Antibody-catalyzed oxidation of Δ9-tetrahydrocannabinol

Article abstract of DOI:10.1021/ja070022m

Marijuana abuse continues to plague society and the lack of effective treatments warrants concern. Catalytic antibodies capable of oxidatively degrading the major psychoactive component of marijuana, Δ⁹- tetrahydrocannabinol (Δ⁹-THC), are presented. The antibodies generate reactive oxygen species from singlet oxygen (¹O ₂*), using riboflavin (vitamin B₂) and visible light as the ¹O₂*source. Cannabitriol was identified as the major degradation product of this reaction, demonstrating the ability of an antibody to catalyze a complex chemical transformation with therapeutic implications for treating marijuana abuse.

Full text of DOI:10.1021/ja070022m

Published on Web 03/03/2007
Antibody-Catalyzed Oxidation of ∆9-Tetrahydrocannabinol
Andrew P. Brogan,† Lisa M. Eubanks,† George F. Koob,‡ Tobin J. Dickerson,*,† and
Kim D. Janda*,†
Contribution from the Departments of Chemistry and Immunology, The Skaggs Institute for
Chemical Biology, Worm Institute for Research and Medicine, and the Committee on the
Neurobiology of AddictiVe Disorders, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, California 92037
Received January 2, 2007; E-mail: kdjanda@scripps.edu; tobin@scripps.edu
Abstract: Marijuana abuse continues to plague society and the lack of effective treatments warrants concern.
Catalytic antibodies capable of oxidatively degrading the major psychoactive component of marijuana, ∆9-
tetrahydrocannabinol (∆9-THC), are presented. The antibodies generate reactive oxygen species from singlet
oxygen (1O2*), using riboflavin (vitamin B2) and visible light as the 1O2* source. Cannabitriol was identified
as the major degradation product of this reaction, demonstrating the ability of an antibody to catalyze a
complex chemical transformation with therapeutic implications for treating marijuana abuse.
Introduction
ated by catalytic antibodies, primarily using transition state
analogues or reactive immunization.9 Several cocaine esterase
∆9-Tetrahydrocannabinol (∆9-THC) is the active component
of marijuana and the most commonly abused illicit drug in the
U.S.1,2 Marijuana use often leads to abuse of other illicit drugs
as postulated by the “cannabis gateway hypothesis”3 and
supported by clinical evidence and numerous population
surveys.4-6 Furthermore, exposure to ∆9-THC increases opiate
self-administration in an animal model by specifically altering
the endogenous opioid system, providing a molecular basis for
the gateway hypothesis.7 Currently, there are no clinical
treatments for marijuana abuse. A promising approach to combat
drug abuse is immunopharmacotherapy, where active or passive
immunizations are administered to bind the target drug before
it can reach its cognate receptor. Using this strategy, approaches
to treat cocaine, nicotine, PCP, and methamphetamine abuse
have been reported.8 However, this method is limited by the
need for stoichiometric amounts of antibody to bind the target
drug; in contrast, a catalytic antibody capable of decomposing
the drug presents a more advantageous substoichiometric system.
A wide range of chemical transformations have been acceler-
antibodies have been generated by replacing the cocaine benzyl
ester with a phosphonate ester as a mimic of the hydrolysis
transition state.10-12 Unfortunately, no obvious route for an
antibody-catalyzed reaction is apparent when examining the
structure of ∆9-THC; although, the C9-C10 olefin is expected
to be susceptible to oxidation. Oxidation reactions have proven
difficult to program into the antibody combining site; however,
a new paradigm in antibody catalysis was recently reported by
Lerner and co-workers, where all antibodies were found to have
the intrinsic ability to destroy their corresponding antigen by
generating reactive oxygen species (ROS) from singlet oxygen
(1O2*).13-17 Examples of potential ROS generated by antibodies
include hydrogen peroxide (H2O2), ozone (O3), and superoxide
(O2-), enabling a range of different reactions. We recently
reported a potential therapeutic application of this phenomenon
by demonstrating the antibody-catalyzed oxidative degradation
of nicotine into putatively pharmacologically inactive products
using visible light and riboflavin (vitamin B2), a known
photosensitizer, as the singlet oxygen source.18 On the basis of
† Departments of Chemistry and Immunology, The Skaggs Institute for
Chemical Biology, and the Worm Institute for Research and Medicine.
‡ Committee on the Neurobiology of Addictive Disorders.
(1) Substance Abuse and Mental Health SerVices Administration. Results from
the 2001 National Household SurVey on Drug Abuse. Summary of National
Findings; NHSDA Series H-17, DHHS Publication SMA 02-3758; Office
of Applied Studies, U.S. Department of Health and Human Services:
Rockville, MD, 2002; Vol. I.
(9) Xu, Y.; Yamamoto, N.; Janda, K. D. Bioorg. Med. Chem. 2004, 12, 5247-
5268.
(10) Landry, D. W.; Zhao, K.; Yang, G. X.; Glickman, M.; Georgiadis, T. M.
Science 1993, 259, 1899-1901.
(11) Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.
A.; Zhao, K.; Landry, D. W. J. Am. Chem. Soc. 1996, 118, 5881-5890.
(12) Matsushita, M.; Hoffman, T. Z.; Ashley, J. A.; Zhou, B.; Wirsching, P.;
Janda, K. D. Bioorg. Med. Chem. Lett. 2001, 11, 87-90.
(2) Johnston, L. D.; O’Malley, P. M.; Bachman, J. G. Monitoring the Future
National SurVey Results on Drug Use, 1975-2002; National Institute on
Drug Abuse: Bethesda, MD, 2003.
(13) Wentworth, A. D.; Jones, L. H.; Wentworth, P., Jr.; Janda, K. D.; Lerner,
R. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10930-10935.
(14) Wentworth, P., Jr.; Jones, L. H.; Wentworth, A. D.; Zhu, X.; Larsen, N.
A.; Wilson, I. A.; Xu, X.; Goddard, W. A., III; Janda, K. D.; Eschenmoser,
A.; Lerner, R. A. Science 2001, 293, 1806-1811.
(3) Kandel, D. Science 1975, 190, 912-914.
(4) Kandel, D. Stages and Pathways of Drug InVolVement: Examining the
Gateway Hypothesis; Cambridge University Press: Cambridge, U.K., 2002.
(5) Fergusson, D. M.; Boden, J. M.; Horwood, L. J. Addiction 2006, 101, 556-
569.
(15) Wentworth, P., Jr.; Nieva, J.; Takeuchi, C.; Galve, R.; Wentworth, A. D.;
Dilley, R. B.; DeLaria, G. A.; Saven, A.; Babior, B. M.; Janda, K. D.;
Eschenmoser, A.; Lerner, R. A. Science 2003, 302, 1053-1056.
(16) Wentworth, P., Jr.; Wentworth, A. D.; Zhu, X.; Wilson, I. A.; Janda, K.
D.; Eschenmoser, A.; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 1490-1493.
(6) Kandel, D. B. JAMA, J. Am. Med. Assoc. 2003, 289, 482-483.
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32, 607-615.
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DiscoV. Technol. 2004, 1, 77-89.
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R. A.; Wilson, I. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2247-2252.
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3698
J. AM. CHEM. SOC. 2007, 129, 3698-3702
10.1021/ja070022m CCC: $37.00 © 2007 American Chemical Society

Oxidation of
∆
9-Tetrahydrocannabinol
A R T I C L E S
Figure 1. Structures of ∆9-tetrahydrocannabinol (∆9-THC) and TCB and TCF haptens.
Figure 2. Possible products of the antibody-catalyzed degradation ∆9-THC. (A) Primary metabolic products of ∆9-THC, 11-hydroxy-∆9-tetrahydrocannabinol
1, and 11-nor-9-carboxy-∆9-tetrahydrocannabinol 2; (B) postulated chemistry and degradation products of the antibody-catalyzed degradation ∆9-THC.
this precedent, two panels of ∆9-THC-binding antibodies19 were
examined in the present study, and all antibodies tested were
found to catalyze the oxidative degradation of ∆9-THC. The
major product from this transformation was identified using
extensive spectroscopic analysis and an unexpected antibody-
catalyzed reaction was revealed.
the TCF hapten has an aromatized C ring and additional amide
bonds in the linker, sacrificing structural similarity in an attempt
to elicit stronger immune responses.12 The TCB hapten elicited
tight binding antibodies with adequate serum titers, and several
monoclonal antibodies from this panel possessed excellent
affinity for ∆9-THC (TCB25G1, Kd ) 0.23 µM). As expected,
higher serum titers were observed from TCF immunization;
however, weaker ∆9-THC binding was observed for monoclonal
antibodies from this panel (Kd > 100 µM), presumably because
the TCF hapten deviates too greatly from the structure of ∆9-
THC. A screen using both panels was conducted by monitoring
∆9-THC concentration over time. Analogous to our previous
studies, the weaker binding TCF panel (20 antibodies) provided
more proficient catalysts, while the tighter binding TCB panel
(5 antibodies) also catalyzed the degradation of ∆9-THC, albeit
less proficiently.
The antibody-catalyzed oxidative degradation of ∆9-THC led
to the formation of one major product and multiple minor
products. Using the limited amount of major product obtained
from the antibody screen, a molecular weight of 346 was
measured by mass spectrometry corresponding to the addition
of two oxygen atoms. The major urinary metabolites of ∆9-
THC in humans are 11-hydroxy-∆9-tetrahydrocannabinol 1 and
Results and Discussion
Considering that no reaction was observed in our previous
study using tight binding nicotine antibodies (Kd,app e 1 µM)
and only weaker binding antibodies (Kd,app ) µM to mM)
generated against a less congruent hapten were found to catalyze
the oxidative degradation of the drug,18 we were particularly
interested in a range of ∆9-THC binding affinities in designing
a screen for the antibody-catalyzed oxidative degradation of ∆9-
THC. The structural nuances of the TCB and TCF haptens
provided monoclonal antibodies with a wide affinity range for
∆9-THC and highlight two strategies employed in hapten design
(Figure 1).19 The TCB hapten is highly analogous to ∆9-THC
with the linker positioned distal to any portion of the molecule
anticipated to be essential for antibody recognition. In contrast,
(18) Dickerson, T. J.; Yamamoto, N.; Janda, K. D. Bioorg. Med. Chem. 2004,
12, 4981-4987.
(19) Qi, L.; Yamamoto, N.; Meijler, M. M.; Altobell, L. J.; Koob, G. F.;
Wirsching, P.; Janda, K. D. J. Med. Chem. 2005, 48, 7389-7399.
9
J. AM. CHEM. SOC. VOL. 129, NO. 12, 2007 3699

A R T I C L E S
Brogan et al.
Figure 3. (A) Key 1H-1H ROESY correlations for cannabitriol; (B) key 1H-13C HMBC correlations for cannabitriol; (C) proposed antibody-catalyzed
transformation of ∆9-THC to cannabitriol.
11-nor-9-carboxy-∆9-tetrahydrocannabinol 2;20-22 however, nei-
ther of these compounds have a molecular weight corresponding
to the unknown oxidation product. Given the potential for
antibody-generated ROS, we also considered possible oxidation
reactions, and in particular, the oxidation of the C9-C10 olefin.
Ozonolysis of this olefin generates ketoaldehyde 3 that could
subsequently undergo an intermolecular aldol reaction to yield
â-hydroxyketone 4 or react with the neighboring phenol
resulting in hemiacetal 5 (Figure 2). Another possibility is a
[2+2] cycloaddition reaction with 1O2* to yield the correspond-
ing dioxetane 6.
To identify the major product from the antibody-catalyzed
degradation of ∆9-THC, significant quantities were needed for
spectroscopic analysis. Traditional synthetic organic chemistry
techniques were employed by exposing ∆9-THC to ozone and
hydrogen peroxide in an attempt to mimic the chemistry
occurring in the antibody-catalyzed reaction and also provide
insight into oxidants involved. Ozonolysis of ∆9-THC provided
no identifiable products; however, trace amounts of the major
product from the antibody-catalyzed degradation of ∆9-THC
were observed in the hydrogen peroxide reaction. In light of
this limited success, we increased the scale of the antibody
catalyzed reaction 100-fold and extended the time of the
reaction. Throughout the course of the reaction additional
aliquots of ∆9-THC and riboflavin were added to maximize
product yield.
With greater quantities of product now available, 1D and 2D
NMR techniques were used to characterize the molecular
composition of the antibody-catalyzed oxidation product. H
1
NMR revealed clear resonances for the hydrocarbon tail and
the A-ring protons, and 13C NMR verified that this portion of
the molecule was intact. Three singlets corresponding to the
C6 and C9 methyl groups remained along with multiplets for
the C7 and C8 protons. Importantly, the C10 olefin proton and
the protons on the tertiary carbons C6a and C10a were absent,
and a new singlet at δ 4.2 emerged. In the 13C NMR, two signals
in the aromatic region of the spectrum at δ 122 and 136 in
addition to resonances for the A ring were observed. The lack
of 13C NMR carbonyl signals eliminated the proposed ozonolysis
product 3 and the subsequent aldol and hemiacetal products 4
and 5. Resonances corresponding to dioxetane 6 were also
inconsistent with the 1D and 2D NMR data, particularly the
disconnect between the C7 and C8 protons and any other portion
1
of the molecule in the H-1H COSY spectrum.
Deviating from our initial assumption of simple single-step
oxidation reactions, we began to consider multistep pathways
involving multiple oxidants and, after further examination of
the NMR data, the oxidized ∆9-THC analogue cannabitriol was
considered as a possible product from the antibody-catalyzed
degradation of ∆9-THC (Figure 3). Cannibitriol is a native
component of marijuana and was first isolated and characterized
by von Spulak et al.23 in 1968, with the stereochemistry later
(20) Huestis, M. A. Handb. Exp. Pharmacol. 2005, 168, 657-690.
(21) Grotenhermen, F. Clin. Pharmacokinet. 2003, 42, 327-360.
(22) McGilveray, I. J. Pain Res. Manage. 2005, 10, 15A-22A.
(23) Von Spulak, F.; Claussen, U.; Fehlhaber, H. W.; Korte, F. Tetrahedron
1968, 24, 5379-5383.
9
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Oxidation of
∆
9-Tetrahydrocannabinol
A R T I C L E S
assigned.24-26 The limited NMR data from these studies are
consistent with the major product from the antibody-catalyzed
Table 1. Kinetic Parameters for the Antibody-Catalyzed
Conversion of ∆9-THC to Cannabitriol
1
1
cat (min-
)
k
cat/kuncat
KM
(µM)
k
cat/KM (min-1 µM-
)
1
oxidation of ∆9-THC. The singlet at δ 4.2 in the H NMR
antibody
k
spectrum corresponds to the C10 proton of cannabitriol, and
the 13C NMR signals at δ 122 and 136 correspond to the C6a-
C10a double bond. The newly formed quaternary center at C9
and the C6a-C10a olefin of cannabitriol provide clarity for the
disconnect between the C7 and C8 protons and any other portion
TCF-26C12 0.999
1.97 × 104
1.97 × 104
1.97 × 104
275
394
595
3.63 × 10-3
2.54 × 10-3
1.68 × 10-3
TCF-23C4
TCF-25G5
none
1.00
1.00
5.08 × 10-5
1
of the molecule in the H-1H COSY spectrum. The additional
mixture did not affect the rate of cannabitriol formation. This
suggests that free hydrogen peroxide or superoxide radical are
not involved in the oxidation of ∆9-THC, although the possibility
that either oxidant is sequestered from the bulk water and
utilized by the antibody cannot be discounted.16,17 In total, the
regio- and stereospecificity of the product suggests that the
oxidation steps are catalyzed by the antibody, whereas, the
epoxide ring opening and elimination reactions may occur in
the bulk water.
hydroxyl groups of cannabitriol generate stereogenic centers at
C9 and C10 and, through the use of a 1H-1H ROESY
experiment, the stereochemistry was assigned as cis by through-
space correlations between the C9-methyl protons and the C10
proton. Key correlations stemming from the oxidation of the C
ring between the C6 methyl, C7, C8, and C10 protons and the
1
C6a-C10a olefin were observed by H-13C HMBC, and the
anticipated 1H-13C HMQC and 1H-13C HMBC correlations
further supported the identity of the unknown as cannabitriol.
The identification of cannabitriol as the major product from
the antibody-catalyzed oxidative degradation of ∆9-THC led us
to explore the chemistry involved in this transformation (Figure
3C). A Schenck ene reaction between ∆9-THC and singlet
oxygen was envisioned as the first step. Experimental and
theoretical studies of the Schenk ene reaction mechanism are
most consistent with perepoxide intermediate 7, which differs
distinctly from the typical pericyclic ene reaction mechanism
using an olefin instead of 1O2*.27 Empirical data indicates that
proton abstraction by the perepoxide nucleophilic oxygen occurs
on the more highly substituted side of the olefin, which is the
proton on C8 or C10a. However, selectivity for the proton on
C10a is favored since the nascent olefin in 8 is in conjugation
with the aromatic A ring, generating a more thermodynamically
stable product. This initial reaction also sets the stereochemistry
at C9 since perepoxide formation must occur on the less
hindered side of the alkene, dictated by the stereochemistry of
∆9-THC, enabling the nucleophilic perepoxide oxygen to be
proximal to the C10a proton. A second oxidant is then needed
in an epoxidation reaction of the newly formed olefin at C10-
C10a. Since there are few steric constraints, in bulk solution,
the stereochemistry of epoxide 9 is dependent upon whether
the subsequent epoxide ring opening reaction occurs at the
thermodynamically favored C10a or the kinetically favored C10
position. Cis epoxidation, relative to the C9 hydroxyl, requires
thermodynamic epoxide ring opening and subsequent elimina-
tion in order to obtain cannabitriol in the proper stereochemical
configuration, whereas trans epoxidation requires kinetic epoxide
ring opening and subsequent elimination. Unfortunately, ad-
ditional clarity in the origin of the stereochemistry at C10 cannot
be deduced from the elimination reaction of tetraol 10 since a
putative tertiary carbocation at C10a favors an E1 mechanism
with no syn or anti selectivity. In addition we cannot rule out
the possibility that the antibody influences this reaction, causing
one pathway to be favored.28-30 Interestingly, the addition of
catalase and superoxide dismutase (1 µM each) to the reaction
The kinetics of cannabitriol formation was measured for the
three most proficient catalysts (TCF-26C12, TCF-23C4, and
TCF-25G5). The high-energy states of photochemical reactions
result in multiple products and, in order for the antibody-
catalyzed transformation to dominate, a high concentration of
antibody relative to the substrate is required for accurate
measurements, invalidating the usual Michaelis-Menten ap-
proximation that free and total substrate concentrations are
essentially equal. Therefore, in order to calculate the kcat and
KM for the antibody-catalyzed formation of cannabitriol (eq 1)
assuming saturating riboflavin conditions, the concentration of
the antibody-substrate complex [AS] was explicitly determined
(eq 2), where Vo is the initial reaction velocity, [A] ) [A]t -
[AS], and [S] ) [S]f ) [S]t - [AS].31
Vo ) kcat[AS]
(1)
[AS] )
([S] + [A] + K ) - ([S] + [A] + K )2 - 4[S] [A]
x
t
t
M
t
t
M
t
t
(2)
2
Each antibody measured catalyzed cannabitriol formation with
approximately the same rate (Table 1). In examining the
therapeutic potential of the antibody-catalyzed degradation of
∆9-THC, it is important to note that cannabitriol is not the only
product from this reaction. Therefore, the kcat for cannabitriol
formation (1.0 min-1) is a conservative estimate of the rate of
drug degradation. The hydrophobic nature of ∆9-THC and
accumulation in fatty tissue leads to an extended half-life (t1/2
)
in vivo of more than 20 h.22,32 Theoretical calculations using a
50 mg/kg dose of TCF-26C12 provide a blood concentration
of approximately 5 µM, assuming that blood mass is equal to
7% of total body weight. An average dose of ∆9-THC (one
3.55% marijuana cigarette) leads to a peak blood concentration
of approximately 0.5 µM; therefore, the antibody would be
operating under substrate limiting conditions ([S]t , KM) and
the rate equation can be simplified and a new rate constant
(24) ElSohly, M. A.; El-Feraly, F. S.; Turner, C. E. Lloydia 1977, 40, 275-
280.
(29) Matsushita, H.; Yamamoto, N.; Meijler, M. M.; Wirsching, P.; Lerner, R.
A.; Matsushita, M.; Janda, K. D. Mol. Biosyst. 2005, 1, 303-306.
(30) Sinha, S. C.; Keinan, E.; Reymond, J. L. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 11910-11913.
(25) ElSohly, M. A.; Boeren, E. G.; Turner, C. E. Experientia 1978, 34, 1127-
1128.
(26) McPhail, A. T.; ElSohly, H. N.; Turner, C. E.; ElSohly, M. A. J. Nat.
Prod. 1984, 47, 138-142.
(31) Segel, I. H. Enzyme Kinetics; John Wiley & Sons: New York, 1975; pp
72-77.
(27) For a review of the Schenck ene reaction see: Prein, M.; Adam, W. Angew.
Chem., Int. Ed. Engl. 1996, 35, 477-494.
(32) Gustafson, R. A.; Kim, I.; Stout, P. R.; Klette, K. L.; George, M. P.;
Moolchan, E. T.; Levine, B.; Huestis, M. A. J. Anal. Toxicol. 2004, 28,
160-167.
(28) Janda, K. D.; Shevlin, C. G.; Lerner, R. A. Science 1993, 259, 490-493.
9
J. AM. CHEM. SOC. VOL. 129, NO. 12, 2007 3701

A R T I C L E S
Brogan et al.
derived, kAb ) 1.82 × 10-2 min-1 (eq 3). Substituting kAb in
the standard first-order half-life equation provides a t1/2 of 38
min (eq 4). Thus, using the rate of cannabitriol formation as a
conservative estimate, the normal metabolic half-life for ∆9-
THC can be reduced by more than 30-fold utilizing TCF-26C12.
an Agilent ESI-TOF mass spectrometer. HPLC assays were performed
on a Hitachi HPLC (model L-2130) equipped with a column oven
(model L-2300) and UV detector (model L-2420) using Vydac RP-C18
columns.
HPLC Assay for the Conversion of ∆9-THC to Cannabitriol. The
rate constants for the antibody-catalyzed oxidative conversion of ∆9-
THC to cannabitriol were determined by the method of initial rates
under pseudo-first-order conditions. Reactions were initiated by adding
a solution of ∆9-THC (50-400 µM) to phosphate buffer (PBS, pH
7.4) containing an antibody from the TCF or TCB panel (20 µM) and
riboflavin (60 µM) to a final volume of 140 µL (5% DMSO). The
reaction vessels were irradiated on a white light transilluminator (2.6
mW/cm2) at 4 °C for various amounts of time, and aliquots (20 µL)
were removed and injected onto the previously described HPLC system,
equipped with a guard column (isocratic mobile phase: 51% A (water
with 0.1% TFA) and 49% B (acetonitrile with 0.09% TFA), solvent
flow rate of 1 mL min-1; UV detection at 240 nm; 50 °C). Each assay
was performed in duplicate, and data were quantified by interpolation
of peak areas relative to a standard curve.
Synthesis of Cannabitriol via the Antibody Catalyzed Oxidation
of ∆9-THC. A solution of ∆9-THC (5 mM), antibodies from the TCF
and TCB panels (∼14 µM), and riboflavin (90 µM) in phosphate buffer
(PBS, pH 7.4) (5% DMSO) was irradiated on a white light transillu-
minator (2.6 mW/cm2) at 4 °C. The reaction was monitored by the
previously described HPLC assay, and additional aliquots of ∆9-THC
and riboflavin were added twice daily for a period of 4 days. The
reaction mixture was extracted with diethyl ether and the combined
organic layers were concentrated. The crude mixture was isolated by
preparative HPLC (53% A (water with 0.1% TFA) and 47% B
(acetonitrile with 0.09% TFA), solvent flow rate of 10 mL min-1; UV
detection at 240 nm; retention time of cannabitriol, 35.3 min). Fractions
were collected at 0.5 min intervals in test tubes containing 1 mL of
diethyl ether and 100 µL of saturated NaHCO3. The fractions containing
cannabitriol were combined, separated, and further extracted with
diethyl ether (2×). The organic layers were combined and dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. This procedure
was performed twice for a total of 360 µg (the amount of cannabitriol
was determined by NMR using 2,5-dimethylfuran as an internal
standard).37 1H NMR (CDCl3) δ: 0.88 (t, J ) 6.4 Hz, 3 H, H3-5′),
1.24 (s, 3 H, CH3-6), 1.26-1.36 (m, 4 H, H2-4′, 3′H2), 1.40 (s, 3 H,
CH3-9), 1.46 (s, 3 H, CH3-6), 1.58 (m, 2 H, H2-2′), 1.77-1.80 (m, 2
H, H2-8), 2.15 (dt, J ) 3.9, 19.0 Hz, 1 H, HH′-7), 2.39-2.46 (m, 1 H,
HH′-7), 2.47 (t, J ) 7.8 Hz, 2 H, H2-1′), 4.20 (s, 1 H, H-10), 6.30 (s,
1 H, H-1 or H-4), 6.35 (s, 1 H, H-1 or H-4). 13C NMR (CDCl3) δ:
14.0 (C5′), 22.5 (C7), 22.6 (C4′), 23.5 (C6-CH3), 25.1 (C9-CH3), 25.3
(C6-CH3), 29.1 (C8), 30.5 (C2′), 31.4 (C3′), 35.6 (C1′), 70.4 (C9), 72.4
(C10), 76.5 (C6), 108.9 (C1a), 109.2 (C2 or C4), 110.9 (C2 or C4),
122.1 (C10a), 136.1 (C6a), 144.8 (C3), 152.3 (C1 or C4a), 153.7 (C1
or C4a). All structural assignments were in agreement with the 1H-1H
COSY, 1H-1H NOESY, 1H-1H ROESY, 1H-13C HMQC, 1H-13C HMBC,
and 13C APT data. MS (electrospray): m/z 347 [M+1]+. HRMS
(electrospray): m/z calcd for C21H31O4, 347.2217 [M+1]+; found,
347.2205
kcat[A]t[S]t kcat[A]t[S]t
Vo )
≈
≈ kAb[S]t,
where kAb
KM
KM + [S]t
k
)
cat[A]t (3)
Km
ln 2
kAb
t1/2
)
(4)
The antibody-catalyzed oxidative degradation of ∆9-THC
yields a structurally complex natural product that may have
pharmacological activity. The physiological effects of ∆9-THC
and other cannabinoids stem from ligand binding to a family
of cannabinoid receptors found primarily on central and
peripheral neurons (CB1) and immune cells (CB2).33 There is
no reported pharmacological data for cannabitriol; therefore, the
cytotoxicity of cannabitriol was measured in SH-SY5Y human
neuroblastoma cells to examine CB1-mediated neurotoxicity.34
In this assay, ∆9-THC and cannabitriol were found to be
cytotoxic at similar concentrations (EC50 ) 5 µM). However,
the addition of two hydroxyl groups makes cannabitriol
significantly more polar than ∆9-THC by decreasing the ClogP
nearly 3 orders of magnitude (∆9-THC ClogP ) 7.2, cannabitriol
ClogP ) 4.4), thus reducing blood-brain barrier permeability.35
Furthermore, the primary metabolic pathway of ∆9-THC
involves hydroxylation of the C ring, providing modification
sites for glucoronidation. Although cannabitriol has cytotoxicity
comparable to ∆9-THC, the antibody-catalyzed degradation aids
the natural metabolic pathways and is expected to facilitate
elimination of the drug.
The conversion of ∆9-THC to cannabitriol represents an
antibody-catalyzed transformation that likely involves two
different oxidants in a multistep chemical process. Catalysis is
not programmed into the antibody by hapten design, unlike
previous catalytic antibodies that employ transition-state ana-
logues or reactive immunization, but rather is an inherent
property of the antibody itself. This is significant considering
1
the role O2*-mediated oxidation may play in the respective
therapeutic outcomes for the increasing number of monoclonal
antibodies in the clinic and under development.36 Furthermore,
by specifically targeting the ability of an antibody to generate
ROS from 1O2*, we demonstrate the potential of the antibody-
catalyzed oxidation of ∆9-THC as a treatment for marijuana
abuse.
Acknowledgment. This work was supported by The Skaggs
Institute for Chemical Biology, the National Institute on Drug
Abuse (Grant DA 15700 to K.D.J.), and NIH Kirchstein
postdoctoral fellowships (DA 18507 to A.P.B. and AI 62014
to L.M.E.).
Materials and Methods
General Procedures. All reagents were purchased from Sigma-
Aldrich or Fisher and used as received. 1H NMR and 13C NMR spectra
were recorded on a Bruker DRX-600 spectrometer equipped with a
cryogenic probe at 600 and 150 MHz, respectively. Electrospray
ionization (ESI) mass spectrometry experiments were performed on
Supporting Information Available: Synthesis of ∆9-THC,
full HMBC correlations, and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
(33) Pertwee, R. G. Handb. Exp. Pharmacol. 2005, 168, 1-51.
(34) Klegeris, A.; Bissonnette, C. J.; McGeer, P. L. Br. J. Pharmacol. 2003,
139, 775-786.
(35) Ecker, G. F.; Noe, C. R. Curr. Med. Chem 2004, 11, 1617-1628.
(36) AdV. Drug DeliVery ReV. 2006, 58, 631-766.
JA070022M
(37) Gerritz, S. W.; Sefler, A. M. J. Comb. Chem 2000, 2, 39-41.
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3702 J. AM. CHEM. SOC. VOL. 129, NO. 12, 2007