Enantiodiscrimination of methamphetamine by circular dichroism using a porphyrin tweezer

Article abstract of DOI:10.1002/chir.20752

Using exciton-coupled circular dichroism (ECCD) spectroscopy, our lab was able to differentiate between the two enantiomers of methamphetamine using a commercially available porphyrin tweezer as an achiral host. The host-guest complex formed with (1)-(S)-methamphetamine produced a negative bisignate-shaped ECCD spectrum, whereas the complex formed with (2)-(R)-methamphetamine produced a positive one. This sensitive technique could serve as an alternative method for the enantiodiscrimination of chiral methamphetamine, a commonly abused drug in the United States.

Full text of DOI:10.1002/chir.20752

CHIRALITY 22:398–402 (2010)
Enantiodiscrimination of Methamphetamine by Circular
Dichroism Using a Porphyrin Tweezer
MARCUS ANDERSON, KATIE WILCOX, MIKE GUERICKE, HAN CHU, MARK V. WILSON,
*
ERIN WILSON, KERRY LUCAS*, AND ANDREA E. HOLMES
Department of Chemistry, Doane College, Crete, Nebraska
ABSTRACT
Using exciton-coupled circular dichroism (ECCD) spectroscopy, our
lab was able to differentiate between the two enantiomers of methamphetamine using a
commercially available porphyrin tweezer as an achiral host. The host–guest complex
formed with (1)-(S)-methamphetamine produced a negative bisignate-shaped ECCD
spectrum, whereas the complex formed with (2)-(R)-methamphetamine produced a
positive one. This sensitive technique could serve as an alternative method for the enan-
tiodiscrimination of chiral methamphetamine, a commonly abused drug in the United
C
VV
States. Chirality 22:398–402, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: chiral drugs; enantiomers; ECCD; bisignate-shaped CD spectrum;
color changes
INTRODUCTION
substance.16 The study described here applies the porphyrin
tweezer method to determine the enantiomeric excess and
absolute configuration of the predominant methamphet-
amine enantiomer in a sample.
Many drugs, including both pharmaceuticals and
narcotics, are only effective if they contain specific stereo-
genic centers. Enantiomers of these chiral drugs frequently
exhibit different physiological characteristics. Methamphet-
amine is an interesting example of a drug in which one enan-
tiomer is used therapeutically, whereas the other causes
detrimental side effects. Both enantiomers of methamphet-
amine are available in the United States. (1)-(S)-Metham-
phetamine is a prescribed medicine that is often abused as a
narcotic because it acts as a potent stimulant and provides a
‘‘high’’ feeling. Overuse of this drug can lead to paranoia,
self-destruction, obsessive skin-picking, damage to heart
valves, poor dental health, and increased risk of strokes.1,2
(2)-(R)-Methamphetamine, which does not cause a ‘‘high,’’
is found in over-the-counter medications such as the Vicks1
Vapor Inhaler1 [active ingredient per inhaler: l-methamphet-
amine 50 mg (nasal decongestant)].3 In Nebraska where
Doane College is located, (1)-(S)-methamphetamine-related
crimes are five times the national average,4 and it is one of
the most commonly abused drugs in the United States.5
Many sensing methods have been established, such as gas
chromatography–mass spectrometry (GC-MS),6–9 ion trap
mobility spectrometry,10,11 fluorescence detection after solid-
phase extraction,12 HPLC tandem mass spectrometry,13 elec-
trokinetic chromatography,14 high-performance thin-layer
chromatography,15 and immunoassays.10 However, most of
these sensing methods may not be able to distinguish
between enantiomers of chiral drugs. There is a need for
more reliable simple spectroscopy methods of distinguish-
ing the enantiomers of methamphetamine because of man-
dated regulatory enantiomeric purity of methamphetamine,
which is a schedule II Drug Enforcement Agency controlled
C
A nonempirical circular dichroic approach, using exci-
ton-coupled circular dichroism (ECCD), to determine the
absolute configuration of primary amines has been
reported.17–20 Berova and coworkers used ECCD to deter-
mine the absolute stereoconfiguration for a variety of chi-
ral amines.18,21,22 Supramolecular complexes of the amines
with pentanediol-linked zinc porphyrins were formed and
referred to as ‘‘zinc porphyrin tweezers.’’22
Figure 1 demonstrates the macrocyclic 1:1 host–guest
complex that we utilized for our purposes to distinguish
between chiral abused drugs like (1)-(S)- and (2)-(R)-
methamphetamines, which are secondary amines with ab-
sorbance maxima below the wavelength cutoff for many
commonly used solvents. Binding occurs first between the
methamphetamine and the carrier molecule, an N-Boc-pro-
tected carboxylic acid, and then to the bidentate porphyrin
tweezer. The resulting 1:1 host–guest complex generates
an ECCD spectrum in the visible region, the sign of which
corresponds to the absolute configuration of the chiral
amine.
Contract grant sponsor: NIH (INBRE Program of the National Center for
Research Resources); Contract grant number: P20 RR016469.
Contract grant sponsor: NSF; Contract grant numbers: DUE-0633462,
CHE-0747949.
*Correspondence to: Andrea E. Holmes or Kerry Lucas, Doane College,
1014 Boswell Ave, Crete, NE 68333. E-mail: kerry.lucas@doane.edu or
E-mail: andrea.holmes@doane.edu
Received for publication 25 August 2008; Accepted 1 May 2009
DOI: 10.1002/chir.20752
Published online 2 July 2009 in Wiley InterScience
(www.interscience.wiley.com).
VV
2009 Wiley-Liss, Inc.

ENANTIODISCRIMINATION OF METHAMPHETAMINE
399
was redissolved in 5 ml of methylene chloride (CH2Cl2)
and washed twice with 5 ml of aqueous sodium bicarbon-
ate (NaHCO3). The organic layer was then removed and
dried with anhydrous sodium sulfate (Na2SO4). Thin-layer
chromatography (TLC) analysis was performed in pure
ethyl acetate to confirm isolation of the product.
The carrier-drug conjugate was purified using silica gel
column chromatography with pure ethyl acetate as the
mobile phase. Fractions were analyzed using TLC and
1
product was verified by mass spectrometry and NMR. H
NMR (500 MHz, CDCl3) d 5 1.25 (d, 3H, CH3ÀÀ), 1.46
(s, 9H, t-Bu), 2.59 (d, 2H, CH2-phen), 3.00 (s, 3H, CH3N),
4.05 (m, 1H, CH), 4.23 (d, 2H, pyr-CH2NH), 6.54 (s, 1H,
H
H
arom), 6.93 (dd, 2H, Harom), 7.12–7.34 (m, 2H, Harom, 2H,
pyr), 8.45(d, 1H, Hpyr). Mass spectrometry showed a
mass of 284.03 (calculated 283.37).
Deprotection of the Carrier-Drug Conjugate
To remove the Boc protecting group, the purified car-
rier-drug conjugate was treated with 5 ml of 10% trifluoro-
acetic acid (TFA, in CH2Cl2) at room temperature for 2 h.
All solvent was subsequently evaporated under reduced
pressure using a rotary evaporator. Structure was verified
1
by NMR. H NMR (90 MHz, CDCl3) d 5 1.2–1.4 (d, 3H,
Fig. 1. (a) Macrocyclic 1:1 host–guest complex formed between guest
and host, orienting porphyrins in a (b) clockwise or counterclockwise
twist depending on the stereoconfiguration of the chiral amine. (c) This
twist can be measured by ECCD, making a nonempirical determination of
the absolute configuration possible. (d) Expanded structure of porphyrin
host.
CH3ÀÀ), 2.6–2.8 (d, 2H, CH2-phen), 2.9–3.1 (s, 3H, CH3N),
3.8–4.2 (m, 1H, CH and 2H, pyr-CH2NH), 6.6–6.7 (bs, 1H,
H
arom), 6.8–6.9 (bm, 2H, Harom), 7.0–7.3 (bm, 2H, Harom
and 2H, Hpyr), 8.4–8.5 (bm, 1H, Hpyr). b, broad.
Preparation of Porphyrin Tweezer for Spectrophotometry
Deprotected carrier-drug conjugate (1 mg) was dis-
solved in a minimal amount of methanol (ꢀ3 ml) and a
saturating amount of sodium carbonate was added. All
MATERIALS AND METHODS
Materials and General Procedures
Unless noted, all materials were obtained from commer-
cial suppliers and used without further purification. 1H
NMR spectra were obtained on either a 90 MHz Anasazi
EFT-90 or a 500 MHz Bruker Avance NMR spectrometer
and are reported in parts per million relative to TMS. UV–
vis spectra were recorded on a Cary-50 spectrophotometer
and are reported as kmax [nm] (emax [mM21 cm21]). The
CD spectra were recorded on a Jasco J-815 spectrophotom-
eter and reported as molar ellipticity (8 M21 cm21) at
room temperature in hexane using 10-mm cuvettes.
solvent was evaporated using
a rotary evaporator.
Anhydrous CH2Cl2 (1 ml) was added to yield a 3.5 mM
solution of carrier-drug conjugate. A 0.1 mM solution of
pentamethylene bis[4-(10,15,20-triphenylporphin-5-yl)ben-
zoate]dizinc(II) (TCI America, hereafter referred to as
‘‘tweezer’’) was prepared in CH2Cl2. A 25 ll sample of
tweezer was titrated with carrier-drug conjugate to
ensure total saturation. UV–vis and CD spectra of the
tweezer-conjugate complex and the tweezer solution
alone were recorded.
Synthesis and Purification of 4-[( tert-
Butoxycarbonylamino)methyl]pyridine-2-carboxylic acid
Methamphetamine Conjugate
Determination of Sensitivity
A series of samples were prepared with decreasing con-
centrations of carrier-drug conjugate, leaving final tweezer
concentration at 1.25 lM. The limit of detection for this
technique was assumed to be where the signal-to-noise
ratio was equal to 3.
For synthesis of the drug-carrier conjugate, a solution
was prepared with 1 equiv of 4-[(tert-butoxycarbonylami-
no)methyl]pyridine-2-carboxylic acid (TCI America, here-
after referred to as ‘‘carrier’’), 1.2 equiv of benzotriazol-
1-yloxytris(dimethylamino)phosphonium hexafluorophos-
phate (BOP), 1.2 equiv of diisopropylethylamine (DIPEA),
and 0.95 equiv of the enantiomerically pure free base
Enantiomeric Excess
Nine solutions of enantiomeric excess values ranging
narcotic (14 mg) in 3 ml of tetrahydrofuran. Methamphet- from 100% (1)-(S)-methamphetamine-carrier conjugate to
amine was converted to its free base form, when neces- 100% (2)-(R)-methamphetamine-carrier conjugate were
sary, according to Sellers and Rigdon.23 The solution was prepared in hexane, using a final drug-carrier conjugate
stirred overnight at room temperature and all solvents concentration of 400 lM and a final tweezer concentration
were evaporated using a rotary evaporator. The product of 1.25 lM. The resultant spectra were corrected for
Chirality DOI 10.1002/chir

400
ANDERSON ET AL.
Scheme 1. (1)-(S)-Methamphetamine was derivatized with an N-Boc-protected carrier, which was then removed with 10% TFA. Similar steps were
performed with (2)-(R)-methamphetamine.
background and smoothed using a Savitzki-Golay smooth- versa. This would mean a negative couplet in the CD spec-
ing filter with an 11 point window. The calibration curve trum of (1)-(S)-methamphetamine is due to the spatial ori-
was generated by reading e at 433.1 nm for all spectra.
entation of the net transition dipole from the Soret bands
in the porphyrins with a counterclockwise twist, corre-
sponding to the S configuration of the chiral center.18 The
opposite applies to the positive couplet of (2)-(R)-metham-
phetamine considering the amide moiety as the highest
RESULTS AND DISCUSSION
Methamphetamine is often mislabeled using the D/L priority group and the aromatic moieties as the second
notation (note the uppercase letters).24,25 Rotation of line- priority.
arly polarized light in the clockwise direction indicates a
To determine whether the absolute configuration of
positive (1) or dextrorotary (D) rotation, whereas in the methamphetamine leads to the predicted CD couplets, the
counterclockwise direction a negative (2) or levorotary chiral narcotics (1)-(S) and (2)-(R)-methamphetamine
(L) rotation. The D/L method is primarily used to denote were derivatized with an N-Boc-protected carrier as shown
the absolute configuration of sugars and amino acids. It is in Scheme 1. The use of the freebase form of the drug is
more appropriate to use the (S) and (R) nomenclature for necessary to ensure a high yield. The N-Boc protection is
other chiral substrates. In accordance with the guidelines necessary to avoid intramolecular amidation of the carrier.
of the International Union of Pure and Applied Che- Exact amounts of DIPEA are also important because
mistry (IUPAC), we will label L-methamphetamine as (2)- excess (1.5 equiv or more) can lead to undesired side
(R)-methamphetamine and D-methamphetamine as (1)- products such as amidation with DIPEA instead of
(S)-methamphetamine.26 We predicted the sign of the CD methamphetamine. The resulting carrier-conjugate was
couplet based on the absolute stereochemistry of the deprotected with TFA and added to the zinc porphyrin
methamphetamine isomers. Preceding literature20,27 indi- tweezer in hexane. The absolute configurations, struc-
cated that the absolute configuration of the chiral center tures, and predicted sign of the CD couplets for each
can be used to forecast the sign of the CD couplet or vice porphyrin tweezer complex are summarized in Table 1.
TABLE 1. Summary of nomenclature, configuration, structure, absolute configuration based on priorities in Newman
projections, and predicted sign by CD
Name
Structure
Twist
Predicted CD couplet
(2)-(R)-Methamphetamine
1-methamphentamine
(1)-(S)-Methamphetamine
d-methamphetamine
Chirality DOI 10.1002/chir

ENANTIODISCRIMINATION OF METHAMPHETAMINE
401
when reading from right to left, a determination of the
absolute configuration at the chiral carbon can be made.
The electronic transition moment of the porphyrin tweezer
became twisted in a right-handed or left-handed fashion
depending on the absolute configuration of the metham-
phetamine. For example, (1)-(S)-methamphetamine
resulted in a preferred conformation of porphyrins in a
counterclockwise twist, leading to a negative couplet in
CD, and the enantiomer led to the mirror image. This com-
plex generated a strong ECCD spectrum centered at 422
nm. Although any chiral substrates that are able to react
with the carrier molecule could give similar results, this is
the first example of using a porphyrin tweezer with respect
to enantiodiscrimination of addictive psychostimulants.
Our data confirm that configurational assignment of chiral
methamphetamine using a porphyrin tweezer is possible
by ECCD. Previously, this tweezer method was not recom-
mended for acyclic secondary amines.21 However, the deri-
vatization of methamphetamine with 4[(tert-butoxycarbo-
nylamino)methyl]pyridine-2-carboxylic acid was easily
reproduced without any variations in synthetic yield or chi-
roptical properties. Thus, the porphyrin-derivatized chiral
complexes serve as promising reporters of stereochemistry
at the carbon adjacent to a secondary amine. Because of
the strong amplitude (A value > 120) of the CD couplets
with nearly equal strengths, we do not believe that racemi-
zation occurred during the derivatization step.
Fig. 2. (a) Determination of enantiomeric excess: circular dichroism
spectra of methamphetamine in hexane at 258C. Concentrations were
determined gravimetrically. (b) UV–vis spectra of porphyrin tweezer alone
and porphyrin tweezer (1)-(S)-methamphetamine conjugate and its enan-
tiomer, in hexane. Inset: UV spectrum of (1)-(S)-methamphetamine (50
lM) in water and of water only.
The assessment of enantiopurity was illustrated with
methamphetamine. Both enantiomers of methamphetamine
were derivatized with the carrier molecule, and the meas-
urements of CDs of solutions containing different e.e. are
The porphyrins’ physical and optical properties make shown in Figure 2a. Although the CD amplitude (A value) is
equal for both enantiomers, the Cotton effect is negative
when the (S) isomer predominates, positive when the (R)
them an excellent choice for spectrophotometric studies.
They have known transition moments, absorbance in the
visible region, large extinction coefficients, potential for isomer predominates, and zero when the solution is com-
pletely racemic. Figure 3 shows a calibration curve with an
excellent linear agreement between De and e.e. (R2 > 0.99).
Also noteworthy, the UV–vis spectrum of the bound
large CD amplitudes, UV–vis detection at low concentra-
tion, stability in air, and easy preparation with commer-
cially available starting materials.27 This allowed us to suc-
cessfully use the porphyrin tweezers for our methamphet- methamphetamine-carrier conjugate complex shows a
large bathochromatic shift when compared with the
unbound porphyrin, clearly indicating the binding of the
amine drug study. For example, the absorbance maximum
for methamphetamine (kmax 5 206 nm in water, Fig. 2b,
inset) was below the wavelength cutoff for many com- conjugate to the porphyrin tweezer (Fig. 2b). This batho-
chromatic shift was also accompanied by a visible color
change from light pink to yellow and an increase in the
monly used solvents (i.e., dichloromethane kc 5 235 nm,
acetone kc 5 330 nm) limiting the solvent selection for
our studies. One of the advantages of the porphyrin extinction coefficient (Fig. 2b).
tweezer system is the ability to eliminate this problem,
allowing structural studies in various solvents. This is par-
ticularly attractive for chiral amines containing chromo-
phores that absorb in the UV region. These chromophores
would not overlap with the porphyrin transitions, and
therefore, a clear spectroscopic analysis window with high
sensitivity (because of high extinction coefficients) is
provided between 400 and 450 nm.
As seen in Figure 2a, the CD spectra of the porphyrin
tweezers containing methamphetamine are split. The CD
of the porphyrin tweezer and the deprotected carrier with
tweezer (data not shown) are both silent, whereas the addi-
tion of (1)-(S)-methamphetamine or (2)-(R)-methamphet-
amine-carrier conjugate to the tweezer produced a bisig-
Fig. 3. Plot showing linear regression between % e.e. of methamphet-
nate spectrum (Fig. 2a). Based on the sign of the couplet, amine carrier and De in hexane at 258C at k 5 nm, R2 > 0.99.
Chirality DOI 10.1002/chir

402
ANDERSON ET AL.
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ratio greater than 3 at a concentration of 500 ng/ml. This
is equivalent to a methamphetamine concentration of 263
ng/ml because of the added mass of the carrier. The Sub-
stance Abuse and Mental Health Services Administration
(SAMHSA) cutoff concentration for amphetamines is
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CONCLUSIONS
Using CD spectroscopy and a porphyrin tweezer host–
guest complex, differentiation between the two enantio-
mers of methamphetamine was accomplished. (1)-(S)-meth-
amphetamine produced a negative couplet, whereas (2)-
(R)-methamphetamine produced a positive one. The addi-
tion of the drug-carrier conjugate to the porphyrin tweezer
prompted a bathochromic shift and change in absorbance.
This shift was visibly detectable as well; the porphyrin
tweezer was light pink in solution before the addition of
the methamphetamine-carrier conjugate but slightly yel-
low after the host–guest complex was formed. The micro-
molar sensitivity of this method offers a widely applicable,
nonempirical technique for chirality sensing of abused chi-
ral substances that have absorbance maxima below the
solvent cutoff. This method could serve as an alternative
method for the enantiodiscrimination and determination of
enantioexcess of chiral methamphetamine.
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ACKNOWLEDGMENTS
The authors thank the American Chemical Society for
funding two Project SEED students, one of whom is a
coauthor of this article (H.C.), and the University of
Nebraska Medical Center Mass Spectrometry and the Pro-
teomics Core Facility for performing the mass spectrome-
try analysis.
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