Epimerization of cyclic alanyl-alanine in basic solutions

Article abstract of DOI:10.13005/ojc/300103

Alanine anhydrides (Cyclo-(Ala-Ala)) are the simplest dipeptides that have two chiral centers and three diastereomers: Cyclo-(L-Ala-L-Ala), Cyclo-(D-Ala-D-Ala), and Cyclo-(L-Ala-D-Ala). Analysis of the epimerization of these peptides may throw light on the development of homochirality in proteins. We show that the epimerization rate of Cyclo-(L-Ala-L-Ala) and Cyclo-(D-Ala-D-Ala) is higher than that of Cyclo-(L-Ala-D-Ala), while the ring-opening rates of Cyclo-(L-Ala-L-Ala) and Cyclo-(D-Ala-D-Ala) arelower than that of Cyclo-(L-Ala-D-Ala) in basic aqueous solutions. The total reaction resulted in the preferred stability of Cyclo-(L-Ala-L-Ala) and Cyclo-(D-Ala-D-Ala) to Cyclo-(D-Ala-L-Ala).

Full text of DOI:10.13005/ojc/300103

ISSN: 0970-020 X
CODEN: OJCHEG
014, Vol. 30, No. (1):
Pg. 23-30
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
2
www.orientjchem.org
Epimerization of Cyclic Alanyl-Alanine in Basic Solutions
1
,2
2
TORATANE MUNEgUMI *,TAkESHI FUJIMOTO ,
2
2
MICHIOTAkADA and NOꢀOMINAgASHIMA
1
Department of Science Education, Naruto University of Education, Naruto,
Tokushima 772-8502, Japan.
2
Department of Materials Chemistry and Bioengineering, Oyama National College of Technology,
Oyama, Tochigi 323-0806, Japan.
http://dx.doi.org/10.13005/ojc/300103
(Received: November 30, 2013; Accepted: January 17, 2014)
ABSTRACT
Alanine anhydrides (Cyclo-(Ala-Ala)) are the simplest dipeptides that have two chiral centers
and three diastereomers:Cyclo-(L-Ala-L-Ala), Cyclo-(D-Ala-D-Ala), and Cyclo-(L-Ala-D-Ala).Analysis
of the epimerization of these peptides may throw light on the development of homochirality in proteins.
We show that the epimerization rate of Cyclo-(L-Ala-L-Ala) and Cyclo-(D-Ala-D-Ala) is higher than
that of Cyclo-(L-Ala-D-Ala), while the ring-opening rates of Cyclo-(L-Ala-L-Ala) and Cyclo-(D-Ala-D-
Ala) arelower than that of Cyclo-(L-Ala-D-Ala) in basic aqueous solutions.The total reaction resulted
in the preferred stability of Cyclo-(L-Ala-L-Ala) and Cyclo-(D-Ala-D-Ala) to Cyclo-(D-Ala-L-Ala).
key words: Cyclic dipeptide, Epimerization, Ring-opening reaction, Homochirality.
INTRODUCTION
original peptides composed of all L-amino acid
10
residues . A typical example is the epimerization in
aspartic residue in a-crystalline form, although this
Epimerization reactions produce
1
11
diastereomers or epimers , while racemization is
the transformation between enantiomers. However,
epimerization is actually an inversion .
2
–5
in some cases ,racemization has been used
to meanboth racemization and epimerization.
Epimerization and racemization in amino acids,
peptides, and proteinshave been investigated from
Ionic mechanisms of racemization and
epimerizationwere proposed by G. G. Smithand
6
, 12–20
co-workers
, whoshowed that the rates of
racemization of peptides were faster than those of
free amino acids.However, this research wasmainly
restricted to the epimerization of linear peptides
and the racemization of 2,5-diketopiperazines
bearing one chiral center, and did not consider
cyclic diastereomeric peptides.Steinberg and0Bada.
2
–11
many viewpoints
. Epimerization of aspartic
residues in proteins may sometimes increase
thehydrophobicity of the proteins, because several
diastereomeric peptides composed of D-aspartic
residues show higher hydrophobicity than the

24
MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
showed that linear peptides containing isoleucine 10mM, pH12; 25mM; pH12.4; 50mM, pH12.7)at
2
1
residue cyclizedto 2,5-diketopiperazines . Cyclic room temperature (25°C). The sample solution
dipeptides have been suggested as easily formed was removed from the reaction mixture containing
2
2
peptides in the chemical evolutionary process
5.3mmol/L of cyclic dipeptidesat a selected time and
and important compounds in the development of analyzed usingthe HPLC described above.Detection
2
3
homochirality , because of theireasy formation was carried out at 210nm UV absorption.
from amino acids and the difference between
diastereomers in hydrophobicity.The topic of this
research is the simple chiral cyclic dipeptides (cyclo-
RESULTS AND DISCUSSION
(
alanyl-alanine):Homo-L-CP (1) and Hetero-CP (2)) Plausible early reactions of cyclic peptides
shown in Figure 1.In particular, the epimerization
The concentrations of the compounds
and ring-opening reactions are investigated in basic illustrated in Figure1 can be determined in the
aqueous solutions.The epimerization of Homo-L-CP equilibrium amongepimerization, ring-opening, and
(
1) and (3) will proceed at first from the hydrogen cyclization reactions. However, the earlier reactions
abstraction step to give solvated anion intermediates of cyclic peptides (Homo-L-CP (1) and Hetero-CP
1
5
(
4) and (5) . These transform to their enantiomers. (2)) may be shown more simply.The rate constant k1
Among the intermediates (4), (5), and the cyclic is much larger than the rate constant k , because the
2
dipeptides (Homo-L-CP (1), Hetero-CP (2), and first pathway from Homo-L-CP (1) to L-Intermediate
1
5
Homo-D-CP (3)), push–pull transition states are (3) is much slower than the second pathway from
suggested.
L-Intermediate (3) to Hetero-CP (2). Therefore, the
first step is the rate-determining stepwhen calculating
the approximate rate constant of the whole pathway
from Homo-L-CP (1) to Hetero-CP (2).Moreover,
the reaction pathways in the left and right sidesin
EXPERIMENTAL
Chemicals
A cyclic dipeptideCyclo-(L-Ala-L- Figure 1are symmetrical and can be simplified to
Ala)(:Homo-L-CP (1)) and a racemic mixture the pathway in Figure 3.
containingCyclo-(L-Ala-L-Ala) (1), Cyclo-(L-Ala-L-
Ala) (2),and Cyclo-(D-Ala-L-Ala) (3) were purchased
Hetero and Homo mean homochiral and
from Sigma Chemicals Inc.Authentic linear peptides, heterochiral, respectively; CP and LP mean cyclic
L-Ala-L-Ala and D-Ala-L-Ala were prepared by the peptide and linear peptide, respectively; DL and LD
22
manner in the literature .
mean D-alanyl-L-alanine and L-alanyl-D-alanine,
respectively.
Instrumentation
A Jasco high-performance liquid The reaction of Homo-L-CP (1) and Hetero-CP
chromatograph composed of an 880-pump, 875-UV, (3) in 1mM NaOH
and a reversed-phase analytical column: Lichrosorb
Figure 4 shows the time course of Homo-L-
R 100 RP-18 (250 × 4.6 mm I.D., Merck) was used CP (1) and Hetero-CP (3)in1mMNaOHat25°C.Both
for analyses. Elution was carried out with a mixture substrates were almost completely recovered and no
of water–acetonitrile (97.5–2.5% (v/v)) at a flow rate other products were found on the chromatograms,
of 0.5 mL/min. The resulting chromatogram was even after 1500min.Cyclicdipeptides Homo-L-CP (1)
recorded by means of a chromatocoda II. Figure and Hetero-CP (3) did not open their rings and did not
2
shows typical chromatograms of the reaction epimerize at all in these conditions. The reaction of
mixtures.
Homo-L-CP (1) and Hetero-CP (3) in 10mM NaOH
Worꢁup of reactions
Figure 5 shows the time course of the
Homo-L-CP (1)15 mg (0.106 mmol) or reaction of Homo-L-CP (1) and Hetero-CP (3) in
an equivalent mixture composed of meso- and 10mMNaOH solution.
racemic cyclic Ala was dissolved in 20 mL of sodium
hydroxide at different concentrations (0.1mM, pH10.0;
Linear peptides were detected up to only
1
.0mM, pH11.0; 2.5mM, pH11.4; 5.0mM,pH11.7; 1.3% at 160min, while cyclic peptides, Homo-L-CP

MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
25
Fiꢂ. 1: Predicted mechanism of the epimerization and rinꢂ-openinꢂ reactions of cyclic-(Ala-Ala)
in aqueous solutions. Hetero and Homo mean homochiral and heterochiral, respectively; CP and
LP mean cyclic peptide and linear peptide, respectively; DL and LD mean D-alanyl-L-alanine and
L-alanyl-D-alanine, respectively.
Fiꢂ. 2:Typical HPLC chromatoꢂrams of the reaction mixture of Homo-L-CP (1) at 5min
and 107 min in 0.05 M NaOH.Column: Lichrosorb R 100 RP-18 (250 × 4.6 mm I.D.);
eluate: water–acetonitrile (97.5–2.5% (v/v)) at a flow rate of 0.5 mL/min. Detection: 210nm

26
MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
O
C
H
H
C
O
O
H
C
O
O
C
H
C
O
C
N
N
C
C
N
N
C
C
N
N
C
H
C
CH
3
H
3
C
H
H
H
3
C
H
CH
3
C
k
1
H
3
H
CH
3
H
D
L
L
L
D
D
k-2
H
H
Hetero-
CP (2)
Homo-L-CP (1)
Homo-D-CP (3)
-H
2
O
k-
k
4
2
+H O
2
-H O k-3
3 2
k +H O
4
H
H
H
H
C
H
O
N
C
H
O
N
C
H
O
N
C
H
O
N
C
CH
H
3
H
H
CH
H
3
L
C
D
C
D
C
L
k
5
CH
3
CH
3
k
-5
N
H
N
H
N
H
N
H
H
3
C
H
H
3
C
H
C
C
L
C
C
D
C
C
L
C
C
D
H
H
3
C
H
3
H C
HO
O
HO
O
HO
O
HO
O
Homo-L-LP (6)
Homo-D-LP (9)
Hetero-DL-
Hetero-LD-LP (8)
LP (7)
Fiꢂ. 3:The simplified pathway of epimerization, rinꢂ-openinꢂ, and cyclization reactions
∆: Homo-L-CP (1); : Hetero-LP (2).
Fiꢂ. 4.Time course of Homo-L-CP (1) and Hetero-CP (3) in 1mM NaOH at 25°C
∆
: HoHꢀ-ꢀꢁCPꢂ(1ꢃ ꢄꢅꢄtHꢀꢁCPꢆ(1£: Homo-L-LP (6) plus Homo-D-LP (9); ∆: Hetero-LP (7) plus (8).
The percentages of totals of Homo-L-LP (6) plus Homo-D-LP (9) and Hetero-LP (7) plus (8) were calculated
on the basis of the initial total amount of cyclic peptides (1) plus (2).
Fiꢂ. 5:Time course of Homo-L-CP (1) and Hetero-CP (3) in 10 mM NaOH at 25 °C

MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
27
(
1) and Hetero-CP (2) gradually decreased.Although The reaction of Homo-L-CP (1) and Hetero-CP
some peaksemerged more quickly; they might be (3) in 25 mM NaOH
from the anion intermediate., epimerization and
The time course of a racemic mixture of
ring–opening reactions were not confirmed under this Homo-L-CP (1) and Hetero-CP (3) is shown in Figure
condition.However, the decrease in the concentration 6 (a), and the time course of Homo-L-CP is shown
of Homo-L-CP (1) and Hetero-CP (2) cannot be in Figure 6 (b).
explained by the increase of only the quickly eluted
peaks. Other reactions like polymerization might
In Figure 6 (a), Homo-L-CP (1) and Hetero-
explain the decrease of the reactants, but further CP (3) decreased gradually, but the decreasing rate
investigation using other chromatography has to be of Hetero-CP (3) was larger than that of Homo-L-CP
carried out.
(1). After ca. 1100min reaction, 50% of Homo-L-CP
1) and 35% of Hetero-CP (3) remained. Hetero-LP
(
Fiꢂ. 6:Time course of the reactions of a racemic mixture containinꢂ Homo-DL-CP plus
Hetero-CP (a (ꢀꢁ Hoꢂo-m-CP ꢃa ꢄꢀ b)ꢂi n(2H (t b)ꢅC ꢆ Hoꢂo-m-CP ꢇa1 ꢈ ꢆ Hetero-CP ba1
£
:Homo-L-LP (6) plus Homo-D-LP (9); ∆: Hetero-LP (7) plus (8).The percentaꢂes of totals of
Homo-L-LP (6) plus Homo-D-LP (9) and Hetero-LP (7) plus (8) were calculated on the
basis of the initial total amount of cyclic peptides (1) plus (2)
Fiꢂ. 7:The time course of the reactions of a racemic mixture containinꢂ
Homo-DL-CP plus Hetero-CP (a) and Homo-L-CP (b) in 50mM NaOH at 25°C
∆ꢆ Hoꢂo-m-CP ꢇa1 ꢈꢆ Hetero-CP ba1 £: Homo-L-LP (6) plus Homo-D-LP (9); ∆: Hetero-LP (7) plus
(
8).The percentaꢂes of total of Homo-L-LP (6) plus Homo-D-LP (9) and Hetero-LP (7) plus (8) were
calculated on the basis of the initial total amount of cyclic peptides (1) plus (2).

28
MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
(
7) plus (8) and Homo-L-LP (6) plus Homo-D-LP (9) The yields of these heterochiral and homochiral linear
increased as the reaction time increased. However, peptides were 40% and 8%, respectively.
Hetero-LP (7) plus (8) increased much more rapidly
than Homo-L-LP (6) plus Homo-D-LP (9).The yields
In Figure 6 (b), the initial reactant Homo-L-
of the linear peptides were calculated from the total CP (1) rapidly decreased and Hetero-CP (3) rapidly
areas of heterochiral linear peptides (7) plus (8) and increased up to 43% at 200min. The increase of
homochiral linear peptides (6) plus (9), because the Hetero-CP (3) indicates the epimerization of Homo-
peaks of the corresponding enantiomers could not be L-CP (1). However, Hetero-CP (3) decreased after
separated by using an achiral column (Lichrosorb). 200min to give 20% recovery at 1700min. The
Fiꢂ. 8. Reaction rate constants of linear dipeptide formation and cyclic dipeptide decrease in 0.05
M NaOH. ∆ꢆ Hoꢂo-m-CP ꢇa1 ꢈ Hetero-CP ba1 £: Homo-L-LP (6) plus Homo-D-LP
(9); D: Hetero-LP (7) plus (8).
Fiꢂ. 9: Comparison of the reaction rate constants of the epimerization and rinꢂ-
openinꢂ reactions of Homo-CP and Hetero-CP in basic aqueous solutions. LL (25mM), DL
(
25mM), LL (50mM), and DL (50mM) are the startinꢂ materials: Homo-L-CP in 25mM
NaOH, Hetero-CP in 25mM NaOH, Homo-L-CP in 50mM NaOH, Hetero-CP in 50mM NaOH

MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
29
decrease of Hetero-CP (3) suggests the ringopening
to afford Hetero-LP (7) plus (8), which increased after
For the reactions using a racemic mixture
composed of Homo-L-CP, Homo-D-CP, and Hetero-
CP, the decrease of these cyclic dipeptides includes
ringopening as well as epimerization from their
diastereomers. The rate constants (kobs= 0.0011 1/
min; 0.0062 1/min in 25mM NaOH) of the decrease
of Hetero-CP were about 1.5 times and 1.9 times
larger than the rate constants (0.00070 1/min;
200min.
The reaction of Homo-L-CP (1) and Hetero-CP
3) in 50 mM NaOH
Figure 7 (a) and (b) shows similar results
(
to those of Figure 6 (a) and (b), respectively.
0.00033 1/min in 50mM NaOH) of the decrease of
Comparing the results in Figure 7 with those
in Figure 6, the decrease of cyclic dipeptides and the
increase of the linear dipeptides are faster than the
results in Figure 6. This suggests that the reactions
accelerated at high pH.
Homo-CP, respectively. However, the rate constant
(kobs) represents the total of the epimerization and
the ringopening of cyclic dipeptides.
On the other hand, the rate constant (kobs
)
of the increase of Hetero-LP was always larger
(1.4 to 6.0 times) than that of Homo-LP. If the rate
constants(k and k ) of the epimerization between
Comparison of the reaction rate constants
Figure 8 shows the rate constant of the
increase of linear dipeptides and the decrease of
cyclic dipeptides in 50 mM NaOH.
5
–5
linear dipeptides as well as the cyclization rate
constants (k–3 and k ) are much smaller than those
–4
(
k and k ) of the ring-opening reactions, the rate
3 4
The vertical axis shows the inverse
logarithm of the concentration of the product
divided by the initial concentration of the reactant for
Homo-L-CP (1) and Hetero-CP (2), and the inverse
logarithm of 1 minus the concentration of the product
divided by the concentration of initial reactant for
Homo-L-LP (6) plus Homo-D-LP (9) and Hetero-LP
constant (kobs) may be approximated byk and k ,
respectively.We can therefore conclude that the rate
3 4
constant (k ) of the ringopening of Hetero-CP is larger
4
than that (k ) of Homo-CP.Under this assumption, the
3
rate constant (k ) of the epimerization of Homo-CP
1
may be larger than that (k ) of Hetero-CP.
–1
(
7) plus (8). The horizontal axis shows the reaction
Although this research demonstrated that
Homo-CP epimerizes faster than Hetero-CP and
that Hetero-CP opens its ring faster than Homo-
CP, more detailed consideration on the cyclization
and epimerization of linear dipeptides may be
necessary to determine the rate constants between
dipeptides.
timein minutes.The plots in each reaction condition
afford the pseudo-first-order reaction constants
(
observed rate constant kobs) as the gradients of
these lines.
The method shown in Figure 8 was also
applied to the results at other NaOH concentrations
to give Figure 9.
CONCLUSION
The negative circular cylinders show the
decrease of the corresponding cyclic dipeptides,
while the positive circular cylinders show the
increase of cyclic dipeptides and linear dipeptides.
For the reactions using Homo-L-CP, the increase of
Hetero-CP may be induced by the epimerization of
This research has demonstrated that
Cyclo-(L-Ala-L-Ala) (:Homo-L-CP (1)) and Cyclo-(D-
Ala-D-Ala)(: Homo-D-CP (3))epimerize faster than
Cyclo-(L-Ala-D-Ala) (: Hetero-CP (2)), and that the
rings of Cyclo-(L-Ala-L-Ala) (: Homo-L-CP (1)) and
Cyclo-(D-Ala-D-Ala) (: Homo-D-CP (3))openmore
slowly than those of Cyclo-(L-Ala-D-Ala) in 25mM
and 50mM NaOH solutions. The total reaction
resulted in the preferred stability of Cyclo-(L-Ala-L-
Ala) and Cyclo-(D-Ala-D-Ala)over Cyclo-(D-Ala-L-
Ala) in these basic solutions.
-
1
Homo-L-CP. The rate constants (kobs= 0.0029 min ;
.0052 1/min in 25mM NaOH) of the increase of
0
Hetero-CP were about 1.9 times and 3.5 times larger
than the rate constants (kobs= 0.00082 1/min;0.0027
1/min in 50mM NaOH) of the decrease of Homo-CP,
respectively.

3
1
0
MUNEGUMI et al., Orient. J. Chem., Vol. 30(1), 23-30 (2014)
REFERENCES
.
M. B. Smith and J. March, March’s Advanced
13.
14.
G. G. Smith, K. M. Williams, and D. M.
Wonnacott, Journal of Organic Chemistry,
43: 1-5 (1978).
G. G. Smith, A. Khatib, and G. S. Reddy,
Journal of American Chemical Society, 105:
293–295 (1983).
Organic Chemistry:Reactions, Mechanisms,
th
and Structure, 6 edition, p. 169, Wiley &
Sons, Inc., Hoboken (2007).
K. A. Kvenvolden and E. Peterson, Science,
69: 1079–1082 (1969).
2
3
4
.
.
.
1
J. L. Bada, B. P. Luyenyk, and J. B. Maynard,
Science, 170: 730–732 (1970).
J. Wehmiller and P. E. Hare, Science, 173:
15.
16.
G.G.Smith andT.Sivakua, Journal of Organic
Chemistry, 48: 628-634 (1983).
E. D. Stroud, D. J. Fife, and G. G. Smith, A
method for the determination of the pKa of the
a-hydrogen in amino acids using racemization
and exchange studies, Journal of Organic
Chemistry, 48: 5368-5369 (1983).
9
07–911 (1971).
5
6
.
.
W. T. Smith Jr., Science, 172: 403 (1971).
K. M. Williams and G. G. Smith, Origins of
Life and Evolutionary Biosphere, 8: 91-144
(
1977).
N. Kriausakul and R. M. Mitterer, Science,
01: 1011–1014 (1978).
17.
18.
R.Baum and G.G.Smith, Journal of American
Chemical Society, 108: 7325-7327 (1986).
G. G. Smith, R. C. Evans, and R. Baum,
Journal of American Chemical Society, 108:
7327-7332 (1986).
G. G. Smith and R. Baum, Journal of Organic
Chemistry, 52: 2248–2255 (1987).
G. G. Smith and G. V. Reddy, Journal of
Organic Chemistry, 54: 4529–4535 (1989).
S. Steinberg and J. L. Bada, Science, 213:
544-545 (1981).
T. Munegumi and A. Shimoyama, Chirality,
15S: 108–115 (2003).
U.Shanker, B.Bhushan, G.Bhattacharjee, and
Kamaluddin, Origins of Life and Evolutionary
Biosphere, 42: 31-45 (2012).
7
8
.
.
2
J. Csapo, Z. Csapo-Kiss, S. Nemethy, S.
Folestad, A.Tivesten, andT.G.Martin, Amino
Acids, 7: 317–325 (1994).
H. Brucker and N. Fujii, eds., D-Amino Acids
in Chemistry, Life Science and Biotechnology,
Wiley-VCH, Zurich (2011).
N. Fujii, Y. Momose, N. Ishii, M. Takita, M.
Akaboshi, and M. Kodama, Mechanisms
of Aging and Development, 107: 347-358
19.
20.
21.
22.
23.
9
1
.
0.
(
1999).
T. Munegumi, Chemistry & Biodiversity, 7:
670–1679 (2010).
G. G. Smith and B. S. De Sol, Science, 207:
65–767 (1980).
1
1
1.
2.
1
7