Synthetic Approach to Argpyrimidine as a Tool for Investigating Nonenzymatic Posttranslational Modification of Proteins

Article abstract of DOI:10.1055/s-0036-1588225

Nonenzymatic posttranslational modifications (nPTMs) of proteins are involved in age-related, metabolic and other diseases and need to be investigated at the molecular level. Here, we describe how we used organic synthesis to enable the study of the effect of argpyrimidine (Apy), an nPTM that forms at arginine residues, on one of its target proteins. We developed an efficient approach to Apy as a universal building block for Fmoc-based solid-phase peptide synthesis that allows for the construction of peptides containing this nPTM in predetermined positions. Moreover, a straightforward one-step synthesis of protecting-group-free Apy was achieved, which enabled the preparation of gram-quantities of this noncanonical amino acid that can serve as a biomarker or a feedstock in construction of Apy-containing proteins via the expanded genetic code methods.

Full text of DOI:10.1055/s-0036-1588225

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M. Matveenko, C. F. W. Becker
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Synthetic Approach to Argpyrimidine as a Tool for Investigating
Nonenzymatic Posttranslational Modification of Proteins
Maria Matveenko
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Christian F. W. Becker*
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University of Vienna, Department of Chemistry, Institute of
Biological Chemistry, Währinger Strasse 38, 1090 Vienna,
Austria
christian.becker@univie.ac.at
Published as part of the Cluster Recent Advances in Protein
and Peptide Synthesis
Received: 24.01.2017
Accepted after revision: 12.03.2017
Published online: 19.04.2017
suitable Apy building block for the construction of a 33-mer
peptide containing this modification instead of Arg188, a
position that is affected in human cancer cells.6b The re-
quired building block had to be suitable for standard auto-
mated Fmoc-strategy solid-phase peptide synthesis (SPPS)
and as such contain a free α-carboxyl moiety, Fmoc-masked
α-amine and side chains that are protected with acid-labile
groups that are resistant to basic conditions used in the re-
peated cycles of Fmoc deprotection. By contrast, the avail-
able syntheses of Apy at that time were limited to providing
protecting-group-free compounds on analytical scale.3,8
Therefore, a novel route had to be established, capable of
delivering multigram quantities of the Apy building block
from readily available materials and using conventional
protecting groups that do not require additional peptide
manipulation.
DOI: 10.1055/s-0036-1588225; Art ID: st-2017-w0066-c
Abstract Nonenzymatic posttranslational modifications (nPTMs) of
proteins are involved in age-related, metabolic and other diseases and
need to be investigated at the molecular level. Here, we describe how
we used organic synthesis to enable the study of the effect of argpyrim-
idine (Apy), an nPTM that forms at arginine residues, on one of its tar-
get proteins. We developed an efficient approach to Apy as a universal
building block for Fmoc-based solid-phase peptide synthesis that allows
for the construction of peptides containing this nPTM in predetermined
positions. Moreover, a straightforward one-step synthesis of protect-
ing-group-free Apy was achieved, which enabled the preparation of
gram-quantities of this noncanonical amino acid that can serve as a
biomarker or a feedstock in construction of Apy-containing proteins via
the expanded genetic code methods.
Key words protein modification, argpyrimidine, amino acid synthesis,
2-aminopyrimidinol, Mitsunobu reaction, SPPS, protein semisynthesis
protein
protein
protein
protein
O
Nonenzymatic posttranslational modifications (nPTMs)
result from the addition of carbonyl-containing metabolites
to nucleophilic side chains of proteins and are involved in
age-related, metabolic, cardiovascular, neurodegenerative
diseases and cancer.1 Despite the lack of enzymatic assis-
tance, these modifications often accumulate site-specifical-
ly,2 and therefore homogeneous modified proteins are
needed to assess the precise impact of nPTM formation.
Here, we focused on argpyrimidine (Apy, Scheme 1), a fluo-
rescent (λem,max = 385 nm) nPTM that forms on arginine res-
idues3 and has been detected in aged and cataract-affected
lenses,4 in amyloid fibers from patients with familial amy-
loidotic polyneuropathy,5 and in human cells and tissues
from diabetic and cancer patients.6 In order to investigate
the structural and functional consequences of Apy forma-
tion on one of its in vivo target proteins, Hsp27, we devised
a semisynthesis approach (Scheme 2)7 that hinged on a
H
O
MG
HN
HN
N
HN
NH2
N
NH3
NH
NH
H2N
N
O
O
OH
Apy (λem,max 385 nm)
HO
Arg
Lys
MG-H1
CEL
Scheme 1 Examples of nPTMs caused by methylglyoxal (MG), a by-
product of glycolysis: arginine-derived MG-hydroimidazolone (MG-H1)
and argpyrimidine (Apy), and lysine-derived carboxyethyllysine (CEL)9
We chose to construct the proposed building block 1 via
a Mitsunobu reaction10 between the known reduced gluta-
mate derivative 2,11 and a suitably protected aminopyrim-
idinol 3 (Scheme 3). The protecting group on the amino
function of the aminopyrimidine intermediate 3 had to
serve an additional and critical role of acidifying the N–H
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

B
M. Matveenko, C. F. W. Becker
Cluster
Synlett
tention to acid catalysis for the condensation reaction. Grat-
ifyingly, using a strong but easy-to-handle methanesulfonic
acid as a solvent, which completely dissolves the substrates
and catalyzes the reaction, a full conversion of guanidine
sulfate (4) into the corresponding 2-aminopyrimidinol
(6)8c,15,16 was achieved, and this material was isolated in
85% yield on multigram scale. While the phenol function
within this last compound was smoothly converted into the
corresponding tert-butyl carbonate (compound 7, 75%), the
next planned step of Boc protection of the aniline moiety
was complicated by the persistent formation of the N,N-bis-
Boc carbamate (e.g., using Boc2O/Et3N/DMAP) due to the
acidity of N–H within the initially formed carbamate. Un-
fortunately, switching to a weaker base pyridine did not im-
prove the desired reaction, while attempts to remove one of
the two N-Boc groups (NaOMe/MeOH)17 or using heat and
high vacuum were also unfruitful, resulting in varying
amounts of the starting material and the completely N-
deprotected compound 7. Fortunately, applying the modi-
fied procedure18 of the established method involving the
addition of two equivalents of NaHMDS,19 the desired inter-
mediate was obtained in 65% yield.
HS
O
Expression
in E. coli
recombinant
Hsp27 (1–172)
synthetic
Hsp27 (174–205)
SPPS
H2N
SR
Apy
O
building
block
native chemical ligation
semisynthetic Hsp27
HN
N
N
R = alkyl or aryl
Apy188
OH
HN
N
N
Apy188
OH
Scheme 2 Semisynthetic approach to Hsp27 with Arg188 Apy modifi-
cation7
hydrogen sufficiently to be amenable to removal by the hy-
drazide anion formed in the Mitsunobu protocol. The aque-
ous pKa of 2-aminopyrimidinol was reported to be ca.
20.5,12 and thus the carbonyl function of a Boc group was
anticipated to reduce it sufficiently for the reaction, partic-
ularly as the related N,N′-bis(tert-butyloxycarbonyl)guani-
dine is a known nucleophile under the Mitsunobu condi-
tions.13
Boc2O (1.8 equiv)
NH
NH2
1) Boc2O (1 equiv)
N
2) NaHMDS (2 equiv)
2 NaOH (aq)
t-BuOH
NH2
NH2
N
N
(75%)
HN
N
OH
OBn
5 (4 equiv)
O
Fmoc
NH
O
Fmoc
NH
MeSO3H
(85%)
0 to 25 °C
(65%)
or
HO
O
• 1/2 H2SO4
Boc2O (3.4 equiv)
t-BuOH, 60 °C
(71%)
Boc
7
NH2
NH2
4
6
HN PG
N
OBn
HN
N
Mitsunobu
O
OBn
Fmoc
HN Boc
N
N
PG
reaction
OH
4
5
N
O
Fmoc
NH
N
N
2
2
O
O
N
condensation
10
O
PG
DIAD, PPh3
or
ADDP, PPh3
3
O
OBn
O
PG = acid-labile and base-stable
protecting group, orthogonal to Bn
AcO
Boc
8
1
O
O
Fmoc
PG
N
N
Boc
NH
Scheme 3 Synthetic approach to the Apy building block for Fmoc-
N
based SPPS
O
O
O
NH
N
O
Boc
9
11
The synthesis began with the construction of the 2-ami-
nopyrimidinol (6) by subjecting readily available guanidine
sulfate (4) to an excess of the known 1,3-diketone 5 (avail-
able in one step from a commercial material),14 in the pres-
ence of NaOAc according to a published method15 (Scheme
4). In our hands, the base-mediated process did not lead to
acceptable yields of the sought-after aminopyrimidinol
(maximum 15% yield). Using inorganic or organic bases of
varying strength (NaOAc, K2CO3, pyridine, NMM, Et3N), in-
creasing the amount of the base-labile diketone reagent to
as much as six equivalents (stepwise addition), and includ-
ing the use of conventional or microwave heating (up to
150 °C) in several solvents did not improve the yield of the
water-soluble product 6. The insolubility of guanidine salts
(sulfate, chloride) in organic solvents, coupled with the in-
stability of the diketone reagent to basic conditions were
the major obstacles in this process. We then turned our at-
Scheme 4 Construction of the 2-aminopyrimidinol core and attempt-
ed Mitsunobu reaction
With compound 8 in hand, we were able to test the pro-
posed Mitsunobu reaction with the primary alcohol 2, ob-
tained in one reduction step from the commercially avail-
able acid Fmoc-Glu-OBn.11 Unfortunately, the intermediate
8 failed to engage in the proposed reaction, even when the
more strongly basic reagent derived from 1,1′-(azodicar-
bonyl)dipiperidine (ADDP)20 was used in place of the stan-
dard diisopropyl azodicarboxylate (DIAD, Scheme 4). In-
stead, only the cyclized material 10 and compound tenta-
tively assigned as 11, the product of the attack of the DIAD-
derived hydrazide anion at the alkoxyphosphonium inter-
mediate,21 were isolated. In another attempt at the desired
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

C
M. Matveenko, C. F. W. Becker
Cluster
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OBn
OBn
OH
piperidine
(20% v/v
in DMF)
1) HBTU (0.95 equiv), DIPEA (2 equiv), DMF
Fmoc
O
N
Fmoc
O
N
R
O
N
Ns
2)
2
NH
NH
NH
NH2
NH
N
AQLGGPEAAKSDETAAK
DIAD
PPh3
0.08 M NaOH
0.66 M CaCl2
p-NsCl
N
N
(73%, 14)
17 (0.4 equiv)
pyridine
N
or
THF
(92%)
i-PrOH/H2O
16 h
(80%)
(65%)
TFA, CH2Cl2
(95%, 15)
N
N
Ns
N
N
Ns
N
N
Ns
O
O
AQLGGPEAAKSDETAAK
HN
Boc
7
Boc
12
O
N
NH
Fmoc
O
HO
HO
Boc 13
16
R = H 14
R = Fmoc 15
N
N
Ns
18
HO
Scheme 5 Successful Mitsunobu reaction and attempted peptide synthesis using the building block 16
alkylation, alcohol 2 was converted into the corresponding
iodide (using I2/Ph3P/imidazole, 97%, see Supporting Infor-
mation for details), but this last compound then failed to
undergo a nucleophilic attack by either amine 7 (combined
with heating and weak amine bases to remove HI) or carba-
mate 8 (combined with strong non-nucleophilic bases to
deprotonate the NH).
product was the unreacted peptide substrate, and only trac-
es of the desired coupled product were present, suggesting
that the free OH group was potentially interfering with the
coupling step.
In an attempt to block the phenol moiety, the two most
common Fmoc-based SPPS side-chain protecting groups
were investigated, tert-butyl (t-Bu) and trityl (Trt). Unfortu-
nately, the phenol function of both the fully assembled
compound 15 and the aminopyrimidinol 6 were resistant to
several alkylating conditions (t-BuBr or TrtCl/amines; Trt-
Cl/ZnCl2/Et3N;23 TrtOH/acids) or the decarboxylative etheri-
fication with Boc2O/Mg(ClO4)2.24 On the other hand, using a
combination of Cs2CO3 or K2CO3 and TrtCl in anhydrous or-
ganic solvents, up to 55% of the monotritylated product
could be isolated following the complete consumption of
the starting material 6. Unfortunately, this material was
identified as the N-Trt compound 19 (Scheme 6), indicating
that in this case the steric hindrance of the neighboring
methyl groups overpowered the nucleophilicity distribu-
tion observed earlier for the Boc protection. The structure
of compound 19 was established unambiguously through a
single-crystal X-ray analysis (Figure 1).
We thus turned our attention to strongly electron-with-
drawing groups for the activation of the amine function to-
ward alkylation. Indeed, p-nitrobenzenesulfonyl (Ns) amide
12 underwent a smooth Mitsunobu reaction to give fully
protected argpyrimidine 13 (Scheme 5). To test the stability
of the Ns group within this last compound, it was submit-
ted to the standard Fmoc-cleavage conditions used in SPPS
(piperidine, 20% v/v in DMF). While the Ns group was un-
touched, the Boc group was cleaved completely to give phe-
nol 14. In the hope that the methyl groups would hinder O-
acylation of this compound while also blocking the electro-
philic substitution at the 3-benzyl position, we decided to
test the free-OH compound as a potential building block for
Fmoc-SPPS. To this end, the O-Boc group was removed us-
ing TFA, and the benzyl ester within the resulting Fmoc-
protected compound 15 had to be cleaved to reveal the free
carboxylic acid. Attempted hydrogenolysis, mediated by
palladium on charcoal, was successful in this respect but re-
sulted in the additional reduction (mixture of partial and
complete) of the aromatic nitro group (see Figure S1 in the
Supporting Information). Fortunately, applying the condi-
tions for preserving the Fmoc group during ester hydroly-
sis,22 the required acid 16 was isolated in 80% yield.
NH2
N
HN
N
Ph3CCl (1.5 equiv)
K2CO3 (1.5 equiv)
N
N
DMF, 25 °C
(55%)
HO
HO
6
19
This material was then submitted to the standard man-
ual peptide coupling procedure (2.5 equiv of the amino acid
16, activated with HBTU or HATU and DIPEA) with the pre-
assembled peptide 17 comprising the C-terminus of Hsp27
and containing the free N-terminal amine (Scheme 5).
Unfortunately, the LC–MS chromatogram of the crude
peptide obtained following the coupling reaction (Figure S2
in the Supporting Information) indicated that the major
Scheme 6 Attempted phenol protection using the trityl group
The ultimate approach (Scheme 7) involved the protec-
tion of the phenol as a TBS ether,26 and substitution of the
nonstandard Ns group with the acid-labile 2,2,4,6,7-pen-
tamethyldihydrobenzofuran-5-sulfonyl (Pbf), which is rou-
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D
M. Matveenko, C. F. W. Becker
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quired the use of corrosive materials such as 12 M HCl or
purification by HPLC, limiting them to small amounts of the
amino acid.3,8a,b After screening several mild reagents (e.g.,
volatile mild acids such as AcOH or hexafluoroisopropanol)
and the use of heat to promote this condensation, the opti-
mum procedure was established that involves the reaction
of 5 with unprotected L-arginine 22 in methanesulfonic
acid (Scheme 8).27 In this way, argpyrimidine is obtained in
up to 82% yield and on gram scale for the first time. The
spectral and physical data derived from this amino acid are
in excellent agreement with the literature values.3,8 In par-
ticular, the characteristic excitation and emission maxima
at 320 and 385 nm, respectively,3 are observed in the fluo-
rescence spectrum of synthetic Apyfree (23, see Figure S3 in
the Supporting Information). With the efficient one-step
synthesis of 23 in hand, it would be interesting to explore
the conversion of this compound into a building block of
type 1. The main concern would be the installation of the
protecting groups given the presence of several reactive
moieties within compound 23.
Figure 1 ORTEP diagram resulting from the X-ray crystal analysis of
1925
OH
OH
O
O
O
tinely applied in Fmoc strategy SPPS. The resulting sulfon-
amide 20 underwent an efficient Mitsunobu reaction (80%,
ca. 4 mmol scale), and the ensuing compound was submit-
ted to gentle hydrogenolysis of the benzyl ester, carefully
avoiding the side reaction of Fmoc group removal, to give
the Apy building block 21 (> 5 g, five steps from guani-
dine).7 This material was successfully utilized, without any
deviations from the standard automated Fmoc-based SPPS
protocols, in the preparation of sufficient quantities of the
required peptide that enabled the semisynthesis of Hsp27
bearing the Apy188 modification.7
O
NH2
NH2
5
AcO
MeSO3H
(65–82%)
NH
NH
N
HN
N
NH2
Apyfree (23)
Arg (22)
HO
Scheme 8 Optimized synthesis of argpyrimidine as a free amino acid
In summary, we have developed an efficient strategy to
assemble a universal Apy building block applicable to man-
ual or automated Fmoc-based SPPS, which allows for the
synthesis of peptides and proteins containing this nPTM in
predetermined positions. Such compounds enable the in-
vestigation of the impact of nPTMs on the molecular prop-
erties of the affected proteins. In addition, the spectroscop-
ic features of Apy and its relatively small size may be taken
advantage of by the incorporation as a fluorescent protein
label, with minimal perturbation of the structure and func-
tion of the protein to be investigated. Finally, the straight-
forward one-step procedure for the synthesis of Apy amino
acid has enabled access to multigram quantities of this
valuable material, which can be used as a standard for mon-
itoring soluble nPTM concentrations in biological fluids or
for ribosomal synthesis of Apy-derivatized proteins follow-
ing reprograming of expression systems.
Pbf
OH
1) 2
DIAD, PPh3
(80%)
1) TBSCl
imidazole
(81%)
NH2
N
NH
N
O
Fmoc
NH
N
N
2) PbfCl
pyridine
(76%)
2) H2, Pd/C
(88%)
HO
O
6
TBS 20
N
N
Pbf
N
O
S
O
Pbf =
O
O
21
TBS
Scheme 7 Optimized synthesis of the building block 217
Samples of the free amino acid Apy are also valuable
and have been previously obtained on small scale for ana-
lytical purposes via the global deprotection of 21.7 A more
efficient and straightforward synthetic procedure for this
amino acid had to be developed that could provide multi-
gram quantities of this material. The pioneering published
approaches involved the condensation of diketone 5 or its
analogues with αN-protected or free L-arginine but re-
Funding Information
Austrian Academy of Sciences (APART fellowship)
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

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M. Matveenko, C. F. W. Becker
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Acknowledgment
Chem 2011, 12, 1270. (c) Renard, B.-L.; Boucherle, B.; Maurin, B.;
Molina, M.-C.; Norez, C.; Becq, F.; Décout, J.-L. Eur. J. Med. Chem.
2011, 46, 1935.
We thank Christian Stieger for technical assistance and Alexander
Roller for X-ray crystallographic analysis. Maria Matveenko is a recip-
ient of an APART fellowship of the Austrian Academy of Sciences.
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S
u
p
p
ortiInfogrmoaitn
S
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p
p
ortioInfgrmoaitn
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Cancer Lett. 2006, 241, 309. (f) Gawlowski, T.; Stratmann, B.;
Stork, I.; Engelbrecht, B.; Brodehl, A.; Niehaus, K.; Korfer, R.;
Tschoepe, D.; Milting, H. Horm. Metab. Res. 2009, 41, 594.
(g) Bair, W. B.; Cabello, C. M.; Uchida, K.; Bause, A. S.; Wondrak,
G. T. Melanoma Res. 2010, 20, 85. (h) Oya-Ito, T.; Naito, Y.;
Takagi, T.; Handa, O.; Matsui, H.; Yamada, M.; Shima, K.;
Yoshikawa, T. Biochim. Biophys. Acta 2011, 1812, 769.
An adaptation of the original published procedure was
employed.8a,b A magnetically stirred solution of L-arginine (22,
3.0 g, 17.2 mmol) in methanesulfonic acid (12 mL, 1.5 M) main-
tained at 25 °C was treated with crude diketone 5 (3.5 g, ca. 22
mmol), resulting in a mildly exothermic reaction. Further por-
tions of compound 5 (2 × 2.7 g, ca. 17 mmol each) were added
after 3 and 6 h, respectively. The ensuing dark brown viscous
mixture was stirred for 48 h then cooled to 0 °C and neutralized
by the dropwise addition of NH4OH (ca. 20 mL of a 28–30% aq
solution). The resulting brown-orange mixture (pH ~7) was
stirred at 25 °C for 30 min then diluted with H2O (25 mL) and
loaded, using additional H2O, onto a column of C18-reversed-
phase silica gel (10 × 10 cm) that had been equilibrated with
MeOH then H2O. Elution with 0 → 10 → 20% v/v MeOH–H2O and
concentration of the relevant fractions containing fluorescent
material (Rf = 0.2 in 1:2:7 v/v/v H2O–i-PrOH–EtOAc) afforded
the title compound 23 (2.83 g, 65%) as a white powder. A
portion of this material was lyophilized from TFA (0.1% in H2O)
to obtain compound 23 (zwitterion, white fluffy powder) that
was used for characterization and all spectroscopic measure-
ments; mp 192–196 °C (decomp.) [lit. for HCl salt8a 207 °C
(7) Matveenko, M.; Cichero, E.; Fossa, P.; Becker, C. F. Angew. Chem.
Int. Ed. 2016, 55, 11397.
(8) (a) Sreejayan, N.; Yang, X.; Palanichamy, K.; Dolence, K.; Ren, J.
Eur. J. Pharmacol. 2008, 593, 30. (b) Hellwig, M.; Geissler, S.;
Matthes, R.; Peto, A.; Silow, C.; Brandsch, M.; Henle, T. ChemBio-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F

F
M. Matveenko, C. F. W. Becker
Cluster
Synlett
(decomp.)]. [α]D +28.1 (c 0.3, H2O) [lit.8a +17.5 (c 0.5, 1 M HCl)].
1H NMR (600 MHz, D2O): δ = 3.73 (t, J = 6.1 Hz, 1 H), 3.44 (t, J =
6.8 Hz, 2 H), 2.39 (s, 6 H), 1.93–1.84 (m, 2 H), 1.75–1.60 (m, 2 H).
1H NMR (600 MHz, (CD3)2SO): δ = 8.14 (br s, 2 H, NH2), 7.87 (br
s, 1 H, OH), 6.38 (s, 1 H, NH), 3.86 (t, J = 6.2 Hz, 1 H), 3.18 (dd, J =
12.0, 6.2 Hz, 2 H), 2.18 (s, 6 H), 1.84–1.71 (m, 2 H), 1.63–1.49
(m, 2 H). 13C NMR (150 MHz, D2O): δ = 174.4, 150.8, 137.7, 54.3,
40.4, 27.5, 23.9, 16.8 (due to H/D exchange of the phenolic OH,
the corresponding ipso carbon is not visible in the spectrum).
13C NMR (150 MHz, (CD3)2SO): δ = 171.2, 156.3, 155.1, 138.8,
52.1, 40.3, 27.8, 24.9, 19.0. ESI-HRMS: m/z [M + H]+ calcd for
C
11H18N4O3: 255.1452; found: 255.1448.
On 0.6 mmol scale and using pre-packed C18 cartridges for puri-
fication, compound 23 was obtained in 82% yield.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2017, 28, A–F