Side-chain-modified sulfonic analogues of aspartic and glutamic acids: Synthesis, protection, and incorporation into peptides

Full text of DOI:10.1021/jo9812680

1420
J . Org. Chem. 1999, 64, 1420-1423
Sid e-Ch a in -Mod ified Su lfon ic An a logu es of
Asp a r tic a n d Glu ta m ic Acid s: Syn th esis,
P r otection , a n d In cor p or a tion in to
P ep tid es
Laurent Bischoff, Christelle David,
Bernard Pierre Roques,* and
Marie-Claude Fournie´-Zaluski
F igu r e 1. Completely protected cysteic and homocysteic acids.
Reagents and conditions: (a) benzyl chloroformate, NaOH,
dioxane/H2O ) 1:1, pH ) 9; (b) EtOH, SOCl2 (0.67 molar
equiv), 0 °C to rt, overnight; (c) Cl2, CCl4/EtOH ) 4:1; (d)
neopentyl alcohol, Et3N, CH2Cl2, 0 °C.
De´partement de Pharmacochimie Mole´culaire et Structurale,
INSERM U266, CNRS URA D 1500, Faculte´ des Sciences
Pharmaceutiques et Biologiques, 4, avenue de l'Observatoire,
75270 Paris Cedex 06, France
terested in an even more stable sulfonic ester. Our
attention was focused on the neopentyl sulfonate, which
was first described by Truce et al.5 and recently used as
a protective group for arylsulfonic acids, in parallel with
its neoN-B analog.6
With this protective group in hand, we needed a
straightforward method for its introduction. Direct in-
troduction onto the poorly nucleophilic free sulfonate
would not be advantageous, since it would require a
powerful alkylating agent able to transfer the neopentyl
group, such as neopentyl trifluoromethanesulfonate.
Moreover, turning the sulfonic acid into its chloride would
be effected using reagents unsuitable for peptide syn-
thesis, e.g., PCl5 or SOCl2. Consequently, we chose to
prepare these protected amino acids from cystine and
homocystine, both commercially available as optically
pure enantiomers.
Received J une 29, 1998
There is considerable and still increasing interest in
the synthesis of unnatural amino acids. These compounds
can be incorporated into various peptide effectors, such
as receptor ligands and enzyme inhibitors, to increase
their affinity and/or selectivity. As part of our studies
toward synthetic substrates and inhibitors of aminopep-
tidases,1 we wish to report here our recent work concern-
ing the synthesis of isosteric analogues of aspartic and
glutamic acids, as orthogonally protected synthons suit-
able for peptide synthesis. In the latter compounds, the
side-chain carboxylic acid of aspartic and glutamic acids
was replaced by a sulfonate, leading to cysteic acid (also
named sulfoalanine,2 Sal) and homocysteic acid, respec-
tively. These two acids are commercially available, as
totally unprotected forms. Unfortunately, the high water
solubility of the polar free sulfonate renders most organic
transformations troublesome. Indeed, the presence of a
free sulfonate considerably decreases the solubility in
organic solvents and makes aqueous workup procedures
impossible. Recently, an example of direct incorporation
of N-Boc cysteic acid by SPPS was described with a fairly
low yield.2
For these reasons, we have searched for a hydrophobic
and stable protection for the sulfonic acid group. Sul-
fonamides are very readily accessible, but require drastic
conditions to be deprotected, these methods being un-
compatible with peptide synthesis. Moreover, the widely
used reductive cleavage3 of sulfonamides is aimed at
yielding the amine portion of the sulfonamide and not
the sulfonic acid. Sulfonate esters are generally so labile
that they are employed as powerful alkylating reagents,
such as methyl methanesulfonate. This problem can be
overcome by the use of a bulkier sulfonate, provided that
it is not derived from a tertiary alcohol which would be
likely to result in elimination products. In the 1990s, an
isopropyl sulfonate was reported4 as a protecting group
involved in the synthesis of modified carbohydrates.
However, due to the mildness of its deprotection, i.e., NaI/
acetone or ammonia/methanol treatments, we were in-
Resu lts a n d Discu ssion
The synthesis of totally and orthogonally protected
cysteic and homocysteic acids was acomplished as de-
picted in Figure 1. Starting from commercially available
L-cystine or L-homocystine 1a and 1b, the amine was
protected by a Cbz group and the carboxylate esterified
by ethanol with dropwise addition of SOCl2 at 0 °C. The
disulfide was then subjected to chlorine-mediated oxida-
tive cleavage leading to the corresponding sulfonyl
chloride. The latter was isolated but not purified and
subsequently esterified by neopentyl alcohol in the pres-
ence of triethylamine or pyridine.
The totally protected synthons 3a and 3b were cleanly
obtained on a multigram scale with overall yields of 71%
and 94%, respectively. We then used various deprotection
methods for the acid and the amine groups, as outlined
in Figure 2. The deprotection of the acid was rendered
difficult due to a possible racemization of the chiral center
in alkaline medium,7 the acidity of the R-proton being
enhanced by the electron-withdrawing sulfonyl group.
Moreover, when compound 3a was treated at 0 °C with
aqueous LiOH, unexpected degradation was observed.
For these reasons, we used acidic conditions for the
hydrolysis of the carboxylic ester of 3a and 3b. By
treatment with 3 N HCl in dioxane/H2O (1:1) at 50 °C,
4a and 4b were obtained in good yields. Under these
conditions, the Cbz and neopentyl groups remained
(1) (a) Chauvel, E. N.; Coric, P.; Llorens-Corte`s, C.; Wilk, S.; Roques,
B. P.; Fournie´-Zaluski, M.-C. J . Med. Chem. 1994, 37, 1339; (b) 1994,
37, 2950. (c) David, C.; Bischoff, L.; Meudal, H.; Llorens-Cortes, C.;
Roques, B. P.; Fournie´-Zaluski, M.-C. Lett. Pept. Sci. 1997, 4, 411.
(2) Hashimoto, T.; Kurosawa, K.; Sakura, N. Chem. Pharm. Bull.
1995, 43(7), 1154.
(3) Quaal, K. S.; J i, S.; Kim, Y. M.; Closson, W. D.; Zubieta, J . A. J .
Org. Chem. 1978, 43, 1311.
(4) (a) Musicki, B.; Widlanski, T. S. J . Org. Chem. 1990, 55, 4231.
(b) Musicki, B.; Widlanski, T. S. Tetrahedron Lett. 1991, 32, 1267.
(5) Truce, W. E.; Vrencur, D. J . J . Org. Chem. 1970, 35(4), 1226.
(6) Roberts, J . C.; Gao, H.; Gopalsamy, A.; Kongsjahju, A.; Patch,
R. J . Tetrahedron Lett. 1997, 38(3), 355-358.
(7) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J . Org. Chem. 1982,
47, 1962.
10.1021/jo9812680 CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/27/1999

Notes
J . Org. Chem., Vol. 64, No. 4, 1999 1421
F igu r e 3. Deprotection of the sulfonate group of cysteic and
homocysteic acids.
F igu r e 2. Orthogonal protections of amine and acid moieties
of cysteic and homocysteic acids. Reagents and conditions: (a)
3 N HCl/dioxane:H2O ) 1:1, 97% (4a ), 100% (4b); (b) H2, Pd/
C, Boc2O, AcOEt, 92% (5a ), 90% (5b); (c) H2, Pd/C, HCl, EtOH,
100%; (d) H2, Pd/C, 95% EtOH, 100% (7a ), 99% (7b); (e)
isobutylene, Et2O, cat. H2SO4, 5 d, 33%; (f) Fmoc-OSu or Fmoc-
Cl, Na2CO3, dioxane, H2O, 95% (9a ), 65% (9b).
unchanged, provided that the reaction time did not
exceed 14 h. Indeed, further heating resulted in depro-
tection of both amine and sulfonic acid.
The free carboxylic acid could be reprotected as a tert-
butyl ester (compound 8a ) by means of isobutene in ether
with a catalytic amount of sulfuric acid.8
F igu r e 4. Solid-phase peptidic synthesis of CCK4 sulfonic
analogue. 1: piperidine, NMP. 2: DCC, HOBt, NMP. 3: HF,
m-cresol, then TFA, Et2O/n-heptane ) 1:1.
complete deprotection, when compound 4a was treated
with liquid hydrogen fluoride (5 mL/100 mg) in the
presence of m-cresol (250 µL/100 mg) as a scavenger at
0 °C to room temperature during 1 h. The deprotected
amine was obtained as the hydrofluoride, which was
displaced by TFA. We noticed that upon subsequent
aqueous extraction and lyophylization compound 11a was
quantitatively obtained in the zwitterionnic form, with
no trace of remaining sulfonic ester being observed.
The Cbz group was removed by classical hydrogenoly-
sis in the presence of H2/Pd on charcoal, leading to 7a
and 7b. No poisoning of the catalyst occurred, since the
sulfur is in a high oxidation state. The free amine could
be reprotected, affording the N-Boc and N-Fmoc deriva-
tives, respectively 5a ,b and 9a ,b which are very useful
N-protected amino acids for peptide synthesis. To shorten
the synthesis time of the N-Fmoc derivative, the direct
oxidation of (Fmoc-Cys-OH)2 or (Fmoc-Cys-OEt)2 by
chlorine in CCl4/H2O or CCl4/tBuOH was attempted but
resulted in decomposition products, with loss of the Fmoc
group.
As an illustration of the incorporation of sulfonate-
protected cysteic acid into a peptide, the sulfonic analogue
of CCK4 (Trp-Met-Asp-Phe-NH2, tetragastrin) was pre-
pared, with Asp being replaced by cysteic acid. This
synthesis (Figure 4) was performed either by means of
liquid-phase synthesis (introduction as the N-Cbz com-
pound, coupling steps using BOP/DIEA) or solid-phase
peptidic synthesis (N-Fmoc derivative, Sieber-Amide
resin, coupling steps with DCC/HOBT). In both cases,
good yields of incorporation were obtained, both for the
coupling of N-protected sulfoalanine and for the following
amino acid (Met). In the last step, the sulfonic acid was
deprotected by liquid HF. We noticed that the analogue
was, as expected, much more hydrophilic (according to
HPLC analysis) than CCK4. Indeed, when the HPLC was
run at 25% CH3CN (C18 Kromasil column), CCK4 exhib-
ited a retention time of 13.3 min, versus 10 min for the
sulfonic analogue. This synthesis is a useful example of
an anionic isostere of a carboxylic moiety, aspartic acid
being generally mimicked by serine O-sulfate. In biologi-
All protections and deprotections described herein did
not interfere with the sulfonate which remained un-
changed during the course of the synthesis and did not
induce any racemization of the chiral center.
As far as the sulfonic ester is concerned, its cleavage
was recently described by Roberts et al.6 in the presence
of tetramethylammonium chloride in DMF at 160 °C. We
first checked that these apparently harsh conditions did
not result in degradation or racemization during the
deprotection of the sulfonate moiety, when these sul-
fonate-bearing amino acids were incorporated into short
peptides. Nevertheless, we examined this deprotection
under milder conditions which could be more adapted to
peptide synthesis. We observed (Figure 3) that both
sulfonate and amino groups underwent a clean and
(8) Valerio, R. M.; Alewood, P. F.; J ohns, R. B. Synthesis 1988, 786.

1422 J . Org. Chem., Vol. 64, No. 4, 1999
Notes
NMR (270 MHz, DMSO) 0.88 (9H, s), 1.14 (3H, t, J ) 7.2 Hz),
1.9-2.18 (2H, m), 3.16-3.48 (2H, m), 4.08 (2H, q, J ) 7.2 Hz),
4.14-4.24 (1H, m), 5.0 (2H, s), 7.28-7.34 (5H, m), 7.82 (1H, d,
cal media, hydrolysis of the sulfate often readily takes
place; this drawback is avoided when a sulfonate is used.
3
J ) 8 Hz); HPLC [CH CN/H2O/TFA ) 60:40:0.04] tR ) 14.6 min.
Con clu sion
2(S)-2-[(Ben zyloxyca r bon yl)a m in o]-3-(n eop en tyloxysu l-
fon yl)p r op a n oic Acid (4a ). To a solution of HCl (11.6 N, 52
mL), water (52 mL), and dioxane (104 mL) was added ester 3a
(10.6 g, 26.4 mmol), and the reaction mixture was stirred at 50
°C during 14 h. After evaporation to dryness and coevaporation
with toluene (150 mL), compound 4a (9.6 g, 97%) was cleanly
In summary, we have shown that the alkyl- and
arylsulfonate-protecting neopentyl esters are compatible
with amino acid synthesis and protection, as illustrated
with cysteic and homocysteic acids. As both compounds
are orthogonally protected, they can be used in further
synthetic transformations as well as in peptide synthesis.
1
obtained as a white powder: mp ) 95 °C; H NMR (270 MHz,
DMSO) 0.86 (9H, s), 3.6 and 3.74 (2H, 2 dd, ABX), 3.84 (2H, 2
d, AB), 4.38-4.46 (1H, m), 5.02 (2H, s), 7.2-7.35 (5H, m), 7.8
(1H, d, J ) 8 Hz); TLC Rf ) 0.28, eluent [Et2O/cyclohexane/
HCOOH ) 1:1:0.02]; HPLC [CH3CN/H2O/TFA ) 50:50:0.05] tR
Exp er im en ta l Section
) 12.3 min; [R]20 ) -14.8 (c 1.08, absolute EtOH). Anal. Calcd
D
Gen er a l. NMR spectra were recorded on spectrometers
operating at 270 and 400 MHz, in d6-DMSO or CDCl3. HPLC
analyses were run on a C18 Kromasil reverse-phase column (5
µm, 100 Å), using CH3CN/H2O/TFA as the mobile phase. Cbz,
Boc, and Fmoc groups were introduced by means of standard
procedures described in the literature. Treatment with liquid
HF was performed in a Teflon apparatus.
for C16H23NO7S: C, 51.46; H, 6.21; N, 3.75. Found: C, 51.35; H,
6.10; N, 3.89.
2(S)-2-[(Ben zyloxyca r bon yl)a m in o]-4-(n eop en tyloxysu l-
fon yl)bu ta n oic Acid (4b). According to the procedure described
for 4a , 5.5 g (100%) was obtained from 5.9 g (14.2 mmol) of 3b:
1H NMR (270 MHz, DMSO) 0.88 (9H, s), 1.92-2.16 (2H, m),
3.14-3.48 (2H, m), 3.82 (2H, s), 4.06-4.14 (1H, m), 5.0 (2H, s),
7.26-7.38 (5H, m), 7.54 (1H, d, J ) 8 Hz); TLC Rf ) 0.16, eluent
[Et2O/cyclohexane/HCOOH ) 1:1:0.02]; HPLC [CH3CN/H2O/TFA
) 60:40:0.04] tR ) 6.6 min; MS (ESI) 388 (MH+), 410 (MNa+).
2(S)-2-[(ter t-Bu toxycar bon yl)am in o]-3-(n eopen tyloxysu l-
fon yl)p r op a n oic Acid (5a ). A suspension of compound 4a (480
mg, 1.29 mmol), Et3N (143 mg, 1.41 mmol), Boc2O (282 mg, 1.29
mmol), and 10% Pd on carbon (137 mg, 0.13 mmol) in AcOEt (5
mL) was stirred for 24 h under H2 (1 atm). After removal of the
catalyst and washing with ethyl acetate, the filtrate was washed
with 2 N HCl and brine, dried over Na2SO4, and concentrated
under reduced pressure to afford 5a (400 mg, 92%) as a white
powder: mp 115 °C; 1H NMR (270 MHz, DMSO) 0.9 (9H, s),
1.35 (9H, s), 3.64 (2H, 2dd, ABX), 3.84 (2H, dd, AB), 4.28-4.36
(1H, m), 7.25 (1H, d, J ) 8 Hz); TLC Rf ) 0.27, eluent [Et2O/
cyclohexane/HCOOH ) 1:1:0.02]; HPLC [CH3CN/H2O/TFA ) 60:
40:0.04] tR ) 6.1 min. Anal. Calcd for C13H25NO7S: C, 46.00; H,
7.42; N, 4.13. Found: C, 46.19; H, 7.37; N, 4.14.
(Z-Cys-OE t )2 (2a ). Commercially available (H-Cys-OH)2
purchased from ACROS Organics (14.2 g, 59 mmol) was solu-
bilized in 220 mL of a 1:1 mixture of dioxane and water, and
NaOH (4.73 g, 118 mmol) was added. At 0 °C, a solution of benzyl
chloroformate (23.6 g, 138 mmol) in 40 mL of dioxane was added
dropwise (2 h), as the pH was maintained around 9 by simul-
taneous addition of 1 N NaOH. After 1 h at this pH, the solution
was washed with ether, acidified with 6 N HCl at 0 °C, and
extracted with ethyl acetate. The combined organic layers were
washed with brine and dried over Na2SO4. After concentration
under reduced pressure, the oily residue was stirred in n-heptane
overnight to allow the formation of white crystals which were
filtered off and dried under vaccum (mp ) 68 °C). They were
stirred in ethanol with dropwise addition (3 drops/min of SOCl2
at 0 °C, followed by stirring overnight at rt). After evaporation
of the solvent, the residue was taken up in ethyl acetate, washed
with NaHCO3 and brine, dried over Na2SO4, and concentrated
under reduced pressure. After trituration in n-heptane, white
2(S)-2-[(ter t-Bu toxycar bon yl)am in o]-4-(n eopen tyloxysu l-
fon yl)bu ta n oic Acid (5b). According to the procedure described
for 5a and starting from 184 mg (0.47 mmol) of 4b, 152 mg (90%)
of 5b was obtained: 1H NMR (270 MHz, DMSO) 0.9 (9H, s),
1.34 (9H, s), 1.9-2.15 (2H, m), 3.3-3.48 (2H, m), 3.82 (2H, s),
3.98-4.06 (1H, m), 7.16 (1H, d, J ) 7.6 Hz); TLC Rf ) 0.23,
1
crystals (26.5 g, 79%) were obtained: mp 83 °C; H NMR (270
MHz, DMSO) 1.15 (3H, t, J ) 7.2 Hz), 2.90 and 3.10 (2H, 2 dd,
ABX), 4.08 (2H, q, J ) 7.2 Hz), 4.25-4.34 (1H, m), 4.97 (2H, 2d,
AB), 7.2-7.3 (5H, m); [R]20 ) -67.5 (c 1.32, absolute EtOH).
D
(Z-Hom ocys-OEt)2 (2b). According to the procedure de-
scribed for 2a , 4.1 g (92%) was obtained from 2.0 g (7.5 mmol)
of commercially available (H-Homocys-OH)2, purchased from
Bachem. 2b: 1H NMR (270 MHz, DMSO) 1.14 (3H, t, J ) 7.1
Hz), 1.84-2.1 (2H, m), 2.65-2.75 (2H, m), 3.96-4.16 (3H, m),
5.0 (2H, dd, AB), 7.2-7.38 (5H, m), 7.74 (1H, d, J ) 8.2 Hz);
TLC Rf ) 0.67, eluent [AcOEt/cyclohexane ) 1:1].
eluent [Et2O/cyclohexane/HCOOH ) 1:1:0.02]; [R]20 ) +1.7 (c
D
0.99, absolute EtOH). Anal. Calcd for C14H27NO7S: C, 47.58;
H, 7.70; N, 3.96. Found: C, 47.62; H, 7.79; N, 4.00.
Eth yl 2(S)-2-Am in o-3-(n eopen tyloxysu lfon yl)pr opan oate,
Hyd r och lor id e (6a ). Compound 3a (650 mg, 1.62 mmol) in
absolute EtOH (15 mL) was stirred under 1 atm H2 in the
presence of 12 N HCl (0.2 mL, 2.4 mmol) during 2 h. The catalyst
was filtered off and the filtrate evaporated to dryness and dried
under vacuum to afford the hydrochloride 6a (490 mg, 100%)
as a white powder: 1H NMR (270 MHz, DMSO) 0.9 (9H, s), 1.24
(3H, t, J ) 7.2 Hz), 3.92-3.98 (4H, m), 4.18 (2H, t, J ) 7.2 Hz),
E t h yl 2(S)-2-[(Ben zyloxyca r b on yl)a m in o]-3-(n eop en t -
yloxysu lfon yl)p r op a n oa te (3a ). Compound 2a (15.1 g, 27
mmol) was solubilized in CCl4/EtOH ) 4:1 (135 mL), and gaseous
chlorine was bubbled at 0 °C through the solution during 40
min. The ice bath was removed, and stirring continued for an
additional 20 min. Solvents were thoroughly evaporated, and
the residue was dried under vaccum at 40 °C for 30 min. It was
solubilized in dichloromethane (300 mL). At 0 °C neopentyl
alcohol (5.91 g, 67 mmol) was added, followed by dropwise
addition of Et3N (10.3 g, 74 mmol). After 40 min of stirring at
rt, the reaction mixture was diluted with ether, washed with 2
N HCl, NaHCO3, and brine, dried over Na2SO4, and concentrated
under reduced pressure. Flash chromatography on silica gel
[AcOEt/cyclohexane ) 1:4] afforded pure 3a (15.2 g, 71%) as a
pale yellow oil: 1H NMR (270 MHz, DMSO) 0.85 (9H, s), 1.13
(3H, t, J ) 7.4 Hz), 3.58-3.76 (2H, 2 dd, ABX spectrum), 3.85
(2H, dd, AB spectrum), 4.08 (2H, q, J ) 7.4 Hz), 4.47 (1H, m),
5.00 (2H, s), 7.25-7.35 (5H, m), 7.95 (1H, d, J ) 8.3 Hz); TLC
Rf ) 0.23, eluent [AcOEt/cyclohexane ) 1:4]; HPLC [CH3CN/
4.42 (1H, t, J ) 5.6 Hz), 8.88 (3H, br s); [R]20 ) +7.8 (c 0.94,
D
absolute EtOH); MS (ESI) 268 (M+). Anal. Calcd for C10H22NO5-
SCl: C, 39.54; H, 7.30; N, 4.61. Found: C, 38.82; H, 6.59; N,
4.69.
Eth yl 2(S)-2-Am in o-4-(n eop en tyloxysu lfon yl)bu ta n oa te,
Hyd r och lor id e (6b). According to the procedure described for
6a and starting from 470 mg (1.17 mmol) of 3b, 372 mg (100%)
of 6b was obtained: 1H NMR (270 MHz, DMSO) 0.88 (9H, s),
1.18 (3H, t, J ) 7.4 Hz), 2.15-2.25 (2H, m), 2.46-2.54 (2H, m),
3.85 (2H, s), 4.1-4.24 (3H, m), 8.58 (3H, br s).
2(S)-2-Am in o-3-(n eop en tyloxysu lfon yl)p r op a n oic Acid ,
Hyd r och lor id e (7a ). Compound 4a (665 mg, 1.78 mmol) in
AcOEt (15 mL) was stirred under 1 atm H2 during 36 h. The
catalyst was filtered off and washed with 0.2 mL of concd HCl
diluted in 20 mL of EtOH. The filtrate was evaporated to dryness
and dried under vacuum to afford the hydrochloride 7a (490 mg,
100%) as a white powder: mp 141 °C; 1H NMR (270 MHz,
H2O/TFA ) 50:50:0.05] tR ) 35.4 min; [R]20 ) -20.8 (c 0.99,
D
absolute EtOH). Anal. Calcd for C18H27NO7S: C, 53.85; H, 6.78;
N, 3.49. Found: C, 53.94; H, 6.86; N, 3.49.
E t h yl 2(S)-2-[(Ben zyloxyca r b on yl)a m in o]-3-(n eop en t -
yloxysu lfon yl)bu ta n oa te (3b). Starting from 5.2 g (8.8 mmol)
of compound 2b, 6.6 g (16.4 mmol, 94%) of 3b was obtained: 1H
DMSO) 0.9 (9H, s), 3.48-3.54 (1H, m), 3.92 (2H, 2d, AB); [R]20
D
) -2.4 (c 0.27, absolute EtOH); MS (ESI) 240 (M+). Anal. Calcd

Notes
J . Org. Chem., Vol. 64, No. 4, 1999 1423
for C8H18NO5SCl: C, 34.85; H, 6.21; N, 5.08. Found: C, 36.40;
H, 6.67; N, 5.40.
101 °C; 1H NMR (270 MHz, DMSO) 0.86 (9H, s), 3.56 (2H, 2 dd,
ABX), 3.85 (2H, dd, AB), 4.24 (2H, 2 dd, ABX), 4.36-4.46 (1H,
m), 7.26 (2H, t, 7.8 Hz), 7.38 (2H, t, 7.8 Hz), 7.68 (2H, d, 7.8
Hz), 7.86 (2H, d, 7.8 Hz), 7.88 (1H, d, 8.0 Hz); TLC Rf ) 0.29,
eluent [Et2O/cyclohexane/HCOOH ) 1:1:0.02]; HPLC [CH3CN/
2(S)-2-Am in o-3-(n eopen tyloxysu lfon yl)bu tan oic Acid (7b).
According to the procedure described for 7a except that no HCl
was added, and starting from 834 mg (2.15 mmol) of 4b, 540
1
mg (99%) of 7b was obtained: mp 158 °C; H NMR (270 MHz,
H2O/TFA ) 50:50:0.05] tR ) 36.3 min; [R]20 ) -13.4 (c 0.85,
D
DMSO + TFA) 0.87 (9H, s), 2.11-2.29 (2H, m), 3.36-3.56 (2H,
absolute EtOH). Anal. Calcd for C23H27NO7S: C, 59.86; H, 5.90;
N, 3.04. Found: C, 59.95; H, 5.79; N, 3.00.
m), 3.83 (2H, s), 3.98-4.04 (1H, m), 8.29 (3H, br s); [R]20
)
D
+12.6 (c 0.53, absolute EtOH); MS (ESI) 254 (MH+). Anal. Fits
with C9H19NO5S‚1/2H2O: Calcd, C, 41.21; H, 7.68; N, 5.34.
Found: C, 41.47; H, 7.28; N, 5.37.
2(S)-2-[(9-F lu or en ylm eth yloxyca r bon yl)a m in o]-4-(n eo-
p en tyloxysu lfon yl)bu ta n oic Acid (9b). According to the
procedure described for 9a , followed by flash chromatography
through silica gel (eluent cyclohexane/Et2O/HCOOH ) 1:1:0.02),
and starting from 197 mg (2.15 mmol) of 4b, 240 mg (65%) of
9b was obtained: mp 114 °C; 1H NMR (270 MHz, DMSO + TFA)
0.88 (9H, s), 1.94-2.18 (2H, m), 3.14-3.44 (2H, m), 3.82 (2H,
s), 4.06-4.20 (3H, m), 4.28 (2H, d, J ) 7 Hz), 7.28 (2H, t, J ) 7.
8 Hz), 7.36 (2H, t, J ) 7. 8 Hz), 7.66 (2H, d, J ) 7. 8 Hz), 7.82
(2H, d, J ) 7. 8 Hz); TLC Rf ) 0.24, eluent [Et2O/cyclohexane/
HCOOH ) 1:1:0.02]; HPLC [CH3CN/H2O/TFA ) 60:40:0.04] tR
ter t-b u t yl 2(S)-2-[(Ben zyloxyca r b on yl)a m in o]-3-(n eo-
p en tyloxysu lfon yl)p r op a n oa te (8a ). Compound 4a (1.87 g,
5.01 mmol) was placed into a high-pressure flask. Ether (30 mL)
was added, followed by isobutylene (approximately 30 mL) at
-78 °C and H2SO4 (0.2 mL). After 7 d at rt, the reaction mixture
was cooled and cold water (5 mL) was added. Subsequent
extractive workup followed by flash chromatography on silica
gel (eluent cyclohexane/AcOEt ) 7:3) afforded 8a (704 mg, 33%)
as a pale yellow oil: 1H NMR (270 MHz, CDCl3) 0.88 (9H, s),
1.42 (9H, s), 3.64-3.72 (2H, m), 3.80 (2H, dd, AB), 3.48-3.56
(1H, m), 5.06 (2H, s), 5.68 (1H, d, 8 Hz), 7.28-7.32 (5H, m); TLC
) 14.9 min; [R]20 ) -1.3 (c 1.07, absolute EtOH). Anal. Calcd
D
for C24H29NO7S: C, 60.62; H, 6.15; N, 2.95. Found: C, 60.47; H,
6.24; N, 2.94.
Rf ) 0.53, eluent [AcOEt/cyclohexane ) 3:7]; [R]20 ) -20.3 (c
D
0.91, absolute EtOH). Anal. Calcd for C20H31NO7S: C, 55.93;
H, 7.28; N, 3.26. Found: C, 56.06; H, 7.18; N, 3.19.
Ack n ow led gm en t. This work was supported by
CNRS, INSERM, and Universite´ Rene´ Descartes (Paris
V) fundings. Christelle David is grateful to the MESR
for a grant. Christine Lenoir is gratefully acknowledged
for solid-phase synthesis of the analogue of CCK4.
2(S)-2-[(9-F lu or en ylm eth yloxyca r bon yl)a m in o]-3-(n eo-
p en tyloxysu lfon yl)p r op a n oic Acid (9a ). To a suspension of
7a in the zwitterionic form (1.85 g, 7.73 mmol) in H2O (55 mL)
and dioxane (14 mL) was added at 0 °C Na2CO3‚10H2O (6.64 g)
followed by a solution of Fmoc-Cl (2.20 g, 8.50 mmol) in dioxane
(45 mL). After stirring overnight at rt, the reaction mixture was
acidified with 2 N HCl and extracted with ethyl acetate. The
combined extracts were washed with brine, dried over Na2SO4,
and concentrated under reduced pressure and then dried under
vacuum to afford clean 9a (3.38 g, 95%) as a white powder: mp
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C spectra
of most compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O9812680