Mechanism study on the oligomerization of amino acids into peptides by phosphorus trichloride

Article abstract of DOI:10.1080/10426500701807467

As treated by phosphorus trichloride, amino acids could oligomerize into polypeptides. Based on the results obtained by 31P-NMR and ESI-MS/MS, a possible reaction mechanism was proposed. The mechanism might undergo a penta-coordinated phosphorus intermediat. The activated amino acid was a five-membered cyclic penta-coordinated phosphorus intermediate. The nucleophilic attack of the amino group from an amino acid or peptide on the carbonyl group of intermediate led to the formation of peptide and released one equivalent dichloride phosphoric acid. The repetition of the reaction sequence generated a series of oligopeptides. Copyright Taylor & Francis Group, LLC.

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Mechanism study on the
Oligomerization of Amino Acids
into Peptides by Phosphorus
Trichloride
Wenjie Zhao a , Dongxin Zhao a & Kui Lu a
a School of Chemistry and Chemical Engineering,
Henan University of Technology, Zhengzhou, P. R.
China
Version of record first published: 21 Jun 2008.
To cite this article: Wenjie Zhao , Dongxin Zhao & Kui Lu (2008): Mechanism study
on the Oligomerization of Amino Acids into Peptides by Phosphorus Trichloride,
Phosphorus, Sulfur, and Silicon and the Related Elements, 183:2-3, 691-698
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Phosphorus, Sulfur, and Silicon, 183:691–698, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701807467
Mechanism study on the Oligomerization of Amino Acids
into Peptides by Phosphorus Trichloride
Wenjie Zhao, Dongxin Zhao, and Kui Lu
School of Chemistry and Chemical Engineering, Henan University of
Technology, Zhengzhou, P. R. China
As treated by phosphorus trichloride, amino acids could oligomerize into polypep-
tides. Based on the results obtained by 31 P-NMR and ESI-MS/MS, a possible reac-
tion mechanism was proposed. The mechanism might undergo a penta-coordinated
phosphorus intermediat. The activated amino acid was a five-membered cyclic
penta-coordinated phosphorus intermediate. The nucleophilic attack of the amino
group from an amino acid or peptide on the carbonyl group of intermediate led to
the formation of peptide and released one equivalent dichloride phosphoric acid.
The repetition of the reaction sequence generated a series of oligopeptides.
Keywords Mechanism; penta-coordinated phosphorus; peptides; phosphorus trichloride
INTRODUCTION
More and more experimental results implied that phosphorus might
play an important role in the formation of peptides in a prebiotic
condition. The polymerization of free and substituted amino acid
phosphor-anhydrides, which are energy-rich monomers that permit the
formation of peptide bonds, were studied.1−4 Rabinowitz5 proposed a
phosphoryl group intermediate mechanism when glycine and alanine
were treated with cyclic polyphosphate and dipeptides were obtained.
Baba and Tsuhako6,7 studied the reaction with high performance liquid
chromatography (HPLC) and 31P-NMR, and a five-membered cyclic car-
boxylic acid-phosphoric acid mix anhydride intermediate was proposed.
N-phosphoryl α-amino acids and N,O-bis(trimethylsilyl)-α-amino acids
The authors would like to thank the financial supports from the Chinese National
Science Foundation (No. 20272055, 20572016), Henan Province Science Foundation
for Prominent Youth (No. 0312000900), and Office of Education of Henan Province
(No. 2006KYCX017).
Address correspondence to Lu Kui, School of Chemistry and Chemical Engineer-
ing, Henan University of Technology, Zhengzhou 450052, P. R. China. E-mail: lucky-
luke@haut.edu.cn
691

692
W. Zhao et al.
could self-activate to give peptides, and the reaction mechanism under-
went a five-membered cyclic penta-coordinated phosphoric-carboxylic
mixed anhydride intermediate.8−10 Our recent study showed that α-
amino acids could be assembled into homopeptide libraries with the
assistance of phosphorus oxychloride or phosphorus pentachloride.11,12
We report herein that α-amino acids treated with phosphorus trichlo-
ride can oligomerize into polypeptides in polar aprotic solvent. The re-
action mechanism was studied by 31P NMR and electrospray ionization
mass spectrometry (ESI-MS/MS), and a five-membered cyclic penta-
coordinated phosphorus intermediate of amino acid was proposed.
EXPERIMENTAL
General Preparation of Oligopeptides
5.0 mmol amino acids were mixed with 5.0 mmol phosphorus trichloride
in 15 mL acetonitrile and stirred at a proper temperature. The reaction
was monitored by 31P NMR. Another parallel experiment was quenched
by adding 10 mL water or alcohol to the reaction mixture, which was
determined by ESI-MS/MS.
Mass Spectrometry
Mass spectra were performed on a Bruker Esquire 3000 plus ion trap
spectrometer equipped with a gas nebulizer probe capable of analyz-
ing ions up to m/z 6000. The samples were dissolved in water or al-
cohols were ionized by electrospray ionization (ESI) and continuously
infused into the ESI chamber at a flow rate of 4 L/min by a Cole-Parmer
74900 syringe pump (Cole-Parmer Instrument Co.). Ions were gated
into the ion trap for scan by using injection times of 50 ms. All the ex-
periments were acquired in positive ion mode.
31P NMR
31P NMR spectra were performed on a Bruker DPX-400 spectrometer,
using 85% H3PO4 as an external standard. 31P-NMR spectra were per-
formed on a Bruker AC 200 p spectrometer for N,O-bis(trimethylsilyl)-
alanine reacted by phosphorus trichloride, using 85% H3PO4 as an ex-
ternal standard.

Amino Acids into Peptides by Phosphorus Trichloride
693
FIGURE 1 Positive ESI-MS spectrum of peptides from phosphorus trichloride
reacting with L-Val for 48 h and then quenched with methanol.
RESULTS AND DISCUSSION
Analysis of the Oligomers
After quenching the reaction of L-valine (L-Val) with phosphorus
trichloride by H2O or various alcohols, the corresponding peptides and
peptide esters were obtained, respectively. The structures of these prod-
ucts were confirmed by ESI-MS/MS. Figure 1 shows a series of mass
peaks (m/z 132+99n, n = 1–10) corresponding to oliogmeric products
of the reaction of L-Val with phosphorus trichloride for 48 h quenched
with methanol. We found that not only the amounts but the length of
the peptides increased with prolonged reaction time. Oligopeptides that
were formed for other amino acids were also determined by positive ion
ESI-MS and the results are listed in Table I. The protonated molecu-
lar ions corresponding to oligopeptides from monomer to nonamer were
observed.
Mechanism of Amino Acids Mediated Oligomerization
into Peptides by Phosphorus Trichloride
The ESI-MS/MS results described previously show that the length of
the peptides was dependent on reaction time. Amino acids could hardly
form any peptide in the absence of phosphorus trichloride at room tem-
perature, which indicated that phosphorus trichloride must be partici-
pated in these reactions. 31P NMR spectroscopy is one of the most impor-
tant tools for monitoring the reaction mechanistic course of phosphorus
compounds, and it can be used to investigate the chemical and stere-
ochemical behavior of reaction intermediates that are difficult or even

Amino Acids into Peptides by Phosphorus Trichloride
695
FIGURE 2 The stack 31P NMR spectra of L-Val oligomerization into peptides
mediated by phosphorus trichloride.
impossible by isolation. The progress of the reaction was monitored by
31P NMR spectroscopy, as is shown in Figure 2.
At the beginning, the starting material 1 (δ 220) quickly reacted with
amino acids and 2 (δ 178), 3 (δ 5.38), and 4 (δ–3 to −7) were obtained.
1 and 2 are tri-coordinated phosphorus compounds, while 3 and 4 are
tetra-coordinated phosphorus compounds. As the reaction continued,
the amount of 1, 2, and 3 gradually decreased while the amount of 4
increased, as is shown in Figure 2.
31P NMR Stack results seem to indicate a possible mechanism,
as shown in Scheme 1. The reaction of amino acids with phospho-
rus trichloride led to 2, then 2 isomerized into a penta-coordinated
phosphonic-carboxylic mixed anhydride 6. Anhydride 6 was considered
to be the activated amino acid. Nucleophilic attack of the secondary
amino group from another amino acid at the carbonyl of 6, dipeptide
was formed and one equivalent dichloride phosphoric acid molecule
left. Repetition of this sequence of reactions generated successively
longer peptides. The oligomerization reaction continues until the ac-
tivated amino acids were used up. 2 could also reversibly covert to
3. As shown in Figure 3, trimethyl phosphate and N-(O,O-bimethyl)-
phosphoryl-valine methyl ester were observed in a positive ESI-MS
spectrum of phosphorus trichloride reacting with L-Val for 1 h at 0◦C
then quenched with methanol, which is consistent with the conclusion

696
W. Zhao et al.
SCHEME 1 The possible mechanism of self-assembly into peptide for amino
acids mediated by phosphorus trichloride.
that the tetra-coordinated phosphorus compound 3 was obtained. The
mass number at m/z 141 corresponds to trimethyl phosphate and
m/z 240 corresponds to N-(O,O-bimethyl)-phosphoryl-valine methyl es-
ter; thestructure was conformed by MS/MS. The protonated N-(O,O-
bimethyl)-phosphoryl-valine methyl ester could yield ions [M+1–32]+,
[M+1–28]+ by stepwise loss of methanol and carbon monoxide. The
amount of 3 gradually decreased, which indicated that 3 participated
the formation of peptides, but 3 could not be a nucleophilic attacked
by another amino acid directly. These results implied that there might
exist an equilibrium between 2 and 3. Among these steps, the activa-
tion of the amino acid is the key step. However, the penta-coordinated

Amino Acids into Peptides by Phosphorus Trichloride
697
FIGURE 3 Positive ESI-MS spectrum of L-Val reacting with phosphorus
trichloride for 1h at 0 then quenched with methanol.
phosphorus intermediate is unstable and only exists as instantaneous
intermediate. There is no signal of penta-coordinated phosphorus in-
termediate appeared in the stack 31P NMR spectra. The process of
N,O-bis(trimethylsilyl)-α-alanine reacting with phosphorus trichloride
in dichloromethane was monitored and the signal of penta-coordinated
phosphorus intermediate (δ -68.5, δ -68.3) appeared in 31P NMR spec-
troscopy, as is shown in Figure 4.
FIGURE 4 The 31P NMR spectrum of penta-coordinated phosphorus from N,O-
bis(trimethylsilyl)-α-alanine reacting with phosphorus trichloride.

698
W. Zhao et al.
The proposed mechanism that only amino acids, mediated by
phosphorus trichloride, could form five-membered cyclic phosphonic-
carboxylic anhydride and assemble into peptides suggests that
phosphorus should play important roles in the prebiotic synthesis of
polypeptide and biosynthesis of protein. On the other hand, phosphorus
trichloride may have potential application for polypeptides synthesis.
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