Polypeptide formation by heating N-t-butyloxycarbonyl acidic amino acid derivatives

Article abstract of DOI:10.14233/ajchem.2014.16185

An acid labile N-protecting group for amino acids, t-butyloxycarbonyl (Boc) group has deprotected at elevated temperatures. The study describes an application of the lability on heating to synthesis of polypeptides from acidic amino acids. t-Butyloxycarbonyl-acidic amino acids (aspartic acid, glutamic acid and β-aminoglutaric acid) and their anhydrides were heated at the higher temperatures than their melting points. Anhydrides of t-butyloxycarbonyl-aspartic acid and t-butyloxycarbonyl-β-aminoglutaric acid gave polypeptides. Thermal analyses of the substrates clarified the pathway of the polypeptide formation.

Full text of DOI:10.14233/ajchem.2014.16185

Asian Journal of Chemistry; Vol. 26, No. 15 (2014), 4716-4722
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.16185
Polypeptide Formation by Heating N-t-Butyloxycarbonyl Acidic Amino Acid Derivatives
1,*
2
2
T. MUNEGUMI , Y-QING MENG and K. HARADA
1Department of Science Education, Naruto University of Education, Naruto, Tokushima 772-8502, Japan
2Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
*Corresponding author: Tel: +81 886876418, Fax: +81 886876022; E-mail: tmunegumi@naruto-u.ac.jp
Received: 13 August 2013;
Accepted: 16 December 2013;
Published online: 16 July 2014;
AJC-15558
An acid labile N-protecting group for amino acids, t-butyloxycarbonyl (Boc) group has deprotected at elevated temperatures. The study
describes an application of the lability on heating to synthesis of polypeptides from acidic amino acids. t-Butyloxycarbonyl-acidic amino
acids (aspartic acid, glutamic acid and β-aminoglutaric acid) and their anhydrides were heated at the higher temperatures than their
melting points.Anhydrides of t-butyloxycarbonyl-aspartic acid and t-butyloxycarbonyl-β-aminoglutaric acid gave polypeptides. Thermal
analyses of the substrates clarified the pathway of the polypeptide formation.
Keywords: Acidic amino acid, Heating, t-Butyloxycarbonyl group, Thermal analysis.
4 h' heating at 130 °C). The purpose of the protection with N-
t-butyloxylcarbonyl group is to achieve melting at the lower
temperatures. Under the reaction conditions, t-butyloxycar-
bonyl-L-Asp anhydride seemed to release carbon dioxide and
2-butene gradually to afford L-Asp anhydride, which was
suggested to react itself to give polyaspartic acid. However,
detailed mechanism and experimental procedure have not been
reported.
INTRODUCTION
Aspartic acid and glutamic acid can be found as compo-
nents in proteins and the research on these amino acids has
been performed in the viewpoints of peptide synthesis using
prebiotic1-8, conventional organic chemistry9 and molecular
biological methods10, as well as their chemical11 and physio-
logical properties12.
Free aspartic acid1-3 and malic acid mono-ammonium
salt4,5 gave polyaspartic acid by heating without solvents at
elevated temperatures or by microwave induction6,8. The
mechanism of dehydration from aspartic acid to polypeptides
has been reported in the literatures1-3,13. The reaction mixture
during heating aspartic acid was monitored13 by infrared
spectrophotometry, thermal gravitmetry and differential
thermal analysis. The residues after the heating reactions
showed IR absorption corresponding to peptide formation
(amide I and amide II). The DTA record of the heating reaction
showed that the decrease in weight was consistent with the
point of endothermal peak due to the formation of peptide
bonds on the DTA line. However, these heating reactions have
been carried out at the higher temperature than 180 °C, at which
anhydrous polyaspartic acid formation and racemization will
proceed. The lower temperature seems to be suitable to depress
such side reactions.
This research describes the mechanism and experimental
procedure of the polypeptide formation at the lower temperatures
using acidic N-t-butyloxylcarbonyl-amino acids (t-butyloxy-
carbonyl-amino acids) and their anhydrides as shown in Fig. 1.
EXPERIMENTAL
A Hitachi 835 amino acid (Hitachi, Tokyo, Japan) analyser
was used for amino acid analysis. A Hitachi Model 260-50
infrared spectrophotometer (Hitachi, Tokyo, Japan) was used
for the record of infrared spectra.A Shimadzu DT-40 (Shimadzu,
Kyoto, Japan) was used for thermal analysis.A JOEL EX-270
NMR system (Joel, Tokyo, Japan) was used for measurement
1
of H NMR spectra. Molecular weight stimulation was
performed using a high performance liquid chromatography
system composed of an ultraviolet detector, Jasco Uvdec-100-
V UV Spectrophotometer and a flow pump, Jasco TRI Rotor-
V equipped with a Sephadex G-25F column (450 × 10 mm
I.D.). The flow rate was 0.15 mL/min (0.1 M sodium phosphate,
pH 6.8) and the absorbance of the samples was detected at
230 nm. The peaks on the chromatograms were integrated with
a SIC Chromatocoda. Enantiomer separation of amino acids
In the previous report14, we described heating reactions
of N-t-butyloxyl-carbonyl-aspartic acid (t-butyloxycarbonyl-
L-Asp) anhydride at 130 °C to afford polyaspartic acid with a
little amount of racemized residues (D/L ratio: 7.5 % during

Vol. 26, No. 15 (2014)
Polypeptide Formation by Heating N-t-Butyloxycarbonyl Acidic Amino Acid Derivatives 4717
was performed with a Hitachi 163 gas chromatograph equipped
with a chiral glass capillary column (Chirasil-Val15-17), 25m ×
0.3 mm I.D.). Nitrogen was used as a carrier gas at a flow rate
30 mL/min. The temperature was programmed from 80 to
170 °C at a rate of 4 °C/min. The detection was carried out
with a flame ionization detector. Optical rotation of the
synthetic compounds from L-aspartic acid was determined
with a Jasco DIP-181 Digital Polarimeter.
reaction solution was filtered and the resulted filtrate was
evaporated in vacuo to give a crude crystal (10.1g, 94 %),
which was recrystallized with acetone-petroleum ether to give
9.8 g (91 %). m.p. 122-124 °C.
N-t-Butyloxycarbonyl-amino acid anhydrides: Com-
pounds 3b, 3c, 3d were prepared from the corresponding N-t-
butyloxycarbonyl-amino acids (2a-d) in the similar manner
of the preparation of 3a. 3b: Yield, 94 %. m.p. 137-139 °C.
[α]D2538.7 (c = 1, acetic acid). 3c:Yield, 88 %. m.p. 104-106 °C.
3d: Yield, 83 %. m.p. 160-162 °C.
Amino acids (1a-c): DL-aspartic acids (1a), L-aspartic
acid (1b) and DL-glutamic acid (1c) were purchased from
Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). Di-
t-butyloxycarbonyl-carbonate was purchased from Watanabe
Chemical Industries, LTD. (Hiroshima, Japan). Glutaconic acid
(trans-2-pentendioic acid) was purchased from Sigma-Aldrich.
β-Amino glutaric acid (1d): To a solution of glutaconic
acid (5.2 g, 20 mmol) in 15 mL water was added concentrated
ammonia (5.6 mL, 40 mmol) to give a crystal at 0 °C. The
crystal was recrystallized with water to give 5 g glutaconic
acid mono-ammonium (85 %). m.p. 179-181 °C. Elemental
analysis calcd. for C5H9NO4: C, 40.81; H, 6.16; N, 9.52 %.
Found: C, 40.62; H, 6.18; N, 9.52 %. Glutaconic acid mono-
ammonium (0.294 g, 2 mmol) was heated at 170 °C for 2 h
under nitrogen stream. To the resulted mixture was added
methanol to a suspension, which was filtered to give β-amino
glutaric acid (0.093 g, 32 %). m.p. 285 °C (dec.). Elemental
analysis: Calcd. for C5H9NO4: C, 40.81; H, 6.16; N, 9.52 %.
Found: C, 40.73; H, 6.24; N, 9.52 %. 1H NMR (1M-DCl/D2O):
δ = 2.95 (4H, d), 4.07 ppm (1H, t). IR: 3200, 1720, 1600,
1490, 1410-1320 cm-1.
Heating reaction of N-t-butyloxycarbonyl-amino acids
(2a-d) and N-t-butyloxycarbonyl-amino acid anhydrides
(3a-d): The compounds 2a-d (2 mmol) and 3a-d (2 mmol)
were put into each test tube (180 mm × 16 mm I.D.), which
was heated under a nitrogen stream in a silicone oil bath
controlled at a constant temperature. After heating, the reaction
mixture was weighed and 180 mg of each sample was dissolved
in 0.5 M acetic acid (5 mL). The solution was loaded onto a
Sephadex-10 (120 mm × 1.8 mm I.D.). The elution was carried
out with 0.5 M acetic acid and the eluted fractions were
collected by a fraction collector, monitoring UV absorption
of each fraction at 230 nm. Remaining samples of the resulted
residue was injected to Sephadex G-25F for molecular weight
analysis.
General procedure of thermal analysis: Samples of t-
butyloxycarbonyl-amino acids (2a-d) and their anhydrides (3a-
d) (8-13 mg, weighed to within 0.1 mg) were placed in the
aluminum sample pans of the α-aluminum reference disk was
placed in the center of the second aluminum pan. The tempe-
rature of the heating chamber was programmed from 27 to
400 °C using a heating rate of 10 °C/min, under a nitrogen
flow (50 mL/min).
N-t-Butyloxycarbonyl-amino acids (2a-d): A typical
preparation procedure for t-butyloxycarbonyl-DL-aspartic acid
(2a) is shown as follows: To a mixture of DL-aspartic acid
(1a) (13.3 g, 0.10 mmol), 4 M sodium hydroxide (80 mL,
0.20 mmol), pure water (20 mL) and 1, 4-dioxane (100 mL),
was added dropwise a solution containing di-t-butyloxy-
dicarbonate (24 g, 0.11 mol) over stirring at 10-20 °C. And
then 1,4-dioxane (40 mL) with 4M-sodium hydroxide (40 mL)
was added. After overnight stirring at room temperature, the
reaction mixture was evaporated in vacuo to about 100 mL
solution, which was cooled and acidified with 5 % potassium
hydrogen sulfate to pH 2. The resulted solution was extracted
with ethyl acetate (40 mL × 3) and the combined ethyl acetate
layer was washed with a little water. The obtained ethyl acetate
solution was dried with anhydrous magnesium sulphate and
evaporated in vacuo to give 21.4 g (92 %). m.p. 145-146 °C.
IR: 3450, 3000-2700, 1740-1680, 1500, 1440 cm-1.
RESULTS AND DISCUSSION
Heating reaction of N-t-butyloxycarbonyl-amino acids
and amino acid recovery: N-t-Butyloxycarbonyl-L-alanine
(Boc-L-Ala) (2e), N-t-butyloxycarbonyl-L-valine (Boc-L-Val)
(2f) and N-t-butyloxy-carbonyl-L-aspartic acid (Boc-L-Asp)
(2b) were heated at each temperature and for 1 or 2 h to free
corresponding amino acids as shown in Table-1.
TABLE-1
FREE AMINO ACID RECOVERY AFTER HEATING
N-t-BUTYLOXYCARBONYL-AMINO ACIDS
Free
Reaction conditions
m.p.
(°C)
amino acid
recovery
(%)
Boc-amino acid
Temp.
(°C)
Time
(h)
N-t-Butyloxycarbonyl-amino acids: Compunds 2b, 2c,
2d were prepared from the corresponding amino acids (1a-d)
in the similar manner of the preparation of 2a. 2b: Yield, 78
%. m.p. 119-121 °C. [α]D25-4.5 (c = 1, methanol). 2c: Yield,
89 %. m.p. 125-128 °C. 2d: Yield, 86 %. m.p. 139-142 °C.
N-t-Butyloxycarbonyl-amino acid anhydrides (3a-d):
A typical preparation procedure for N-t-butyloxycarbonyl-DL-
aspartic acid anhydride (3a) is shown as follows: N-t-butyloxy-
carbonyl-DL-aspartic acid (11.8 g, 50 mmol) was dissolved
in 150 mL ethyl acetate. To this cooled solution at 0 °C was
added a dicyclohexylcarbodiimide (11.4 g, 55 mmol) dissolved
in 50 mL ethyl acetate. The reaction was carried out over
stirring for 1 h at 0 °C and for 24 h at room temperature. The
Boc-L-Asp (2b)
Boc-L-Ala (2e)
Boc-L-Val (2f)
119-120
83-84
80
130
140
140
1
2
2
81
80
70
The results show a kind of elimination from t-butyloxy-
carbonyl-amino acid proceeded to give 2-butene (6), carbon
dioxide (7) and corresponding amino acids (8b, 8e, 8f) as
shown in Fig. 2.
The results are obtained by just short time heating, but
further longer reactions are not monitored. This study describes
further longer reactions of t-butyloxycarbonyl-acidic amino
acids (2a-d). Heating reaction of 2e, 2f and other neutral

4718 Munegumi et al.
Asian J. Chem.
CH3
H3C C O C O C O C CH3
CH3 O CH3
CH3
CH3
C
H2C
COOH
H2
H2C
COOH
n
n
O
H2
C
H
N
H3C
O
C
O
C
H
C
COOH
H2N
C
H
COOH
m
m
NaOH aq./1,4-dioxane
CH3
2a-d
1a-d
O
C
∆
CH3
H2C
O
C
n
DCC
H2
C
H
N
O
H3C
C
O
C
O
C
H
4a-d
m
CH3
∆
3a-d
5a-d
Compound
m
n
Amino acid
Configuration
Aspartic acid (Asp)
Aspartic acid (Asp)
Glutamic acid (Glu)
2a, 3a
2b, 3b
2c, 3c
2d, 3d
DL
L
0
0
0
1
1
DCC:Dicyclohexylcarbodiimide
1
2
1
DL
β
-Amino-glutaric acid (
β
-Glu) Non
Fig. 1. Preparation of t-butyloxycarbony-amino acids (2a-d) and their anhydrides (3a-d)
H3C
CO2
C
CH2
+
CH3
R
7
R
6
H3C
H
N
H3C
C
O
C
O
C
H
COOH
H2N
C
H
COOH
CH3
8b, 8e, 8f
2b, 2e, 2f
8b: R=-CH2COOH (L-Asp)
8e: R=-CH3 (L-Ala)
2b: R=-CH2COOH (L-Asp)
2e: R=-CH3 (L-Ala)
2f: R=-CH(CH3)2 (L-Val)
8f: R=-CH(CH3)2 (L-Val)
Fig. 2. Plausible degradation pathway of t-butyloxycarbony-amino acids (2b, 2e, 2f)
t-butyloxycarbonyl-amino acids will be discussed in other
opportunity.
spectrum of heating product of t-butyloxycarbonyl-β-amino-
glutaric acid (2c). The spectrum was identical to that of β-
aminoglutaric acid (1c). Fig. 5 shows IR spectrum of a heating
product of t-butyloxycarbonyl-DL-glutamic acid (2d). The
spectrum was identical to that of pyroglutamic acid.
Heating reactions of t-butyloxycarbonyl-acidic amino acids
(2a, 2c, 2d)
(1) IR spectrum of the products of heating t-butyloxy-
carbonyl-amino acids (2a, 2c, 2d): Fig. 3 shows a heating
product of t-butyloxycarbonyl-amino acid 2a due to imide
structure, which suggests the formation of anhydro-poly-
aspartic acid through polyaspartic acid during the heating
reaction. This is supported by the observation in the products
of heating reactions of aspartic acids10. Fig. 4 shows an IR
(2) Thermal analyses of t-butyloxycarbonyl-amino
acids (2a, 2d): Fig. 6 shows differential thermal analysis and
thermal gravitmetry of t-butyloxycarbonyl-amino acid (2a).
The peak (A) on DTA line corresponds to the endothermal
peak due to melting of the sample, because the decrease in
weight was not observed until the peak (A). However, with
the peak (A), the weight of the sample rapidly decreased as

Vol. 26, No. 15 (2014)
Polypeptide Formation by Heating N-t-Butyloxycarbonyl Acidic Amino Acid Derivatives 4719
shown in TG line. The weight loss is due to 44.6 % of the total
weight of the sample (12.3 mg) after peaks B and C, which
seem to be the decomposition of t-butyloxycarbonyl-group.
The weight loss of t-butyloxycarbonyl-group is 43.3 %, which
almost equal to 44.6 %. During the peak D reveals on the
DTA line, 14 % weight loss due to the weight of two water
molecules (15.4 %) was observed. The last two broad peaks
mean gradual degradation. The degradation is composed of
three steps after melting.
70
60
50
40
Imide (1790 cm–1)
30
20
10
0
a
TG
–44.6 %
B
Imide (1710 cm–1)
DTA
C
0 3600 3200 2800 2400 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100
Fig. 3. IR spectrum of the product obtained by heating Boc-DL-Asp at
150 °C for 4 h
–14.0 %
b
c
60
50
40
30
20
10
D
A
0
100.0
200.0
Temperature (°C)
300.0
400.0
Fig. 6. Thermal analysis of Boc-DL-Asp (2a)
Fig. 7 shows differential analysis and thermal gravitmetry
of t-butyloxycarbonyl-amino acid (2d). The peak (A) on DTA
line corresponds to the endothermal peak due to melting of
the sample, because the decrease in weight was not observed
until the peak (A). However, with the peak (A), the weight of
the sample rapidly decreased as shown in TG line. The weight
loss is due to 43.9 % of the total weight of the sample (11.8
mg) after peaks B and C, which seem to be the decomposition
of t-butyloxycarbonyl-group. The weight loss of t-butyloxy-
carbonyl-group is 43.9 %, which almost equal to 40.9 %.
However, the three step weight losses on the TG line as shown
in thermal analysis of 2a (Fig. 6) was not observed in that of
2d. After melting, TG line is composed of two step weight
losses and the later step includes whole degradation without
clear dehydration step.
0
0 3600 3200 2800 2400 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100
Fig. 4. IR spectrum of the product obtained by heating t-butyloxycarbony-
β-aminoglutaric acid at 150 °C for 4 h
80
70
60
50
40
Table-2 shows the summarized results of heating reactions
of Boc-amino acids (2a, 2c, 2d).
a
TG
–43.9 %
B
C
DTA
Amide-I
COOH
30
20
10
0
b
c
A
D
0 3600 3200 2800 2400 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100
0
100.0
200.0
Temperature (°C)
300.0
400.0
Fig. 5. IR spectrum of the product obtained by heating Boc-DL-Glu at
150 °C for 4 h
Fig. 7. Thermal analysis of t-butyloxycarbony-β-aminoglutaric acid

4720 Munegumi et al.
Asian J. Chem.
(a) Poly-Asp
TABLE-2
REACTION CONDITIONS OF HEATING
REACTION OF BOC-AMINO ACIDS
Boc-amino acid
m.p. (°C)
145-146
Temp. (°C)
150
Time (h)
4
12
4
2a
150
(b) Poly-β-NH2-glu
170
170
12
4
125-128
139-142
130
2c
150
4
2d
Amide-II
–COOH
Heating reactions of t-butyloxycarbonyl-acidic amino acids
anhydrides (3a-d)
Amide-I
(1) Observation of reaction mixture upon heating: The
prepared anhydrides of t-butyloxycarbonyl-acidic amino acids
(3a-d) were heated at different temperatures for each as shown
in Table-3. The common process of morphologic change during
the heating reaction is as follows: At first the crystal of the
sample melted to a liquid, which released bubbles and then
solidified again. Substrates 3a-b gave polyaspartic acid and
3d gave poly-β-amino-glutaric acid. The proof of the peptide
structure of the product from substrate 3a and 3d is shown in
the IR of Fig. 8. However, the product from substrate 3b gave
identical IR spectrum to that of pyroglutamic acid (Fig. 9).
The formation mechanism can be explained by self-cyclization
shown in Fig. 10.
2000
1800
1600
Wavelength (cm–1)
1400
1200
Fig. 8. IR spectrum of polyaspartic acid and poly-β-aminoglutaric acid
(a) p-Glu obtained by heating of Boc-DL-Glu anhydride (110°C, 1 h)
(b) Authentic p-Glu
The free amino group by the destruction of t-butyloxy-
carbonyl-group attacked α-carboxyl carbon to afford a five-
membered ring compound, which is pyroglutamic acid.
(2) Thermal analyses of Boc-amino acids anhydrides
(3a, 3c, 3d): Although thermal analysis of Boc-amino acid
anhydrides (3a, 3b, 3d) was carried out, the data did not give
us meaningful information because of very noisy lines of DTA
and TA. The lines look a vibration probably caused by a rapid
bubbling of removing gasses.
Amide-I
COOH
4800 4000 3200 2400 1900 1700 1500 1300 1100 900 700 500
Wavelength (cm–1)
Fig. 9. IR spectrum of the product obtained by heating Boc-DL-Glu and
standard pyro-glutamic acid
TABLE-3
HEATING REACTION OF Boc-AMINO ACID ANHYDRIDES (3a-c)
Reaction conditions
Boc-amino
acid anhydride
Amino acid
recovery (%)
m.p. (°C )
122-124
Product
Yield (%)
D/L ratio (%)
Temp. (°C )
130
130
130
140
150
130
140
140
140
150
160
110
110
110
140
170
165
165
165
175
185
Time (h)
1
4
91
89
93
89
90
90
89
89
93
89
93
93
85
92
83
75
90
81
90
85
88
87
99
97
97
95
93
87
91
89
95
94
-
-
-
3a
3b
12
1
Polyaspa
-
-
1
-
1
10
14
14
17
14
18
-
1
4
135-137
Polyaspa
12
1
1
1
4
-
-
3c
104-106
160-162
12
1
Polyaspa
Polyaspa
-
-
-
-
1
-
-
1
-
-
4
-
-
3d
12
1
-
-
-
-
1
-
-
aPolyaspartic acid; bPyroglutamic acid; cPoly-β-amino glutaric acid

Vol. 26, No. 15 (2014)
Polypeptide Formation by Heating N-t-Butyloxycarbonyl Acidic Amino Acid Derivatives 4721
H3
H3
C
C
the product by heating reaction at 130 °C for 1 h as shown in
Table-3. The longer reaction time and the higher reaction
+
CH2
CO2
O
O
temperature gave a little higher racemization (D/L ratio: 14 to
18 %).
CH3
C
O
C
H
N
H2N
O
H3C
O
O
CH3
3c
O
O
L-Asp
H
N
NH2
H
O
H
O
O
O
OH
O
Fig. 10. Plausible mechanism of the formation of pyroglutamic acid from
Boc-DL-Glu anhydride
D-Asp
Elementary analysis for some products obtained by
heating 3a, 3b and 3d was carried out. Polyaspartic acid (5a)
by heting 3a at 130 °C for 1 h; calcd. for C4H5NO3·0.4H2O: C,
39.28; H, 4.78; N, 11.45 %. Found: C, 39.22; H, 5.04; N,
11.00 %. Polyaspartic acid (5b) by heating 3b at 130 °C for
1 h: calcd. for C4H5NO3·0.3H2O: C, 39.87; H, 4.68; N, 11.62 %.
Found: C, 40.08; H, 5.03; N, 11.08 %. Poly-β-aminoglutaric
acid (5d) by heating 165 °C for 1 h: calcd. for C4H5NO3·0.3H2O:
C, 44.64; H, 5.69; N, 10.41 %. Found: C, 44.64; H, 5.85; N,
10.48 %. Data of the elementary analysis almost agree with
those of the calculated values based on the corresponding
amino acid residue.
0
5
10
Time (min)
15
20
Fig. 12. Gas chromatogram of DL-aspartic acid. Aspartic acids were
derivertized to N-trifluoroacetyl-aspartic acid 2-propyl esters, which
were injected to gas chromatograph equipped with a chiral glass
capillary column, Chirasil-Val
Conclusions
• t-Butyloxycarbonyl-acidic amino acids (DL-, L-aspartic
acid, DL-glutamic acid and β-amino glutaric acid) 2a-d and
their anhydrides 3a-d were heated at the higher temperatures
than their melting points to give corresponding main products
as follows: Polyaspartic acid from compounds 2a-b and 3a-b;
pyroglutamic acid from compounds 2c, 3c; β-amino glutaric acid
from 2d; poly-β-aminoglutaric acid from 3d.
(3) GPC of mixture of heating reaction: Fig. 11 shows
a gel permeation chromatography of the product obtained by
heating t-butyloxycarbonyl-DL-Asp anhydride (3a). The peak
of the polypeptide shows that the averaged molecular weight
is in the range between 3,000 and 12,400 Da. The product
from 3b and 3d gave similar molecular weight.
• Averaged molecular weight of polyaspartic acid was
estimated as 3000 to 12,400 Da with a gel permeation chro-
matography.
Blue dextran (MW = 1,000,000)
Cytochrome-C(MW = 12,400)
Poly-Dl-Asp
• Simultaneous measurements of thermal gravitmetry
(TG) and differential thermal analysis showed the gradual
weight decrease of compounds 2b-d corresponding to 2-butene,
carbon dioxide and water molecules.
Insulin-A, B(MW = 3,000)
Asp-Asp (MW = 270)
• Polyaspartic acid synthesized upon heating t-butyloxy-
carbonyl-L-Asp anhydride (3b) showed a slight degree of
racemization (D/L: 10 %, for 1 h at 130 °C).
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20
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60 80 100 120 140 160 180 200 220 240
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Fig. 11. Gel permeation chromatography (Sephadex G-25F) of the product
obtained by heating Boc-DL-Asp anhydride
(4) D/L ratio of the product from heating reaction of
3b: Hydrolyzates of the heating products were evaporated in
vacuo to give residues containing amino acids. The products
were derivatized15-17 to N-trifluoroacetyl-amino acid 2-propyl
esters, which were injected to a gas chromatography equipped
with a chiral capillary column. A typical chromatograph is
shown in Fig. 12. About 10 % D/L ratio was observed from
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