A peptide/oligourea/azapeptide hybrid that adopts a hairpin turn

Full text of DOI:10.1021/jo980969u

276
J . Org. Chem. 1999, 64, 276-281
tures.9 Lokey and Iverson have reported conceptually
A P ep tid e/Oligou r ea /Aza p ep tid e Hybr id
related polyaromatics that adopt unique π-stacked struc-
Th a t Ad op ts a Ha ir p in Tu r n
tures.10
In this paper, we report the synthesis and structural
studies of peptidomimetic compound 1, a hybrid that
adopts an intramolecularly hydrogen-bonded hairpin
turn. Components of 1 were chosen from the arsenal of
building blocks introduced in the many recent reports of
unnatural oligomers.1,11 These monomeric building blocks
have been used to create a variety of homooligomers
including peptoids,6,12 vinylogous polypeptides,5d â-pep-
tides,2,3 â-peptoids,13 oligocarbamates,14 oligoureas,15-17
azatides,18 ureapeptoids,19,20 oligothioureas,21 oligosul-
fonamides,4,22,23 and oligoethoxyformacetals.24 We antici-
pate that a continuing direction in the unnatural oligo-
mer area will be to mix and match these building blocks
to create structured hybrids, as we demonstrate with 1.
Michael J . Soth and J ames S. Nowick*
Department of Chemistry, University of California, Irvine,
California 92697-2025
Received May 21, 1998
In tr od u ction
The creation of compounds in which noncovalent forces
stabilize secondary structure is an exciting new focus of
peptidomimetic chemistry.1 Seebach et al. and Gellman
et al. have introduced â-peptides, oligomers of â-amino
acids, that adopt intramolecularly hydrogen-bonded he-
lices.2,3 Gennari et al. have reported that sulfonylpeptides
can adopt hydrogen-bonded turns.4 A number of labora-
tories, including our own, have reported compounds that
adopt linear strand conformations.5 Zuckermann et al.
have recently reported helical peptoids.6
These compounds are oligomers built from one type of
monomer. A growing number of reports focus on struc-
tured peptidomimetic compounds built from two or more
types of monomers. Schreiber et al. have reported “hy-
brids” of peptides and vinylogous peptides, one of which
adopts a turn structure and another of which adopts a
helical structure.5d Hamilton et al. have reported helical
hybrids containing oligoanthranilamides.5b,7 We have
been hybridizing various oligomer types to create artifi-
cial â-sheets.8 Gellman et al. have recently prepared
â-peptide/depsipeptide hybrids that adopt sheet struc-
Hairpin 1 is a hybrid of three oligomer types:
a
peptide, an oligourea,15-17 and an azapeptide (Scheme
(8) (a) Smith, E. M.; Holmes, D. L.; Shaka, A. J .; Nowick, J . S. J .
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* To whom correspondence should be addressed. E-mail: jsnowick@
uci.edu.
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10.1021/jo980969u CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/19/1998

Notes
J . Org. Chem., Vol. 64, No. 1, 1999 277
Sch em e 1
Sch em e 2. Syn th esis of Ha ir p in 1a
1).18,25 The design of 1 relies on the propensity of ureas
linked by two-carbon spacers to adopt hydrogen-bonded
turn structures.8,17,26 The expected turn conformation of
1 has side chains in relative positions similar to those in
a peptide â-turn, an important element of protein struc-
ture and a popular target for mimicry.27 Hairpin turns
are of special interest28,29 because they can serve as
nucleators of antiparallel â-sheet structure; in addition,
hairpins are often sites for molecular recognition of
proteins.30
a
Key: (a) (i) Boc2O, MeOH, (ii) CbzCl, CH2Cl2/saturated
aqueous NaOH, (iii) HCl, MeOH (68%); (b) (i) COCl2, CH2Cl2/
saturated aqueous NaHCO3, (ii) L-prolinol, CH2Cl2/saturated
aqueous NaHCO3 (92%); (c) (i) MsCl, THF, 0 °C, (ii) NaN3, DMF,
64 °C (60%); (d) PPh3, H2O, THF, ∆; (e) (CH3)2CO, NaCNBH3,
MeOH; (f) L-leucine methyl ester isocyanate, CH2Cl2 (76%, three
steps); (g) MeNH2, MeOH (93%).
in methanol to furnish the hydrochloride salt 2.31 Al-
though this protection procedure involves three steps, the
overall process is quick and convenient, requiring only a
few hours. The Cbz-protected hydrazine 2 was converted
to the corresponding carbamoyl chloride 3,17a,32 and the
crude carbamoyl chloride was coupled with L-prolinol to
generate alcohol 4, which was sufficiently pure to be used
without purification. Alcohol 4 was converted to the
corresponding mesylate by reaction with methanesulfonyl
chloride, and the mesylate was converted to azide 5 by
reaction with sodium azide. Azide 5 was reduced under
Staudinger conditions, the resulting primary amine 6 was
reductively alkylated with acetone to introduce an iso-
propyl substituent, and the reductive alkylation product
(7) was coupled with L-leucine methyl ester isocyanate
to generate diurea 8.33 Diurea 8 was converted to hairpin
1 by aminolysis with methylamine.
Resu lts a n d Discu ssion
Syn th esis of Ha ir p in 1. The synthesis of hairpin 1
is shown in Scheme 2. Methylhydrazine was Boc-
protected on its substituted end, the remaining end was
Cbz-protected, and the Boc group was removed with HCl
(23) de Bont, D. B. A.; Moree, W. J .; Liskamp, R. M. J . Biorg. Med.
Chem. 1996, 4, 667.
(24) Hall, D. G.; Schultz, P. G. Tetrahedron Lett. 1997, 38, 7825.
(25) For a review on azapeptides, see: Gante, J . Synthesis 1989,
405.
(26) Nowick, J . S.; Abdi, M.; Bellamo, K.; Love, J . A.; Martinez, E.
J .; Noronha, G.; Smith, E. M.; Ziller, J . W. J . Am. Chem. Soc. 1995,
117, 89.
(27) (a) Giannis, A.; Kolter, T. Angew. Chem., Int. Ed. Engl. 1993,
32, 1244. (b) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bo¨s, M.; Cook.
C. M.; Fry, D. C.; Graves, B. J .; Hatada, M.; Hill, D. E.; Kahn, M.;
Madison, V. S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall, J .; Vincent, G.
P.; Voss, M. E. J . Med. Chem. 1993, 36, 3039. (c) Rose, G. D.; Gierasch,
L. M.; Smith, J . A. Adv. Protein Chem. 1985, 37, 1.
Str u ctu r a l Stu d ies of Ha ir p in 1. 1H NMR chemical
shift, variable-temperature (VT), and nuclear Overhauser
effect (NOE) studies provide evidence that 1 adopts a
hairpin conformation in CDCl3 solution. In the chemical
shift studies, compounds 9 and 10 were used as controls
for the upper (leucine) and lower (methylhydrazine)
(28) Blanco, F.; Ramirez-Alvarado, M.; Serrano, L. Curr. Opin.
Struct. Biol. 1998, 8, 107.
(29) (a) Schopfer, U.; Stahl, M.; Brandl, T.; Hoffmann, R. W. Angew.
Chem., Int. Ed. Engl. 1997, 36, 1745. (b) Gardner, R. R.; Liang, G.-B.;
Gellman, S. H. J . Am. Chem. Soc. 1995, 117, 3280.
(30) Recent examples: (a) Bjo¨rk, I.; Brieditis, I.; Raub-Segall, E.;
Pol, E.; Håkansson, K.; Abrahamson, M. Biochemistry 1996, 35, 10720.
(b) Machleidt, W.; Na¨gler, D. K.; Assfalg-Machleidt, I.; Stubbs, M. T.;
Fritz, H.; Auerswald, E. A. FEBS Lett. 1995, 361, 185. (c) Auerswald,
E. A.; Na¨gler, D. K.; Assfalg-Machleidt, I.; Stubbs, M. T.; Machleidt,
W.; Fritz, H. FEBS Lett. 1995, 361, 179. (d) Ilag, L. L.; Lo¨nnerberg,
P.; Persson, H.; Iba´n˜ez, C. F. J . Biol. Chem. 1994, 269, 19941. (e)
Mizuno, K.; Inoue, H.; Hagiya, M.; Shimizu, S.; Nose, T.; Shimohigashi,
Y.; Nakamura, T. J . Biol. Chem. 1994, 269, 1131.
(31) A similar but less efficient preparation of 2 has been reported:
Dutta, A. S.; Morley, J . S. J . Chem. Soc., Perkin Trans. 1 1975, 1712.
(32) Andre´, F.; Marraud, M.; Boussard, G.; Didierjean, C.; Aubry,
A. Tetrahedron Lett. 1996, 37, 183.
(33) (a) Nowick, J . S.; Holmes, D. L.; Noronha, G.; Smith, E. M.;
Nguyen, T. M.; Huang, S. L. J . Org. Chem. 1996, 61, 3929. (b) Nowick,
J . S.; Powell, N. A.; Nguyen, T. M.; Noronha, G. J . Org. Chem. 1992,
57, 7364.

278 J . Org. Chem., Vol. 64, No. 1, 1999
Notes
Ta ble 1. NOESY Cr oss-P ea k s Obser ved for Ha ir p in 1a -c
proton
Ha
Hb
Hc
Hd
He
NOESY cross-peaksd
Hb (w), Hh (m), Hi′ (m), Hl (w), Hm/m′/n (m)
Ha (w), Hd (w), He (w), He′ (w), Hm/m′/n (w)
Hj (w), Hl (s), Hm/m′/n (w), Hp (s)
Hb (w)
Hb (w), Hf/f ′ (m), Hh (w)
Hb (w), Hf/f ′ (m)
He (m), He′ (m), Hg′ (m)
Hh (m)
Hf/f ′ (m), Hi (m), Hj (w), Hk (m)
Ha (m), He (w), Hg (m), Hi (m), Hi′ (m)
Hg′ (m), Hh (m), Hk (m), Hk′ (m)
Ha (m), Hh (m)
Hc (w), Hg′ (w), Hk (s), Hk′ (s)
Hg′ (m), Hi (m), Hj (s), Hk′ (s)
Hi (m), Hj (s), Hk (s)
Ha (w), Hc (s), Hm/m′/n (s), Ho (m), Ho′ (m)
Ha (m), Hc (w), Hb (w), Hl (s)
Hl (m), Hm/m′/n (s)
He′
Hf/Hf ′
Hg
Hg′
Hh
Hi
e
Hi′
Hj
Hk
Hk′
Hl
F igu r e 1. NH chemical shifts for hairpin 1 and controls 9
and 10 (1 mM solution in CDCl3, 295 K).
e
strands of 1, respectively (Figure 1).34 1H NMR spectra
for 1, 9, and 10 were acquired at 1 mM in CDCl3 solution,
at which concentration these compounds are negligibly
self-associated.35 The spectrum for control 10 shows two
conformers, with two different NH chemical shift values
(6.59 and 6.35 ppm in 1 mM CDCl3). These conformers
presumably have extended (urea carbonyl trans to the
hydrazine methyl) and folded (urea carbonyl cis to the
hydrazine methyl) conformations.25,36
H
m/Hm′/Hn
Ho
Ho′
Hp
Hl (m), Hm/m′/n (s)
Hc (s)
These 1H NMR chemical shift studies indicate that the
urea and hydrazine NH protons of 1 are intramolecularly
hydrogen bonded in 1 mM CDCl3 solution (Figure 1). The
urea resonance of 1 (Ha) is shifted 1.7 ppm downfield from
the corresponding resonance of control 9. The hydrazine
resonance of 1 (Hb) is shifted 2.1-2.3 ppm downfield from
the corresponding resonance of control 10. In contrast,
the amide resonance of 1 (Hc) is shifted only 0.2 ppm
downfield from the corresponding resonance of control
9. These downfield shifts are consistent with a hairpin
structure in which the urea and hydrazine NH protons
are intramolecularly hydrogen bonded, and the amide
NH proton is not hydrogen bonded.
a
b
10 mM solution in CDCl3, 295 K. Protons of the benzyl group
do not show any cross-peaks to protons of the rest of the molecule
and are not listed in this table. c Geminal NOEs are not listed.
d
Cross-peaks were identified as strong (s), medium (m), or weak
(w) on the basis of their relative intensities. e These protons have
overlapping resonances. f For this structure, Hm was arbitrarily
chosen from the Hm/m′/n set of overlapping resonances; the NOESY
cross-peak with Hb may be for any or all of these protons, without
changing conclusions on interstrand proximity.
1H NMR VT studies offer additional insight on the
hydrogen-bonding experienced by the NH protons of 1.
In 1 mM chloroform solution over a range of 294-330 K,
1
the H NMR chemical shifts of the urea resonance (Ha)
Two-dimensional proton magnetic resonance nuclear
Overhauser effect (NOESY) studies provide compelling
evidence for the hairpin turn conformation of 1 (Table
1).35 Most significantly, both the urea proton (Ha) and
the leucine â-/γ-protons (Hm, Hm′, and/or Hn) show NOE
cross-peaks with the hydrazine proton (Hb).38,39 These
NOEs demonstrate the proximity of the leucine and
hydrazine strands. Ha also shows NOE cross-peaks with
backbone protons Hh and Hi′, but not with any protons
of the isopropyl group. These NOEs provide evidence for
the turn portion of the hairpin.
and the amide resonance (Hc) show a small temperature
dependence (-0.4 and -1.8 ppb/K, respectively). In a
noncompetitive solvent, a small temperature dependence
indicates that a proton is either completely hydrogen-
bonded or completely non-hydrogen-bonded.8b,37 The chemi-
cal shift of the hydrazine resonance (Hb) shows a large
temperature dependence (-13.3 ppb/K). In a noncompeti-
tive solvent, a large temperature dependence indicates
that a proton participates in an equilibrium between
hydrogen-bonded and non-hydrogen-bonded states.8b,37
The VT and chemical shift data collectively indicate that
the urea proton is completely hydrogen bonded, the
amide proton is not hydrogen bonded, and the hydrazine
proton is largely, but not completely, hydrogen bonded.
The leucine R-proton (Hl) shows a strong NOE cross-
peak with the amide proton (Hc) and only a weak NOE
cross-peak with the urea proton (Ha). This NOE pattern
is consistent with an extended strand conformation for
(34) Compound 9 has been previously reported: Insaf, S.; Nowick,
J . S. J . Am. Chem. Soc. 1997, 119, 10903.
(38) The 1H NMR resonances of the leucine â-CH2 and γ-CH overlap.
For this reason, it is not possible to distinguish which of these leucine
side-chain protons are involved in NOEs.
(39) The hydrazine NHb resonance was broad, and all NOESY cross-
peaks involving this resonance were weak. To corroborate these weak
NOEs, we performed one-dimensional difference NOE experiments.
These experiments were performed in triplicate to ensure that all
enhancements were real and not subtraction artifacts. The same NOEs
were seen as in the NOESY experiment. Upon irradiation of NHb, the
(35) 1H NMR chemical shift studies, in which the chemical shifts of
the amide and hydrazine NH’s of 1 were measured at varying
concentrations (1-100 mM range) in CDCl3 solution, indicate that 1
self-associates with an estimated Kdim of 5 M-1. Thus, 1 is ca. 1% self-
associated in 1 mM solution and ca. 10% self-associated in 10 mM
solution (NOE studies were performed at 10 mM).
(36) Greenlee, W. J .; Thorsett, E. D.; Springer, J . P.; Patchett, A.
A. Biochem. Biophys. Res. Commun. 1984, 122, 791.
(37) Stevens, E. S.; Sugawara, N.; Bonora, G. M.; Toniolo, C. J . Am.
Chem. Soc. 1980, 102, 7048.
following enhancements were observed: urea NHa (1%), leucine â-CHm
/
â-CHm′/γ-CHn (0.8%), hydrazine C(Hd)3 (1.7%), pyrrolidine CHe (1.9%),
and pyrrolidine CHe′ (1.7%).

Notes
J . Org. Chem., Vol. 64, No. 1, 1999 279
F igu r e 2. Possible minor conformation of 1.
the leucine portion of 1. Diastereotopic protons Hi and
Hi′ exhibit different sets of NOEs: Hi shows NOE cross-
peaks with the isopropyl methyl groups (Hk and Hk′) and
with pyrrolidine ring proton Hg, while Hi′ shows an NOE
cross-peak with the urea proton (Ha). This observation
suggests that Hi and Hi′ are in completely different
environments and is consistent with the proposed turn
conformation, in which these protons are on different
faces of a hydrogen-bonded ring.
Three NOEs are not consistent with a hairpin turn
structure. The hydrazine proton (Hb) shows weak NOE
cross-peaks with pyrrolidine ring protons He and He′.
These NOEs suggest the conformer shown in Figure 2;
this conformer must be minor, because the chemical shift
studies indicate that the hydrazine NH is hydrogen-
bonded.40 The amide proton (Hc) shows an NOE cross-
peak with isopropyl proton Hj. This NOE is very weak,
but it furthers the point that nonhairpin conformations
are present.
To gain further insight into the structure of the major
conformer of 1, we attempted to generate a model using
MacroModel41 and the AMBER*42 force field. This force
field has poor parameters for the diacylhydrazine func-
tional group and did not allow the generation of a local
minimum of hairpin 1 in which both hydrogen bonds
were present. We attribute the failure to achieve a
hydrogen-bonded conformation to the force field’s strong
torsional bias toward a planar geometry of the diacylhy-
drazine functional group. Crystallographic studies of
related methylhydrazine-pyrrolidine ureas32,36 and theo-
retical studies of diacylhydrazines43 indicate that these
groups should adopt nonplanar conformations. When we
constrained the diacylhydrazine group to a twisted
geometry, a hairpin was obtained in which both hydrogen
bonds were present (Figure 3). This model is consistent
with the NOEs attributed to the major conformer.
F igu r e 3. Model of hairpin 1 in a minimum energy conforma-
tion (local minimum). The benzyl group and leucine side chain
are truncated for clarity. The model was generated using
MacroModel V5.5 and the AMBER* force field; the diacylhy-
drazine group was constrained to a twisted (90°) geometry by
constraining its C(O)-N-N-C(O), C(O)-N-N-H, CH3-N-
N-C(O), and CH3-N-N-H torsions.
tidomimetic compounds. Three different types of oligo-
mers were hybridized to create 1. The chemical shift, VT,
and NOESY experiments indicate that 1 adopts a tight
simple turn (containing one intramolecular hydrogen
bond), with a large population also adopting a hairpin
turn (containing two intramolecular hydrogen bonds).
We are currently developing solid-phase syntheses for
analogues of 1. This structure is a promising scaffold for
combinatorial chemistry, since it is made in a modular
synthesis from readily varied building blocks. We will
report structurally and biologically interesting analogues
as they are discovered.
Exp er im en ta l Section
Gen er a l Meth od s. Commercially available reagents and
solvents were used without further purification. Tetrahydrofuran
was distilled from sodium and benzophenone under nitrogen.
Phosgene was obtained from Fluka as a 20% (1.93 M) solution
in toluene. High-resolution mass spectra (HRMS) were obtained
by electron ionization (EI) at 70 eV, chemical ionization (CI)
using isobutane or ammonia, or liquid secondary ion mass
spectrometry (LSIMS) of samples in a m-nitrobenzyl alcohol
matrix bombarded with Cs+ ions at 25 kV (instrumental varia-
tion σ ) (2 mmu). Combustion analyses were performed by
Desert Analytics, Tucson, AZ.
NOESY and NOE studies on hairpin 1 were performed using
a 10 mM sample in CDCl3 (solvent was predried over basic
alumina) that was degassed by three freeze-pump-thaw cycles
Con clu sion
on
a high-vacuum line (<0.001 mmHg) and sealed under
vacuum. Peak assignments used in the interpretation of NOE
data were based on COSY spectra.
This work demonstrates the potential of a “mix and
match” strategy toward the creation of structured pep-
P r otected Hyd r a zin e 2.31 A solution of methyl hydrazine
(1.4 mL, 26 mmol) and di-tert-butyl dicarbonate (4.32 g, 19.8
mmol) in 25 mL of methanol was stirred for 30 min and then
concentrated by rotary evaporation to 2.99 g of a yellow liquid.
Dichloromethane (20 mL) and 20 mL of a 1 M NaOH solution
were added, and the mixture was rapidly stirred as benzyl
chloroformate (2.8 mL, 20 mmol) was added in one portion. The
mixture was rapidly stirred for 1 h, the layers were separated,
and the organic layer was sequentially washed with 20 mL of a
1 M HCl solution, 20 mL of water, and 20 mL of a saturated
aqueous NaCl solution, dried over MgSO4, filtered, and concen-
trated by rotary evaporation to 5.12 g of a yellow oil. The oil
was dissolved in 40 mL of methanol, and the solution was chilled
in an ice bath for ca. 15 min. Acetyl chloride (10 mL) was added
(40) Most of the peaks in the 1H NMR spectra of 1 are ill-resolved,
suggesting that the rate of interconversion between conformations is
intermediate or slow on the NMR time scale. This possibility appears
very likely considering that the 1H NMR spectra for control 10 show
two conformers.
(41) Macromodel V5.5: Mohamadi, F.; Richards, N. G. J .; Guida,
W. C.; Liskamp, R.; Lipton, M.; Caulfield, C.; Chang, G.; Hendrickson,
T.; Still, W. C. J . Comput. Chem. 1990, 11, 440.
(42) (a) McDonald, D. Q.; Still, W. C. Tetrahedron Lett. 1992, 33,
7743. (b) McDonald, D. Q.; Still, W. C. Tetrahedron Lett. 1992, 33, 7747.
(43) (a) Reynolds, C. H.; Hormann, R. E. J . Am. Chem. Soc. 1996,
118, 9395. (b) Graybill, T. L.; Ross, M. J .; Gauvin, B. R.; Gregory, J .
S.; Harris, A. L.; Ator, M. A.; Rinker, J . M.; Dolle, R. E. Bioorg. Med.
Chem. Lett. 1992, 2, 1375.

280 J . Org. Chem., Vol. 64, No. 1, 1999
Notes
over 30 s, the ice bath was removed, and the solution was stirred
for 20 min. The solution was then concentrated by rotary
evaporation to a white solid, which was recrystallized from 15
mL of 2-propanol to afford 2.89 g (68%) of 2 as white flakes:
mp 165-166 °C; IR (KBr) 3462, 3145, 3007, 2962, 2789, 2700,
of a 1 N NaOH solution, and then extracted with six 20 mL
portions of dichloromethane. The combined dichloromethane
layers were dried over MgSO4, filtered, and concentrated by
rotary evaporation to afford 0.324 g (87%) of amine 6 as a
colorless oil, which was used without further purification: 1H
NMR (300 MHz, CDCl3) δ 8.05 (br s, 1 H), 7.34 (s, 5 H), 5.14 (s,
2 H), 4.03 (m, 1 H), 3.38-3.36 (m, 1 H), 3.24-3.19 (m, 1 H),
3.01 (s, 3 H), 2.85-2.78 (m, 1 H), 2.60-2.56 (m, 1 H), 1.89-1.95
(m, 1 H), 1.77-1.46 (m, 5 H).
1
2416, 1741, 1551 cm-1; H NMR (500 MHz, DMSO-d6) δ 11.48
(br s, 2 H), 10.80 (s, 1 H), 7.44-7.37 (m, 5 H), 5.19 (s, 2 H), 2.73
(s, 3 H); 13C NMR (500 MHz, DMSO-d6) δ 154.8, 135.6, 128.5,
128.3, 128.1, 67.2, 35.6; HRMS (EI) m/e for C9H12N2O2 (M)+,
calcd 180.0900, found 180.0896. Anal. Calcd for C9H13ClN2O2:
C, 49.89; H, 6.05; Cl, 16.36; N, 12.93. Found: C, 49.80; H, 6.36;
Cl, 16.04; N, 12.85.
Alcoh ol 4. A 1.93 M solution of phosgene in toluene (12 mL,
24 mmol) was added to a rapidly stirred, ice-cooled mixture of
hydrazine 2 (1.02 g, 4.72 mmol), 25 mL of dichloromethane, and
25 mL of a saturated aqueous NaHCO3 solution. The mixture
was rapidly stirred at 0 °C for 20 min. The layers were separated,
the aqueous layer was extracted with 25 mL of dichloromethane,
and the combined organic layers were dried over MgSO4, filtered,
and concentrated by rotary evaporation to afford 1.121 g (4.63
mmol) of crude carbamoyl chloride 3, which was used im-
mediately.
To a solution of amine 6 (0.324 g, 1.06 mmol) in 10 mL of
methanol were added acetone (0.80 mL, 11 mmol) and sodium
cyanoborohydride (0.065 g, 1.03 mmol). After 21 h, the colorless
solution was concentrated by rotary evaporation to a colorless
oil, which was partitioned between 20 mL of diethyl ether and
20 mL of a saturated aqueous NaCl solution. The aqueous layer
was extracted with 20 mL of diethyl ether, and the combined
ether layers were dried over MgSO4, filtered, and concentrated
by rotary evaporation to afford 0.364 g (98%) of amine 7 as a
colorless oil, which was used without further purification: 1H
NMR (300 MHz, CDCl3) δ 7.36 (appar s, 6 H), 5.16 (s, 2 H), 4.12
(br s, 1 H), 3.40 (br s, 1 H), 3.36-3.29 (m, 1 H), 3.03 (s, 3 H),
2.85 (br s, 2 H), 2.72 (appar d, J ) 3.7 Hz, 2 H), 2.06-2.01 (m,
1 H), 1.83-1.80 (m, 1 H), 1.70-1.62 (m, 2 H), 1.08 (br s, 6 H).
A 1.93 M solution of phosgene in toluene (1.6 mL, 3.1 mmol)
was added to a rapidly stirred, ice-cooled mixture of L-leucine
methyl ester hydrochloride (0.271 g, 1.49 mmol), 10 mL of
dichloromethane, and 10 mL of a saturated aqueous NaHCO3
solution. The mixture was rapidly stirred for 20 min. The layers
were separated, the aqueous layer was extracted with 10 mL of
dichloromethane, and the combined organic layers were dried
over MgSO4, filtered, and concentrated by rotary evaporation
to a colorless oil (crude leucine methyl ester isocyanate). The
oil was dissolved in 10 mL of dichloromethane, and the solution
was added to amine 7 (0.364 g, 1.04 mmol). After 21 h, the
colorless solution was concentrated by rotary evaporation, and
the resultant oil was purified by column chromatography (2:1
EtOAc/hexanes) to afford 0.481 g (76% three steps) of 8 as a
The oil was dissolved in 25 mL of dichloromethane and 25
mL of a saturated aqueous NaHCO3 solution. L-Prolinol (0.483
g, 4.78 mmol) was added in 5 mL of dichloromethane, and the
mixture was rapidly stirred for 24 h. The layers were separated,
the aqueous layer was extracted with 25 mL of dichloromethane,
and the combined organic layers were dried over MgSO4, filtered,
and concentrated by rotary evaporation to afford 1.34 g (92%)
of 4 as a colorless oil: IR (neat) 3431, 3244, 1722, 1632 cm-1
;
1H NMR (300 MHz, CDCl3) δ 7.72 (s, 1 H), 7.34 (s, 5 H), 5.14 (s,
2 H), 4.10-4.00 (m, 1 H), 3.81 (br s, 1 H), 3.67 (br s, 1 H), 3.40-
3.36 (m, 2 H), 3.22-3.18 (m, 1 H), 3.00 (s, 3 H), 1.95-1.82 (m,
1 H), 1.76-1.68 (m, 1 H), 1.64-1.62 (m, 2 H); 13C NMR (500
MHz, CDCl3) δ 161.5, 155.3, 135.5, 128.6, 128.5, 128.3, 67.8, 64.0,
61.0, 49.8, 39.1, 27.5, 25.2; HRMS (CI) m/e for C15H22N3O4 (M
+ H)+, calcd 308.1611, found 308.1597. An analytical sample
was further purified by column chromatography (2:1 EtOAc/
hexanes). Anal. Calcd for C15H21N3O4: C, 58.62; H, 6.89; N,
13.67. Found: C, 58.36; H, 7.05; N, 13.37.
foamy white solid: IR (CH2Cl2) 3415, 3316, 1740, 1642 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.45 (br s, 1 H), 7.36 (s, 5 H), 6.96
(br s, 1 H), 5.19 (d, AB pattern, J ) 11.5 Hz, 1 H), 5.14 (d, AB
pattern, J ) 11.8 Hz, 1 H), 4.42 (br s, 1 H), 4.37-4.35 (m, 1 H),
4.24 (br s, 1 H), 3.70 (s, 3 H), 3.45-3.33 (m, 2 H), 3.25 (br s, 1
H), 3.04 (s, 3 H), 2.70 (br s, 1 H), 2.20-2.13 (m, 1 H), 1.84-1.75
(m, 4 H), 1.58-1.44 (m, 2 H), 1.14 (appar d, J ) 5.4 Hz, 3 H),
1.03 (br s, 3 H), 0.94 (appar d, J ) 6.5 Hz, 3 H), 0.91 (m, 3 H);
13C NMR (500 MHz, CDCl3) δ 176.0, 161.7, 158.8, 155.1, 135.8,
128.5, 128.4, 128.2, 67.5, 58.7, 52.7, 51.9, 49.5, 46.6, 46.0, 40.3,
38.5, 31.1, 25.5, 24.8, 23.1, 21.5, 21.4, 20.2; HRMS (LSIMS) m/e
for C26H42N5O6 (M + H)+, calcd 520.3137, found 520.3134. Anal.
Calcd for C26H41N5O6: C, 60.10; H, 7.95; N, 13.48. Found: C,
60.08; H, 7.94; N, 13.56.
Azid e 5. A solution of alcohol 4 (0.605 g, 1.97 mmol), 20 mL
of tetrahydrofuran, and triethylamine (0.830 mL, 5.95 mmol)
was chilled in an ice bath for ca. 15 min. Methanesulfonyl
chloride (0.305 mL, 3.94 mmol) was added in one portion; a white
precipitate immediately started to form. The suspension was
stirred for 20 min, diluted with 60 mL of dichloromethane, and
sequentially washed with two 50 mL portions of a saturated
aqueous NaHCO3 solution and 50 mL of a saturated aqueous
NaCl solution. The dichloromethane layer was dried over
MgSO4, filtered into a round-bottomed flask, and concentrated
by rotary evaporation to 0.783 g (2.0 mmol crude) of a colorless
viscous oil. To the flask were added sodium azide (0.769 g, 11.8
mmol) and 20 mL of dimethylformamide, and the suspension
was stirred, heating to 64 °C, for 16 h. The suspension was
diluted with 50 mL of diethyl ether and then sequentially
washed with 100 mL of water, three 50 mL portions of water,
and 50 mL of a saturated aqueous NaCl solution. The ether layer
was dried over MgSO4, filtered, and concentrated by rotary
evaporation to 0.469 g of a pale yellow oil, which was purified
by column chromatography (1.5:1 hexanes/EtOAc), affording
0.391 g (60%) of 5 as a colorless oil: IR (neat) 3248, 2102, 1736,
Ha ir p in 1. Urea 8 (0.154 g, 0.297 mmol) was dissolved in 5
mL of a methylamine in methanol solution (10 M, 5 mmol). After
4 days, the colorless solution was concentrated by rotary
evaporation to 0.151 g of a glassy solid, which was further
purified by column chromatography (19:1 EtOAc/MeOH) to
afford 0.143 g (93%) of 1 as a foamy white solid: IR (5 mM
CHCl3) 3448, 3303, 1735, 1660, 1645 cm-1; 1H NMR (500 MHz,
CDCl3) δ 8.86 (br s, 1 H), 7.36-7.30 (m, 5 H), 6.69 (s, 1 H), 6.38
(s, 1 H), 5.22 (d, AB pattern, J ) 12.1 Hz, 1 H), 5.15 (d, AB
pattern, J ) 12.1 Hz, 1 H), 4.37-4.33 (m, 2 H), 4.20-4.10 (m, 1
H), 3.43 (br s, 1 H), 3.29-3.20 (m, 1 H), 3.23 (dd, J ) 15.5 Hz,
6.8 Hz, 1 H), 3.07 (s, 3 H), 2.89 (appar d, J ) 13.4 Hz, 1 H), 2.77
(d, J ) 4.8 Hz, 3 H), 1.99-1.97 (m, 1 H), 1.84-1.79 (m, 2 H),
1.60-1.53 (m, 3 H), 1.53-1.46 (m, 1 H), 1.19 (d, J ) 6.1 Hz, 3
H), 1.05 (d, J ) 5.8 Hz, 3 H), 0.85 (d, J ) 4.5 Hz, 3 H), 0.81 (d,
J ) 4.7 Hz, 3 H); 13C NMR (500 MHz, CDCl3) δ 175.2, 162.5,
159.2, 155.9, 135.9, 128.4, 128.2, 128.1, 67.2, 59.1, 53.2, 49.5,
47.1, 46.9, 40.7, 38.1, 29.6, 26.2, 25.4, 24.8, 22.9, 21.9, 21.1, 19.8;
HRMS (LSIMS) m/e for C26H43N6O5 (M + H)+, calcd 519.3298,
found 519.3297. Anal. Calcd for C26H42N6O5: C, 60.21; H, 8.16;
N, 16.20. Found: C, 60.03; H, 8.24; N, 16.12.
1
1632 cm-1; H NMR (300 MHz, CDCl3) δ 7.26 (s, 5 H), 6.82 (s,
1 H), 5.17 (s, 2 H), 4.16 (br s, 1 H), 3.56 (br s, 1 H), 3.43 (br s, 1
H), 3.36-3.29 (m, 1 H), 3.24 (appar d, J ) 11.5 Hz, 1 H), 3.05
(s, 3 H), 2.04-1.98 (m, 1 H), 1.85-1.81 (m, 1 H), 1.73-1.81 (m,
2 H); 13C NMR (500 MHz, CDCl3) δ 160.7, 155.2, 135.6, 128.5,
128.4, 128.2, 67.6, 58.1, 52.4, 49.6, 38.9, 28.0, 25.3; HRMS
(LSIMS) m/e for C15H21N6O3 (M + H)+, calcd 333.1677, found
333.1686. Anal. Calcd for C15H20N6O3: C, 55.16; H, 6.94; N,
24.12. Found: C, 54.88; H, 6.92; N, 23.93.
Ur ea 8. A solution of azide 5 (0.405 g, 1.22 mmol), 10 mL of
tetrahydrofuran, triphenylphosphine (0.638 g, 2.43 mmol), and
water (0.045 mL, 2.5 mmol) was heated to reflux, stirred for 6
h, and then concentrated by rotary evaporation to a pale yellow
liquid, which was dissolved in 25 mL of diethyl ether and 25
mL of a 0.1 N HCl solution. The aqueous layer was extracted
with two 25 mL portions of diethyl ether, made basic with 5 mL
Con tr ol 10. A 1.93 M solution of phosgene in toluene (1.0
mL, 1.93 mmol) was added to a rapidly stirred mixture of
hydrazine 2 (0.100 g, 0.461 mmol), 5 mL of dichloromethane,
and 5 mL of a saturated aqueous NaHCO3 solution. The mixture
was vigorously stirred for 30 min. The layers were separated,

Notes
J . Org. Chem., Vol. 64, No. 1, 1999 281
and the aqueous layer was further extracted with 5 mL of
dichloromethane. The combined organic layers were dried over
MgSO4, filtered, and concentrated by rotary evaporation to a
colorless liquid (carbamoyl chloride 3). To the liquid was added
5 mL of dichloromethane, 5 mL of a saturated aqueous NaHCO3
solution, and pyrrolidine (0.077 mL, 0.926 mmol). The mixture
was vigorously stirred for 24 h, and then the layers were
separated. The dichloromethane layer was sequentially washed
with 5 mL of a 1 M HCl solution and 5 mL of a saturated
aqueous NaCl solution, dried over MgSO4, filtered, and concen-
trated by rotary evaporation to afford 0.106 g (83%) of 10 as a
colorless oil, which slowly solidified to a white solid: mp 69-70
°C; IR (5 mM CHCl3) 3427, 3377, 1741, 1645 cm-1; 1H NMR (500
MHz, 5 mM CDCl3) δ 7.36 (5 H), 6.61 (br s, 0.6 H), 6.38 (br s,
0.4 H), 5.18 (br s, 2 H), 3.35 (br s, 4 H), 3.05 (s, 3 H), 1.78 (br s,
4 H); 13C NMR (500 MHz, CDCl3) δ 160.5, 155.4, 135.8, 128.5,
128.4, 128.2, 67.7, 67.5, 58.5, 48.0, 39.2, 25.4; HRMS (CI) m/e
for C14H20N3O3 (M + H)+, calcd 278.1506, found 278.1496. Anal.
Calcd for C14H19N3O3: C, 60.63; H, 6.91; N, 15.15. Found: C,
60.91; H, 6.58; N, 15.11.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health Grant GM-49076. J .S.N.
thanks the following agencies for support in the form
of awards: the National Science Foundation (Presiden-
tial Faculty Fellow Award), the Camille and Henry
Dreyfus Foundation (Teacher-Scholar Award), and the
Alfred P. Sloan Foundation (Alfred P. Sloan Research
Fellowship).
Su p p or tin g In for m a tion Ava ila ble: One-dimensional,
1
COSY, and NOESY H NMR spectra for hairpin 1 (7 pages).
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