The urea-dipeptides show stronger H-bonding propensity to nucleate β-sheetlike assembly than natural sequence

Article abstract of DOI:10.1016/j.tet.2009.07.048

In this article, we report the distinct solution behavior of a set of urea-dipeptides to that of natural sequence. The urea-dipeptides adopt β-folding conformations and form into β-sheetlike assembly in chloroform. Most surprisedly, the urea-dipeptides tend to form interpeptide H-bonding interactions even at a concentration of as low as 0.1 mM, while the natural sequence shows H-bonding propensity at a concentration of about 7 mM, indicating that the urea-dipeptides show much stronger H-bonding propensity to nucleate formation of β-sheetlike assembly than the natural sequence. CD spectra reveal that the investigated urea-dipeptides have two negative CD bands, respectively, around 217 nm and 224 nm, supporting the β-folding conformations and in turn formation of β-sheetlike assembly. The β-sheetlike assembly is also confirmed by the XRD reflections, which give two typical d-spacings of 12.7 and 4.8 A?, respectively, corresponding to stacking periodicity of the β-sheets and the spacing between peptide backbones running orthogonal to the β-sheet axis.

Full text of DOI:10.1016/j.tet.2009.07.048

Tetrahedron 65 (2009) 8269–8276
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
The urea-dipeptides show stronger H-bonding propensity to nucleate
assembly than natural sequence
b-sheetlike
,
,
a
,
,
Damei Ke a b, Chuanlang Zhan a , Xiao Li b, Alexander D.Q. Li c, Jiannian Yao a
*
*
a Beijing National Laboratory of Molecular Science (BNLMS), Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China
b Graduate School, Chinese Academy of Sciences, Beijing 100039, PR China
c Department of Chemistry, Washington State University, Pullman, WA 99164, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In this article, we report the distinct solution behavior of a set of urea-dipeptides to that of natural se-
Received 27 April 2009
Received in revised form 20 July 2009
Accepted 22 July 2009
quence. The urea-dipeptides adopt
b-folding conformations and form into b-sheetlike assembly in
chloroform. Most surprisedly, the urea-dipeptides tend to form interpeptide H-bonding interactions even
at a concentration of as low as 0.1 mM, while the natural sequence shows H-bonding propensity at
a concentration of about 7 mM, indicating that the urea-dipeptides show much stronger H-bonding
Available online 25 July 2009
propensity to nucleate formation of
that the investigated urea-dipeptides have two negative CD bands, respectively, around 217 nm and
224 nm, supporting the -folding conformations and in turn formation of -sheetlike assembly. The
-sheetlike assembly is also confirmed by the XRD reflections, which give two typical d-spacings of 12.7
and 4.8 Å, respectively, corresponding to stacking periodicity of the -sheets and the spacing between
peptide backbones running orthogonal to the -sheet axis.
b-sheetlike assembly than the natural sequence. CD spectra reveal
b
b
b
b
b
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
structures in solution and solid states,11–14 and the cyclo-oligoureas
can assemble into nanotubes.17
H-bonds play a vital role in forming protein secondary struc-
Alternatively, nature also selects the urea moiety as a H-bonding
functionality, for examples, the naturally occurring linear tetra-
tures such as
a
-helices,
b-sheets, and turn structures. They are also
a central feature in protein
b
-sheet interactions and protein–pro-
peptides and cyclic peptides, such as GE 20372 A and B,18 and (S)-
a-
tein interactions. However, natural short peptides generally do not
participate in specific H-bonds, due to the high flexibility of peptide
main chain and weak interstrand H-bonding interactions between
the peptide amides.
and (R)-b
-MAPI,19–22 monzamide A and B,23 Schizopeptin 791,24
brunsvicamides (BVA) A–C,25 and psymbamide.26 These naturally
occurring compounds all contain an unusual urea-dipeptide seg-
ment, in which two amino acid residues are anti-parallel linked at
the amine terminals to form a urea unit.20 Inspired by these, a set of
peptidomimetics, which bear a urea-dipeptide segment were de-
veloped20,21,27,28 and were shown to be efficient protease in-
hibitors.18–22,27–30
Compared with the amide, the urea unit possesses elegant hy-
drogen-bond-forming capacity between –C]O and –NH units.
Moreover, it may provide one more H-bond, and thus may cause
additional affinity in molecular recognition. Based on these con-
siderations, the urea has been widely used as a hydrogen-bonding
functionality either in supramolecular constructions,1–7 potential
Similar to the interesting structural features and in turn the
highly strong and stable intermolecular H-bonds of imide-di-
peptides,31 which have been created by us, the urea-dipeptide seg-
ment also introduces the same chemical structural characters
unmatched in the natural sequence (Scheme 1), including (i) self-
pairing H-bonds; (ii) a peptide polindron sequence; (iii) top-
ochemical symmetry (C2 symmetry); (iv) different orientation of the
two side-chains. Expectedly, all these structural characters may also
show positive or negative influences on the H-bonding-forming
capacity and recognition affinity, and thus may result in a different
H-bonding pattern to the natural sequence, as do the imide-di-
peptides observed by us.31 However, what is the difference and how
the distinct structural characters influence on H-bonding
inhibitors for the epoxide hydrolase,8 mimicking
b
-sheets,9 or
peptide backbone mimetics, for examples, the N,N0-linked lin-
ear-10–16 and cyclo-oligoureas developed by replacing the amide
with urea unit.17 Several reports have shown that the linear-oli-
goureas bearing proteinogenic side-chains can form stable helical
* Corresponding authors. Tel.: þ86 10 82617312; fax: þ86 10 82616517 (C.Z.); tel./
fax: þ86 10 82616517 (J.Y.).
E-mail addresses: clzhan@iccas.ac.cn (C. Zhan), jnyao@iccas.ac.cn (J. Yao).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.07.048

8270
Urea-dipeptide
D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
NO2
O
O
Natural peptide
O
g)
OH
R
O
R
O
H
H
O
R
O
N
N
H
9
10
R1
N
N
R2
N
H
H
N
N
O
O
H
H
O
i)
O
R
h)
OH
N
H
O
O
1a: R= -CH3, R1= R2= -CH3
N
H2N
2: R = i-Bu
O
H
O
O
1b: R= i-Bu, R1= R2= -CH3
13
11
12
1c: R= i-Bu, R1= -CH3, R2= -phenyl
1d: R= i-Bu, R1= R2= -phenyl
O
k)
j)
O
2
Scheme 1. Chemical structures of the urea-dipeptides (1) and a selected natural
sequence (2).
N
H
7 b
O
14
NO2
Scheme 3. Synthesis of the natural sequence 2. Reaction conditions: (g) 4-nitro-
phenol/DCC, DCM, 90%; (h) acetonitrile/rt, 90–95%; (i) NaOH, EtOH/H2O, w95%; (j)
4-nitrophenol/DCC, DCM, 80–90%; (k) acetonitrile (containing 5% methanol), rt, 76%.
interactions are unknown yet. Accordingly, we synthesized a set of
urea-dipeptides (1)dthe shortest urea-linked peptidomimetics and
a natural sequence 2. A comparison study of the solution behavior
between 1 and 2 indicates that the urea-dipeptides show much
The dipeptide 231b was synthesized by following a procedure as
shown in Scheme 3. 1-Propyl acid was first activated by using
stronger H-bonding propensity to nucleate the b-sheetlike assembly.
4-nitrophenoyl and then reacted with N-unprotected
ethyl ester (11) at rt to give N-ethyl-carbomoyl -leucine ethyl ester
(12), which was further hydrolyzed to N-ethyl-carbomoyl -leucine
L-leucine
L
2. Results and discussion
L
(13) by using sodium hydroxide in 1:1 ethanol/water. Then, car-
boxylic acid group of 13 was again functionalized with 4-nitro-
phenoyl to yield 14. Reaction of 14 with 7b at rt in acetonitrile
afforded the expected dipeptide 2.
2.1. Synthesis of the dipeptides
The urea-dipeptides 1 and the selected natural sequence 2 were
successfully synthesized from the commercial amino acids by fol-
lowing 5–6 steps, as shown in Schemes 2 and 3. We selected
4-nitrophenyl as an activating group31a,32 to convert either car-
2.2. Solubility
boxylic acid to amide or amine to urea. The commercial
L-alanine or
Compound 1a is insoluble in chloroform, while 1b shows im-
proved solubility in chloroform with a value of w3 mM at rt. The
concentration of 1b can reach up to 20 mM upon heating, while the
compound may precipitate within several minutes, depending on
the concentrations, for example, 1b precipitates within about 3 min
when cooling the 20 mM chloroform solution. This ‘temporal’ sol-
ubility makes it be possible to measure the 1H NMR spectra ap-
proximately. The solubility of the urea-dipeptides can be further
improved largely by replacing one or two n-propyl (n-Pr) groups
L-leucine (3) was first converted into N-Boc-protected amino acid
(4) in 10:1 water/THF mixture, in which about 10% THF was used to
dissolve (Boc)2O to afford a homologous reaction solution. Next,
1-propyl amine or 2-phenylethyl amine in acetonitrile efficiently
replaced the activating 4-nitrophenoyl group in 5 at rt to give 6.
Reacting the Boc-deprotected 7 and 4-nitrophenyl chloroformate
yielded compound 8. Finally, coupling between compounds 7 and 8
in acetonitrile at rt successfully yielded the urea-dipeptides 1 as
white precipitate.
with phenylethyl (fe) units, for example, the solubility of both 1c
and 1d can reach up than 70 mM at rt.
R
R
R
OH
Boc
O
Boc
OH
H2N
N
N
2.3. Interpeptide H-bonding interactions
H
O
H
O
O
a)
R
b)
NO2
3a: R=-CH3
4a: R=-CH3
4b: R=-i-Bu
5a: R=-CH3
In general, the amide-protons engaged in intermolecular H-
bonds give a characteristic of nonlinear upfield shift with an
increase of the concentration, whereas, the chemical shifts of in-
tramolecularly H-bonded amide-protons show an nearly
independence on concentration.
3b: R=-i-Bu
5b: R=-i-Bu
H
H
R
Boc
c)
6a: R=-CH3, R1=-CH3
6b: R=-i-Bu, R1=-CH3
6c: R=-i-Bu, R1=-phenyl
N
N
R1
N
R1
H2N
H
O
O
d)
7a: R=-CH3, R1=-CH3
7b: R=-i-Bu, R1=-CH3
7c: R=-i-Bu, R1=-phenyl
Figure 1a reveals that compounds 1b–1d exhibit a typically
nonlinear concentration dependence of the d–NHs, characteristically
indicating intermolecularly H-bonded-NHs, and therefore sug-
O2N
gesting the
b-sheetlike aggregates of the urea-dipeptides. As
O
H
N
R
e)
R1
O
N
expected, the amide-protons of 1b–1d all show a distinct concen-
tration-dependent behavior to that of compound 2 (Fig. 1b). At
a low concentration level, typically, 1–7 mM, the amide-protons in
the natural peptide 2 do not show any downfield shift with the
increase of concentration, suggesting that the amide-protons re-
main most likely free of H-bonds. Conversely, the d–NHs of 1b–1d all
show a dramatical increase as the increase of concentration, re-
vealing that the urea-dipeptides hold much stronger H-bonding
propensity than the natural sequence. Such a strong H-bonding
propensity is supported by the fact that no plateau was observed
even when the concentration is down to 0.1 mM, a concentration
being about two orders of magnitude lower than that concentration
(about 7 mM) for the natural sequence starting to show H-bonding
H
O
8a: R=-CH3, R1=-CH3
8b: R=-i-Bu, R1=-CH3
8c: R=-i-Bu, R1=-phenyl
f)
f)
7b
7c
8c
8c
7a
7b
+
+
1c
1d
8a
8b
+
+
1a
1b
f)
f)
Scheme 2. Synthesis of the urea-dipeptides (1). Reaction conditions: (a) (Boc)2O, KOH,
H2O/THF (10:1), 50 ꢀC, 85–95%; (b) 4-nitrophenol/DCC, DCM, 80–90%; (c) 1-propyl
amine or 2-phenylethyl amine, acetonitrile, 90–95%; (d) TFA, DCM, w100%; (e) 4-nitro-
phenyl chloroformate, THF,
tetrahydrofuran; DCC, N,N0-dicyclohexyl carbodiimide; DCM, dichloromethane; TFA,
0
ꢀC, 60–70%; (f) acetonitrile, rt, 50–80%. Note: THF,
trifluoroacetic acid.

D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
8271
a high concentration level (typically >10 mM). These results in-
dicate that the dipeptide chains adopt -folding conformations at
high concentration levels, at which the peptide molecules form
matured -sheetlike assembly (typically, >10 mM).
(a)
7.5
7.0
6.5
6.0
5.5
5.0
4.5
b
b
2.4.2. NOE spectra. The NOE experiment is an ideal tool to char-
acterize formation of -sheetlike structures. In -sheetlike assem-
bly, interstrand NOE signals are generally observed between
amide-protons and -protons of opposite residues, and that be-
b
b
R
R
O
O
R
R
H
N
H
N
a
R1
R1
N
H
N
H
R2
tween side-chains and between backbone protons and side-chain
protons. Other evidences include intramolecular NOE contacts in
a pleated peptide chain. These NOE signals characterize that the
peptide chains are intermolecularly H-bonded together to form
O
O
O
O
H
N
H
N
N
H
N
H
R2
60
b-sheetlike assembly. As shown by NOE experiment of 1d (Fig. 2),
for example, the strong interstrand NOE coupling between the
urea- and amide-NHs and NOEs from side-chains to backbones
0
10
20
30
Concentrations (mM)
40
50
demonstrate the formation of the
molecular NOE signals between the amide-NHs and
that between the e- C-protons and the phenyl-protons all in-
dicate the -folding conformations of the peptide chains, further
confirming the -sheetlike assembly. In addition, the -sheetlike
assembly is consistent with the fact that the presence of phenyl
b
-sheetlike assembly. The intra-
a
-protons and
(b)
f
a
7.5
7.0
6.5
6.0
5.5
b
b
b
-NH2
p–
p
stacks in 1c or 1d intensifies as concentration increases (Fig. 3).
-NH1
-NH3
ppm
2
O
O
R
H
O
O
O
O
N
H
N
H
N
N
H
N
H
N
H
N
H
R
R
R
R
O
O
1
O
R
2
4
6
3
H
N
H
N
α
N
H
N
H
0
10
20
30
40
Concentrations (mM)
50
60
β
Figure 1. (a) Concentration-dependent d–NHs of the amide-protons and urea-protons
of 1b (- and C), 1c (+ and ) and 1d (6 and 7). Inset shows self-pairing and
-sheetlike assembly of 1 in CDCl3. (b) Concentration-dependent d–NHs of the amide-
protons of 2 in CDCl3 (þ, , and ꢁ). Inset shows NH numbers in dipeptide 2, assigned
by both COSY and NOE spectra, as compared, it also shows the d–NHs of the amide-NHs
of 1b–1d at different concentrations.
q
b
propensity. When concentration exceeds 10 mM, the amide-pro-
tons of 1b–1d nearly reach their plateau, indicating maturation of
interstrand H-bonding interactions and b-sheetlike aggregates. The
8
amide-protons of 2, however, continuously increase higher and
higher, suggesting the propensity to form H-bonds at this high
concentration level.
8
7
6
5
4
3
2
1
ppm
Figure 2. NOESY spectrum of 1d in CDCl3 solution carried out at 74 mM and 25 ꢀ
C
(mixing time 0.8 s). The interstrand NOE is indicated by arrow, the intrastrand NOE is
labeled by unfilled circles, and NOEs of side-chain to backbone are pointed by unfilled
squares.
2.4. Characterizations of the b-sheetlike assembly
3
2.4.1. JHNa coupling constants. The magnitude of the 3JHNa coupling
2.4.3. CD spectra. CD is a main and direct evidence to characterize
formation of the b-sheetlike assembly. In general, peptides with
constant for a peptide residue is dependent on the
therefore on the local conformation of the peptide chain.33
-folding conformation generally gives a 3JHNa coupling constant
f
-angle (N–Ca),
natural sequences forming
b-sheet assembly give a negative CD
A
b
band around 216 nm.35 As shown in Figure 4, CD spectra of 1b–1d
in the range of 8–10 Hz, while an unstructured random coil con-
conducted in chloroform solutions all show a negative CD band
around 217 nm, suggesting the formation of the b-sheetlike as-
sembly. In addition, all three compounds also yield a negative CD
band around 225 nm. This CD signal is corresponding to the ab-
3
formation yields a JHN coupling constant ranging from 5.8 to
a
7.3 Hz. Additionally, Wishart et al.34 have produced a simple
method for secondary structure determination by analyzing the
difference between the
and for the same residue type in a ‘random coil’ conformation.
Accordingly, -strands consistently show greater chemical shifts
than that of the random coil. In our cases, the -protons of 1b–1d all
a
-proton chemical shift for each residue
sorption band peaking at 220 nm originated from the n/p tran-
sitions of the urea and/or amide units35 (most likely, it is originated
from the urea unit since the natural sequence and reported natural
peptides do not show this CD band. However, the originity needs to
b
a
show a characteristically nonlinear shift downfield from 4.17 ppm
(a chemical shift relative to the leucine residue type in random coil
be investigated in more details). This fact suggests that the n/
p
transitions of the urea-dipeptides adopting -folding conforma-
b
conformations) to 4.30 ppm when the concentration increases. And
tions yields two negative CD bands around 217 and 225 nm. This is
surprised. Another interesting thing is that the intensity of the
3
the
a
-protons of 1b–1d give a JHN coupling constant of 8.0 Hz at
a

8272
D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
strong propensity of the urea-dipeptides to nucleate the formation
of the -sheetlike assembly.
phenyl-protons of 1c
phenyl-protons of 1d
7.17
7.16
7.15
7.14
7.13
b
2.4.4. XRD characteristics. X-ray diffraction (XRD) experiments
may give better insights into the structural information of the
b
-sheet assembly. The XRD scattering shown in Figure 6 carried out
by depositing the 1b/CHCl3 solution, for example, onto the Si sub-
strate shows a strong and a weak reflections, respectively, at 2
¼7.0
q
and 18.4ꢀ, which are corresponding to two d-spacings of 12.7 and
4.8 Å. Other reflections in the range of 2
the higher or lower orders of these two reflections. The d-values of
q
¼5–35ꢀ can be assigned to
12.7 and 4.8 Å are, respectively, relative to the stacking periodicity
of the
ning orthogonal to the
-sheet structures of 1b.36
b
-sheets and the spacing between peptide backbones run-
b
-sheet axis, typically characteristic of the
b
0
10
20
30
40
Concentrations (mM)
50
60
Figure 3. Concentration-dependent d–NHs of the phenyl-protons of 1c and 1d.
7.0°
0.9
0.6
0.3
-12
18.4°
1b 3 mM
-24
-36
1c 10 mM
1d 10 mM
220
240
Wavelength (nm)
260
280
10
20
30
2θ
Figure 4. UV–vis and CD spectra of 1b–1d in the chloroform solutions.
Figure 6. XRD scattering of 1b deposited onto the Si substrate.
2.5. Asymmetrical b-sheetlike assembly
225 nm signal is dependent on both the structure and concentra-
tion. For examples, 1c has a much stronger 225 nm CD signal than
that at 217 nm, while 1d with a same concentration (10 mM) shows
a reversal intensity of the 225 band over the 217 nm band (Fig. 4).
Concentration-dependent CD experiments shown in Figure 5 reveal
that the intensity of the 225 nm CD signal of 1c, for example, de-
creases more quickly than that of the 217 nm signal with decreasing
the concentration. In particular, the existence of the 217 nm signal
at a concentration of as low as 0.5 mM indicates again that the
Particularly, 1c bears asymmetrical terminal groups at two ends,
while, the NMR evidences indicate that it tends to form ‘asym-
metrical’ b-sheetlike assembly, in which the urea-dipeptide mole-
cules self-organize mainly in a parallel fashion, not in the anti-
parallel way or both (Fig. 7). The first evidence comes from the
0
05.mM
4 mM
6 mM
8 mM
10 mM
-15
-30
224.6 nm
217.5 nm
220
240
Wavelength (nm)
260
280
Figure 7. Part of COSY (a) and NOE spectra (b) of 1c conducted at 60 mM and rt;
Figure 5. CD spectra of 1c at different concentrations.
parallel (c) and anti-parallel (d) self-organizations and the NOE contacts.

D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
8273
concentration-dependent behavior of the amide-NHs. The two
amide-NHs attached at the e and n-Pr ends, respectively, give two
distinct triplets in the NMR spectra at high concentration levels,
which are undoubtedly assignable to the amide-NH, respectively,
7.2
6.8
6.4
6.0
5.6
5.2
f
linking with the n-Pr (at downfield) or
COSY spectrum (Fig. 7a). As further indicated in Figure 8, the Dda-
representing the difference between the chemical shifts of
fe group (at upfield) by the
mide-NH
Adding
Methanol (5%)
these two amide-NHs shows an increase with the concentration.
This fact suggests that the aggregate process into the -sheetlike
b
assembly may produce different chemical environments around
the end amide-protons.
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0
5
10
Concentrations (mM)
15
20
25
Figure 9. Concentration-dependent d–NHs of the amide- and urea-NHs of 1d from
a 20 mM solution of 5% of CH3OH/CDCl3 (v/v) diluted by using either 5% CH3OH/CDCl3
q
(A and >) or pure CDCl3 ( and +). As compared, it also shows concentration-de-
pendent d–NHs of the amide- (,) and urea-NHs (: and 7) of 1d from a 23 mM CDCl3
solution diluted with pure CDCl3.
methanol in the solution remains constantly. When the solution is
further diluted using pure CDCl3, however, all the –NHs show
a large upfield shift as the chloroform molecules have replaced
some methanol molecules, effectively reducing H-bonding
opportunities.
0
10
20
30
40
Concentrations (mM)
50
60
3. Conclusions
Figure 8. A plot of the Ddamide-NH (difference between the chemical shifts of the am-
ide-NH at the e end and that at the n-Pr end) of 1c versus concentrations.
f
The solution NMR and CD studies demonstrate that the urea-
dipeptides adopt
solvents, and self-organize into the
surprisingly, the urea-dipeptides show much stronger H-bonding
propensity to nucleate the formation of the -sheetlike assembly
than the natural sequence. Additionally, the urea-dipeptides show
very different solution behavior to the imide-dipeptides31 although
they have similar structural features. These distinct features of the
H-bonding nature suggest that the urea-dipeptide segment may be
b
-folding conformations in non-H-bonding
In order to get an insight into this, we conducted the NOE ex-
periment. As shownin Figure 7b, the NOE spectrum shows very strong
b-sheetlike assembly. Most
NOE signals both between the
the e end and between the n-Pr-
the n-Pr end. While the NOE signal between
amide-protons at the n-Pr end and that between the n-Pr-
protons and amide-protons at the e end are very weak. This
f
e-
a
a
C-protons and amide-protons at
C-protons and amide-protons at
e- C-protons and
C-
b
f
f
a
a
f
fact illustrates the parallel to parallel self-organization of the
urea-dipeptide 1c as the dominant type of aggregates. Obvi-
an alternative interesting linear b-mimetic motif to be incorporated
with the imide-dipeptide segment together to generate new pep-
tidomimetics, which contain both the urea- and imide-dipeptide
segments, which is undergoing in our lab.
ously, the
side of the assembled ribbon-like structure would produce dif-
ferent environments for the amide-NHs linked with the e unit
to that ones attached to the n-Pr group. The above results
suggest that the phenyl stacking may play role in
-sheetlike
p–p stacks between the phenyl-units residing at one
f
4. Experimental part
p–
p
a
directing molecules of 1c into the ‘asymmetrical’
b
4.1. Apparatus and materials
assembly.
NMR measurements. NMR spectra were recorded on Brucker
instruments (300, 400, or 600 kHz), NOESY and COSY spectra were
made at rt on the 600 kHz instrument.
2.6. Disruption of b-sheetlike assembly
H-bonding solvents such as methanol effectively disrupt the
-sheetlike assembly, even only several percent of methanol. For
Mass spectrometry. ESI-mass spectra (ESI¼electro spray ioniza-
tion) were obtained with a Shimadzu LC–MS 2010 mass spec-
trometer. A proper solvent such as dichloromethane, acetonitrile,
methanol, water, was used for dissolving the sample.
CD spectrometry. CD spectra were obtained on a JASCO model
J-815 spectropolarimeter. Each CD spectrum was obtained by in-
tegrating three repeated scans with a scan rate of 500 nm/min and
was background-corrected with a 0.1 mm quartz cell.
b
example, addition of 5% methanol into a 20 mM 1d/CDCl3 solution
resulted in complete disruption of the intermolecular H-bonds and
formation of new competitive H-bonds between methanol and
–NHs, which displayed an obvious upfield shift for the urea-protons
and a little downfield shift for the amide-protons (Fig. 9). This
upfield shift of the urea-protons or slightly downfield shift of the
amide-protons almost remains well if the solution is further diluted
using 5% methanol/CDCl3 (v/v), indicating the initial addition of
methanol have completely consumed the interstrand H-bonds, and
the competitive H-bonding opportunities between the dipeptide
and methanol molecules are also kept well by the further addition
of methanol in 5% methanol/CDCl3 since the concentration of
XRD measurements. XRD measurements were performed by
using a X-ray powder diffraction (XRD, Bruker D8 Focus) with Cu K
a
as the radiation source (
40 mA.
l¼1.5418 Å) and operated at 40 kV and
Materials. Starting materials are all commercially available re-
agents and solvents used as received except for statements.

8274
D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
Tetrahydrofuran (THF) was distilled on sodium. Dichloromethane
and acetonitrile were dried on anhydrate Na2CO3. Reactions were
monitored by thin-layer chromatography on pre-coated silica gel
plates (Yantai Shi Huagxue Gongye Yanjiusuo) and visualized using
UV irradiation (254 nm) or colored by iodine. Flash chromatogra-
phy was performed on silica gel H60 (Giandao Haiyang
Huagongchang).
leucine), 1.02 (d, 6H, –CH3, 3J¼4.4 Hz). 13C NMR
d (ppm, CDCl3,
100 MHz): 171.8, 155.6, 155.4, 145.6, 125.4, 122.5, 80.5, 55.9, 41.3,
28.4, 23.0, 21.9. ESI-MS: 375 (þNaþ).
4.2.2. General procedure for synthesis of 6. 2 mmol of 5 was dis-
solved in 10 mL of dried CH3CN, excess amine was injected inside,
and stirred overnight. Then, acetonitrile was removed under vac-
uum and the residue was re-dissolved in 10 mL of dichloromethane
and washed, respectively, using satd Na2CO3 aqueous and water
(10 mL, ꢁ3) and then dried with anhydrate Na2SO4. After removal
of organic solvent, the residue was applied to flash chromatography
to afford 6 as a white solid.
4.2. Synthesis of the dipeptides
4.2.1. General procedure for synthesis of N-Boc-amino acid
(4). 10 mmol of commercial amino acid and 11 mmol of KOH were
dissolved in a mixture of water (40 mL) and THF (4 mL). To it
11 mmol of di-tert-butyl-dicarbonate was added, and the resultant
solution was allowed to react at 50 ꢀC. 2–3 h later, the reaction
mixture was cooled down to rt and stirred overnight. Then,11 mL of
1 M HCl was added dropwise to adjust pH value of the solution to
about 5. The solution was extracted with ethyl acetate (50 mL, ꢁ3)
and dichloromethane (50 mL, ꢁ3), respectively. The organic phases
were mixed and dried with anhydrate Na2SO4. Removal of organic
solvents afforded expected product.
4.2.2.1. tert-Butyl N-[(S)-1-propylcarbamoyl-ethyl]carbamate
(6a). 620 mg (2 mmol) of 5a and 330 mL (7.8 mmol) of 1-propyl
amine were mixed in 10 mL of CH3CN. The reaction residue was
applied to flash chromatography with petrol ether/ethyl
acetate¼4:1 as eluents to afford 6a as a white solid (420 mg,
1.8 mmol, 91%, Rf¼0.2). 1H NMR
d (ppm, CDCl3, 400 MHz): 6.61 (s,
1H, amide-H), 5.33 (s, 1H, carbamate-H, 3J¼7.2 Hz), 4.21 (t, 1H,
a
-proton, 3J¼5.0 Hz), 3.25 (q, 2H, propyl-CH2, 3J¼6.4 Hz), 1.53 (m,
2H, propyl-CH2, 3J¼7.2 Hz), 1.46 (s, 9H, Boc-Hs), 1.38 (d, 3H, –CH3,
3J¼7.0 Hz), 0.94 (t, 3H, propyl-CH3, 3J¼7.4 Hz). 13C NMR
d (ppm,
4.2.1.1. N-Boc-
L
-alanine acid (4a). 4.5 g (50 mmol) of
L-alanine and
CDCl3, 100 MHz): 172.7, 155.6, 79.9, 50.0, 41.1, 29.7, 22.8, 18.6, 11.3.
3.8 g (55 mmol) of KOH, 13.0 g (55 mmol) of di-tert-butyl-dicar-
bonate were mixed in a mixture of water (200 mL) and THF
(20 mL). The reaction afforded 4a as an oil-like solid (9 g, 47 mmol,
ESI-MS: 253 (þNaþ).
4.2.2.2. tert-Butyl N-[(S)-3-methyl-1-propylcarbamoyl-butyl]carba-
mate (6b). 705 mg (2 mmol) of 5b and 330 mL (7.8 mmol) of
95%). 1H NMR
d
(ppm, CDCl3, 400 MHz): 10.66 (s, broad,1H, acid-H),
5.08 (s, 1H, carbamate-H), 4.35 (s, 1H,
a
-proton), 1.53 (d, 3H, –CH3,
1-propyl amine were mixed in 10 mL of CH3CN. The reaction resi-
due was applied to flash chromatography with petrol ether/ethyl
acetate¼5:1 as eluents to afford 6b as a white solid (510 mg,
3J¼7.6 Hz), 1.45 (s, 9H, Boc-Hs). ESI-MS: 188 (ꢂHþ), 189, 211 (ꢂHþ
and þNaþ), 227 (ꢂHþ and þKþ).
1.9 mmol, 94%, Rf¼0.2). 1H NMR
d (ppm, CDCl3, 400 MHz): 6.15 (s,
4.2.1.2. N-Boc-
L
-leucine acid (4b). 1.31 g (10 mmol) of
L-leucine,
1H, amide-H), 4.84 (d, 1H, carbamate-H, 3J¼8.8 Hz), 4.06 (t, 1H,
0.75 g (11 mmol) of KOH, and 2.4 g (11 mmol) of di-tert-butyl-
dicarbonate were dissolved in a mixture of water (40 mL) and THF
(4 mL). The reaction afforded 4b as an oil-like solid (2.2 g, 9.0 mmol,
a
-proton, 3J¼7.4 Hz), 3.20 (q, 2H, propyl-CH2, 3J¼6.4 Hz), 1.67 (s, 2H,
–CH2), 1.60–1.43 (m, 3H, propyl-CH2 and –CH, 3J¼7.2 Hz), 1.40 (s,
9H, Boc-Hs), 0.89–0.83 (q, 9H, propyl-CH3 and –CH3, 3J¼7.2 Hz). 13
C
90%). 1H NMR
4.91 (s, 1H, carbamate-H), 4.31 (s, 1H,
d
(ppm, CDCl3, 400 MHz): 6.20 (s, broad, 1H, acid-H),
NMR d (ppm, CDCl3, 100 MHz): 172.5, 155.8, 79.9, 53.1, 41.3, 41.1,
a
-proton), 1.76–1.60 (m, 2H,
28.3, 25.0, 22.9, 22.1, 11.3. ESI-MS: 295 (þNaþ).
–CH2, 3J¼6.4 Hz), 1.59–1.49 (m, 1H, –CH, 3J¼6.6 Hz),1.44 (s, 9H, Boc-
Hs), 0.96 (d, 6H, –CH3, 3J¼6.4 Hz). ESI-MS: 230 (ꢂHþ), 231, 253
(ꢂHþ and þNaþ), 269 (ꢂHþ and þKþ).
4.2.2.3. tert-Butyl N-[(S)-3-methyl-1-phenethyl-carbamoyl-butyl]car-
bamate (6c). 705 mg (2 mmol) of 5c and 1 mL (8.5 mmol) of
2-phenylethyl amine were mixed in 10 mL of CH3CN. The reaction
residue was applied to flash chromatography with petrol ether/ethyl
acetate¼5:1 as eluents to afford 6c as a white solid (600 mg,
4.2.1.3. 4-Nitrophenoyl N-Boc-L-alanine ester (5a). 1.9 g (10 mmol)
of 4a, 1.7 g (12 mmol) of 4-nitrophenol, and 2.8 g (12 mmol) of DCC
were mixed with 100 mL of CH2Cl2 and stirred overnight. Then, the
precipitate was filtered out and the solvents of the filtration were
removed. The resultant residue was applied to flash chromatogra-
phy with petrol ether/dichloromethane/ethyl acetate¼10:2:1 as
eluents to afford 5a as a light-yellow solid (2.6 g, 8.4 mmol, 85%,
1.8 mmol, 90%, Rf¼0.2). 1H NMR
d (ppm, CDCl3, 300 MHz): 7.30–7.14
(m, 5H, phenyl-Hs, 3J¼6.8 Hz), 6.62 (s, 1H, amide-H), 5.18 (d, 1H,
carbamate-H, 3J¼7.3 Hz), 4.09 (s, 1H,
a-proton), 3.54–3.50 (m, 1H,
phenylethyl-CH2, 3J¼6.8 Hz), 3.42–3.37 (m, 1H, phenylethyl-CH2,
3J¼6.1 Hz), 2.80–2.75 (t, 2H, phenylethyl-CH2, 3J¼7.1 Hz), 1.58 (m,
2H, –CH2, 3J¼7.0 Hz), 1.46 (m, 1H, –CH, 3J¼7.9 Hz), 1.40 (s, 9H, Boc-
Rf¼0.2). 1H NMR
d (ppm, CDCl3, 400 MHz): 8.30 (d, 2H, phenyl-Hs,
3J¼9.0 Hz), 7.32 (d, 2H, phenyl-Hs, 3J¼9.0 Hz), 5.11 (s, 1H, carba-
Hs), 0.89 (d, 6H, –CH3, 3J¼5.4 Hz). 13C NMR
d (ppm, CDCl3, 100 MHz):
mate-H), 4.54 (t, 1H,
a
-proton, 3J¼7.1 and 6.5 Hz), 1.58 (d, 3H, –CH3,
172.8,155.8,138.8,128.8,128.6,126.5, 79.8, 53.2, 41.6, 40.7, 35.7, 28.3,
3J¼7.4 Hz), 1.47 (s, 9H, Boc-Hs). 13C NMR
d
(ppm, CDCl3, 100 MHz):
24.8, 22.9, 22.1. ESI-MS: 357 (þNaþ).
171.4, 155.3, 155.2, 145.6, 125.4, 122.4, 80.5, 49.7, 28.4, 18.1. ESI-MS:
333 (þNaþ).
4.2.3. General procedure for synthesis of 7. 0.5 mmol of 6 was dis-
solved in dichloromethane (10 mL) and 1 mL of TFA was added. 2 h
later, 10 mLꢁ3 of satd Na2CO3 aqueous and water were, re-
spectively, used to wash the solution. Organic phase was collected
and dried with anhydrate Na2SO4. The solvents were removed to
afford 7 as an oil-like solid. The residue was used for further syn-
thesis without any other purification.
4.2.1.4. 4-Nitrophenoyl N-Boc-L-leucine ester (5b). 2.3 g (10 mmol)
of 4b, 1.7 g (12 mmol) of 4-nitrophenol, and 2.8 g (12 mmol) of DCC
were mixed with 100 mL of CH2Cl2 and stirred overnight. After the
precipitate was filtered out and the solvents of the filtration were
removed, the residue was applied to flash chromatography with
petrol ether/dichloromethane/ethyl acetate¼10:4:1 as eluents to
afford 5b as a light-yellow solid (2.8 g, 8 mmol, 80%, Rf¼0.2).1H NMR
4.2.4. General procedure for synthesis of 8. The above obtained 7
was mixed with 10 mL of dried THF and 200 mL of DIEA, then,
a solution of 120 mg of 4-nitrophenyl chloroformate in 10 mL of
THF was added dropwise at 0 ꢀC and then stirred overnight. After
d
(ppm, CDCl3, 400 MHz): 8.28 (d, 2H, phenyl-Hs, 3J¼8.9 Hz), 7.31 (d,
2H, phenyl-Hs, 3J¼8.9 Hz), 4.93 (s, 1H, carbamate-H), 4.51 (s, 1H,
a
-
proton), 1.69 (m, 2H, –CH2, 3J¼6.4 Hz), 1.47 (s, 10H, Boc-Hs, –CH of

D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
8275
removal of organic solvent, the residue was applied to flash chro-
matography to afford 8.
3JHN ¼8.0 Hz), 3.22–3.12 (m, 4H, n-Pr-
a
C–Hs, 3J¼6.4 Hz), 1.66–1.56
a
(m, 4H,
b
-Hs of Leu, 3J¼5–7 Hz), 1.53–1.46 (m, 6H,
g-Hs of Leu and
n-Pr-bC–Hs), 0.91–0.88 (t, 18H, d-Hs of Leu and n-Pr-gC–Hs,
4.2.4.1. 4-Nitrophenyl N-[(S)-1-propylcarbamoyl-ethyl]carbamate
(8a). The reaction residue was applied to flash chromatography
with petrol ether/dichloromethane/ethyl acetate¼1:1:1 as eluents
to afford 8a as a light-yellow solid (130 mg, 0.9 mmol, 90%, Rf¼0.2).
3J¼6.8 Hz). Elementary Analysis for C19H38N4O3: Calcd C, 61.59; H,
10.34; N, 15.12; Exp. C, 61.89; H, 10.30; N, 14.86; ESI-MS: 371 (þHþ),
393 (þNaþ).
1H NMR
d
(ppm, CDCl3, 400 MHz): 8.27 (d, 2H, 4-nitro-phenyl-Hs,
4.2.4.6. Urea-dipeptide (1c). 90 mg (0.52 mmol) of 7b and 220 mg
(0.55 mmol) of 8c were dissolved in 5 mL of dried CH3CN and stirred
at rt overnight. A day later, the white precipitate was collected by
filtration and washed with water to afford urea-dipeptide 1c
3J¼8.7 Hz), 7.31 (d, 2H, 4-nitro-phenyl-Hs, 3J¼8.8 Hz), 6.10 (s, 1H,
amide-H), 5.86 (s, 1H, carbamate-H), 4.31 (q, 1H,
a-proton,
3J¼7.0 Hz), 3.35 (q, 2H, propyl-CH2, 3J¼6.6 Hz), 1.6–1.5 (m, 5H,
propyl-CH2 and –CH3), 1.04 (t, 3H, propyl-CH3, 3J¼7.3 Hz). 13C NMR
(60 mg, 0.14) as a white solid with a yield of 27%. 1H NMR
d (ppm,
d
(ppm, CDCl3,100 MHz): 171.4,155.7,152.7,144.9,125.2, 122.1, 50.8,
CDCl3, 400 MHz, 13 mM): 7.28–7.13 (m, 5H, ph-Hs, 3J¼7.6 Hz), 7.04
41.5, 22.8, 19.2, 11.3. ESI-MS: 296 (þHþ), 318 (þNaþ).
(s, 1H, amide-NH), 7.01(s, 1H, amide-NH), 6.27–6.21 (quartet, 2H,
urea-NHs, 3J¼8.8 and 8.4 Hz), 4.28–4.23 (sextet, 2H,
a-proton,
4.2.4.2. 4-Nitrophenyl N-[(S)-1-propylcarbamoyl-ethyl]carbamate
(8b). The reaction residue was applied to flash chromatography
with petrol ether/dichloromethane/ethyl acetate¼2:1:1 as elu-
ents to afford 8b as a light-yellow solid (140 mg, 0.4 mmol, 84%,
3JHN ¼7.8 Hz), 3.57–3.48 (m, 1H, phenylethyl-
a
C–Hs, 3J¼7.8 Hz),
a
3.42–3.34 (m, 1H, phenylethyl-
a
C–Hs, 3J¼8.0 Hz), 3.23–3.09 (m, 2H,
n-propyl-Hs, 3J¼8.0 Hz), 2.81–2.78 (m, 2H, phenylethyl-
b
C–Hs,
C–
C–Hs,
3J¼7.4 Hz), 1.63–1.46 (m, 8H,
b
-Hs of Leu,
g
-Hs of Leu, and n-Pr-
b
Rf¼0.2). 1H NMR
d
(ppm, CDCl3, 400 MHz): 8.26 (d, 2H, 4-nitro-
Hs, 3J¼3–8 Hz), 0.91–0.85 (q, 15H,
d-Hs of Leu and n-Pr-g
phenyl-Hs, 3J¼9.1 Hz), 7.32 (d, 2H, 4-nitro-phenyl-Hs, 3J¼9.1 Hz),
3J¼6.0, 3J¼6.8 Hz). 13C NMR
d (ppm, CDCl3, 100 MHz): 172.9, 157.2,
5.80 (s, 1H, amide-H), 5.84 (s, 1H, 4-nitrophenyl-carbamate-H,
138.1, 127.7, 127.4, 125.3, 57.4 (weak), 51.7, 40.9, 40.8, 40.2, 39.8, 34.7,
30.9 (weak), 28.7 (weak), 23.9, 23.8, 21.8, 21.7, 21.6, 17.4 (weak), 13.1
(weak), 10.4. ESI-MS: 433 (þHþ), 455 (þNaþ).
3J¼8.3 Hz), 4.2–4.13 (q, 1H,
a
-proton, 3J¼6.3 Hz), 3.35–3.20 (m,
2H, propyl-CH2, 3J¼6.0 Hz), 1.7–1.6 (m, 2H, –CH2 of leucine,
3J¼4.5 Hz), 1.6–1.5 (m, 3H, propyl-CH2 and –CH), 1.00 (d, 3H,
propyl-CH3, 3J¼5.7 Hz), 0.91 (t, 6H, –CH3, 3J¼5.2 Hz). 13C NMR
4.2.4.7. Urea-dipeptide (1d). 120 mg (0.51 mmol) of 7c and 220 mg
(0.55 mmol) of 8c were dissolved in 5 mL of dried CH3CN and
stirred at rt overnight. A day later, the white precipitate was col-
lected by filtration and washed with water to afford urea-dipeptide
1d (90 mg, 0.18 mmol) as a white solid with a yield of 36%. 1H NMR
d
(ppm, CDCl3, 100 MHz): 172.0, 155.8, 153.3, 144.7, 125.1, 122.0,
54.0, 41.9, 41.4, 24.8, 22.8, 22.6, 22.2, 11.3. ESI-MS: 338 (þHþ),
360 (þNaþ).
4.2.4.3. 4-Nitrophenyl N-[(S)-3-methyl-1-phenethyl-carbamoyl-bu-
tyl]carbamate (8c). The reaction residue was applied to flash
d (ppm, CDCl3, 400 MHz, 20 mM): 7.26–7.13 (m, 12H, ph-Hs and
amide-NHs, 3J¼7.2 Hz), 6.38 (d, 2H, urea-NHs, 3J¼8.4 Hz), 4.31–4.24
3
chromatography
with
petrol
ether/dichloromethane/ethyl
(q, 2H,
ethyl-
a
-proton, JHN ¼8.0 Hz), 3.54–3.49 (quartet, 2H, phenyl-
a
acetate¼2:1:1 as eluents to afford 8c as a light-yellow solid
a
C–Hs, 3J¼7.0 Hz), 3.38–3.23 (m, 2H, phenylethyl-
aC–Hs,
(180 mg, 0.5 mmol, 91%, Rf¼0.2). 1H NMR
d
(ppm, CDCl3,
3J¼6.8 Hz), 2.81–2.78 (m, 4H, phenylethyl-
b
C–Hs, 3J¼7.4 Hz), 1.58–
400 MHz): 8.18 (d, 2H, 4-nitro-phenyl-Hs, 3J¼9.2 Hz), 7.35–7.24
(m, 5H, phenyl-Hs, 3J¼5.9 Hz), 6.90 (d, 2H, 4-nitro-phenyl-Hs,
3J¼9.2 Hz), 6.05 (d, 1H, 4-nitrophenyl-carbamate-H, 3J¼6.9 Hz),
1.50 (m, 4H,
b
-Hs of Leu, 3J¼6–7 Hz), 1.44–1.41 (m, 2H,
g-Hs of Leu,
3J¼6.8 Hz), 0.84–0.82 (q, 12H,
d
-Hs of Leu, 3J¼6.5, 3J¼2.8 Hz). 13C
NMR d (ppm, CDCl3,100 MHz): 172.8,157.1, 138.0, 127.7, 127.4, 125.3,
5.86 (s, 1H, amide-H), 4.12 (q, 1H,
a
-proton, 3J¼7.1 Hz), 3.70–3.59
57.4 (weak), 51.7, 40.9, 39.8, 34.7, 28.7 (weak), 23.8, 21.7, 21.6, 17.4
(m, 2H, phenylethyl-CH2, 3J¼6.8 Hz), 2.98–2.89 (t, 2H, phenylethyl-
CH2, 3J¼6.8 Hz), 1.72 (m, 2H, –CH2, 3J¼4.5 Hz), 1.40–1.30 (m, 1H,
–CH, 3J¼6.7 Hz), 0.97–0.86 (m, 6H, –CH3, 3J¼5.7 Hz). 13C NMR
(weak). TOF-MS: 494. ESI-MS: 495 (þHþ), 517 (þNaþ).
4.2.5. 4-Nitro-phenyl n-propyl ester (10). 1.4 mL (20 mmol) of n-
propyl acid (9), 1.2 g (10 mmol) of 4-nitrophenol, and 2.1 g
(10 mmol) of DCC were mixed with 100 mL of CH2Cl2 and stirred
overnight. Then the precipitate was filtered out. After removal of
organic solvents, the residue was applied to flash chromatography
with petrol ether/dichloromethane¼4:1 as eluents to afford 10 as
a white solid (1.7 g, 8.7 mmol, Rf¼0.2) with a yield of 87%. 1H NMR
d
(ppm, CDCl3, 100 MHz): 174.1, 161.3, 157.0, 142.2, 138.5, 129.0,
128.5, 126.7, 126.3, 115.7, 55.6, 40.9, 39.7, 33.9, 25.3, 23.0. 21.6. ESI-
MS: 400 (þHþ), 422 (þNaþ).
4.2.4.4. Urea-dipeptide (1a). 70 mg (0.54 mmol) of 7a and 170 mg
(0.58 mmol) of 8a were dissolved in 5 mL of dried CH3CN and
stirred at rt overnight. A day later, the white precipitate was col-
lected by filtration and washed with water to afford urea-dipeptide
1a (130 mg, 0.45 mmol) as a white solid with a yield of 84%. 1H NMR
d
(ppm, CDCl3, 400 MHz): 8.18 (d, 2H, phenyl-Hs, 3J¼8.8 Hz), 7.16 (d,
2H, phenyl-Hs, 3J¼8.8 Hz), 2.51 (quartet, 2H, –CH2, 3J¼7.6 Hz), 1.14
(d, 3H, –CH3, 3J¼7.8 Hz). 13C NMR
d (ppm, CDCl3, 100 MHz): 171.8,
d
(ppm, DMSO-d6, 400 MHz): 7.81 (t, 2H, amide-NHs, 3J¼5.2 Hz),
155.4, 145.0, 124.9, 122.3, 27.4, 8.6. ESI-MS: 195, 218 (þNaþ).
6.27 (d, 2H, urea-NHs, 3J¼7.2 Hz), 4.08 (quintet, 2H,
a-proton,
3J¼7.2 Hz), 2.99, (m, 4H, n-Pr-
a
C–Hs, 3J¼6.0 Hz), 1.38 (m, 4H, n-Pr-
4.2.6. Ethyl N-ethyl-carbonyl-
(3.6 mmol) of 10, 0.6 g (3.1 mmol) of ethyl L
L
-leucine acid ester (12). 0.7 g
-leucine acid ester hydro-
b
C–Hs, 3J¼7.2 Hz), 1.13 (6H, –CH3, 3J¼6.8 Hz), 0.82 (t, 6H, –CH3,
3J¼7.7 Hz) (see below). 13C NMR
d
(ppm, DMSO-d6,100 MHz): 173.4,
chloride (11), and 2.0 mL of triethyl amine were dissolved in 5 mL of
dried CH3CN and stirred overnight. After CH3CN was removed under
vacuum, the residue was re-dissolved into 10 mL of dichloro-
methane and washed using satd Na2CO3 aqueous (10 mLꢁ3) and
water (10 mLꢁ3), Then the collected organic phase was dried with
anhydrate Na2SO4. The solvents were removed and the resulted
residue was applied to flash chromatography with petrol ether/ethyl
acetate¼5:1 as eluents to afford 12 as a light-yellow oil-like solid
157.1, 49.1, w40 (overlapping with DMSO), 22.8, 20.2, 11.8. ESI-MS:
287(þHþ), 309 (þNaþ).
4.2.4.5. Urea-dipeptide (1b). 90 mg (0.52 mmol) of 7b and 190 mg
(0.56 mmol) of 8b were dissolved in 5 mL of dried CH3CN and
stirred at rt overnight. A day later, the white precipitate was col-
lected by filtration and washed with water to afford urea-dipeptide
1b (80 mg, 0.22 mmol) as a white solid with a yield of 42%. 1H NMR
(0.6 g, 2.8 mmol, Rf¼0.2) with a yield of 90%. 1H NMR
d
(ppm, CDCl3,
-proton,
d
(ppm, CDCl3, 400 MHz, 20 mM): 7.12 (s, broad, 2H, amide-NHs),
400 MHz): 5.84 (d, 1H, amide-H, 3J¼7.3 Hz), 4.62 (m, 1H,
a
6.38 (d, 2H, urea-NHs, 3J¼8.0 Hz), 4.29–4.23 (q, 2H,
a-proton,
3J¼4.0 Hz), 4.18 (quartet, 2H, –CH2, 3J¼7.2 Hz), 2.25 (quartet, 2H,

8276
D. Ke et al. / Tetrahedron 65 (2009) 8269–8276
–CH2, 3J¼7.8 Hz), 1.66 (m, 2H, –CH2, 3J¼6.4 Hz), 1.53 (m, 1H, –CH,
3J¼8.0 Hz), 1.29 (t, 3H, –CH3, 3J¼7.2 Hz), 1.17 (t, 3H, –CH3, 3J¼7.8 Hz),
References and notes
0.95 (quartet, 6H, –CH3, 3J¼5.6 Hz). 13C NMR
d (ppm, CDCl3,
1. (a) Zhao, X.; Chang, Y.-L.; Fowler, F. W.; Lauher, J. W. J. Am. Chem. Soc. 1990, 112,
6627–6634; (b) Cbang, Y.-L.; West, M.-A.; Fowler, F. W.; Lauher, J. W. J. Am.
Chem. Soc. 1993, 115, 5991–6000; (c) Coe, S.; Kane, J. J.; Nguyen, T. L.; Toledo, L.
M.; Wininger, E.; Fowler, F. W.; Lauher, J. W. J. Am. Chem. Soc. 1997, 119, 86–93.
2. Castellano, R. K.; Kim, B. H.; Rebek, J., Jr. J. Am. Chem. Soc. 1997, 119, 12671–
12672.
3. Rincon, A. M.; Prados, P.; de Mendoza, J. J. Am. Chem. Soc. 2001, 123, 3493–3498.
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100 MHz): 173.7, 173.3, 61.0, 50.4, 41.3, 29.1, 24.6, 22.6, 21.7, 13.9, 9.6.
ESI-MS: 215, 238 (þNaþ).
4.2.7. N-Ethyl-carbonyl-L-leucine acid (13). 600 mg (2.8 mmol) of
12 and 240 mg (6 mmol) of NaOH were dissolved in 5 mL of
C2H5OH and 5 mL of water and stirred for a week. Then the solution
was neutralized using 1 M HCl to pH¼3.0. 10 mLꢁ3 of dichloro-
methane was then used for extraction. The organic phases were
collected and dried with anhydrate Na2SO4. The solvents were re-
moved to afford 13 as a white solid (500 mg, 2.7 mmol) with a yield
5. Vos, M. R. J.; Jardl, G. E.; Pallas, A. L.; Breurken, M.; van Asselen, O. L. J.; Bomans,
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of 96%. 1H NMR
d (ppm, CDCl3, 300 MHz): 8.92 (s, broad, 1H, acid-
H), 5.97 (d, 1H, amide-H, 3J¼7.2 Hz), 4.62 (m, 1H,
a-proton,
3J¼6.0 Hz), 2.29 (quartet, 2H, –CH2, 3J¼7.2 Hz), 1.75 (m, 2H, –CH2,
3J¼6.0 Hz),1.60 (m,1H, –CH, 3J¼8.8 Hz),1.17 (t, 3H, –CH3, 3J¼7.5 Hz),
0.95 (quartet, 6H, –CH3, 3J¼5.6 Hz). ESI-MS: 186 (ꢂHþ), 187, 209
(ꢂHþ and þNaþ), 225 (ꢂHþ and þKþ).
4.2.8. 4-Nitro-phenyl N-ethyl-carbonyl-
L
-leucine acid ester
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(14). 500 mg (2.7 mmol) of 13, 560 mg (4 mmol) of 4-nitro-
phenol, and 800 mg (4 mmol) of DCC were mixed with 20 mL of
CH2Cl2 and stirred overnight. Then the precipitate was filtered
out. After removal of organic solvents, the residue was applied to
flash chromatography with petrol ether/dichloromethane/ethyl
acetate¼10:10:1 as eluents to afford 14 as a light-yellow solid
(700 mg, 2.3 mmol, Rf¼0.2) with a yield of 85%. 1H NMR
d (ppm,
CDCl3, 400 MHz): 8.30 (d, 2H, phenyl-Hs, 3J¼9.0 Hz), 7.31 (d, 2H,
phenyl-Hs, 3J¼9.0 Hz), 5.78 (d, 2H, amide-H, 3J¼6.9 Hz), 4.83 (m,
1H,
a
-proton, 3J¼9.6, 5.4, 8.4 Hz), 2.33 (quartet, 2H, –CH2,
3J¼7.6 Hz), 1.81 (m, 2H, –CH2, 3J¼6.4 Hz), 1.22 (t, 3H, –CH3,
3J¼7.8 Hz), 1.17 (t, 1H, –CH, 3J¼7.8 Hz), 1.05 (t, 6H, –CH3,
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3J¼5.6 Hz). 13C NMR
d (ppm, CDCl3, 100 MHz): 173.9, 171.2, 155.2,
145.6, 125.3, 122.4, 51.1, 41.1, 31.0, 25.1, 22.9, 21.9, 9.6. ESI-MS: 309
(þHþ), 331 (þNaþ), 347 (þKþ).
4.2.9. Dipeptide 2. 156 mg (0.51 mmol) of 14, 90 mg (0.52 mmol) of
7b, and 2.0 mL of triethyl amine were dissolved in 2 mL of CH3CN
(containing 5% of methanol) and stirred overnight. After CH3CN was
removed under vacuum, the residue was re-dissolved into 10 mL of
dichloromethane and washed using satd Na2CO3 aqueous water
(respectively, 10 mLꢁ3). The organic phases were collected and
then dried with anhydrate Na2SO4. The solvents were removed and
the resulted residue was applied to flash chromatography with
petrol ether/dichloromethane/ethyl acetate¼5:5:1 as eluents to
afford 2 as a white solid (135 mg, 0.39 mmol, Rf¼0.2) with a yield of
17. Semetey, V.; Didierjean, C.; Briand, J.-P.; Aubry, A.; Guichard, G. Angew. Chem.,
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76%. 1H NMR
d (ppm, CDCl3, 400 MHz, 24 mM): 6.77–6.75 (d, 1H,
amide-NH, No. 2, 3J¼8.1 Hz), 6.36 (t, 1H, amide-NH, No. 1,
3J¼8.0 Hz), 6.11–6.09 (d, 1H, amide-NH, No. 3, 3J¼7.9 Hz), 4.54–4.46
3
(m, 1H,
a
-proton, JHN ¼8.4 Hz), 4.43–4.36 (m, 1H,
a-proton,
a
3JHN ¼8.4 Hz), 3.22–3.16 (q, 2H, n-Pr-
a
C–Hs, 3J¼6.6 Hz), 2.26–2.20
a
(q, 2H, –CH2, 3J¼6.0 Hz), 1.80–1.45 (m, 8H,
b-Hs of Leu, g-Hs of Leu,
and n-Pr-
b
C–Hs, 3J¼3–8 Hz), 1.17–1.12 (t, 3H, –CH3, 3J¼7.5 Hz),
0.93–0.88 (q, 15H,
d
-Hs of Leu and n-Pr-
g
C–Hs, 3 J¼6.0, 3J¼6.8 Hz).
13C NMR
d
(ppm, CDCl3, 100 MHz): 174.0, 172.3, 171.6, 51.9, 51.6,
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ESI-MS: 341, 364 (þNaþ).
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We thank NSFC (Nos. 20872145 and 20733006), the Chinese
Academy of Sciences, the National Research Fund for Fundamental
Key Project 973 (2006CB806200, 2007CB936401), and the CAS/
SAFEA International Partnership Program for Creative Research
Teams.