Investigation of reaction mechanism of amino acids and phosphorus trichloride by 31P NMR and ESI-MS/MS

Article abstract of DOI:10.1002/cjoc.201190219

The reaction of amino acids and phosphorus trichloride in THF was studied by ³¹P NMR tracing and ESI-MS/MS. A series of hydridophoranes and cyclic dipeptides were obtained. The reaction presented interesting diversity and the reaction mechanism was proposed. The mechanism suggests that phosphorus plays an important role in the synthesis of amino acid hydridophorane and cyclic dipeptides. The results also show that ³¹P NMR and ESI-MS/MS are useful tools for the investigation of reaction mechanism. Copyright

Full text of DOI:10.1002/cjoc.201190219

FULL PAPER
Investigation of Reaction Mechanism of Amino Acids and
Phosphorus Trichloride by 31P NMR and ESI-MS/MS
Guo, Yanchuna(郭艳春)
Cao, Shuxia*,a(曹书霞)
Xie, Yalia(谢娅黎)
Zong, Xiangkuna(宗向坤)
Zhao, Yufen*,a,b,c(赵玉芬)
a Key Laboratory of Chemical Biology and Organic Chemistry, Department of Chemistry, Zhengzhou University,
Zhengzhou, Henan 450052, China
b College of Chemistry and Chemical Engineering and The Key Laboratory for Chemical Biology of Fujian Prov-
ince, Xiamen University, Xiamen, Fujian 361005, China
c Key Laboratory for Bioorganic Phosphorus Chemistry of Ministry of Education, Department of Chemistry, School
of Life Sciences and Engineering, Tsinghua University, Beijing 100084, China
The reaction of amino acids and phosphorus trichloride in THF was studied by 31P NMR tracing and
ESI-MS/MS. A series of hydridophoranes and cyclic dipeptides were obtained. The reaction presented interesting
diversity and the reaction mechanism was proposed. The mechanism suggests that phosphorus plays an important
role in the synthesis of amino acid hydridophorane and cyclic dipeptides. The results also show that 31P NMR and
ESI-MS/MS are useful tools for the investigation of reaction mechanism.
Keywords 31P NMR, ESI-MS/MS, mechanism, amino acid, phosphorus trichloride
Introduction
added into reaction system to neutralize hydrochloric
acid produced, amino acids could mainly transform to
amino acid hydridophorane, 3,8-dialkyl-1,6-dioxa-4,9-
diaza-5λ5-phospha-spiro[4.4]-nonane-2,7-dione in IU-
PAC name.17 Therefore, the reaction of unprotected
amino acids assisted by phosphorus trichloride pre-
sented interesting diversity as shown in Scheme 1.
It is well known that phosphorus reagents are im-
portant condensation reagents for peptide formation in
prebiotic evolution and in the process of life. As early as
1965, Fox1 has used polyphosphoric acid as condensa-
tion reagents in the polycondensation reaction of amino
acids. In 1969, polyphosphate was used to investigate
the polycondensation reaction of amino acid in water,
and a phosphoryl group intermediate mechanism was
proposed by Rabinowitz et al.2,3 Furthermore, a
five-member cyclic carboxylic-phosphoric mixed anhy-
dride intermediate was proposed by studying the reac-
tion with HPLC, ion-exchange chromatograph (IEC), IR
spectrum and 31P NMR.4-8 Moreover, it was discovered
that N-phosphoryl-α-amino acids and N,O-bis(tri-
methylsilyl)-α-amino acids could self-activate to pro-
The product, cyclic dipeptide, is an important com-
pound exhibiting a great variety of biological activi-
ties.18,19 Another product, hydridophorane, is a class of
special pentacoordinate phosphorus compounds, and has
many special chemical properties. Many studies showed
that it can be used to synthesize a number of important
compounds.20 However, in terms of the reaction mecha-
nisms of amino acids and phosphorus trichloride, even
though much is known about some reaction mechanisms
involving the influence of phosphorus reagent, only few
papers have been published to describe the reaction
mechanisms about the amino acids and inorganic phos-
phorus regents.17,21 The reaction mechanism of amino
acids and phosphorus trichloride in acetonitrile to pro-
duce oligopeptides has been proposed,21 and that of
producing hydridophorane in glycol dimethyl ether was
investigated by 31P NMR monitoring method.17 Fur-
thermore, it was found that in different solvents, the
triphenyldichlorophosphane gave different chemical
shifts reflected different structures, such as ionic species
or non-ionic species.22 Sometimes, inorganic phospho-
duce peptides, and the reaction underwent
a
five-member cyclic pentacoordinated phosphoric-
carboxylic mixed anhydride intermediate.9-11 Recent
studies showed that α-amino acids could assemble into
homopeptide libraries with the assistance of inorganic
phosphorus reagents,12-14 and the oligopeptide libraries
constructed by this method could be transformed to
conjugated and phosphoryl oligopeptide libraries.15,16
Further studies of our group showed that α-amino acids
could assemble into not only linear polypeptides but
also cyclic dipeptide with the assistance of phosphorus
trichloride. Furthermore, when triethylamine (TEA) was
*
E-mail: csx@zzu.edu.cn; Tel.: 0086-0371-67767050; Fax: 0086-0371-67767051
Received July 27, 2010; revised October 25, 2010; accepted February 20, 2011.
Project supported by the National Natural Science Foundation of China (Nos. 20672104, 20732004)
Chin. J. Chem. 2011, 29, 1173— 1179
© 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPER
Scheme 1 The reaction of amino acids and phosphorus trichloride
rus regents could even react with some solvent to pro-
duce new compounds.23 Thus, the solvent should be
considered when the reaction mechanisms involving
phosphorus regents are investigated. Therefore, the re-
actions of unprotected amino acids and phosphorus tri-
chloride in the same solvent, tetrahydrofuran (THF),
were studied and optimized. Six cyclic dipeptides and
six amino acid hydridophoranes were synthesized by
this method. The reaction mechanisms of amino acids
and phosphorus trichloride in THF were investigated by
31P NMR tracing and ESI-MS/MS. In order to rational-
ize the structure of reaction intermediate, the energies of
the transition states and the reaction intermediate were
estimated using the B3LYP/6-31G(d,p) model.
Then triethylamine (Et3N) (30 mmol) in THF (5 mL)
was added dropwise to the solution and the mixture was
stirred and refluxed for 2 h. After evaporation of the
solvent, H2O was added and the solution was filtered
and the solid was washed with water (10 mL×3). The
crude hydridophorane was obtained as a pair of di-
astereoisomer and the crude products were analyzed by
HPLC. Analytical sample was obtained by recrystalliza-
tion from acetone and petroleum ether as a white solid.
The yield of hydridophorane was 55%—76%. The
1
structures of hydridophoranes were characterized by H
NMR, 13C NMR, ESI-MS and IR methods.
Mass spectrometry
Mass spectra analysis was performed by a Bruker
Esquire 3000 (Bruker Dalton, Germany) ion trap mass
spectrometer that interfaced an ESI source. Ionization
was carried out using the following setting of the ESI:
nebulizer gas flow 48.265 kPa, dry gas 4 L/min, dry
temperature 300 ℃, capillary voltage 4000 V. Calibra-
Experimental
General preparation of cylic peptides
All chemicals and materials were of analytical grade.
Solvents used were freshly purified and dried by stan-
dard procedures. Each L-amino acid (20 mmol) was
dissolved in THF (30 mL), and PCl3 (10 mmol) was
added in batches. Then the mixture was stirred and re-
fluxed for 2 h. After evaporation of the solvent, H2O
was added and the solution was adjusted to pH 7—8
with saturated aqueous NaHCO3. Then the reaction
mixture was filtered and the solid was washed with wa-
ter (10 mL×3). The crude cyclic dipeptide was ob-
tained and analyzed by HPLC. Analytical sample was
obtained by column chromatography [silica gel,
V(methanol)∶V(ethyl acetate)＝1∶19 or 1∶4) or re-
crystallization from acetone and petroleum ether as
white solid. The yield of cyclic dipeptide was 37%—
78%. The structures of cyclic dipeptides were charac-
tion of m/z was performed using
a
standard
ESI-tuning-mix. Scan range was m/z 15—600 and scan
resolution was normal [13000 (m/z)/s]. MSn spectra
were obtained by CID experiments with helium after
isolation of the appropriate precursor ions. High resolu-
tion mass spectra were performed on an
ESI-Q-TOF-MS spectrometer (Micromass, England).
31P NMR experiment
31P NMR spectra were performed on a Bruker
DPX-400 MHz spectrometer, using 85% H3PO4 as an
external standard.
Computational methods
1
terized by H NMR, 13C NMR, ESI-MS, HRMS and IR
Using density functional theory, the geometries of
the transition states and intermediates in part reaction
channel were optimized at the B3LYP/6-31G(d,p) level.
All theoretical calculations were performed using the
GAUSSIAN0324 suite of software.
methods.
General preparation of hydridophorane
Each L-amino acid (20 mmol) was dissolved in THF
(30 mL), and PCl3 (10 mmol) was added in batches.
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Investigation of Reaction Mechanism of Amino Acids and Phosphorus Trichloride
Results and discussion
of a CO and a substituent molecule. As far as the frag-
mentation of leucine hydridophorane is concerned, in
positive mode, the signal at m/z 291＋corresponds to the
intact protonated molecule [M＋H] , and the peak at
m/z 263 can be attributed to a fragment ion formed by
the decarbonylation pathway. Furthermore, the [M＋H
－CO]＋ ion at m/z 263 successively extrudes a meta-
phosphorous acid to form [M＋H－CO－HPO2]＋ ion at
m/z 199, which is in good agreement with the reported
fragmentation pathways.29 In addition to the ions of
products, there are also some peaks of reaction interme-
diates. The ion at m/z 238 should correspond to O- or N-
(O,O-bimethyl)-phosphoryl leucine. They have similar
fragmentation pathways in ESI-MS/MS. In negative
mode, they are all pron－e to eliminate one methanol to
form [M－H－CH3OH] ion at m/z 206, and then they
can successively lose CO and another methanol to form
[M－H－CH3OH－CO]－ ion at m/z 178 and [M－H－
2CH3OH－CO]－ ion at m/z 146. Taborsky et al. have
proved that phosphoryl groups linked to the phospho-
protein phosvitin may be shifted from hydroxyl to
amino groups of the protein by changing the pH valve
of the solution. In the range of pH 8.5—11, the
O-terminal phosphoryl amino acids could transform into
N-terminal phosphoryl amino acids.30 Accordingly, the
pH valves of reaction systems were detected. The pH
valves of reaction system without Et3N were 3—4, and
the pH valves of reaction system with Et3N were 5—6.
Therefore, the ion at m/z 238 should be deduced to be
O-(O,O-bimethyl)-phosphoryl leucine. The peak at m/z
176 is assumed to correspond to deprotonated molecule
ion of cyclic phosphorylate amino acid. The ion at m/z
Analysis of the reaction product by ESI-MS/MS
Since not only the molecular mass but also the
structure information could be acquired by ESI-MS/MS,
now ESI-MS/MS has been proven to be a valuable
technique in monitoring the reactions and tracing the
intermediates.25-27 Therefore, the reaction firstly was
detected by ESI-MS/MS. The reaction of L-leucine
(L-Leu) and PCl3 in THF was chosen as an example.
After diluting the reaction solution by methanol and
injecting to the ESI-MS, the reaction solution after 1 h
was detected by ESI-MS. Some useful information was
observed from the spectra in negative mode. The corre-
sponding product cyclic dipeptide at m/z 225 and hy-
dridophorane at m/z 289 were observed respectively.
Besides the products, some reaction intermediates and
byproducts were also observed in the spectra. It is well
known that ESI-MS/MS is a powerful tool to identify
the structures of the unknown compounds. The struc-
tures of the products and the reaction intermediates were
confirmed by positive and negative ESI-MS/MS and the
data are listed in Table 1. In terms of cyclic dipeptide, in
positive mode, the molecule ion at m/z 227 firstly loses
CO to produce [M＋H－CO]＋ ion at m/z 199. Subse-
quently, an eliminati＋on of amino acid residue forms [M
＋H－CO－residue] ion at m/z 86. In negative mode,
the molecule ion at m/z 225 always tends to eliminate
thei－r substituent radical to form radical ions [M－H－
R•] and [M－H－2R•]－ at m/z 168 and 111, respec-
•
tively, as the literature reported.28 The [M－H－CO－
(R＋H)]－ ion at m/z 139 is produced by successive loss
Table 1 ESI-MS/MS data of reaction products and intermediates
Compound
Number
Mode
MS＋
Precurser ion
227.0 (6)
198.8 (8)
225.0 (5)
168.0 (9)
Fragment ion (relative abundance/%)
198.8 (100), 86.1 (36)
86.1 (100)
31P NMR δ
Cyclic L-Leu
dipeptide
1
MS－
167.7 (52), 138.7 (100), 110.7 (11)
110.8 (100)
(product)
MS+
MS－
291.0 (72)
263.0 (61)
289.0 (4)
263 (100), 199 (2)
199 (100), 178 (2)
157.6 (100), 129.7 (18)
129.6 (100)
－67.4
－67.91
(e, f)
2
L-Leu hydridophorane
O-Phosphoryl-leucinea
(product)
157.6 (22)
MS－
238.0 (6)
206.0 (8)
205.6 (100), 177.9 (15), 79.0 (6)
177.6 (100)
－5.36
(c)
3
4
178.0 (4)
145.6 (100)
R＝CH2CH(CH3)2
R＝CH2CH(CH3)2
1.81
MS＋
178.0 (5)
121.8 (81), 92.9 (100)
(h)
a The Cl－ ion of compound c has displaced by methoxyl group in the ESI-MS spectrum, because of choosing methanol as the solvent.
Chin. J. Chem. 2011, 29, 1173— 1179
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Guo et al.
FULL PAPER
133 should contain two chlorine atoms, since its char-
acteristic isotope peaks are at m/z 135 and m/z 137. It is
speculated to be the deprotonated molecular ion of
phosphoric acid dichloride.
and signal d gradually increased, which means a by-
product containing phosphorous gradually increased
along with the reaction. The results seem to indicate a
possible mechanism, as shown in Scheme 2. As the re-
action goes on, the signal of reactants a and b decreased
slowly. Moreover, O-(dichloride)-phosphoryl leucine at
signal c appeared and then disappeared as shown in
Figure 1. The N-terminal of amino acid attacks the acti-
vated carbonyl of O-(dichloride)-phosphoryl leucine to
produce the reaction intermediate I and eliminate phos-
phoric acid dichloride, whose signal is at d (δ＝－5.86).
The reaction intermediate I quickly closes the ring and
converts to amino acid cyclic dipeptide accompanying
by elimination of another phosphoric acid dichloride.
Therefore, the signal d corresponding to phosphoric acid
dichloride increased. It is also observed that chemical
shift of signal d has changed from δ －5.86 to －6.21
as the reaction going on from 1 h to 2 d. The change of
pH of the reaction system induced the chemical shift of
signal d, as the literature reported.21 Furthermore, the
results of ESI-MS are consistent with the reaction
mechanism proposed from 31P NMR stack spectra.
Mechanism of amino acid and PCl3 to produce cyclic
dipeptides
L-Leu (0.15 mmol) was dissolved in 1 mL THF in an
NMR tube, and then PCl3 (0.075 mmol) was added. The
reaction was monitored by 31P NMR spectroscopy and
the results are shown in Figure 1.
By the detection of HPLC and TLC, it is recognized
that a pair of cis-trans-isomers of cyclic dipeptide were
produced. Two cis-trans-isomer, cis- and trans-cyclic
dipeptide, were acquired respectively by column
chromatogram and recrystallization, and their structures
Figure 1 The stack 31P NMR spectra of L-leucine and PCl3 in
THF.
It has been reported that N,O-bis(trimethylsilyl)-
amion acid or amino acid could hardly produce any
peptide in the absence of phosphorus reagents at room
temperature.11,21 The result implied that phosphorus re-
agents must mediate the reactions of peptide formation.
As shown in Figure 1, at the beginning of the reaction,
there are three signals in the 31P NMR spectrum at δ
218.33 (a), 177.63 (b) and －5.36 (c). After 1 h, some
new 31P NMR signals at δ －5.86 (d), －67.40 (e) and
－67.91 (f) appeared. As the reaction continued, the
amounts of signals a, b and c gradually decreased while
the amount of signals d, e and f increased. The signals e
and f has been identified as a pair of amino acid hydri-
dophoranes which will be discussed below.
1
were identified by H NMR, 13C NMR, IR and HRMS.
As literature reported,31,32 the racemization of cyclic
dipeptides took place mainly after cyclization. Possible
reason of racemization was proposed to be keto-enol
tautomerism resulted from deprotonation from α-carbon.
Therefore, cis- and trans-cyclic dipeptides were all ob-
tained in the reaction system.
Mechanism of L-amino acids assembling into hydri-
dophorane directed by PCl3
31P NMR was also used to monitor the reaction of
L-leucine and phosphorus trichloride in THF to synthe-
size hydridophorane. L-Leu (0.15 mmol) was dissolved
in 1 mL THF, and then 0.075 mmol PCl3 and 0.225
mmol Et3N were added into the NMR tube. The result
by monitoring 31P NMR spectra is shown in Figure 2.
Similar to cyclic dipeptide system, a (δ＝218.23), b
(δ＝177.83), and c (δ＝－5.40) could be observed
clearly in the THF solution. They correspond to PCl3,
hydroxyl phosphorus dichloride and O-terminal-
phosphoryl leucine respectively. After adding Et3N, the
signals a, b and c disappeared and new signals g (δ＝
－9.12) and h (δ＝1.81) appeared. After 0.5 h, a couple
of new signals e (δ＝－67.28) and f (δ＝－67.79)
emerged. As the reaction continued, during the period of
time 0.5 to 16 h, the signals g and h increased with re-
spect to e and f and then, the signals g and h gradually
decreased and converted into the signals of e and f,
which corresponded to the reaction products, a pair of
amino acid hydridophoranes.
Since the product, cyclic dipeptide, could not be ob-
served in 31P NMR spectra, only reaction intermediates
and byproducts could be deduced from the stack spectra.
The signals at d, e and f containing phosphrous gradu-
ally increased while the reactant at a and b continually
decreased until it disappeared. It is known that the sig-
nal a (δ＝218.33) corresponds to PCl3. Since PCl3 hy-
drolyzed and oxidized easily when exposed in atmos-
phere, thus hydroxyl phosphorus dichloride b (δ＝
177.63) is considered to exist at the same time. Then
hydrolyzed and oxidized product of PCl3 quickly re-
acted with amino acid and transformed into c (δ＝
－ 5.36). Since the signal c is in the range of
tetra-coordinated phosphorus compounds and the pH
valves of reaction systems were detected to be 3—4, the
signals c was speculated to be the O-terminal-pho-
sphoryl leucine, which was also observed in the ESI-MS
spectrum. However, the signal c rapidly disappeared
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Chin. J. Chem. 2011, 29, 1173— 1179

Investigation of Reaction Mechanism of Amino Acids and Phosphorus Trichloride
Scheme 2 The possible mechanism of the reaction of L-Leucine and PCl3
pared with the literature.33 Therefore, it was observed
that the signal g gradually decreased, while the signal h
gradually increased.
After adding Et3N, the pH valves of reaction systems
has changed to 5—6, so it was considered that the
formed phosphonyl leucine is O-terminal-phosphonyl
leucine (g1) or N-terminal-phosphonyl leucine (g2) as
shown in Scheme 3. By studying the chemical combina-
tion of phosphonic acid dichloride with the amino acid
using the GAUSSIAN03 suite of programs, all struc-
tures were optimized by employing the hybrid density
functional B3LYP method34,35 and 6-31G(d,p) basis set.
Moreover, the zero-point energies and energies of g1,
g2, TS1, TS2, h and HCl were calculated at the
B3LYP/6-31G (d, p) level, as shown in Table 2. Amino
acid can initiate the reaction through two different posi-
tions because amino acid has two different functional
groups attached to the phosphoryl. The presence of two
reaction sites gives rise to two distinct channels, result-
ing in two different product O-terminal-phosphonyl leu-
cine (g1) or N-terminal-phosphonyl leucine (g2). Then
compound g1 or g2 generated h by cyclization as shown
in Scheme 3. TS1 results from the N-terminal of g1 ap-
proaching to phosphonyl and TS2 results from the
O-terminal of g2 approaching to phosphonyl (Figure3).
The energy of transition state TS1 is 89.04 kJ/mol lower
than the energy of the TS2 by comparing the energy ∆E
of TS1 to TS2. Therefore, we speculated that the path-
way from the N-terminal of g1 approaching to generate
compound h is more favorite.
Figure 2 The stack 31P NMR spectra of L-Leu assembling into
hydridophorane directed by PCl3 at room temperature.
It is reported that the signal of the analogue of com-
pound 4 (Table 1, when R＝H), appeared at δ 10.39
using TFA as solvent in 31P NMR spectroscopy.33 The
literature also reported that, in different solvents, the
triphenyldichlorophosphane gave different chemical
shifts reflecting different structures, such as ionic forms
or non-ionic forms.22 Therefore, we detected the 31P
NMR spectrum of reaction solution in TFA. The results
show that all signals shifted to low field. For example,
the signal h (δ＝1.81) shifted to 10.60, and the signal g
(δ＝－9.12) shifted to －0.59. The signals of com-
pound 2, leucine hydridophoranes, at e (δ＝－67.28)
and f (δ＝－67.79) shifted to δ －62.72 and －62.21,
respectively. As shown in Scheme 2, when Et3N was
added, phosphonic acid dichloride, which was the oxi-
dized product of hydroxyl phosphorus dichloride (b),
could react with leucine to produce phosphonyl leucine
(g) by removing one molecule HCl. While triethylamine
hydrochloric acid salt was produced. The Et3N could
liberate hydrochloric acid again and lead to the forma-
tion of compound 4 as shown in Table 1. The signal of
compound 4 should be signal h in the Figure 2 com-
Moreover, from the difference of two structures of
O-terminal-phosphoryl leucine c and g in Scheme 2, the
signal of O-terminal-phosphoryl leucine (c) in cyclic
dipeptide system at c (δp＝－5.36) should be lower field
than that of O-terminal-phosphonyl leucine (g) in
Chin. J. Chem. 2011, 29, 1173— 1179
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1177

Guo et al.
FULL PAPER
Scheme 3 The mechanism of formation of compound h
Table 2 The zero-point energies and energies of g1, g2, TS1,
TS2, h and HCl at the B3LYP/6-31G(d,p) level (unit: a.u.)
eliminating H2O molecule.
Conclusion
Species
ZPE
Energy
g1
g2
TS1
TS2
h
0.114442
0.114645
0.114277
0.108852
0.101757
0.006718
1200.455425
1200.460826
1200.428980
1200.400312
739.664515
460.794059
The reaction process, in which L-amino acid could
produce amino acid hydridophoranes and cyclic dipep-
tides mediated by PCl3, was traced by 31P NMR and
ESI-MS/MS. The mechanism was proposed and the
reaction presented the interesting diversity. The mecha-
nism suggests that phosphorus plays important roles in
the formation of pentacoordinate phosphorus com-
pounds and cyclic peptides. On the other hand, the
mechanism investigation of the reaction reveals the di-
versity of amino acid reactions and is helpful for cyclic
peptides and hydridophoranes synthesis. The results
also show that 31P NMR and ESI-MS/MS are useful
tools for the investigation of reaction mechanism.
－
HCl
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Figure 3 The structures and the geometrical parameters of TS1
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unit: Å).
2
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Investigation of Reaction Mechanism of Amino Acids and Phosphorus Trichloride
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