Solid-phase synthesis for novel nerve agent adducted nonapeptides as biomarkers

Article abstract of DOI:10.1016/j.tetlet.2017.02.017

An efficient synthesis of d₅-VX adducted nonapeptide and d₁₅-GD adducted nonapeptide via solid-phase approach has been developed. The deuterated peptides could be used as the isotope-labeled internal standard for LC-MS/MS detecting the BuChE-OPNA biomarkers. This method also offers an access to the synthesis and detection of other phosphorylated nonapeptides.

Full text of DOI:10.1016/j.tetlet.2017.02.017

Accepted Manuscript
Solid-Phase Synthesis for Novel Nerve Agent Adducted Nonapeptides as Bio-
markers
Xinhai Li, Ling Yuan, Qinggang Wang, Longhui Liang, Guilan Huang, Xiaosen
Li, Chunhong Zhang, Shilei Liu, Jingquan Liu
PII:
S0040-4039(17)30181-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.02.017
TETL 48628
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 December 2016
5 February 2017
7 February 2017
Please cite this article as: Li, X., Yuan, L., Wang, Q., Liang, L., Huang, G., Li, X., Zhang, C., Liu, S., Liu, J., Solid-
Phase Synthesis for Novel Nerve Agent Adducted Nonapeptides as Biomarkers, Tetrahedron Letters (2017), doi:
http://dx.doi.org/10.1016/j.tetlet.2017.02.017
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The deuterated nonapeptides synthesized could be used as the internal standard
for LC-MS/MS isotope-dilution detection for OPNAs exposure.
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Xinhai Lia, b , Ling Yuana, b, Qinggang Wang c , Longhui Liang a, b , Guilan Huang a, b , Chunhong Zhanga, b
,
Xiaosen Li a, b , Shilei Liua, b,  and Jingquan Liua, b, 

1
Tetrahedron Letters
Solid-Phase Synthesis for Novel Nerve Agent Adducted Nonapeptides as
Biomarkers
Xinhai Lia, b , Ling Yuan a, b, Qinggang Wang c , Longhui Liang a, b , Guilan Huang a, b , Xiaosen Li a, b , Chunhong Zhang
a, b , Shilei Liua, b,  and Jingquan Liu a, b, 
a
State key Laboratory of NBC Protection for Civilian, Beijing 102205, China.
Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing, 102205, China.
Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, 266101
b
c
Qingdao, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
An efficient synthesis of d5-VX adducted nonapeptide and d15-GD adducted nonapeptide
via solid-phase approach has been developed. The deuterated peptides could be used as the
isotope-labeled internal standard for LC-MS/MS detecting the BuChE-OPNA biomarkers.
This method also offers an access to the synthesis and detection of other phosphorylated
nonapeptides.
Available online
Keywords:
Keyword_1 d5-VX
.
2016 Elsevier Ltd. All rights reserved.
Keyword_2 d15-GD
Keyword_3 nonapeptide
Keyword_4 solid-phase
Keyword_5 BuChE-OPNA biomarkers
———
 Corresponding author. Tel.: + (86)-10-6675-8062; fax: +(86)-10-6976-5318; e-mail: liushilei402@263.net
 Corresponding author. Tel.: + (86)-10-6675-8062; fax: +(86)-10-6976-5318; e-mail: jingquan@lacricd.com

2
Tetrahedron
1. Introduction
of the isotope-labeled internal standard nonapeptides. Herein, we
envisioned to synthesize brand new d5-VX and d15-GD
nonapeptides with unique alkoxy moieties (Figure 1) which could
be used as the isotope-labeled internal standard of BuChE-
OPNAs biomarkers via LC-MS/MS detecting.
Organophosphates (OP compounds) with anti-cholinesterase
activity have been widely used as pesticides and incidentally as
chemical warfare agents. Among the OP compounds,
organophosphorus nerve agents (OPNAs) are a series of
alkylphosphonic esters compound with signature phosphorus-
carbon bond which could inhibit the enzymatic activity of
acetylcholin esterase (AChE)1 upon even low levels of exposure.
As is known, The AChE is a neurotransmitter responsible for the
conduction of nerve impulses at cholinergic synapses, which
plays an important role in the hydrolysis of acetylcholine in
vivo. Acute toxic effects of OPNAs exposure are due to AChE
inhibition caused by phosphylation of a serine residue in the
active site of the enzyme which will result in uncontrolled
increase of acetylcholine that overstimulates cholinergic
receptors. This will cause muscle twitching, miosis, convulsions,
respiratory arrest, seizures, and even death. 2 Because of the high
toxicity and proliferation in terrorist attacks and warfare, OPNAs
is still one of the most potential threats to human life and modern
societies, e.g. sarin or its similar compounds attack in Tokyo,
Matsumoto and Syrian.3 Therefore, new rapid, sensitive, and
reliable methods are urgently needed for retrospective detection
of exposure to and handling of these agents.
O
P
F
O
O
P
F
iPr
iPr
N
Et
P
Me
O
O
S
Me
O
tBu
iPr
Me
GB
GD
VX
FGESAGAAS
FGESAGAAS
FGESAGAAS
CD3
P
P
P
Me
OR
Me
O
Me
OC2D5
O
O
O
CD3
R = Et, iPr, pinacolyl
BuChE-OPNAs
others?
D3C
CD3
d5 -VX nonapeptide d15 -GD nonapeptide
this work this work
Figure 1
uctures of prominent organophosphorus nerve
agents, their nonapeptides derived from the pepsin digestion of
the BChE-OPNA adducts, the isotope-labeled internal standard
of d5-VX nonapeptide and d15-GD nonapetide.
Basically, two approaches have been explored to detect
exposure to OPNAs involving mass spectrometry technology.
The conventional method developed from 1990s is based on
measurement of the OPNAs hydrolysis products in vivo, e.g., O-
alkyl methylphosphonicacids. Methods for analysis of these
compounds are based on GC-MS or on LC-MS4. However, the
serious shortcoming of these method is that the hydrolysis
products can’t be detained in vivo for a relatively long life-time.
Actually, it is estimated that ∼90% of the compounds will be
excreted in urinary within 2-3 days which increase the difficulty
in the analysis of the trace exposure to OPNAs5. In contrast, the
techniques used nowadays relying on enzymatic digestion of the
adducts combined by cholinesterase and OPNAs followed by
LC-MS/MS analysis provide better limits of detection, greater
specificity, and afford positive identification of the OPNAs. As a
kind of the cholinesterase and stoichiometric bioscavenger of
OPNAs, the butyrylcholine esterase (BuChE) which is readily
available in serum could form a relatively stable covalent adduct
combined with OPNAs has a lifetime for at least 16 days6 and
mainly exists in blood. Thus, the advantage of the BuChE
aforementioned make it an ideal biomarker for monitoring
OPNAs exposure.
2. Synthesis procedures for the d5-VX nonapeptide and d15-
GD nonapetide
The synthesis procedures of the d5-VX adducted nonapeptide
and d15-GD adducted nonapeptide are presented in Figure 2 and 3，
respectively. As the starting material to synthesize the d5-VX
adducted nonapeptide, dimethyl methylphosphonate was treated
with 2.5 equivalent sulfoxide chloride at 140 ºC for 6 hours,
affording methylphosphonic dichloride 28 in the 90% yield.
Subsequent monoesterfication with d6-Ethanol and triethylamine
lead to d5-Ethyl methylphosphonic monocloridate 3 in 36%
yield9. The chemical 3 must be purified by carefully distillation
under vacuum to prevent the undesired polymerization. d5-Fmoc-
serine(O-ethyl methylphosphonate) benzyl ester 410 was obtained
in the 60% yield by reacting with the protected serine 6 in the
presence of DMAP and triethylamine. Four equivalent of the
intermediate 3 was used during the esterfication because of the
low reactivity. Finally, target molecule 5, which is the important
precursor before Solid-Phase synthesis, was formed by the Pd/C
hydrogenation of 410 with 80% yield.
While the synthetic procedures of the d15-GD adducted
nonapeptide started from phosphorus trichloride. Phosphorus
trichloride was treated with three equivalent d4-methanol in -15
oC to afford d9-trimethyl phosphite in 40% yield. d9-Dimethyl
methylphosphonate was obtained in 95% yield via the subsequent
Arbuzov rearrangement reaction triggered by adding 1% d3-
iodomethane11. d9-Dimethyl methylphosphonate was treated with
exess equivalent sulfoxide chloride at 140 ºC for 6 hours,
affording d3-methylphosphonic dichloride 11 in 90% yield8.
Intermediate 13 was obtained in the 50% yield via bi-molecular
reduction of d6-acetone in amalgam formed by mercuric chloride
and magnesium turnings12. Subsequent Pinacol rearrangement
reaction with 3 mol/L sulfuric acid lead to d12-3,3-dimethyl-2-
butanone 14 in 60% yield13. The chemical 15 was formed in 80%
yield by potassium borohydride reduction. Subsequent
monoesterfication with intermediate and triethylamine lead to
d15-Pinacolyl methylphosphonic monocloridate 16 in 60%
yield14. d15-Fmoc-serine(O-pinacolyl methylphosphonate) benzyl
ester 1710 was obtained in the 60% yield by reacting with the
protected serine 6 in the presence of DMAP and triethylamine.
Also four equivalent of the intermediate 16 was used during the
With the US military codes GB, GD and VX respectively,
there are three predominate compounds among the OPNAs
presented as follows: O-isopropyl methylphosphonofluridate, O-
pinacolyl methylphosphonofluridate and O-ethyl, S-2-
diisopropyl-amino ethyl methylphosphonothiolate. The structure
of these three compounds are shown in Figure 1. As the
biomarkers formed by OPNAs and BuChE can be converted to
the phosphorylated nonapeptide7 compounds with similar
structures through the pepsin digestion (Figure 1), only the
obvious structural difference are shown between the peptides
originated from VX and GD in alkoxy moieties.
In recent years, the developed method for the verification of
the OPNAs exposure was based on high performance liquid
chromatography isotope dilution tandem mass spectrometry
quantitative detection of the phosphorylated nonapeptide.
Although the analysis of the nonapeptides biomarkers in blood
samples plays an important role in forensic investigations of the
alleged use of nerve agents currently, to the best of our
knowledge, there is no literature reporting the synthesis method

3
esterfication because of the low reactivity. Finally, target
molecule 18, which is the important precursor before Solid-Phase
synthesis, was formed by the Pd/C hydrogenation of 1710 with
80% yield .
Fmoc protection of other residues without side-chain protection.
Cleavage from the resin was achieved by treating the peptide
resin with 1% TFA–CH2Cl2 containing 2% triisopropylsilane
overnight at room temperature. The crude product of the
phosphonylated peptide which could be precipitated by
anhydrous ether, was further purified by reversed-phase HPLC
on a C18 column (0.05% TFA–water–2% acetonitrile). Peptide
purity was >90% and was verified by analysis of HPLC-DAD,
NMR and high resolution mass spectrometry.
O
P
O
P
O
P
b
a
OMe
OC2D5
O
Cl
OMe
Cl
Cl
2
3
1
P
OC2D5
OBn
O
O
OH
g
f
h
D C P OCD
3
D C P Cl
3
PCl
P(OCD )
3
O
3
3 3
c
Cl
OCD
3
OBn
3
8
9
10
11
FmocHN
O
FmocHN
O
4
6
D C
3
O
D C
3
OH
CD
HO
OH
CD
O
O
k
i
j
D C
D C
D C
3
3
3
3
P
OC2D5
D C
3
CD
3
D C
3
CD
D C
3
D C
CD
3
3
3
3
d
FGESAGAAS
O
e
12
13
14
15
P
O
7
OC2D5
OH
CD
3
O
P
CD
3
FmocHN
D C
O
CD
3
3
O
P
O
CD
3
CD
l
3
m
3
O
5
CD
D C
O
11 15
3
CD
3
(a) SOCl2 (2.5 equiv.), 140 oC, 90% yield; (b) C2D5OD (1 eqiuv.),
Et3N, bezene, 36% yield; (c) reagent 6 (0.25 equiv.), Et3N, DMAP,
bezene, 0 C, 75% yield; (d) H2, 10% Pd/C, THF/MeOH, 80% yield;
Cl
CD
OBn
3
FmocHN
O
17
16
o
D C
3
(e) manual Solid-phase peptide synthesis procedure
O
FGESAGAAS
CD
3
D C
3
P
O
CD
3
P
o
CD
3
n
3
Figure 2 Synthesis route of d5-Vx nonapeptide
D C
3
O
O
CD
O
CD
3
Solid-phase peptide synthesis was done manually from C-
D C
3
CD
3
OH
FmocHN
terminal to N-terminal to enhance the
. The
19
-Soman
18
O
d
15
d5-VX adducted nonapeptide was synthesized using a universal
method for peptides preparation on a Fmoc-Ser (tBu)-Wang resin
and HBTU/DIPEA activation. Compared with the method
reported before10, it takes less time for the resin elution to obtain
the target peptide and the method could be used in practical
synthesis. Protected amino acids used in the peptide synthesis
included Fmoc-glutamic acid with the tert-butyl ester on the
sidechain carboxylic acid and Fmoc protection of the other
residues without side-chain protection. Piperidine/DMF (25%)
was used to de-protect the Fmoc group. The rest of the amino
acids residues were induced onto the resin in sequence by
HBTU/DIPEA activation. Kaiser test was used to ensure the
connection with right amino acid residue upon every step. After
this point, cleavage from the resin was achieved by treating the
peptide resin with the solution containing 68.5% TFA, 10% 1,2-
ethanedithiol, 10% thioanisole, 5% phenol, 3.5% double-distilled
water and 1% triisopropylsilane for 3 h at room temperature. The
crude product of the phosphonylated peptide which could be
precipitated by anhydrous ether, was further purified by reversed-
phase HPLC on a C18 column (0.05% TFA–water–2%
acetonitrile). Peptide purity was >90% and was verified by
HPLC-DAD, NMR and high resolution mass spectrometry
analysis.
nonapeptide
(f) CD3OD (1 equiv.), -15 oC, 40% yield; (g) CD3I(1% eqiuv.) , 50 oC,
95% yield; (h) SOCl2 (2.5 equiv.), 140 oC, 90% yield; (i) magnesium
amalgam, 50~60 oC, 50% yield; (j) 3 mol/L sulfuric acid, 107 oC , 60%
yield; (k) potassium borohydride (1 equiv.), 80 oC, 80% yield; (l) reagent
15 (1 equiv.), Et3N, bezene, 60% yield; (m) reagent 6 (0.25 equiv.), Et3N,
DMAP, bezene, 0 oC, 75% yield; (n) H2, 10% Pd/C, THF/MeOH, 80%
yield; (o) manual Solid-phase peptide synthesis procedure
Figure 3 Synthesis route of d15-GD nonapeptide
Phe
Gly
Glu
Ser
Ala
Gly
Ala
Ala
Ser
OH
CO H
CO H
2
Bn
O
O
Me
O
Me
O
H
H
H
H
N
N
N
N
H N
N
H
N
N
N
2
2
H
H
H
Me
O
O
O
O
O
P
Me
O
OC D
2 5
Figure 4 Structures of the d5-VX adducted nonapeptide and fragmentation
sites denoted with dashed lines.
Phe
Gly
Glu
Ser
Ala
Gly
Ala
Ala
Ser
CO2H
OH
Bn
O
O
Me
O
Me
O
H
H
N
H
N
H
N
N
H2N
N
H
N
H
N
N
CO2H
H
H
O
O
O
O
Me
However, the d15-GD adducted nonapeptide was failed to be
obtained via the Solid-phase peptide synthesis procedure for the
d5-VX adducted nonapeptide. Maybe the Pinacolyl moiety is
susceptible to elimination or readily to leave in 68.5% TFA and
this phenomenon would be in accord with the study that the GD-
BuChE adduct is quite unstable in vivo and easy to lose the
pinacolyl moiety which is called “aging” process6a. Alternatively,
we employed the reaction condition containing 2-chlorotrityl
resin and 1% TFA instead10.Protected amino acids used in the
peptide synthesis included Fmoc-glutamic acid with the
isopropyl phenyl ester on the side chain carboxylic acid and
O
P
CD3
CD3
O
O
Me
CD3
D3C
Figure 5 Structures of the d15-GD adducted nonapeptide and fragmentation
sites are denoted with dashed lines.

4
Tetrahedron
alkyl phosphonic momochloridate to substitute for the isotope
3. The mass spectrometry fragment pathway of the d5-VX
labeling peptides to synthesize the target nonapeptides. They can
be used as brand new internal standards for the retrospective
analysis after the OPNAs exposure. This method may also find
applications in the synthesis and detection of other
phosphorylated nonapeptides.
nonapeptide and d15-GD nonapetide
The fragmentation of the d5-VX and d15-GD adducted
nonapeptides (Figure 4 and Figure 5) was observed by Precursor-
to-Product Ion Transitions Monitored using Multiple Reaction
Monitoring (MRM). Interestingly, The mass spectrometry
fragment pathways of the d5-VX and d15-GD nonapetide were
identified with the isotopically enriched nonapeptides biomarker
in the similar structure7c. The protonated, peptide-adduct
fragments show losses of the adducted nerve agent, the C-
terminal serine and the C-terminal alanine. It showed the same
fragmentation pattern as the d5-VX-BuChE and d15-GD-BuChE
nonapeptide7c. As is showed in the Figure 6 and 7, the precursor
ion for the chemical 7 (m/z 907.3968) exhibited product ions
derived from a loss of 129 Da that corresponded to the loss of the
d5-VX moiety (m/z 778.3364); further losses of the terminal
serine (m/z 673.2932), and the alanine (m/z 602.2554) residue
were also exhibited. While the precursor ion for the chemical 19
(m/z 973.5225) exhibited product ions derived from a loss of 195
Da that corresponds to the loss of the d15-GD moiety (m/z
778.3371); further losses of the terminal serine (m/z 673.2937),
and the alanine (m/z 602.2562) residue were also exhibited.
Additionally, we find the dehydration impurities for d15-GD
adducted nonapeptides. As is metioned in reference10, the
compound generated via the elimination of OP-serine and
addition of piperidine process was found ( calcd for C38H58N10O13:
863.4258 ).
Acknowledgments
The work was supported by the State Key Laboratory of NBC
Protection for Civilian (grant no. SKLNBC2014-06).
References and notes
1.
(a) John H.; Worek F. Anal. Bioanal. Chem. 2008, 391, 97-116.
(b) Fidder, A.; Hulst, A. G.; Noort, D.; de Ruiter, R.; van der
Schans, M. J.; Benschop, H. P.; Langenberg, J. P. Chem. Res.
Toxicol. 2002, 15, 582−583.
2.
3.
Abney. Carter W.; Knaack. L. S. Jennifer; Ali. I. Ahmed A.;
Johnson. C. Rudolph. Chem. Res. Toxicol. 2013, 26, 775-782.
(a) Morita, H.; Yanagisawa, N.; Nakajima, T.; Shimizu, M.;
Hirabayashi, H.; Okudera, H.; Nohara, M.; Midorikawa, Y.;
Mimura, S. Lancet. 1995, 346, 290−293. (b) Nakajima, T.; Ohta,
S.; Morita, H.; Midorikawa, Y.; Mimura, S.; Yanagisawa, N. J.
Epidemiol. 1998, 8, 33−34. (c) Suzuki, T.; Morita, H.; Ono, K.;
Maekawa, K.; Nagai, R.; Yazaki, Y. Lancet. 1995, 345, 980-981.
(d) Yan Long, Chen Jia, Xu Bin, Guo Lei, Xie Yan, Tang Jijun, ,
Xie Jianwei. J. Chromatogr. A. 2016, 1450, 86-93.
4.
(a) Shih, M. L.; Smith, J. R.; McMonagle, J. D.; Dolzine, T. W.;
Gresham, V. C. Biol. Mass Spectrom. 1991, 20, 717-723. (b)
Black, R. M.; Clarke, R. J.; Read, R. W.; Reid, M. T. J. J.
Chromatogr. A. 1994, 662, 301-321. (c) Tørnes, J. A. Rapid.
Commun. Mass Spectrom. 1996, 10, 878-882. (c) Black, R. M.;
Read, R. W. J. Chromatogr. A. 1997, 759, 79-92. (d) Black, R. M.;
Read, R. W. J. Chromatogr. A. 1998, 794, 233-244. (d) Noort, D.;
Hulst, A. G.; Platenburg, D. H. J. M.; Polhuijs, M.; Benschop, H.
P. Arch. Toxicol. 1998, 72, 671-675.
5.
6.
Riches. J.; Morton. I.; Read. R. W.; Black. R. M. J. Chromatogr.
B. 2005, 816, 251-258.
(a) Bao Yi; Liu Qin; Chen Jia; Lin Ying; Wu Bidong; Xie Jianwei.
J. Chromatogr. A. 2012, 1229, 164-171. (b) Noort. D.; Benschop.
H. P.; Black. R. M. Toxicol. Appl. Pharmacol. 2002, 184, 116–
126.
Figure 6 Structures of the d5-VX adducted nonapeptide and
fragmentation sites denoted with dashed lines.
7.
(a) Knaack. S. Jennifer.; Zhou. Yingtao.; Abney. W. Carter.; Jacob.
T. Justin.; Prezioso. M. Samantha.; Hardy. Katelyn.; Lemire. W.
Sharon.; Thomas. Jerry.; Johnson. C. Rudolph. Anal. Chem. 2012,
84, 9470−9477. (b) Carter. D. Melissa.; Crow. S. Brian.;
Pantazides. G. Brooke.; Watson. M. Caroline.; Thomas. D. Jerry.;
Blake. Thomas A.; Johnson. C. Rudolph. Anal. Chem. 2013, 85,
11106−11111. (c) Sporty. L. S. Jennifer; Lemire. W. Sharon;
Jakubowski. M. Edward; Renner. A. Julie.; Evans. A. Ronald.;
Williams. F. Robert.; Schmidt. G. Jurgen.; van der Schans. J.
Marcel.; Noort Daan.; Johnson. C. Rudolph. Anal. Chem. 2010, 82,
6593–6600.
(a) He, Zheng-Jie.; Wang, You-Ming.; Tang, Chu-Chi.
Phosphorus, Sulfur and Silicon and Related Elements. 1997, 127,
59-66. (b) Bennet, Andrew J.; Kovach, Ildikkom.; Bibbs, Jeffrey
A. J. Am. Chem. Soc. 1989, 111, 6224-6427.
(a) Hudson; Keay. J. Am. Chem. Soc. 1956, 2463-2466. (b) Struck.
J. Am. Chem. Soc. 1966, 9, 231-234.
4. Conclusion
Figure 7 Structures of the d15-GD adducted nonapeptide and
8.
9.
fragmentation sites denoted with dashed lines.
In summary, the synthesis approaches of d5-VX and d15-GD
adducted nonapeptide via solid-phase has been developed. The
MS/MS fracture manners of the d5-VX and d15-GD nonapeptides
are as same as the VX-BuChE nonapeptide7c. So the brand new
compound d5-VX and d15-GD peptide could be used as the
substitute of the isotope-labeled internal standard reported before
for LC-MS/MS to monitor the OPNAs exposure. Additionally,
the synthesis material d6-Ethanol and d12-3,3-Dimethyl-2-butanol
used in the procedure has the property of the easier availability
than the deuterated amino acid used in the former literature5. The
superiority of the nonapeptides described in the paper is that the
convenience in the synthesis procedure. Although the procedures
involve in many steps, the synthesis for the previously reported
internal standards may also follow it actually except for the d15-
10. Mary MacDonald.; Marion Lanier.; John Cashman. Synlett. 2010,
13, 1951-1954.
11. (a) Arbuzov
B A. Pure Appl. Chem. 1964, 9, 307. (b)
Bhattacharya A K, Thyagarajan G. Chem. Rev. 1981, 81, 415.
12. Roger Adams; E. W. Adams. Org. Synth. 1925, 5, 87.
13. G. A. Hill; E. W. Flosdorf. Org. Synth. 1925, 5, 91.
14. (a) Pelchowicz, Z. J. Chem. Soc. 1961, 238-240. (b) Balthazor, T.
M. J. Chem. Soc. 1980, 45, 530.
15. The target compound d5-VX adducted nonapeptide was
synthesized via the Solid-phase peptide synthesis manually from
C-terminal to N-terminal to enhance the
. The
peptides were synthesized using Fmoc chemistry on a Fmoc-Ser
(tBu)-Wang resin and HBTU/DIPEA activation. Piperidine/DMF
(25%) was used to de-protect the Fmoc group. The rest of the
amino acid residues were induced onto the resin in sequence by
HBTU/DIPEA activation. Kaiser test was used to ensure the
connection with right amino acid residue upon every step. After
pinacolyl
methylphosphonic
monochloridate
synthesis.
Additionally to our knowledge, there is no literature reporting the
synthesis method to these compouds, and we used the deuterated

5
this point, cleavage from the resin was achieved by treating the
peptide resin with the solution containing 68.5% TFA, 10% 1,2-
ethanedithiol, 10% thioanisole, 5% phenol, 3.5% double-distilled
water and 1% triisopropylsilane for 3h at room temperature. The
crude product of the phosphorylated peptide which could be
precipitated by anhydrous ether, was further purified by reversed-
phase HPLC on a C18 column (0.05% TFA–water–2%
16. The target compound d15-GD adducted nonapeptide was
synthesized using Fmoc chemistry on a 2-chlorotrityl resin and
HBTU/DIPEA 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 protection
of other residues without side-chain protection. Cleavage from the
resin was achieved by treating the peptide resin with 1% TFA–
CH2Cl2 containing 2% triisopropylsilane overnight at room
temperature. The crude product of the phosphonylated peptide
which could be precipitated by anhydrous ether, was further
purified by reversed-phase HPLC on a C18 column (0.05% TFA–
water–2% acetonitrile). Peptide purity was >90% and was verified
by analysis of HPLC-DAD, NMR and high resolution mass
spectrometry. 1H NMR (599.7 MHz, D2O): δ =1.28 (m, 9H), δ
=1.95 (m, 5H), δ = 2.36 (t, J = 7.1 Hz, 2H), δ =3.13 (m, 3H), δ =
3.75-3.87 (m, 7H), δ = 4.19-4.31 (m, 8H), δ = 7.18 (d, J = 7.1 Hz,
2H), δ = 7.29 (m, 3H), 31P{1H} (242.8 MHz, D2O): δ = 34.52,
34.60, 34.95, 35.01. ESI+-HRMS: m/z (M+1)+ calcd for
C40H49D15PN9O16: 973.5223 ; found: 973.5225.
acetonitrile). Peptide purity was>90% by analysis of HPLC-DAD.
It was also verified by NMR and high resolution mass
spectrometry. ESI+-HRMS data showed the molecular weight
907.3968 as the formular C36H51D5PN9O16 and the 1H NMR
(599.7 MHz, D2O) data was showed as follows: δ =1.28 (t, J = 7.4
Hz, 9H), δ = 1.47 (d, 2JH-C-P = 17.6 Hz, 3H), δ = 1.80-2.11 (m, 6H),
δ = 2.37 (t, J = 7.1 Hz, 2H), δ = 3.12 (m, 3H), δ = 3.75-3.88 (m,
6H), δ = 4.20-4.35 (m, 9H), δ = 4.55 (s, 1H), δ = 7.18 (d, J = 7.2
Hz, 2H), δ = 7.26-7.31 (m, 3H). While 13C{1H} NMR (150.8 MHz,
D2O) data was showed as follows: δ = 177.1, 175.0, 174.7, 174.6,
173.5, 173.3, 170.9, 170.7, 169.8, 169.6, 133.6, 129.3, 129.1,
128.0, 64.2, 64.1, 61.2, 61.1, 55.1, 54.4, 53.45, 53.40, 53.35, 53.1,
49.9, 49.5, 49.4, 42.2, 36.7, 30.0, 26.1, 16.7, 16,4, 9.4, 8.5. 31P{1H}
NMR (242.8 MHz, D2O) data: δ = 35.9.

6
Tetrahedron
Highlights in the paper
1. The two target compounds reported in the paper are brand new.
4. We present a new practical synthesis method for the d5-VX nonapeptides.
3. The mass spectrometry fragment pathways are identical as the biomarkers.
4. They are new deuterated internal standards in the analysis of OPNAs exposure.