Solid-Phase Synthesis of the Aged-Nonapeptide-Nerve-Agent Adduct of Butyrylcholinesterase as Reference Materials for Analytical Verification

Article abstract of DOI:10.1002/hlca.201700198

Two pathways were developed and investigated for the synthesis of the ‘aged’-nonapeptide nerve-agent bioadduct of human butyrylcholinesterase (BuChE). Considering the fast ageing of nerve-agent adducts of BuChE in patients and biomedical samples this target molecule is of paramount relevance for quantitative analysis with respect to the Chemical Weapons Convention. Two approaches using a precursor bearing a hydroxyl on its phosphonyl moiety and a benzyl protected precursor were considered. Several impurities were identified and circumvented during the optimization of the peptide synthesis step. The ‘aged’-nonapeptide adduct was successfully synthesized by solid-phase-peptide-synthesis (SPPS).

Full text of DOI:10.1002/hlca.201700198

Article Type: Full Paper
Solid-phase Synthesis of the Aged-Nonapeptide-Nerve-Agent Adduct of Butyrylcholinesterase as
Reference Materials for Analytical Verification
†,‡
†
‡
Andreas Bielmann , Christophe Curty , Christian G. Bochet*
†
Spiez Laboratory, Austrasse, CH-3700 Spiez, Switzerland
‡
Department of Chemistry, University of Fribourg, Chemin du Musée 9, CH-1700 Fribourg,
Switzerland
christian.bochet@unifr.ch
Abstract
Two pathways were developed and investigated for the synthesis of the “aged”-nonapeptide nerve-
agent bioadduct of human butyrylcholinesterase (BuChE). Considering the fast ageing of nerve-agent
adducts of BuChE in patients and biomedical samples this target molecule is of paramount relevance
for quantitative analysis with respect to the Chemical Weapons Convention. Two approaches using a
precursor bearing a hydroxyl on its phosphonyl moiety and a benzyl protected precursor were
considered. Several impurities were identified and circumvented during the optimization of the
peptide synthesis step. The “aged”-nonapeptide adduct was successfully synthesized by solid-phase-
peptide-synthesis (SPPS) <deleted for the first time>.
O
R
O
P O
O
P OH
O
OH
SPPS
OPG2
Phosphylation
1
OPG2
PG HN
1
PG HN
FGESAGAAS
O
O
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doi: 10.1002/hlca.201700198
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Introduction
Nerve agents (NAs) are among the most toxic chemical warfare agents (CWAs). Developed in the
1
930’s, they have been used on several occasions, e.g. during the Iran-Iraq war, in the Tokyo
[
1-6]
subway, in Syria (2013 & 2017), or recently in Malaysia. The development, production, use and
stockpile of CWAs is prohibited by the Chemical Weapons Convention (CWC) since 1997. The CWC
is enforced by the Organisation for the Prohibition of Chemical Weapons (OPCW) with the help of its
network of designated laboratories. Despite the efforts of the OPCW to destroy the stockpiled
CWAs they still pose a threat, especially for the general public in the context of terrorist attacks.
[
7]
[
8]
The structure of the most relevant nerve agents is given in Figure 1. They are organophosphorus
compound (OPs) which consist of a phosphoester and a leaving group. The differentiation between
G- and V-series nerve agents is of historical nature while structurally their main difference consists of
the leaving groups.
Figure 1. Structures of the nerve agents GA (Tabun), GB (Sarin), GD (Soman), GF (Cyclosarin), VX ,
CVX (Chinese VX) and VR (Russian VX).
Nerve agents are strong acetylcholinesterase (AChE) and butyrylcholinesterase inhibitors. The LD50
can be as low as 8.4 µg/kg (VX, in rats, subcutaneous). Symptoms of poisoning are miosis, nausea,
[
9]
cramps, loss of muscle control, paralysis and loss of consciousness. Death usually occurs through
[
10]
respiratory paralysis. Standard therapy consists of symptomatic treatment with atropine and
reactivation of inhibited acetylcholinesterase with oximes. Common reactivators are pyridinium
aldoxims (2-PAM, obidoxime) and dipyridinium aldoximes (TMB-4, HI-6, LüH-6). However all of them
are permanently charged which prevents them of passing the blood-brain-barrier and therefore they
provide limited protection for the central nervous system. The reactivators show an agent-specific
effectiveness and no broadband reactivator is available today. Survivors might suffer from long-term
[
11-15]
neuropathic effects.
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Using bioadducts to detect usage of nerve-agent is attractive, due to their relatively long half-life
(
several days to several weeks). On the other hand, degradation in environmental samples is fast
[
10]
and free metabolites in blood or urine get excreted within 48-72 h.
The most relevant bioadducts in blood formed between nerve agents and proteins are AChE (on Ser-
[
16,17]
2
03), BuChE (on Ser-198) and albumin (on Tyr-411) adducts.
BuChE adducts are the most
popular analytical targets, since their abundance is ten times greater than those of AChE and BuChE
[
16,18]
reacts 500 times faster with nerve agents than albumin.
NA bioadducts of AChE and BuChE undergo an “ageing” process which leads to the loss of the O-alkyl
chain of the phosphyl group (Scheme 1). This process can occur spontaneously and is due to
[
19]
enzymatic activity in the body. The rate of ageing depends on the ester group, e.g. the half-life of
[
10,20]
ageing is 2-6 min for soman-, 3-5 h for sarin-, 13 h for tabun- and 48 h for VX-adducts of AChE.
After ageing has occurred the phosphylated enzymes cannot be reactivated since electrostatic
[
21]
repulsion between reactivators and the oxyanion prevent nucleophilic attacks. Further the
electrostatic interaction between the oxyanion and the His of the enzymatic pocket strongly
[
22]
stabilizes the aged structure. Therefore detection methods which rely on fluoride reactivation e.g.
[
23,24]
detecting reactivated OPs by GC-MS, cannot work after ageing.
bioadducts is highly relevant.
Hence the ability to detect aged
O
O
P O
O
P OH
O
-
HOiPr
BuChE
BuChE
Scheme 1. Enzyme mediated “ageing” of the sarin-butyrylcholinesterase adduct.
Modern detection methods rely on LC-MS/MS for the detection of nerve-agent bioadducts. BuChE is
isolated from plasma with immunomagnetic beads and digested by pepsin to yield a specific
nonapeptide with the sequence FGES(MPX)AGAAS, where MPX denotes the phosphyl moiety, which
[
16,25-28]
is then detected by tandem LC-MS/MS.
With this method, quantitative analysis is possible but
reference materials are needed, especially for the “aged” nonapeptide adducts (Figure 2).
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OH
O
O
O
O
O
O
H
H
H
H
H N
2
N
N
N
N
N
N
H
N
N
OH
H
H
H
O
O
O
HO
12
HO
O
O
P OH
O
O
O
O
O
O
H
H
N
H
H
N
H N
2
N
N
N
N
N
N
OH
H
H
H
H
O
O
O
O
HO
16 & 16'
HO
O
Figure 2. Non-phosphylated nonapeptide 12 and “aged” nerve-agent bioadduct 16 after pepsin
digestion of phosphylated human BuChE.
Posttranslational modification of proteins by phosphylation plays a key role in regulating signaling
processes and protein-protein interactions in the body, therefore synthesis of phosphopeptides has
[
29]
been of interest for a while. The use of phosphylated serines in SPPS is a challenge since the
phosphyl group is decomposed by strong acids and prone to β-elimination when in contact with
bases. When a building block approach is used, the phosphyl group should be protected to lower
such reactivity. As an alternative approach, peptides can be phosphylated post synthetically which
[
30-33]
requires unprotected side chain hydroxyls to be built in.
In this work, we report the first synthesis of the aged nerve-agent nonapeptide adduct using a
building block approach, in which first serine is phosphonylated and then used in solid-phase-
peptide-synthesis. The phosphonylation of serine is based on work carried out in our laboratory.
[
34]
A
similar approach was very recently published for the synthesis of deuterium-labelled soman- and VX-
[
35]
nonapeptides as reference materials.
Results and Discussion
Two routes to synthesize the aged nonapeptided 16 were investigated (routes A and B). For route A
we chose Fmoc-Ser(MPA)-OH 1 as a precursor to be used in SPPS. Since it was unclear whether or
not the hydroxyl(methyl)phosphonyl group would lead to side reactions during SPPS, a second route
(route B) was investigated in parallel, using a protecting group on the phosphonyl moiety. Fmoc-
Ser(MPBn)-OH 2 was chosen, since we expected the benzyl group to be deprotected together with
the other protecting groups at the release step of the peptide from the resin after SPPS.
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Commercially available Fmoc-Ser-OH 3 was protected using benzyl alcohol and thionyl chloride
[
36]
giving Fmoc-Ser-OBn 4 with 82% yield (Scheme 2). 4 was treated with methyl phosphonic
dichloride (MPDC) 5 using DABCO/DMAP as bases with twofold and 0.2 fold excess respectively. This
base combination has proven to be crucial to prevent the loss of the phosphonyl group by β–
elimination. The intermediate Fmoc-Ser(methyl phosphonic chloride)-OBn 6 was hydrolyzed directly
on the column during purification by reversed phase flash chromatography yielding 7 (purity 87%, P
NMR). The benzyl group was then selectively deprotected by hydrogenolysis with 1,4-
cyclohexadiene and Pd/C yielding 1 in mediocre purity. Further purification of 1 by flash
chromatography led to considerable losses; hence it was used without further purification for SPPS.
OH
O
OH
O
N
H
O
3
SOCl2
BnOH
82%
O
P
Cl
MPDC 5
DABCO / DMAP
OH
O
O
O
O
67%
O
O
N
H
O
N
H
O
O
4
6
Reversed phase flash chromatography
O
O
P
OH
OH
P
OH
O
Cyclohexadiene
Pd / C
O
O
O
O
88%
O
N
O
N
H
H
O
O
1
7
Scheme 2. Synthesis of the precursor to route A.
Since 2 already contains a benzyl group on the phosphonyl, an orthogonal protecting group for the
acid group had to be found. tert-Butyl was assumed to be cleavable while leaving the P-OBn group
intact under weak acidic conditions. A screening was made to find the mildest conditions to
hydrolyze commercially available Fmoc-Ser-tBu 8 with trifluoroacetic acid. The tBu group was
cleaved within five minutes when the TFA concentrations in the range from 80% to 5% were used. At
concentrations below 5% the reaction slowed down considerably, taking 30 min at 4% and >210 min
at 3%. For concentrations of 2% and 1% full conversion was not reached after 24 h.
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The synthesis of 2 started with MPDC 5 which was esterified with benzylic alcohol and DMAP to give
methyl phosphonic acid dibenzyl ester 9 (Scheme 3) with excellent purity.
BnOH
DMAP
O
O
P
O
P
DABCO
4%
P
O
Cl
O
9
71%
OH
Cl
O
5
10
9
Fmoc-Ser-OtBu 8
PyBOB / DIPEA
63%
O
P
O
O
P
O
O
O
O
O
5
% TFA in DCM
OH
O
O
N
O
N
99%
H
H
O
O
2
11
Scheme 3. Synthesis of the precursor to route B.
Under basic conditions one benzyl group of 9 was cleaved to give monoester 10 (purity 98%, P
[
37]
NMR). 10 was then coupled to 8 using PyBOP and DIPEA as coupling agents yielding
phosphonylated serine 11 in 63% yield and 90% purity (P NMR). Two diastereoisomers were
generated due to the chiral center at the phosphorous. 11 was exposed to the best cleavage
conditions found in the screening which was shown to be 5% TFA in DCM. These were the mildest
conditions in which full deprotection would occur after 5 min. Deprotection of 11 was slower than of
8
, and the reaction was extended to two days at room temperature. To our satisfaction these
conditions left the benzyl group intact and 2 could be obtained in 99% yield after purification. 2
again was present as a mixture of two diastereoisomers due to the chiral center at the P center. In
contrast to 1, purification of 2 by flash chromatography occurred without extensive loss and the
product was gained in high purity. An alternative deprotection procedure using aqueous phosphoric
[
38]
acid was unsuccessfully attempted.
In order to fine-tune our SPPS procedure, non-phosphylated nonapeptide 12 (which is also a helpful
reference material in the development of biomedical analysis methods), was synthesized and the
procedure scaled up. Pre-loaded resins were used and 20% piperidine in DMF was used for Fmoc-
deprotection. For deprotection and cleavage from the resin a cleavage mixture consisting of
TFA:TIS:H O=95:2.5:2.5 was used. The best work-up method proved to be precipitation in cold
2
diethyl ether and subsequent centrifugation yielding the nonapeptide in pure form, while extraction
or flash chromatography gave inferior results in terms of yield and purity.
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First attempts in employing 1 in solid-phase-peptide-synthesis using microwave (mw) heating (65 °C,
min) to speed up amino acid coupling proved unsuccessful, and led to β-elimination of the
phosphonyl group and produced the dehydroalanin-containing peptide 13 as the major product
Scheme 4).
5
(
O
R
P O
O
FGE
AGAAS
N
H H
O
Base-
O
R
-
P O
O-
FGE
AGAAS
N
H
O
13
Scheme 4. β-Elimination of the phosphonyl group under mw and basic conditions.
Therefore, all subsequent peptide syntheses were performed at room temperature. Small scale
experiments (0.01 mmol) showed that the introduction of phosphonylated serine into the peptide
chain was slower than the introduction of regular amino acids. In fact, deletion peptide 14 (Figure 3)
was found to be the major product together with deletion peptide 15 in an almost 1:1 ratio. While
1
4 lacked the phosphylated serine, 15 lacked the last two amino acids of the sequence.
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O
O
P OH
O
P OH
O
FGEAGAAS
ESAGAAS
FESAGAAS
14
15
17
O
P O
N
HN
H
N
O
AGAAS
N
H
FGESAGAAS
HO
O
O
22
23
Figure 3. Side products of peptide synthesis with phosphonylated Ser and piperidine as base during
the deprotection step. 14: deletion product without Ser(MPA) incorporated. 15: deletion product
with missing Phe and Gly. 17: deletion product with missing Gly (not observed). 22: cyclic peptide
after phosphonamidation side reaction. 23: trapping product with the base.
The desired product 16 from route A was only found in traces. Prolonging the coupling time of the
phosphonylated serine to 120 min prevented 14 from occurring. Switching the base for the
deprotection step of SPPS from 20% piperidine in DMF to 5% piperazine in DMF completely
prevented the formation of 15. From these experiments, the absence of a deletion peptide 17 which
would contain Phe but not the second Gly and the fact that Phe and Gly only missed if Ser(MPA) was
incorporated we concluded that the base is crucial for the formation of side product 15 and the
formation follows an unusual mechanism. The proposed mechanism is shown in Scheme 5.
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O
P
OH
O
O
H
O
N
N
N
AGAAS(OtBu)-Resin
H
O
O
O
O
O
O
Piperidine
O
H
N
AGAAS(OtBu)-Resin
H N
2
N
O
P
H
O-
O
O
O
O
P
O
P
O
H
N
O
O
H N
2
O
N
AGAAS(OtBu)-Resin
H
H
H N
2
N
O
20
N
AGAAS(OtBu)-Resin
H
O
18
O
O
DIC / Oxyma
O
O
O
OEt
O
O
P
N
P
O
O
CN
HN
-Oxyma
O
H
O
N
O
AGAAS(OtBu)-Resin
N
H
O
H
H N
2
N
O
O
N
AGAAS(OtBu)-Resin
19
H
O
21
O
O
TFA:TIS:H O
2
O
P
OH
O
O
H
H N
2
N
N
AGAAS-OH
15
H
O
HO
O
Scheme 5. Proposed mechanism for the deletion product 15.
We hypothesized that during the harsher conditions of the piperidine deprotection method, the
growing peptide chain may end up as the cyclic phosphonamidate 19, either by reaction of 20 with
the coupling agents mixture, or via an activation pathway which might involve pyrophosphonate
formation. The intramolecular cyclisation is faster than the intermolecular peptide bond formation,
and the formation of the cycle prevents further elongation of the peptide chain. Under the harsh
acidic cleavage and deprotection conditions after SPPS the cycle opens again and gives 15. After
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+
scanning for cycle 22 in the HR/LC-MS data, its mass [M+H] = 652.2338 could be found as a minor
byproduct (ca. 4%) of the crude peptide. Our proposition is further supported by the fact that if a
protection group is used on P-OH, no deletion product 15 was found, because no cyclic
phosphonamidate could be formed. The proposed mechanism is similar to the findings of Amedjkoh,
who investigated the phosphonamidation reaction mechanism with standard peptide coupling
agents, including DIC and Oxyma and Kitamura, who used protected α-amino phosphonic acids and
[
39,40]
coupling agents to synthesize α-amino phosphonic acid oligomers by solid-phase synthesis.
However, in our system we could not confirm the formation of pyrophosphonate 18 as it was
observed by Amedjkoh.
Using the optimized protocol 16 from route B could nevertheless be obtained as the major product
however with low purity (79%, P NMR).
For the SPPS following route B, the prolonged coupling time at room temperature for 2 was already
applied and therefore byproduct 14 was not observed. Using 20% piperidine in DMF for Fmoc-
deprotection gave desired product 16 as the major product but led to significant amount of
piperidinyl peptide 23 and small quantities of elimination product 13 were observed as well. The
[
41]
formation of piperidinyl peptides is a known side reaction with phosphopeptides. Switching to the
optimized protocol (5% piperazine in DMF) prevented the occurrence of 23. Interestingly the P-OBn
group was cleaved with the same cleavage cocktail which was used to deprotect the tBu protecting
groups of the first Ser and Glu and to cleavage the peptide from the resin (Scheme 6). This optimized
protocol yielded 16 after a simple work-up step in very good purity (>95% P NMR) and as a single
stereoisomer.
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O
P
R
O
O
O
OH
O
N
H
O
SPPS
O
P
R
O
O
O
O
O
O
O
H
H
H
H
N
H N
2
N
N
N
N
N
H
N
N
O
Resin
H
H
H
O
O
O
O
O
O
O
TFA:TIS:H O
2
95:2.5:2.5
16
Scheme 6. Precursors 1 and 2 in SPPS (1 R=H, 2 R=Bn). Cleavage from the resin and deprotection of
the side chains and phosphonyl group in a one-pot reaction.
Neither side products 15 nor 17 could be found in any syntheses using 2. This can be explained by
the protection of the P-OH group and further supports our proposition of the side reaction
mechanism.
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Figure 4. HRMS and HRMS/MS show the fragmentation pattern of 16.
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+
HRMS measurement of 16 obtained by route B clearly shows the molecular ion peak at m/z [M+H]
+
8
74.3328 and the sodium adduct at m/z [M+Na] 896.3138 (Figure 4). The first fragment in the
MS/MS analysis shows the loss of methyl phosphonic acid while generating a β-elimination type
peptide fragment. The molecular ion further fragmentizes by subsequent loss of Ser, Ala, Ala, Gly
and Ala from the C-terminus on. The aged nonapeptide shows the same fragmentation pattern as
[
42]
non-aged nonapeptide nerve-agent adducts.
Conclusion
1
Two routes to synthesize the aged nonapeptide 16 using different protecting group strategies were
developed and investigated in detail. The synthesis for the precursor for route A consists of three
steps with an overall yield of 48%. The synthesis for the precursor of route B consists of four steps
with an overall yield of 42%. While both pathways have a similar yield in the final step, the route B
through protected phosphonyl serine delivers better purity. When possible the side products were
identified and mechanisms for their generation proposed. Several side reactions could be identified
when using the standard Fmoc-deprotection method for SPPS with 20% piperidine in DMF during the
deprotection step. An optimized protocol for the use of phosphylated serine in SPPS using piperazine
in the deprotection step was developed which takes into account the slower coupling to the peptide
chain as well as the thermo- and base lability of the phosphonyl group. The optimizations applied in
this work can also be used for the synthesis of other phosphonylated peptides which suffer from the
same labilities.
Experimental Part
All reactions, except the SPPS and synthesis of 2, were carried out under Ar atmosphere in dried
glassware. Chemicals were obtained from standard commercial sources like Sigma-Aldrich, Roth,
Alfa Aesar and Tokyo Chemical Industry Co. and were used without further purification. Amino acids
were obtained from Sigma Aldrich and Aroz Technologies. Resins for SPPS were obtained from Iris
Biotech, Sigma-Aldrich and Protein Technologies.
Solid-phase-peptide-synthesis was performed on an Alstra automated peptide synthesizer from
Biotage. Flash chromatography was done on an Isolera One coupled to a Dalton mass detector from
1
13
1
31
1
Biotage. H NMR (400 MHz), C NMR { H} (100 MHz) and P NMR { H} (162 MHz) were measured
with a Bruker Avance III HD 400 MHz Nano Bay in CDCl or D O. The chemical shifts (δ) are indicated
3
2
in ppm with respect to the internal standard TMS or the solvent peak. Coupling constants (J) are
indicated in Hertz (Hz) and the spins are indicated with letters s = singlet, bs = broad singlet, d =
doublet, t = triplet, q = quartet and m = multiplet. LC-MS (ESI) measurements were performed on an
1
Reference material 16 was used for the development of analytical methods at Spiez Laboratory to
detect the aged nonapeptide and led to the discovery of aged nonapeptide in sarin spiked blood
nd
samples during the 2 biomedical proficiency test conducted by OPCW.
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Agilent Technologies 1290 Infinity LC coupled to a Bruker Daltonics maXis plus UHR QTof system. The
column used in the LC was a Sigma-Aldrich Discovery HS C18 (150 mm x 2.1 mm, particle size 5 µm).
H O with 5mM NH Ac and MeOH with 5mM NH Ac were used as eluent with a flow rate of 0.6
2
4
4
mL/min. Melting points were measured on a Büchi 535. IR were measured on a Bruker Tensor II
equipped with a Golden Gate diamond ATR system.
Benzyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)serinate (Fmoc-Ser-OBn) 4. Fmoc-Ser-OH (5.5 g, 16.8
mmol, 1 equiv) was dissolved in 100 mL DCM and benzyl alcohol (8.7 mL, 84 mmol, 5 equiv) was
added. The solution was cooled to 0 °C. Thionyl chloride (1.8 mL, 25 mmol, 1.5 equiv) was added
dropwise and the solution was stirred for 1.5 h at 0 °C. Another portion of thionyl chloride (1.8 mL,
2
5 mmol, 1.5 equiv) was added to the reaction. The ice bath was renewed after the second addition.
Then the reaction was stirred over night while slowly warming up to room temperature (r.t.). The
reaction was cooled to 0 °C, quenched with sat. NaHCO solution (200 mL) and extracted with EtOAc
3
(
2x200 mL). The organic layer was washed with brine (3x200 mL) and dried over Na SO . After
2
4
filtration, the solvent was removed under reduced pressure. The crude product was purified by flash
chromatography (Isolera One, SNAP Ultra 100g, 1 column volume (CV) EtOAc : Hexane = 1 : 3,
gradient during 10 CV up to 100% EtOAc). The fractions containing the product were collected and
the solvent removed under reduced pressure. The crude mixture was dissolved in DCM (12.5 mL)
and precipitated in cold Hexane (600 mL). The solid was collected, dried under reduced pressure and
1
then lyophilized. 5.77 g (82% yield) of product was obtained as a white solid. H NMR (400 MHz,
CDCl , δ) 7.75 (d, 2H, J=8), 7.58 (d, 2H, J=8), 7.38-7.29 (m, 9H), 5.79 (d, 1H, J=8), 5.20 (s, 2H), 4.49-
3
1
3
1
4
1
5
.38 (m, 3H), 4.21-4.18 (m, 1H), 4.02-3.90 (dm, 2 H, J=18) C NMR { H} (100 MHz, CDCl , δ) 170.3,
3
56.2, 143.7, 141.3, 128.7,135.0 128.7, 128.5, 128.2, 127.8, 127.0,, 125.0, 120.0, 67.6, 67.2, 63.4,
+
6.1, 47.1. HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for C H NO 269.132477; Found 269.132503. FTIR
25
23
5
(neat): 3315, 3065, 3037, 2948, 2889, 2361, 2341, 1734, 1686, 1609, 1537, 1500, 1478, 1449, 1393,
1
5
339, 1254, 1217, 1193, 1104, 1084, 1045, 1030, 977, 907, 757, 135, 696, 647, 621, 604, 587, 571,
42, 523, 505, 482, 470, 464, 443 cm . Mp 97-98 °C.
-
1
Benzyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(hydroxy(methyl)phosphonyl)-serinate (Fmoc-
Ser(MPA)-OBn) 7. Methyl phosphonic dichloride (1.14 g, 4.3 mmol, 2 equiv, 0.6 mL) in DCM (52 mL)
was given in a three-neck round bottom flask equipped with a magnetic stirrer and a dropping
funnel. After cooling to 0 °C, a solution of Fmoc-Ser-OBn 4 (1.8 g, 4.3 mmol, 1 equiv), DABCO (0.96 g,
8
.6 mmol, 2 equiv) and DMAP (0.1 g, 0.86 mmol, 0.2 equiv) in DCM (45 mL) was added dropwise.
After the addition, the ice bath was removed and the reaction warmed to r.t. The reaction process
was monitored by TLC (EtOAc : Hexan = 1 : 1). After 2.5 h the mixture was filtered and the filtrate
concentrated. It was further purified by reverse phase flash chromatography (SNAP Ultra C-18 60 g,
MeCN : H O = 1 : 9, gradient up to 100% MeCN). The fractions containing the product were collected
2
and the solvent removed under reduced pressure. 1.44 g product (67% yield) was obtained as a
1
white solid. H NMR (400 MHz, CDCl , δ) 9.16 (bs, 1H), 7.74 (d, 2H, J=8), 7.57 (d, 2H, J=8), 7.38-7.26
3
(
m, 9H), 6.03 (dd, 1H, , J=64, J=8), 5.13-4.97 (m, 2H), 4.60-4.48 (m, 2H), 4.41-4.25 (m, 2H), 4.21-4.13
13
1
(m, 2H), 1.54-1.46 (m, 3H) C NMR { H} (100 MHz, CDCl , δ) 170.4, 155.9, 143.8, 141.3, 136.2, 135.6,
3
31
1
28.8, 128.8, 128.5, 127.9, 127.7, 127.0, 125.2, 119.9, 68.0, 67.3 54.3, 47.0, 11.7, 11.5, 11.0, 10.2 P
This article is protected by copyright. All rights reserved.

1
+
NMR { H} (162 MHz, CDCl , δ) 35.05 (87%), 34.27 (13%). HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for
3
C H NO P 496.151965; Found 496.151811. FTIR (neat): 3432, 3062, 3038, 2959, 2352, 1728, 1513,
26
26
7
1
8
506, 1450, 1392, 1338, 1313, 1250, 1211, 1192, 1167, 1081, 1057, 1033, 998, 935, 897, 878, 830,
13, 788, 758, 728, 696, 655, 645, 621 cm . Mp 112-114 °C.
-
1
N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-O-(hydroxy(methyl)phosphonyl)serine (Fmoc-Ser(MPA)-
OH) 1. Fmoc-Ser(MPA)-OBn 7 (1.5 g, 3 mmol, 1 equiv) was dissolved in THF (16 mL) and MeOH (16
mL) was added. Pd/C 10% (1.9 g, 18 mmol, 6 equiv) and 1,4-cyclohexadiene ( 4.2 mL,45 mmol, 15
equiv) were added and the mixture was stirred for 2 h under Ar. The reaction was monitored by TLC
(THF : MeOH = 2 : 1). Another portion of 1,4-cyclohexadiene (3.4 mL, 40 mmol, 10 equiv) was added
after 2h. After another hour of stirring, the reaction mixture was filtered through Celite and the
Celite was rinsed with MeOH (100 mL). The solvent was removed under reduced pressure and then
lyophilized. The product was used without further purification. 1.07 g (88 %) product was obtained.
To obtain better spectral data the product was further purified by flash (SNAP Ultra 100 g, 100%
DCM (1CV), to 5% MeOH (1CV), to 30% MeOH (1CV), to 50% MeOH (10CV)). The fractions containing
the product were collected and the solvent removed under reduced pressure. 0.59 mg (48%)
1
product was obtained after purification as a white foam. H NMR (400 MHz, CDCl , δ) 10.34 (bs, 1H),
3
7
.77-7.69 (m, 2H), 7.57-7.7.45 (m, 2H), 7.38-7.25 (m, 4H), 6.20 (s, 1H), 4.70-4.14 (m, 5H), 1.59-1.27
13
1
(m, 3H) C NMR { H} (100 MHz, CDCl , δ) 172.6, 156.2, 143.8, 141.3, 127.8, 127.1, 125.2, 119.9, 67.5,
3
31
1
6
5.5, 54.6, 46.9,11.8, 10.3 P-NMR { H} (162 MHz, δ) 34.17 (87%), 32.57 (13%). HRMS (ESI/Q-TOF)
+
m/z: [M+H] Calcd for C H NO P 406.105015; Found 406.105056. FTIR (neat): 3306, 3063, 2929,
19
20
7
2
7
359, 2341, 1714, 1518, 1449, 1337, 1314, 1247, 1209, 1193, 1079, 1056, 1031, 985, 902, 821, 775,
58, 737, 697, 668, 645, 620 cm . Mp 57-58 °C.
-
1
Dibenzyl methyl phosphonate (MPDBn) 9. Methyl phosphonic dichloride (10 g, 71.8 mmol, 1 equiv)
and benzene (12 mL) were mixed and poured in a dry 250 mL three-neck round-bottom flask
immersed in an ice bath which was equipped with a dropping funnel. In an Erlenmeyer flask, DMAP
(19.3 g, 158 mmol, 2 equiv), BnOH (16.4 mL, 158 mmol, 2 equiv) and benzene (98 mL) were mixed.
The mixture was added dropwise to the MPDC solution at 0 °C. After the addition the ice bath was
removed and the reaction was stirred at r.t. for 1h. Hexane (50 mL) was added and stirring was
continued for 1h. The product was separated by suction filtration through Al O which was
2
3
thoroughly washed with hexane and the solvent was removed under reduced pressure. 14.8 g (71%
1
yield) was obtained. H NMR (400 MHz, CDCl , δ) 7.37-7.34 (m, 10H), 5.08-4.94 (dm, 4H, J=40), 1.47
3
13
1
31
1
(
d, 3H, J=16) C NMR { H} (100 MHz, CDCl , δ) 136.4, 129.7-127.0, 67.1, 12.4, 11.0 P NMR { H} (162
MHz, CDCl , δ) 31.74 (99%). HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for C H O P 277.098807; Found
3
+
3
15 17
3
2
1
5
77.098585. FTIR (neat): 3037, 3032, 2953, 2926, 2890, 2359, 2325, 1716, 1700, 1650, 1606, 1568,
497, 1455, 1418, 1379, 1311, 1237, 1213, 1081, 1035, 993, 918, 897, 828, 729, 695, 649, 638, 592,
23, 507, 494, 475, 467, 462, 440, 432 413 cm .
-
1
This article is protected by copyright. All rights reserved.

Benzyl hydrogen phosphonate (MPBnA) 10. MPDBn 9 (1 g, 3.62 mmol, 1 equiv) was given in toluene
12.5 mL) under Ar and DABCO (400 mg, 3.62 mmol, 1 equiv) was added. The mixture was stirred
(
under reflux for 2 d. The solvent was evaporated, the residue retaken in 5% HCl (40 mL) and
extracted with EtOAc (2x 40 mL). The organic phases were collected and the solvent removed under
1
reduced pressure. 640 mg (94% yield) product was obtained as a yellowish oil. H NMR (400 MHz,
1
3
1
CDCl , δ) 7.39-7.32 (m, 5H), 5.04 (d, 2H, J=4), 1.52 (d, 3H, J=20) C NMR { H} (100MHz, CDCl , δ)
3
3
31
1
1
36.2, 128.6, 128.4, 127.8, 127.7, 127.0, 66.5, 12.6, 11.2 P NMR { H} (162 MHz, CDCl , δ) 34.65
98%). HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for C H O P 187.051857; Found 187.051974. FTIR
3
+
(
8
11
3
(
9
neat): 3091, 3065, 3033, 2891, 2659, 2357, 2326, 1738, 1498, 1417, 1381, 1363, 1313, 1213, 1081,
95, 904, 824, 734, 698, 631 cm .
-
1
tert-Butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((benzyloxy)(methyl)phosphonyl) serinate
Fmoc-Ser(MPBn)-OtBu) 11. To a solution of Fmoc-Ser-OtBu (1 g, 2.6 mmol, 1 equiv) was added
MPBnA 10 (0.48 g, 3 mmol, 1 equiv) and PyBoB (1.63 g, 3.2 mmol, 1.2 equiv) in DCM (16 mL). DIPEA
1.8 mL, 1.35 g, 10.4 mmol, 4 equiv) was added slowly while the reaction mixture was immersed in
(
(
an ice bath. The reaction was stirred at r.t. for 24 h. The solvent was removed under reduced
pressure and the crude further purified by flash chromatography (SNAP Ultra 100 g, EtOAc : Hexane,
gradient from 10% EtOAc up to 100% EtOAc). The fractions containing the product were collected
and the solvent removed under reduced pressure. 903 mg (63% yield) of product was obtained as a
1
yellow oil. H NMR (400 MHz, CDCl , δ) 7.76 (d, 2H, J=8), 7.61 (d, 2H, J=8), 7.36-7.29 (m, 9H), 5.87
3
(
dd, 1H, J=8, J=40), 5.09-4.94 (m, 2H), 4.45-4.35 (m, 2H), 4.23-4.22 (m, 1H), 4.13-4.11 (m, 2H), 1.50-
13
1
1
1
{
.46 (m, 12H) C NMR { H} (100 MHz, CDCl , δ) 171.1, 155.8, 143.8, 141.2, 136.0, 128.4, 128.6,
3
31
28.6, 127.9, 127.1, 125.1, 119.9, 83.1, 67.4, 67.1, 65.5, 54.8, 47.0, 27.9, 12.0, 10.5 (d, J=48) P NMR
1
+
H} (162 MHz, CDCl , δ) 32.27 (39%), 32.18 (51%). HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for
3
C H NO P 552.214566; Found 552.214766. FTIR (neat): 3270, 3065, 3037, 2979, 2951, 2896, 2358,
30
34
7
2
7
337, 1720, 1537, 1478, 1451, 1393, 1369, 1343, 1311, 1240, 1155, 1081, 1047, 999, 920, 843, 786,
60, 740, 697, 631, 622 cm .
-1
N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-O-((benzyloxy)(methyl)phosphonyl)serine (Fmoc-
Ser(MPBn)-OH) 2. Fmoc-Ser(MPBn)-OtBu 11 (1.5 g, 2.7 mmol, 1 equiv) was added to a 5% TFA
solution in DCM (125 mL) and stirred for 48 hours. The solvent was removed under reduced
pressure. The crude mixture was diluted with DCM (3 mL) and further purified by flash
chromatography (SNAP Ultra C-18, 60 g, MeCN : H O, gradient from 12%-100% MeCN). The fractions
2
containing the product were collected and the solvent removed under reduced pressure. 1.33 g
(99%) product was obtained as a light brownish solid.
1
H NMR (400 MHz, CDCl , δ) 10.48 (bs, 1H), 7.72 (d, 2H, J=8), 7.57 (d, 2H, J=8), 7.36-7.25 (m, 9H),
3
6
1
1
.07 (dt, 1H, J=8, J=40), 5.09-5.00 (m, 2H), 4.56-4.52 (m, 2H), 4.39-4.27 (m, 2H), 4.19-4.16 (m, 1H),
13
1
.49 (d, 3H, J=16) C NMR { H} (100 MHz, δ) 168.8, 154.2, 141.9, 139.4, 133.7, 126.9, 126.9, 126.3,
31
1
26.2, 125.9, 123.3, 118.1, 66.2, 66.2, 65.9, 52.5, 45.2, 9.7, 8.2 P NMR { H} (162 MHz, CDCl , δ)
3
This article is protected by copyright. All rights reserved.

+
3
4
1
3.57 (54%), 41.01 (41%). HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for C H NO P 496.151965; Found
26
26
7
96.151628. FTIR (neat): 3413, 3316, 3064, 3036, 2953, 2927, 2895, 2539, 2359, 2341, 1715, 1609,
516, 1478, 1450, 1414, 1381, 1313, 1294, 1194, 1079, 997, 918, 830, 759, 736, 696, 645, 621 cm .
-
1
Mp 50-52 °C.
Screening of the tBu group hydrolysis
5
mL of the cleavage cocktail was added to Fmoc-Ser-OtBu (0.1 g, 0.26 mmol, 1 equiv) and the time
until full conversion was monitored by mass spectrometry (direct injection in the Dalton mass
detector).
SPPS general procedure
The synthesis was performed on an Alstra automated peptide synthesizer from Biotage. For 0.01
mmol scale the 5 mL reactor vial was used, for 0.1-0.25 mmol scale the 10 mmol vial and for 1.2
mmol scale the 30 mL reactor vial was used. Amino acids and coupling agents were used in fourfold
excess for the synthesis of the unlabeled nonapeptide and fivefold excess for the phosphonylated
peptides. Their stock solutions were prepared with 0.5 M concentration in DMF. Phosphonylated
serines were used in five-and-a-half-fold excess and in 0.5 M solutions in DMF. DIC and Oxyma were
used as coupling agents in fourfold or fivefold excess respectively. The following protected amino
acids were used: Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-
Phe-OH. Preloaded Ser(OtBu)-HMPB or Ser(OtBu)-2-CT resin was swollen in DMC at r.t. for 60 min.
The couplings were performed at r.t. for 60 min (120 min for phosphonylated serines), followed by
four washing steps with DMF. The deprotection step was done twice (3 min and 15 min) with 5%
piperazine in DMF at r.t. followed by four washing steps with DMF. At the end of the synthesis the
resin was washed ten times (6xDCM, then MeOH, DCM, MeOH, DCM). The peptide was cleaved from
the resin and deprotected using TFA:TIS:H O=95:2.5:2.5 as cleavage cocktail for 1h. The resin was
2
filtered off and washed twice with TFA. The filtrate was concentrated and precipitated in ice cold
diethyl ether. After centrifugation and decantation, another portion of cold diethyl ether was added.
The centrifuge tube was agitated to wash the peptide and centrifuged again. This last washing step
was repeated a total of three times. After decantation, MeCN (1 mL) was added to the residue and it
was agitated. The peptide was solved in H O (1 mL) and lyophilized.
2
This article is protected by copyright. All rights reserved.

Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser 12. The synthesis was performed according to the general
procedure for SPPS. Fmoc-Ser(OtBu) was used. The scale was 1.2 mmol and the 30 mL reactor vial
was used. Ser(OtBu)-2-CT-resin (1.76 g, 1.2 mmol, 0.68 mmol/g loading, Iris Biotech) was used. 20%
piperidine instead of 5% piperazine was used in the deprotection step. Fourfold excess of amino
1
acids and coupling agents were used. 1 g (99%) product was obtained as a white fluffy solid. H NMR
(
400 MHz, D O, δ) 7.34-7.22 (m, 5H), 4.38-4.21 (m, 6H), 3.94-3.77 (m, 8H), 3.62 (S, 2H), 3.12-3.09 (m,
2
13
1
2
1
4
7
1
1
H), 2.42-2.36 (m, 2H), 1.94-1.88 (dm, 2H, J=48), 1.33-1.31 (m, 9H) C NMR { H} (100 MHz, D O, δ)
2
77.0, 175.4, 174.8-170.7, 169.7, 133.7, 129.3, 128.0, 61.0, 60.6, 54.7, 54.4, 52.9, 49.9, 49.5, 49.5,
+
2.3, 42.2, 36.7, 29.9, 26.1, 16.9-16.3. HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for C H N O
33
49
9
14
96.347174; Found 796.347951. FTIR (neat): 3284, 3070, 2933, 2636, 1696, 1661, 1622, 1518, 1450,
-
1
416, 1339, 1312, 1195, 1059, 1032, 1000, 981, 904, 834, 798, 758, 738, 726, 698, 642, 621 cm . Mp
72 °C dec.
Phe-Gly-Glu-Ser-O-(hydroxy(methyl)phosphonyl)-Ala-Gly-Ala-Ala-Ser synthesized by route A 16.
The synthesis was performed according to the general procedure for SPPS. Ser(OtBu)-HMPB-resin
(
222 mg, 0.3-0.6 mmol/g loading, Sigma-Aldrich) and Fmoc-Ser(MPA)-OH 1 (223 mg, 0.55 mmol, 5.5
equiv) were used. The scale of the synthesis was 0.1 mmol and the 10 mL reactor vial was used. 47
1
mg product was obtained as a white fluffy solid. H NMR (400 MHz, D O, δ) 7.34-.7.22 (m, 5H), 4.45-
2
4
1
.21 (m, 7H), 4.08-3.80 (m, 9H), 3.50 (s, 2H), 3.18-3.12 (m, 2H), 2.18-1.80 (m, 2H), 1.34-1.32 (m, 9H),
31
1
+
.21 (d, 3H, J=20) P NMR { H} (162 MHz, D O, δ) 27.78 (76%). HRMS (ESI/Q-TOF) m/z: [M+H] Calcd
2
for C H N O P 874.334240; Found 874.3337.
34
52
9
16
Phe-Gly-Glu-Ser-O-(hydroxy(methyl)phosphonyl)-Ala-Gly-Ala-Ala-Ser synthesized by route B 16.
The synthesis was performed according to the general procedure for SPPS. Ser(OtBu)-HMPB-resin
(223 mg, 0.3-0.6 mmol/g loading, Sigma-Aldrich) and Fmoc-Ser(MPBn)-OH 2 (273 mg, 0.55 mmol, 5.5
equiv) were used. The scale of the synthesis was 0.1 mmol and the 10 mL reactor vial was used. 43.4
1
mg product was obtained as a white fluffy solid. H NMR (400 MHz, D O, δ) 7.37-7.22 (m, 5H), 4.46-
2
4
1
1
4
.21 (m, 7H), 4.08-3.78 (m, 7H), 3.22-3.08 (m, 2H), 2.44-2.40 (m, 2H), 2.10-1.90 (dm, 2H, J=60), 1.34-
13
1
.31 (m, 9H), 1.20 (d, 3H, J=16) C NMR { H} (100 MHz, D O, δ) 177.0, 175.4, 174.8, 174.7, 173.5,
2
73.2, 171.2, 171.0, 170.9, 169.7, 133.7, 129.4, 128.0, 69.6, 62.8, 61.0, 54.8, 54.4, 53.1, 50.2, 49.8,
31
1
9.5, 42.4, 42.2, 36.8, 29.9, 25.9, 16.9-16.3, 11.6, 10.3 P NMR { H} (162MHz, D O, δ) 27.66 (95.5%).
2
+
HRMS (ESI/Q-TOF) m/z: [M+H] Calcd for C34H52N9O16P 874.334240; Found 874.332770. FTIR
(neat): 3427, 326, 3074, 3947, 2450, 2287, 2163, 1728, 1714, 1697, 1682, 1651, 1623, 1520, 1504,
1
7
452, 1417, 1394, 1343, 1311, 1255, 1198, 1183, 1089, 1060, 1032, 1005, 966, 929, 885, 834, 813,
95, 760, 74, 728, 699, 661, 622, 618 cm . Mp 161-164 °C dec.
-
1
Associated content
1
13
31
H-NMR, C-NMR, P-NMR, IR and MS spectra, as well as chromatograms are located in the
supporting information.
This article is protected by copyright. All rights reserved.

Author information
Corresponding Author
*
E-mail: christian.bochet@unifr.ch
Notes
The authors declare no competing financial interests.
Acknowledgements
We thank the Swiss Federal Office for Civil Protection FOCP for funding this project. Special thanks
go to Dr. Martin Schär for measuring all the MS samples.
th
We dedicate this paper to the 20 anniversary of OPCW.
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