A practical and convenient synthesis of the protease inhibitor epibestatin

Article abstract of DOI:10.1055/s-0029-1218771

A convenient synthesis of the protease inhibitor epibestatin, a useful component in protease inhibition cocktails for use in proteomics research, is described. The synthesis sequence consists of seven steps, starting from phenylacetaldehyde, yielding enantiopure epibestatin in 8% overall yield. A regioselective Mitsunobu transformation of a diol is the key step in the sequence. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0029-1218771

PAPER
2039
A Practical and Convenient Synthesis of the Protease Inhibitor Epibestatin
Synthesisof
E
pibe
n
statin ja Richter, Christian Hedberg*
Max-Planck Institute of Molecular Physiology, Department of Chemical Biology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany
Fax +49(231)1332498; E-mail: Christian.Hedberg@mpi-dortmund.mpg.de
Received 14 January 2010; revised 29 March 2010
OH
2
O
OH
O
4
H
H
Abstract: A convenient synthesis of the protease inhibitor
epibestatin, a useful component in protease inhibition cocktails for
use in proteomics research, is described. The synthesis sequence
consists of seven steps, starting from phenylacetaldehyde, yielding
enantiopure epibestatin in 8% overall yield. A regioselective
Mitsunobu transformation of a diol is the key step in the sequence.
3
1
N
2'
N
1'
4'
OH
5''
OH
⋅HCl
NH2
O
NH2
O
3'
⋅HCl
5'
epibestatin (1)
bestatin (2)
Key words: amino acids, asymmetric synthesis, osmium, dihy-
droxylation, Mitsunobu reaction
OBn
OH
OBn
OBn
O
HO
HO
HO
O
O
O
NHBoc
N3
Commercial ‘protease inhibitor cocktails’ are composed
of varying amounts of natural product derived protease in-
hibitors like aprotinin,1a leupeptin,1b bestatin1c (2,
Scheme 1), and pepstatin,1d depending on the applica-
tion.1e These inhibitor mixtures have been optimized to
maintain and preserve protein functionality following cell
lysis by reducing the amount of protease-dependent pro-
teolysis. It has been found beneficial by investigators in
the field to spike the inhibitor cocktail with epibestatin1c
(1, Scheme 1), in order to increase the spectrum of pro-
tease inhibition.1f Epibestatin is the generic name for the
dipeptide, which consists of L-leucine and the unusual a-
hydroxy-b-amino acid (2R,3R)-3-(2-hydroxy-4-phe-
nyl)butanoic acid, a synthetic derivative1g of the natural
product bestatin isolated from Streptomyces olivoreticu-
lithe.1c So far, epibestatin has only been sporadically and
scarcely available from commercial sources.2a Recently,
Park and co-workers reported the synthesis of enantiopure
epibestatin by a chiral pool approach, starting from D-glu-
cono-d-lactone.2b In order to secure the availability of
epibestatin for our in house proteomics work, we have de-
signed and developed a practical and atom-economical
synthesis route to enantiopure epibestatin based on a
Sharpless dihydroxylation.
OH
4
9
6
5
Scheme 1 Epibestatin (1), bestatin (2) (top), and a retrosynthetic
simplification (bottom)
gether with benzyl ester were chosen as orthogonal pro-
tective groups.
The synthetic strategy used is delineated in Scheme 2. We
obtained the isomerically pure E-olefin 4 containing less
than 2% Z-isomer from Horner–Wadworths–Emmons
(HWE) reaction between 2-phenylacetaldehyde (3) and
benzyl 2-(diethoxyphosphoryl)acetate employing MeMgBr
as base.4 Initial attempts with benzyl(triphenylphosphor-
anylidene)acetate and phenylacetaldehyde (3) yielded a
4:1 E/Z-mixture displaying difficult separation character-
istics on preparative scale. Olefin 4 was then subjected to
Sharpless dihydroxylation under standard conditions,5
which gave the diol 5 in 64% isolated yield and 88% ee as
determined by chiral HPLC. Notably, this reaction hardly
went to completion under reaction conditions resulting in
high enantiomeric excess (0 °C, methanesulfonamide). In
addition, the ee (88%) was lower than what we expected
to reach with AD-mix, which could not be further opti-
mized under conditions that gave reasonable conversion.
However, the enantiomeric impurity could be removed as
its diastereomer after coupling to L-leucine at a later stage
in the synthesis. In line with the work of Ko, we anticipat-
ed to substitute the b-hydroxy group of the ester 5 with in-
version of configuration under Mitsunobu conditions
employing the azide anion as nucleophile. In our hands,
when using the originally described conditions (2 equiv of
HN3 in benzene) only starting material was recovered. We
undertook an extensive screening and optimization of the
reaction conditions investigating different azide sources
(diphenylphosphoryl azide, HN3, azidotrimethylsilane,
and 1,1,3,3-tetramethylguanidinium azide).6 Upon vary-
We envisaged a synthetic approach based on the findings
of Ko, who reported in 2002 that the b-hydroxy function
of a,b-dihydroxy esters can be regioselective substituted
at the b-carbon with inversion employing various nucleo-
philes under Mitsunobu conditions.3 A retrosynthetic sim-
plification of epibestatin based on the above mentioned
finding led to the Boc-protected phenylaminobutyric acid
benzyl ester (8), which originates from the Mitsunobu
substitution reaction of diol 5 via azide 6 (Scheme 1, low).
Acid-labile functionalities (Boc and tert-butyl ester) to-
SYNTHESIS 2010, No. 12, pp 2039–2042
x
x.
x
x
.
2
0
1
0
Advanced online publication: 05.05.2010
DOI: 10.1055/s-0029-1218771; Art ID: T01210SS
© Georg Thieme Verlag Stuttgart · New York

2040
A. Richter, C. Hedberg
PAPER
OH
ing reaction temperature, stoichiometry and addition or-
der, we finally settled on protocol including
O
OBn
OBn
a
I)
II)
O
preformation of the DEAD:Ph3P adduct, followed by the
addition of the substrate and subsequent addition of a rel-
atively large excess of HN3 in benzene. In the end, this
modified protocol yielded the desired b-substituted prod-
uct 6 exclusively in 40% yield (67% b.r.s.m.)7 together
with the elimination product 7 in 20% yield. Residual
40% were unconverted starting material, which could be
easily recovered on a preparative scale. Further attempts
did not significantly improve the outcome. In comparison
to Ko’s work,3 our substrate is not benzylic (more readily
substituted) and more sterically impaired, which makes it
considerably less reactive in comparison to the published
examples. After purification, 6 was reduced under
Staudinger conditions employing triphenylphosphine in
THF–water and directly transferred to the subsequent
Boc-protection step. Treatment of the residue from the
previous step with di-tert-butyl dicarbonate in 1,4-diox-
ane–i-PrOH gave the carbamate 8 in good yield (84%) af-
ter purification. Removal of the benzyl ester by
hydrogenolysis in methanol over Pearlman’s catalyst un-
der 5 bar of hydrogen provided the free acid 9 in quantita-
tive yield.8 With this compound in hand, we were able to
build up the desired epibestatin (1) by direct coupling with
L-leucine tert-butyl ester using (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate9
(PyBOP) as a mild and effective coupling reagent, result-
ing in the protected epibestatin 10 in 87% yield after puri-
fication. When employing PyBOP as a coupling reagent,
the unprotected hydroxyl function in 9 did not cause any
problems. After column chromatography, the diastereo-
meric excess of compound 10 was found to be above 98%
OH
O
60%
64%
3
4
5
OH
O
OBn
OBn
IV)
84%
III)
+
N3
O
O
40%
(67% b.r.s.m.)
6
7
O
OH
OH
O
OBn
V)
quant.
VI)
OH
NHBoc
NHBoc
87%
8
9
O
H
N
OH
O
O
H
N
Ot-Bu
VII)
66%
OH
OH
NH2
⋅HCl
O
NHBoc
10
1
Scheme 2 Synthesis of epibestatin (1). Reagents and conditions: I)
benzyl 2-(diethoxyphosphoryl)acetate (0.9 equiv), THF, MeMgBr
(0.9 equiv), r.t., then reflux, 2.5 h; II) AD-mixa, t-BuOH–H2O,
MeSO2NH2 (1.0 equiv), 0 °C, 14 h; III) Ph3P (1.2 equiv), THF,
DEAD (1.3 equiv), HN3 (10 equiv) in benzene, 0 °C to r.t., overnight;
IV) 1. Ph3P (1.7 equiv), THF–H2O, r.t., 13 h, 2. 1,4-dioxane–i-PrOH,
Boc2O, r.t., 16 h; V) Pearlman’s catalyst (20% Pd) (0.70 equiv), Me-
OH, 5 bar H2, r.t. 16 h; VI) L-leucine tert-butyl ester hydrochloride
(1.30 equiv), CH2Cl2, PyBOP (1.2 equiv), DIEA (2.4 equiv), 0 °C to
r.t., 16 h; VII) dioxane–H2O (10:1), HCl (gas), 0 °C to r.t., overnight.
DEAD: diethyl azodicarboxylate; Boc2O: di-tert-butyl dicarbonate;
PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluo-
rophosphate; DIEA: N,N-diisopropylethylamine.
400 MHz, a Bruker AMX 400 MHz, or Bruker DRX 500 MHz ma-
chine and are reported in ppm (d). Assignment of the 1H NMR sig-
nals in the final product 1 was conducted according to the given
numbering shown in Scheme 1. Melting points were measured us-
ing a Büchi 530 machine and are uncorrected. Optical rotations
were acquired using a Schmidt-Hänsch Polartronic HH8 instru-
ment. Sample concentration c is given in g/100 mL. HRMS spectra
were obtained either on an Accela/LTQ Orbitrap (ESI) or a TRACE
GC Ultra/DSF (EI) machine (Thermo Scientific). RP-HPLC was
carried out on a Hewlett Packard Series 1100 using a Machery-
Nagel column (CC 125/4 Nucleodur C18 Gravity, 3 mm).
1
dr by H NMR spectroscopy. Global deprotection was
performed by HCl (gas) saturation of a solution of 10 in
dioxane–water (10:1). This solvent mixture was used to
ensure the solubility of the Boc-deprotected tert-butyl
ester intermediate, which is not soluble enough in HCl-
saturated anhydrous dioxane. Lyophilization, followed by
trituration with diethyl ether yielded epibestatin hydro-
chloride (1) as an analytically pure white solid in 66%
yield. Co-injection on RP-HPLC with an authentic sample
(Sigma) of epibestatin gave a single peak under both iso-
cratic and gradient elution.
Benzyl (E)-4-Phenylbut-2-enoate (4)
To a stirred solution of benzyl 2-(diethoxyphosphoryl)acetate (10.0
g, 34.9 mmol, 1.0 equiv) in THF (452 mL) under argon was added
dropwise MeMgBr (1.0 M in THF, 34.9 mL, 34.9 mmol, 1.0 equiv).
The mixture was stirred at r.t. for 15 min, followed by the dropwise
addition of 2-phenylacetaldehyde (3; 4.73 mL, 38.3 mmol, 1.1
equiv). Subsequently, the reaction mixture was heated to reflux for
2.5 h. After cooling to r.t., the reaction was quenched by the addi-
tion of sat. aq NH4Cl (200 mL). The mixture was subsequently ex-
tracted with Et2O (3 × 50 mL). The combined organic layers were
washed with brine (100 mL), dried (MgSO4), and concentrated in
vacuo. Purification by column chromatography on silica gel [pen-
tane–CH2Cl2 (1:1) to pentane–EtOAc (9:1)] provided the desired E-
isomer 4 as a slightly yellow wax; yield: 5.27 g (60%); Rf = 0.80
(cyclohexane–EtOAc, 9:1).
In summary, we have described here a stereoselective
synthesis of epibestatin hydrochloride (1) involving 7 re-
action steps with an (unoptimized) overall yield of 8%,
delivering the desired enantiopure compound in >98%
diastereomeric excess as its commonly used hydrochlo-
ride salt.
Reagents, solvents and the starting materials were purchased from
Aldrich, ABCR, or Acros. All reactions were carried out using an-
hydrous solvents and under inert gas atmosphere unless otherwise
specified. THF, MeOH, and benzene were purchased as anhydrous
solvents from Aldrich in sure-seal bottles and used without any fur-
ther treatment. CH2Cl2 was freshly distilled over CaH2. Silica gel
used for column chromatography (35–70 mm) was purchased from
Acros. 1H and 13C NMR spectra were acquired on a Varian Mercury
1H NMR (400 MHz, CDCl3): d = 3.43 (dd, J = 1.5, 6.8 Hz, 2 H, H-
4), 5.07 (s, 2 H, OCH2Ph), 5.76 (dt, J = 1.6, 15.6 Hz, 1 H, H-2),
7.35–7.00 (m, 11 H, H-3, Harom).
Synthesis 2010, No. 12, 2039–2042 © Thieme Stuttgart · New York

PAPER
Synthesis of Epibestatin
2041
13C NMR (101 MHz, CDCl3): d = 38.7, 66.3, 122.2, 126.8, 128.3,
128.4 (2), 128.7 (2), 128.9 (2), 129.0 (2), 136.2, 137.7, 148.1, 166.4.
Benzyl (2R,3R)-3-(tert-Butoxycarbonylamino)-2-hydroxy-4-
phenylbutanoate (8)
To a solution of 6 (0.402 g, 1.29 mmol, 1.0 equiv) in THF (1.0 mL)
was added Ph3P (0.587 g, 2.19 mmol, 1.7 equiv), followed by H2O
(0.2 mL). The mixture was stirred at r.t. under argon for 13 h and
subsequently evaporated. The residue was dissolved in 1,4-dioxane
(3.5 mL)–i-PrOH (1.0 mL) and di-tert-butyl dicarbonate (0.310 g,
1.42 mmol, 1.1 equiv) was added. The reaction mixture was stirred
at r.t. until no further change by TLC could be detected (16 h). The
solvent was evaporated and the crude product was purified by col-
umn chromatography on silica gel (cyclohexane–EtOAc, 7:3) to
give the desired carbamate 8 as a colorless solid; yield: 0.418 g
(84%); mp 125–126 °C; [a]D20 –9.5 (c 0.63, CHCl3) at 88% ee;
Rf = 0.41 (cyclohexane–EtOAc, 7:3).
1H NMR (500 MHz, CDCl3): d = 1.35 (s, 9 H, t-C4H9), 2.66 (m, 1
H, H-4), 2.73 (m, 1 H, H-4), 3.30 (br s, 1 H, OH), 4.29 (d, J = 6.0
Hz, 1 H, H-3), 4.37 (br s, 1 H, H-2), 4.80 (d, J = 8.0 Hz, 1 H, NH),
5.00 (d, J = 12.0 Hz, 1 H, OCH2Ph), 5.08 (d, J = 12.0 Hz, 1 H,
OCH2Ph), 7.10–7.09 (m, 2 Harom), 7.27–7.15 (m, 3 Harom), 7.38–7.34
(m, 5 Harom).
HRMS (EI): m/z [M]+ calcd for C17H16O2: 252.1145; found:
252.1141.
Benzyl (2R,3S)-2,3-Dihydroxy-4-phenylobutanoate (5)
AD-mix-a (29.2 g) was dissolved in t-BuOH–H2O (1:1, 200 mL) re-
sulting in a clear two-phase system. MeSO2NH2 (1.98 g, 20.9 mmol,
1.0 equiv) was added to this suspension. The mixture was cooled to
0 °C and a solution of the olefin 4 (5.26 g, 20.9 mmol, 1.0 equiv) in
t-BuOH–H2O (1:1, 10 mL) was slowly added and the reaction mix-
ture was stirred at 0 °C. After 36 h, no starting material was detect-
able (GC-MS). The reaction was quenched by adding Na2S2O5 (14.3
g) at 0 °C. After stirring for an additional 30 min, the mixture was
evaporated to dryness and the residue was partitioned between H2O
(100 mL) and EtOAc (50 mL). The aqueous layer was extracted
with EtOAc (2 × 50 mL). The combined organic layers were
washed with brine (80 mL), dried (MgSO4), and evaporated. Purifi-
cation by column chromatography (silica gel, cyclohexane–EtOAc,
7:3) provided the desired product 5 as a white low-melting wax;
yield: 3.82 g (64%); [a]D20 –21.4 at 88% ee (c 0.56, CHCl3);
Rf = 0.17 (cyclohexane–EtOAc, 7:3). Enantiomeric excess was de-
termined to be 88% by chiral HPLC; Chiralpak IC column (0.46
cm × 25 cm) [CH2Cl2–EtOH (100:2)–n-hexane (60:40), 0.5 mL/
min]: tR (minor) = 15.0 min, tR (major) = 19.6 min.
1H NMR (400 MHz, CDCl3): d = 1.99 (br s, 1 H, OH), 2.93 (s, 1 H,
H-4), 2.95 (s, 1 H, H-4), 3.09 (br s, 1 H, OH), 4.17 (m, 1 H, H-2),
4.12 (m, 1 H, H-3), 5.23 (s, 2 H, OCH2Ph), 7.28–7.21 (m, 3 Harom),
7.37–7.30 (m, 7 Harom).
13C NMR (101 MHz, CDCl3): d = 40.3, 67.9, 72.2, 73.6, 126.9 (2),
128.5 (2), 128.6, 128.8 (3), 129.6 (2), 135.1, 137.5, 173.5.
HRMS (ESI): m/z [M + H]+ calcd for C17H19O4: 287.12779; found:
287.12798; m/z [M + NH4]+ calcd for C17H22NO4: 304.1543; found:
304.1546; m/z [M + Na]+ calcd for C17H18O4 + Na: 309.10973;
found: 309.10985.
13C NMR (101 MHz, CDCl3): d = 28.4 (3), 35.9, 54.6, 67.8, 72.8,
79.9, 126.7 (2), 128.5 (2), 128.9 (2), 128.9 (2), 129.6 (2), 135.0,
137.3, 155.6, 172.7.
HRMS (ESI): m/z [M + H]+ calcd for C22H28NO5: 386.1962; found:
386.1964; m/z [M + Na]+calcd for C22H27NO5 + Na: 408.1781;
found: 408.1782.
(2R,3R)-3-(tert-Butoxycarbonylamino)-2-hydroxy-4-phenyl-
butanoic Acid (9)
To a solution of the protected a-hydroxy-b-amino acid 8 (1.15 g,
2.97 mmol, 1.0 equiv) in MeOH (155 mL) was added Pearlman’s
catalyst (1.11 g, 20% Pd, 2.08 mmol, 0.7 equiv) and the suspension
was stirred under H2 (5 bar) at r.t. until the starting material was
consumed (16 h). The catalyst was filtered off over a pad of Celite,
which was subsequently washed with MeOH (50 mL). The filtrate
was evaporated to dryness to give the carboxylic acid 9 as a color-
less solid; yield: 0.877 g (quant); mp 136–138 °C for material with
88% ee [Lit.8 mp 147–148 °C for enantiopure 2S,3S-isomer];
[a]D20 –1.6 (c 0.56, MeOH) {Lit.8 [a]D20 –2.1 (c 1.0, MeOH) for
enantiopure 2R,3R-isomer}; Rf = 0.54 (CH2Cl2–MeOH–AcOH,
50:10:1).
Anal. Calcd for C17H18O4: C, 71.3; H, 6.3. Found: C, 71.3, H, 6.8.
Benzyl (2R,3R)-3-Azido-2-hydroxy-4-phenylbutanoate (6)
In a flame-dried Schlenk tube equipped with a large magnetic stir-
ring bar, Ph3P (0.789 g, 3.01 mmol, 1.2 equiv) was dissolved in an-
hyd THF (5 mL) under argon. Diethyl azodicarboxylate (0.516 mL,
3.26 mmol, 1.3 equiv) was added to this solution at 0 °C. The diol 5
(0.718 g, 2.51 mmol, 1.0 equiv) dissolved in THF (1 mL) was added
to the mixture followed by HN3 in benzene (10 mL, approx. 2.50 M,
25.1 mmol, 10 equiv). The ice bath was removed and the mixture
was stirred overnight at r.t. The reaction was quenched by the addi-
tion of sat. aq NaHCO3 (20 mL) and extracted with EtOAc (3 × 20
mL). The combined organic layers were washed with brine (50
mL), dried (MgSO4), and evaporated. Purification by column chro-
matography (silica gel, cyclohexane–EtOAc, 9:1 to 1:1) provided
azide 6 as a slightly yellow wax, followed by starting material 5
(40%), and elimination product 7 (20%); yield: 0.316 g (40%);
[a]D20 +36.6 at 88% ee (c 0.63, CHCl3); Rf = 0.56 (cyclohexane–
EtOAc, 7:3).
1H NMR (500 MHz, CD3OD): d = 1.32 (s, 9 H, t-C4H9), 2.80 (dd,
J = 3.7, 14.2 Hz, 1 H, H-4), 2.72 (m, 1 H, H-4), 4.15 (m, 1 H, H-3),
4.21 (d, J = 3.9 Hz, 1 H, H-2), 7.16 (m, 1 Harom); 7.27–7.20 (m, 4
Harom).
13C NMR (126 MHz, CD3OD): d = 28.7 (3), 36.4, 56.3, 74.3, 80.1,
127.2, 129.2 (2), 130.4 (2), 139.9, 157.7, 176.0.
HRMS (ESI): m/z [M + H]+ calcd for C15H22NO5: 296.1493; found:
296.1494; m/z [M + Na]+ calcd for C15H21O5N + Na: 318.1312;
found: 318.1314.
(2R,3R)-[2-Hydroxy-3-(tert-butoxycarbonylamino)-4-phe-
nyl]butanoylleucine tert-Butyl Ester (10)
To a solution of the a-hydroxy-b-amino acid 9 (0.859 g, 2.91 mmol,
1.0 equiv) in CH2Cl2 (70 mL) at 0 °C under argon was added L-leu-
cine tert-butyl ester hydrochloride (0.781 g, 3.49 mmol, 1.3 equiv),
followed by PyBOP (1.82 g, 3.49 mmol, 1.2 equiv). N,N-Diisopro-
pylethylamine (1.20 mL, 6.98 mmol, 2.4 equiv) was added drop-
wise to the well-stirred reaction mixture. The mixture was stirred
for 16 h at r.t. until completeness, diluted with H2O (30 mL), and ex-
tracted with CH2Cl2 (3 × 30 mL). The combined organic layers
were washed with brine (100 mL), dried (MgSO4), and evaporated.
Purification by column chromatography on silica gel (pentane–
EtOAc, 7:3) provided the desired product 10 as a single diastereo-
1H NMR (400 MHz, CDCl3): d = 2.82 (dd, J = 5.8, 14.1 Hz, 1 H, H-
4), 2.93 (dd, J = 9.0, 14.1 Hz, 1 H, H-4), 3.16 (d, J = 5.4 Hz, 1 H,
OH), 3.84 (ddd, J = 3.0, 5.8, 9.0 Hz, 1 H, H-3), 4.38 (dd, J = 3.0,
5.2 Hz, 1 H, H-2), 5.16 (d, J = 12.0 Hz, 1 H, OCH2Ph), 5.21 (d,
J = 12.0 Hz, 1H, OCH2Ph), 7.14–7.11 (m, 2 Harom), 7.28–7.21 (m, 3
Harom), 7.42–7.35 (m, 5 Harom).
13C NMR (101 MHz, CDCl3): d = 35.7, 66.0, 68.3, 73.0, 127.1,
128.8 (2), 128.9 (2), 128.9 (2), 129.1, 129.4 (2), 134.7, 136.9, 171.9.
HRMS (ESI): m/z [M + Na]+calcd for C17H17N3O3 + Na: 334.1162;
found: 334.1164.
Synthesis 2010, No. 12, 2039–2042 © Thieme Stuttgart · New York

2042
A. Richter, C. Hedberg
PAPER
mer; yield: 1.18 g (87%); mp 170–172 °C; [a]D20 +40.7 (c 0.63,
CHCl3); Rf = 0.38 (cyclohexane–EtOAc, 7:3).
Acknowledgment
A.R. is grateful for financial support from Fonds der Chemischen
Industrie through a Kekule fellowship; C.H. thanks AstraZeneca
AB (Sweden) for financial support. A.R. and C.H. acknowledge
Prof. Herbert Waldmann, Max-Planck Institute for Molecular
Physiology, for his unconditional support.
1H NMR (500 MHz, CDCl3): d = 1.02 (d, J = 3.6 Hz, 3 H, H-5¢),
1.03 (d, J = 3.6 Hz, 3 H, H-5¢¢), 1.47 (s, 9 H, t-C4H9), 1.53 (s, 9 H,
t-C4H9), 1.62 (m, 1 H, H-4¢), 1.79–1.69 (m, 2 H, H-3¢), 3.11 (m, 1
H, H-4), 3.01 (m, 1 H, H-4), 4.07 (br s, 1 H, H-3), 4.38 (br s, 1 H,
H-2), 4.57 (m, 1 H, H-2¢), 5.08 (d, J = 7.1 Hz, 1 H, NH), 5.42 (br s,
1 H, OH), 7.29–7.21 (m, 3 Harom), 7.36–7.30 (m, 3 H, 2 Harom, NH).
13C NMR (126 MHz, CDCl3): d = 22.1, 23.0, 25.1, 28.2 (3), 28.4
(3), 35.3, 41.6, 51.3, 57.6, 75.5, 80.8, 81.9, 126.6, 128.6 (2), 129.5
(2), 138.4, 158.2, 171.5, 171.8.
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Rich, D. H.; Moon, B. J.; Harbeson, S. J. Med. Chem. 1984,
27, 417. (g) For the original description of epibestatin, see:
Nishizawa, R.; Saino, T. J. Med. Chem. 1977, 20, 510.
(2) (a) Epibestatin is available in very limited quantities from
Sigma, ordering number E0381. (b) Lee, J. H.; Kim, J. H.;
Lee, B. W.; Seo, W. D.; Yang, M. S.; Park, K. H. Bull.
Korean Chem. Soc. 2006, 27, 1211.
HRMS (ESI): m/z [M + H]+ calcd for C25H41N2O6: 465.2959; found:
465.2955; m/z [M + Na]+ calcd for C25H40N2O6 + Na: 487.2779;
found: 487.2772.
Epibestatin Hydrochloride (1) [(2R,3R)-(2-Hydroxy-3-amino-4-
phenyl)butanoylleucine Hydrochloride]
The protected dipeptide 10 (1.17 g, 2.51 mmol) was dissolved in di-
oxane–H2O (10:1, 55 mL) at r.t. HCl gas was bubbled through this
solution under ice bath cooling until saturation was reached. The ice
bath was removed and the solution was stirred for 18 h at r.t. The
reaction mixture was evaporated to dryness at r.t. and dried for 3 h
under high vacuum. The slightly yellow solid was triturated with
Et2O, the resulting white solid was collected by filtration and
washed carefully with Et2O. The solid was dried under high vacuum
to constant weight, thus yielding the desired epibestatin hydrochlo-
ride (1) as a white solid; yield: 0.569 g (66%); 98% dr by RP-HPLC;
CC 125/4 Nucleodur C18-Gravity, 3 mm, MeOH–H2O (25:75) for
50 min, then to 100:0 over 20 min, at 0.5 mL/min): tR
(epibestatin) = 20.9 min, tR (diastereomer) = 68.0 min.
HCl salt: mp 228–230 °C; [a]D20 +5.9 (c 0.38, H2O); mp of an
authentic sample of HCl salt (Sigma) 227–229 °C.
Free amino acid: [a]D20 +28.8 (c 0.83, AcOH) {Lit.2b [a]D20 +30.3 (c
0.85, AcOH) for free amino acid}.
1H NMR (400 MHz, D2O): d = 0.94 (d, J = 6.0 Hz, 3 H, H-5¢), 0.97
(d, J = 6.0 Hz, 3 H, H-5¢¢), 1.76–1.67 (m, 3 H, H-4¢, H-3¢), 2.96 (dd,
J = 10.1, 14.6 Hz, 1 H, H-4), 3.09 (dd, J = 5.2, 14.8 Hz, 1 H, H-4),
4.01 (m, 1 H, H-3), 4.34 (m, 1 H, H-2¢), 4.57 (m, 1 H, H-2), 7.34–
7.33 (m, 2 Harom), 7.46–7.39 (m, 3 Harom).
13C NMR (126 MHz, D2O): d = 21.1, 22.6, 24.9, 33.0, 39.8, 51.9,
55.5, 70.6, 128.1, 129.5 (2), 129.8 (2), 135.5, 172.8, 176.6.
(3) Ko, S. J. Org. Chem. 2002, 67, 2689.
(4) (a) Wadsworth, W.; Emmons, W. J. Am. Chem. Soc. 1961,
83, 1733. (b) Claridge, T. D. W.; Davies, S. G.; Lee, J. A.;
Nicholson, R. L.; Roberts, P. M.; Russell, A. J.; Smith, A.
D.; Toms, S. M. Org. Lett. 2008, 10, 5437.
(5) (a) Jacobsen, E.; Marko, I.; Mungall, W.; Schroder, G.;
Sharpless, K. J. Am. Chem. Soc. 1988, 110, 1968.
(b) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino,
G. A.; Hartung, J.; Jeong, K. S.; Kwong, H. L.; Morikawa,
K.; Wang, Z. M.; Xu, D. Q.; Zhang, X. L. J. Org. Chem.
1992, 57, 2768.
(6) Swamy, K. C. K.; Kumar, N. N. B.; Balaraman, E.; Kumar,
K. V. P. P. Chem. Rev. 2009, 109, 2551.
(7) The structural verification of 5 was based on the
characteristic 2D NMR cross peaks (COSY, HMBC) after
conversion of the azide functionality into the corresponding
N-acetamide derivative.
HRMS (ESI): m/z [M + H]+ calcd for C16H25N2O4: 309.1809; found:
309.1811.
(8) Compound 9 and its enantiomer have been reported
previously in literature. For a recent synthesis, see: Masaya,
K.; Matsumoto, J.; Yukifumi, N. Tetrahedron: Asymmetry
2002, 13, 1201.
Anal. Calcd for C16H25ClN2O4: C, 55.7; H, 7.3; N, 8.1. Found: C,
56.2; H, 7.3; N, 7.1.
(9) Montalbetti, C.; Falque, V. Tetrahedron 2005, 61, 10827.
Synthesis 2010, No. 12, 2039–2042 © Thieme Stuttgart · New York