Synthesis of nonproteinogenic amino acids to probe lantibiotic biosynthesis

Article abstract of DOI:10.1021/jo051182o

The synthesis of six nonproteinogenic amino acids appropriately protected for Fmoc-based solid-phase peptide synthesis is described. These amino acids are (2S,3A)-vinylthreonine, (2S)-(E)-2-amino-5-fluoro-pent-3-enoic acid (fluoroallylglycine), (S)-β

Full text of DOI:10.1021/jo051182o

Synthesis of Nonproteinogenic Amino Acids To Probe Lantibiotic
Biosynthesis
Xingang Zhang, Weijuan Ni, and Wilfred A. van der Donk*
Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana-Champaign,
600 South Mathews Avenue, Urbana, Illinois 61801
vddonk@scs.uiuc.edu
Received June 11, 2005
The synthesis of six nonproteinogenic amino acids appropriately protected for Fmoc-based solid-
phase peptide synthesis is described. These amino acids are (2S,3R)-vinylthreonine, (2S)-(E)-2-
amino-5-fluoro-pent-3-enoic acid (fluoroallylglycine), (S)-â2-homoserine, (S) and (R)-â3-homo-
cysteine, and (2R,3R)-methylcysteine. Once incorporated into peptides, these compounds were
envisioned to serve as alternative substrates for the lantibiotic synthases that dehydrate serine
and threonine residues in their peptide substrates and catalyze the subsequent intramolecular
Michael-type addition of cysteines to the dehydroamino acids.
SCHEME 1
Introduction
Nonproteinogenic amino acids with highly functional-
ized side chains are frequently found as constituents of
biologically important peptides. As a result, there has
been significant interest in the synthesis, biosynthesis,
and biological activities of these compounds.1 In addition,
â-amino acids have gained recent attention both for their
natural occurrence and as building blocks for oligomers
with well-defined folding behavior.2 The so-called lanti-
biotics are a group of ribosomally synthesized and post-
translationally modified peptide antibiotics.3 These modi-
(1) For examples, see: (a) Walsh, C. T. Antibiotics: Actions, Origins,
Resistance; ASM Press: Washington, DC, 2003. (b) Williams, R. M.
Synthesis of Optically Active Alpha-Amino Acids; Pergamon: Oxford,
1989. (c) Amino Acids, Peptides, and Proteins; Young, G. T., Ed.; The
Chemical Society: London, 1968-1971. (d) Amino Acids, Peptides, and
Proteins; Sheppard, R. C., Ed.; The Chemical Society: London, 1972-
1981.
fications involve dehydrations of Ser and Thr residues
followed by intramolecular Michael-type additions of Cys
residues to the dehydroamino acids in a regio- and
stereoselective fashion (Scheme 1). The enzymatic system
that carries out these modifications during the biosyn-
thesis of lacticin 481 has been recently reconstituted in
vitro.4 The enzyme was shown to display a high degree
of substrate promiscuity, opening the possibility of
introducing nonproteinogenic amino acids into its peptide
(2) For reviews, see: (a) Seebach, D.; Matthews, J. L. Chem.
Commun. 1997, 2015. (b) Gellman, S. H. Acc. Chem. Res. 1998, 31,
173. (c) Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr. Med.
Chem. 1999, 6, 905. (d) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F.
Chem. Rev. 2001, 101, 3219. (e) Hill, D. J.; Mio, M. J.; Prince, R. B.;
Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101, 3893. (f) Seebach,
D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodiver. 2004, 1, 1111. (g)
Enantioselective Synthesis of â-Amino Acids; Juaristi, E., Ed.; John
Wiley & Sons: New York, 1997.
(3) Chatterjee, C.; Paul, M.; van Der Donk, W. A. Chem. Rev. 2005,
105, 633.
10.1021/jo051182o CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/28/2005
J. Org. Chem. 2005, 70, 6685-6692
6685

Zhang et al.
CHART 1
SCHEME 3
SCHEME 2
substrate. Since the ribosomal origin of the lantibiotics
limits in vivo engineering of their structures to the 20-
22 proteinogenic amino acids, a synthetic approach would
greatly expand the functional and structural diversity
that can be incorporated into lantibiotics via the bio-
synthetic enzymes. We report here the preparation of a
series of nonproteinogenic amino acids designed to in-
vestigate this premise. These compounds are the vinyl-
threonine derivative 1, fluoroallylglycine 2, the â-amino
acids 3-5, and cysteine derivative 6 (Chart 1). All
compounds were appropriately protected for use in Fmoc-
based solid-phase peptide synthesis (SPPS).
Once incorporated into the peptide substrate for the
dehydratase, amino acids 1-3 were envisioned as pos-
sible Ser or Thr analogues that would serve to further
investigate the substrate specificity of the dehydration
reaction (Scheme 2). Similarly, the â3-homocysteines 4
and 5 and 3-methylcysteine 6 were anticipated as po-
tential alternative nucleophiles for the cyclase catalyzed
Michael-type addition. In the case of 6 this would provide
(2R,3R,6S)-3-methyllanthionine instead of (2S,3S,6R)-3-
methyllanthionine, which has been found in all lanti-
biotics characterized to date.3
erate yields and/or stereoselectivity and utilized protect-
ing groups that are not directly amenable for SPPS.5
Hence, we explored alternative routes toward the syn-
thesis of 1. Protected amino acids 1 and 2 were both
accessed from the D-Garner aldehyde6 7. Vinyllithium
was combined with anhydrous zinc bromide in diethyl
ether followed by addition of aldehyde 7 to produce
alcohol 8 as a solid with 5:1 syn-diastereoselectivity in
90% yield (Scheme 3).7c Recrystallization of the crude
reaction mixture produced syn-8 in 24:1 dr. The recrys-
tallized allylic alcohol was converted to the corresponding
tert-butyl ether 9 in 78% yield by treatment with tert-
butyl trichloroacetimidate and a catalytic amount of
boron trifluoride etherate in cyclohexane at room tem-
perature. Initial attempts to deprotect the isopropylidene
and Boc groups of compound 9 with 10% HCl, followed
by protection of the resulting free amino group with
FmocCl, afforded compound 13 in low yield (32%).
Byproduct 10 resulting from cleavage of the tert-butyl
ether was also formed in the deprotection step. A survey
of a series of reaction conditions showed that treatment
with 1 N HCl-dioxane at room temperature for 24 h
maximized the desired product, although significant
amounts of 10 were still formed. The crude mixture of
Results and Discussion
Synthesis of Vinylthreonine 1, (E)-2-Amino-5-
fluoro-pent-3-enoic Acid 2, and â2-Homoserine 3. A
number of elegant approaches have been described for
the asymmetric synthesis of various â-hydroxy R-amino
acids, but the previous methods for the asymmetric
synthesis of derivatives of vinylthreonine produced mod-
(5) For the asymmetric synthesis of vinyl-threonine derivatives,
see: (a) Bold, G.; Duthaler, R. O.; Riediker, M. Angew. Chem., Int.
Ed. Engl. 1989, 28, 497. (b) Ohfune, Y.; Nishio, H. Tetrahedron Lett.
1984, 25, 4133. (c) Blaskovich, M. A., Evindar, G.; Rose, N. G. W.;
Wilkinson, S.; Luo, Y.; Lajoie, G. A. J. Org. Chem. 1998, 63, 3631.
(6) Garner, P.; Park, J. M. Org. Synth. 1991, 70, 18.
(7) (a) Williams, L.; Zhang, Z.; Shao, F.; Carroll, P. J.; Joullie, M.
M. Tetrahedron 1996, 52, 11673. (b) Coleman, R. S.; Carpenter, A. J.
Tetrahedron Lett. 1992, 33, 1697. (c) Takahata, H.; Banba, Y.; Ouchi,
H.; Nemoto, H. Org. Lett. 2003, 5, 2527.
(4) Xie, L.; Miller, L.; Chatterjee, C.; Averin, O.; Kelleher, N. L.;
van der Donk, W. A. Science 2004, 303, 679.
6686 J. Org. Chem., Vol. 70, No. 17, 2005

Synthesis of Ser and Cys Analogs
SCHEME 4
SCHEME 5
compounds 10 and 11 was treated with FmocCl and
NaHCO3 to provide compound 13 in 45% yield (2 steps),
along with 29% (2 steps) of compound 12. Compounds
12 and 13 were separated by silica gel chromatography.
To increase the overall yield, compound 12 was trans-
formed into 13 in three steps (Scheme 3). Subsequent
oxidization of alcohol 13 with Jones reagent afforded the
protected target amino acid 1 in 76% yield.
modified Evans auxiliary was used and excellent stereo-
selectivity was obtained. We applied this methodology to
prepare the â2-homoserine derivative 20. The hydroxyl
group of compound 20 was protected as the tert-butyl
ether typically used for Fmoc-based solid-phase peptide
synthesis in the presence of isobutylene and catalytic H2-
SO4 in CH2Cl2. Removal of the chiral auxiliary from
compound 21 with LiOH and H2O2 proceeded smoothly
(Scheme 5), but as previously observed,12 the phthaloyl
group is not stable under the reaction conditions and ring
opening took place to give compound 22 in 95% yield.
Rather than opting for other deprotection conditions that
leave the phthaloyl group intact but that lead to an
erosion in er,11c we chose to reinstall the phthaloyl group
with CDI in THF12 to afford compound 23 in 95% yield.
Subsequent hydrazinolysis of the phthaloyl group pro-
ceeded smoothly, and the product mixture was carried
on without further purification. Protection of the result-
ing amino group with FmocOSu and NaHCO3 in THF
and H2O afforded amino acid 3 in 60% yield (2 steps).
Synthesis of â3-Homocysteines 4 and 5 and
(2R,3R)-Methylcysteine 6. Since the stereochemical
requirement for acceptance of â-amino acids by the
lantibiotic synthases is unknown, both enantiomers of
â3-homocysteine were prepared. The preparation of the
(R)-isomer followed the Arndt-Eistert homologation
protocol developed by Seebach and co-workers13a using
the S-tert-butyl disulfide protecting group for the thiol
functionality of L-Fmoc-cysteine (Scheme 6). After forma-
tion of diazoketone 24, silver-mediated Wolf rearrange-
For the preparation of fluorinated amino acid 2, the
(E)-R,â-unsaturated aldehyde 15 was prepared by Wittig
reaction of aldehyde 7 with (triphenyl-phosphanylidene)-
acetaldehyde in 86% yield (Scheme 4).8 DIBAL-H reduc-
tion of aldehyde 15 provided allylic alcohol 16 in 69%
yield. With this key intermediate in hand we turned our
attention to the introduction of fluorine. A wide variety
of fluorinating reagents are available.9 We opted for the
use of diethylaminosulfur trifluoride (DAST), as it is a
highly effective reagent for the direct, one-step, and high-
yielding conversion of alcohols into fluorides under mild
conditions. Treatment of allylic alcohol 16 with DAST
afforded fluorinated compound 17 in 56% yield. The
moderate yield is probably due to competitive SN2′
reaction,10 although this product was not isolated. Depro-
tection of the isopropylidene and Boc groups of compound
17 with 4 N HCl-dioxane, followed by protection of the
free amino group with FmocCl and NaHCO3, afforded
compound 18 in 80% yield (2 steps). Oxidization of 18
with Jones reagent afforded the target amino acid 2 in
low yield 32%. Attempts to improve the yield using either
PDC or the Dess-Martin reagent failed to give the
desired target compound 2. Under the latter conditions,
the conjugated product 19 was formed instead of pro-
tected amino acid 2.
For the preparation of Fmoc-â2-homoserine(But)-OH
3, the common approach of conjugate addition of nitrogen
nucleophiles to R,â-unsaturated carboxylic acid deriva-
tives is not practical. Several years ago, Seebach and co-
workers developed an asymmetric synthesis method to
access â2-homoserine derivatives.11 In this method, a
(11) (a) Hintermann, T.; Seebach, D. Helv. Chim. Acta 1998, 81,
2093. (b) Seebach, D.; Kimerlin, T.; Sebesta, R.; Cmpo, M. A.; Beck, A.
K. Tetrahedron 2004, 60, 7455. (c) Lelais, G.; Micuch, P.; Josien-
Lefebvre, D.; Rossi, F.; Seebach, D. Helv. Chim. Acta 2004, 87, 3131.
(12) Gademann, K.; Kimmerlin, T.; Hoyer, D.; Seebach, D. J. Med.
Chem. 2001, 44, 2460.
(13) For syntheses of â2-homocysteine with other protecting groups
that are not desirable for our purposes, see: (a) Rossi, F.; Lelais, G.;
Seebach, D. Helv. Chim. Acta 2003, 86, 2653. (b) Seebach, D.; Jacobi,
A.; Rueping, M.; Gademann, K.; Ernst, M.; Jaun, B. Helv. Chim. Acta
2000, 83, 2115.
(8) Jurgens, A. R. Tetrahedron Lett. 1992, 33, 4727.
(9) Wilkinson, J. A. Chem. Rev. 1992, 92, 505.
(10) Tellier, F.; Sauvetre, R. Tetrahedron Lett. 1991, 32, 5963.
J. Org. Chem, Vol. 70, No. 17, 2005 6687

Zhang et al.
SCHEME 6
SCHEME 7
ment14 resulted in the desired product but in disappoint-
ing yield (48%). However, when the rearrangement was
induced by photolysis, the protected â3-homocysteine
derivative 4 was obtained in excellent yield (82%). This
protected â3-amino acid can be incorporated into the
N-terminal position of peptides by Fmoc-based SPPS and
provides, after global deprotection, a disulfide-masked
N-terminal thiol that can be used immediately for
expressed protein ligation4,15 to provide the desired 51-
mer substrate analogues for LctM.
The same protocol can in principle be used for the
preparation of (S)-â3-homocysteine, but this route suffers
from the high cost of Fmoc-D-Cys(StBu) (1 g, $225,
BACHEM 2005 catalog). Hence an alternative route was
developed starting from L-aspartic acid. The advanced
intermediate 25 was obtained in three steps16 from
commercially available N-R-Boc-L-aspartic acid â-tert-
butyl ester N-hydroxysuccinimide ester (Boc-Asp(OtBu)-
OSu, Scheme 7). Treatment of 25 with sodium methoxide
in methanol quantitatively yielded the corresponding
thiol,17 which was converted to intermediate 26 (Scheme
7). Without further purification, treatment of activated
compound 26 with tert-butylthiol in the presence of
triethylamine gave the tert-butyl disulfide 27 in 51% yield
from 25. Subsequent removal of the Boc group with TFA
in methylene chloride followed by protection of the
primary amine using Fmoc-OSu yielded the target com-
pound 5 in 79% yield.
76% yield (Scheme 7). The benzyl group was subse-
quently removed using silicon tetrachloride in the pres-
ence of diphenylsulfoxide and trifluoro acetic acid19 to
provide the desired (2R,3R)-methylcystine for immediate
use in solid-phase peptide synthesis19 and subsequent
expressed protein ligation.
In summary, nonproteinogenic amino acids 1-6 were
prepared using stereoselective syntheses. The incorpora-
tion of these amino acids into the 51-mer peptide sub-
strate for lacticin 481 synthase is currently in progress.
Experimental Section
(4R,1′R)-4-(1-Hydroxy-allyl)-2,2-dimethyl-oxazolidine-
3-carboxylic Acid tert-Butyl Ester (syn-8). MeLi (1.6 M in
Et2O, 42.36 mL, 67.77 mmol) was added to a solution of
tetravinyltin (3.05 mL, 16.94 mmol) in Et2O (127 mL) at 0 °C
and the mixture was stirred for 15 min at the same temper-
ature. Then ZnBr2 (15.24 g, 67.77 mmol) was added to the
mixture and stirred for 1 h at room temperature. The resulting
solution of vinyl zinc bromide in Et2O was slowly added to a
suspension of 7 (3.88 g, 16.94 mmol) and ZnBr2 (3.82 g, 16.94
mmol) in Et2O (43 mL) at -78 °C. Then the reaction mixture
was warmed to room temperature and stirred for 2 h. The
reaction mixture was cooled to 0 °C, and saturated aqueous
NH4Cl was added. The organic layer was separated and the
aqueous layer was extracted with ether (40 mL × 3). The
combined organic phases were washed with brine, dried over
Na2SO4, and concentrated. The residue was purified by column
chromatography on silica gel (hexane/EtOAc ) 3:1, Rf ) 0.3)
to give a mixture of syn and anti 8 (syn:anti ) 5:1) (3.90 g,
90%) as a white solid. Recrystallization of the solid from a
solution of hexane/EtOAc (5:1) gave syn-8 (2.89 g, 74 %) as a
For the preparation of disulfide 6, benzyl-protected
methylcysteine derivative 28 was prepared from L-
threonine using previously developed aziridine methodol-
ogy.18 Acidic removal of both the carboxybenzyl group and
hydrolysis of the methyl ester, followed by protection of
the primary amine with Fmoc, provided compound 29 in
(14) Ellmerer-Muller, E. P.; Brossner, D.; Maslouh, N.; Tako, A.
Helv. Chim. Acta 1998, 81, 1998.
(15) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705.
(16) Bergeron, R. J.; Wiegand, J.; Weimar, W. R.; Vinson, J. R. T.;
Bussenius, J.; Yao, G. W.; McManis, J. S. J. Med. Chem. 1999, 42, 95.
(17) Rietman, B. H.; Peters, R. F. R.; Tesser, G. I. Synth. Commun.
1994, 24, 1323.
(18) (a) Nakajima, K.; Oda, H.; Okawa, K. Bull. Chem. Soc. Jpn.
1983, 56, 520. (b) Wakamiya, T.; Shimbo, K.; Shiba, T.; Nakajima, K.;
Neya, M.; Okawa, K. Bull. Chem. Soc. Jpn. 1982, 55, 3878 (c) Zhou,
H.; van der Donk, W. A. Org. Lett. 2002, 4, 1335. For a new improved
method to these types of compounds that was reported while this
manuscript was under review, see: Narayan, R. S.; VanNieuwenhze,
M. S. Org. Lett. 2005, 7, 2655.
white solid: mp 80-81 °C (lit. 80-80.5 °C); [R]20 ) 47.07 (c
D
1.25, CHCl3) (lit. 48.9 (c 1.25, CHCl3)); 1H NMR (400 MHz,
CDCl3) δ 5.80 (m, 1H), 5.32 (d, J ) 17.2 Hz, 1H), 5.21 (d, J )
(19) Zhu, Y.; Gieselman, M. D.; Zhou, H.; Averin, O.; van der Donk,
W. A. Org. Biomol. Chem. 2003, 1, 3304.
6688 J. Org. Chem., Vol. 70, No. 17, 2005

Synthesis of Ser and Cys Analogs
10.0 Hz, 1H), 4.34 (br, 1H), 4.20 (br, 1H), 3.96 (m, 1H), 3.91-
3.87 (m, 2H), 1.56 (s, 3H), 1.48 (s, 12H); 13C NMR (100 MHz,
CDCl3) δ 155.1, 137.7, 117.9, 94.3, 81.4, 76.1, 64.5, 61.8, 28.3,
27.1, 24.2; MS m/z 258 (M+ + H+, 0.24), 202 (5.9), 144 (9.1),
100 (27.2), 57 (100).
organic layers were separated and the aqueous layer was
extracted with ether (3 × 30 mL). The combined organic layers
were dried over Na2SO4, filtered, and concentrated. The
residue was purified by column chromatography on silica gel
(hexane/EtOAc ) 3:1) to give compound 13 (1.020 g, 45%, two
steps, Rf ) 0.24) as a foam and compound 12 (551 mg, 28.5%,
two steps, Rf ) 0.1) as a solid.
(4R,1′R)-4-(1-tert-Butoxy-allyl)-2,2-dimethyl-oxazolidine-
3-carboxylic Acid tert-Butyl Ester (9). To a solution of
syn-8 (2.60 g, 10.12 mmol) in cyclohexane (20 mL) was added
tert-butyl-2,2,2-trichloroacetimide (4.09 mL, 23.16 mmol), fol-
lowed by BF3‚Et2O (202 µL) at room temperature. After
stirring for 2.5 days, the reaction was quenched with NaHCO3,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (hexane/EtOAc ) 10:1, Rf ) 0.4)
to give product 9 (1.341 g) as a clear oil. Elution of the column
with hexane/EtOAc 2:1 recovered reactant syn-8 (1.993 g). The
recovered compound syn-8 was dissolved in cyclohexane (15.5
mL) again. tert-Butyl-2,2,2-trichloroacetimide (2.74 mL) and
BF3‚Et2O (155 uL) were added to the reaction mixture. After
stirring for 2.5 days at room temperature, the reaction was
quenched with NaHCO3, filtered, and concentrated. The
residue was purified by column chromatography on silica gel
(hexane/EtOAc ) 10:1) to give product 9 (523 mg) as a clear
oil and recovered reactant syn-8 (1.257 g) (hexane/EtOAc )
2:1). The recovered compound syn-8 was dissolved in cyclo-
hexane (8 mL) once more. tert-Butyl-2,2,2-trichloroacetimide
(2.2 mL) and BF3‚Et2O (83 uL) were added to the reaction
mixture. After stirring for 2.5 days at room temperature, the
reaction was quenched with NaHCO3, filtered, and concen-
trated. The residue was purified by column chromatography
on silica gel (hexane/EtOAc ) 12:1, Rf ) 0.3) to give product
9 (605 mg) as a clear oil. The overall product 9 from this
Compound 13: [R]20 ) -3.72 (c 0.835, CHCl3); 1H NMR
D
(400 MHz, CDCl3) δ 7.76 (d, J ) 7.6 Hz, 2H), 7.60 (d, J ) 7.6
Hz, 2H), 7.40 (t, J ) 7.6 Hz, 2H), 7.32 (t, J ) 7.6 Hz, 2H),
5.89-5.85 (m, 1H), 5.29 (d, J ) 17.2 Hz, 1H), 5.26 (m, 1H),
5.18 (d, J ) 10.8 Hz, 1H), 4.44 (dd, J ) 10.4, 6.8 Hz, 1H), 4.36
(dd, J ) 10.4, 6.8 Hz, 1H), 4.28-4.22 (m, 2H), 3.71 (br, 3H),
2.71 (br, 1H), 1.22 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 156.9,
144.1, 141.5, 139.0, 127.9, 127.3, 125.3, 120.2, 116.6, 75.4, 72.6,
67.0, 63.3, 56.6, 47.5, 28.8; IR (thin film) 3437, 3377, 1713,
1605, 1505 cm-1; MS m/z (ESI) 418 (M+ + Na+), 396 (M+
+
H+); HRMS calcd for C24H29NO4 (M+ + Na+) 418.1994, found
418.2010.
Compound 12: mp 79-82 °C; [R]20D ) 3.06 (c 0.82, CHCl3);
1H NMR (400 MHz, CDCl3) δ 7.76 (d, J ) 7.6 Hz, 2H), 7.58 (d,
J ) 7.6 Hz, 2H), 7.40 (t, J ) 7.2 Hz, 2H), 7.30 (t, J ) 7.2 Hz,
2H), 5.91-5.84 (m, 1H), 5.46 (d, J ) 8.4 Hz, 1H), 5.35 (d, J )
17.2 Hz, 1H), 5.22 (d, J ) 10.4 Hz, 1H), 4.46-4.42 (m, 2H),
4.36 (m, 1H), 4.20 (t, J ) 6.8 Hz, 1H), 3.81 (br, 2H), 3.74-3.73
(m, 1H), 3.14 (br, 2H); 13C NMR (100 MHz, CDCl3) δ 157.2,
144.0, 141.5, 137.6, 128.0, 127.3, 125.3, 120.2, 116.8, 72.8, 67.1,
63.9, 55.7, 47.4; IR (thin film) 3400, 3067, 1694 cm-1; MS m/z
(ESI) 340.1 (M+ + H+); HRMS calcd for C20H21NO4 (M+ + Na+)
362.1368, found 362.1348.
Carbonic Acid Allyl Ester (2S,3R)-2-(9H-Fluoren-9-
ylmethoxycarbonylamino)-3-hydroxy-pent-4-enyl Ester
(12a). To a solution of compound 12 (100 mg, 0.295 mmol) in
CH2Cl2 (4 mL) and dry pyridine (72 µL, 3.0 equiv) was added
allyl chloroformate (57 µL) at 0 °C. Then the reaction was
warmed to room temperature and stirred for 24 h. Water was
added, the organic layer was separated and the aqueous layer
was extracted with EtOAc. The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated. The
residue was purified by column chromatography on silica gel
(hexane/EtOAc ) 2:1, Rf ) 0.4) to give compound 12a (102
procedure was 3.121 g (yield 78%): [R]20 ) 43.86 (c 0.93,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.85 (m, 1H), 5.23-5.13
(m, 2H), 4.51 and 4.38 (t, J ) 5.6 Hz, 1H), 4.06 (ddd, J ) 17.2,
9.6, 1.6 Hz, 1H), 3.93 and 3.81 (m, 1H), 3.88 (dd, J ) 8.8, 6.4
Hz, 1H), 1.53-1.39 (m, 15 H), 1.17 and 1.14 (s, 9H); note that
this compound exists as different rotamers at 25 °C; 13C NMR
(100 MHz, CDCl3) δ 152.6 and 152.1, 137.3 and 137.1, 116.7
and 116.4, 94.6 and 93.9, 79.9 and 79.8, 74.5 and 74.3, 71.4
and 70.6, 63.6 and 63.3, 61.1 and 60.5, 28.7, 28.4, 26.4 and
25.5, 24.4 and 23.0; IR (thin film, cm-1) 3076, 1704, 1694, 1642;
MS m/z (ESI) 336.4 (M+ + Na+), 314.5 (M+ + H+). Anal. Calcd
for C17H31NO4: C, 65.14; H, 9.97; N, 4.47. Found: C, 65.07;
H, 10.12; N, 4.63
mg, 82%) as a white solid: mp 102-105 °C; [R]20 ) 5.61 (c
D
1
0.425, CHCl3); H NMR (400 MHz, CDCl3) δ 7.76 (d, J ) 7.6
Hz, 2H), 7.57 (d, J ) 7.6 Hz, 2H), 7.40 (t, J ) 7.6 Hz, 2H),
7.31 (td, J ) 7.6, 0.8 Hz, 2H), 5.96-5.86 (m, 2H), 5.36 (m, 2H),
5.29-5.24 (m, 3H), 4.63 (d, J ) 6.0 Hz, 2H), 4.45-4.34 (m,
3H), 4.30-4.20 (m, 3H), 3.98 (m, 1H), 2.51 (d, J ) 3.6 Hz, 1H);
13C NMR (100 MHz, CDCl3) δ 156.7, 155.3, 144.0, 141.5, 137.0,
131.5, 128.0, 127.3, 125.3, 120.2, 119.5, 117.3, 71.0, 69.1, 67.2,
66.8, 53.8, 47.4; IR (thin film) 3400, 3067, 1747, 1723, 1701,
1522 cm-1; MS m/z (ESI) 446.2 (M+ + Na+), 430.2 (M+ + Li+),
424.2 (M+ + H+); HRMS calcd for C24H26NO6 (M+ + H+)
424.1760, found 424.1761.
(2R,3R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-
hydroxy-tert-butyl-pent-4-enoic Acid (13). Method A. A
solution of compound 9 (107 mg, 0.342 mmol) in MeOH (6 mL)
and aqueous 10% HCl (3 mL) was heated to 40 °C. After
stirring for 3 h, the reaction mixture was diluted with CH2-
Cl2. The organic layers were separated, and the aqueous layer
was extracted with EtOAc (20 mL × 3). The combined organic
layers were washed with saturated aqueous NaHCO3, dried
over Na2SO4, filtered, and concentrated to give crude amino
alcohol 11 (38 mg) as a clear oil, which was used without
further purification. The crude amino alcohol was dissolved
in THF/H2O (2:1, 3 mL). FmocCl (72 mg, 0.257 mmol) was
added, followed by NaHCO3 (42 mg, 0.5 mmol) at room
temperature. After stirring for 24 h, the reaction mixture was
diluted with ether. The organic layers were separated and the
aqueous layer was extracted with ether (20 mL × 3). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated. The residue was purified by column chromatog-
raphy on silica gel (hexane/EtOAc ) 3:1, Rf ) 0.24) to give
compound 13 (43 mg, 32%, two steps) as a foam.
Carbonic Acid Allyl Ester (2S,3R)-3-tert-Butoxy-2-(9H-
fluoren-9-ylmethoxycarbonylamino)-pent-4-enyl Ester
(14). To a solution of compound 12a (54 mg, 0.128 mmol) in
CH2Cl2 (2 mL) was added a catalytic amount H2SO4 (5 µL) at
0 °C. Then isobutylene was bubbled through the solution for
15 min. The reaction was sealed and stirred overnight. The
reaction mixture was diluted with ethyl acetate and washed
with water and brine, dried over Na2SO4, filtered, and con-
centrated. The residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc ) 4:1, Rf ) 0.6) to give
compound 14 (40 mg, 66%): [R]20 ) -4.20 (c 0.58, CHCl3);
D
1H NMR (400 MHz, CDCl3) δ 7.76 (d, J ) 7.2 Hz, 2H), 7.58 (d,
J ) 7.2 Hz, 2H), 7.40 (t, J ) 7.2 Hz, 2H), 7.31 (td, J ) 7.6, 3.2
Hz, 2H), 5.91-5.80 (m, 2H), 5.35 (dd, J ) 16.8, 1.2 Hz, 1H),
5.28 (dt, J ) 12.9 Hz, 1.2 Hz, 1H), 5.26 (dd, J ) 10.4, 0.8 Hz,
1H), 5.17 (dt, J ) 10.4, 1.2 Hz, 1H), 5.12 (d, J ) 8.8 Hz, 1H),
4.63 (dt, J ) 6.0 Hz, 1.2 Hz, 2H), 4.44-4.34 (m, 2H), 4.24-
4.17 (m, 4H), 3.94-3.93 (m, 1H), 1.19 (s, 9H); 13C NMR (100
MHz, CDCl3) δ 156.1, 154.7, 143.9, 141.3, 138.4, 131.4, 127.7,
Method B. Compound 9 (1.785 g, 5.702 mmol) was dis-
solved in 1 N HCl-dioxane (60 mL) at room temperature. The
reaction mixture was stirred overnight. Volatiles were re-
moved, the residue was diluted with THF and concentrated
again. The residue was dissolved in THF/H2O (2:1, 75 mL).
FmocCl (2.246 g, 8.017 mmol) was added, followed by NaHCO3
(1.106 g, 13.167 mmol) at room temperature. After stirring for
24 h, the reaction mixture was diluted with EtOAc. The
J. Org. Chem, Vol. 70, No. 17, 2005 6689

Zhang et al.
127.0, 125.1, 120.0, 119.1, 116.6, 74.9, 70.4, 68.6, 66.8, 66.3,
54.1, 47.2, 28.5; IR (thin film) 3424, 3339, 3068, 1748, 1731,
(S)-((1E)-3-Fluoro-propenyl)-2,2-dimethyl-oxazolidine-
3-carboxylic Acid tert-Butyl Ester (17). To a cooled solution
(-78 °C) of DAST (0.8 mL, 6 mmol) in dry CH2Cl2 (20 mL)
was added dropwise a solution of alcohol 16 (514 mg, 2 mmol)
in dry CH2Cl2 (20 mL) over a period of 1 h. After the reaction
mixture was stirred at -78 °C for 3 h, water was added. The
organic layer was separated and the aqueous layer was
extracted with CH2Cl2. The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concen-
trated. The residue was purified by column chromatography
on silica gel (hexane/EtOAc ) 7:1, Rf ) 0.3) to give compound
17 (292 mg, 56%) as a yellow oil: [R]20D ) 4.53 (c 0.99, CHCl3);
1H NMR (400 MHz, CDCl3) δ 5.78 (m, 2H), 4.85 (dd, J ) 48.6,
4.4 Hz, 2H), 4.44-4.30 (m, 1H), 4.04 (dd, J ) 8.4, 6.8 Hz, 1H),
3.75 (d, J ) 8.4 Hz, 1H), 1.61-1.38 (m, 15H); 13C NMR (100
MHz, acetone-d6) δ 152.3, 135.0 (d, J ) 11.4 Hz), 127.2 (d, J
) 16.7 Hz), 94.3, 83.2 (d, J ) 160.0 Hz), 79.7, 68.5, 59.2, 28.5,
26.8, 23.7; 19F NMR (376 MHz, CDCl3) δ -212.9 and -214.1
(t, J ) 48.6 Hz), note that this compound exists as different
rotamers at 25 °C; IR (thin film) 2981, 1694, 1386, 1367, 1100
cm-1; MS m/z (ESI) 282.3 (M+ + Na+), 260.2 (M+ + H+); HRMS
calcd for C13H22NO3F (M+ + Na+) 282.1481, found 282.1490.
1715, 1651 cm-1; MS m/z (ESI) 502 (M+ + Na+), 480 (M+
+
H+); HRMS calcd for C28H34NO6 (M+ + H+) 480.2386, found
480.2362.
(2R,3R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-
hydroxy-tert-butyl-pent-4-enoic Acid (13). To a solution
of compound 14 (15 mg, 0.031 mmol) and (Ph3P)4Pd (2 mg) in
THF (0.5 mL) was added morpholine (3 µL, 0.037 mmol) at
room temperature under N2. The reaction mixture was stirred
for 30 min. Ethyl acetate was added, the resulting mixture
was washed with 1 N HCl, and the aqueous layer was
extracted with EtOAc. The combined organic layers were
washed with saturated NaHCO3, dried over Na2SO4, concen-
trated. The residue was purified by column chromatography
on silica gel (hexane/EtOAc ) 3:1, Rf ) 0.2) to give compound
13 (8 mg, 70%) as a foam.
(2S,3R)-3-tert-Butoxy-2-(9H-fluoren-9-ylmethoxycar-
bonylamino)-pent-4-enoic Acid (1). To a solution of com-
pound 13 (0.79 g, 2.01 mmol) in acetone (56 mL) was added
Jones reagent (1.0 M in H2O, 6.02 mL) at 0 °C. After stirring
for 1 h, the reaction mixture was warmed to room temperature
and stirred for an additional 5 h. Then the reaction was
quenched with 2-propanol (1.2 mL). After the resulting mixture
was stirred for 2 h, water was added. The organic layer was
separated and the aqueous layer was extracted with EtOAc.
The combined organic layers were washed with brine, dried
over Na2SO4, filtered, and concentrated. The residue was
purified by preparative TLC (MeOH/MeOH ) 20:1, Rf ) 0.1)
to give amino acid 1 (624 mg, 76%) as a foam: mp 64 °C (foam);
[R]20D ) 9.12 (c 1.05, MeOH); 1H NMR (400 MHz, MeOH-d4) δ
7.78 (d, J ) 7.6 Hz, 2H), 7.65 (m, 2H), 7.38 (t, J ) 7.2 Hz,
2H), 7.29 (td, J ) 7.2, 0.8 Hz, 2H), 6.62 (d, J ) 9.6 Hz, 1H),
5.91-5.85 (m, 1H), 5.34 (dd, J ) 17.2, 1.2 Hz, 1H), 5.16 (dd, J
) 10.8, 1.2 Hz, 1H), 4.62 (m, 1H), 4.36-4.28 (m, 2H), 4.24-
4.20 (m, 2H), 1.18 (s, 9H); 13C NMR (100 MHz, MeOH-d4) δ
174.0, 158.6, 145.3, 145.1, 142.6, 140.2, 128.8, 128.2, 126.3,
126.0, 120.9, 116.8, 76.0, 74.2, 68.2, 60.7, 28.8; IR (thin film)
3429, 3294, 3073, 1724, 1715, 1607 cm-1; MS m/z (ESI) 410.2
(M+ + H+); HRMS calcd for C24H27NO5 (M+ + Na+) 432.1787,
found 432.1796.
(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-fluoro-
pent-3-enoic Acid (18). Compound 17 (287 mg, 1.108 mmol)
was dissolved in 4 N HCl-dioxane (2.5 mL) at room temper-
ature. After stirring for 45 min, the reaction mixture was
concentrated. The residue was dissolved in THF and evapo-
rated again to give crude amino alcohol, which was used
without further purification. The crude amino alcohol was
dissolved in dioxane/H2O (1:1, 16 mL). FmocCl (434 mg, 1.551
mmol) was added, followed by NaHCO3 (215 mg, 2.659 mmol).
The reaction mixture was stirred overnight. Ethyl acetate was
added, the organic layers were separated, and the aqueous
layer was extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over Na2SO4, filtered,
and concentrated. The residue was purified by column chro-
matography on silica gel (hexane/EtOAc ) 1:1, Rf ) 0.3) to
give compound 18 (302 mg, 80%) as a white solid: mp 137-
1
139 °C; [R]20 ) -14.94 (c 0.29, MeOH); H NMR (400 MHz,
D
MeOH-d4) δ 7.77 (d, J ) 7.6 Hz, 2H), 7.64 (d, J ) 6.8 Hz, 2H),
7.37 (t, J ) 7.6 Hz, 2H), 7.29 (td, J ) 7.6, 1.2 Hz, 2H), 5.81
(br, 2H), 4.82 (dd, J ) 47.6, 3.2 Hz, 2H), 4.42-4.35 (m, 2H),
4.20-4.17 (m, 2H), 3.54-3.52 (m, 2H); 13C NMR (100 MHz,
MeOH-d4) δ 157.2, 144.1 (d, J ) 10.9 Hz), 141.4, 131.9 (d, J )
10.9 Hz), 127.6, 126.9, 126.6 (d, J ) 17.1 Hz), 125.0, 119.7,
82.4 (d, J ) 162.6 Hz), 66.5, 63.7 (d, J ) 2.3 Hz), 54.5, 47.3;
19F NMR (376 MHz, MeOH-d4) δ -215.5 (t, J ) 47.6 Hz); IR
(thin film) 3315, 1684, 1589, 1542 cm-1; MS m/z (ESI) 364.1
(M+ + Na+), 348.2 (M+ + Li+), 342.1 (M+ + H+); HRMS calcd
for C20H20NO3F (M+ + Na+) 364.1325, found 364.1328. Anal.
Calcd for C20H20NO3F: C, 70.37; H, 5.91; N, 4.10; Found: C,
70.06; H, 5.96; N, 4.18.
(S)-2,2-Dimethyl-4-((1E)-3-oxo-propenyl)-oxazolidine-
3-carboxylic Acid tert-Butyl Ester (15). Triphenylphos-
phoranylidene-acetaldehyde (320 mg, 1 mmol) was added to
a solution of D-Garner aldehyde 7 (229 mg, 1 mmol) in dry
toluene (12 mL). The reaction mixture was heated to 70 °C.
After stirring for 24 h, the solution was concentrated and the
residue was purified by column chromatography on nutralized
silica gel (hexane/EtOAc/Et3N ) 70/20/1, Rf ) 0.3) to give R,â-
unsaturated aldehyde 15 (220 mg, 86%) as an ample oil: 1H
NMR (400 MHz, CDCl3) δ 9.55 (d, J ) 17.2 Hz, 1H), 6.71 (td,
J ) 15.6, 5.7 Hz, 1H), 6.16 (m, 1H), 4.66-4.51 (m, 1H), 4.15-
4.05 (m, 1H), 3.85-3.82 (m, 1H), 1.63-1.40 (m, 15H).
(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-fluoro-
pent-3-enoic Acid (2). Jones Oxidation. To a solution of
compound 18 (58 mg, 0.170 mmol) in acetone (4.7 mL) was
added Jones reagent (1.0 M, 0.51 mL, 0.510 mmol) at 0 °C.
Then the reaction was warmed to room temperature and
stirred overnight. The reaction was quenched with 0.5 mL of
2-propanol. After the resulting mixture was stirred for 2 h,
water was added. The aqueous layer was extracted with ethyl
acetate. The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel (CH2Cl2/
MeOH ) 4:1, Rf ) 0.4) to give compound 2 (19 mg, 32%) as a
(S)-((1E)-3-Hydroxy-propenyl)-2,2-dimethyl-oxazolidine-
3-carboxylic Acid tert-Butyl Ester (16). To a solution of
aldehyde 15 (220 mg, 0.863 mmol) in dry THF (4 mL) was
added DIBAl-H (1.2 mL, 1.2 mmol, 1.3 equiv, 1.0 M in hexanes)
within 10 min at -75 °C. After the reaction mixture was
stirred at the same temperature for 2 h, it was slowly warmed
to -40 °C and stirred for 1 h. Then the resulting reaction
mixture was cooled to -75 °C, and 0.2 mL of MeOH was slowly
added so as to keep the internal temperature below -70 °C.
The resulting mixture was poured into 0.1 M HCl, the organic
layers were separated, and the aqueous layer was extracted
with EtOAc. The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated. The residue was
purified by column chromatography on silica gel (hexane/
EtOAc ) 2:1, Rf ) 0.3) to give alcohol 16 (154 mg, 69%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) δ 5.78-5.64 (m, 2H),
4.40-4.28 (m, 1H), 4.13 (t, J ) 4.8 Hz, 2H), 4.02 (dd, J ) 8.8,
6.0 Hz, 1H), 3.72 (dd, J ) 8.8, 2.0 Hz, 1H), 1.59-1.41 (m, 15H);
white solid: mp 129-132 °C; [R]20 ) -5.67 (c 0.35, CHCl3);
D
1H NMR (400 MHz, MeOH-d4) δ 7.76 (d, J ) 7.6 Hz, 2H), 7.64
(t, J ) 6.4 Hz, 2H), 7.36 (t, J ) 7.6 Hz, 2H), 7.28 (t, J ) 7.6
Hz, 2H), 6.01 (dt, J ) 15.6 Hz, 4.0 Hz, 1H), 5.86 (m, 1H), 4.82
(dd, J ) 47.2, 5.6 Hz, 2H), 4.67 (br, 1H), 4.40-4.31 (m, 2H),
4.19 (t, J ) 6.8 Hz, 1H); 13C NMR (100 MHz, MeOH-d4) δ
176.4, 156.8, 144.1 (d, J ) 23.5 Hz), 141.4, 131.6 (d, J ) 11.3
Hz), 127.6, 127.0, 125.6 (d, J ) 15.9 Hz), 125.0, 119.7, 82.4 (d,
IR (thin film) 3435, 1696 cm-1
.
6690 J. Org. Chem., Vol. 70, No. 17, 2005

Synthesis of Ser and Cys Analogs
J ) 161.6 Hz), 66.6, 57.6, 47.2; 19F NMR (376 MHz, MeOH-
d4) δ -215.9 (td, J ) 47.2, 12.0 Hz); IR (thin film) 3392, 3073,
3035, 1694, 1682, 1586 cm-1; MS m/z (ESI) 378.1 (M+ + Na+),
362.2 (M+ + Li+); HRMS calcd for C20H18NO4F (M+ + Na+)
378.1118, found 378.1124.
3574, 3472, 3138, 1774, 1715, 1610; MS m/z (ESI) 328.1 (M+
+ Na+), 306.1 (M+ + H+); HRMS calcd for C16H20NO5 + H+
306.1341, found 306.1338. Anal. Calcd for C34H36N2O6: C,
62.94; H, 6.27; N,4.59. Found: C, 62.44; H, 6.15; N, 4.58.
(S)-2-tert-Butoxymethyl-3-(9H-fluoren-9-ylmethoxy-
carbonylamino)-propionic Acid (3). Compound 23 (335 mg,
1.1 mmol) was dissolved in EtOH (13 mL). Hydrazine (140 µL)
was added and the reaction mixture was heated to reflux. After
being stirred for 30 min under reflux, the reaction was cooled
to room temperature and the solvent was evaporated to give
a white solid. Then the solid was suspended in CH2Cl2, and
the solvent was filtered. The organic layer was discarded, and
the solid (370 mg) was used to do the next step without
purification. The solid was dissolved in THF/H2O (1/1, 40 mL),
the reaction mixture was cooled to 0 °C, and FmocOSu (450
mg, 1.32 mmol) was added, followed by NaHCO3 (914 mg, 11
mmol). After the reaction mixture was stirred overnight, the
solvent was removed, the residue was dissolved in 3 mL of
10% NaHCO3, and the resulting solution was washed with
Et2O. The aqueous layer was acidified to pH ) 1 with 1 N
HCl and extracted with EtOAc. The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and
concentrated. The residue was purified with silica gel column
chromatography (hexane/EtOAc ) 2:1 then CH2Cl2/MeOH )
16:1, Rf ) 0.1) to give product 3 (262 mg, 60%, two steps) as a
foam: [R]20D ) 7.32 (c 0.68, CHCl3); 1H NMR (400 MHz, CDCl3)
δ 7.79 (d, J ) 7.2 Hz, 2H), 7.64 (d, J ) 8.0 Hz, 2H), 7.38 (t, J
) 7.2 Hz, 2H), 7.30 (t, J ) 7.2 Hz, 2H), 7.10 (t, J ) 5.6 Hz,
1H), 4.33 (d, J ) 7.0 Hz, 2H), 4.20 (t, J ) 7.0 Hz, 1H), 3.58 (d,
J ) 5.6 Hz, 2H), 3.41 -3.32 (m, 2H), 2.72 (m, 1H), 1.16 (s,
9H); 13C NMR (100 MHz, CDCl3) δ 176.8, 158.7, 145.3, 142.6,
128.8, 128.1, 162.2, 120.9, 74.2, 67.7, 61.9, 48.4, 48.0, 41.0, 27.7;
IR (thin film, cm-1) 339, 3073, 1718, 1708, 1525; MS m/z (ESI)
420.2 (M+ + Na+), 398.2 (M+ + H+); HRMS calcd for C23H28-
NO5 + H+ 398.1967, found 398.1985.
2-[(2S)-2-tert-Butoxymethyl-3-((4R)-4-isopropyl-2-oxo-
5,5-diphenyl-oxazolidin-3-yl)-3-oxo-propyl]-isoindole-1,3-
dione (21). To a solution of compound 20 (631 mg, 1.23 mmol)
in CH2Cl2 (20 mL) was added a catalytic amount of H2SO4 (41
µL). The resulting mixture was cooled to 0 °C, isobutylene was
bubbled into the solution for 15 min, and then the reaction
mixture was allowed to warm to room temperature and stirred
overnight. The mixture was diluted with CH2Cl2 and was
washed with water and brine successively, dried over Na2SO4,
filtered, and concentrated. The residue was purified by silica
gel column chromatography (hexane/EtOAc ) 2:1, Rf ) 0.5)
to give product 21 (532 mg, 76%) as a white solid: mp 151-
1
153 °C; [R]20 ) 128.8 (c 0.945, CHCl3); H NMR (400 MHz,
D
CDCl3) δ 7.75-7.73 (m, 2H), 7.69-7.66 (m, 2H), 7.42-7.25 (m,
7H), 7.21-7.12 (m, 3H), 5.49 (d, J ) 2.8 Hz, 1H), 4.38-4.33
(m, 1H), 3.96-3.85 (m, 2H), 3.78 (dd, J ) 8.8 Hz, 4.4 Hz, 1H),
3.67 (dd, J ) 8.8 Hz, 5.6 Hz, 1H), 1.96-1.89 (m, 1H), 1.12 (s,
9H), 0.93 (d, J ) 7.2 Hz, 3H), 0.74 (d, J ) 6.4 Hz, 3H); 13C
NMR (100 MHz, CDCl3) δ 172.0, 167.8, 152.5, 142.1, 138.4,
133.7, 131.9, 128.7, 128.33, 128.26, 127.7, 125.8, 125.6, 123.2,
89.0, 73.3, 64.0, 61.4, 43.5, 36.5, 30.2, 27.3, 21.7, 15.8; IR (thin
film, cm-1) 3057, 1779, 1716, 1610; MS m/z (ESI) 591.2 (M+
+
Na+), 569.6 (M+ + H+). Anal. Calcd for C34H36N2O6: C, 71.81;
H, 6.38; N,4.93. Found: C, 71.75; H, 6.43; N, 5.06.
(S)-N-(3-tert-Butoxy-2-carboxy-propyl)-phthalamic Acid
(22). To a solution of compound 21 (1.32 g, 2.32 mmol) in THF/
H2O (125 mL, 2.4:1) was added H2O2 (30% aqueous 955 µL,
0.4 mmol) at 0 °C. After the mixture was stirred for 5 min,
LiOH‚H2O (198 mg, 4.65 mmol) was added. The mixture was
stirred at the same temperature for 3 h. Na2SO3 (1.16 g) was
added at 0 °C, and the reaction mixture was stirred for 30
min. THF was evaporated and 16 mL of Et2O was added. The
suspension was stirred for about 15 min and the solid was
filtered and washed with 1 mL of 1 N NaOH (16 mL), H2O
(16 mL), Et2O (10 mL), and pentane (10 mL) successively to
give an auxiliary 600 mg (recovery 92%). The filtrate was
diluted with EtOAc and the aqueous layer was acidified to pH
2 with 1 M HCl, which was extracted with EtOAc. The
combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated to give compound 22 (713
mg, 95%) as a sticky oil: 1H NMR (400 MHz, CD3OD) δ 7.95
(dt, J ) 8.0 Hz, 1H), 7.59 (td, J ) 7.6 Hz, 1.2 Hz, 1H), 7.52
(td, J ) 7.6 Hz, 1.6 Hz, 1H), 7.42 (dd, J ) 7.6 Hz, 1.2 Hz, 1H),
3.72-3.67 (m, 2H), 3.61-3.58 (m, 2H), 3.61-3.58 (m, 2H),
2.95-2.94 (m, 1H), 1.19 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
176.4, 173.0, 169.2, 139.9, 133.0, 131.3, 130.7, 130.5, 128.8,
74.2, 62.1, 46.9, 39.9, 27.7; IR (thin film) 3569-2620 (br), 1772,
1716, 1557; MS m/z (ESI) 346.1 (M+ + Na+), 324.1 (M+ + H+);
HRMS calcd for C16H22NO6 + H+ 324.1447, found 324.1446.
(S)-2-tert-Butoxymethyl-3-(1,3-dioxo-1,3-dihydro-isoin-
dol-2-yl)-propionic Acid (23). To a solution of compound 22
(375 mg, 1.162 mmol) in dry THF (23 mL) was added
1,1-carbonyl diimidazole (CDI, 570 mg, 3.486 mmol) at room
temperature. The reaction mixture was stirred for 47 h and
then was concentrated. The residue was redissolved in 10%
NaHCO3 (16 mL). Then the aqueous solution was acidified to
pH 1 with 1 N HCl. The resulting precipitate was extracted
with EtOAc, and the combined organic layers were washed
with brine, dried over Na2SO4, filtered, and concentrated. The
residue was purified by silica gel column chromatography
(CH2Cl2/MeOH ) 12:1, Rf ) 0.4) to give product 23 (337 mg,
95%) as a white solid: mp 121-124 °C; [R]20D ) -2.04 (c 0.595,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.86-7.24 (m, 2H), 7.72-
7.70 (m, 2H), 4.09 (dd, J ) 14.4 Hz, 6.8 Hz, 1H), 3.95 (dd, J )
14.4 Hz, 8.0 Hz, 1H), 3.62 (d, J ) 5.6 Hz, 2H), 3.20-3.16 (m,
1H), 1.12 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 175.9, 168.4,
134.3, 132.2, 123.6, 74.3, 60.6, 44.5, 37.0, 27.4; IR (thin film)
(R)-Diazomethyl Ketone 24. To a solution of FmocCys-
(StBu)OH (86 mg, 0.2 mmol) in THF (-15 °C) was added NMM
(15.6 µL, 0.2 mmol), followed by the addition of methyl
chloroformate (22 µL, 0.2 mmol). The mixture was stirred
under a N2 atmosphere at -15 °C for 2 h. Filtration gave the
crude anhydride, which was slowly treated with diazo methane
in ether solution at -5 °C. The reaction mixture was allowed
to warm to room temperature and left overnight. A couple of
drops of HOAc were added to quench the reaction. The mixture
was washed with saturated aqueous NaHCO3 (15 mL), NH4-
Cl (15 mL), dried over MgSO4, and concentrated under reduced
pressure. Purification over silica gel gave the desired product
24 (EtOAc/Hexane ) 1:1, Rf ) 0.65) as a white solid (74 mg,
76%): [R]20 ) -48.1 (c 0.86, CHCl3); 1H NMR (500 MHz,
D
CDCl3) δ 1.30 (s, 9 H), 3.06 (m, 2H), 4.21 (t, 1 H, J ) 6.5 Hz),
4.44-4.52 (m, 3 H), 5.45 (br, 1 H), 5.76 (d, 1 H, J ) 8.0), 7.29-
7.40 (m, 4H), 7.60 (m, 2H), 7.75 (d, 2 H, J ) 7.5 Hz); 13C NMR
(125 MHz CDCl3) δ 29.7, 41.9, 47.7, 48.3, 54.8, 57.1, 60.3,
66.8, 119.9, 119.9, 125.0, 127.0, 127.6, 141.2, 141.3, 143.5,
143.6, 155.7, 191.9; IR (thin film, cm-1) 3306, 2959, 2108, 1362;
MS m/z (ESI) 427.9 (M+ - N2 + H), 478.0 (M+ + Na); HRMS
(ESI+) calcd for (M+ - N2 + H) C23H25N3NaS2 478.1235, found
478.1219.
(R)-4-tert-Butyldisulfanyl-3-(9H-fluoren-9-ylmethoxy-
carbonylamino)-butyric Acid (4). Method A. A mixture of
AgNO3 (3.3 mg, 0.02 mmol) and compound 24 (81.1 mg, 0.18
mmol) was dissolved in 9.6 mL of THF/H2O (5:1) with the
exclusion of light at -15 °C, followed by the addition of TEA
(78 µL, 0.52 mmol). The mixture was stirred overnight at -15
°C, and the reaction mixture was acidified by addition of 10%
aqueous citric acid (2 mL). The reaction mixture was diluted
with EtOAc (30 mL), washed with brine, and concentrated.
Purification over silica gel gave the desired product 4 (CH3-
OH/DCM ) 1:10, Rf ) 0.3) as a foamlike white solid.
Method B. A solution of diazoketone 24 (74 mg, 0.17 mmol)
in 9.6 mL of degassed THF/H2O (5:1) was irradiated with long
wavelength UV light (Hg lamp) under nitrogen for 3 h. The
J. Org. Chem, Vol. 70, No. 17, 2005 6691

Zhang et al.
reaction mixture was diluted with EtOAc (30 mL), washed
with brine and concentrated. Purification over silica gel gave
the desired product 4 (CH3OH/DCM ) 1:10, Rf ) 0.3) as a
(m, 4 H), 5.47-5.49 (m, 1 H), 7.29-7.41 (m, 4H), 7.41 (d, 2H,
J ) 7.6 Hz), 7.75 (d, 2 H, J ) 7.6 Hz); 13C NMR (CDCl3) δ
29.8, 36.8, 43.4, 47.1, 47.7, 48.2, 66.9, 1199, 125.1, 127.0, 127.6,
141.1, 143.8, 155.6, 176.4; IR (thin film, cm-1) 3323, 3066,
2961, 1713, 1518, 1450, 1248, 1044; MS m/z (ESI) 446.1 (M+
+ H); HRMS (ESI+) calcd for (M+ + H) C23H28NO4S2 446.1460,
found 446.1460.
foamlike white solid (56 mg, 82%): [R]20 ) -8.80 (c 0.8,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.32 (s, 9 H), 2.77-3.2.99
(m, 4 H), 4.22-4.40 (m, 4 H), 5.45(br, 1 H), 7.26-7.41 (m, 4H),
7.41 (d, 2H, J ) 7.6 Hz), 7.75 (d, 2 H, J ) 7.6 Hz); 13C NMR
(100 MHz CDCl3) δ 29.7, 36.8, 43.5, 47.1, 47.7, 66.9, 119.9,
125.1, 127.0, 127.6, 141.2, 143.8, 155.6, 176.2; IR (thin film,
cm-1) 3324, 3066, 2961, 1713, 1517, 1451, 1248; MS m/z (ESI)
446.1 (M+ + H); HRMS (ESI+) calcd for (M+ + H) C23H28NO4S2
446.1460, found 446.1445.
(2R,3R)-3-Benzylsulfanyl-2-(9H-fluoren-9-ylmethoxy-
carbonylamino)-butyric Acid (29). Compound 28 (0.965 g,
2.69 mmol) was dissolved in HOAc (9.5 mL) and HCl (6 M,
9.5 mL). The reaction mixture was refluxed at 100 °C for 9 h
and concentrated. The residue was lyophilized to give a white
solid. The solid was washed twice with EtOAc. The resulting
salt was dissolved in a mixture of THF (9.5 mL) and H2O (9.5
mL), and to this solution was added FmocOSu (1.0 g, 2.96
mmol), followed by the addition of NaHCO3 (2.26 g, 16.9 mmol).
The reaction mixture was stirred at room temperature over-
night and extracted with Et2O. The basic aqueous layer was
acidified to pH around 2 with 3 N HCl and extracted times
with EtOAc (3 × 100 mL). The combined EtOAc layers were
washed with brine, dried over MgSO4, and concentrated.
Purification by silica gel chromatography (CH3OH/CH2Cl2 )
1:10, Rf ) 0.3) afforded the desired product 29 (0.9015 g, 76%)
as a white solid: [R]20D ) +41.2, (c 2.0, CHCl3); mp 64-66 °C;
1H NMR (500 MHz, CD3OD + CDCl3) δ 1.26 (d, 3 H, J ) 7.0
Hz), 2.82 (m, 1 H), 3.18 (d, 1 H, J ) 6.4 Hz), 3.76 (Abq, 2H),
4.23 (t, 1H, J ) 7.0 Hz), 4.29-4.44 (m, 3H), 7.20-7.39 (m, 9H),
7.64-7.78 (m, 4H); IR (thin film, cm-1) 3307, 3064, 3030, 2967,
2927, 1718, 1513, 1449, 1233; 13C NMR (CDCl3) δ 18.5, 34.8,
41.6, 46.3, 58.1, 66.3, 119.1, 124.3, 126.3, 126.9, 127.6, 128.1,
137.4, 140.5, 143.0, 143.2, 156.4, 172.8; HRMS (ESI+) calcd
for (M+ + Na) C26H25NO4NaS 470.1402, found 410.1387.
(2R,3R)-3-[2-Carboxy-2-(9H-fluoren-9-ylmethoxy-
carbonylamino)-1-methyl-ethyl disulfanyl]-2-(9H-fluo-
ren-9-ylmethoxycarbonylamino)-butyric Acid (6). SiCl4
(1.6 mL, 13.5 mmol) and Ph2SO (0.683 g, 3.38 mmol) in 3 mL
of TFA were added to compound 29 (0.318 g, 0.677 mmol) at
0 °C. The resulting mixture was stirred for 1 h and diluted
with Et2O (30 mL). The solution was treated with saturated
aqueous NaHCO3 (50 mL). The aqueous layer was then
acidified to pH ∼1 and extracted with EtOAc (3 × 100 mL).
The combined EtOAc layers was dried over MgSO4 and
concentrated. Purification by silica gel chromatography (CH3-
OH/CH2Cl2 ) 1:2, Rf ) 0.3) gave title compound (97 mg, 40%)
(S)-3-tert-Butoxycarbonylamino-4-mercapto-butyric
Acid tert-Butyl Ester (25a). Compound 25 (261 mg, 0.783
mmol) was dissolved in methanol solution (0.1 M NaOCH3 in
54 mL CH3OH) and the solution was stirred at room temper-
ature under nitrogen for 15 min. The mixture was acidified
with acetic acid and extracted with ethyl acetate. The ethyl
acetate layer was washed with water, dried over MgSO4, and
concentrated, and the product was dried under reduced
pressure to give the crude thiol product 25a, which was used
immediately in the next step reaction without further purifica-
tion: 1H NMR (500 MHz, CDCl3) δ 1.43 and 1.42 (2 s, 18 H),
2.48-2.68 (m, 4 H), 4.02 (m, 1H), 5.28 (br, 1 H).
(S)-(Methoxycarbonylsulfenyl)-3-tert-butoxycarbon-
ylamino-4-mercapto-butyric Acid tert-Butyl Ester (26).
To a stirred ice-cold solution of methoxycarbonylsulfenyl
chloride (0.14 mL, 1.56 mmol) in methanol (5 mL) was added
dropwise a solution of the thiol 25a (0.783 mmol) in methanol
(4 mL) that was prepared as described above. The clear
solution was stirred for 2 h at 0 °C. Then the methanol was
evaporated under reduced pressure and a white crystalline
residue 26 was obtained: 1H NMR (500 MHz, CDCl3) δ 1.43
and 1.49 (2 s, 18 H), 2.61 (m, 1 H), 2.70 (m, 1 H), 2.96-3.08
(m, 2 H), 3.88 (s, 3 H), 4.05 (m, 1H), 5.26 (m, 1 H). Compound
26 was used immediately in the next step without further
purification.
(1-tert-Butyldisulfanylmethyl-5,5-dimethyl-3-oxo-hexyl)-
carbamic Acid tert-Butyl Ester (27). To a solution of tert-
butyl mercaptan (0.17 mL, 0.16 mmol) and TEA (0.1 mL, 0.72
mmol) in methanol (13 mL) was added a solution of compound
26 in methanol (10 mL). The mixture was stirred at room
temperature for 20 min and concentrated. The residue was
dissolved in EtOAc and washed with water, brine, dried over
MgSO4 and concentrated. Purification over silica gel gave
desired product 27 (EtOAc/hexane ) 1:2, Rf ) 0.6) as colorless
oil in 51% yield over three steps (from compound 25 to
compound 27): 1H NMR (500 MHz, CDCl3) δ 1.31 (s, 9 H),
1.39 (s, 9 H), 1.40 (s, 9 H), 2.55-2.62 (m, 2 H), 2.85-2.98 (m,
2 H), 4.10 (m, 1 H), 5.09-5.11 (m, 1 H); 13C NMR (125 MHz
CDCl3) δ 27.8, 27.9, 28.1, 28.2, 29.3, 29.5, 29.7, 29.8, 38.1, 44.3,
47.5, 47.8, 79.2, 80.9, 154.8, 170.6; IR (thin film, cm-1) 3366,
2972, 2932, 1717, 1500, 1366, 1248, 1161; HRMS (ESI+) calcd
for (M+ + H) C17H34NO4S2 380.1929, found 380.1927.
as a white solid: [R]20 ) -27.3, (c 1.0, CHCl3); mp 180-182
D
1
°C; H NMR (400 MHz, CDCl3) δ 1.31 (d, 3 H, J ) 7.2 Hz),
3.62 (m, 1 H), 4.23 (t, J ) 7.0 Hz), 4.32-4.65 (m, 3H), 7.20-
7.39 (m, 4H), 7.50-7.78 (m, 4H); 13C NMR (DMSO-d6) δ 17.5,
46.6, 47.6, 58.0, 66.0, 120.2, 124.3, 125.4, 125.5, 126.1, 127.2,
129.6, 129.8, 130.8, 131.2, 131.4, 131.9, 132.6, 140.8, 143.8,
156.1, 171.7; IR (thin film, cm-1) 3306, 3066, 2965, 2926, 1693,
1517, 1449, 1210; LRMS (ESI+) calcd for (M+) C38H36N2O8S2
712.2, found 712.9; HRMS (ESI+) calcd for (M+ + H)
C38H37N2O8S2 713.1991, found 713.1975.
4-tert-Butyldisulfanyl-3-(9H-fluoren-9-ylmethoxycar-
bonylamino)-butyric Acid tert-Butyl Ester (5). Compound
27 (0.105 g, 0.28 mmol) was dissolved in a solution of TFA/
CH2Cl2 (2 mL/2 mL) and stirred at room temperature for 4 h.
After removal of solvent, the residue was dissolved in a
solution of THF/H2O (2 mL/2 mL), followed by the addition of
FmocOSu (0.104 g, 0.28 mmol) and NaHCO3 (237 mg, 2.8
mmol). The resulting mixture was stirred overnight and was
adjusted to ∼pH 3 with 6 N HCl. The mixture was diluted
with EtOAc and the layers were separated. The aqueous phase
was extracted further with EtOAc. The combined EtOAc
extracts were concentrated. Purification over silica gel gave
desired product 5 (CH3OH/CH2Cl2 ) 1:10, Rf ) 0.3) as a white
solid (60 mg, 79%): [R]20D ) +7.17 (c 0.8, CHCl3); 1H NMR (400
MHz, CDCl3) δ 1.33 (s, 9 H), 2.77-3.02 (m, 4 H), 4.20-4.87
Acknowledgment. This work was supported by the
National Institutes of Health (GM58822). W.A.V. is an
Alfred P. Sloan Fellow and Camille Dreyfus Teacher-
Scholar. NMR spectra were obtained in the Varian
Oxford Instrument Center for Excellence, funded in part
by the W. M. Keck Foundation, NIH (PHS 1 S10
RR10444), and NSF (CHE 96-10502).
1
Supporting Information Available: Copies of H NMR
and 13C NMR spectra of all unknown compounds. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
JO051182O
6692 J. Org. Chem., Vol. 70, No. 17, 2005