Synthesis of catecholamine conjugates with nitrogen-centered bionucleophiles

Article abstract of DOI:10.1016/j.bioorg.2012.05.002

The enzymatic (tyrosinase) and chemical (NaIO₄, Ag₂O or Fremys's salt) oxidation of biologically relevant catecholamines, such as dopamine (DA), N-acetyldopamine (NADA) and the Ecstasy metabolites (α-MeDA and N-Me-α-MeDA) generates t

Full text of DOI:10.1016/j.bioorg.2012.05.002

Bioorganic Chemistry 44 (2012) 19–24
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of catecholamine conjugates with nitrogen-centered bionucleophiles
a,
Filipa Siopa a, Alice S. Pereira a, Luísa M. Ferreira a, M. Matilde Marques b, , Paula S. Branco
⇑
⇑
a Chemistry Department, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
b Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1049-001 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 January 2012
Available online 19 June 2012
The enzymatic (tyrosinase) and chemical (NaIO4, Ag2O or Frémys’s salt) oxidation of biologically relevant
catecholamines, such as dopamine (DA), N-acetyldopamine (NADA) and the Ecstasy metabolites
(a-MeDA and N-Me-a-MeDA) generates the corresponding o-quinone which can be trapped with
nitrogen bionucleophiles such as N-acetyl-histidine and imidazole in a regioselective reaction that takes
place predominantly at the 6-position of the catecholamine.
Keywords:
Dopamine
Ecstasy
Ó 2012 Elsevier Inc. All rights reserved.
Catecholamines
Quinones
Amino acids
Nitrogen adducts
1. Introduction
the quinones [12,13]. Some syntheses of sulfhydryl-dopamine con-
jugates, involving the enzymatic oxidation of DA, have been re-
The catecholamine dopamine (DA), 1 is an important neuro-
transmitter involved in the pathogenesis of Parkinson’s disease
(PD) [1,2], a degenerative neurological disorder characterized by
rigidity, hypokinesia and tremor. A combination of factors have
been proposed to explain this condition [3–7], in particular the for-
mation of neurotoxic cysteinyl–catecholamine conjugates [8]. The
primary metabolic pathway of catecholamines is oxidation to their
catechol–quinones and semiquinones. These species are toxic
intermediates that can cause cellular lesions at neutral pH via
intermolecular and/or intramolecular Michael addition; they can
also generate toxic reactive oxygen species (ROS) via redox cycling.
Both mechanisms can lead to toxicity. Some authors reported the
affinity of N-acetyldopamine (NADA, 2) to nucleophilic groups in
cuticular proteins of insects, with direct evidence of covalent bind-
ing obtained by solid-state NMR [9]. Moreover, DA and serotonin
can produce covalent modification of cellular proteins, and possi-
bly even cell degeneration, in the presence of high levels of metal
ions [10]. Furthermore, both antitumor and toxic properties of sev-
eral catecholamines have been attributed to the inhibition of crit-
ical enzymes (e.g., DNA polymerase) by quinone-mediated
covalent binding [11]. Indeed, inactivation of enzymes/proteins
by o-quinones is presumably due to nucleophilic addition reactions
of sulfhydryl or amino groups present in these macromolecules to
ported [14–17]. Other methods are more demanding, such as
those involving the electrochemical generation of oxidized species
[18–23]. An additional drawback of electrochemical methods is
that the initially formed adducts are further oxidized to their cor-
responding quinones at the applied potential, leading to complex
mixtures. Other strategies have relied on the ability of metal ions
[e.g., Fe(II/III) or Mn(II)] to catalyze the oxidation of DA in the pres-
ence of
L-cysteine [24]. 3,4-Methylenedioxymethamphetamine
(MDMA or Ecstasy) is a widely abused, psychoactive recreational
drug. There is growing evidence that the neurotoxic profile of
MDMA is greatly dependent upon its hepatic metabolism, which
produces highly reactive derivatives, such as catecholamines, cate-
cholamine thioethers, and quinones. Our group has previously as-
sessed the toxicity of MDMA and some of its catecholamines
metabolites, namely a-MeDA (3), N-Me-a-MeDA (4) and their cor-
responding glutathione (GSH) and N-acetylcysteine (NAcCys) con-
jugates, in rat cortical neuronal cultures [25,26]. To better
understand the role of specific catecholamines in biological pro-
cesses, the synthesis and structural elucidation of catecholamines
adducts of amino acids, nucleosides and small peptides is of ut-
most significance. However, the synthesis of catecholamine conju-
gates with nitrogen bionucleophiles is difficult, due to the less
nucleophilic nature of these compounds when compared with
GSH or NAcCys. Hawley and co-workers [27,28] reported the elec-
trochemical oxidation of NADA- and N-a-alanyldopamine-gener-
⇑
Corresponding authors. Address: CQE, Complexo I, IST, Av. Rovisco Pais, 1049-
001 Lisboa, Portugal. Fax: +351 21 8464455 (M. Matilde Marques), fax: +351 21
2948550 (P.S. Branco).
ated quinones with two nitrogen-centered nucleophiles,
imidazole (Imid) and N-acetyl-histidine (NAcHis), to model
cuticular proteins containing histidinyl residues. They reported
the predominant products from both nucleophiles to be C6 adducts
E-mail addresses:
matilde.marques@ist.utl.pt (M. Matilde Marques), paula.
branco@fct.unl.pt (P.S. Branco).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.05.002

20
F. Siopa et al. / Bioorganic Chemistry 44 (2012) 19–24
Table 1
of NADA and N-a-alanyldopamine. We describe herein for the first
Reaction conditions and yields for catecholamine conjugates with the nucleophiles
NAcHis and Imid.
time the chemical and enzymatic synthesis of nitrogen adducts of
relevant catecholamines (1–4).
Reaction conditions
Products
Isolated
yield (%)
Oxidant/Nuc/catecholamine
Ty/NAcHis (10 eq.)/2a
2-NAcHis–NADA (8)
6-NAcHis–NADA (7)
6-NAcHis–NADA (7)
2
16
10
4
2. Results
NaIO4/NAcHis (10 eq.)/2
Frémy’s salt/NAcHis (5 eq.)/2 6-NAcHis–NADA (7)
When subjecting 1 to enzymatic oxidation with tyrosinase
using our previously reported method [25], followed by quenching
of the respective o-quinone (5) with nitrogen nucleophiles, such as
imidazole or the amino acids lysine (Lys), histidine (His), N-acetyl-
lysine (NAcLys), NAcHis, and tryptophan (Trp) only intractable
mixtures were obtained. Chemical oxidation with NaIO4, Ag2O
and Frémys’s salt similarly produced a black residue, probably cor-
responding to melanin, a dark polymeric pigment stemming from
intramolecular amine cyclization of the quinone and ensuing poly-
merization (Scheme 1). Tse et al. [29], reported that the intramo-
lecular cyclization of 1 is faster than any intermolecular reaction,
except when sulfur nucleophiles (e.g., Cys and GSH) are present. In-
deed, the reaction of 1 with NAcCys was reported to be 106-fold
faster than with NAcHis [17]. In order to restrict this unwanted
reaction we focused on 2, where the presence of the N-acetyl pro-
tecting group was expected to prevent the pathway of intramolec-
Ag2O/NAcHis (5 eq.)/2
Ty/Imid (10 eq.)/2
NaIO4/Imid (10 eq.)/2
NaIO4/NAcHis (5 eq.)/3
NaIO4/NAcHis (10 eq.)/4
6-NAcHis–NADA (7)
6-Imid–NADA (9)
6-Imid–NADA (9)
6-NAcHis–NAc-a-MeDA (10)
6-NAcHis–NAc-NMe-
2
15
10
5
a-MeDA
8
(11)
a
Ty, tyrosinase.
the lifetime of 5 in 50 mM phosphate (pH 7.4) was 30 min at
0.47 M but decreased to 3 min at 1.40 M. Taking into consider-
l
l
ation the short lifetime of the quinones, the nucleophiles were
added to the reaction mixture 5 min after starting the generation
of the o-quinone, regardless of the specific (enzymatic or chemical)
oxidation procedure.
ular cyclization and subsequent polymerization.
A further
consideration was that the ionizable groups of Lys or His predom-
inate in a zwitterionic form, involving protonated (and therefore
non-nucleophilic) nitrogens, under our experimental conditions
(pH 7.4). To ensure that the progress of the reactions would not
be hampered by protonation of potentially nucleophilic sites, we
used the N-protected amino acids NAcHis and NAcLys. The inter-
mediate o-quinone 5 should in principle undergo a 1,4 or 1,6 inter-
molecular Michael addition in the presence of nucleophiles,
although reaction on the external chain of the quinone-methide
(6) could not be excluded (Scheme 1). The products from reaction
with the nucleophiles NAcHis and Imid were found only in trace
amounts. This may be attributed in part to the fact that the qui-
none intermediates are short-lived species. Indeed, we conducted
a kinetic spectrophotometric study, which clearly indicated that
isomerization of 5 (k 390 nm) to 6 (k 500 nm) occurs spontane-
ously (see ESI). These results agree with previous reports [30,31],
An additional observation was that the yield of the catechol-
amine conjugate, 6-(N-acetyl)-histidine-N-acetyldopamine (6-
NAcHis–NADA, 7) could be improved (from trace amounts to 16%)
if, prior to addition of the nucleophile, the enzyme was removed
by gel chromatography filtration. Furthermore, these conditions al-
lowed the isolation of a small amount of the isomer at the C2 posi-
tion, 2-(N-acetyl)histidine-N-acetyldopamine (2-NAcHis–NADA, 8)
(Table 1). This procedure involving enzyme separation avoids fur-
ther oxidation of the initially formed adducts, which are typically
more prone to oxidation than the parent catechols [33]. As a further
advantage, we measured the activity of the recovered enzyme and
concluded that it remained active and thus reusable. The full struc-
tural characterization of the isolated reaction products showed that
the nitrogen adducts resulted from a 1,4-Michael addition reaction
to C6, the most electropositive site on the o-quinone ring
(Scheme 2). This contrasts with the regioselectivity observed in
reactions with sulfur nucleophiles, where the attack occurs pre-
dominantly at the C5 of the o-quinone although minor products
resulting from attack at the C2 position were observed by some
although some authors observed
a slower decomposition of
NADA–quinone by NMR [31,32]. We had definite evidence that
the rate of disappearance of 5 was concentration-dependent. Thus,
Scheme 1. Proposed pathway for catecholamine metabolism, yielding reactive intermediates capable of alkylating cellular components or generating reactive oxygen
species, ultimately leading to toxicity.

F. Siopa et al. / Bioorganic Chemistry 44 (2012) 19–24
21
Scheme 2. Proposed mechanism for the conjugated Michael-addition of the nitrogen nucleophiles NAcHis and Imid, to biologically relevant catecholamines.
authors [15,17,23,34,35]. It has been proposed that base catalysis
by the neighboring oxygen on C4 enhances the nucleophilicity of
the sulfur compound for attack on C5 [36]. However, a similar argu-
ment (proximity of the oxygen on C3) might be used regarding at-
tack on C2, which is minor with sulfur nucleophiles. This suggests
that other factors (e.g., relative softness/hardness of the electro-
philic sites on the quinones, stereochemical hindrance) may be at
play. Interestingly, in contrast with 6-NAcHis–NADA (7), the NMR
spectra of 2-NAcHis–NADA (8) indicated the presence of two con-
yields (Table 1). Despite the difficulty found in the synthesis of
these compounds, which arose from the instability of the o-quinone
and lower reactivity of the nucleophiles, the method presented
herein uses a less demanding protocol that those reported in the lit-
erature for other catecholamines [27,36–38] and allows the isola-
tion of adducts in acceptable yields, amenable to complete
structural analysis and future use as biomarkers. It should be noted
that while some of the 1H NMR signals for adducts 10 and 11 were
duplicated (cf. Section 4 and Supplementary material), this does not
compromise their characterization. Indeed, since catechols 3 and 4
were used as racemates, both 10 and 11 were produced as mixtures
of two diastereomers [6-(N-acetyl)-S-histidine-(R,S)-N-acetyl-
formers. We observed
a partial, temperature-dependent and
reversible, merging of the duplicated signals, consistent with re-
stricted rotation, presumably through the histidine–NADA bond.
This restriction may have been induced by an intramolecular
hydrogen bond (e.g., involving the hydroxyl on C3 and the histidine
nitrogen bound to NADA), which is not possible in adduct 7. Com-
pound 7 and 6-N-imidazole–N-acetyldopamine (6-Imid–NADA, 9)
have previously been reported as products from the electrochemi-
cal oxidation of 2 [27,28]. However, we question the structural
characterization of 9 reported therein. Indeed, we performed a full
characterization of 9 by two-dimensional NMR spectroscopy and
found discrepancies regarding the reported assignments, namely
a >1 ppm difference for the imidazole H20 (cf. Electronic Supporting
Information (ESI) for the spectroscopic data). From analysis of
Table 1 it can be concluded that the best result was obtained with
tyrosinase and the most discouraging one with Ag2O. With NaIO4
the yields were only slightly lower than with tyrosinase, suggesting
that the mechanism for quinone formation, either via simultaneous
transfer of two-electrons (tyrosinase) or consecutive transfer of
one-electron with formation of a semiquinone intermediate does
not affect the progress of the reaction (Scheme 1). Upon reaction
of NADA-quinone with a series of other amino acids only adducts
derived from reaction with valine and tryptophan could be detect-
able by electrospray ionization mass spectrometry (ESI-MS). Given
the results obtained with NADA–quinone, and with the purpose of
obtaining nitrogen adducts from the Ecstasy metabolites 3 and 4,
we generated the quinones from 3 and 4 with NaIO4 and selected
NAcHis as a representative, and biologically relevant, nitrogen
nucleophile. In view of the instability of these compounds, which
are prone to intramolecular amine cyclization of the quinone
and subsequent polymerization when bearing a free amino group,
the nitrogen was protected by acetylation prior to reaction with
a
-methyldopamine and 6-(N-acetyl)-S-histidine-(R,S)-N-acetyl-
N-methyl- -methyldopamine, respectively], which were indistin-
a
guishable (and therefore unseparable) in our HPLC conditions.
The N-acetyl protecting groups could be easily removed by hydro-
lysis, thus allowing the synthesis of the dopamine conjugates,
otherwise impossible to achieve. Indeed, on hydrolysis under acidic
conditions compounds 7, 8, and 9 gave the DA-nitrogen adducts 6-
N-histidine-dopamine (6-His-DA, 12), 6-N-histidine-N-acetyldop-
amine (6-His-NADA, 13), and 6-N-imidazole-dopamine (6-Imid-
DA, 14), in 27%, 21%, and 40% yield, respectively (Scheme 2). Only
6-His-NADA (13) was identified as a partially deacetylated com-
pound. We have not attempted any deacetylation of 8 due to the
low yield of the isolated compound. Although the S-enantiomer of
MDMA is more potent than the R-enantiomer in producing the typ-
ical effects that are characteristic of Ecstasy, MDMA and related
compounds are consumed as racemates. Despite the limited infor-
mation, the rates of elimination of both enantiomers are expected
to be different [39]. Likewise, little is known about the relative sus-
ceptibilities of the enantiomers of 3 and 4 to enzyme-mediated oxi-
dation to the corresponding quinones. A kinetic in vitro study with
mushroom tyrosinase, using enantiomorphs of several diphenols
(e.g., L-, D, and D,L-dopa; L-, D, and D,L-a-methyldopa), demonstrated
that the enzyme has stereospecificity in its affinity towards the sub-
strates (as measured by Km) but not in the reaction rate (Vmax) of
those substrates [40]. It remains to be established whether stereo-
specific oxidation and adduct formation rates will occur in vivo.
3. Conclusion
NAcHis. The adducts 6-(N-acetyl)-histidine–N-acetyl-
a-methyl-
dopamine (10) and 6-(N-acetyl)-histidine–N-acetyl-N-methyl-
methyldopamine (11), formed by chemical oxidation with NaIO4
of catecholamines 3 and 4 respectively, were obtained in modest
a
-
We report herein for the first time the enzymatic and chemical
synthesis of important catecholamine adducts with nitrogen
nucleophiles using a less demanding protocol than those described

22
F. Siopa et al. / Bioorganic Chemistry 44 (2012) 19–24
in the literature. Although the adducts were obtained in modest
yields, full spectroscopic analysis was possible and proved that
the reaction is regioselective, with the modification taking place
predominantly at the 6-position of the catecholamines. Modifica-
tion of peptides and proteins occurs in all eukaryotic systems
and the complete elucidation of macromolecules modified by cat-
echolamines is crucial to understand the role of catecholamines in
several biological processes. Although mass spectrometric tech-
niques are becoming routine in analyzing these modifications, de-
tailed structural characterization remains difficult when dealing
with unknown complex samples; toward that end, the availability
of synthetic standards, such as those described in the present man-
uscript, is crucial. Should these adducts prove to be biologically rel-
evant, their potential clinical use as (pre)disease biomarkers could
represent an important breakthrough in the treatment and preven-
tion of neurodegenerative diseases.
1.84 (3H, s, NAcHis-COCH3), 2.12–2.23 and 2.39–2.42 (2H, m,
H7), 2.74–2.99 (2H, m, NAcHis-Hb), 3.01–3.14 (2H, m, H8), 4.28
(1H, dd, J = 8.6 and 5.2 Hz, NAcHis-Ha), 4.38 (1H, dd, J = 8.2 and
4.7 Hz, NAcHis-Ha0), 6.81 and 6.87 (1H, s, H6), 6.88 (1H, s, H5),
7.38 and 7.41 (1H, s, NAcHis-H5), 8.76 (1H, s, NAcHis-H2) ppm.
13C NMR (D2O) d: 22.6 (COCH3 + NAcHis-COCH3), 26.1 (NAcHis-
Cb), 30.3 (C7), 40.7 (C8), 52.8 (NAcHis-Ca), 116.5 (C6), 118.5 (C5),
119.2 (NAcHis-C5), 123.1 (C1), 127.9 (C2), 131.5 (NAcHis-C4),
135.8 (NAcHis-C2), 143.6 (C3/4), 146.7 (C3/4), 163.6 (NAcHis-
CO), 173.8 (NAcHis-COCH3 + COCH3) ppm. MS(ESI) m/z: 391
[MH]+.
4.2.3. 6-Imid–NADA (9)
Yellow oil (4.0 mg, 15%). UV/vis (kmax, nm): 229 and 283.5. 1H
NMR (D2O) d: 1.78 (3H, s, COCH3), 2.45 (2H, t, J = 6.6 Hz, H7),
3.11 (2H, t, J = 6.5 Hz, H8), 6.84 (1H, s, H5), 6.85 (1H, s, H2), 7.54
(2H, s, Imid-H5 + H4), 8.82 (1H, s, Imid-H2) ppm. 13C NMR (D2O)
d: 22.1 (COCH3), 29.6 (C7), 40.1 (C8), 114.7 (C5), 117.8 (C2),
120.1 (Imid-C4/5), 124.4 (Imid-C4/5), 126.4 (C6), 127.6 (C1),
135.9 (Imid-C2), 143.5 (C3/4), 146.6 (C3/4), 174.3 (COCH3) ppm.
MS(ESI) m/z: 284 [M+Na]+, 262 [MH]+.
4. Experimental
4.1. Materials
NAcLys, NAcHis, Imid, DA (hydrochloride salt), and mushroom
tyrosinase (5370 U/mg of solid; EC 1.14.18.1), were obtained from
Sigma–AldrichÒ (Steinheim, Germany) and used as received, unless
4.3. General procedure for the chemical oxidation of catecholamines,
followed by addition of the nitrogen nucleophile
specified.
a-MeDA (3), and N-Me-a-MeDA (4) were synthesized
and purified as described previously [25].
4.3.1. Using NaIO4 or Frémy’s salt as oxidant
To a solution of catecholamine (5.5 mg, 1 eq.) in 10 mL of phos-
phate buffer (pH 7.4, 50 mM) at r.t. was added 1 eq. of NaIO4 or
Frémy’s salt (weighed under inert atmosphere) and then a solution
of 5 eq. of the nucleophile in 2 mL phosphate buffer (pH 7.4,
50 mM). After stirring for 3 h the reaction was stopped by addition
of 1 mL of 88% formic acid. The reaction mixture was carefully
evaporated under reduced pressure and the compound purified
by HPLC (see ESI).
4.2. General procedure for the enzymatic oxidation of catecholamines,
followed by addition of the nitrogen nucleophile
Mushroom tyrosinase (0.2 mg, 1074 units) was added to a solu-
tion of catecholamine (20 mg) in 1 mL phosphate buffer (pH 7.4,
50 mM) at r.t. The color changed to yellow, indicating the forma-
tion of the corresponding o-quinone. The enzyme was removed
from the reaction mixture using a 5 mL HitrapTM desalting column
(GE HeathcareÒ), eluted with water (20 mL). The yellow solution of
catecholamine–quinone was then added dropwise to a solution of
NAcHis (10 eq.) or Imid (10 eq.) in phosphate buffer (15 mL, pH 7.4,
50 mM), and the reaction mixture was stirred for 1 h. At the termi-
nus of the reaction 1 mL of 88% formic acid was added, and the
reaction mixture was carefully evaporated under reduced pressure.
The resulting orange oil was dissolved in water and purified by re-
verse-phase (RP-18) modified silica gel column chromatography,
using water (100 mL) and 50% MeOH (100 mL) as eluent. The frac-
tion containing the product eluted with 50% MeOH and was puri-
fied by HPLC (see ESI).
4.3.2. Using Ag2O as oxidant
To a solution of 2 (102 lmol) in 6 mL of DMF were added 10 eq.
of Ag2O. The color changed to yellow, indicating the formation of
the corresponding o-quinone. After vortexing (5 min) to remove
the excess of oxidant, the nucleophile, NAcHis (5 eq.) in 30 mL of
50 mM phosphate buffer (pH 7.4) was slowly added to the o-qui-
none solution. After stirring for 3 h the reaction was stopped by
addition of 1 mL of 88% formic acid. The reaction mixture was care-
fully evaporated under reduced pressure, using toluene to help
removing the DMF, and the compound was purified by HPLC (see
ESI).
4.2.1. 6-NAcHis–NADA (7)
Yellow oil (6.4 mg, 16%). UV/vis (kmax, nm): 286. 1H NMR (D2O,
d): 1.78 (3H, s, COCH3), 1.91 (3H, s, NAcHis-COCH3), 2.36–2.44 (2H,
m, H7), 3.05–3.15 (3H, m, H8 + NAcHis-Hba), 3.30 (1H, dd, J = 15.4
4.3.3. 6-(N-acetyl)-histidine-(R,S)-N-acetyl-a-methyldopamine (10)
Yellow oil (0.5 mg, 5%, with NaIO4). UV/vis (kmax, nm): 284. 1H
NMR (D2O) d: 0.99 (3H, d, J = 7.1 Hz, H9), 1.81 (3H, s, COCH3),
1.99 (3H, s, NAcHis-COCH3), 2.84 (2H, s, H7), 2.99 (2H, s, NAcHis-
Hb), 3.33 (1H, m, H8), 6.87 (2H, s, H2 + H5), 7.43 (1H, s, NAcHis-
H5), 7.91 (1H, s, NAcHis-H50), 8.80 (1H, s, NAcHis-H2), ppm. MS(E-
SI) m/z: 405 [MH]+.
and 5.0 Hz, NAcHis-Hbb), ca. 4.70 (NAcHis-Ha), 6.80 (2H, s,
H2 + H5), 7.38 (1H, s, NAcHis-H5), 8.74 (1H, s, NAcHis-H2) ppm.
13C NMR (D2O) d: 22.1 (NADA-COCH3 + NAcHis-COCH3), 26.8 (NA-
cHis-Cb), 29.7 (C7), 39.9 (C8), 52.2 (NAcHis-Ca), 114.7 (C5), 117.9
(C2), 123.5 (NAcHis-C5), 126.4 (C6), 127.5 (C1), 130.1 (NAcHis-
C4), 135.9 (NAcHis-C2), 143.6 (C3/4), 146.6 (C3/4), 163.6 (NA-
cHis-CO), 173.7 (NAcHis-COCH3), 174.3 (NADA-COCH3) ppm.
MS(ESI) m/z: 391 [MH]+.
4.3.4. 6-(N-acetyl)-histidine-(R,S)-N-acetyl-N-methyl-a-
methyldopamine (11)
Yellow oil (0.8 mg, 8%, with NaIO4). UV/vis (kmax, nm): 223 and
284. 1H NMR (D2O) d: 0.99–1.02 (3H, m, Hz, H9), 1.15 (3H, m, H90),
1.89 (3H, s, COCH3), 1.97 (3H, s, NAcHis-COCH3), 2.18 (2H, s, H7),
2.52 (2H, m, NAcHis-Hb), 2.75 (3H, s, N-CH3), 3.30 (1H, m, H8),
6.82 (2H, s, H2 + H5), 7.46 (1H, s, NAcHis-H5), 8.74 (1H, s, NA-
cHis-H2) ppm. MS(ESI) m/z: 419 [MH]+.
4.2.2. 2-NAcHis–NADA (8)
Yellow oil (0.5 mg, 1.3%). Two conformers were detected by 1H
NMR at room temperature, possibly due to restricted rotation in-
duced by an intramolecular hydrogen bond). UV/vis (kmax, nm):
283.5. 1H NMR (D2O) d: 1.77 and 1.78 (3H, s, COCH3), 1.83 and

F. Siopa et al. / Bioorganic Chemistry 44 (2012) 19–24
23
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4.4.1. 6-His–DA (12)
Yellow oil (2.3 mg, 27%). UV/vis (kmax, nm): 214 and 284. 1H
NMR (D2O) d: 2.61 (2H, t, J = 7.7 Hz, H7), 2.98 (2H, t, J = 7.7 Hz,
H8), 3.26 (1H, dd, J = 15.4 and 7.4 Hz, His-Hba), 3.35 (1H, dd,
J = 15.4 and 5.8 Hz, His-Hbb), 4.04 (1H, t, J = 7.7 Hz, His-Ha), 6.89
(1H, s, H2/5), 6.90 (1H, s, H2/5), 7.48 (1H, s, His-H5), 8.82 (1H, s,
His-H2) ppm. 13C NMR (D2O) d: 26.1 (His-Cb), 27.6 (C7), 39.9
(C8), 53.7 (His-Ca), 115.1 (C5), 117.4 (C2), 122.9 (His-C5), 125.0
(C6), 126.3 (C1), 128.9 (His-C4), 136.4 (His-C2), 144.2 (C3/4),
147.1 (C3/4), 163.2 (His-CO) ppm. MS(ESI) m/z: 307 [MH]+. HRMS-
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4.4.2. 6-His–NADA (13)
Yellow oil (2 mg, 21%). UV/vis (kmax, nm): 211.4 and 284.1. 1H
NMR (D2O) d: 1.78 (3H, s, COCH3), 2.46 (2H, t, J = 6.7 Hz, H7),
3.09 (2H, t, J = 6.6 Hz, H8), 3.34 (2H, d, J = 6.5 Hz, His-Hb), 4.10
(1H, t, J = 6.6 Hz, His-H
7.48 (1H, s, His-H5), 8.81 (1H, s, His-H2) ppm. 13C NMR (D2O) d:
22.1 (COCH3), 26.0 (His-Cb), 29.7 (C7), 40.1 (C8), 55.4 (His-C ),
114.8 (C5), 117.6 (C2), 123.1 (His-C5), 126.4 (C6), 127.5 (C1),
128.5 (His-C4), 136.5 (His-C2), 143.6 (C3/4), 146.7 (C3/4), 163.2
(His-CO), 174.3 (COCH3) ppm. MS(ESI) m/z: 349 [MH]+. HRMSESI
m/z calcd. for C16H20N4O5 [MH]+: 349.1512; obtained 349.1506.
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a
4.4.3. 6-Imid-DA (14)
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This work has been supported by Fundação para a Ciência e a
Tecnologia (FCT) through Grants No. PEst-C/EQB/LA0006/2011
and PEst-OE/QUI/UI0100/2011. Filipa Siopa also thanks FCT for
the award of a doctoral fellowship (SFRH/BD/39171/2007). Thanks
are also due to the Portuguese NMR and MS networks (FCT-UNL
and IST nodes) for providing access to the facilities.
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2012.05.
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