Amino acid dependent formation of phosphate anhydrides in water mediated by carbonyl sulfide

Article abstract of DOI:10.1021/ja056036e

Carbonyl sulfide (COS), a component of volcanic gas emissions and interstellar gas clouds, is shown to be an efficient condensing agent in the context of phosphate chemistry in aqueous solutions. We report that high-energy aminoacyl-phosphate anhydrides a

Full text of DOI:10.1021/ja056036e

Published on Web 12/07/2005
Amino Acid Dependent Formation of Phosphate Anhydrides in Water
Mediated by Carbonyl Sulfide
Luke J. Leman,† Leslie E. Orgel,‡ and M. Reza Ghadiri*,†
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and The Salk Institute for Biological Studies,
P.O. Box 85800, San Diego, California 92186
Received September 1, 2005; E-mail: ghadiri@scripps.edu
Scheme 1. Phosphate Activations Mediated by Carbonyl Sulfide
Carbonyl sulfide (COS) is a component of volcanic gas emissions
on present-day Earth,1 a major constituent of the atmosphere of
Venus,2 and one of the molecules observed in interstellar gas
clouds.3 It brings about the efficient formation of oligopeptides from
amino acids under a variety of potentially prebiotic conditions.4
Here we investigate the reactions of COS in the context of phos-
phate activation in neutral aqueous solutions. We show that COS
is an effective reagent for the synthesis of aminoacyl phosphates
from amino acids and inorganic phosphate or aminoacyl adenylates
from amino acids and adenylic acid. We further show that the mixed
anhydrides of amino acids and inorganic phosphate, in the presence
of certain divalent ions, are phosphorylating agents, producing
pyrophosphate from phosphate. The amino acid dependent activa-
tions of phosphate reported here, which occur under mild aqueous
conditions in parallel with the production of peptides, suggest that
peptide synthesis and phosphoryl transfer reactions might have
shared a common activated precursor on the prebiotic Earth.
The reaction between inorganic phosphate and an aminoacyl
N-carboxyanhydride5 (NCA, 3, Scheme 1) has recently been shown
to generate a mixed aminoacyl-phosphate anhydride (4).6 Despite
indications from the literature that concentrated phosphate solutions
can rapidly hydrolyze COS gas to CO2 and H2S,7 we wondered if
N-carboxyanhydrides might still form in the presence of phosphate
and then react with phosphate to yield an aminoacyl-phosphate
anhydride. Our studies indicate that mixed aminoacyl-phosphate
anhydrides (4) are rapidly produced from amino acids (1) and
inorganic phosphate in the presence of COS and ferricyanidesa
potentially prebiotic8 oxidizing agent that greatly enhances the
efficiency of COS-mediated peptide synthesis4 (Table 1, Figures 1
and S1). Yields of the aminoacyl-phosphate anhydrides were as
high as 25% (Table 1). The participation of aminoacyl thiocarbam-
ate 2 as an intermediate in the formation of aminoacyl-phosphate
anhydride 4 was confirmed by initiating a reaction with an authentic
sample of L-phenylalanine thiocarbamate 2 without COS (Scheme
1). Further, in the absence of amino acid or COS, no phosphate
anhydrides were observed. Product identities were established by
comparison of 31P NMR chemical shifts (Figure S4) with those of
authentic samples.6,9 Product yields depended markedly on the
concentration of phosphate in the reaction mixture (Figure S3);
maximum yields were obtained at phosphate concentrations of ∼500
mM. The optimal pH value of ∼7.5 for the formation of 4 (Figure
S3) is somewhat lower than the optimal pH value of ∼9.0 for COS-
mediated peptide synthesis.4 The reaction of amino acids with NCA
at near-neutral pH values is less favorable than that at higher pH
values as a result of protonation of the R-amine. Also, the most
abundant phosphate ion at pH 7.5 is the dianion (HPO42-), which
is reportedly the reactive species in attacking the NCA.6 The
Table 1. COS-Mediated Formation of Aminoacyl-Phosphate
Anhydrides (4) for Various Amino Acids (1)a
amino acid (1)
time
AA
−anhydride (4)
(20 mM)
final pH
(min)
(% yield)
L-arginine
7.1
7.2
7.2
7.2
7.2
7.2
7.2
7.1
7.2
25
35
30
20
50
85
30
45
30
26
L-phenylalanine
L-glutamic acid
L-aspartic acid
glycine
L-alanine
L-cysteine
24
23
22
19
14
6.0
L-serine
L-histidine
not observed
not observed
a Reactions were carried out in bis-Tris propane buffer (pH 7.5, 400 mM)
at 25 °C. Yields were determined by integration of 31P NMR peaks.
reaction of L-phenylalanine (Table 1) still generated a ∼6% yield
of dipeptide (7), a ∼1% yield of tripeptide, and a trace of
tetrapeptide (observed by HPLC-MS) in just 35 min.
In principle, aminoacyl-phosphate anhydrides could act as
acylating agents10 and/or as phosphorylating agents6,11,12 by reacting
via C-O or P-O bond cleavage, respectively. We have found that
aminoacyl-phosphate anhydrides, in the presence of certain alkaline
earth metal ions that form phosphate precipitates, can generate
pyrophosphate (5) in yields of up to 30% based on the yield of
† The Scripps Research Institute.
‡ The Salk Institute for Biological Studies.
9
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J. AM. CHEM. SOC. 2006, 128, 20-21
10.1021/ja056036e CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Table 3. Aminoacyl Adenylate (6) Formation Induced by COSa
amino acid (1)
time
AA
−adenylate (6)
(20 mM)
initial/final pH
(min)
(% yield)
glycine
L-arginine
7.0/6.7
7.0/6.8
7.0/6.7
7.0/6.8
7.0/6.9
7.0/6.8
7.0/6.8
110
150
105
160
180
120
120
12
8
7
7
6
5
L-phenylalanine
L-aspartic acid
L-alanine
L-glutamic acid
L-serine
not observed
Figure 1. 31P NMR spectra illustrating phosphate anhydride formations.
(a) Formation of L-phenylalanine phosphate (4) in a reaction as described
in Table 1. (b) Formation of L-alanyl phosphate (4) and its conversion to
pyrophosphate (5) in a reaction as described in Table 2. PhPO4* is phenyl
phosphate, an internal concentration standard added after quenching the
reaction. The differences in chemical shift for the two spectra result from
pH differences in the two samples.9
a Reactions contained PIPES buffer (pH 7.0, 400 mM) at 25 °C. Yields
were determined by integration of 31P NMR peaks using 0.2% phosphoric
acid in a sealed capillary tube as an external concentration standard.
yields are complicated by their hydrolytic instability, yields of
∼10% have been observed for several amino acids (Table 3). In
the case of phenylalanine, product identities were confirmed using
reported 31P NMR chemical shift values14 and by comparison of
HPLC-MS and 31P NMR profiles with those of an authentic
sample.9,15
Table 2. Pyrophosphate (5) Formation under Various Conditionsa
The studies reported here suggest that COS, a simple volcanic
gas, could have mediated both phosphoryl transfer and peptide
synthesis reactions via a single intermediate, aminoacyl N-carboxy-
anhydride, under mild aqueous conditions on the prebiotic Earth.
amino acid (1)
additive
time
(h)
pyrophosphate (5)
(20 mM)
(100 mM)
(% yield)b
L-glutamic acid
L-glutamic acid
L-glutamic acid
L-glutamic acid
L-glutamic acid
L-glutamic acid
L-arginine
L-phenylalanine
L-alanine
L-aspartic acid
glycine
CaCl2
27
37
46
46
46
46
21
27
40
40
40
40
30 (5.4)
12 (2.2)
2 (0.4)
hydroxylapatitec
SrCl2
Acknowledgment. We thank NASA Exobiology (NAG5-12160)
for financial support, and NSF for a Predoctoral Fellowship (L.J.L.).
BaCl2
MgCl2
PbCl2
CaCl2
CaCl2
CaCl2
CaCl2
CaCl2
CaCl2
6 (1.1)
not observed
not observed
23 (4.1)
13 (2.3)
18 (3.2)
32 (5.8)
17 (3.1)
5.0 (0.9)
Note Added in Proof. An account of reactions between NCA
and nucleotides has recently been reported. See: Biron, J.-P.;
Parkes, A. L.; Pascal, R.; Sutherland, J. D. Angew. Chem., Int. Ed.
2005, 44, 6731-6734.
Supporting Information Available: Experimental procedures, 31
P
L-histidine
NMR spectra, plots of reaction time course, product formation versus
pH and phosphate concentration, and product characterizations. This
material is available free of charge via the Internet at http://pubs.acs.org.
a Reactions contained PIPES buffer (pH 7.5, 200 mM) at 25 °C. b Yields
were determined by 31P NMR integration and are based on the maximum
concentration of intermediate 4. The values in parentheses represent yields
based on amino acid. c Hydroxylapatite is a naturally occurring CaPO4
mineral; 150 mM NaH2PO4 was used in this reaction.
References
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Aminoacyl adenylates (6) are critical intermediates in contem-
porary protein biosynthesis and would be produced if NCA reacted
with the AMP in the same way as it does with inorganic phosphate.
Indeed, adenylates (6) were generated with a variety of amino acids
under similar conditions to those employed for the formation of 4
(Figure S5). While accurate determinations of aminoacyl adenylate
(15) Luo, L.; Walsh, C. T. Biochemistry 2001, 40, 5329-37.
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