Synthesis of novel diketopiperazine derivative and obsevation of self-assembled structure

Article abstract of DOI:10.3987/COM-09-11762

An N-monomethylated unsymmetrical diketopiperazine was synthesized from D-p-hydroxyphenylglycine and sarcosine, and condensed with trans-1,4-cyclohexanedicarboxylic acid to obtain the ester having diketopiperazine moieties at the both termini. Atomic force microscope measurement indicated that the ester formed a supramolecular structure aligned in a circular pattern based on hydrogen bonding between the amide groups of the diketopiperazine moieties.

Full text of DOI:10.3987/COM-09-11762

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HETEROCYCLES, Vol. 78, No. 10, 2009, pp. 2523 - 2530. © The Japan Institute of Heterocyclic Chemistry
Received, 21st May, 2009, Accepted, 30th June, 2009, Published online, 1st July, 2009
DOI: 10.3987/COM-09-11762
SYNTHESIS OF NOVEL DIKETOPIPERAZINE DERIVATIVE AND
OBSEVATION OF SELF-ASSEMBLED STRUCTURE
Yosuke Ohta,a Kayo Terada,a Toshio Masuda,b and Fumio Sanda*a
a
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto
University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan
b
Department of Environmental and Biotechnological Frontier Engineering,
Faculty of Engineering, Fukui University of Technology, 3-6-1 Gakuen, Fukui
910-8505, Japan
Abstract – An N-monomethylated unsymmetrical diketopiperazine was
synthesized from D-p-hydroxyphenylglycine and sarcosine, and condensed with
trans-1,4-cyclohexanedicarboxylic acid to obtain the ester having
diketopiperazine moieties at the both termini. Atomic force microscope
measurement indicated that the ester formed a supramolecular structure aligned in
a circular pattern based on hydrogen bonding between the amide groups of the
diketopiperazine moieties.
INTRODUCTION
Diketopiperazine (DKP), the smallest cyclic peptide, has two s-cis amide groups that form tandem
hydrogen-bonding strands.1–5 This nature enables DKPs to take regulated higher order structures such
as liquid crystalline6 and microcapsules7 by appropriately modifying the side chains. Phenylalanine-,
aspartic and glutamic acid-based DKPs serve as oil and ionic liquid gelators8–10 due to the strong ability
to form intermolecular hydrogen bonding even in the presence of large amount of solvents. In the field
of polymer chemistry, several attempts have been made to utilize DKPs as components of polymers that
largely interact with polymeric and monomeric compounds. We have recently reported the
polycondensation of acidic amino acid DKPs with various diamines and dibromoxylenes to obtain
polyamides and polyesters.11 We have also performed the acyclic diene metathesis polycondensation of
glutamic acid DKP -alkenyl esters with ruthenium catalysts.12 The formed polymers are associated in
the solid and solution states based on hydrogen bonding between the DKP moieties.

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DKP derivatives are commonly poorly soluble in solvents due to the lack of flexibility of the DKP ring,
which is caused by the confinement of the amide groups to the ring, as well as self-assembling based on
hydrogen bonding between the amide groups. We have confirmed that DKPs having long alkyl groups
aggregate based on hydrophobic interaction between the alkyl groups together with hydrogen bonding.13
In this manuscript, we synthesize a novel trans-1,4-cyclohexanedicarboxylate 1 substituted with an
unsymmetrical DKP consisting of D-p-hydroxyphenylglycine and sarcosine (Figure 1), and observe
aggregates at the solid state by atomic force microscope (AFM) measurement.
O
N
H
O
O
O
N
N
O
O
O
H
N
O
1
Figure 1. Structure of compound 1
RESULTS AND DISCUSSION
We had designed DKP–linker–DKP type compounds having DKP moieties at the both termini to
construct supramolecular structures. We selected D-p-hydroxyphenylglycine as one component of DKP,
because it is commercially available and has hydroxy group, which is connectable to a linker part.
Serine and threonine also have hydroxy group and satisfy this demand, but it is considered that phenylene
spacer between the hydroxy group and chiral center of D-p-hydroxyphenylglycine is rigid compared with
methylene and methine spacers of serine and threonine.
Consequently, use of D-p-
hydroxyphenylglycine seems to be more effective for constructing regulated structures. We first tried to
synthesize unsymmetrical DKPs from D-p-hydroxyphenylglycine with several amino acids such as
glycine, alanine, and leucine, but the formed compounds were poorly soluble in organic solvents, and it
was difficult to purify and isolate them. We resolved the solubility problem by employing sarcosine
(N-methylglycine) as another component. As the result, we could obtain a highly pure solvent-soluble
diketopiperazine 5 (Scheme 1). N-Monomethylation was truly effective to enhance the solubility. We
then tried to synthesize DKP–linker–DKP compounds with various linkers including xylylene,
phenyleneethynylene, phenylenesilylene, and ethylenesilylene, but could not obtain the target compounds
with sufficient purities. Compound 1 having trans-1,4-cyclohexanedicarboxylate linker could be
synthesized in the present study.
Scheme 1 illustrates the synthetic route for 1. The amino group of D-p-hydroxyphenylglycine was
protected with Boc to obtain 2, and it was submitted to condensation with sarcosine ethyl ester, which
was prepared by ethyl esterification of N-(tert-butoxycarbonyl)sarcosine (Boc-sarcosine) followed by Boc

HETEROCYCLES, Vol. 78, No. 10, 2009
2525
cleavage with trifluoroacetic acid (TFA).
DMT-MM {4-[4,6-dimethoxy-1,3,5-triazin-2-yl]-4-
methylmorpholinium chloride} was used as a condensation agent to avoid ester formation between the
carboxy and hydroxy groups of 2. DMT-MM can selectively react with amine to form amide with
carboxylic acid in the presence of alcohol.14 Then the terminal Boc group of formed dipeptide 4 was
removed with TFA, and the resulting TFA salt was heated in the presence of triethylamine in
mesitylene/2-methyl-1-propanol.
The intramolecular ester-amide exchange releasing ethanol
satisfactorily proceeded to give diketopiperazine 5 in a high yield (81%). Diketopiperazine 5 was
obtained as precipitate during the reaction. The purity of 5 was high enough. It is considered that the
precursor N-nonprotected linear dipeptide was completely soluble in the solvent, while 5 was slightly
soluble in mesitylene/2-methyl-1-propanol mixed solvent. Diketopiperazine 5 was soluble in DMF and
DMSO. Subsequently, 1 was synthesized by the condensation of trans-1,4-cyclohexanedicarboxylic
acid with two equivalents of 5 using EDC•HCl {1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride} as a condensation agent in the presence of DMAP {4-(dimethylamino)pyridine}.
O
O
H
H2N
O
N
OH
OH
(Boc)2O
O
dioxane/H2O
OH
OH
2 100%
D-p-hydroxyphenylglycine
O
O
EtOH
EDC•HCl, DMAP
O
1) TFA, CH2Cl2
OH
O
N
O
N
CH2Cl2
2) 2, Et3N
O
O
DMT-MM, CH2Cl2
3 71%
Boc-sarcosine
O
H
OH
O
N
O
O
O
N
1) TFA, CH2Cl2
2) Et3N
O
O
N
N
H
mesitylene/2-methyl-1-propanol
OH
4
5
40%
81%
O
N
trans-1,4-cyclohexanedicarboxylic acid
H
O
EDC•HCl, DMAP
O
O
N
N
O
O
DMF
O
H
N
O
1
82%
Scheme 1. Synthetic route for compound 1

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Compound 1 was insoluble in common organic solvents except DMSO, although one amide N–H group
was methylated for enhancing the solubility. We measured the circular dichroism spectra of a solution
of 1 in DMSO to examine the possibility of formation of a chiral supramolecular structure, but could not
observe a Cotton effect at all. DMSO possibly breaks hydrogen bonding between the amide groups of 1
molecules to prevent them from association. We then examined the morphology of 1 in the solid state
by AFM. Figure 2 shows the AFM image of a silicon substrate coated with a solution of 1 in DMSO.
It exhibited circular objects together with a linear ribbon-like structure. Judging from the size (diameter:
close to 10 nm, height: close to 1 nm)15 of the circular structure, it seems that molecules of 1 aggregated
on a silicon surface during the process of DMSO evaporation. The linear structure looks like a
self-assembly of the circular objects.
200 nm
Figure 2. AFM image of 1 coated on a silicon wafer from a 0.2 w/v% solution in DMSO.
Considering the spread symmetrical structure of 1 having DKP moieties at the both termini, it is likely
that 1 forms intermolecular hydrogen bonding between the DKP moieties to construct cyclic
supramolecuels observed in the AFM. To elucidate this assumption, we modeled supramolecular
structures consisting of 2–16 units of 1, wherein hydrogen bonding exists between the DKP moieties.
Figure 3 plots the potential energy per unit and diameter of the ring structure calculated with the
molecular mechanics method using the MMFF94 force field.16 We postulated that the dimer would
aggregate in a linear fashion and that the trimer and the tetramer would aggregate based on a single-strand
of hydrogen bonding between DKP moieties to form the ring structures. Oligomers consisting of five
and more units would aggregate based on double-strands of hydrogen bonding to form cyclic structures.
The energy per one unit gradually decreased with the increment of unit number (n), and became saturated
around n = 5. The diameter of the ring increased in proportion to n. For example, the diameter of 10.4

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nm corresponded with a ring consisting of 14 molecules of 1 as illustrated in Figure 4, whose height was
calculated to be 1.1 nm. The value was coincident with the value (1.0 nm) observed by AFM.
Consequently, these data suggest that the AFM-observed ring structure with a diameter of 10.4 nm is
cyclic 14-mer of 1 aggregated by intermolecular hydrogen bonding between the terminal DKP moieties.
It seems that cyclic oligomers larger and smaller than 14-mer also exist judging from the distribution of
ring size observed in Figure 2 and the energy diagram in Figure 3.
Figure 3. Unit number vs. energy per unit and diameter of cyclic supramolecules of 1.
Figure 4. Cyclic 14-mer of 1 optimized by the molecular mechanics calculation. Green dotted lines
represent hydrogen bonds between the DKP amide moieties.
We have demonstrated the synthesis of novel compound 1 having two DKPs consisting of
D-p-hydroxyphenylglycine and sarcosine. AFM measurement and molecular mechanics calculations

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suggested that 1 formed cyclic supramolecular structures based on intermolecular hydrogen bonding
between the terminal DKP moieties on silicon surface. It is expected that further molecular design of a
linker tethering two DKPs leads to variation of supramolecular structures.
EXPERIMENTAL
13
Measurements. 1H and C NMR spectra were recorded on a JEOL EX-400 spectrometer. Melting
points (mp) were measured on a Yanaco micro melting point apparatus. Specific rotations ([]D) were
measured on a JASCO DIP-100 digital polarimeter with a sodium lamp as a light source. Elemental
analysis was done at the Microanalytical Center of Kyoto University. Mass spectra were measured on a
JEOL JMS-HX110A mass spectrometer. A sample for AFM measurement was prepared by dropping a
solution of 1 in DMSO (0.2 w/v%) on a silicon substrate, followed by washing with DMSO and drying in
vacuo. Prior to preparation of the sample, the silicon substrate was treated with a fresh piranha solution
at 100 °C for 1 h to form clean silicon oxide surface, followed by extensive rinsing with deionized water,
and then dried under nitrogen flow. A Nanoscope III (Digital Instruments, Inc.) was used for
tapping-mode AFM to observe phase and height images.
Materials. All the reagents in monomer synthesis were used as purchased without purification. DMF
was distilled over calcium hydride.
Synthesis of 1,4-trans-bis[cyclo(D-p-phenylglycinylsarcosine)]cyclohexane dicarboxylate (1).
N-(tert-Butoxycarbonyl)-D-p-hydroxyphenylglycine (2) Triethylamine (11 mL, 79.0 mmol) was
added to a mixture of D-p-hydroxyphenylglycine (8.84 g, 52.9 mmol) and di-tert-butyl dicarbonate
[(Boc)2O, 11.5 g, 52.9 mmol] in dioxane/H2O (70 mL/70 mL) at 0 °C, and the reaction mixture was
stirred at rt overnight. H2O (100 mL) was added to the resulting mixture, and then it was washed with
EtOAc. The aqueous layer was acidified with 2 M HCl to pH 2, and then extracted with EtOAc. The
organic layer was dried with anhydrous MgSO4 and concentrated on a rotary evaporator to obtain 2 as a
yellow solid quantitatively. 1H NMR (400 MHz, CDCl3): δ 1.40 [s, 9H, –OC(CH3)3], 5.02 [s, 1H,
>NCH<], 5.19 [s, 1H, –ArOH], 5.73 [s, 1H, –NH–], 6.70–7.16 [m, 4H, Ar], 8.28 [br, 1H, –OH].
N-(tert-Butoxycarbonyl)sarcosine ethyl ester (3) EDC•HCl (19.2 g, 100 mmol), DMAP (1.22 g, 9.99
mmol), and EtOH (10 mL, 171 mmol) were added to a solution of N-(tert-butoxycarbonyl)sarcosine (18.9
g, 99.9 mmol) in CH2Cl2 (500 mL) at 0 °C, and then the resulting mixture was stirred at rt overnight.
Then, CH2Cl2 was distilled off using a rotary evaporator, and the residue was dissolved in EtOAc. It
was washed with 0.5 M HCl, saturated NaHCO3 aq., and saturated NaCl aq., and then dried over
anhydrous MgSO4. EtOAc was evaporated off to obtain 3 as a colorless liquid in 71% yield. 1H NMR
(400 MHz, CDCl3): δ 1.27, 1.29 [s, 3H, –CH2CH3], 1.43, 1.47 [s, 9H, –OC(CH3)3], 2.92, 2.94 [s, 3H,
>NCH3], 3.88, 3.97 [s, 2H, >NCH2–], 4.20 [s, 2H, –OCH2–].
N-(tert-Butoxycarbonyl)-D-p-hydroxyphenylglycinylsarcosine ethyl ester (4) Trifluoacetic acid

HETEROCYCLES, Vol. 78, No. 10, 2009
2529
(TFA, 15.0 mL, 202 mmol) was added to a solution of 3 (8.43 g, 38.8mmol) in CH2Cl2 (100 mL) at 0 °C,
and the reaction mixture was stirred at rt overnight. After confirming the complete consumption of 3 by
TLC, CH2Cl2 and TFA were distilled off in vacuo. The residual viscous liquid was dissolved in
EtOAc/CH2Cl2 (200 mL/100 mL). Triethylamine (17 mL, 122 mmol), DMT-MM (Tokuyama Co.),
12.6 g, 38.8 mmol), and 2 (13.4 g, 50.1 mmol) were added to the solution at 0 °C, and the resulting
mixture was stirred at rt overnight. The resulting mixture was concentrated on a rotary evaporator, and
then the residual viscous liquid was dissolved in CH2Cl2 (150 mL). It was washed with 0.5 M HCl,
saturated NaHCO3 aq., and saturated NaCl aq., and then dried over anhydrous MgSO4. CH2Cl2 was
evaporated off, and the residual mass was purified by recrystallization from hexane/EtOAc (1/2, volume
ratio) to obtain 4 as a colorless solid in 40%. Mp 155–161 °C. 1H NMR (400 MHz, CDCl3): δ 1.24 [t,
J = 6.8 Hz, 3H, –CH2CH3], 1.41 [s, 9H, –OC(CH3)3], 2.93, 3.01 [s, 3H, >NCH3], 3.88, 4.34 [d, J = 8.8 Hz,
2H, >NCH2–], 5.54, 5.92 [d, J = 4.0 Hz, 1H, >NCH<], 7.01 [s, 1H, –ArOH], 7.14 [d, J = 4.4 Hz, 1H,
–NH–], 6.74–7.23 [m, 4H, Ar]. 13C NMR (100 MHz, CDCl3): δ 14.07 [CH3CH2–], 28.32 [(CH3)3C–],
36.29 [>NCH3], 50.09 [>NCH2–], 54.67 [>NCH<], 61.38 [CH3CH2–], 80.01 [(CH3)3C–], 115.86, 128.35,
129.19, 155.25 [Ar], 156.35 [–OCONH–], 168.87[>CHCON<], 171.32[–CH2COO–]. Anal. Calcd for
C18H26N2O6: C, 59.00; H, 7.15; N, 7.65. Found: C, 58.87; H, 7.12; N, 7.52.
cyclo(D-p-Hydroxyphenylglycinylsarcosine) (5) TFA (11 mL, 148 mmol) was added to a solution of 4
(5.54 g, 15.1mmol) in CH2Cl2 (100 mL) at 0 °C, and the reaction mixture was stirred at room temperature
overnight. After confirming the complete consumption of 4 by TLC, CH2Cl2 and TFA were distilled off
in vacuo. The residual viscous liquid was dissolved in mesitylene/2-methyl-1-propanol (2/1, volume
ratio), and then triethylamine (5.00 mL, 35.9 mmol) was added to the resulting solution. The resulting
mixture was heated to 110 °C and stirred for 11 h. After standing to cool to rt, a white solid precipitated
was separated by filtration to obtain 5 as a colorless solid in 81%. Mp (decomposed at 250–257 °C).
[]D −41.4° (c 0.10 g/dL, MeOH at rt). 1H NMR (400 MHz, DMSO-d6): δ 2.80 [s, 3H, >NCH3], 3.87,
4.11 [d, J = 8.8 Hz, 2H, >NCH2–], 4.79 [s, 1H, >NCH<], 6.72–7.11 [m, 4H, Ar] , 8.59 [s, 1H, –NH–]
9.48 [s, 1H, –ArOH]. 13C NMR (100 MHz, DMSO-d6): δ 33.16 [>NCH3], 50.96 [>NCH2–], 58.10
[>NCH<], 115.18, 127.97, 129.47, 157.06 [Ar], 164.77[–CH2CO–], 164.96[>NCO–]. Anal. Calcd for
C11H12N2O3: C, 59.99; H, 5.49; N, 12.72. Found: C, 59.80; H, 5.53; N, 12.68. HRMS (M+). Calcd
for C11H12O3N2 (m/z) 220.0848. Found: 220.0847.
1 EDC•HCl (959 mg, 4.99 mmol), 4-(dimethylamino)pyridine (DMAP, 61.0 mg, 0.499 mmol), trans-1,
4-cyclohexanedicarboxylic acid (431 mg, 2.50 mmol) were added to a solution of 5 (1.10 g, 4.99 mmol)
in DMF (90 mL) at 0 °C and the resulting mixture was stirred at rt overnight. The precipitate formed
was separated by filtration and washed with H2O and MeOH to obtain 1 as a colorless solid in 82%. Mp
(decomposed at 225–227 °C). []D –51° (c 0.10 g/dL in DMSO at rt). 1H NMR (400 MHz,
DMSO-d6): δ 1.57–2.64 [m, 8H, –C6H10–], 2.82 [s, 6H, >NCH3], 4.04 [d, J = 9.3 Hz, 4H, >NCH2–], 4.99

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[s, 2H, >NCH<], 7.11–7.38 [m, 8H, Ar] , 8.71 [s, 2H, –NH–]. 13C NMR (100 MHz, DMSO-d6): δ 27.37
[>CH(C2H4)2CH<], 33.24 [>NCH3], 41.43 [>CH(C2H4)2CH<], 50.95 [>NCH2–], 58.06 [>NCH<], 121.86,
128.20, 136.73, 150.17 [Ar], 164.28[–CH2CO–], 164.97[>NCO–], 173.61 [–OCO–]. IR (cm-1, KBr):
3570 (NH), 3425 (NH), 3232 (NH), 2923 (CH), 2864 (CH), 1728 (C=O), 1673 (NHCO), 1510, 1475,
1451, 1321, 1167, 1148, 1013, 804. HRMS (M+). Calcd for C30H32O8N4 (m/z) 577.2298. Found:
577.2296.
Molecular mechanics calculation. The molecular mechanics calculation was carried out with
Wavefunction Inc., Spartan ’06 Windows, using the MMFF94 forcefield.
ACKNOWLEDGEMENTS
We thank Prof. Hirokazu Hasegawa and Mr. Tomohiro Inoue at Kyoto University who assisted to us to
measure AFM.
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