The first expedient entry to the human melanogen 2-S-cysteinyldopa exploiting the anomalous regioselectivity of 3,4-dihydroxycinnamic acid-thiol conjugation

Article abstract of DOI:10.1016/j.tetlet.2007.08.098

The first convenient synthesis of 2-S-cysteinyl-3,4-dihydroxyphenylalanine (2-S-cysteinyldopa) in 30% overall yield is reported, which capitalizes on the anomalous regiochemistry of the oxidative coupling of 3,4-dihydroxycinnamic acid derivatives with thi

Full text of DOI:10.1016/j.tetlet.2007.08.098

Tetrahedron Letters 48 (2007) 7650–7652
The first expedient entry to the human melanogen
2-S-cysteinyldopa exploiting the anomalous regioselectivity
of 3,4-dihydroxycinnamic acid–thiol conjugation
Lucia Panzella, Maria De Lucia, Alessandra Napolitano* and Marco d’Ischia
Department of Organic Chemistry and Biochemistry, University of Naples Federico II,
Complesso Universitario Monte S. Angelo, I-80126 Naples, Italy
Received 22 June 2007; accepted 29 August 2007
Available online 31 August 2007
Abstract—The first convenient synthesis of 2-S-cysteinyl-3,4-dihydroxyphenylalanine (2-S-cysteinyldopa) in 30% overall yield is
reported, which capitalizes on the anomalous regiochemistry of the oxidative coupling of 3,4-dihydroxycinnamic acid derivatives
with thiol compounds, leading to 2-S rather than the usual 5-S conjugates.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The cysteinyldihydroxyphenylalanines (or cysteinyldo-
pas), for example, 1, 2, are central melanocyte metabo-
lites involved in the biosynthesis of pheomelanins, the
characteristic pigments of red-haired, fair-complexioned
individuals at high risk for melanoma and other skin
cancers.1 Besides being recruited for pheomelanin syn-
thesis, these metabolites are partly excreted into body
fluids and are among the best diagnostic markers of dis-
seminated malignant melanoma.2 Moreover, their metal
chelating3 and antioxidant properties4 point to impor-
tant, though still unclear, functional roles. Availability
of cysteinyldopa isomers on a gram-scale is therefore
central for addressing a number of chemical, biological
and clinical issues relating, for example, to the structure
and photoprotective/photosensitizing properties of
pheomelanins, to the functional significance of circulat-
ing melanogens, and to their role in the etiopathogenesis
of malignant melanoma.
Current synthetic methodologies involve oxidative con-
jugation of cysteine with 3,4-dihydroxyphenylalanine
(dopa) or a derivative mediated by a range of oxidizing
systems, including cerium ammonium nitrate (CAN) in
sulfuric acid,5 silver oxide in methanol,6 Fe2+-EDTA/
H2O2 or tyrosinase8 in neutral aqueous buffers. As a
7
rule, 5-S-isomer 19 is obtained as the major product
(14–70% isolated yields), reflecting the dominant regio-
chemical course of the oxidative conjugation of
4-alkylcatechols with thiols,10,11 whereas 2-S isomer 2
is formed in much lower amounts (<5% in our hands,
despite slightly higher claims in the literature). To date,
separation from the reaction mixtures provides the only
means of obtaining small amounts of 2 for biological
studies and/or analytical purposes. This, however,
requires a lengthy and cumbersome procedure involving
repeated ion-exchange column chromatography with
HCl gradients.5–8 In fact, the regiochemical issue in
catechol–thiol conjugation has so far prevented a
gram-scale preparation of 2, despite the importance of
this isomer in pheomelanin build-up.12
COOH
NH2
HO
HOOC
NH2
HO
S
S
COOH
NH2
HO
HO
Our entry to this goal relied on the observation that caf-
feic acid and related 3,4-dihydroxyphenylpropenoic acid
derivatives react with thiol compounds under oxidative
conditions to give mainly 2-S-conjugates,13–15 instead
of the usual 5-S-conjugates that are formed with simple
4-alkylcatechols, such as dopa, dopamine, and 4-methyl-
catechol.1,10,11 This anomalous regiochemistry has been
rationalized in terms of the effects of the conjugated pro-
penoate chain enhancing the coefficient of the LUMO of
H2N
COOH
1
2
Keywords: Cysteinyldopas; Thiol conjugation; Pheomelanin; 3,4-Di-
hydroxycinnamic acid derivatives.
Corresponding author. Tel.: +39 081674133; fax: +39 081674393;
e-mail: alesnapo@unina.it
*
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.08.098

L. Panzella et al. / Tetrahedron Letters 48 (2007) 7650–7652
7651
O
O
ii
HO
HO
i
AcO
AcO
COOH
NHAc
HO
HO
H
O
N
+
3
AcHN
COOH
Scheme 1. Synthesis of 3 from 3,4-dihydroxybenzaldehyde and N-acetylglycine. Reagents and conditions: (i) Acetic anhydride, 120 ꢁC, 5 h; (ii) 0.2 M
HCl, under reflux, 1 h.
HOOC
NHAc
HOOC
NHAc
HOOC
NHAc
i
ii
iii
S
S
2
+
3
COOH
NHAc
COOH
NHAc
HO
HO
HO
HO
HS
4
Scheme 2. Oxidative conjugation of 3 with N-acetylcysteine and stereoselective reduction of the adduct. Reagents and conditions: (i) Tyrosinase
(50 U/mL), rt, 2 h; (ii) H2, 3 mol % chiral catalyst, rt, 50 atm, 72 h; (iii) 3 M HCl reflux, 5 h.
the corresponding o-quinones at the 2-position of the
quinone ring.15 Capitalizing on this observation, a syn-
thetic procedure was devised, which revolves around
an amino-substituted 3,4-dihydroxyphenylpropenoic
acid, which would expectedly form a 2-S-adduct by
oxidative conjugation with L-cysteine. Stereoselective
reduction of the propenoate moiety would then afford
the desired product 2 with both amino acids in the nat-
ural L-configurations.
starting material with respect to residual cysteine con-
tamination proved critical because of catalyst poisoning,
and hence a chromatographic purification prior to
hydrogenation proved necessary. Under 50 atm hydro-
gen pressure, with a 3 mol % catalyst loading and meth-
anol as the solvent, the reaction was complete within
72 h (Scheme 2). Acid hydrolysis of the hydrogenation
mixture under reflux eventually afforded 2 in 90% yield
with a diastereoisomeric excess of 90% by HPLC analy-
sis.21 Product identity was secured by comparison with
an authentic sample,6,8 while purity was checked by
NMR analysis.21
Accordingly, 2-acetylamino-3-(3,4-dihydroxyphenyl)-
propenoic acid (3) was prepared by a Knoevenagel-type
condensation of 3,4-dihydroxybenzaldehyde with
N-acetylglycine in acetic anhydride at 120 ꢁC followed
by hydrolysis of the ethyl acetate extracts containing
the oxazolone intermediate (Scheme 1).16 After the
removal of some unreacted starting material by ethyl
ether extraction, product 3 (>95% purity) was isolated
in 60% yield.17
In conclusion, we have developed the first direct synthe-
sis of the human melanogen 2 by a convenient three-step
synthesis in overall 30% yield. This is by far the highest
yield for this biologically and clinically relevant amino
acid, which has so far been obtained only as a minor side
product in the synthesis of 1. The procedure is simple,
efficient, scaleable and is expected to open up the door-
way to novel insights into pheomelanin structure and
functions. Moreover, the approach proposed in this
study may be of value as a general access route to syn-
thetic targets featuring ‘anomalous’ 2-S thiol–catechol
conjugate substructures.
Several oxidants were then screened to install the cys-
teinyl moiety onto the desired position of 3. To this
aim, N-acetylcysteine was preferably used in the place
of cysteine to prevent intramolecular cyclization of the
amino group on the quinone produced by oxidation of
3. Under a variety of conditions, however, di- and tri-
cysteinyl adducts were invariably obtained as main
products because of the increased ease to oxidation of
the initial S-conjugate with respect to the starting mate-
rial. This difficulty was overcome by using commercially
available mushroom tyrosinase as the oxidant. Thus, in
line with our expectations, enzyme-promoted oxidation
of 3 in the presence of 2 M equiv of N-acetylcysteine
in aqueous buffer at pH 7.418 resulted in a complete
consumption of the starting material after 2 h with the
formation of the desired (Z)-2-S-cysteinyl-3-(3,4-dihydro-
xyphenyl)-2-acetylaminopropenoic acid (4),19 isolated in
55% yields by preparative HPLC (Scheme 2).
Acknowledgments
This work was carried out with financial support from
Italian MiUR. We thank the ‘Centro Interdipartimen-
tale di Metodologie Chimico-Fisiche’ (CIMCF, Univer-
sity of Naples) for NMR facilities, and Miss Silvana
Corsani for technical assistance.
References and notes
Asymmetric hydrogenation of the Z-double bond in 4
was achieved in the presence of a rhodium chiral biphos-
phine catalyst20 in a Parr apparatus.21 Purity of the
1. Prota, G. Fort. Chem. Org. Nat. 1995, 64, 94–148.
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L. Panzella et al. / Tetrahedron Letters 48 (2007) 7650–7652
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(10 lm particle size 250 · 22 mm Econosil C18, eluant as
above, 30 mL/min) to afford 4 (460 mg, 55% yield). Purity
>95% as determined by H NMR analysis.
1
3. Napolitano, A.; Memoli, S.; Nappi, A. J.; d’Ischia, M.;
Prota, G. Biochim. Biophys. Acta 1996, 1291, 75–82.
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9. The common system for catecholamine nomenclature was
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carbons of the aromatic ring.
10. Zhang, F.; Dryhurst, G. Bioorg. Chem. 1995, 23, 193–216.
11. Ito, S.; Palumbo, A.; Prota, G. Experientia 1985, 41, 960–
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12. Ito, S.; Wakamatsu, K. In The Pigmentary System:
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310.
19. (Z)-2-Acetylamino-3-[2-(2-acetylamino-2-carboxyethylthio)-
3,4-dihydroxyphenyl]propenoic acid (4). HR ESI+/MS:
found m/z 399.1049 ([M+H]+), calcd for C16H19N2O10
m/z 399.1040; UV kmax (CH3OH) 257, 314; [a]D +44 (c
0.25, CH3OH); 1H NMR (DMSO-d6) d (ppm) 1.82 (3H, s,
COCH3cys), 1.87 (3H, s, COCH3), 2.85 (1H, dd, J = 13.2,
10.0 Hz, –SCH2), 3.32 (1H, dd, J = 13.2, 4.4 Hz, –SCH2),
3.98 (1H, m, –CH2CH), 6.80 (1H, d, J = 8.4 Hz, H-60),
6.96 (1H, d, J = 8.4 Hz, H-50), 7.63 (1H, s, H-3) 8.16 (1H,
d, J = 8.0 Hz, NHCOCH3cys), 9.11 (1H, s, NHCOCH3);
13C NMR (DMSO-d6) d (ppm) 22.5 (COCH3cys), 22.6
(COCH3), 35.2 (–SCH2), 52.2 (CHCH2), 115.6 (C-50),
120.0 (C-20), 120.4 (C-60), 126.0 (C-2), 128.7 (C-10), 131.5
(C-3), 146.3 (C-40), 147.2 (C-30), 166.8 (C-1), 169.5
(–NHCO), 169.9 (–NHCOcys), 172.6 (COOHcys). Proton
and carbon resonance assignment follows from 2D NMR
experiments.
20. Vineyard, B. D.; Knowles, W. S.; Sabacky, M. J.;
Bachman, G. L.; Weinkauff, D. J. J. Am. Chem. Soc.
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21. To a 100 mL hydrogenation bomb were added a solution
of 4 (800 mg) in methanol (30 mL) and cyclooctadiene-
1,5[(R,R)1,2-ethanediylbis(o-methoxyphenyl)phenylphos-
phine]rhodium tetrafluoroborate (44 mg, Acros Organics).
The solution was purged by filling and evacuating with N2
and finally with H2, and then allowed to stand under
stirring at 50 atm for 72 h. After the removal of the solvent
the residue was taken up in 3 M HCl (50 mL) and taken
under reflux for 5 h under an argon atmosphere. Removal
of the catalyst by filtration and evaporation of the acid
gave a pale yellow oil, which was dissolved in ethanol and
added to ethyl acetate to afford 2 as a colorless powder.6,8
Diastereoisomeric excess was determined by HPLC anal-
ysis using a Synergi Hydro-RP 80A column (250 ·
4.6 mm, 4 lm) using 0.1% trifluoroacetic acid/methanol
99:1 (0.7 mL/min) as the eluant, detection wavelength
280 nm. Retention times of 2 and its diastereoisomer
under these conditions were 9.9 and 10.8 min, respectively.
1H NMR (D2O): d (ppm) 3.18 (1H, dd, J = 14.4, 9.2 Hz,
–CHCH2), 3.31 (1H, dd, J = 14.8, 4.4 Hz, CH2cys), 3.45
(1H, dd, J = 14.8, 7.2 Hz, CH2cys), 3.66 (1H, dd, J = 14.4,
6.0 Hz, –CHCH2), 4.12 (1H, dd, J = 7.2, 4.4 Hz, CHcys),
4.23 (1H, dd, J = 9.2, 6.0 Hz, CH), 6.84 (1H, d,
J = 8.0 Hz, H-6), 6.96 (1H, d, J = 8.0 Hz, H-5); 13C
NMR (D2O): d 36.1 (–CHCH2), 36.4 (CH2cys), 54.1
(CHcys), 55.9 (–CHCH2), 118.8 (C-5), 119.7 (C-2), 124.3
(C-6), 132.0 (C-1), 145.6 (C-4), 148.6 (C-3), 172.0
(COOHcys), 173.3 (COOH). Proton and carbon resonance
assignment follows from 2D NMR experiments.
13. Richard, F. C.; Goupy, P. M.; Nicolas, J. J.; Lacombe, J.;
Pavia, A. A. J. Agric. Food Chem. 1991, 39, 841–847.
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17. The reported procedure16 was followed with slight mod-
ifications. 3,4-Dihydroxybenzaldehyde (1.0 g, 7.3 mmol),
N-acetylglycine (1 g, 8.5 mmol), and sodium acetate (2.3 g,
28 mmol) were heated at 120 ꢁC in acetic anhydride
(7.8 mL) under stirring. After 5 h the reaction mixture
was diluted with 0.1 M phosphate buffer (pH 7.4) and
extracted with ethyl acetate. The residue obtained from the
organic layers was taken up in 0.2 M HCl (74 mL) and
refluxed for 1 h. After ethyl ether washing the mixture was
taken to dryness to afford 316 (1.0 g, 60% yield) as
yellowish powder (>95% pure by HPLC and 1H NMR
analyses). ESI+/MS: m/z 238 [M+H]+, 260 [M+Na]+.
18. A solution of 3 (500 mg, 2.1 mmol) in 0.01 M phosphate
buffer (pH 7.4) (1.15 L) was sequentially treated with N-
acetyl-L-cysteine (688 mg, 4.2 mmol) and mushroom
tyrosinase (50 U/mL) and the mixture was taken under
vigorous stirring at rt. After 2 h a complete consumption
of the starting product was obtained (HPLC evidence,
0.1 M formic acid/methanol 88:12) and the mixture was
acidified to pH 3 and fractionated by preparative HPLC