Solid-phase synthesis of phosphonylated peptides

Article abstract of DOI:10.1055/s-0030-1258132

We report the solid-phase syntheses of two series of phosphonylated peptides using Fmoc-protected amino acids. The peptides corresponded to regions containing phosphonylated Ser195 in the active site of butyrylcholinesterase and Tyr411 of human serum albu

Full text of DOI:10.1055/s-0030-1258132

LETTER
1951
Solid-Phase Synthesis of Phosphonylated Peptides
Solid-Phase
Synthesis
a
of Phospho
r
nylated
y
Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA
Fax +1(858)4589311; E-mail: mmacdonald9@gmail.com
Received 5 March 2010
is an obvious need for a quick and easy method to detect
OP while in the field.
Abstract: We report the solid-phase syntheses of two series of
phosphonylated peptides using Fmoc-protected amino acids. The
peptides corresponded to regions containing phosphonylated
Ser195 in the active site of butyrylcholinesterase and Tyr411 of
human serum albumin. The phosphonylated Fmoc-protected amino
acids were used in solid-phase peptide synthesis to prepare the pep-
tides. Fmoc-serine and Fmoc-tyrosine with benzyl ester protection
were treated with alkyl methylphosphonic monochloridates to
phosphonylate the side-chain hydroxy groups. The phosphonylated
peptides were designed to mimic the protein region after exposure
of the proteins to organophosphorus agents.
Monoclonal antibodies against hydrolyzed agent (meth-
ylphosphonic acid) have been developed that display
comparable sensitivity to previously reported spectromet-
ric techniques.4 An alternative method of detection is to
analyze for OP-adducted proteins in physiological fluids
using monoclonal antibodies targeting adducted regions
of those proteins. Serum proteins that bind OPs include
butyrylcholinesterase (BuChE) and human serum albu-
min (HSA).5 OPs covalently modify BuChE at the active
site serine 195 and they modify HSA at tyrosine 411 lo-
cated on the protein surface. Other surface tyrosines are
affected, but tyrosine 411 is predominantly phosphonylat-
ed.6 OP-modified proteins retain the alkyl group that dis-
tinguishes the agent used. Ideally, monoclonal antibodies
against BuChE and HSA adducted to each OP nerve agent
would provide information about which agents were
present. Haptens used to elicit antibody production are
phosphonylated peptides mimicking affected regions of
BuChE and HSA. Peptide sequences for each protein re-
gion are shown in Table 1.
Key words: organophosphonates, nerve agent, phosphonylated
peptides, peptide synthesis, solid phase
Organophosphate (OP) moieties are the core structure of
many nerve agents.1 Their mechanism of action is the
phosphonylation of hydroxy side chains of serine, threo-
nine, and tyrosine residues in proteins thereby interfering
with protein function. OPs are well known to inhibit ace-
tylcholinesterase (AChE) and produce cholinergic toxici-
ty. However, additional noncholinergic targets have also
been identified.2 Four commonly known OP nerve agents
are shown in Figure 1.
Table 1 Peptide Sequences with C-Terminal Aminocaproic Acid for
Human BuChE and Serum Albumin
Because of OPs toxicity and its potential use in future
chemical warfare and terrorist attacks, a reliable and effi-
cient method to detect OPs would be of considerable in-
terest. Current methods of nerve-agent detection utilize
gas chromatography protocols that require lengthy sample
preparation and heavy equipment. Furthermore, nerve
agents and their metabolites are not stable indefinitely,
and this type of analysis is therefore problematic.3 There
Protein
BuChE
Peptide sequencea
H2N-Lys-Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-
Ala-Ala-Aca-COOH
HSA
H2N-Leu-Val-Arg-Tyr-Thr-Lys-Lys-Val-Pro-Gln-Aca-
COOH
a Aca = aminocaproic acid.
While there are many syntheses of phosphorylated pep-
tides reported in the literature,7 there are relatively few
syntheses of phosphonylated peptides. In these accounts,
the unmodified peptide was first assembled by solid-
phase synthesis followed by phosphonylation of the
serine, threonine, or tyrosine side chain by treatment with
a phosphorus(III) reagent–oxidation step while the pep-
tide was still on the resin.8 This method required differen-
tial side-chain protection of other serine, threonine, and
tyrosine residues if present in the sequence because they
would also be modified by phosphorus reagents. To our
knowledge, there are no reported syntheses that utilize
O
P
O
P
O
P
O
O
O
F
F
F
sarin (1)
soman (2)
GF (3)
O
P
N
S
O
VX (4)
Figure 1 Structures of prominent organophosphorus nerve agents
SYNLETT 2010, No. 13, pp 1951–1954
x
x
.x
x
.2
0
1
0
Advanced online publication: 09.07.2010
DOI: 10.1055/s-0030-1258132; Art ID: S01110ST
© Georg Thieme Verlag Stuttgart · New York

1952
M. MacDonald et al.
LETTER
phosphonylated amino acids in solid-phase peptide syn- nal amino acid products were also characterized by high-
theses to produce a phosphonylated peptide. An advan- resolution mass spectrometry. In accord with a literature
tage of this method is that the number of side-chain procedure, compounds 5–8 were prepared using a one-
protecting groups is decreased thereby decreasing possi- step process from methylphosphonic dichloride, and
ble side reactions during their removal. Herein we de- crude products were purified by vacuum distillation.9
1
scribe the use of phosphonylated amino acids that are These products were characterized by H NMR and 31P
incorporated into peptides corresponding to selected re- NMR spectroscopy. Experimental procedures with com-
gions of BuChE and HSA adducted by sarin, soman, GF, pound characterization are available in the Supporting In-
and VX at serine 195 and tyrosine 411, respectively.
formation. Caution: While these compounds are not
classified as nerve agents, proper precautions must be tak-
en when handling them.
O
O
P
The synthesis of Fmoc-protected phosphonylated serine
and Fmoc-protected phosphonylated tyrosine is depicted
in Scheme 1. Fmoc-serine and Fmoc-tyrosine benzyl es-
ters without side-chain protection were treated with phos-
phorus reagents 5–8 in the presence of DMAP and
triethylamine.10,11 Unreacted phosphorus monochloridate
was first quenched by addition of isopropanol, then the re-
action mixture was concentrated in vacuo. After purifica-
tion by chromatography on silica gel, the benzyl ester was
removed by hydrogenolysis to form the carboxylic acid.
This preparation produced a mixture of stereoisomers at
the phosphorus atom. The starting amino acids were enan-
tiopure so the phosphonylated products were isolated as
diastereomeric mixtures and incorporated into peptide
synthesis.
P
O
O
Me
Me
Me
Cl
Cl
5
6
O
O
P
P
O
O
Me
Cl
Cl
7
8
Figure 2 Monochloridate analogues of nerve agents used to modify
serine and tyrosine side chains
O
OR
P
Me
O
There was some concern about the lability of Fmoc
groups and phosphonylated serine side chains to hydro-
genolysis. The hydrogenation was done following a liter-
ature procedure that described the preparation of side-
chain-modified serine residues.10 Product yields ranged
from 41–87% after the removal of benzyl esters. We did
not explore other ester protection such as tert-butyl ester-
ification because we did not encounter any problem with
the hydrogenation of the benzyl ester. There was undoubt-
edly some elimination of the phosphonate from serine to
form dehydroalanine during the reaction based on TLC of
the crude reaction mixture. However, the corresponding
dehydroalanine was not the major product. Yields from
hydrogenation of the serine analogs ranged from 47–87%.
X
5–8, Et3N,
CH2Cl2
Fmoc Ser CO2Bn
OBn
or
Fmoc
N
H
Fmoc Tyr CO2Bn
O
9a–d, 10a–d
9a X = CH2, R = i-Pr
10a X = CH2C6H4, R = i-Pr
10b X = CH2C6H4, R = pinacolyl
10c X = CH2C6H4, R = Cy
10d X = CH2C6H4, R = Et
9b X = CH2, R = pinacolyl
9c X = CH2, R = Cy
9d X = CH2, R = Et
O
OR
P
Me
O
X
H2, 10% Pd/C
THF, MeOH
OH
Fmoc
N
Solid-phase peptide synthesis was done using an Ad-
vanced ChemTech ACT-3926 peptide synthesizer. The
peptides were synthesized using Fmoc chemistry on a 2-
chlorotrityl resin and HBTU/HOBt activation. Protected
amino acids used in the peptide synthesis included Fmoc-
glutamic acid with the isopropyl phenyl ester on the side-
chain carboxylic acid and Fmoc-lysine with Boc protec-
tion of the amine side chain. Nonadducted serine and thre-
onine residues were used without side-chain protection.
Peptide synthesis was done on an automated synthesizer
up to the residue preceding the phosphonylated amino
acid. After this point, couplings were done manually to
ensure the highest coupling efficiency. Cleavage from the
resin was achieved by treating the peptide resin with 1%
TFA–CH2Cl2 containing 2% triisopropylsilane overnight
at room temperature. Crude phosphonylated peptide prod-
ucts were purified by reversed-phase HPLC on a C18 col-
umn (0.1% TFA water–acetonitrile). Peptide purity was
H
O
11a–d, 12a–d
11a X = CH2, R = i-Pr
11b X = CH2, R = pinacolyl
11c X = CH2, R = Cy
11d X = CH2, R = Et
12a X = CH2C6H4, R = i-Pr
12b X = CH2C6H4, R = pinacolyl
12c X = CH2C6H4, R = Cy
12d X = CH2C6H4, R = Et
Scheme 1
Phosphonylated Fmoc-serine and Fmoc-tyrosine were
prepared and then incorporated into the solid-phase syn-
thesis of the requisite peptides as described in Scheme 1.
Protected serine and tyrosine were treated with alkyl
methylphosphonic monochloridates 5–8 (Figure 2) to af-
ford phosphonylated protected amino acids 9a–d and
1
10a–d. All compounds were characterized by H NMR
and 31P NMR spectroscopy. Analysis of amino acid inter-
mediates included low resolution mass spectrometry. Fi-
Synlett 2010, No. 13, 1951–1954 © Thieme Stuttgart · New York

LETTER
Solid-Phase Synthesis of Phosphonylated Peptides
1953
>90% and was verified by analytical HPLC and mass peridine for Fmoc deprotection of the OP-modified serine
spectrometry.
since piperizine was listed as an alternative base for Fmoc
deprotection.13 Similar byproducts were not isolated from
the synthesis of the HSA peptides because tyrosine does
not undergo a dehydration reaction.
Serine is especially susceptible to elimination of phospho-
nates followed by addition of water to regenerate the side-
chain hydroxy group.12 Retention of the phosphonate
throughout synthesis, cleavage from the resin, and purifi-
cation was verified by electrospray mass spectrometry.
Calculated masses and confirmed masses for each peptide
are shown in Tables 2 and 3. Peptides mimicking the
BuChE active site are shown in Table 2 while peptides
mimicking the sequence around tyrosine 411 of HSA are
shown in Table 3. The observed masses agree with the
calculated masses leading us to conclude that the correct
peptides were synthesized and isolated.
O
OR
P
Me
O
O
N
N
H
N
N
N
H
H
H
O
O
OP-serine
piperidinylalanine
Scheme 2 Elimination of OP-serine and addition of piperidine to
form piperidinylalanine
Table 2 Phosphonylated Peptides with C-Terminal Aminocaproic
Acid Corresponding to Active Site Phosphonylated BuChE
In summary, we developed a new method to prepare phos-
phonylated peptides by solid-phase synthesis. This meth-
od incorporates serine and tyrosine amino acids with
phosphonylated side chains into the synthesis. This elim-
inates the use of orthogonal protecting groups if multiple
amino acids with hydroxy side chains are in the sequence.
This can be the case with procedures to phosphonylate
peptides while they are still on the resin. We also showed
that peptide phosphonates can also withstand acidic con-
ditions to cleave peptides from the resin. In conclusion,
the synthesis of phosphonylated peptides is efficient and
applicable to the elaboration of serine-, threonine-, or ty-
rosine-containing peptides.
Peptide Name
#
Phosphonate ester MW
MW
calcd
(R)
obs.
13
14
15
16
BuChE-sarin
isopropyl
pinacolyl
cyclohexyl
ethyl
1470.8
1513.4
1511.3
1457.2
1470.6
1512.7
1510.7
1456.6
BuChE-soman
BuChE-GF
BuChE-VX
Table 3 Phosphonylated Peptides with C-Terminal Aminocaproic
Acid Corresponding to Phosphonylated HSA
Peptide
#
Name
Phosphonate ester MW
MW
calcd
(R)
obs.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett. It includes
the syntheses and analytical data for all the compounds described as
well as the HPLC chromatograms for peptides 13–20.
17
18
19
20
HSA-sarin
HSA-soman
HSA-GF
isopropyl
pinacolyl
cyclohexyl
ethyl
1465.4
1507.4
1505.3
1451.3
1465.7
1507.8
1505.8
1451.7
Acknowledgment
HSA-VX
We thank Michael Freydkin of Biopeptide, Inc. (San Diego, CA) for
the synthesis and purification of the phosphonylated peptides. The
work described herein was funded by the National Institutes of
Health CounterACT Program through the National Institute of Neu-
rological Disorders and Stroke (Award # U01 NS058038). Its con-
tents are solely the responsibility of the authors and do not
necessarily represent the official views of the federal government.
During the synthesis of the BuChE-OP peptides 14 and
15, a peptide byproduct was also formed. Because this
byproduct consistently gave the same mass of 1418 mass
units and same HPLC retention time from syntheses of
different OP-BuChE peptides, we assumed that it was the
same byproduct from each synthesis. A recent account de-
scribed a survey of alternate Fmoc deprotection strategies
to suppress elimination of phosphorylated serine side
chains.13 Based on this report we surmised that the
byproduct arose from elimination of the phosphonyl por-
tion of the serine residue during Fmoc deprotection using
20% piperidine–DMF to give a dehydroalanine-contain-
ing peptide. Addition of piperidine to dehydroalanine
formed piperidinylalanine in place of the phosphonylated
serine (Scheme 2). The calculated mass of the BuChE
peptide with piperinylalanine replacement was also 1418
mass units supporting our belief. Phosphorylated serine is
prone to elimination in the presence of 20% piperidine–
DMF especially when in the N-terminal position of a pep-
tide. This was resolved by using piperazine in place of pi-
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