An operationally simple and efficient synthesis of orthogonally protected L-threo-β-hydroxyasparagine

Article abstract of DOI:10.1055/s-2007-982544

A synthesis of orthogonally protected L-threo-β-hydroxyasparagine from L-aspartic acid is reported. Iodocyclization of 3-benzoylaminoaspartic acid provided an intermediate oxazoline dicarboxylate that was efficiently hydrolyzed to L-threo-β-hydroxyasparti

Full text of DOI:10.1055/s-2007-982544

LETTER
1513
An Operationally Simple and Efficient Synthesis of Orthogonally Protected
L-threo-b-Hydroxyasparagine
Synthesis of
O
rtho
i
gonally
k
Protecte
d
L
o
-
threo
-
b
-Hyd
m
r
oxyasparagine ari Guzmán-Martínez, Michael S. VanNieuwenhze*
Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Fax +1(858)8220386; E-mail: msv@ucsd.edu
Received 22 March 2007
cient synthetic route to the nonproteinogenic amino acid
Abstract: A synthesis of orthogonally protected L-threo-b-hy-
L-threo-b-OH-Asn (Figure 1). The isomers of b-OH-Asn
droxyasparagine from L-aspartic acid is reported. Iodocyclization of
3-benzoylaminoaspartic acid provided an intermediate oxazoline
dicarboxylate that was efficiently hydrolyzed to L-threo-b-hy-
were originally isolated from human urine or synthesized
as a racemate that was separated via resolution.7,8 Several
droxyaspartic acid. The synthetic route for conversion of the free b- synthetic procedures have been published for the enantio-
hydroxy-a-amino acid into the target compound is highly efficient
selective synthesis of the isomers of b-hydroxyaspartic
acid9–14 and erythro-b-hydroxyasparagine.15 In contrast,
and amenable to the preparation of various orthogonally protected
asparagine derivatives, on a multigram scale.
there are few descriptions of an asymmetric synthesis of
threo-b-hydroxyasparagine.16,17 Boger et al. reported a
Key words: asymmetric synthesis, amino acids, amides, peptides,
protecting groups
procedure that relies upon the Sharpless asymmetric ami-
nohydroxylation reaction of (E)-4-methoxycinnamate,
where the methoxy-substituted aromatic ring serves as
masking group for the side chain carboxyl group.16 Lectka
also reported a procedure involving ring-opening of
appropriately derivatized b-lactams deriving from a cin-
chona alkaloid catalyzed asymmetric [2+2] cycloaddition
reaction of ketenes and imines.17 We sought to develop a
synthetic route to threo-hydroxyasparagine derivatives
that would take advantage of readily available members of
the chiral pool. Herein, we describe an efficient alterna-
tive asymmetric synthesis of orthogonally protected L-
threo-FmocNH-b-OH-Asn(Tr)-OH (2) for use in our
synthesis of lysobactin.
Lysobactin (katanosin B) is a cyclic depsipeptide antibiot-
ic that was isolated by the Shionogi Research Laboratories
from a producing organism related to the genus
Cytophaga.1,2 Lysobactin was also isolated from a species
of Lysobacter by workers at the Squibb Institute.3–5 In a
recent evaluation, lysobactin displayed very good antibac-
terial activity against methicillin-resistant Staphylococcus
aureus (MRSA), as well as vancomycin-resistant enter-
cocci, with minimum inhibitory concentrations (MIC’s)
ranging from 0.39–0.78 mg/mL.6 Given this promising
antibacterial activity, lysobactin has emerged as an attrac-
tive target for chemical synthesis.
Our synthetic route (Scheme 1) relied upon the highly ste-
reoselective conversion of L-aspartic acid to L-threo-b-
hydroxyaspartic acid (3), as reported by Cardillo.9 A two-
step sequence was used to convert 3 into Boc-protected
During the course of our ongoing investigation into the
synthesis of lysobactin (1), and structurally related dep-
sipeptide antibiotics, we required a dependable and effi-
TrNOC
HO
OH
H2NOC
O
OH
O
H
N
H
N
H
N
NHFmoc
O
HN
O
O
N
H
O
H2N
O
O
2
HO
OH
O
R
O
NH
O
HN
H
N
H
HO
N
N
H
O
O
Me
NH
1 lysobactin
R =
N
NH2
H
Figure 1 Structure of lysobactin
SYNLETT 2007, No. 10, pp 1513–1516
1
8
.0
6
.2
0
0
7
Advanced online publication: 07.06.2007
DOI: 10.1055/s-2007-982544; Art ID: S01407ST
© Georg Thieme Verlag Stuttgart · New York

1514
A. Guzmán-Martínez, M. S. VanNieuwenhze
LETTER
Cl–
+NH3
1) HCl
MeOH
O
NHBoc
OH
O
NH3
reflux, 3 h
OH
MeO
HO
MeOH
r.t., 2 d
90%
2) Boc2O
10% Na2CO3
dioxane
OH
O
OH
O
3
4
62% overall
1) 4 N HCl
in dioxane
O
NHBoc
OH
O
NHBoc
OBn
BnBr, NaHCO3
DMF
H2N
H2N
2) Fmoc-OSuc
NaHCO3
dioxane, H2O
87% overall
86%
OH
O
OH
O
5
6
O
NHFmoc
O
NHFmoc
Tr-OH
H2SO4 (cat)
OR
OBn
TrHN
H2N
Ac2O, AcOH
78%
OH
O
OH
O
8, R = Bn
2, R = H
H2, Pd/C
EtOH
90%
7
Scheme 1 Synthesis of orthogonally protected b-hydroxyasparagine
monoester 4. Exposure to acidic methanol at reflux, to ity for the synthesis of these valuable building blocks on a
provide the side chain monoester,18,19 followed by Boc- multigram scale. The protecting group scheme employed
protection of the free amino group provided 4 in 62% for the synthesis of 2 was selected in order to maximize its
overall yield. Direct aminolysis of methyl ester 4 (NH3, utility in our synthetic efforts directed toward lysobactin.
MeOH, r.t., 48 h, 90%) provided carboxamide 5. The free Efforts toward the synthesis of lysobactin, and related
carboxyl group was efficiently protected as its benzyl es- depsipeptide antibiotics, are in progress and will be pre-
ter (BnBr, NaHCO3, DMF) and provided 6 in 86% yield. sented in due course.
At this juncture, the protecting group scheme we en-
visaged for our total synthesis of lysobactin required ex-
change of the amine protective groups. Thus, removal of
Acknowledgment
Financial support provided by the National Institutes of Health (AI
059327), The Hellman Foundation, and the Regents of the Univer-
sity of California, is gratefully acknowledged. MSV also thanks Eli
Lilly and Company for support in the form of an Eli Lilly and Com-
pany New Faculty Award. AGM thanks the University of California
at San Diego for support in the form of GAANN Fellowship.
the Boc group (4 N HCl, dioxane) and reprotection of the
free amino group (Fmoc-OSu, NaHCO3, dioxane–H2O) as
its Fmoc carbamate provided the Fmoc-protected benzyl
ester 7 in 87% overall yield.
Unprotected carboxamide groups of asparagine residues
are susceptible to various side reactions during peptide
coupling, for example, dehydration to nitriles and intra-
molecular cyclization onto activated carboxyl groups to
provide succinimide by-products. In an effort to avoid
these unwanted side reactions during the course of our
synthetic effort toward lysobactin, a trityl protecting
group was introduced onto the side chain carboxamide.
The trityl group was readily installed (TrOH, cat. H2SO4,
Ac2O, AcOH, 78%) using the Boger modification16 of the
protocol initially reported by Sieber and co-workers.20 Fi-
nally, careful hydrogenolysis of benzyl ester 8 (H2, Pd/C,
EtOH, 90%) provided the target compound L-threo-
Fmoc-b-OH-Asn(Tr)-OH.21
References and Notes
(1) Shoji, J.; Hinoo, H.; Matsumoto, K.; Hattori, T.; Yoshida, T.;
Matsuura, S.; Kondo, E. J. Antibiot. 1988, 41, 713.
(2) Kato, T.; Hinoo, H.; Terui, Y.; Kikuchi, J.; Shoji, J. J.
Antibiot. 1988, 41, 719.
(3) Bonner, D. P.; O’Sullivan, J. O.; Tanaka, S. K.; Clark, J. M.;
Whitney, R. R. J. Antibiot. 1988, 41, 1745.
(4) O’Sullivan, J.; McCullough, J. E.; Tymiak, A. A.; Kirsch, D.
R.; Trejo, W. H.; Principe, P. A. J. Antibiot. 1988, 41, 1740.
(5) Tymiak, A. A.; McCormick, T. J.; Unger, S. E. J. Org.
Chem. 1989, 54, 1149.
(6) Maki, H.; Miura, K.; Yamano, Y. Antimicrob. Agents
Chemother. 2001, 45, 1823.
(7) Okai, H.; Izumiya, N. Bull. Chem. Soc. Jpn. 1969, 42, 3550.
(8) Singerman, A.; Liwschitz, Y. Tetrahedron Lett. 1968, 4733.
(9) Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Tomasini, C.
Synlett 1999, 1727.
(10) Cho, G. Y.; Ko, S. Y. J. Org. Chem. 1999, 64, 8745.
(11) De Angelis, M.; Campiani, G. Tetrahedron Lett. 2004, 45,
2355.
In summary, we have accomplished an asymmetric syn-
thesis of an orthogonally protected version of L-threo-b-
hydroxyasparagine. The synthetic route utilizes a precur-
sor that is readily available from the chiral pool. The syn-
thetic sequence is sufficiently flexible such that other
protecting group schemes could be utilized. The reaction
sequence is also quite efficient and provides the possibil-
Synlett 2007, No. 10, 1513–1516 © Thieme Stuttgart · New York

LETTER
Synthesis of Orthogonally Protected L-threo-b-Hydroxyasparagine
1515
(12) Deng, J.; Hamada, Y.; Shioiri, T. J. Am. Chem. Soc. 1995,
117, 7824.
(13) Dudding, T.; Hafez, A. M.; Taggi, A. E.; Wagerle, T. R.;
Lectka, T. Org. Lett. 2002, 4, 387.
(14) Hanessian, S.; Vanasse, B. Can. J. Chem. 1993, 71, 1401.
(15) Tohdo, K.; Hamada, Y.; Shioiri, T. Synlett 1994, 247.
(16) Boger, D. L.; Lee, R. J.; Bounaud, P.-Y.; Meier, P. J. Org.
Chem. 2000, 65, 6770.
(17) Hafez, A. M.; Dudding, T.; Wagerle, T. G.; Shah, M. H.;
Taggi, A. E.; Lectka, T. J. Org. Chem. 2003, 68, 5819.
(18) Liwschitz, Y.; Singerman, A. J. Chem. Soc. C 1967, 1696.
(19) Mattingly, P. G.; Miller, M. J.; Cooper, R. D. G.; Daugherty,
B. W. J. Org. Chem. 1983, 48, 3556.
(20) Sieber, P.; Riniker, B. Tetrahedron Lett. 1991, 32, 739.
(21) L-threo-BocNH-b-OH-Asp(OMe)-OH (4): L-threo-b-
Hydroxyaspartic acid (1.00 g, 5.41 mmol) was dissolved in
a solution of concentrated HCl (0.89 mL, 10.8 mmol) and
MeOH (18.0 mL) at 0 °C. The reaction was heated at reflux
for 3 h, then cooled to r.t., and concentrated in vacuo. The
crude L-threo-b-OH-Asp(OMe)-OH was triturated with
Et2O, collected by filtration, and used without further
purification in the following reaction. An aqueous solution
of 10% Na2CO3 (18.0 mL) was added to L-threo-b-OH-
Asp(OMe)-OH and the resulting mixture was cooled in an
iced bath. A solution of Boc2O (3.65 g, 16.2 mmol) in
dioxane (18.0 mL) was added dropwise to the reaction
mixture and the resulting solution was stirred overnight at r.t.
After concentration by rotary evaporation, the residue was
dissolved in EtOAc (150 mL) and washed with 1 N HCl
solution (3 × 100 mL). The organic layer was separated,
dried over Na2SO4, filtered, and concentrated in vacuo. Flash
chromatography (SiO2, 35% EtOAc–hexanes followed by
10–25% MeOH–CHCl3) provided 4 (0.88 g, 62%) as a
foamy white solid; mp 109–110 °C; [a]23D +22 (c = 1.00,
MeOH). 1H NMR (500 MHz, CD3OD): d = 4.79 (s, 1 H),
4.50 (s, 1 H), 3.77 (s, 3 H), 1.46 (s, 9 H). 13C NMR (125
MHz, CD3OD): d = 174.2, 170.2, 155.1, 80.4, 73.0, 59.0,
52.8, 28.7. IR (film): 3388, 2981, 2934, 2858, 1739, 1699,
1608, 1512, 1393, 1367, 1251, 1167, 1107, 1061 cm–1.
HR–EI–MS: m/z [M+] calcd for C10H17O7N: 263.1000;
found: 263.1001.
MeOH–CHCl3), which provided 6 (1.06 g, 86%) as a
crystalline solid; mp 135–136 °C; [a]23D –17 (c = 1.0,
CHCl3). 1H NMR (400 MHz, CD3OD): d = 7.30–7.38 (m, 5
H), 5.19 (s, 2 H), 4.70 (d, J = 2.0 Hz, 1 H), 4.59 (d, J = 2.5
Hz, 1 H), 1.40 (s, 9 H). 13C NMR (125 MHz, CD3OD): d =
176.2, 171.7, 157.8, 136.9, 129.3, 129.2, 129.0, 128.9, 80.8,
72.7, 68.2, 58.1, 28.7. IR (film): 3358, 2979, 1685, 1653,
1559 cm–1. HR–EI–MS: m/z [M+] calcd for C16H22O6N2:
338.1472; found: 338.1471.
L-threo-FmocNH-b-OH-Asn-OBn (7): L-threo-BocNH-b-
OH-Asn-OBn (6; 0.740 g, 2.19 mmol) was dissolved in 4 N
HCl–dioxane (16.4 mL) and stirred at r.t. for 2 h. The
solution was concentrated by rotary evaporation and dried
under high vacuum. The residue obtained was dissolved in
50% dioxane–H2O (32.4 mL, 1:1) and the resulting solution
was cooled to 0 °C. Fmoc-OSuc (1.14 g, 3.28 mmol) and
NaHCO3 (0.368 g, 4.37 mmol) were added to the solution
and the resulting mixture was stirred at r.t. for 12 h. The
reaction solution was diluted with EtOAc (100 mL) and sat.
aq NaHCO3 (150 mL). The aqueous layer was separated and
extracted with EtOAc (2 × 75 mL). The combined organic
extracts were dried over Na2SO4 and concentrated by rotary
evaporation. The crude product was dissolved in the minimal
amount of 20% MeOH–CHCl3 solution. Precipitation with
hexanes and collection of the solid by filtration provided 7 as
a white solid; mp 173–174 °C; [a]23D –18 (c = 0.80, CHCl3).
1H NMR (400 MHz, CD3OD): d = 7.78 (d, J = 7.2 Hz, 2 H),
7.64 (d, J = 6.0 Hz, 2 H), 7.26–7.38 (m, 9 H), 5.21 (d, J = 3.2
Hz, 2 H), 4.64 (d, J = 2.4 Hz, 1 H), 4.35 (dd, J = 3.6, 6.0 Hz,
1 H), 4.22 (m, 2 H). 13C NMR (125 MHz, CD3OD): d =
176.4, 171.7, 158.8, 145.2, 145.1, 142.5, 142.4, 137.0,
129.5, 129.2, 129.1, 128.8, 128.7, 128.2, 128.1, 126.3,
126.2, 120.1, 72.8, 68.4, 68.3, 58.6, 48.2. IR (film): 3328,
1724, 1695, 1655, 1539, 1451, 1299, 1255, 1109 cm–1. HR–
EI–MS: m/z [M + H+] calcd for C26H24O6N2: 460.1629;
found: 460.1638.
L-threo-FmocNH-b-OH-Asn(Tr)-OBn (8): A solution of
L-threo-FmocNH-b-OH-Asn-OBn (7; 0.863 g, 1.87 mmol)
and Tr-OH (5.05 g, 18.7 mmol) in HOAc (6.52 mL) was
heated to 50 °C and treated with concd H2SO4 (65 mL, 1.12
mmol) and Ac2O (0.443 mL, 4.69 mmol). The resulting
mixture was stirred at 50 °C for 2.5 h. After cooling to r.t.,
the reaction solution was diluted with EtOAc (100 mL) and
sat. aq NaHCO3 (150 mL). The organic layer was separated
and the aqueous layer was extracted with EtOAc (2 × 50
mL). The combined organic layers were dried over Na2SO4
and concentrated in vacuo. Purification by flash
chromatography (SiO2, 15% → 30% EtOAc–hexanes)
provided 8 (1.03g, 1.46 mmol, 78%) as a white solid; mp 74–
75 °C; [a]23D –14 (c = 0.93, CHCl3). 1H NMR (400 MHz,
CD3OD): d = 7.79 (d, J = 3.2 Hz, 1 H), 7.77 (d, J = 3.2 Hz,
1 H), 7.67 (d, J = 7.6 Hz, 1 H), 7.62 (d, J = 7.2 Hz, 1 H),
7.17–7.38 (m, 24 H), 5.20 (s, 2 H), 4.81 (d, J = 2.0 Hz, 1 H),
4.65 (d, J = 2..0 Hz, 1 H), 4.51 (dd, J = 4.0, 9.6 Hz, 1 H), 4.17
(m, 2 H), 13C NMR (125 MHz, CHCl3): d = 172.2, 171.8,
158.9, 145.7, 145.3, 145.0, 142.5, 142.4, 137.1, 129.9,
129.7, 129.5, 129.2, 129.0, 128.9, 128.8, 128.7, 128.2,
128.1, 126.5, 126.2, 120.9, 73.4, 71.5, 68.6, 68.3, 58.6, 48.2.
IR (film): 3347, 3061, 3022, 2915, 2522, 1723, 1709, 1670,
1493, 1451, 1333, 1218 cm–1. HR–EI–MS: m/z [M+] calcd
for C45H38O6N2: 702.2724; found: 702.2721.
L-threo-FmocNH-b-OH-Asn(Tr)-OH (2): Pd/C (0.086 g)
was cautiously added to a solution of L-threo-FmocNH-b-
OH-Asn(Tr)-OBn (8; 0.860 g 1.22 mmol) in EtOH (12.2
mL). The solution was placed under an atmosphere of
hydrogen gas for 1 h. The catalyst was removed by filtration
through Celite and the filter cake was washed with EtOH.
L-threo-BocNH-b-OH-Asn (5): A sample of L-threo-
BocNH-b-OH-Asp(OMe)-OH (4; 1.30 g, 4.94 mmol) was
dissolved in MeOH (16.5 mL) and treated with NH3 gas until
the solution became saturated. The resulting mixture was
stirred at r.t. for 3 d. The reaction was concentrated by rotary
evaporation and the crude material was purified by flash
chromatography (SiO2, 25% → 50% MeOH–CHCl3), which
provided 5 (1.10 g, 90%) as a crystalline solid; mp 165–
166 °C; [a]23D –11 (c = 1.0, MeOH). 1H NMR (400 MHz,
CD3OD): d = 4.63 (s, 1 H), 4.48 (s, 1 H), 1.40 (s, 9 H).
13C NMR (125 MHz, CD3OD): d = 177.8, 177.7, 157.6, 80.4,
73.6, 59.4, 28.8. IR (KBr): 3369, 2980, 2933, 1694, 1602,
1510, 1395, 1251, 1168, 1109, 1102, 1059, 1029 cm–1.
HR–EI–MS: m/z [M+] calcd for C9H16O6N2: 249.1003;
found: 249.1002.
L-threo-BocNH-b-OH-Asn-OBn (6): A solution of L-threo-
BocNH-b-OH-Asn (5; 0.910 g, 3.67 mmol) in DMF (18.3
mL) at 0 °C was treated with NaHCO3 (0.616 g, 7.33 mmol)
and benzyl bromide (1.74 mL, 14.3 mmol). The resulting
solution was stirred at 0 °C for 2 h and then for 24 h at r.t.
The solution was cooled to 0 °C and H2O (100 mL) was
slowly added. The resulting mixture was extracted with
EtOAc (3 × 50 mL). The combined organic extracts were
washed with H2O (100 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuo. The crude product
was purified by flash chromatography (SiO2, 0% → 5%
Synlett 2007, No. 10, 1513–1516 © Thieme Stuttgart · New York

1516
A. Guzmán-Martínez, M. S. VanNieuwenhze
LETTER
The combined filtrates were concentrated in vacuo and
purified by flash chromatography (SiO2, 25% → 50%
MeOH–CHCl3), which provided 2 (0.674g, 90%) as a white
solid; mp 189–190 °C (dec.); [a]23D –17 [c = 1.00, MeOH–
CHCl3 (1:1)]. 1H NMR (400 MHz, DMSO-d6): d = 8.29 (s, 1
H), 7.88 (d, J = 7.5 Hz, 2 H), 7.76 (d, J = 6.5 Hz, 1 H), 7.65
(d, J = 7.0 Hz, 1 H), 7.38 (q, J = 5.0, 7.0 Hz, 2 H), 7.08–7.27
(m, 18 H), 4.52 (d, J = 12 Hz, 1 H), 4.35 (m, 3 H), 4.17 (t,
J = 6.5 Hz, 1 H). 13C NMR (DMSO, 125 MHz): d = 173.8,
171.0, 156.1, 144.7, 143.8, 143.7, 140.7, 128.4, 127.7,
127.1, 126.7, 125.3, 120.1, 72.8, 69.1, 65.8, 57.7, 46.7. IR
(KBr): 3387, 3062, 2926, 2857, 1954, 1695, 1601, 1510,
1448, 1404, 1328, 1248, 1106, 1058 cm–1. HR–EI–MS:
m/z [M+] calcd for C38H32O6N2: 612.2255; found: 612.2256.
Synlett 2007, No. 10, 1513–1516 © Thieme Stuttgart · New York

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