Controlled synthesis of labelled 3-L-chlorotyrosine-[ring- 13C6] and of 3,5-L-dichlorotyrosine-[ring- 13C6]

Article abstract of DOI:10.1002/jlcr.882

Pure 3-L-chlorotyrosine-[ring-¹³C₆] is prepared by chlorination of the 5-oxazolidinone of L-tyrosine-[ring-¹³C ₆] with SO₂Cl₂ in CH₃COOH-Et ₂O and successive one-pot regeneration of the protected aminoacidic functions by BCl₃ in dichloromethane. Copyright

Full text of DOI:10.1002/jlcr.882

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 935–945.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.882
Research Article
Controlled synthesis of labelled 3-l-chlorotyrosine-
[ring-13C6] and of 3,5-l-dichlorotyrosine-[ring-13C6]
Pietro Allevi1,*, Paola Olivero2 and Mario Anastasia2
1
"
Dipartimento di Medicina, Chirurgia e Odontoiatria, Universita di Milano, via A. Di
Rudini 8, I-20142 Milano, Italy
2
"
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di
Milano, via Saldini 50, I-20133 Milano, Italy
Summary
Pure 3-l-chlorotyrosine-[ring-13C6] is prepared by chlorination of the 5-oxazolidinone
of l-tyrosine-[ring-13C6] with SO2Cl2 in CH3COOH-Et2O and successive
one-pot regeneration of the protected aminoacidic functions by BCl3 in dichloro-
methane. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: 3-l-chlorotyrosine-[ring-13C6]; 3,5-l-dichlorotyrosine-[ring-13C6]; Sul-
phuryl chloride; l-tyrosine-[ring-13C6]; Myeloperoxydase
Introduction
Hypochlorous acid, generated by the heme enzyme myeloperoxydase,1 is a
highly reactive species that participates in both oxidation2,3 and chlorination
reactions4 of various biological targets and converts some residues of
l-tyrosine 1a, present in peptides, to residues of 3-chloro-l-tyrosine 2a4,5
and, to a minor extent, of 3,5-dichloro-l-tyrosine 3a (Fig. 1).6
Because of this action of the hypochlorous acid, myeloperoxydase has
been implicated in the tissue damage that occurs in numerous diseases that
involve inflammatory cells.7 The chlorinated amino acids 3-chloro-l-tyrosine
and 3,5-dichloro-l-tyrosine are found intact in various human fluids and
organs and therefore are believed to be important markers for probing the
involvement of oxidation by myeloperoxydase in the pathology of particular
diseases.8
"
*Correspondence to: P. Allevi, Dipartimento di Medicina, Chirurgia e Odontoiatria, Universita di Milano,
"
via A. Di Rudinı 8, I-20142 Milano, Italy. E-mail: pietro.allevi@unimi.it
"
Contract/grant sponsor: Italian MIUR (Ministero dell’Istruzione, dell’Universita e della Ricerca); contract/
grant number: 2002-2003
Copyright # 2004 John Wiley & Sons, Ltd.
Received 14 June 2004
Revised 21 July 2004
Accepted 23 July 2004

936
P. ALLEVI ET AL.
-
-
-
COO
COO
COO
+
+
+
NH3
NH3
NH3
Cl
Cl
Cl
OH
OH
OH
2
1
3
a: ring-12C6
b: ring-13C6
Figure 1. Tyrosine and chlorinated analogues
An accurate quantification of 3-chloro-l-tyrosine and of 3,5-dichloro-l-
tyrosine in peptides of various biological substrates requires the use of gas
chromatography-mass spectrometry (GC-MS) and dilution of the biological
material with isotopically pure 3-chloro-l-tyrosine-[ring-13C6] 2b and of 3,5-
dichloro-l-tyrosine-[ring-13C6] 3b as internal standards. Isotopomers 2b and
3b are now commonly obtained by direct halogenation of the free amino acid
l-tyrosine-[ring-13C6] 1b and successive purification by preparative HPLC.6,9–11
Moreover, despite the great interest in these amino acids no convenient
synthetic method is available other than on a very small scale. The reported
methods involve reactions in which very small amounts (1–2 mg) of 3-chloro-
l-tyrosine-[ring-13C6] 2b are prepared and purified by HPLC from the
3,5-dichloro-l-tyrosine-[ring-13C6] 3b9–11 which always accompanies the
monochloroderivative. On the other hand, when less than an equivalent of
the chlorinating agent is used, some tyrosine accompanies the mono and
dichlorinated amino acids and these must be separated by preparative HPLC.6
This therefore reduces the availability of 3-l-chlorotyrosine-[ring-13C6] for
future biological studies. Fortunately, the 3,5-dichloro-l-tyrosine-[ring-13C6]
can be obtained in good yield by exhaustive chlorination, at temperature
higher than 408C, of the free amino acid l-tyrosine-[ring-13C6].12
In this paper, we report a convenient method which overcomes the above
difficulties and allows us to obtain both 3-chloro- and 3,5-dichloro-l-tyrosine-
[ring-13C6] 2b and 3b in a pure state and in sufficient quantities that enable the
compounds to be used for both analytical, biological and chemical studies
amount suitable not only for analytical purposes but also for other biological
or chemical studies.
Results and discussion
Our initial procedure for the preparation of 3-chloro-l-tyrosine-[ring-13C6] 2b
(Scheme 1) exploits the observation reported by Yu et al.12 and by Masilamani
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 935–945

CONTROLLED SYNTHESIS
937
CO2CH3
CO2CH3
CO2CH3
NH2
NH2
a
NH2
b
Cl
Cl
Cl
OH
OH
OH
4
5
2b
Scheme 1. a: SO2Cl2, AcOH-Et2O, 08C, 1 h; b: K2CO3, MeOH, 258C, 12 h
and Rogic,13 that sulphuryl chloride (SO2Cl2), at low temperature, provides a
route to the efficient aromatic monochlorination of phenolic amino esters or
phenols.
In fact, treatment of the methyl ester14 4 of the commercially available l-
tyrosine-[ring-13C6] with one equivalent of SO2Cl2, in acetic acid and diethyl
ether, at 08C, afforded the 3-chloro-l-tyrosine ester 5 in nearly pure form.
However, in agreement with the literature,12 variable amounts of the
dichlorinated compound were also formed. As reported by Yu et al.,12 the
amount of dichlorinated product decreases if the reaction is performed below
258C and with a slow addition of the chlorinating agent. In order to ensure this
condition, we dissolved SO2Cl2 in diethyl ether and added the solution
previously cooled at ꢀ108C to the amino acid cooled to 0–58C. Under these
conditions, the monochloroderivative 5 was obtained in constantly high yields
(75%) but accompanied by some dichlorinated compound (4–5% by HPLC).
The monochloroderivative 5 was then treated with potassium carbonate in
aqueous methanol to afford the 3-chloro-l-tyrosine-[ring-13C6] 2b, which was
isolated by HPLC.y
To obtain pure 3-chloro-l-tyrosine-[ring-13C6] 2b, and simplify the
purification process, we decided to set up a second procedure based on the
chlorination of a completely protected tyrosine. For this, our recent work on
the chemistry of benzyloxycarbonyl-5-oxazolidininones was of immediate
utility.15–18 In our work,16 under controlled conditions,17 the tyrosine methyl
ester 4 (Scheme 2) could be transformed into the 5-oxazolidinone-[ring-13C6] 7,
having both the aminoacidic functions suitably protected. This was obtained
by transforming 4 into the benzyloxycarbonyl ester 6 and reacting it with
paraformaldehyde in toluene, in the presence of p-toluenesulphonic acid.16,17
The obtained 5-oxazolidinone 7 was then chlorinated, using sulphuryl chloride
y For the determination of the optical purity, each amino acid (3 mg) was converted into the corresponding
ester hydrochloride salt by treatment with methanolic HCl (0.2 ml; 258C, 12 h). The mixture was then
evaporated and the residue was then treated with a solution of trifluoracetic anhydride and trifluoracetic
acid (0.2 ml, 1:1, v:v), and analysed by GLC on a chiral column (see ‘General’ section).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 935–945

938
P. ALLEVI ET AL.
O
O
N
O
O
N
-
COOH
NHZ
COO
CO2CH3
NHZ
b
+
NH3
Z
Z
e
d
a
c
4
Cl
Cl
OH
OH
9
OH
1b
OH
6
OH
7
f
f
8
-
COO
z = benzyloxycarbonyl
+
NH3
Cl
OH
2b
Scheme 2. a: PhCH2O COCl, aqueous NaHCO3, 258C, 5 h; b: paraformalde-
hyde, p-TsOH, toluene, 808C, 30 min; c: SO2Cl2, AcOH-Et2O, 08C, 1 h; d:
NaHCO3, MeOH–H2O, reflux, 10 min; e: H2, Pd/C, EtOH; f: BCl3,
dichloromethane, 258C, 1 h
and the controlled conditions used above for the methyl ester 4.12 From the
reaction the monochloroderivative-[ring-13C6] 8 was obtained accompanied by
trace amounts of the corresponding less polar 3,5-dichloroderivative, from
which it can be separated by rapid chromatography on silica. Starting from the
oxazolidinone 8, we first attempted to obtain 2b, by regeneration of the
aminoacidic functions in two steps, using a mild hydrolytic procedure to open
the 5-oxazolidinone ring by treatment with aqueous NaHCO137,18 and
subjecting the obtained carbobenzyloxy acid 9 to hydrogenolysis cleavage of
the carbobenzyloxy group. Unfortunately, while the opening of the 5-
oxazolidinone 8 to the acid 9 occurs with satisfactory yields, the hydro-
genolysis of 9 causes, under all conditions used (different solvents
and catalyst), the loss of the chlorine19 substituent with the formation of
the starting l-tyrosine-[ring-13C6] 1b. A better and more satisfactory result
was, however, obtained, by performing the regeneration of the amino group
by means of BCl3 in dichloromethane, according to our recent protocol.18
Thus we performed in one-pot the simultaneous cleavage of the 5-
oxazolidinone ring and of the carbobenzyloxy group and obtained the desired
3-chloro-l-tyrosine-[ring-13C6] 2b uncontaminated by the 3,5-dichloro homo-
logous.
The second desired compound, the 3,5-dichloro-l-tyrosine-[ring-13C6] 3b
was obtained by simple exhaustive chlorination of the tyrosine 1b with SO2Cl2
(3 molar equivalents) at 258C (Scheme 3).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 935–945

CONTROLLED SYNTHESIS
939
-
-
COO
COO
+
+
NH3
NH3
a
Cl
Cl
OH
3
OH
1
Scheme 3. a: SO2Cl2, sulpholane-Et2O, 258C, 24 h
In conclusion we have devised a simple route to prepare the 3-chloro-l-
tyrosine-[ring-13C6] 2b and the 3,5-dichloro-l-tyrosine-[ring-13C6] 3b in pure
form and in sufficient quantity to be used as internal standards in the
quantification of the isotopomers formed by in vivo action of the heme enzyme
myeloperoxydase.
Experimental
General
Nuclear magnetic resonance spectra were recorded at 298 K on a Bruker AM-
1
500 spectrometer, operating at 500.13 MHz for H and 125.76 MHz for 13C.
Chemical shifts are reported in parts per million (ppm, d units) relative to
1
CHCl3, fixed at 7.24 ppm, or to HDO, fixed at 4.54 ppm, for the H spectra
and relative to dioxane, fixed at 67.60 ppm, for the 13C spectra.20 1H-NMR
and 13C-NMR data are tabulated in the following order: number of protons,
multiplicity (s, singlet; d, doublet; t, triplet; bs, broad singlet; m, multiplet),
coupling constant(s) in Hz, assignment of proton(s).
Optical rotations were taken at 258C on a Perkin-Elmer 241 polarimeter
and [a]D values are given in 10ꢀ1 deg cm2/g1. Chiral GCL analyses were
carried out on a Hewlett-Packard 5890 gas chromatograph equipped with a
octakis(3-O-butyryl-2,6-di-O-pentyl)-g-cyclodextrin (Lipodex E)21 capillary
column (25 m, 0.25 mm ID, purchased from Macherey-Nagel). The
HPLC system was equipped with a RP-18 column (LiChroCART,
125 mm, 4 mm ID, 5 mm purchased from Merck) eluted with H2O/MeOH/
AcOH 95:5:1 v/v at 1 ml/min; the detection was performed at 276 nm. Mass
spectra were obtained using a Finnigan LCQdeca (ThermoQuest) ion trap
mass spectrometer fitted with an electrospray source (ESI). All reactions
were monitored by HPLC and/or by thin-layer chromatography (TLC)
carried out on 0.25 mm E. Merck silica gel plates (60 F254) using UV light,
50% sulphuric acid or 0.2% ninhydrin in ethanol and heated as developing
agent. E. Merck 230-400 mesh silica gel was used for flash column chromato-
graphy.22
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 935–945

940
P. ALLEVI ET AL.
Preparation of the (2S)-2-amino-3-(4-hydroxyphenyl-[ring-13C6])-propionic
acid methyl ester (4) hydrochloride
Thionyl chloride (80 mg; 0.66 mmol) was added to methanol (2 ml) cooled at
ꢀ158C and the solution was kept for 1 h at this temperature. Then, crystalline
l-tyrosine-[ring-13C6] (100 mg; 5.4 mmol) was added and the suspension was
stirred at room temperature for 24 h, when a clear solution formed. The
solvent was evaporated, the residue was tritured with methanol and the solvent
removed again under reduced pressure. The crude product was dried under
high vacuum and crystallized from methanol-diisopropyl ether to afford, after
filtration, the title ester 4 as hydrochloride in quantitative yields (122 mg,
96.6%): mp 189–1928C; [a]D22=+71 (c 1, py), [(lit.14 mp 190–1918C);
[a]D19=+70.9 (c 1, py)]; ESI/MS (positive) m/z: 202.3 (M+H+); H-NMR
1
(D2O): d 7.16 (2H, d, J ¼ 158:0, 20-H2), 6.91 (2H, d, J ¼ 160:7, 30-H2), 4.38
(1H, m, 2-H), 3.84 (3H, s, OCH3), 3.26 (1H, m, 3-Ha), 3.17 (1H, m, 3-Hb);
13C-NMR (D2O): d 170.8 (s, CO2CH3), 155.8 (dt, J ¼ 64:8 and 7.6, 40-C),
131.4 (ddd, J ¼ 57:2, 57.2 and 6.2, 20-C and 60-C), 126.0 (dt, J ¼ 57:2 and 8.6,
10-C), 116.5 (ddd, J ¼ 64:8, 57.2 and 6.7, 30-C and 50-C), 54.8 (s, OCH3),
54.1 (s, 2-C), 35.3 (d, J ¼ 44:8, 3-C). Analytically calculated for
12C4 13C6H14ClNO3: C 53.05, H 5.94, Cl 14.92, N 5.89. Found: C 52.99,
H 6.09, Cl 15.18, N 5.75.
Preparation of the 3-chloro-l-tyrosine-[ring-13C6] (2b), via the 3-chloro-l-
tyrosine-[ring-13C6] methyl ester (5) hydrochloride
l-tyrosine-[ring-13C6] methyl ester 4 hydrochloride (80 mg; 0.34 mmol) was
suspended in a mixture of AcOH-Et2O (1–0.5 ml) and cooled at 0–58C. A
solution of SO2Cl2 (27.3 ml; 46 mg; 0.34 mmol) in Et2O (0.5 ml), cooled at
ꢀ108C, was then slowly added. The reaction mixture was stirred for 1 h at 08C,
then the solvent was evaporated under a steam of nitrogen and the residue was
crystallized from methanol-diisopropyl ether to afford the 3-chloro-l-tyrosine-
[ring-13C6] methyl ester 5 (as hydrochloride), a white solid (69 mg, 75%)
containing some 3,5-dichloro-l-tyrosine-[ring-13C6] (4–5%, HPLC). The
product gave: mp 168–1698C; ESI/MS (positive) m/z (relative intensity):
1
238.1 (31, [37Cl]M+H+), 236.1 (100, [35Cl]M+H+); H-NMR (D2O): d 7.33
(1H, d, J ¼ 164:2, 20-H), 7.12 (1H, dd, J ¼ 158:4 and 7.9, 60-H), 7.04 (1H, dd,
J ¼ 160:5 and 7.9, 50-H), 4.42 (1H, m, 2-H), 3.88 (3H, s, OCH3), 3.29 (1H, dd,
J ¼ 14:9 and 5.0, 3-Ha), 3.19 (1H, dd, J ¼ 14:9 and 7.7, 3-Hb). The obtained
product was then dissolved in a saturated methanolic solution of potassium
carbonate (2.5 ml) and stirred at room temperature for 12 h. Then the solvent
was evaporated and the residue recovered with water and applied to a Dowex
50 ꢁ 8-200 cation-exchange resin column, activated with 2 M HCl and washed
with water. After washing with water, the amino acid was recovered by eluting
Copyright # 2004 John Wiley & Sons, Ltd.
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CONTROLLED SYNTHESIS
941
with 0.5 M ammonia solution.23 The eluates containing the amino acids were
collected to afford 2b (45.2 mg, 80%) that showed a major peak in HPLC
(tr ¼ 8:8 min; 95%) accompanied by a minor peak (tr=22.3 min; 5%)
corresponding to the dichloroderivative 3b. Purification of a sample by
preparative HPLC gave pure 2b : [a]D22=ꢀ23.57 (c 1, H2O); ESI/MS
(negative) m/z (relative intensity): 441.0 (60, [35Cl]M+[37Cl]M-H+), 439.0
(100, [35Cl]M+[35Cl]M-H+), 222.0 (27, [37Cl]M-H+), 220.3 (85, [35Cl]M-H+);
1H-NMR (D2O): d 7.23–6.49 (3H, m, 20-H, 50-H and 60-H), 4.26 (1H, dd,
J ¼ 7:7 and 5.6, 2-H), 3.21 (1H, dd, J ¼ 14:9 and 5.6, 3-Ha), 3.08 (1H, dd,
J ¼ 14:9 and 7.7, 3-Hb); 13C-NMR (D2O): d 171.9 (s, 1-C), 152.4 (dd, J ¼ 68:7
and 64.8, 40-C), 131.4 (dd, J ¼ 64:8 and 57.2, 20-C), 129.9 (ddd, J ¼ 57:2, 57.2
and 7.6, 60-C), 128.2 (ddd, J ¼ 57:2, 57.2 and 7.6, 10-C), 121.4 (ddd, J ¼ 74:4,
67.7 and 6.7, 30-C), 118.1 (ddd, J ¼ 63:9, 58.2 and 5.7, 50-C), 56.7 (s, 2-H), 36.0
(d, J ¼ 43:9, 3-H).
Preparation of the 3-chloro-l-tyrosine-ring-[ring-13C6] (2b), via the chlorina-
tion of benzyl 4-(4-hydroxybenzyl-[ring-13C6])-5-oxo-1,3-oxazolidine-3-car-
boxylate (7)
(a) Benzyloxycarbonylation
of
the
(2S)-2-amino-3-(4-hydroxyphenyl-
[ring-13C6])-propionic acid methyl ester (4) hydrochloride. The methyl ester
hydrochloride 4 (60 mg; 0.25 mmol) was dissolved in an aqueous solution of
NaHCO3 (2 ml, 0.3 M) and the solution cooled to 08C. Then, benzyl
chloroformate (45 ml, 0.32 mmol) was added under stirring and the solution
kept at room temperature for 5 h. After extraction with ethyl acetate, the
organic layers were then washed with water and dried over Na2SO4.
Evaporation of the solvent under reduced pressure afforded a crude oil which,
after column chromatography (eluting with hexane/ethyl acetate, 60/40, v/v),
gave the pure methyl (2S)-2-benzyloxycarbonylamino-3-(4-hydroxyphenyl-
[ring-13C6])propanoate 6 (40.1 mg, 48.0%): mp 90.5–91.58C, [(lit.24 mp 91–
928C)]; [a]D22=+37.8 (c 1, CHCl3); ESI/MS (negative) m/z (relative
1
intensity): 669.1 (100, M+M-H+), 334.3 (72, M-H+); H-NMR (CDCl3): d
7.36–7.29 (5H, m, aromatics), 6.92 (2H, dd, J ¼ 155:6 and 7.5, 20-H and 60-H),
6.69 (2H, dd, J ¼ 161:7 and 6.0, 30-H and 50-H), 5.37 (1H, d, J ¼ 8:2, NH),
5.11 (1H, d, J ¼ 12:3, A part of a AB system, NHCO2CHHC6H5), 5.07 (1H, d,
J ¼ 12:3, B part of a AB system, NHCO2CHHC6H5), 4.63 (1H, ddd, J ¼ 8:2,
6.1 and 5.4, 2-H), 3.71 (3H, s, OCH3), 3.06 (1H, dd, J ¼ 13:9 and 5.4, 3-Ha),
2.98 (1H, dd, J ¼ 13:9 and 6.1, 3-Hb); 13C-NMR (CDCl3): d 172.43 (s,
COOCH3), 156.0 (dt, J= 64.8 and 9.5, 40-C), 131.1 (ddd, J ¼ 57:2, 57.2 and
5.7, 20-C and 60-C), 127.7 (dt, J ¼ 57:2 and 9.5, 10-C), 116.2 (ddd, J ¼ 64:8,
64.8 and 5.7, 30-C and 50-C), 67.1 (s, OCH2Ph), 54.9 (s, OCH3), 52.4 (s, 2-C),
37.5 (s, 3-C). Analytically calculated for 12C12 13C6H19NO5: C 65.64, H 5.81, N
4.25. Found: C 65.42, H 5.72, N 4.54.
Copyright # 2004 John Wiley & Sons, Ltd.
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942
P. ALLEVI ET AL.
(b) Preparation of benzyl 4-(4-hydroxybenzyl-[ring-13C6])-5-oxo-1,3-oxazoli-
dine-3-carboxylate (7). A mixture of methyl ester 6 (80 mg; 0.24 mmol),
p-toluenesulfonic acid (22.8 mg; 0.12 mmol) and toluene (3 ml) was placed in a
flask. Then paraformaldehyde (18 mg; 0.6 mmol) was added to the solution,
which was heated to 808C. After 30 min the reaction was stopped, the solution
was washed with aqueous NaHCO3 and extracted with AcOEt; the organic
layer was dried over anhydrous Na2SO4. After evaporation of the solvent
under reduced pressure, a crude oil was obtained that, after column
chromatography (eluting with dichloromethane/acetone, 100/0.5, v/v), gave
the pure 7 (44.8 mg, 56.0%); mp 1118C; [a]D22=+208.0 (c 1, CHCl3) [(lit.25
mp 110–1128C; [a]D22=+206.0 (c 1, CHCl3)]; ESI/MS (positive) m/z (relative
intensity): 753.6 (100, M+M+2CH3OH+Na+), 388.3 (72, M+CH3OH+-
1
Na+); H-NMR (DMSO-d6): d 7.41–7.36 (5H, m, aromatics), 6.78 (2H, dd,
J ¼ 158:5 and 7.6, aromatic), 6.62 (2H, dd, J ¼ 161:0 and 7.6, aromatic), 5.26
(1H, d, J ¼ 3:7, OCHHN), 5.20 (2H, m, OCH2Ph), 4.54 (1H, bs, COCHN),
4.45 (1H, bs, OCHHN), 3.09 (1H, m, CHCHHAr), 2.91 (1H, dd, J ¼ 13:9 and
13C6H17NO5: C 66.05, H
12
2.5, CHCHHAr). Analytically calculated for
C
12
5.23, N 4.28. Found: C 65.88, H 5.33, N 4.11.
(c) Chlorination of the benzyl 4-(4-hydroxybenzyl-[ring-13C6])-5-oxo-1,3-
oxazolidine-3-carboxylate (7). The ester 7 (54 mg; 0.162 mmol) was suspended
in a mixture of AcOH-Et2O (240 ml–1.1 ml) and the whole cooled at 0–58C. To
this mixture, SO2Cl2 (13.8 ml; 0.180 mmol) dissolved into Et2O (0.60 ml), and
cooled to ꢀ108C, was then slowly added. The formed solution was then stirred
at 08C for 1 h and then diluted with an aqueous solution of NaHCO3. The
mixture was then extracted with AcOEt, washed with water and dried over
Na2SO4. Evaporation of the solvent, under reduced pressure, afforded a crude
residue that, after column chromatography (eluting with hexane/ethyl acetate,
75/25, v/v), gave pure benzyl 4-(3-chloro-4-hydroxybenzyl-ring-13C6)-5-oxo-
1,3-oxazolidine-3-carboxylate
8
(40.5 mg,
68.0%):
mp
81–828C;
[a]D22=+167.7 (c 1, CHCl3); ESI/MS (positive) m/z (relative intensity):
424.1 (35, [37Cl]M+CH3OH+Na+), 422.1 (100, [35Cl]M+CH3OH+Na+);
1H-NMR (DMSO-d6): d 7.39–7.32 (5H, m, aromatics), 6.95 (1H, d, J ¼ 163:2,
aromatic), 6.82 (1H, dd, J ¼ 163:5 and 8.2, aromatic), 6.75 (1H, ddd,
J ¼ 158:9, 8.1 and 1.3, aromatic), 5.29 (1H, d, J ¼ 4:0, OCHHN), 5.16 (2H,
m, OCH2Ph), 4.63 (1H, bs, COCHN), 4.55 (1H, m, OCHHN), 3.08 (1H, m,
CHCHHAr), 2.92 (1H, dd, J ¼ 14:1 and 3.5, CHCHHAr). Analytically
calculated for 12C12 13C6H16ClNO5: C 59.76, H 4.46, Cl 9.80, N 3.87. Found: C
60.01, H 4.22, Cl 9.86, N 3.91.
(d) Regeneration of the aminoacidic function of 8, preparation of 3-chloro-l-
tyrosine-[ring-13C6] (2b). The oxazolidinone 8 (80 mg; 0.22 mmol) was
dissolved in dichloromethane (8 ml) and treated at 258C with BCl3 (0.6 ml of
Copyright # 2004 John Wiley & Sons, Ltd.
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CONTROLLED SYNTHESIS
943
a 1 M solution in dichloromethane; 0.64 mmol). After 1 h, the solution was
diluted with H2O, and applied on a Dowex 50 ꢁ 8-200 cation-exchange resin
column and processed as described above in the saponification of the methyl
ester 5. The eluates containing the amino acid were collected to afford pure 2b
(24.4 mg, 50.1%) that showed a single peak in HPLC (tr=8.8 min) and all
physicochemical properties reported above.
(e) Preparation of (2S)-2-benzyloxycarbonylamino-3-(3-chloro-4-hydroxy-
8
pheny-[ring-13C6])-propanoic acid 9. The oxazolidinone
(30 mg;
0.081 mmol) was dissolved in a saturated solution of NaHCO3 in MeOH-
H2O (1:1 v/v) (2 ml) and refluxed for 10 min.15 Then the solution was acidified,
extracted with AcOEt and the organic layers were washed with water and
dried over Na2SO4. Evaporation of the solvent, under reduced pressure,
afforded the acid 9 (24.5 mg, 85.0%): mp 140–1418C; [a]D22=+33.0 (c 1,
CHCl3); ESI/MS (negative) m/z (relative intensity): 712.8 (6,
[37Cl]M+[37Cl]M-H+), 710.8 (31, [37Cl]M+[35Cl]M-H+), 708.8 (51,
[35Cl]M+[35Cl]M-H+), 355.8 (30, [37Cl]M-H+), 353.8 (100, [35Cl]M-H+);
1H-NMR (CDCl3): d 7.34–7.26 (5H, m, aromatics), 7.08 (1H, d, J ¼ 158:0, 60-
H), 6.90 (2H, dd, J ¼ 165:9 and 8.6, 20-H and 50-H), 5.28 (1H, d, J ¼ 1:23,
NH), 5.10 (1H, d, J ¼ 11:93, A part of a AB system, NHCO2CHHC6H5), 5.04
(1H, d, J ¼ 11:93, B part of a AB system, NHCO2CHHC6H5), 4.59 (1H, m, 2-
H), 3.08 (1H, m, CHCHHAr), 2.96 (1H, m, CHCHHAr); 13C-NMR (CDCl3):
d 150.6 (ddd, J ¼ 71:5, 70.5 and 6.7, 40-C), 119.9 (ddd, J ¼ 71:5, 67.7 and 5.7,
30-C), 116.3 (ddd, J ¼ 58:2, 10.5 and 5.7, 50-C), 130.3-128.0 (m, overlapping,
13C6H13ClNO5: C 58.38,
12
10-C, 20-C and 60C). Analytically calculated for
C
11
H 4.61, Cl 10.14, N 4.00. Found: C 58.63, H 4.73, Cl 10.05, N 3.83.
(f) Regeneration of 3-chloro-l-tyrosine-[ring-13C6] (2b) from (2S)-2-benzy-
acid
loxycarbonylamino-3-(3-chloro-4-hydroxypheny-ring-13C6)-propanoic
(9). The 5-oxazolidinone 9 (31 mg; 0.087 mmol) was dissolved in dichlor-
omethane (2 ml) and treated at 258C with BCl3 (440 ml of a 1 M solution in
dichloromethane; 0.44 mmol). After 1 h the solution was diluted with H2O,
and applied on a Dowex 50 ꢁ 8-200 cation-exchange resin column to be
recovered as described above. The eluates containing the amino acid were
collected to afford pure 2b (9.6 mg, 50.0%) with all physicochemical properties
reported above for 2b, purified by HPLC.
Preparation of the 3,5-dichloro-l-tyrosine-[ring-13C6] (3b)
l-tyrosine-[ring-13C6] 1b (50 mg; 0.27 mmol) was suspended in sulpholane
(1 ml) and the mixture was stirred at 258C under argon. Then SO2Cl2 (0.1 ml;
1.23 mmol) dissolved in diethyl ether (1.0 ml) was added dropwise. After
addition, a solution gradually formed which was stirred for 24 h at 238C. Then,
Copyright # 2004 John Wiley & Sons, Ltd.
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944
P. ALLEVI ET AL.
anhydrous dichloromethane was added until precipitation of a white solid was
complete; then it was filtered and dried under vacuum to give the title
compound 3b as the hydrochloride (69.5 mg, 88.0%). The compound gave a
single spot in TLC, eluting with a solution of CHCl3/MeOH/NH3 (6/4/1; v/v/
v), Rf=0.26, and shows a single peak in HPLC (tr ¼ 22:3 min): mp 205–
2068C, (from AcOH); ½aꢂD22=+2.9 (c 1, H2O); ESI/MS (negative) m/z
(relative intensity): 258.2 (10, [37Cl2]M-H+), 256.1 (69, [35Cl,37Cl]M-H+),
254.2 (100, [35Cl2]M-H+); 1H-NMR (D2O): d 7.27 (2 H, d, J ¼ 162:8, 20-H and
60-H), 4.24 (1 H, dd, J ¼ 7:5 and 5.7, 2-H), 3.24 (1 H, dd, J ¼ 14:9 and 5.7, 3-
Ha), 3.12 (1 H, dd, J ¼ 14:9 and 7.5, 3-Hb); 13C-NMR (D2O): d 171.9 (s, 1-C),
148.2 (t, J ¼ 72:5, 40-C), 130.0 (ddd, J ¼ 62:9, 59.1 and 9.5, 20-C and 60-C),
128.1 (dt, J ¼ 59:1 and 3.8, 10-C), 122.8 (ddd, J=30-C and 50-C), 54.6 (s, 2-C),
35.1 (d, J ¼ 42:9, 3-C). Analytically calculated for 12C3 13C6H10Cl3NO3: C
38.99, H 3.45, Cl 36.36, N 4.79. Found: C 39.10, H 3.61, Cl 36.45, N 4.70.
Acknowledgements
This work was supported financially by the Italian MIUR (Ministero
"
dell’Istruzione, dell’Universita e della Ricerca).
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