Water soluble synthetic dieptide-based biodegradable nanoporous materials

Article abstract of DOI:10.1039/b822607e

Two water-soluble synthetic dipeptides, β-alanyl-l-phenylglycine and its retro analogue l-phenylglycyl-β-alanine form a new class of dipeptide-based nanoporous materials, which are composed of a hybrid of α and β-amino acids. These materials adsorb N₂ gas with adsorption capacity of 173 cc g^-1 and 71 cc g^-1 and BET surface area of 56.76 m²g^-1 and 41.73 m²g^-1 for these dipeptides, respectively. Moreover, these nanoporous materials are found to be biodegradable towards soil bacterial consortium making them an interesting class of eco-friendly dipeptide-based nanoporous materials.

Full text of DOI:10.1039/b822607e

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PAPER
www.rsc.org/greenchem | Green Chemistry
Water soluble synthetic dieptide-based biodegradable
nanoporous materials†
Samit Guha,a Tushar Chakrabortyb and Arindam Banerjee*a
Received 17th December 2008, Accepted 20th April 2009
First published as an Advance Article on the web 15th May 2009
DOI: 10.1039/b822607e
Two water-soluble synthetic dipeptides, b-alanyl-L-phenylglycine and its retro analogue
L-phenylglycyl-b-alanine form a new class of dipeptide-based nanoporous materials, which are
composed of a hybrid of a and b-amino acids. These materials adsorb N2 gas with adsorption
capacity of 173 cc g-1 and 71 cc g-1 and BET surface area of 56.76 m2g-1 and 41.73 m2g-1 for these
dipeptides, respectively. Moreover, these nanoporous materials are found to be biodegradable
towards soil bacterial consortium making them an interesting class of eco-friendly
dipeptide-based nanoporous materials.
It is also very challenging to degrade non-protein amino acid
Introduction
based peptides. Seebach and coworkers have reported that soil
bacterial consortium11 can grow with b-amino acid contain-
ing peptides as the sole carbon and energy source.12 More-
over, two aminopeptidases capable of degrading N-terminal
b-alanine containing peptides, namely, L-aminopeptidase-D-
alanine-esterase/amidase (DmpA) from Ochrobactrum anthropi
and b-Ala-Xaa dipeptidase BapA from Pseudomonas sp.
MCI3434 (Ps BapA), have been identified and characterized.13
In this paper we report, a new class of water soluble dipeptide-
based nanoporous materials, which are composed of a hybrid
of b,a-amino acids having sequences b-alanyl-L-phenylglycine
(b-Ala-L-Phg, 1) and its retro analogue L-phenylglycyl-b-alanine
(L-Phg-b-Ala, 2). b-Alanine (b-Ala) is a non-coded naturally
occuring b-amino acid and it has a role in a variety of biological
processes.14 It is believed to be a neurotransmitter in the central
nervous system, binding to receptor sites common to glycine
and g -amino butyric acid (GABA), and acting in the visual
system.14a b-Ala is one of the constituents of the naturally
occuring dipeptides anserine and carnosine.14c,d a-Phenylglycine
(Phg) is a non protein a-amino acid. Its molecular structure is
more rigid than naturally occuring a-phenylalanine (Phe) with
a lower degree of rotational freedom, as it has one CH2 unit less
than phenylalanine. Nanoporous materials formed by dipeptides
1 and 2 are structurally different from the previously reported
dipeptide-based nanoporous materials,5,6 as they are composed
of both a and b-amino acids instead of just a-amino acids.
Moreover, these hybrid b,a-amino acid based nanoporous
materials are found to be biodegradable towards a consortium
of bacteria,11 making them a novel, ecofriendly, new class of
nanoporous materials. These compounds also adsorb nitrogen
gas.2a–e,j
Nanoporous materials based on purely inorganic materials such
as zeolites1 (aluminosilicates) and hybrid materials, consisting of
both organic and inorganic counterparts such as metal–organic
frameworks (MOFs),2 have been extensively studied. These
porous materials can be used in gas storage, chiral recognition,
molecular separation, ion exchange, catalysis, and in sensors.3
However, zeolites and MOFs are not based on purely organic
molecules. There are a few examples of nanoporous materials
based on purely organic molecules and these are termed as
organo zeolites.4–6 These include cyclic bis-urea based porous
materials that bind reversibly with guest molecules,4 Gorbitz’s
Val-Ala and Phe-Phe class structures formed from hydropho-
bic dipeptides5 and Ripmeester’s dipeptide-based microporous
(nanoporous) materials that can adsorb inert gas such as
Xe.6 Recently Sozzani and co-workers have reported dipeptide-
based nanoporous materials and investigated their absorption,
separation, and storage of gases such as methane, carbon
dioxide, and hydrogen.7 Some dipeptide-based compounds are
able to host small organic molecules.5f,8 A recent report describes
that a polymeric sorbent has been used for very efficient removal
of H2S from a mixture of gases for hydrogen purification.9
Nanoporous materials based on water soluble peptide-based
molecules have advantages over purely inorganic and hybrid
materials, because they are of biological origin and generally
non-toxic, eco-friendly and may be used as nanobiomaterials.
Environmentally benign nanomaterials are an emerging field of
current research.10 However, none of the previously mentioned
organic nanoporous materials are experimentally proven as
biodegredable nanomaterials.
aDepartment of Biological Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata, 700 032, India.
E-mail: arindam.bolpur@yahoo.co.in, bcab@iacs.res.in;
Fax: (+)91-332473-2805
Results and discussion
Two water soluble dipeptides, where b-alanine and L-
phenylglycine are used as constituents, b-Ala-L-Phg (1), and
its retro analogue L-Phg-b-Ala (2) (Fig. 1a and b) have been
synthesized by conventional solution-phase methodology,15,16
bDepartment of Cell Biology and Physiology, Indian Institute of
Chemical Biology, Jadavpur, Kolkata, 700 032, India
† Electronic supplementary information (ESI) available: Spectra and
biodegradation data for dipeptides 1 and 2. See DOI: 10.1039/b822607e
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14.2◦, 16.5◦, 20◦, 21.3◦, 21.9◦, 24◦, 24.4◦, 28◦ and dipeptide
2 has peaks at 2q = 5.6◦, 7.6◦, 11.5◦, 16◦, 20.8◦, 21.9◦, 23◦,
24.3◦, 24.7◦, 26.8◦, 28.5◦, 29.7◦. These two dipeptides are retro
analogues with each other and their XRPD patterns are slightly
different. The sharp lines in the XRPD patterns (Fig. 2) suggest
the crystalline behavior of these two compounds. The above
mentioned peaks give an indication that nanopores may be
present in these structures.2h,17 To confirm the nanoporous struc-
tures we further investigated using TEM and gas adsorption
experiments.
Transmission electron microscopic (TEM) studies
To prove their porous architecture we have studied Transmission
electron microscopy (TEM). TEM studies of dipeptides 1 and 2
were carried out using an aqueous solution of the corresponding
compounds (3 mg mL-1) on a carbon coated copper grid
(300 mesh) by slow evaporation and vacuum drying at 30 ◦C for
2 days. The TEM images and electron diffraction patterns clearly
indicate that the formation of nanoporous architecture with
˚
˚
˚
˚
dimensions of 5.8 A ¥ 6.4 A and 3.2 A ¥ 3.2 A for dipeptides 1 and
2 respectively (Fig. 3 and Fig. 4). TEM images clearly show that
dipeptides 1 and 2 form an ordered porous framework. Selected
area electron diffraction pattern of these porous architectures
show sharp spots. This suggests that these porous structures are
crystalline in nature. Energy dispersive X-ray (EDX) analyses
of nanoporous structures indicate the presence of C, N and O
(ESI† Fig. S1a and b). C, N and O come from the dipeptide.
Fig. 1 The molecular structures of the dipeptides (a) 1 and (b) 2.
purified, characterized, and studied. Gorbitz’s hydrophobic
dipeptide-based microporous materials5 are based exclusively
˚
on a-amino acids with pore diameters in the range of 3–10 A. In
contrast to the previous work, two reported dipeptide-based
microporous materials are composed of a hybrid of b and
a-amino acids, where one of the components is a b-amino
acid (b-alanine) and the other component is an a-amino acid
(L-phenylglycine) with varying pore size. These pore sizes are in
˚
the range of 6.4–3.2 A indicating that they are comparable to
the previously reported dipeptide-based nanoporous materials.
X-ray powder diffraction (XRPD) studies
The synthesized dipeptides 1 and 2 were separately grounded to
a powder and examined by X-ray powder diffraction (XRPD)
(Fig. 2). Dipeptide 1 has peaks at 2q = 7◦, 10.6◦, 11.6◦, 12.1◦,
Fig. 3 TEM image of dipeptide (a) 1 showing the formation of an
ordered nanoporous structure; enlarged portion of the figure (a) is shown
in figure (b). (c) Electron diffraction pattern of the porous structure
formed by dipeptide 1 shows sharp spots, indicating its crystalline
behaviour.
Fig. 2 XRPD patterns of the dipeptides (a) 1 and (b) 2 synthesized
(ground at room temperature).
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1 and 2 were also confirmed by the N2 gas adsorption studies.
From the nitrogen adsorption at low P/P0 we observed the
following pore size distribution of the sample using the Horvath–
Kawajoe (H–K) method (micropore analysis). The pore size
distribution curve of dipeptide 1 showed two peaks centered at
˚
˚
5.8 A and 6.4 A (inset Fig. 5a) indicating that dipeptide 1 forms
a nanoporous architecture. The pore size distribution curve of
˚
dipeptide 2 showed one peak centered at 3.2 A (inset Fig. 5b)
also indicating that dipeptide 2 forms a nanoporous architecture.
From the Brunauer–Emmett–Teller (BET) equation, the BET
surface areas were calculated as 56.76 m2g-1 and 41.73 m2g-1 for
dipeptides 1 and 2, respectively (Fig. 6). Prior to the analyses
these samples were degassed at 40 ◦C for 5 h.
Fig. 4 TEM image of (a) dipeptide 2 showing the formation of an
ordered nanoporous structure. (b) Sharp spots in the selected area
electron diffraction pattern of the porous structure formed by dipeptide
2 indicate its crystallinity.
Nitrogen gas adsorption experiments
To prove the porous architecture of dipeptides 1 and 2 we also
carried out N2 gas adsorption experiments. The N2 adsorption–
desorption isotherms (77 K)2a–e,j of dipeptides 1 and 2 are shown
in Fig. 5 and are close to the type I adsorption isotherm typical
for a nanoporous material.2a–e,j,4c Gas adsorption data show
that the adsorption capacities of dipeptides 1 and 2 are about
173 cc g-1 and 71 cc g-1, respectively. Pore sizes of dipeptides
Fig. 6 BET equation shows that the BET surface areas of dipeptides
(a) 1 and (b) 2 are 56.76 m2g-1 and 41.73 m2g-1, respectively.
From TEM images, XRPD patterns and N2 gas adsorption
data it can be said that these reported dipeptides form crystalline
nanoporous structures. However, the pore sizes of these two
dipeptides varied slightly.
Thermogravimetric analysis (TGA) and differential thermal
analysis (DTA) experiments
Dipeptides 1 and 2 showed significant thermal stability in the
solid state, as demonstrated by TGA-DTA experiments. The
TGA-DTA experiments showed that no solvent molecules are
present in the crystal lattice, which is also confirmed from
their elemental data analyses. These dipeptides showed no
decomposition,◦phase transiti◦ons or mass loss up to their melting
points of ~270 C and ~240 C, respectively (Fig. 7). The high
melting points of these dipeptides indicate that the nanoporous
frameworks are highly stable at a high temperature.
Fig. 5 N2 gas adsorption isotherms (type I), adsorption , desorption
for dipeptides (a) 1 and (b) 2. Adsorption capacities are 173 cc g-1
and 71 cc g-1 for 1 and 2, respectively. Insets represent the pore size
distribution curves showing pore sizes of 5.8 A ¥ 6.4 A and 3.2 A ¥ 3.2 A
of dipeptides 1 and 2, respectively.
˚
˚
˚
˚
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Fig. 8 Time dependent biodegradation of dipeptides 1 and 2 at 0 h, 24 h
(1 d), 7 d and 14 d after treatment with bacterial consortium quantified
through ESI-MS data. Intensity of the molecular ion peak for dieptides
1 and 2, [M + H]+ gradually decreases with respect to time.
by colony formation assay on agar plates (ESI† Fig. S4). The
growth of bacteria in the dipeptide containing medium was
observed in parallel with the depletion of each dipeptide in the
medium, and indicated biodegradation as well as utilization of
the nanomaterial for subsistence (Fig. 9). Dipeptides 1 and 2
were utilized easily by the bacterial consortium, which is also
confirmed from their respective mass spectra. Viability was
tested with 1% peptone in M9 media19 where the bacteria give
good growth. However, bacterial consortia from other sources
may be tried to challenge the recalcitrant nature of this material.
The population of bacteria utilizing dipeptide was found to be
Fig. 7 TGA-DTA curves of the dipeptides (a) 1 and (b) 2. It is clear
from these figures that no solvent molecule is present within the crystal
lattice.
Biodegradability test
Soil bacterial population can utilize and degrade many natural
and synthetic materials, and they often do it as a social network,
or community. Such communities can be selected from a pre-
existing population. We wanted to test this hypothesis against
the dipeptide nanoporous materials described here because
it is challenging to degrade these non-protein amino acid
based dipeptides. Biodegradation of the dipeptide nanoporous
materials was performed using a soil derived culturable bacterial
consortium.18 The bacterial consortium was selected in two
steps. First, in a low level non-specific peptide as nutrient and
then in the specific dipeptide in same medium. After selection
on a specific dipeptide, the growth of the selected bacterial
population in the presence of a dipeptide as the major carbon
and nitrogen source was measured in comparison with a control
without bacteria. The growth was monitored for a period of
eight weeks at 30 ◦C. Growth was measured from optical density
(OD) taken at 595 nm. The degradation of the nanoporous
materials based on dipeptides 1 and 2 have been detected using
mass spectrometric analysis of these nanomaterials containing
soil bacterial consortium with respect to different time intervals
(0 h, 24 h, 7 d, 14 d). It was observed that intensity of the
peak corresponding to the [M + H]+ (where M = molecular
ion of dipeptides 1 and 2) gradually decreased with respect to
time and after 14 d the intensity was diminished to a great
extent leading to the appearance of a tiny peak of [M + H]+
in the spectrum (Fig. 8) (ESI† Fig. S2 and S3). This clearly
indicates that dipeptide nanomaterials have been used as food
for soil bacterial consortium. The growth was further confirmed
Fig. 9 The growth of bacterial consortium on dipeptides, where series
2 and 3 represent dipeptides 1 and 2, respectively. Series 1 represents the
same consortium grown on 1% peptone in M9 medium and series 4 is
the same as series 2, but uninoculated to serve as background control.
OD595 was plotted against time, (1, 2, 3, 4, 5, 6 time scale represents time
0 h, 6 h, 12 h, 24 h, 7 d, 14 d). Growth remained stalled and did not
decline much for at least 4 weeks.
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of a mixed variety, and the dominant Pseudomonas sp. isolated
from the population failed to utilize or degrade the dipeptide
in isolation see Fig. 10. Apparently, the mixed population is
needed for its utilization. However, further studies will be needed
to prove this point conclusively. The solubility and uptake of
dipeptide may not be the decisive factor in these bacterial
subsistence and degradation phenomena. The two dipeptides
are water soluble. This result indicates that some bacterial
populations can subsist on the peptide–nanomaterial, which
makes these nanomaterials environmentally safe. It is expected
that a soil bacterial population would be able to degrade
such material once disposed to the environment. Recently,
bacteria subsisting on antibiotics have been isolated from soil,
and shown to be phylogenetically diverse.20 The same may
hold for nanomaterials formed from peptides, carbohydrates,
hydrocarbon and nucleic acids.
and desorption of N2 gas. Moreover, the biodegradibility of
these nanobiomaterials towards soil bacterial consortium and
water solubility makes them interesting candidates in green
nanobiotechnology. Our results clearly indicate that the reported
dipeptides composed of a hybrid of a and b-amino acids are
amenable to biodegradation and that a soil bacterial consortium
was able to utilize these dipeptides as sole carbon and en-
ergy sources. Water soluble, biodegradable short peptide-based
nanoporous materials can be regarded as a new entry in the
plethora of nanoporous materials, which are environmentally
safe and can be used as eco-friendly nanobiomaterials.
Experimental section
Dipeptide synthesis
The dipeptides 1 and 2 were synthesized by conventional
solution-phase methods using racemization free fragment con-
densation strategy. The Boc group was used for N-terminal
protection and the C-terminus was protected as a methyl
ester. Couplings were mediated by dicyclohexylcarbodiimide/
1-hydroxybenzotriazole (DCC/HOBt). Methyl ester deprotec-
tion was performed via the saponification method, and the Boc
group was deprotected by 98% formic acid. All the intermediates
were characterized by 300 MHz 1H NMR and mass spectrome-
try. The final compounds were fully characterized by 300 MHz
1H NMR spectroscopy, 13C NMR spectroscopy, DEPT 135,
mass spectrometry.
(a) Boc-b-Ala-OH. See reference 16.
(b) Boc-Phg-OH. A solution of L-phenylglycine (4.53 g,
30 mmol) in a mixture of dioxane (60 mL), water (30 mL),
and 1 N NaOH (30 mL) was stirred and cooled in an ice-water
bath. Di-tert-butylpyrocarbonate (7.2 g, 33 mmol) was added,
and stirring was continued at room temperature for 6 h. Then
the solution was concentrated in a vacuum to about 20–30 mL,
cooled in an ice-water bath, covered with a layer of ethyl acetate
(about 50 mL) and acidified with a dilute solution of KHSO4
to pH 2–3 (Congo red). The aqueous phase was extracted with
ethyl acetate, and this operation was carried out repeatedly. The
ethyl acetate extracts were pooled, washed with water, dried
over anhydrous Na2SO4, and evaporated in a vacuum. The pure
material was obtained.
Fig. 10 The growth of a clonally selected Pseudomonas sp., a dominant
member of the same bacterial consortia failed to utilize or degrade the
dipeptide, where series 2 and 3 represent dipeptides 1 and 2, respectively.
Series 1 represents the same consortium grown on 1% peptone (control)
in M9 medium and series 4 is the same as series 2, but uninoculated to
serve as the background control. OD595 was plotted against time, (1, 2,
3, 4, 5, 6, 7, 8 time scale represents time 0 h, 6 h, 12 h, 24 h, 7 d, 14 d, 30
d, 60 d).
Comparison between hydrophobic micropous materials, Ala-Val,
Val-Ala with these reported dipeptides
Yield: 6.58 g (26.2 mmol, 87%).
The reported dipeptides are structurally different from the
previously reported Ala-Val and Val-Ala dipeptides6 because
the reported dipeptides are composed of both a and b-amino
acid residues instead of just a-amino acids. However, all these
compounds are nanoporous materials. Moreover, the reported
dipeptides 1 and 2 are experimentally proven to be biodegradable
nanoporous materials.
Anal. Calcd for C13H17NO4 (251): C, 62.14; H, 6.82; N, 5.57.
Found: C, 62.19; H, 6.75; N 5.59%.
(c) Boc-b-Ala(1)-Phg(2)-OMe. 3.78 g (20 mmol) of Boc-
b-Ala-OH was dissolved in 10 mL of DMF in an ice-water
bath. H-Phg-OMe was isolated from 8.07 g (40 mmol) of the
corresponding methyl ester hydrochloride by neutralization,
subsequent extraction with ethyl acetate, and ethyl acetate
extract was concentrated to 10 mL. It was then added to the
reaction mixture, followed immediately by 4.12 g (20 mmol)
of dicyclohexylcarbodiimide (DCC) and 2.7 g (20 mmol) of
HOBt. The reaction mixture was allowed to come to room
temperature and stirred for 3 d. The residue was taken up in
ethyl acetate (40 mL) and dicyclohexylurea (DCU) was filtered
off. The organic layer was washed with 1 N HCl (3 ¥ 30 mL),
brine (1 ¥ 30 mL), 1 M sodium carbonate (3 ¥ 30 mL),
Conclusions
In conclusion this study indicates a new class of self-assembling
water soluble dipeptide-based nanoporous environmentally be-
nign materials in which the constituent dipeptides are made up of
a hybrid of a and b-amino acid residues, instead of convention-
ally used dipeptides containing only a-amino acid residues.5,6
These nanoporous materials have been used for adsorption
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brine (2 ¥ 30 mL), dried over anhydrous sodium sulfate, and
evaporated in a vacuum. A white material was obtained.
Yield: 5.99 g (17.8 mmol, 89%).
1H NMR (300 MHz, CDCl3): d = 7.31–7.36 (m, 5H, Phg(2)
phenyl ring protons), 6.64 (d, J = 7.1 Hz, 1H, Phg(2) NH), 5.55
(d, J = 7.1 Hz, 1H, Phg(2) Ca H), 5.16 (br, 1H, b-Ala(1) NH),
3.73 (s, 3H, -OCH3), 3.39 (q, J = 6.3 Hz, 2H, b-Ala(1) Cb Hs);
2.47 (t, J = 6.3 Hz, 2H, b-Ala(1) Ca Hs), 1.42 (s, 9H, Boc CH3).
HRMS m/z 359.3267 [M + Na]+
(f) Boc-Phg(1)-b-Ala(2)-OH. To 5.05 g (15 mmol) of Boc-
Phg(1)-b-Ala(2)-OMe were added 35 mL MeOH and 25 mL
2 N NaOH, and the progress of saponification was monitored
by thin-layer chromatography (TLC). The reaction mixture was
stirred. After 10 h, methanol was removed under a vacuum, the
residue was taken in 50 mL of water, washed with diethyl ether
(2 ¥ 50 mL). Then the pH of the aqueous layer was adjusted to
2 using 1 N HCl, and it was extracted with ethyl acetate (3 ¥
50 mL). The extracts were pooled, dried over anhydrous sodium
sulfate, and evaporated in a vacuum. A white crystalline material
was obtained.
Anal. Calcd for C17H24N2O5 (336): C, 60.70; H, 7.19; N 8.33.
Found: C, 60.76; H, 7.11; N, 8.39%.
Yield: 4.13 g (12.8 mmol, 85%).
(d) Boc-Phg(1)-b-Ala(2)-OMe. 5.02 g (20 mmol) of Boc-
Phg-OH was dissolved in 10 mL of DMF in an ice-water
bath. H-b-Ala-OMe was isolated from 5.58 g (40 mmol) of
the corresponding methyl ester hydrochloride by neutralization,
subsequent extraction with ethyl acetate, and ethyl acetate
extract was concentrated to 10 mL. It was then added to the
reaction mixture, followed immediately by 4.12 g (20 mmol)
of dicyclohexylcarbodiimide (DCC) and 2.7 g (20 mmol) of
HOBt. The reaction mixture was allowed to come to room
temperature and stirred for 3 d. The residue was taken up in
ethyl acetate (40 mL) and dicyclohexylurea (DCU) was filtered
off. The organic layer was washed with 1 N HCl (3 ¥ 30 mL),
brine (1 ¥ 30 mL), 1 M sodium carbonate (3 ¥ 30 mL), brine (2 ¥
30 mL), dried over anhydrous sodium sulfate, and evaporated in
a vacuum. A white material was obtained.
1H NMR (300 MHz, DMSO-d6): d = 12.38 (br, 1H, -COOH);
8.20 (br, 1H, Phg(1) NH), 7.24–7.37 (m, 5H, Phg(1) phenyl ring
protons), 7.19 (br, 1H, b-Ala(2) NH), 5.13 (d, J = 7 Hz, 1H,
Phg(1) Ca H), 3.21 - 3.23 (m, 2H, b-Ala(2) Cb Hs); 2.34 (t,
J = 6 Hz, 2H, b-Ala(2) Ca Hs); 1.34 (s, 9H, Boc CH3).
HRMS m/z 345.0036 [M + Na]+, 667.0282 [2M + Na]+.
Anal. Calcd for C16H22N2O5 (322): C, 59.61; H, 6.88; N, 8.69.
Found: C, 59.54; H, 6.83; N, 8.75%.
(g) H3N+-b-Ala(1)-Phg(2)-COO- (Dipeptide 1). To 3.22 g
(10 mmol) of Boc-b-Ala(1)-Phg(2)-OH was added 4 mL of 98%
formic acid, and the removal of the Boc group was monitored
by TLC. After 8 h, formic acid was removed under a vacuum.
The residue was taken in water (20 mL) and washed with diethyl
ether (2 ¥ 30 mL). The pH of the aqueous solution was then
adjusted to 8 with 30% aqueous NH3. The aqueous portion was
evaporated in a vacuum to yield dipeptide 1 as white solid.
Yield: 1.64 g (7.4 mmol, 74%). m.p. 270 ◦C
Yield: 5.68 g (16.9 mmol, 84.5%).
1H NMR (300 MHz, CDCl3): d = 7.30–7.34 (m, 5H, Phg(1)
phenyl ring protons), 6.57 (br, 1H, b-Ala(2) NH), 5.86 (d,
J = 6.5 Hz, 1H, Phg(1) NH), 5.12 (d, J = 6.5 Hz, 1H, Phg(1)
Ca H), 3.59 (s, 3H, -OCH3), 3.46 (q, J = 5.9 Hz, 2H, b-Ala(2)
Cb Hs), 2.46 (t, J = 6 Hz, 2H, b-Ala(2) Ca Hs); 1.40 (s, 9H, Boc
CH3).
1H NMR (300 MHz, D2O): d = 7.24–7.33 (m, 5H, Phg(2)
phenyl ring protons), 5.08 (s, 1H, Phg(2) Ca H), 3.13 (t,
J = 6.7 Hz, 2H, b-Ala(1) Cb Hs); 2.61 (t, J = 6.7 Hz, 2H,
b-Ala(1) Ca Hs).
13C NMR (75 MHz, D2O): d = 177.123 (C of COO-), 171.570
(C of CONH), 138.427 (tertiary C of phenyl ring), 129.357 (m-
C’s of phenyl ring), 128.542 (p-C of phenyl ring), 127.783 (o-C’s
of phenyl ring), 60.129 (a-C of Phg), 36.023 (a-C of b-Ala),
32.214 (b-C of b-Ala).
HRMS m/z 337.0668 [M + H]+, 359.0295 [M + Na]+
Anal. Calcd for C17H24N2O5 (336): C, 60.70; H, 7.19; N 8.33.
Found: C, 60.61; H, 7.23; N, 8.29%.
(e) Boc-b-Ala(1)-Phg(2)-OH. To 5.05 g (15 mmol) of Boc-
b-Ala(1)-Phg(2)-OMe were added 35 mL MeOH and 25 mL
2 N NaOH, and the progress of saponification was monitored
by thin-layer chromatography (TLC). The reaction mixture was
stirred. After 10 h, methanol was removed under a vacuum, the
residue was taken in 50 mL of water, washed with diethyl ether
(2 ¥ 50 mL). Then the pH of the aqueous layer was adjusted to
2 using 1 N HCl, and it was extracted with ethyl acetate (3 ¥
50 mL). The extracts were pooled, dried over anhydrous sodium
sulfate, and evaporated in a vacuum. A white crystalline material
was obtained.
DEPT 135 (D2O): d = 177.123, 171.570 and 138.427 (disap-
pear); 129.357, 128.542, 127.783, 60.129 (positive); 36.023 and
32.214 (negative).
HRMS m/z 223.0015 [M + H]+, 244.9728 [M + Na]+, 466.9553
[2M + Na]+.
Anal. Calcd for C11H14N2O3.H2O (222): C, 59.45; H, 6.35; N,
12.61.
Found: C, 59.39; H, 6.41; N, 12.72%.
(h) H3N+-Phg(1)-b-Ala(2)-COO- (Dipeptide 2). To 3.22 g
(10 mmol) of Boc Phg(1)-b-Ala(2)-OH was added 4 mL of 98%
formic acid, and the removal of the Boc group was monitored
by TLC. After 8 h, formic acid was removed under a vacuum.
The residue was taken in water (20 mL) and washed with diethyl
ether (2 ¥ 30 mL). The pH of the aqueous solution was then
adjusted to 8 with 30% aqueous NH3. The aqueous portion was
evaporated in a vacuum to yield dipeptide 1 as white solid.
Yield: 1.60 g (7.2 mmol, 72%). m.p. 240 ◦C
Yield: 3.99 g (12.4 mmol, 83%).
1H NMR (300 MHz, DMSO-d6): d = 12.32 (br, 1H, -COOH),
8.54 (d, J = 7.4 Hz, 1H, Phg(2) NH), 7.25–7.32 (m, 5H, Phg(2)
phenyl ring protons), 6.62 (br, 1H, b-Ala(1) NH), 5.25 (d,
J = 7.4 Hz, 1H, Phg(2) Ca H), 3.02–3.08 (q, 2H, J = 6.7 Hz,
b-Ala(1) Cb Hs); 2.28 (t, J = 6.7 Hz, 2H, b-Ala(1) Ca Hs); 1.30
(s, 9H, Boc CH3).
HRMS m/z 345.3268 [M + Na]+
Anal. Calcd for C16H22N2O5 (322): C, 59.61; H, 6.88; N, 8.69.
Found: C, 59.52; H, 6.93; N, 8.61%.
1H NMR (300 MHz, D2O): d = 7.36–7.43 (m, 5H, Phg(2)
phenyl ring protons), 4.99 (s, 1H, Phg(2) Ca H), 3.27–3.39 (m,
2H, b-Ala(1) Cb Hs); 2.21–2.27 (m, 2H, b-Ala(1) Ca Hs).
1144 | Green Chem., 2009, 11, 1139–1145
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13C NMR (75 MHz, D2O): d = 180.117 (C of COO-), 168.748
(C of CONH), 134.480 (tertiary C of phenyl ring), 130.717 (m-
C’s of phenyl ring), 129.813 (p-C of phenyl ring), 128.375 (o-C’s
of phenyl ring), 58.899 (a-C of Phg), 37.339 (a-C of b-Ala),
36.624 (b-C of b-Ala).
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HRMS m/z 222.9990 [M + H]+, 244.9732 [M + Na]+, 445.0010
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NMR experiments. All 300 MHz NMR studies were carried
out on a Bru¨ker DPX 300 MHz spectrometer at 300 K.
Mass spectrometry. Mass spectra were recorded on a Qtof
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Acknowledgements
S. G. wishes to acknoeledge the CSIR, New Delhi, India, for
financial assistance. We gratefully acknowledge the nanoscience
and nanotechnology initiative (DST project).
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