Fmoc-based solid-phase synthesis of adenylylated peptides using diester-type adenylylated amino acid derivatives

Article abstract of DOI:10.1016/j.tetlet.2012.04.063

Phosphodiester-type adenylylated (AMPylated) Ser, Thr, and Tyr derivatives were developed for Fmoc solid phase peptide synthesis of AMPylated peptides. One-pot/sequential reaction consisting of condensation of an N-nonprotected adenosine derivative and Fm

Full text of DOI:10.1016/j.tetlet.2012.04.063

Tetrahedron Letters 53 (2012) 3429–3432
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Tetrahedron Letters
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Fmoc-based solid-phase synthesis of adenylylated peptides using diester-type
adenylylated amino acid derivatives
⇑
Keiji Ogura, Akira Shigenaga, Koji Ebisuno, Hiroko Hirakawa, Akira Otaka
Institute of Health Bioscience and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphodiester-type adenylylated (AMPylated) Ser, Thr, and Tyr derivatives were developed for Fmoc
solid phase peptide synthesis of AMPylated peptides. One-pot/sequential reaction consisting of conden-
sation of an N-nonprotected adenosine derivative and Fmoc-Ser/Thr/Tyr-OAllyl using allyl-N,N-dii-
sopropylchlorophosphoramidite and subsequent oxidation with m-chloroperbenzoic acid gave
phosphotriester-type AMPylated Ser/Thr/Tyr derivatives. After Pd(0)-mediated deprotection of allyl
groups, the resulting phosphodiester-type AMPylated Ser/Thr/Tyr derivatives were successfully incorpo-
rated into peptides by standard Fmoc solid phase peptide synthesis without significant side reactions
including dehydroalanine formation.
Received 22 March 2012
Revised 10 April 2012
Accepted 13 April 2012
Available online 21 April 2012
Keywords:
AMPylation
AMPylated amino acid
Phosphodiester amino acid
Phosphoramidite method
Fmoc solid-phase peptide synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
Introduction
by coupling of a protected adenosine derivative and subsequent
oxidation of the resulting H-phosphonate-adenosine.5 However,
Nucleotidylation of the hydroxyl functionality of proteins has
been recognized as an important posttranslational modification.
Among such nucleotidylations, uridinylylation or guanylylation of
viral proteins found in RNA viruses has been reported to be in-
volved in the initiation of genome replication.1 Very recently, Fic
(filamentation induced by cyclic AMP) domain proteins from two
pathogenic bacteria have been reported to use ATP to catalyze
the addition of adenosine monophosphate (AMP) to a tyrosine or
threonine residue of Rho-family GTPases RhoA, Rac1 and Cdc42,
leading to modulation of downstream signaling events (Fig. 1).2,3
Such AMP addition or adenylylation is referred to as ‘AMPyla-
tion’ and is predicted to be functionally similar to other posttrans-
lational modifications including phosphorylation and acetylation
with respect to transient modification of protein functions. The
presence of a Fic protein in higher eukaryotes, including humans,
and AMPylation catalyzed by a non-Fic protein (Legionella protein
DrrA) have also been reported.4 However, the physiological roles
of AMPylation as a newly emerging posttranslational modification
have remained elusive. We have therefore studied the synthesis of
AMPylated peptides to provide AMPylated materials for uncover-
ing the biological significance of AMPylation events.
application of this protocol to AMPylated Tyr peptides has yet to
be achieved. Synthetic approaches using nucleotidylated Fmoc
amino acid building blocks have been applied to the preparation
of uridinylylated, guanylylated, and AMPylated Tyr and uridinyly-
lated Ser peptides using nucleotidylated phosphotriester-type ami-
no acid derivatives.6 Very recently, Hedberg and co-workers
reported the synthesis of AMPylated Tyr7 and Ser/Thr peptides8
using phosphotriester- and phosphodiester-type protected AMPy-
lated amino acids, respectively. The logical extension of the triest-
er-type compounds used in AMPylated Tyr peptide synthesis to the
Fmoc-based synthesis of AMPylated Ser/Thr peptides initially
OH
Tyr/Thr/(Ser)
Protein
NH2
ATP
AMP
N
N
N
AMP
Phospho-
diesterase
transferase
N
O
Pyrophosphate
(PPi)
Recently, solid-phase synthesis of AMPylated Ser/Thr peptides
was achieved using an H-phosphonate approach involving phos-
phonylation of the hydroxyl group on a Ser/Thr residue followed
O
O
H2O
P
O
O
OH OH
Tyr/Thr/(Ser)
Protein
⇑
Corresponding author. Tel.: +81 88 633 7283; fax: +81 88 633 9505.
E-mail address: aotaka@ph.tokushima-u.ac.jp (A. Otaka).
Figure 1. Adenosine monophosphorylation (AMPylation) of proteins.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.04.063

3430
K. Ogura et al. / Tetrahedron Letters 53 (2012) 3429–3432
failed, and gave b-elimination products; this led to the use of dies-
ter-type compounds. These unproductive studies using phospho-
triester-type compounds have been well documented in the
synthesis of phosphopeptides by Fmoc protocols. Phosphodiester-
type amino acid derivatives have therefore frequently been used
in Fmoc solid-phase peptide synthesis (Fmoc SPPS), and have been
very successful in the synthesis of phosphopeptides (Fig. 2, 1–3).9
In particular, phosphoserine- and phosphothreonine-containing
peptides have been successfully synthesized using monobenzyl-
protected phoshoamino acids as diester-type derivatives.
NH2
N
NH2
N
N
N
N
N
N
N
O
O
O
P
O
P
O
O
O
O
R
HO
O
CO2H
CO2H
Fmoc
O
HO
O
HN
On the basis of this knowledge, we planned to synthesize three
types of AMPylated peptides using diester-type AMPylated Ser/
Thr/ Tyr derivatives (4, 5, and 6) by Fmoc methodology (Fig. 3).
HN
O
Fmoc
4: R = H
6
(diester-type AMPylated serine)
(diester-type
AMPylated tyrosine)
Results and Discussion
5: R = Me
(diester-type AMPylated threonine)
Initially, we attempted the synthesis of the desired phosphodi-
ester-type compounds using temporary methyl protection on the
phosphotriester unit. In our previous synthetic work on phospho-
peptides, we realized that one methyl group on the phosphotries-
ter unit can be easily removed under acidic conditions.10 However,
final deprotection of the methyl group on an AMPylated Ser deriv-
ative did not proceed efficiently. We attempted to use temporary
allyl protection, which can be removed using Pd(0) reagents. The
synthetic routes for Fmoc-diester-type AMPylated amino acids
are shown in Scheme 1. In our protocol, 20,30-O-isopropylidenead-
enosine 7 was reacted with a slight excess of allyl-N,N-diisopropyl-
chloro-phosphoramidite11 8 (1.1 eqiv) in CH2Cl2 in the presence of
triethylamine at room temperature for 2 h to yield ally-(2030-O-iso-
propylideneadenosin-50-yl)phosphoramidite 9. Without purifica-
tion of the resulting phosphoramidite reagent, an equimolar
mixture of Fmoc-Ser-OAllyl 10 and 1H-tetrazole was added to
the reaction mixture with additional stirring at room temperature
for 2 h. Then, oxidation with m-chloroperbenzoic acid (mCPBA)
was performed to give crude fully protected AMPylated Ser deriv-
ative 12. Column chromatographic purification of the crude mate-
rials on silica-gel afforded pure triester-type AMPylated Ser
derivative 12 as a diastereomeric mixture in 44% isolated yield
based on starting adenosine derivative 7. Without requiring an
intermediate purification step, two-step condensation, and subse-
quent oxidation proceeded efficiently to yield the desired
materials.
Figure 3. Diester-type AMPylated amino acid derivatives synthesized in this work.
NH2
NH2
(A)
N
N
N
N
N
N
N
N
AllylO
N
HO
P
O
O
O
AllyO
Cl
P
N
O
O
O
O
8
7
9
Et3N, CH2Cl2
NH2
N
N
N
N
Fmoc-Ser-OAllyl
O
10
O
P
mCPBA
1H-tetrazole
CH2Cl2
O
O
AllylO
CO2Allyl
Fmoc
O
11
HN
NH2
N
N
N
N
In previous papers, 6,6-N,N-diBoc or 6-N-benzoyl group was
used for N-protection in the nucleobase part.6c,7,8 However, we
doubted the necessity of protecting the 6-amino group on adenine
during Fmoc-based SPPS using nucleophilic piperidine,12 that is,
partial loss of the acyl-type protection was anticipated. Further-
more, the accidentally generated 6-amino group is expected to
show low nucleophilicity and not to be involved in the acylation
reactions commonly used in peptide chain elongation steps. In
our synthesis of AMPylated amino acids, the N-non-protected
adenosine derivative 7, which is easily obtainable from major
O
10 mol % Pd(PPh3)4
O
P
4
(65%
1.1 equiv N-methylaniline
O
O
after HPLC
purification)
THF
AllylO
O
CO2Allyl
O
12 (Isolated yield 44%
HN
Fmoc
from 7 in one-pot
reactions)
NH2
N
(B)
N
N
N
1) 9 (7 + 8),
Fmoc-Thr-OAllyl 13
or
Fmoc-Tyr-OAllyl 14
O
P
1H-tetrazole, CH2Cl2
O
diester unit
2) mCPBA, CH2Cl2
O
O
O
P
R
O
BnO
HO
CO2H
Fmoc
CO2H
Fmoc
O
P
AllylO
R
O
BnO
HO
HN
15: R = Fmoc-Thr-OAllyl 48% from 7
16: R = Fmoc-Tyr-OAllyl 42% from 7
HN
O
1: R = H
10 mol % Pd(PPh3)4
3
5 from 15 (46% after HPLC purification)
6 from 16 (64% after HPLC purification)
(diester-type phosphoserine)
(diester-type phosphotyrosine)
1.1 equiv N-methylaniline
THF
2: R = Me
(diester-type phosphothreonine)
Scheme 1. Synthetic routes to AMPylated amino acid derivatives. (A) AMPylated
serine 4; (B) AMPylated threonine 5 and tyrosine 6.
Figure 2. Phosphodiester-type amino acids compatible with Fmoc SPPS.

K. Ogura et al. / Tetrahedron Letters 53 (2012) 3429–3432
3431
Having obtained the requisite AMPylated amino acid derivatives
4, 5, and 6, we then tried to incorporate these derivatives into model
peptide sequences. The AMPylated Tyr derivative 6 was first in-
stalled in a model peptide sequence (H-EVY⁄VPTVFENY-NH2: the
asterisk indicates the AMPylated residue) found in RhoA GTPase.
Fmoc-SPPS was conducted on NovaSyn TGR resinÒ (amine content:
0.25 mmol/g) as shown in Scheme 2. Before incorporation of the
AMPylated derivatives, peptide chain elongation was performed
by coupling of the Fmoc amino acid (5.0 equiv) using diisopropylcar-
bodiimide (DIPCDI) (5.0 equiv) and 1-hydroxybenzotriazole mono-
hydrate (HOBtꢀH2O) (5.0 equiv) in DMF, and removal of the Fmoc
group with 20% piperidine in DMF. Incorporation of 6 (2.0 equiv)
was achieved using O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetram-
ethyluronium hexafluorophosphate (HATU) (1.9 equiv) in the
presence of diisopropylethylamine (DIPEA) (4.0 equiv) with the
aim of achieving high coupling efficiency.13,14 The following cou-
plings were performed using a combination of Fmoc amino acid
(A)
Fmoc SPPS
H-VPT(tBu)VFE(OtBu)-
N(Trt)Y(tBu)
H2N
1) Fmoc-AA-OH, DIPCDI, HOBt
for condensation
NovaSyn
TGR resin
2) 20% piperidine in DMF
for Fmoc removal
1) HATU/DIPEA coupling for 6
H-E(OtBu)VY*VPT(tBu)V-
FE(OtBu)N(Trt)Y(tBu)
2) HBTU/DIPEA coupling for V and E
17: Y* = AMPylated Tyr residue
H-EVY*VPTVFENY-NH2
18: Y* = AMPylated Tyr residue
TFA-Et3SiH-H2O (95:2.5:2.5, v/v)
at room temperature for 90 min
H-EVYVPS*VFENY-NH2
19: S* = AMPylated Ser residue
(B)
H2N
Similar protocols to
those employed in A
by the use
of 4 or 5
H-EVYVPT*VFENY-NH2
20: T* = AMPylated Thr residue
(5.0 equiv),
O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HBTU) (4.9 equiv), and DIPEA (5.0 equiv)
in DMF.14 Final deprotection of the completed resin 17 with TFA–
Et3SiH–H2O (95:2.5:2.5, v/v) at room temperature for 90 min gave
the crude deprotected peptide 18. Analysis of the crude materials
by HPLC indicated that Fmoc SPPS of AMPylated Tyr peptide using
6 proceeded successfully without significant side reactions attribut-
able to non-protection of the adenine moiety (Fig. 4-A). HPLC purifi-
cation of crude peptides yielded pure peptide in 26% isolated yield
based on the starting resin.
Synthetic protocols similar to those used for synthesis of the
AMPylated Tyr peptide 18 were applied to the syntheses of model
peptides 19 and 20 (H-EVYVPX⁄VFENY-NH2: 19: X⁄ = AMPlyated
Ser; 20: X⁄ = AMPylated Thr) (Scheme 2-B). Fmoc SPPS followed
by acidic deprotection gave the crude desired peptides 19 and 20
with reasonable purities (Fig. 4-B and C: 17 and 21% yields after
HPLC purification, respectively). Formation of a dehydroalanine
Scheme 2. Outline for the Fmoc SPPS for AMPylated peptides using derivatives 4, 5,
and 6.
commercial suppliers, was therefore used. The steps in the synthe-
sis of 6-N-non-protected derivatives (4, 5, and 6) were not accom-
panied by significant side reactions.
Next, the resulting phosphotriester derivative 12 was subjected
to Pd(0)-mediated deprotection of the two allyl groups in the pres-
ence of N-methylaniline in THF. After reaction for 2 h at room tem-
perature, addition of EtOAc to the reaction mixture gave a yellow
soild. After sequential washing of the resulting solid with EtOAc
and MeOH, the desired diester-type AMPylated Ser derivative 4,
which is adequate for subsequent synthetic purpose, was obtained
in nearly quantitative yield. For analytical purposes, reversed-
phase HPLC purification gave purified 4 in 65% isolated yield.
Similar one-pot synthetic protocols were applied to Fmoc-
Thr-OAllyl 13 and Fmoc-Tyr-OAllyl 14 to yield the corresponding
fully protected triester-type AMPylated Thr and Tyr derivatives
(15 and 16), respectively. Deprotection of phophotriesters 15 and
16 with Pd(0) in the presence of N-methylaniline, followed by pre-
cipitation with EtOAc and subsequent washing steps, afforded the
Fmoc-SPPS compatible AMPylated amino acid derivatives 5 and 6,
respectively. Reversed-phase HPLC purifications of the crude
materials gave pure samples in 46% (5) and 64% (6) isolated yields
(Scheme 1-B).
(DAla) residue from a diester-type uridinylylated Ser residue was
reported by van der Marel and co-workers.6a
In this work, the N-Fmoc-O-o-chlorophenyl-phosphotriester-
type amino acid 21 was incorporated on the N-terminal position
of a model peptide using a standard coupling method under
slightly acidic conditions to prevent b-elimination (Fig. 5).
The resulting resin was deprotected with a sequence of reagent
systems: (i) tetrabutylammonium fluoride (TBAF)–pyridine–DMF–
H2O (for removal of the o-chlorophenyl group on the phosphate);
(ii) 20% piperidine in DMF; (iii) TFA–triisopropylsilane–H2O–
Figure 4. HPLC analyses of crude AMPylated amino acid-containing peptides. (A) AMPylated Tyr peptide 18; (B) AMPylated Ser peptide 19; (C) AMPylated Thr peptide 20. ⁄N-
terminal Glu-deleted peptide. HPLC conditions: Cosmosil 5C18 AR-II column (4.6 ꢁ 250 mm) with a linear gradient of 0.1% TFA–MeCN 0.1% TFA aq (10:90–60:40 over 30 min)
at a flow rate 1.0 mL/min, detection at 220 nm.

3432
K. Ogura et al. / Tetrahedron Letters 53 (2012) 3429–3432
O
that diester-type AMPylated Ser and Thr derivatives (4 and 5) are
O
N
Fmoc-HN
useful building blocks for Fmoc-based synthesis of AMPylated Ser
and Thr peptides. At this stage, we do not know the possible reason
for the different behaviors of uridinylylated and AMPylated pep-
tides with respect to piperidine treatment.
OH
O
HN
O
P
O
O
O
In conclusion, we achieved the Fmoc-based SPPS of AMPylated
Ser, Thr, and Tyr peptides using phosphodiester-type amino acid
derivatives 4, 5, and 6. In our synthetic schemes for phosphotries-
ter-type intermediates 12, 15, and 16, one-pot/sequential conden-
sations using a phosphoramidite reagent are possible. A robust
check of contamination by dehydroalanine-containing byproducts
in the crude AMPylated Ser materials using reference peptides
clearly showed that the diester-type AMPylated derivatives serve
as building blocks of great value in the preparation of AMPylated
Ser and Thr peptides. These synthetic protocols will help in future
research on AMPylation of proteins.
O
Cl
OAc OAc
Removal by aqueous TBAF
21
Figure 5. Uridinylylated Ser derivative 21 used in uridinylylated peptide.6a
OH
Boc-E(OBzl)VY(tBu)VP-Ser-VFE(OtBu)N(Trt)Y(tBu)-resin 23
Ac2O-pyridine in DMF
OAc
Acknowledgments
Boc-E(OBzl)VY(tBu)VP-Ser-VFE(OtBu)N(Trt)Y(tBu)-resin 24
This research was supported in part by a Grant-in-Aid for Scien-
tific Research (KAKENHI). A.O. and A.S. are grateful for research
grants from the Takeda Science Foundation.
TBAF in DMF
Boc-EVY(tBu)VP-ΔAla-VFE(OtBu)N(Trt)Y(tBu)-resin 25
TFA-based deprotection
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
04.063.
H-EVYVP-ΔAla-VFENY-NH2 22
Scheme 3. Synthetic route for dehydroalanine peptide 22.
References and notes
PhOH–1,2-ethanedithiol (EDT); and (iv) concentrated ammonia in
1,4-dioxane (for removal of the acetyl group of 20,30-O-acetyluri-
dine). The major product obtained from the attempted four-step
1. (a) Puustinen, P.; Mäkinen, K. J. Biol. Chem. 2004, 279, 38103–38110; (b) Belliot,
G.; Sosnovtsev, S. V.; Chang, K.-O.; McPhie, P.; McPhie, P.; Green, K. Y. Virology
2008, 374, 33–49; (c) Pan, J.; Lin, L.; Tao, Y. J. Virology 2009, 395, 87–96.
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A.; Orth, K. Curr. Opin. Microbiol. 2010, 13, 34–40.
deprotection was a dehydroalanine (DAla)-containing peptide. In
contrast, subjection of the same model resin to three-step depro-
tection protocols consisting of (i) the aqueous TBAF system, (ii)
the TFA system, and (iii) the concentrated NH3 system yielded
the desired uridinylylated Ser peptide without the formation of
3. Although AMPylation on serine has yet to be found but is expected to occur, in
analogy to intracellular phosphorylation.
D
Ala-peptide. On the basis of these experimental results, the
incompatibility of the serine phosphodiester function with the
piperidine treatment required for Fmoc removal was addressed.
Similar to our approach, Hedberg and co-workers, who used phos-
phodiester-type AMP amino acids, did not mention the contamina-
tion of the dehydroalanine unit in their final synthetic peptide
although they paid attention to b-elimination during the coupling
step under basic conditions.8 We therefore tried to prepare a refer-
4. Muller, M. P.; Peters, H.; Blumer, J.; Blankenfeldt, W.; Goody, R. S.; Itzen, A.
Science 2010, 329, 946–949.
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6. (a) Kriek, N.; Filippov, D. V.; van den Elst, H.; Meeuwenoord, N. J.; Tesser, G. I.;
van Boom, J. H.; van der Marel, G. A. Tetrahedron 2003, 59, 1589–1597; (b)
Kriek, N.; Meeuwenoord, N. J.; van den Elst, H.; Heus, H. A.; van der Marel, G. A.;
Filippov Org, D. V. Biomol. Chem. 2006, 4, 3576–3586; (c) van Noort, G. J. V.;
Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V. J. Org. Chem. 2010, 75,
5733–5736.
ence peptide 22 (H-EVYVP-DAla-VFENY-NH2) and to check its
7. Smit, C.; Blümer, J.; Eerland, M. F.; Albers, M. F.; Müller, M. P.; Goody, R. S.;
Itzen, A.; Hedberg, C. Angew. Chem. Int. Ed. 2011, 50, 9200–9204.
8. Albers, M. F.; van Vliet, B.; Hedberg, C. Org. Lett. 2011, 13, 6014–6017.
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Chem. Soc. Jpn. 1996, 69, 465–468.
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14. For incorporation of diester-type phosphoamino acid building blocks followed
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contamination in the AMPylated Ser materials (Scheme 3). On Nov-
asyn TGR resinÒ, we constructed protected peptide chain 23 using
standard Fmoc amino acids, except for Fmoc-Ser-OH and Boc-
Glu(OBzl)-OH for the dehydroalanine and N-terminal positions,
respectively. The hydroxyl group of the Ser residue in the protected
resin (Boc-E(OBzl)VY(tBu)VP-Ser-VFE(OtBu)N(Trt)Y(tBu)-resin 23)
was acetylated using excess acetic anhydride and pyridine in DMF.
The resulting acetylated resin 24 was treated with TBAF (1 M in
toluene, 100 equiv) in DMF,15 giving b-elimination of the acety-
lated Ser residue and removal of the benzyl group on the Glu
residue. Exposure of the treated resin 25 to TFA–Et3SiH–H2O
(95:2.5:2.5, v/v) at room temperature for 90 min, followed by HPLC
purification, afforded the reference
DAla-containing peptide 22.
15. Ramapanicker, R.; Mishra, R.; Chandrasekaran, S. J. Pept. Sci. 2010, 16, 123–125.
HPLC analysis of the crude AMPylated Ser materials showed no
contamination by 22 (Fig. 4-B). This observation clearly indicated