamine; MS,
mass spectrometry; OVA, ovalbumin; RIA, radioimmunoassay;
Sulfo-SMCC, sulfosuccinimidyl 4-[N-maleimidomethyl]cycloh-
exane-1-carboxylate
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