An improved stereoselective synthesis of L-alanosine

Article abstract of DOI:10.1002/ejoc.200400508

An improved, stereoselective synthesis of the natural, non-proteogenic amino acid L-alanosine has been developed, starting from the readily available and cheap substrate L-serine, in six steps and 49% overall yield. The process is very efficient, is suitable for large-scale production, and affords L-alanosine with properties comparable with those of the natural substance. In addition, the structural assignment concerning some previously reported synthetic alkylated derivatives of the natural amino acid has been definitively confirmed. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400508

FULL PAPER
An Improved Stereoselective Synthesis of -Alanosine
L
Paolo Strazzolini,*[a] Maria Grazia Dall’Arche,[a] Maura Zossi,[a] and Andrea Pavsler[a]
Keywords: Amino acids / Antitumour agents / Asymmetric synthesis / Chiral pool / Structure elucidation
An improved, stereoselective synthesis of the natural, non-
proteogenic amino acid -alanosine has been developed,
starting from the readily available and cheap substrate -ser-
ine, in six steps and 49% overall yield. The process is very
efficient, is suitable for large-scale production, and affords
ural substance. In addition, the structural assignment con-
cerning some previously reported synthetic alkylated deriv-
atives of the natural amino acid has been definitively con-
firmed.
L
L
L-
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
alanosine with properties comparable with those of the nat- Germany, 2004)
Introduction
-Alanosine [(S)-3-(hydroxynitrosoamino)alanine (1);
Scheme 1],[1] which is an antitumour antibiotic produced by
fermentation of Streptomyces alanosinicus n. sp. ATCC
15710,[2] has the structure of an α-amino acid with an 
Scheme 2
configuration, and was the first naturally occurring sub-
stance found to contain an N-nitroso-hydroxylamino
group.[3] Like many other amino acids, 1 exists as a zwit-
cyclic zwitterion (2, Scheme 2), which is more stable than
terion, where the role of the more acidic function (pKa Ͻ
1) is taken by the N-nitroso-hydroxylamino group,[3] which
is known to exist as an equilibrium of two tautomers,[4] na-
mely the hydroxynitrosoamino (1a, Scheme 1) and the diim-
ide N-oxide (1b) forms.[5] In the solid state, 1 has been
shown to be present as the diimide oxide tautomer 1b,[6]
and this configuration has been considered likely to be fav-
oured also in solution, taking into account the reported
structural assignment concerning a number of alkylation
products derived from 1 (see below).[7] This circumstance
might explain the preferential formation of a six-membered
the corresponding five-membered one (3), and could also
be responsible for the difficulties encountered in N-acyl-
ation of 1,[7] as well as for the anomalous behaviour ob-
served, compared with that shown by other α-amino ac-
ids.[8]
-Alanosine exhibits a number of interesting biological
and pharmacological properties,[1] including antiviral and
antimicrobial activity, immunosuppressive and anti-ar-
thritis action, as well as insect growth regulatory function,[9]
but its anti-cancer activity, which is associated with its
ability to interfere with the metabolism of aspartic acid,[10]
has appeared since the beginning to be by far the most at-
tractive one. In view of the renewed interest concerning the
applications of 1 in cancer therapy,[11] and in the course of
a study aimed at obtaining new -alanosine derivatives with
potential anti-cancer activity, we needed to be able to pro-
duce large amounts of 1 by chemical synthesis as we were
unavailable to obtain it by a fermentation process.[3]
A number of syntheses of 1 have been proposed in the
literature to date; however, the majority either only produce
the amino acid in its racemic form,[12] or require tedious
enantiomeric separations in order to afford the active iso-
mer.[13] Recently, a stereoselective process taking the start-
ing material from the chiral pool[14] appeared as a Japanese
patent,[15] but it does not seem promising in view of a large-
scale application. We therefore decided to develop a method
for producing 1 which could offer acceptable safety and
Scheme 1
[a]
Department of Chemical Sciences and Technologies, University
of Udine,
Via del Cotonificio 108, 33100 Udine, Italy
Fax: (internat.) ϩ 39-0432-558-803
E-mail: strazzolini@dstc.uniud.it
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DOI: 10.1002/ejoc.200400508
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An Improved Stereoselective Synthesis of L-Alanosine
FULL PAPER
economical standards, as well as being suitable for potential in MeCN (Scheme 4) to obtain 7 in very good isolated yield
scale-up.
(80%), essentially comparable with those reported em-
ploying WSCs.[23]
Results and Discussion
Among the variety of methods available for the synthesis
of pure stereoisomers of chiral amino acids, the strategy
based on the elaboration of α-amino acid precursors ap-
pears, when applicable, the most convenient in terms of
feasibility, simplicity and low costs.[16] In this context, the
naturally occurring chiral amino acid serine represents, for
the organic chemist, an attractive synthetic template as both
isomers are readily available and cheap starting materi-
als.[17] Nevertheless, many of the possible synthetic ap-
proaches starting from -serine (4) that were potentially
able to afford the important intermediate (S)-2-amino-3-
(hydroxyamino)propanoic acid (5) proved to be inad-
Scheme 4
When we tried to repeat the cyclisation reaction in order
equate,[18] due to the risk of incurring competitive elimin- to prepare the azetidinone intermediate 8 (Scheme 3),[15]
ations,[19] as well as unwanted epimerisations and intramol- using classical Mitsunobu conditions,[24] in preliminary
ecular cyclisations;[18] moreover, even the use of reactive de- experiments we succeeded in obtaining the desired com-
rivatives of 4, like activated aziridines[20] or β-lactones,[21] pound, but in quite unsatisfactory yield due to difficulties
often resulted in the formation of isomeric mixtures. For encountered in separating it from the reduced counterpart
these reasons, the more straightforward strategy suggested (DEADH2) of diethyl azodicarboxylate (DEAD). This sep-
in a recent patent,[15] based on the intermediacy of an azeti- aration, which requires column chromatography,[25] is not
dinone derivative of 4, even if not satisfying as such (vide suitable for large-scale production; moreover, it must be
infra), seemed to prove the most promising synthetic ap- pointed out that DEAD is a rather expensive, potentially
proach (Scheme 3).
unstable chemical whose use in large batches is best avoided
Therefore, -serine (4) was converted, in nearly quantitat- for safety reasons. Therefore, we looked for alternative con-
ive yield and according to a previously described pro- ditions to perform the cyclisation and decided to apply the
cedure,[22] into the corresponding N-(phenylmethyloxy)car- elegant reaction proposed by Miller,[23] which consists of
bonyl derivative 6, which was, in turn, transformed into the treating 7 with PPh3, CCl4 and TEA in MeCN (Scheme 4),
corresponding O-protected hydroxylamide 7. The con- and affords the azetidinone 8 in very high isolated yield
ditions reported in the literature for this reaction after a simple workup requiring just a fast passage through
(Scheme 3)[15] involve the use of expensive water-soluble a SiO2 column in order to separate the formed OPPh3.
carbodiimides (WSCs), which is impractical for large-scale Alternate conditions that do not require any purification by
processes. Even though the employment of the much more SiO2 treatment have also been reported.[26]
convenient dicyclohexylcarbodiimide (DCC) had been dis-
Subsequent hydrolytic ring-opening to give quantitatively
couraged for this application,[23] it was possible to set up the protected hydroxyamino derivative 9 (Scheme 5), per-
suitable and very simple conditions to perform the reaction formed with LiOH in THF/H2O,[27] proceeded smoothly in
between 6 and O-(phenylmethyl)hydroxylamine, using DCC accordance with the literature,[15] whereas the following de-
Scheme 3
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P. Strazzolini, M. G. Dall’Arche, M. Zossi, A. Pavsler
FULL PAPER
protection step was somehow disappointing. In fact, when yield (Scheme 6) and with properties comparable with those
an aqueous HCl/AcOH solution of 9 was hydrogenolysed of the natural amino acid and an undetectable ash content.
in the presence of 5% Pd/C, at room temperature and at- The optical purity of 1 (S configuration) was confirmed by
mospheric pressure, careful monitoring of the composition measuring its specific rotation {[α]2D0 ϭ Ϫ45.0 (c ϭ 0.5, 0.1
of the reaction mixture (1H NMR) showed that the com-  NaOH)}, which was found to be equal to the values pre-
plete conversion of substrate 9 gave the expected fully de- viously reported.[3,13c]
protected intermediate 5 (Scheme 5), accompanied by a
The availability of the protected intermediate 9 offered
consistent amount (35%) of the over-reduction product (S)- the possibility to unequivocally verify the correct structural
2,3-diaminopropanoic acid (10). The hydrogenolytic sus- assignment previously attributed to some products obtained
ceptibility of the NϪO bond in hydroxylamines has been by alkylation of a masked derivative of 1.[7] It is well-
well documented.[19,27,28]
known[4,5] that alkylation products of N-nitroso-hy-
droxylamines (12, Scheme 7) may exist in two isomeric
arrangements, namely 13 and 14. In the course of a study
directed towards the synthesis of some derivatives of 1,[7]
(S)-3-(hydroxy-nitrosoamino)-N-[(phenylmethyloxy)-
carbonyl]alanine (N-Z--alanosine, 15) was protected at the
acidic functions by dialkylation with alkyl halides in the
presence of TEA. In one case, when the alkylating agent
was phenylmethyl bromide, the structure assigned to the
predominant, if not exclusive, isomer formed was, on the
basis of chemical and physico-chemical properties, the diim-
ide N-oxide one (16, Scheme 8), rather than the N-nitroso
form 17. In order to definitively confirm that attribution,
compound 9 was esterified with phenylmethyl bromide in
benzene and in the presence of DBU (Scheme 9),[30] and the
obtained dibenzylated derivative 18 was subsequently N-ni-
trosated under the aprotic conditions reported above, to af-
ford the previously unknown N-nitroso derivative 17, whose
properties proved very different to those of compound 16,
thus unequivocally confirming the structural assignment
given in the literature.[7]
Scheme 5
In order to overcome this drawback, and also taking into
account the observed,[7] unexpected resistance exhibited by
the N-nitroso-hydroxylamine function in the course of hy-
drogenolytic dealkylation, we decided to anticipate the
nitrosation step, which was smoothly accomplished under
aprotic conditions[29] by addition of a moderate excess of n-
butyl nitrite (nBuONO) in CH2Cl2, affording the intermedi-
ate 11 in quantitative yield (Scheme 6). Surprisingly en-
ough, compound 11 (as its sodium salt) could be eventually
deprotected under hydrogenolytic conditions (5% Pd/C,
H2O, 24 h, at moderate pressure and 30 °C) without suffer-
ing any over-reduction to give -alanosine (1) in fairly good
Scheme 7
Scheme 6
Scheme 8
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An Improved Stereoselective Synthesis of L-Alanosine
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are reported in ppm relative to the central line of the CDCl3 triplet
(δ ϭ 77.00 ppm). When D2O was the solvent, spectral references
were used and are indicated where appropriate. Coupling constants
are given in Hz. Some 1H NMR multiplets are characterised by the
term app (apparent): this refers only to their appearance and may
be an oversimplification. MS measurements were carried out with a
Fisons TRIO-2000 apparatus, working in the positive-ion electron
impact mode (70 eV), by direct introduction of the sample into the
ion source and heating from 50 up to 300 °C. The five most intense
peaks and the molecular peak (when detectable) for each individual
compound with bracketed intensity values are reported.
Scheme 9
(S)-N-[(Phenylmethyloxy)carbonyl]serine (6): Compound 6 was pre-
pared in 87% yield, according to a previously described pro-
cedure.[22]
Conclusion
(S)-{2-[(Phenylmethyloxy)amino]-1-(hydroxymethyl)-2-oxoethyl}-
carbamic Acid Phenylmethyl Ester (7):[25] A solution of N,N-di-
cyclohexylcarbodiimide (DCC, 4.28 g, 21.0 mmol) in CH3CN
(80 mL) was slowly added dropwise, at 0 °C, into a well-stirred
solution of 6 (4.78 g, 20.0 mmol) and O-(phenylmethyl)hydrox-
ylamine (2.46 g, 20.0 mmol) in CH3CN (80 mL), and the reaction
mixture was stirred for 4 h at 0 °C and overnight at room tempera-
ture. After completion of the reaction, the obtained white precipi-
tate (N,N-dicyclohexylurea; DCU) was separated by filtration and
the resulting clear reaction mixture concentrated to a small volume
under reduced pressure. After the addition of Et2O, a crystalline
product formed, which was collected by filtration, washed with
Et2O and dried for 30 min at 40 °C under vacuum. White solid
(80% yield): m.p. (from CH3CN/Et2O) 127Ϫ129 °C. [α]2D0 ϭ Ϫ27.5
(c ϭ 2.0, CH3OH). IR (pellet): ν˜ ϭ 3425 br. m, 3230 m, 1670 s,
1539 s, 1456 w, 1294 m, 1247 m, 1100 w, 1073 w, 1044 w, 914 w,
737 s, 696 m, 637 w cmϪ1. 1H NMR: δ ϭ 9.66 (s, 1 H, OϪNHϪCϭ
O), 7.38Ϫ7.25 (m, 10 H, C6H5), 5.96 (d, J ϭ 7.8 Hz, 1 H,
CϪNHϪCϭO), 5.01 (s, 2 H, PhϪCH2), 4.83 (s, 2 H, PhϪCH2),
4.11 (m, 1 H, CH2ϪCH), 3.86 (m, 1 H, OH), 3.61 (m, 2 H,
CHϪCH2) ppm. 13C NMR: δ ϭ 168.5 (OϪNϪCϭO), 156.5
(NϪCϭO), 135.7, 134.8, 129.3, 128.9, 128.6 (2 overlapped signals),
128.3, 128.0, 78.4, 67.4, 62.3, 53.5 ppm. MS (50 °C): m/z ϭ 91
(100), 79 (56), 108 (55), 77 (43), 107 (41). C18H20N2O5 (344.37):
calcd. C 62.78, H 5.85, N 8.14; found C 62.65, H 5.86, N 8.12.
In view of the renewed interest concerning -alanosine
and its applications in cancer therapy, an improved, stereo-
selective chemical synthesis has been developed, allowing
the isolation of the pure amino acid starting from cheap
and readily available -serine in six steps and 49% overall
yield. This process is characterised by its simplicity and low
cost, and is safe, eco-friendly and potentially suitable for
large-scale applications. Moreover, the present work has
firmly established the correctness of the structural assign-
ment concerning some previously reported alkyl derivatives
of -alanosine, thus casting more light on the chemistry of
N-nitroso-hydroxylamines.
Experimental Section
General Remarks: Unless otherwise specified, reagents and solvents
are commercially available (Aldrich Italia, Milano, Italy) and were
used as received. Commercial PPh3 was carefully dried prior to use
by keeping it over P2O5, in vacuo at 70 °C for 1 h. Anhydrous CCl4
and CH3CN were obtained by heating under reflux over P2O5, fol-
lowed by distillation under an inert atmosphere. Anhydrous NEt3 (S)-[1-(Phenylmethyloxy)-2-oxo-3-azetidinyl]carbamic Acid Phe-
(TEA) was obtained by distillation after prolonged treatment with
nylmethyl Ester (8):[23,25] A solution of PPh3 (4.51 g, 17.2 mmol) in
CaH2. O-(Phenylmethyl)hydroxylamine was prepared as described dry CH3CN (33 mL) was added dropwise, at room temperature and
elsewhere.[31] Compounds 5, 7, 8 and 9 have been already described
in the literature (see below). TLC analyses and column chromatog-
raphy were performed on silica gel 60 from Merck (Darmstadt,
Germany) under suitable conditions. The course of all the de-
scribed reactions was monitored by TLC and by a parallel accurate
1H NMR quantitative evaluation. Melting points (uncorrected)
were previously determined in open-ended capillary tubes by using
a Mettler FP 61 automatic system, and subsequently visually con-
firmed by a Büchi 512 apparatus. Elemental analyses were obtained
by a CarloϪErba CHN/OS 1106 analyzer for all isolated com-
pounds and found to be satisfactory. The ash content in 1 was
determined by weighing the residue left by a suitable sample after
mineralisation (1 h at 800 °C). Optical activities were measured at
20 °C on a Atago Polax-D polarimeter at 589 nm in a 1.0-dm tube.
IR spectra were recorded on a Nicolet FTIR Magna 550 spectro-
photometer using the KBr technique. Unless otherwise indicated,
under an inert atmosphere, to a stirred solution of 7 (5.64 g,
16.4 mmol) in dry CH3CN (50 mL), containing CCl4 (1.70 mL,
17.6 mmol) and anhydrous triethylamine (TEA, 3.50 mL,
25.1 mmol). The resultant clear reaction mixture was stirred for
24 h at room temperature. After completion of the reaction, the
obtained white precipitate (TEA·HCl and Ph3PO) was separated
by filtration and the resulting clear reaction mixture concentrated
under reduced pressure. The residue was redissolved in EtOAc
(350 mL), washed with 10% aqueous Na2SO4 (3 ϫ 170 mL), dried
over anhydrous Na2SO4, filtered, concentrated to a small volume
at reduced pressure and passed through a short SiO2 column by
elution with hexane/EtOAc (1:1, v/v). The fractions containing the
product of interest were combined and the solvent was evaporated
off, yielding a solid residue which was collected and dried for
30 min at 40 °C under vacuum. White solid (87% yield): m.p. (from
hexane/EtOAc) 91Ϫ92 °C. [α]2D0 ϭ Ϫ12.5 (c ϭ 1.0, CH3OH). IR
1H and 13C NMR spectra were recorded in CDCl3 on a Bruker (pellet): ν˜ ϭ 3309 br. m, 3065 w, 2967 w, 1802 s, 1703 s, 1543 s,
AC-200 spectrometer at 200 and 50 MHz, respectively. The proton
chemical shifts are reported in ppm on the δ scale relative to TMS
1455 w, 1337 w, 1272 s, 1227 w, 1132 w, 1077 m, 970 s, 913 w, 854
1
w, 755 m, 699 w, 578 w cmϪ1. H NMR: δ ϭ 7.47Ϫ7.27 (m, 10 H,
as an internal reference (δ ϭ 0.00 ppm); the carbon chemical shifts C6H5), 5.57 (d, J ϭ 6.8 Hz, 1 H, NH), 5.08 (s, 2 H, PhϪCH2), 4.95
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P. Strazzolini, M. G. Dall’Arche, M. Zossi, A. Pavsler
FULL PAPER
(s, 2 H, PhϪCH2), 4.55 (sym m, J ϭ 6.8, JBX ϭ 4.5, JAX ϭ 2.0 Hz,
(S)-2,3-Diaminopropanoic Acid (10) Dihydrochloride: 1H NMR
1 H, CH2ϪCH), 3.51 (app t, JAB ϭ 4.8, JBX ϭ 4.5 Hz, 1 H, (D2O, spectral reference ϭ 2853.00 Hz): δ ϭ 4.29 (dd, JAX ϭ 5.9,
CHϪCH2), 3.24 (dd, JAB ϭ 4.8, JAX ϭ 2.0 Hz, 1 H, CHϪCH2) BX ϭ 7.8 Hz, 1 H, CH2ϪCH), 3.48 (dd, JAB ϭ 13.6, JBX ϭ 7.8 Hz,
ppm. 13C NMR: δ ϭ 162.4 (CϭO), 155.6 (NϪCϭO), 135.8, 134.6, 1 H, CHϪCH2), 3.42 (dd, JAB ϭ 13.6, JAX ϭ 5.9 Hz, 1 H,
129.2, 129.1, 128.7, 128.5, 128.3, 128.1, 77.9, 67.3, 54.1, 53.6 ppm.
CHϪCH2) ppm. 13C NMR (D2O, spectral reference
J
ϭ
MS (50 °C): m/z ϭ 91 (100), 92 (9), 77 (7), 108 (7), 79 (6). 3313.00 Hz): δ ϭ 171.4 (CϭO), 52.1 (CH2), 40.7 (CH) ppm.
C18H18N2O4 (326.35): calcd. C 66.25, H 5.56, N 8.58; found C
66.14, H 5.58, N 8.57.
(S)-3-[Nitroso(phenylmethyloxy)amino]-N-[(phenylmethyloxy)-
carbonyl]alanine (11): Butyl nitrite (3.10 g, 30.0 mmol) was added
dropwise to a solution of 9 (3.44 g, 10.0 mmol) in CH2Cl2 (6.5 mL)
and the resultant clear, yellow mixture was stirred for 1 h at room
temperature in the dark. After completion of the reaction, the sol-
vent was evaporated off under reduced pressure and the residue
was treated with Et2O until crystallisation occurred. The obtained
product was collected by filtration, washed with Et2O and dried
for 30 min at 40 °C under vacuum. Yellowish solid (99% yield):
m.p. (from Et2O) 79Ϫ80 °C. [α]2D0 ϭ Ϫ15.0 (c ϭ 1.0, CHCl3). IR
(pellet): ν˜ ϭ 3420 m, 3039 br. s, 1757 m, 1743 m, 1670 s, 1531 s,
1448 m, 1425 m, 1408 w, 1311 w, 1278 m, 1199 s, 1117 w, 1071 m,
1015 w, 909 w, 876 w, 753 m, 744 w, 697 s, 611 w cmϪ1. 1H NMR:
δ ϭ 10.39 (br. s, 1 H, COOH), 7.36Ϫ7.27 (m, 10 H, C6H5), 5.59
(d, J ϭ 7.5 Hz, 1 H, NH), 5.07 (s, 2 H, PhϪCH2), 4.94 (s, 2 H,
PhϪCH2), 4.64 (sym m, J ϭ 7.5, JAX ϭ 4.3, JBX ϭ 3.9 Hz, 1 H,
CH2ϪCH), 4.53 (dd, JAB ϭ 13.6, JBX ϭ 3.9 Hz, 1 H, CHϪCH2),
4.39 (dd, JAB ϭ 13.6, JAX ϭ 4.3 Hz, 1 H, CHϪCH2) ppm. 13C
NMR: δ ϭ 173.0 (CϭO), 155.8 (NϪCϭO), 135.6, 133.3, 129.6,
129.4, 128.7, 128.5, 128.3, 128.0, 77.4, 67.5, 54.1, 51.8 ppm. MS
(50 °C): m/z ϭ 91 (100), 108 (86), 107 (67), 79 (46), 77 (26), 373(Ͻ1)
[Mϩ]. C18H19N3O6 (373.37): calcd. C 57.91, H 5.13, N 11.26; found
C 57.79, H 5.14, N 11.25.
(S)-3-[(Phenylmethyloxy)amino]-N-[(phenylmethyloxy)carbonyl]-
alanine (9):[15] Compound 9 was obtained according to a described
procedure.[27] Solid LiOH·H2O (0.90 g, 21.4 mmol) was added in
one portion to a stirred solution of 8 (4.52 g, 13.9 mmol) in THF
(70 mL) and H2O (35 mL) and the clear reaction mixture was kept
at room temperature overnight under an inert atmosphere. After
completion of the reaction, the mixture was acidified by addition
of 2  HCl (33 mL), and subsequently extracted with EtOAc (3 ϫ
65 mL). The combined organic phase was washed with H2O
(100 mL), 10% aqueous Na2SO4 (2 ϫ 100 mL), dried over anhy-
drous Na2SO4, filtered and concentrated to dryness, yielding a solid
residue which was collected and dried for 30 min at 60 °C under
vacuum. Yellowish solid (Ͼ99% yield): m.p. (from EtOAc) 73Ϫ75
°C. [α]2D0 ϭ Ϫ20.0 (c ϭ 1.0, CH3OH). IR (pellet): ν˜ ϭ 3309 br. m,
3039 w, 2940 w, 1730 s, 1524 s, 1455 m, 1397 m, 1334 w, 1245 s,
1132 w, 1061 m, 915 w, 786 w, 740 s, 699 m, 578 w cmϪ1. 1H NMR:
δ ϭ 8.18 (br. s, 2 H, COOH ϩ OϪNH), 7.45Ϫ7.14 (m, 10 H,
C6H5), 5.83 (d, J ϭ 7.6 Hz, 1 H, OϭCϪNH), 5.09 (s, 2 H,
PhϪCH2), 4.63 (s, 2 H, PhϪCH2), 4.50 (sym m, J ϭ 7.6, JAX
5.1, JBX ϭ 4.1 Hz, 1 H, CH2ϪCH), 3.43 (dd, JAB ϭ 14.0, JBX
ϭ
ϭ
4.1 Hz, 1 H, CHϪCH2), 3.21 (dd, JAB ϭ 14.0, JAX ϭ 5.1 Hz, 1 H,
CHϪCH2) ppm. 13C NMR: δ ϭ 175.0 (CϭO), 156.2 (NϪCϭO),
137.3, 135.9, 128.5, 128.4, 128.4, 128.2, 128.1, 128.1, 75.9, 67.2,
52.3, 52.1 ppm. MS (80 °C): m/z ϭ 91 (100), 108 (42), 79 (37), 77
(35), 107 (32), 344 (Ͻ1) [Mϩ]. C18H20N2O5 (344.37): calcd. C 62.78,
H 5.85, N 8.14; found C 62.69, H 5.87, N 8.13.
(S)-3-(Hydroxynitrosoamino)alanine (1, l
-alanosine):[3,13c] 5% Pd/C
(444 mg) was added to a solution of 11 (1.87 g, 5.00 mmol) in 0.5
 NaOH (10.0 mL) and the mixture was vigorously stirred under
a H2 atmosphere, at 30 °C and 500 kPa, until complete conversion
of the starting material was evidenced by TLC and 1H NMR analy-
ses (24 h). The reaction mixture was then filtered in order to elimin-
ate the catalyst, concentrated to ca. 5 mL, filtered again and the
pH (7.6) corrected to 5.4 by careful addition of 1  HCl. Absolute
EtOH (10 mL) was added to the solution and the resulting cloudy
mixture was left standing at 0 °C for 12 h, producing a precipitate.
The gummy residue was separated from the mother liquor, treated
with additional absolute EtOH (5 mL) and triturated until a crys-
talline solid was obtained, which was filtered, washed with Et2O,
collected and dried over P2O5 for 30 min at 60 °C under vacuum.
Brownish solid (82% yield): m.p. (from EtOH) 184Ϫ187 °C (de-
comp.). [α]2D0 ϭ Ϫ45.0 (c ϭ 0.5, 0.1  NaOH). IR (pellet): ν˜ ϭ 3437
w, br., 3073 br. m, 1642 s, 1517 m, 1476 w, 1393 m, 1343 w, 1285
w, 1257 m, 1163 w, 1092 w, 1077 m, 952 s, 920 w, 864 m, 814 w,
664 m, 625 w, 549 w cmϪ1. 1H NMR (2  NaOD in D2O, spectral
reference: 2853.00 Hz): δ ϭ 3.49 (dd, JAX ϭ 5.0, JBX ϭ 8.0 Hz, 1
H, CH2ϪCH), 3.80 (dd, JAB ϭ 13.0, JAX ϭ 5.0 Hz, 1 H,
CHϪCH2), 3.87 (dd, JAB ϭ 13.0, JBX ϭ 8.0 Hz, 1 H, CHϪCH2)
ppm. 13C NMR (2  NaOD in D2O, spectral reference ϭ
3313.00 Hz): δ ϭ 181.7 (CϭO), 65.4 (CH2), 56.2 (CH) ppm. MS:
not determined. C3H7N3O4 (149.11): calcd. C 24.17, H 4.73, N
28.18; found C 24.12, H 4.75, N 28.10.
Hydrogenolysis of (S)-3-[(Phenylmethyloxy)amino]-N-[(phenylmeth-
yloxy)carbonyl]alanine (9):[15] 5% Pd/C (35 mg) was added to a
solution of 9 (350 mg, 1.00 mmol) in AcOH (2.5 mL) and 2.0 
HCl (3.0 mL) and the mixture was vigorously stirred under a H2
atmosphere, at room temperature and pressure, until complete con-
version of the starting material was evidenced by TLC and 1H
NMR analyses (72 h). The reaction mixture was then filtered in
order to eliminate the catalyst, diluted with 2.0  HCl (1.0 mL)
and concentrated to dryness, yielding a solid residue which was
collected and dried for 60 min over KOH pellets at 60 °C under
vacuum. A suitable sample was then dissolved in D2O and analysed
1
by H and 13C NMR spectroscopy, which showed the presence of
a mixture of two substances, in the ratio 65:35 (mol/mol). In fact,
besides the expected deprotected hydroxyamino derivative 5 (65%),
obtained as the corresponding dihydrochloride and identified on
the basis of its NMR properties (vide infra), the over-reduction
product (S)-2,3-diaminopropanoic acid (10, 35%) was also present,
also in its dihydrochloride form, which was identified by compari-
son with a commercial authentic sample.
(S)-2-Amino-3-(hydroxyamino)propanoic Acid (5) Dihydrochlo-
ride:[13c,15] 1H NMR (D2O, spectral reference ϭ 2853.00 Hz): δ ϭ
4.42 (dd, JAX ϭ 6.4, JBX ϭ 5.3 Hz, 1 H, CH2ϪCH), 3.38 (dd,
(S)-3-[(Phenylmethyloxy)amino]-N-[(phenylmethyloxy)carbonyl]-
JAB ϭ 14.6, JBX ϭ 5.3 Hz, 1 H, CHϪCH2), 3.73 (dd, JAB ϭ 14.6, alanine Phenylmethyl Ester (18): Compound 18 was obtained essen-
JAX ϭ 6.4 Hz, 1 H, CHϪCH2) ppm. 13C NMR (D2O, spectral tially according to a described procedure.[30] A solution of phe-
reference ϭ 3313.00 Hz): δ ϭ 171.7 (CϭO), 51.8 (CH2), 51.5
nylmethyl bromide (1.11 g, 6.50 mmol) in benzene (15 mL) was ad-
(CH) ppm.
ded dropwise to a solution consisting of 9 and 1,8-diazabicy-
4714
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Eur. J. Org. Chem. 2004, 4710Ϫ4716

An Improved Stereoselective Synthesis of L-Alanosine
FULL PAPER
clo[5.4.0]undec-7-ene (DBU; 0.83 g, 5.40 mmol) in benzene
(35 mL) and the obtained mixture was stirred for 24 h at room
temperature. After this time, the resulting suspension was diluted
with Et2O (100 mL), freed from formed DBU·HBr by filtration,
and the clear filtrate was washed with H2O (100 mL), 1  HCl
(100 mL), 5% aqueous NaHCO3 (100 mL), and 10% aqueous
Na2SO4 (100 mL). It was then dried over anhydrous Na2SO4, fil-
tered and the solvent evaporated off. The obtained oily residue was
submitted to column chromatography (SiO2, hexane/EtOAc), af-
fording pure 18. Colourless liquid (57% yield). [α]2D0 ϭ not deter-
mined. IR (film): ν˜ ϭ 3427 br. m, 3348 br. m, 3039 w, 2927 m, 1723
s, 1497 m, 1454 w, 1386 m, 1346 w, 1200 s, 1168 w, 1055 s, 1027 w,
914 w, 824 w, 741 m, 700 m, 597 w cmϪ1. 1H NMR: δ ϭ 7.42Ϫ7.16
(m, 15 H, C6H5), 5.79 (d, J ϭ 7.8 Hz, 1 H, OϭCϪNH), 5.74 (br.
s, 1 H, OϪNH), 5.12 (s, 2 H, PhϪCH2), 5.08 (s, 2 H, PhϪCH2),
4.61Ϫ4.46 (m, 3 H, CH2ϪCH ϩ PhϪCH2ϪOϪN), 3.47 (dd, JAB ϭ
Acknowledgments
We are indebted to Prof. G. Verardo for MS data collection and to
Dr. P. Martinuzzi for recording the NMR spectra. Thanks are also
due to the University of Udine (F. U. R. D.) for financial support.
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4.4 Hz, 1 H, CHϪCH2) ppm. 13C NMR: δ ϭ 171.1 (CϭO), 155.9
(NϪCϭO), 137.1, 136.1, 135.3, 128.5 (3 overlapped signals), 128.4,
128.31, 128.28, 128.2, 128.1, 127.9, 76.0, 67.2, 67.0, 52.9, 52.7 ppm.
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435 (Ͻ1) [Mϩ]. C25H26N2O5 (434.49): calcd. C 69.11, H 6.03, N
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(S)-3-[(Phenylmethyloxy)nitrosoamino]-N-[(phenylmethyloxy)-
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m.p. (from hexane/Et2O) 63Ϫ65 °C. [α]2D0 ϭ ϩ15.0 (c ϭ 1.0,
CH3OH). IR (film): ν˜ ϭ 3348 br. m, 3039 w, 2954 w, 1736 s, 1715
s, 1531 s, 1442 m, 1374 w, 1310 w, 1283 m, 1235 w, 1109 w, 1067
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1
m, 1029 w, 960 w, 910 m, 875 w, 741 m, 697 m, 602 w cmϪ1. H
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NMR: δ ϭ 7.38Ϫ7.21 (m, 15 H, C6H5), 5.51 (d, J ϭ 7.4 Hz, 1 H,
NH), 5.08 (s, 2 H, PhϪCH2), 5.08 (d, JAB ϭ 12.1 Hz, 1 H,
PhϪCH2), 5.01 (d, JAB ϭ 12.1 Hz, 1 H, PhϪCH2), 4.90 (s, 2 H,
PhϪCH2), 4.81Ϫ4.74 (m, 3 H, CHϪCH2 ϩ CH2ϪCH) ppm. 13C
NMR: δ ϭ 168.9 (CϭO), 155.5 (NϪCϭO), 135.8, 134.5, 133.5,
129.6, 129.3, 128.63 (2 overlapped signals), 128.61, 128.5 (2 over-
lapped signals), 128.2, 128.0, 77.3, 68.0, 67.3, 54.3, 52.2 ppm. MS
(100 °C): m/z ϭ 91 (100), 108 (78), 107 (70), 106 (64), 105 (64), 463
(Ͻ1) [Mϩ]. C25H25N3O6 (463.49): calcd. C 64.79, H 5.44, N 9.07;
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[15]
[16]
(S)-N-[(Phenylmethyloxy)carbonyl]-3-[(phenylmethyloxy)-NNO-
azoxy]alanine Phenylmethyl Ester (16): Compound 16 was prepared
as previously reported.[7] Brownish solid (48% yield): m.p. (from
hexane/Et2O) 78Ϫ80 °C. [α]2D0 ϭ not determined. IR (film): ν˜ ϭ
3330 br. m, 3035 w, 2934 w, 1741 s, 1689 s, 1521 s, 1456 w, 1413 w,
1392 w, 1348 m, 1290 w, 1221 s, 1053 m, 1021 w, 955 w, 913 w, 782
w, 740 m, 697 m, 588 w cmϪ1. 1H NMR: δ ϭ 7.42Ϫ7.23 (m, 15 H,
C6H5), 5.77 (d, J ϭ 7.5 Hz, 1 H, NH), 5.23Ϫ5.08 [m (2 overlapped
signals of AB systems), 4 H, PhϪCH2], 5.09 (s, 2 H, PhϪCH2),
4.74 (sym m, J ϭ 7.5, JAX ϭ 4.1, JBX ϭ 3.7 Hz,1 H, CH2ϪCH),
4.61 (dd, JAB ϭ 13.3, JBX ϭ 3.7 Hz, 2 H, CHϪCH2), 4.49 (dd,
JAB ϭ 13.3, JAX ϭ 4.1 Hz, 1 H, CHϪCH2) ppm. 13C NMR: δ ϭ
168.3 (CϭO), 155.7 (NϪCϭO), 135.8, 135.1, 134.7, 128.8Ϫ128.0
(complex), 76.0, 68.0, 67.2, 63.1, 51.6 ppm. MS (100 °C): m/z ϭ 91
(100), 159 (21), 107 (17), 92 (16), 187 (15). C25H25N3O6 (463.49):
calcd. C 64.79, H 5.44, N 9.07; found C 64.70, H 5.44, N 9.06.
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Eur. J. Org. Chem. 2004, 4710Ϫ4716
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4715

P. Strazzolini, M. G. Dall’Arche, M. Zossi, A. Pavsler
FULL PAPER
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Received July 20, 2004
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Eur. J. Org. Chem. 2004, 4710Ϫ4716