Regio- and diastereo-selectivity of montmorillonite-catalyzed oligomerization of racemic adenosine 5′-phosphorimidazolide

Article abstract of DOI:10.1080/15257770801954955

Clay is a possible candidate for an effective catalyst in prebiotic chemical evolution of biomolecules. Montmorillonite was reported to effectively catalyze oligomerization of racemic adenosine 5′-phosphorimidazolide (DL-ImpA). In the oligomerization reaction, considerable amounts of cyclic dimers as well as linear dimers were produced in the oligomerization reactions. To assess the regio- and diastereo-selectivities of the oligomerization reaction, the dimer products including cyclic dimers were completely identified by means of enzymatic degradation reactions of the products. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770801954955

Nucleosides, Nucleotides and Nucleic Acids, 27:421–430, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770801954955
REGIO- AND DIASTEREO-SELECTIVITY OF
MONTMORILLONITE-CATALYZED OLIGOMERIZATION OF RACEMIC
ADENOSINE 5ꢀ-PHOSPHORIMIDAZOLIDE
Hidehito Urata, Mami Fujimori, Chie Aono, Tomomi Yamakawa,
Emi Harada, and Masao Akagi
Osaka University of Pharmaceutical Sciences, 4-20-1, Nasahara, Takatsuki, Osaka, Japan
Clay is a possible candidate for an effective catalyst in prebiotic chemical evolution of
2
biomolecules. Montmorillonite was reported to effectively catalyze oligomerization of racemic adeno-
sine 5 ꢁ-phosphorimidazolide (DL-ImpA). In the oligomerization reaction, considerable amounts of
cyclic dimers as well as linear dimers were produced in the oligomerization reactions. To assess the
regio- and diastereo-selectivities of the oligomerization reaction, the dimer products including cyclic
dimers were completely identified by means of enzymatic degradation reactions of the products.
Keywords RNA; mononucleotide; racemate; clay-catalyzed oligomerization; cyclic nu-
cleotide
INTRODUCTION
Biomolecules such as amino acids, sugars, and nucleic acids are chiral,
and the chirality of biomolecules is thought to have been essential for
the origins of life.[1,2] Indeed, the homochirality of biomolecules plays an
important role for the specificity in the recognition between them.[3] Since
the discovery of ribozymes,[4,5] RNA is thought to have been one of the
important precursors of life.[6] Under prebiotic conditions, biomolecules
such as nucleotides are thought to have been formed as a racemate.
Considering the chemical evolution of RNA, there are serious problems how
racemic nucleotides have been oligomerized, and how the homochirality of
RNA has been established.[7]
Received 30 July 2007; accepted 31 January 2008.
We thank the Sumitomo Foundation 2003 (Japan) for financial support.
Address correspondence to Hidehito Urata, Osaka University of Pharmaceutical Sciences 4-20-1
Nasahara, Takatsuki, Osaka569-1094, Japan E-mail: urata@gly.oups.ac.jp
421

422
H. Urata et al.
ꢁ
ꢁ
FIGURE 1 Structures of D-, L-ImpA (upper) and D-homochiral cyclic pA3 pA3 (bottom).
Template-directed oligomerization of activated mononucleotide D-
ImpA (Figure 1) effectively yielded polynucleotides,[8] but was unsuccessful
for oligomerization of L-ImpA and racemic ImpA.[9] Since Ferris and
coworkers have reported effective oligomerization of D-ImpA on montmo-
rillonite clay,[10,11] we noticed the achirality of montmorillonite and its po-
tential to catalyze oligomerization for L-ImpA or racemic ImpA as effective
as for D-ImpA. Then, we found that montmorillonite serves an effective
catalyst for the oligomerization of L-ImpA and racemic ImpA.[12] Inde-
pendently, Ferris and coworkers have also reported the montmorillonite-
catalyzed reaction of racemic ImpA.[13] Although the formation of con-
siderable amounts of cyclic dimers (Figure 1) were observed in the re-
action, the identification of cyclic dimers formed by the oligomerization
of racemic ImpA was not reported because of the possibility of formation
of various kinds of regio-, enantio-, and diastereo-isomers. Very recently,
Ferris and coworkers have reported the identification of homochiral D,D-
ꢁ
ꢁ
ꢁ
ꢁ
cyclic pA3 pA3 and heterochiral cyclic pA3 pA3 as well as linear dimers
produced by the montmorillonite-catalyzed oligomerization of racemic
ImpA.[14] However, under their experimental conditions to suppress the
formation of longer oligomers, they haveꢁ found neither the regio-isomers
ꢁ
of the phosphodiester bond of cyclic pA3 pA3 nor HEPES-pApA, which is
an adduct of the dimer with HEPES used as a buffer. We found that the
HEPES-pApA adduct shows the same chromatographic behavior with one
of the cyclic dimers in reversed phase HPLC. The chromatographic overlap
of HEPES-pApA and cyclic dimers makes the identification of them difficult,
leading to decrease of the accuracy of quantitative product analysis. To solve

Montmorillonite-Catalyzed Oligomerization
423
such difficulty, we employed imidazole as a buffer in place of HEPES to
avoid formation of HEPES-pApA. In this article, we report on the complete
identification of the dimer products, including the first identification
ꢁ
ꢁ
of homochiral cyclic pA2 pA3 produced by the montmorillonite-catalyzed
oligomerization of racemic ImpA.
EXPERIMENTAL SECTION
General
D- and L-ImpAs were synthesized by a literature procedure from
adenosine 5ꢁ-phosphoric acid,[8] of which L-isomer was synthesized by
phosphorylation[15] of L-adenosine.[6] Calf intestine alkaline phosphatase
and RNase T2 was purchased from Takara Bio Inc. (Shiga, Japan) and
Sigma (St. Louis, MO, USA), respectively. Reversed phase HPLC was
˚
performed on a column of µBondasphere 5C18 100A with a linear gradient
of acetonitrile (0–10%) in 50 mM potassium phosphate (pH 4.0) by a
Shimadzu LC-10A system. Anion exchange HPLC was performed on a
column of TSK GEL DNA-NPR (TOSOH) with a linear gradient of NaClO4
(0–0.15 M/30 minutes) in 20 mM Tris-HCl (pH 9.0).
Oligomerization of ImpA
To 50 mg of Na+-montmorillonite, 14 mM ImpA solution containing 0.2
M NaCl, 75 mM MgCl2, 0.1 M HEPES or imidazole (pH 8.0) was added. After
the pH was adjusted to 8.0 by adding diluted NaOH or HCl, respectively,
reactions were stand for 7 days at 25◦C.
Alkaline Phosphatase Treatment
A solution of dimers (0.2 OD units/200 µL) containing 10 mM MgCl2,
50 mM Tris-HCl (pH 8.0) was incubated with 0.4 µL of calf intestine alkaline
phosphatase (0.5 units/µL) at 37◦C for 3 hours. The mixture was filtered
through Ultrafree-MC 5000 NMWL filter unit (Millipore) and was analyzed
by a reversed phase HPLC.
RNase T2 Treatment
A solution of dimers (0.015 OD units/12 µL) was added 0.2 M ammo-
nium acetate (pH 5.0) 1.5 µL and RNase T2 (0.1 units/µL) 1.5 µL, and was
incubated at 37◦C for 3 hours. After addition of 10 mM CuSO4 (1.65 µL),
the mixture was filtered through Ultrafree-MC 5000 NMWL filter unit and
was analyzed by a reversed phase HPLC.

424
H. Urata et al.
FIGURE 2 An anion exchange HPLC chromatogram of montmorillonite-catalyzed oligomerization
products of racemic ImpA.
RESULTS AND DISCUSSION
Oligomerization reactions were carried out by adding 50 mg of Na+-
montmorillonite to a solution (1 mL) containing 14 mM racemic ImpA, 0.2
M NaCl, 75 mM MgCl2, 0.1 M HEPES buffer (pH 8.0) at 25◦C for 7 days. The
reaction efficiently proceeded to give oligomers up to 8-mers (Figure 2).
To identify the dimer products (Figure 3), the dimer fraction was collected
by using anion exchange HPLC and desalted. Figure 4 shows the HPLC
chromatograms of the dimer fraction; (a) anion exchange, (b) reversed
phase HPLC chromatograms, and (c) a HPLC chromatogram of the dephos-
phorylated product of the dimer fraction with alkaline phosphatase. This
enzyme shows an enough dephosphorylation activity for L-nucleotides as
well as for D-nucleotides,[16,17] and the only isomers of pApA among possible
dimer products (Figure 3) are converted to 5ꢁ-dephosphorylated dimers.
The peaks for the four linear dimers shifted to more lipophilic region
(C–F) by this enzyme, however dimers A and B were not affected. This
result indicates that dimers C–F are the regio- and diastereo-isomers of ApA
produced from the corresponding pApA isomers. These dimers C–F were
ꢁ
easily identified as homochiral (D,D- and L,L-) A2 pA, heterochiral (D,L-
ꢁ
ꢁ
and L,D-) A2 pA, homochiral (D,D- and L,L-) A3 pA and heterochiral (D,L-
ꢁ
and L,D-) A3 pA, respectively by comparison with authentic samples.[16,17]
On the other hand, dimers A and B should be cyclic dimers or 5ꢁ-capped
dimers such as AppApA or HEPES-pApA (Figure 3). In the preliminary
degradation experiments of dimer A with alkaline hydrolysis followed by
alkaline phosphatase treatment, dimer A was found to contain HEPES-pApA
as well as cyclic dimers by detecting HEPES-pA.[18]

Montmorillonite-Catalyzed Oligomerization
425
FIGURE 3 Possible dimer products formed by the condensation reaction of D-ImpA catalyzed by Na+-
montmorillonite. In the reaction of racemic ImpA, additional enantio- and diastereo-isomers are formed.
Alternatively, we carried out the oligomerization reaction of racemic
ImpA under the similar condition by using imidazole buffer in place of
HEPES buffer to avoid forming HEPES-pApA. Both the reactions pro-
ceed similarly, and the yields of oligomers produced by each reaction
are compared in Table 1. After isolation of the dimer fraction from the
reaction in imidazole buffer, it was analyzed by an anion exchange HPLC
FIGURE 4 HPLC analyses of the dimer fraction produced by clay-catalyzed oligomerization of racemic
ImpA in HEPES buffer. (a) Anion exchange, and reversed phase HPLC chromatograms (b) before and
(c) after alkaline phosphatase treatment of the dimer fraction.

426
H. Urata et al.
TABLE 1 Yields of oligomers produced by montmorillonite-catalyzed oligomerization of
D,L-ImpAa
Oligomers (%)
Buffer
1 mer
2 mer
3 mer
4 mer
5 mer
6 mer
7 mer
8 mer
HEPES
Imidazole
47.8
47.0
29.4
30.1
14.2
13.3
5.53
5.73
2.01
2.28
0.75
1.02
0.28
0.42
0.06
0.11
aReactions were carried out at 25◦C for 7 days. D,L-ImpA concentration was 14 mM (Each
enantiomer concentration is 7 mM) in 0.2 M NaCl, 75 mM MgCl2, and 0.1 M buffer, pH 8.0.
Percentages listed are the uncorrected HPLC absorbance readings.
(Figure 5a). The chromatogram lacks a peak at 7.9 minutes, which was
observed in the dimer fraction from the reaction in HEPES buffer (Figure
4a). It was proved that this peak corresponds to HEPES-pApA by degrada-
tion experiments (data not shown). Figure 5b and 5c shows reversed phase
HPLC chromatograms of the dimer fraction and its dephosphorylated
product after alkaline phosphatase treatment, respectively. Dimers C–F were
readily assigned to the isomers of ApA as described above.
FIGURE 5 HPLC analyses of the dimer fraction produced by clay-catalyzed oligomerization of racemic
ImpA in imidazole buffer. (a) Anion exchange, and reversed phase HPLC chromatograms (b) before
and (c) after alkaline phosphatase treatment of the dimer fraction.

Montmorillonite-Catalyzed Oligomerization
427
FIGURE 6 Reversed phase HPLC analyses of RNase T2 followed by alkaline phosphatase (AP) treatment
of dimer A.
In this case, degradation experiments with alkaline hydrolysis followed
by alkaline phosphatase treatment demonstrated that dimers A and B
contained neither AppApA nor HEPES-pApA. Therefore, these two peaks
were thought to contain only cyclic dimers. After isolation of dimers A and
B, we carried out RNase T2 treatment followed by alkaline phosphatase
treatment to characterize these cyclic dimers. Figure 6 shows the result for
the enzymatic digestion of dimer A. As a result, dimer A afforded three
peaks corresponding to dimers A, C, and F. RNaseT2 can cleave the 3ꢁ-5ꢁ
phosphodiester linkage, but can cleave neither the 2ꢁ-5ꢁ phosphodiester
linkage,[19] nor the 3ꢁ-5ꢁ phosphodiester linkage of which 5ꢁ-end side
nucleoside has an L-stereochemistry.[17] The cyclic dimer has ten kinds of
regio-, enantio- and diastereo-isomers (Figure 7). Among them, the isomers
ꢁ
ꢁ
that afford homochiral A2 pA (dimer C) and heterochiral A3 pA (dimer F)
ꢁ
ꢁ
after these treꢁatments are homochiral D,D-cyclic pA2 pA3 and heterochiral
ꢁ
cyclic pA3 pA3 , respectively. Therefore, it is very likely that dimer A consists
ꢁ
ꢁ
of heterochiral cyclic pA3 pA3 as a major constituent and homochiral cyclic
ꢁ
ꢁ
pA2 pA3 as a minor one. It is also plausible that homochiral L,L-cyclic
ꢁ
ꢁ
pA2 pA3 was not hydrolyzed and is still observed as dimer A.
Figure 8 shows the results of the enzymatic digestion with RNase T2 and
alkaline phosphatase for dimer B. Dimer B afforded nearly equal amounts
of two products corresponding to dimer B and 3ꢁ-AMP with RNase T2
treatment. 3ꢁ-AMP was converted to adenosine with alkaline phosphatase
treatment. Amongꢁ the cyclic dimers shown in Figure 7, only homochiral
ꢁ
D,D-cyclic pA3 pA3 affords 3ꢁ-AMP and adenosine with these treatments.
Thus, the unreacted constituent is considered to be homochiral L,L-cyclic
ꢁ
ꢁ
pA3 pA3 .
The percentage of each isomer contained in the dimer fraction
is summarized in Table 2. The homochiral dimers are preferentially

428
H. Urata et al.
FIGURE 7 Regio-, enantio-, and diastereo-isomers of cyclic adenylyladenosine, and their expected
degradationꢁ products with RNaseꢁT2 and alkaline phosphatase (AP) treatments. Since heterochiral cyclic
ꢁ
ꢁ
3
2
pAL3 pAD and cyclic pAL2 pAD are mesomeric, their enantiomers do not exist.
produced (66.0%) relative to the heterochiral ones (33.9%), and the 3ꢁ-5ꢁ
phosphodiester linkages are more efficiently formed (78.3%) than the 2ꢁ-5ꢁ
phosphodiester linkages (21.7%). These proportions are basically similar
to those reported by Ferris and coworkers,[14] although their experimental
FIGURE 8 Reversed phase HPLC analyses of RNase T2 followed by alkaline phosphatase (AP) treatment
of dimer B.

Montmorillonite-Catalyzed Oligomerization
429
TABLE 2 Relative yields of dimers formed by montmorillonite-catalyzed
oligomerization reaction of racemic ImpAa
Products
Yield (%)
ꢁ
D-D & L-L dimer (homochiral)
D-L & L-D dimer (heterochiral)
A2 pA
ꢁ
18.5
9.8
4.0
33.7
9.9
12.4
11.6
A3 pA
ꢁ
ꢁ
ꢁ
cyclic pA2 pA3
ꢁ
cyclic pA3 pA3
ꢁ
A2 pA
ꢁ
A3 pA
ꢁ
ꢁ
cyclic pA3 pA3
aYields were estimated based on HPLC peak area of the dimer fraction after alkaline
phosphatase treatment (Figure 5c).
conditions are considerably low ImpA concentration (0.15 or 0.6 mM) to
suppress the formation of longer oligomers relative to our experimental
conditions (14 mM). It should be noted that the regioselectivity for the
3ꢁ-5ꢁ phosphodiester linkages is relatively low in our conditions although
the homochiral selectivity is very similar in both experimental conditions.
The buffer condition and/or ImpA concentration may affect on the re-
gioselectivity of the reaction. The content of the cyclic dimers reaches
49.3% of the total dimer products. This feature of the reaction may lead
to the some decrease of the oligomerization efficiency for racemic ImpA on
montmorillonite compared with that for D- or L-ImpA. It can be considered
that the formation of the cyclic dimers may serve as the inactivating pathway
of reactive dimers, or that when large amounts of dimers are accumulated,
the formation of cyclic dimers may serve as the storage of the quasi-reactive
form of dimers.
CONCLUSION
In conclusion, we have successfully characterized the cyclic dimers as
well as linear dimers in the montmorillonite-catalyzed oligomerization prod-
ucts of racemic ImpA. We have reported that the diastereo-isomers of ApA
show the different hydrolytic reactivity.[20] The elucidation of the chemical
properties of the cyclic dimers and its difference between the isomers
would be important to understand the characteristics of clay-catalyzed[12,13]
and metal ion-catalyzed[21] oligomerization reactions of racemic mononu-
cleotides.
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