Treatment of relatively concentrated aqueous solutions of O-phospho-serine (50 mM), aspartic acid (100 mM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at their surfaces and catalyze efficient oligomerization even from dilute solutions

Boehler, Christof

Hill, Aubrey R., Jr.

Orgel, Leslie E.

English

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CATALYSIS OF THE OLIGOMERIZATION OF O-PHOSPHO-SERINE,
ASPARTIC ACID, OR GLUTAMIC ACID BY CATIONIC MICELLES
__
CHRISTOF BOHLER, AUBREY R. HILL, JR. and LESLIE E. ORGEL
The Salk Institute for Biological Studies, Post Office Box 85800, San Diego, CA 92186-5800, U.S.A.
(Received 5 May 1995)
Abstract. Treatment of relatively concentrated aqueous solutions of O-phospho-serine (50 raM),
aspartic acid (100 raM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation
of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is
reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium
bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at
their surfaces and catalyze efficient oligomerization even from dilute solutions.
Introduction
Catalysis by micelles in aqueous solution has been studied for more than two
decades. This work has focussed on reactions involving hydrolysis, aminolysis and
nucleophilic substitution reactions (Broxton and Lucas, 1994; Fendler and Fendler,
1975; Menger, 1991; Menger and Fei, 1994) and, more recently, on oxidation (Eng-
bersen et al., 1990; Panigrahi and Mishra, 1993) and decarboxylation (Patel et aL,
1994) reactions. We are aware of only one report of micellar catalysis of a poly-
merization reaction (Armstrong et al., 1978). In this paper we report the catalysis
by cetyltrimethyl ammonium bromide (CTAB) micelles of the polymerization of
activated, negatively-charged amino acids.
Carbonyldiimidazole (CDI), in aqueous solution, brings about the oligomeriza-
tion of amino acids and N-monoalkylated amino acids including glycine, sarcosine,
a!anine, phenylalanine and histidine (Ehler and Orgel, 1976; Ehler et al., 1977).
This method has been used to generate oligopeptides attached to oligonucleotides
by a linker containing an amino group. The latter reaction is readily monitored by
gel-electrophoresis when the amino acids are neutral or cationic, but this method
of analysis fails for negatively-charged amino acids (Zieboll and Orgel, 1994).
The formation of oligopeptides in these reactions proceeds by the initial forma-
tion of an intermediate (I) which is in equilibrium with the N-carboxyanhydride
(II) (Figure 1). A free primary or secondary amino group reacts with (II) to form
an amide bond and leaves a carbamic acid terminus. The latter decarboxylates
rapidly to generate a new amino group (Ehler, 1976). Repetition of this sequence
of reactions generates successively longer peptides attached to the initiating amino
group.
Origins of Life and Evolution of the Biosphere 2.6: 1-5, 1996.
_) 1996 Kluwer Academic Publishers. Printed in the Netherlands.
https://ntrs.nasa.gov/search.jsp?R=19980119837 2020-06-15T23:38:31+00:00Z
2 CHRISTOF BOHLER ET AL.
CDI
H2N- CHR --COzH
O
N_ o Zl - Im R_C
- g.o
0
I II
R = CH2OPO32- O-phoapho-L-serina
R • CH2CO2- L-asparticacid
R = CH2CH2CO 2- L-glutarn_cacid
Fig. 1.
oligopeptides
Materials
Reagent grade chemicals were used throughout. O-phospho-L-serine, L-aspartic
acid, L-glutamic acid and cetyltrimethyl ammonium bromide were purchased
from Sigma Chemical Company. Carbonyldiimidazole was from Aldrich Chemical
Company.
Methods
PREPARATION OF REACTION MIXTURES
Reaction mixtures were prepared by adding an amino acid stock solution at pH 8
to solid CDI in an Eppendorf-tube. To study micelle-catalyzed reactions, CTAB
was added to the amino acid stock solution. The tubes were incubated at 16 °C in
a waterbath for appropriate times. Uncatalyzed reaction mixtures were analyzed
by withdrawing aliquots and diluting them with water. CTAB-eatalyzed reactions
were treated the following way: Equal volumes of the reaction solution and 0.1 M
NaCIO4-solution were mixed in an Eppendorf-tube. Precipitation was observed
immediately. The tubes were vortexed and centrifuged for 5 min at 0 °C and
10,000 rpm. Aliquots of the supernatant were diluted with H20 and analyzed by
HPLC. In every case 0.25/zmole of amino acid was applied to the column.
ANALYSIS OF REACTION MIXTURES
HPLC was carried out on a stainless steel column (0.39 x 30 cm) packed with
RPC-5 material (Joyce et al., 1984). A Waters model 680 automated gradient
controller was used in combination with a Waters model 510 solvent delivery
system. A linear gradient of NaC104 (0 to 0.03 M over 40 min) at a flow rate of
OLIGOMERIZATION OF ANIONIC AMINO ACIDS BY CATIONIC MICELLES
SS 7_tai m10
C2! , se
78 1_
Fig. 2. Polymerization of O-phospho-L-serine (A I-A3), L-aspartic acid (B l-B3) and L-glutamic
acid (CI-C3) activated by carbonyldiimidazole (CDI). Samples were analyzed by HPLC on RPC-5
after 1 day (A and B) or 6 h (C). AI: 50 raM O-phospho-L-serine, 100 mM CDI; A2:5 mM
O-phospho-L-serine, 20 mM CDI, 20 mM CTAB; A3:5 mM O-phospho-L-serine, 20 mM CDI. B 1:
100 mM L-aspartic acid, 200 mM CDI; B2:10 mM L-aspartic acid, 20 mM CDI, 20 mM CTAB;
B3:10 mM L-aspartic acid, 20 mM CDI. C1:100 mM L-glutamic acid, 200 mM CDI; C2:10 mM
L-glutamic acid, 20 mM CDI, 20 mM CTAB; C3:10 mM L-glutamic acid, 20 mM CDI. The lengths
of the oligomers corresponding to selected peaks in each chromatogram are indicated in the figure.
The lengths were determined by co-injection with authentic sample in the case of L-aspartic and
L-glutamie acid and by co-injection with material isolated by paper chromatography in the case of
O-phospho-L-serine.
I ml/min was used to elute the oligomers formed in the reaction. Chromatography
was performed at pH 8 in the presence of 0.002 M Tris-HClO4. The eluate was
monitored at 200 nm using an ABI Analytical Kratos Division model Spectroflow
757 UV detector. Paper chromatography was carded out on Whatman 3MM paper
using a mixture of n-propanol (55%), a concentrated NH4OH solution (10%), and
H20 (35%). Oligopeptides were visualized on the paper with ninhydrin spray (1%
ninhydrin, 4% acetic acid in n-butanol. The paper was heated to 100 °C to visualize
the spots).
Results
We have studied the oligomerization of O-phospho-L-serine by carbonyldiimida-
zole (CDI) in some detail, using high performance liquid chromatography (HPLC)
on RPC-5 to analyze the products (Joyce, Inoue et al., 1984). When a 0.05 M solu-
tion of O-phospho-L-serine is treated with a twofold excess of CDI at pH 8 and 16
4 CHRISTOF BOHLER ET AL.
°C a series of oligomers is revealed by HPLC (Figure 2, A1). Paper chromatogra-
phy of the reaction products reveals a corresponding series of peptides (data not
shown). Analysis by the ninhydrin method shows that the yield of oligomers sub-
stantially exceeds 50%. Co-injection of material eluted from paper chromatograms
or of a commercial sample with the complete reaction mixture allows us to assign
lengths to the products that are separated by HPLC as indicated in Figure 2.
When the concentration of O-phospho-L-serine is reduced to 0.005 M, the
pattern of products detected is very different. The longest detectable oligomer
is now the trimer (Figure 2, A3). However, 0.005 M O-phospho-L-serine in the
presence of 0.02 M CTAB, a concentration above its critical micelle concentration
(Fendler and Fendler, 1975), yields a series of oligomers up to the 7-mer (Figure 2,
A2). Catalysis of the formation of (I) is not involved, since the same results are
obtained if (I) is generated first and CTAB added later.
The striking effect of CTAB micelles described above is not restricted to the
polymerization of O-phospho-L-serine. In less extensive studies we obtained sim-
ilar results with L-aspartic acid and L-glutamic acid (Figure 2, B and C).
Discussion
The micelles formed by CTAB carry positive charges on their surfaces. The use
of a quaternary amine makes it unlikely that the reactants are specifically oriented
on the surface of the micelle and also makes it unlikely that acid-base catalysis is
important. We believe that negatively-charged monomers are concentrated close
to the cationic surface, and that the polyanionic oligomers, once formed, attach to
the surface with an affinity that increases with their length. Thus the N-terminus of
an oligomer is in an environment where the concentration of the activated amino
acid exceeds that in free solution. Consequently the probability that an oligomer
will be extended increases with its length, at least until it is long enough to attach
stably to a micelle.
Micellar catalysis of polymerization reactions has been proposed as an impor-
tant aspect of prebiotic chemistry (Oparin, 1924). We believe this to be the first
description of the catalysis of the polymerization of a negatively charged monomer
on a cationic micelle. We anticipate that the effect will prove a general one for
monomers carrying either a positive or a negative charge, adsorbed on micelles
carrying the opposite charge.
A full description of the CDI-mediated oligomerization of negatively-charged
amino acids, including a discussion of the temperature and concentration depen-
dence of the reaction and of the catalysis of the reaction by metal ions, will be
published in due course.
OLIGOMERIZATION OF ANIONIC AMINO ACIDS BY CATIONIC MICELLES 5
Acknowledgements
This work was supported by NSCORT/EXOBIOLOGY Grant No. NAGW-2881
and Grant No. NAGW-1600 from the National Aeronautics and Space Adminis-
tration. We thank Sylvia Bailey for manuscript preparation.
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Catalysis of the Oligomerization of O-Phospho-Serine, Aspartic Acid, or Glutamic Acid by Cationic Micelles

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