Communications
J . Org. Chem., Vol. 61, No. 22, 1996 7651
To reveal the amino acid functionality, fully protected
5 is treated with hydroxide to produce Nγ-Boc-4-aminopi-
peridine-4-carboxylic acid (8) with concomitant formation
of di-tert-butyl imidodicarbonate (Boc2NH).14 Isolated
yields of Nγ-Boc-protected 8 were often low and contami-
nated with salts, so a one-pot procedure for conversion
of tri-Boc hydantoin 5 to 3 was developed. Thus, the
above cleavage reaction containing 8 is extracted with
ether to remove Boc2NH and then Fmoc-OSu was added
along with NaOH to keep the pH ∼9, giving 3 in 63%
overall yield from 4-piperidone.15,16
With Fmoc-Pip(Boc)-OH (3) in hand, we prepared three
peptides by automated solid-phase Fmoc chemistry on
PAL-PEG-PS support:17 H-Tyr-Pip-Aib-Phe-Leu-NH2 (9)
and the two sequence permutation isomers H-Aib-Aib-
Pip-Lys-Aib-Aib-Pip-Lys-Aib-Aib-NH2 (10) and H-Pip-
Aib-Aib-Lys-Aib-Aib-Lys-Aib-Aib-Pip-NH2 (11). Peptide
9, a Pip analog of a test peptide used by Carpino,18 was
synthesized with HATU in situ activation.18 Crude
peptide 9 was 80% pure by reversed-phase HPLC analy-
sis. Peptides 10 and 11 were prepared in automated
fashion using preformed acid fluorides.19 This method
worked well for peptide 10,8 producing material that was
36% pure by reversed-phase HPLC; however, the syn-
thesis of peptide 11 failed to give full-length product. This
was traced to problems with the initial three C-terminal
RRAA couplings, perhaps due to steric crowding around
the PAL-linker/resin attachment point. This problem
was alleviated by coupling the first three amino acid
fluorides in refluxing CH2Cl2 for extended periods. The
rest of the synthesis went smoothly with standard auto-
mated acid fluoride couplings, giving a crude peptide 11
that was 78% pure by HPLC. All three peptides 9-11
were readily purified to homogeneity by preparative
HPLC.
Peptides 9-11 were readily soluble in water without
any organic modifier present. Circular dichroism (CD)
studies of the 10-mer peptides 10 and 11 in the presence
of SDS micelles showed very high helicity. Peptide 10,
recently reported to have activity against intracellular
pathogens,8 showed a CD indicative of 43% R-helix,8
consistent with its amphipathic design. Peptide 11, de-
signed to be an amphipathic 310-helix, also showed a
highly helical structure in the presence of SDS micelles,
but in contrast had a unique CD spectum with a strong
negative ellipticity at 206 nm (-5000 θ) and a low 222/
206 intensity ratio (0.32), which is strongly indicative of
a 310-helix.7b Detailed studies of solvent effects on the
solution structures of peptides 10 and 11 and related
sequences will be reported elsewhere.20
We have reported the first suitably protected “polar”
RRAA derivative, Fmoc-Pip(Boc)-OH (3), that can be
readily incorporated into peptides. Access to polar pep-
tides with large percentages of RRAAs should provide
fertile ground for the continuing debate of the relative
stabilities of 310- and R-helices in aqueous media.21
Ack n ow led gm en t. We are grateful to Dr. Atsuo
Kuki for helpful discussions at the early stages of this
work. We thank the National Science Foundation, the
Lousiana Education Quality Support Fund (LEQSF)
and the National Institutes of Health for support of this
research via an NSF/EPSCoR grant [(RII/EPSCoR)-
LEQSF(1992-1996)-ADP-01], an NSF grant (CHE-
9500992, to R.P.H.), and an NIH grant (GM 42101, to
Prof. Mary D. Barkley and M.L.M.). We are grateful
also to LEQSF for a Board of Regents Fellowship to
C.L.W. and to the NSF REU Program (CHE-9424021)
for a summer stipend to R.L.G.
(16) P ip er id in e-4-sp ir o-5′-h yd a n toin (4). A solution of NaCN
(17.0 g, 347 mmol) in H2O (50 mL) added dropwise over 5 min to a
solution of 4-piperidone monohydrate hydrochloride (25.0 g, 163 mmol)
and (NH4)2CO3 (chunks, 34.5 g, 359 mmol) in H2O (90 mL) and CH3-
OH (110 mL). An off-white precipitate began to form soon after addition
was complete. The reaction flask was sealed and the suspension stirred
at room temperature for an additional 2 days. The resultant light
yellow precipitate was isolated by filtration and washed with small
portions of H2O until almost pure white: yield 24.0 g (87%); mp >300
°C. A second crop was obtained from the filtrate by evaporation of most
of the solvent and dilution with H2O (100 mL), filtration, and again
repeated washings with H2O: yield 1.5 g, mp >300 °C. Overall yield:
25.5 g (93%). 1H NMR (250 MHz, CD3SOCD3) δ 10.75 (bs, 1H), 8.46
(s, 1H), 2.83 (app dt, J ≈ 13, 4 Hz, 2H), 2.67 (app dt, J ≈ 12, 2 Hz,
2H), 1.67 (app dt, J ≈ 12, 4 Hz, 2H), 1.39-1.34 (m, 2H). 1-Boc-
p ip er id in e-4-sp ir o-5′-(1′,3′-bis-Boc)h yd a n toin (5). In a flask fitted
with an oil bubbler, piperidine hydantoin (4) (15.3 g, 90.5 mmol) was
suspended in DME (450 mL), and Boc2O (102 g, 468 mmol), DMAP
(0.22 g, 1.64 mmol), and Et3N (12.8 mL, 91.9 mmol) were added in
succession. CO2 evolution was vigorous at the initial addition of DMAP
and continued at a steady pace (1 bubble/20-40 s). After 3 h, an
additional portion of DMAP (0.2 g, 1.64 mmol) was added and the
reaction mixture stirred for an additional 18 h. The mixture was
concentrated under reduced pressure to yield a solid that was taken
up in CH2Cl2 (500 mL), washed with 1 N HCl (2 × 75 mL), saturated
aqueous Na2CO3 (1 × 100 mL), and brine (1 × 100 mL), dried over
anhydrous MgSO4, filtered, and concentrated to yield a light creamy
white solid (42.4 g, quantitative): mp 186-190 °C; 1H NMR (250 MHz,
CDCl3) δ 4.18-4.03 (dd, 1H), 3.38 (bt, 1H), 2.67 (dt, J ) 5.3, 13.1 Hz,
1H), 1.77-1.71 (d, 1H), 1.57 (s, 9H), 1.52 (s, 9H), 1.46 (s, 9H). 1-Boc-
p ip er id in e-4-F m oc-a m in o-4-ca r boxylic Acid (3). 1 N NaOH (287
mL, 287 mmol) was added all at once to a suspension of tri-Boc-
hydantoin 5 (15.0 g, 32.0 mmol) in DME (200 mL), resulting in a
homogeneous solution. After 26 h, the resulting light yellow solution
was extracted with Et2O (3 × 75 mL) to remove Boc2NH. The aqueous
layer containing 4-amino-1-Boc-piperidine-4-carboxylic acid (8) from
above was cooled in an ice bath and the pH adjusted to 9.5 with 12 N
HCl. This precooled solution was added dropwise to a chilled mixture
(ice bath) of Fmoc-OSu (16.0 g, 47.4 mmol) in DME (40 mL). A
precipitate formed immediately, and the reaction mixture was allowed
to warm to room temperature, keeping the pH at 9.0-9.5 by addition
of 1 N NaOH; total reaction time 18 h. The DME was removed in vacuo
(<40 °C), and the resultant aqueous layer was extracted with Et2O (2
× 50 mL) to remove unreacted Fmoc-OSu. The aqueous fraction was
chilled in an ice bath and adjusted to pH 4 with 12 N HCl and extracted
with EtOAc (4 × 250 mL). The combined EtOAc layers were washed
with 1 N HCl (100 mL) and brine (100 mL) and dried (Na2SO4) and
the solvent removed in vacuo to yield a light yellow powder (10.8 g,
87%): mp 80-82 °C; 1H NMR (200 MHz, CD3SOCD3) δ 8.30 (s, 1H),
7.85-7.96 (d, 2H), 7.70-7.80 (d, 2H), 7.22-7.54 (m, 4H), 4.19-4.32
(m, 3H), 3.55-3.76 (m, 2H), 2.90-3.09 (m, 2H), 1.91-2.15 (m, 2H),
1.65-1.89 (m, 2H), d 1.39 (s, 9H).
Su p p or tin g In for m a tion Ava ila ble: Futher details on the
synthesis and characterization of 3-11 (6 pages).
J O961594K
(17) P ep tid e Syn th esis. Solid-phase peptide syntheses were per-
formed on a PerSeptive Biosystems 9050 using Fmoc-PAL-PEG-PS
resin (PerSeptive Biosystems, 0.15 mmol/g loading). Peptide 9 was
prepared exactly according to the HATU protocol described by Carpino
(ref 18) on a 0.1 mmol scale (660 mg resin) using HATU (0.4 mmol, 4
equiv), Fmoc-amino acid (0.4 mmol, 4 equiv), and DIEA (0.8 mmol, 8
equiv) in DMF (1.5 mL, 0.27 M amino acid). Syntheses of peptides 10
and 11 were performed using Fmoc-amino acid fluorides (prepared
according to ref 19a) as suggested by Carpino (ref 19b) with protocols,
cleavage and purification as described by us (ref 18), with the exception
that deblocking was performed with DBU-piperidine-DMF (2:20:80;
1 × 1 min, 1 × 10 min). Also, in the case of H-Pip-Aib-Aib-Lys-Aib-
Aib-Lys-Aib-Aib-Pip-NH2 (11), the first three acid fluorides (1.6 mmol,
8 equiv) with DIEA (0.56 mL, 3.2 mmol, 2 equiv) were coupled off the
machine onto the deblocked resin (1.34 g, 0.2 mmol) in refluxing CH2-
Cl2 (10 mL, 0.16 M of amino acid, 0.32 M DIEA) overnight. After the
resin was washed with CH2Cl2 (4 × 30 mL), deblocking was performed
with DBU-piperidine-DMF (2:20:80; 1 × 1 min, 1 × 10 min) and the
resin washed with CH2Cl2 (5 × 30 s) and coupled to the next acid
fluoride using the same method. After the third coupling, the resin
was placed on the instrument and couplings, cleavage, and purification
were accomplished by the standard methods.
(18) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201-203.
(19) (a) Carpino, L. A.; Aalaee-Sudat, D.; Chao, H. G.; De Selms, R.
H. J . Am. Chem. Soc. 1990, 112, 9651-9652. (b) Wenschuh, H.;
Beyermann, M.; Krause, E.; Brudel, M.; Winter, R.; Schumann, M.;
Carpino, L.; Bienert, M. J . Org. Chem. 1994, 59, 3275-3280.
(20) Yokum, T. S.; Gauthier, T. J .; Hammer, R. P.; McLaughlin, M.
L. J . Am. Chem. Soc., submitted.
(21) (a) Smythe, M. L.; Nakaie, C. R.; Marshall, G. R. J . Am. Chem.
Soc. 1995, 117, 10555-10562. (b) Hanson, P.; Martinez, G.; Millhauser,
G.; Formaggio, F.; Crisma, M.; Toniolo, C.; Vita, C. J . Am. Chem. Soc.
1996, 118, 271-272 and references cited therein.