Synthesis of mono-ADP-ribosylated oligopeptides using ribosylated amino acid building blocks

Article abstract of DOI:10.1021/ja910940q

Adenosine diphosphate ribosylation (ADP-ribosylation) is a widely occurring post-translational modification of proteins at nucleophilic side chains of amino acid residues, such as asparagine, glutamic acid, and arginine. Elucidation of the biological role of ADP-ribosylation events would benefit from the availability of well-defined ADP-ribosylated peptides. Main issues in the construction of synthetic ADP-ribosylated peptides involve the availability of protected ribosylated amino acids suitable for peptide synthesis, development of a protective group strategy for peptide fragments compatible with the integrity of the adenosine diphosphate moiety, and an efficient procedure for pyrophosphate formation. In this paper we present a first approach to the chemical synthesis of ADP-ribosylated peptides in solution and on solid support. We describe an efficient synthesis of suitably protected ribosylated asparagine and glutamine building blocks suitable for Fmoc-based peptide synthesis. We further demonstrate a successful application of these ribosylated amino acids in the assembly of three fully synthetic ADP-ribosylated peptides by solution and solid phase approaches.

Full text of DOI:10.1021/ja910940q

Published on Web 03/16/2010
Synthesis of Mono-ADP-Ribosylated Oligopeptides Using
Ribosylated Amino Acid Building Blocks
Gerbrand J. van der Heden van Noort, Maarten G. van der Horst,
Herman S. Overkleeft, Gijsbert A. van der Marel, and Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden UniVersity, PO Box 9502,
2300 RA Leiden, The Netherlands
Received December 29, 2009; E-mail: filippov@chem.leidenuniv.nl
Abstract: Adenosine diphosphate ribosylation (ADP-ribosylation) is a widely occurring post-translational
modification of proteins at nucleophilic side chains of amino acid residues, such as asparagine, glutamic
acid, and arginine. Elucidation of the biological role of ADP-ribosylation events would benefit from the
availability of well-defined ADP-ribosylated peptides. Main issues in the construction of synthetic ADP-
ribosylated peptides involve the availability of protected ribosylated amino acids suitable for peptide synthesis,
development of a protective group strategy for peptide fragments compatible with the integrity of the
adenosine diphosphate moiety, and an efficient procedure for pyrophosphate formation. In this paper we
present a first approach to the chemical synthesis of ADP-ribosylated peptides in solution and on solid
support. We describe an efficient synthesis of suitably protected ribosylated asparagine and glutamine
building blocks suitable for Fmoc-based peptide synthesis. We further demonstrate a successful application
of these ribosylated amino acids in the assembly of three fully synthetic ADP-ribosylated peptides by solution
and solid phase approaches.
Introduction
We envisage that the elucidation of the role of ADP-
ribosylation events would benefit greatly from the availability
of well-defined ADP-ribosylated peptides and analogues thereof.
The synthesis of these compounds has not yet been described,
and we thus made it the subject of our studies.
Adenosine diphosphate ribosylation (ADP-ribosylation) is a
post-translational modification of proteins that is effected by
mono-ADP-ribosyl transferases (MARTs) or poly-ADP-ribosyl
polymerases (PARPs).1 These enzymes function by transferring
ADP-ribose from ꢀ-NAD+ to nucleophilic functional groups
in the side chain of amino acid residues in the target protein, a
process that is accompanied by the release of nicotinamide (see
Figure 1).2 The best studied ADP-ribosylations are related to
the actions of bacteria, such as Vibrio cholera, Bacillus cereus,
Staphylococcus aureus, Corynebacterium diphtheria, and Clostrid-
ium botulinum.3,4 Host proteins playing a role in the immune
response, cell adhesion, and metabolism are ADP-ribosylated
at Asn, Glu, Asp, Arg, or Cys residues, thereby undergoing an
alteration in their functioning.1 Although ADP-ribosylating
enzymes have been identified in many prokaryotic and eukary-
otic species as well as in viruses, the biological processes in
which they partake and their exact mode of action have not
been clarified completely.2 Interestingly it was recently proposed
that inhibiting ADP-ribosylation might be a novel approach to
cancer therapy.5
Main issues in the construction of mono- or poly-ADP-
ribosylated peptides entail the synthesis of protected ribosylated
amino acids that are compatible with peptide synthesis as well
as an appropriate procedure for pyrophosphate formation. In
this paper we present the synthesis of suitably protected
ribosylated asparagine and glutamine building blocks and their
application in the assembly of ADP-ribosylated peptides by
solution and solid phase approaches.
Results and Discussion
The key N-ribosylated Asn and Gln building blocks 6 and 7
(Scheme 1) are provided with the mutually orthogonal TBDPS
and Fmoc protecting groups, allowing introduction of a pyro-
phosphate moiety and elongation of the peptide chain in a
sequential manner. The route of synthesis started with silylation
of the primary alcohol of known ꢀ-D-ribofuranosyl azide 1 and
subsequent acylation of the secondary 2′ and 3′ hydroxyls in 2
to give fully protected azide 3 in near quantitative yield.
Reduction of ꢀ-azide 3 at 10 °C using PtO2/H2 resulted in the
formation of an epimeric hemiaminal mixture. EDC-mediated
coupling of this mixture with either Z-Glu-OBn or Z-Asp-OBn
gave the ribosylated amino acids 4 and 5 as anomeric mixtures
(1) Hassa, P. O.; Haenni, S. S.; Elser, M.; Hottiger, M. O. Microbiol.
Mol. Biol. ReV. 2006, 70, 789–829.
(2) Corda, D.; Di Girolamo, M. EMBO J. 2003, 22, 1953–1958.
(3) Yates, S. P.; Jorgensen, R.; Andersen, G. R.; Merrill, A. R. Trends
Biochem. Sci. 2006, 31, 123–133.
(4) Wilde, C.; Chhatwal, G. S.; Schmalzing, G.; Aktories, K.; Just, I.
J. Biol. Chem. 2001, 276, 9537–9542.
(5) Fong, P. C.; Boss, D. S.; Yap, T. A.; Tutt, A.; Wu, P. J.; Mergui-
Roelvink, M.; Mortimer, P.; Swaisland, H.; Lau, A.; O’Connor, M. J.;
Ashworth, A.; Carmichael, J.; Kaye, S. B.; Schellens, J. H. M.; de
Bono, J. S. N. Engl. J. Med. 2009, 361, 123–134.
(6) 2,3,5-Tri-O-Ac-ꢀ-D-ribofuranosyl azide was prepared according to
Stimac, A.; Kobe, J. Carbohydr. Res. 1992, 232, 359–365, and
subsequently deacetylated using Zemplen conditions.
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Mono-ADP-Ribosylated Oligopeptides
A R T I C L E S
Figure 1. Example of mono-ADP-ribosylation of a protein at an asparagine residue.
Scheme 1. Ribosylated Asn and Gln Fmoc Amino Acids
(R:ꢀ ) 3:1), respectively.7 In addition a minor side reaction
was detected in both cases, originating from migration of the
acetyl group from the 2′ O to the 1′ NH.
After separation by silica gel column chromatography, the
identities of the individual anomers of both asparagine (4) and
glutamine building blocks (5) were established by means of
NOESY-NMR spectroscopy.8
With suitably protected ribosylated Gln and Asn building
blocks in hand, the synthesis of mono-ADP-ribosylated dipeptide
18 in solution was undertaken (see Scheme 2).9 Peptide fragment
18 is derived from the mammalian Rho protein, which is ADP-
ribosylated by the Clostridium botulinum C3 toxin at the Asn-
41 residue.10 The partially protected precursor 17 of target
dipeptide 18 was provided with base labile protecting groups
(Fm, Fmoc, Ac, and Cbz carbonate) to permit a straightforward
alkaline deprotection procedure in the final stage of the synthesis.
The route of synthesis started with the preparation of suitably
protected tyrosine derivative 10 (Scheme 2) by conversion of
commercially available Boc-Tyr(OBn)-OH into Fm ester 8,
followed by hydrogenolysis of the benzyl ether and finally
installation of the Cbz group at the released phenolic hydroxyl
function. Acidic removal of the Boc group in 10 and EDC-
assisted coupling to ribosylated Asn building block 6 resulted
in clean formation of dipeptide 11. After unmasking the 5′-OH
with HF/pyridine both the phosphate and the pyrophosphate
functionality were introduced by adaptation of our previously
published procedure.11 Phosphate monoester 14 was obtained
by phosphitylation of ribosylated dipeptide 12 with bis(p-
methoxybenzyl)-N,N-diisopropylphosphoramidite 13 and activa-
tor dicyanoimidazole (DCI), followed by oxidation of the
intermediate phosphite triester with tBuO2H in nonane and
finally p-methoxybenzyl removal using 3% TFA. Similar
phosphitylation of adenosine 15 and changing the oxidation
conditions to iodine in pyridine afforded the putative intermedi-
ate 16, as an activated phosphorimidazolidate suitable for
pyrophosphate formation. Addition of crude 14 to in situ formed
16 resulted in the partially protected mono-ADP-ribosylated
dipeptide 17, which was purified using RP-HPLC. Removal of
Fm and Fmoc groups using DBU, followed by cleavage of the
remaining acyl functions using aqueous ammonia and purifica-
tion by gel filtration, provided us with 4.1 mg (4.9 µmol) of
the homogeneous target compound 18.
The successful synthesis of the mono-ADP-ribosylated dipep-
tide 18 prompted us to investigate an approach toward elongated
peptide fragments. Having established an efficient solution phase
method for the incorporation of ribosylated amino acid 6 as
well as a suitable solution phase method to prepare phosphate
and pyrophosphate moieties with the aid of phosphitylating agent
13, we were curious to find out whether these methods were
also applicable on solid phase synthesis. Two complementary
procedures are conceivable to introduce the pyrophosphate
(7) Boschelli, D. H.; Powell, D.; Sharky, V.; Semmelhack, M. F.
Tetrahedron Lett. 1989, 30, 1599–1600.
(8) See Supporting information.
(9) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A
Practical Approach; Oxford University Press: New York: 2000.
(10) Sekine, A.; Fujiwara, M.; Narumiya, S. J. Biol. Chem. 1989, 264,
8602–8605.
(11) van der Heden van Noort, G. J.; Verhagen, C. P.; van der Horst, M. G.;
Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V. Org. Lett.
2008, 10, 4461–4464.
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A R T I C L E S
van der Heden van Noort et al.
Scheme 2. Synthesis of Mono-ADP-ribosylated Dipeptide 18
function by reacting a phosphate monoester and an activated
phosphorimidazolidate. The phosphate monoester-equipped pep-
tide can be reacted with the nucleotide phosphorimidazolidate,
or by reversing functionalities, the phosphorimidazolidate of the
ribosylated peptide can be reacted with the phosphate monoester
of the nucleotide. ADP-ribosylated peptides 21 and 25 (see
Scheme 3) were constructed each using a different approach to
on-resin pyrophosphate formation.
We opted to investigate the first procedure for on-resin
pyrophosphate introduction, having the reactive phosphorimi-
dazolidate at the 5′ hydroxyl of immobilized ribosylated peptide
19 and adding the phosphate monoester in solution, on model
hexapeptide 21 (see Scheme 3) containing an ADP-ribosylated
Asn residue. We selected 4-hydroxymethylbenzoic acid (HMBA)
as a suitable linker allowing the removal of the base labile
protective groups and the release of the ADP-ribosylated peptide
from the resin in a single step.12 Hexapeptide 19 was assembled
on commercially available Tentagel resin equipped with the
HMBA linker using a BOP/HOBt Fmoc-based SPPS protocol.9
En route to immobilized 19 the incorporation of ribosylated Asn
building block 6 as well as ensuing repetition of the amino acid
coupling cycle proceeded uneventfully, as was determined by
LC-MS analysis of crude ribosylated peptide, obtained after
cleavage from the solid support and removal of the protecting
groups in 19 on an analytical sample.13 Unmasking of the 5′
hydroxyl of ribose 19 with TEA/HF in pyridine permits the
introduction of the ADP moiety. The activated phosphorimi-
dazolidate was installed at the 5′ hydroxyl of the immobilized
peptide 20 using DCI-assisted phosphitylation with reagent 13
and subsequent oxidation using iodine in pyridine. Addition of
6-N-benzoyl-2′,3′-di-O-isobutyryladeninyl-5′-monophos-
phate11 led to formation of the immobilized and protected ADP-
ribosylated peptide. Next the Dmab group on the glutamic acid
residue side chain was removed by a two-step procedure,14 and
subsequent removal of the remaining protecting groups and
concomitant cleavage from the resin were effected by treatment
with methanolic ammonia to give C-terminal methyl ester 21.
LC-MS analysis of the crude reaction mixture showed besides
the main product 21 the presence of a few side products, of
which the C-terminal carboxamide, the terminal monophosphate,
and the corresponding H-phosphonate could be identified.15
We explain the formation of H-phosphonate by invoking a
partial degradation of the phosphite triester on the resin prior
(13) See Supporting information for LC-MS traces of crude ribosylated
peptides.
(12) Furthermore HMBA is reported to be a good choice to afford thioesters,
which in the future will be convenient in the synthesis of larger
m-ADP-ribosylated peptides or proteins using native chemical ligation.
(14) Conroy, T.; Jolliffe, K. A.; Payne, R. J. Org. Biomol. Chem. 2009, 7,
2255–2258.
(15) Story, S. C.; Aldrich, J. V. Int. J. Pept. Protein Res. 1992, 39, 87–92.
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Mono-ADP-Ribosylated Oligopeptides
A R T I C L E S
Scheme 3. Mono-ADP-ribosylated Peptides 21 and 25
to iodine oxidation; the presence of the monophosphate was
attributed to hydrolysis of the phosphorimidazolidate due to
incomplete coupling of the incoming phosphate monoester.
Nevertheless, 0.8 mg (0.6 µmol) of the pure mono-ADP-
ribosylated hexapeptide 21 could be obtained after two RP-
HPLC cycles, and the identity of the product was ascertained
by 31P NMR spectroscopy and high-resolution mass spectrometry.
In the second procedure to introduce the pyrophosphate
linkage the phosphate monoester was installed at the 5′ hydroxyl
residue of immobilized ribosylated peptide 24, while phospho-
rimidazolidate 16, prepared in solution, is added to the solid
support. This has the potential to be more effective, as unwanted
hydrolysis of the intermediate phosphorimidazolidate can be
counterbalanced by the addition of an excess of 16. We opted
to use hexapeptide 24 (see Scheme 3), containing an ADP-
ribosylated Gln residue. ADP-ribosylated heptapeptide 25 is an
isosteric fragment of the naturally occurring mono-ADP-
ribosylated N-terminus of human histone H2B (Scheme 3).16
The peptide moiety is N-linked to ADP-ribose, whereas in nature
the histone H2B is O-linked via glutamic acid. Precursor
heptapeptide 22 was prepared according to the same procedure
as described for the synthesis of 19. Also in this case the
incorporation of ribosylated Gln building block 7 proceeded
uneventfully.13 Ensuing protecting group manipulations replaced
the trityl protecting group on the serine residues for the base
labile acetyl and released the 5′ hydroxyl of ribose to give
suitably protected peptide 24. On-resin phosphate monoester
formation was attained by phosphitylation with 13 as described
above, oxidation of the intermediate phosphite triester with
tBuO2H, and p-methoxybenzyl removal using 3% TFA. Ensuing
treatment of the resin with an excess of phosphorimidazolidate
16 gave the immobilized protected precursor of target 25.
Complete deprotection and cleavage from the solid support was
effected by treatment with methanolic ammonia. Analysis of
the crude mixture by LC-MS showed besides the main product
25 again the terminal monophosphate and the corresponding
H-phosphonate as side products. Purification of the mixture by
RP-HPLC followed by gel filtration afforded 1.3 mg (1.0 µmol)
of homogeneous ADP-ribosylated heptapeptide 25. The forma-
tion of the peptide H-phosphonate in the course of the
phosphitylation reaction proved to be a significant hindrance
en route to the target ADP-ribosylated peptides, and in order to
suppress this unwanted transformation, we reinvestigated the
phosphitylation reaction of 13 on the immobilized ribosylated
peptide. Unfortunately it proved to be impossible to suppress
the formation of the H-phosphonate by variation of the excess
of 13 and the reaction time. Our attempts to use an alternative
phosphorylation method involving the treatment of the im-
mobilized peptide with POCl3 in the presence of pyridine did
not result in an efficient formation of the targeted phosphate
monoester on resin.17
A number of biologically relevant applications can be
envisaged for the peptides accessible by the methodology
described here. One line of research would be the attachment
of ADP-ribosylated peptides, such as the ones prepared in this
study, to a carrier protein as well-defined haptens to generate
antibodies against the ADP-ribose modification. Such tools
might prove invaluable in the identification of mono-ADP-
ribosylated cellular proteins, an intriguing but little understood
post-translational modification.18 In this fashion peptide 25 may
find use in the discovery of the hitherto unknown ribosylation
on the Gln side chain. In an alternative line of research, ADP-
(17) Zhang, B.; Bailey, V. C.; Potter, B. V. L. J. Org. Chem. 2008, 73,
1693–1703.
(16) Ogata, N.; Ueda, K.; Hayaishi, O. J. Biol. Chem. 1980, 256, 7610–
(18) Di Girolamo, M.; Dani, N.; Stilla, A.; Corda, D. FEBS J. 2005, 272,
7615.
4565–4575.
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A R T I C L E S
van der Heden van Noort et al.
ribosylated peptides 18, 21, and 25 or closely related analogues
represent well-defined substrates for elongation by enzymes of
the PARP family. As certain small peptides are reported to be
accepted as substrates for PARP-1 (nonapeptide) and ADP-
ribose protein lyase (pentapeptide, PE(ADP-ribosyl)PAK), a
better insight into the mechanism of the action of PARP
enzymes can be attained by using synthetic mono-ADP-
ribosylated peptides as substrates in elongation assays.19 Mono-
ADP-ribosylated peptides containing ADP-ribosylated glutamine
(prepared using Fmoc amino acid 7), if indeed accepted by
PARPs as substrates, allow preparation of poly-ADP-ribosylated
proteins containing a stabilized ribose-protein linkage, when
combined with established chemical ligation tactics to extend
the polypeptide chain.20 Such constructs are unattainable by
current biochemical techniques.
allowed us to synthesize well-defined ADP-ribosylated peptides
for the first time and in milligram amounts, which can be useful
for biological experiments. Further efforts will be made to
improve the yield of the immobilized phosphate monoester and
possibly to increase the efficiency in pyrophosphate formation.
Apart from this we will also direct our research toward the
synthesis of other ribosylated amino acids such as Arg, Cys,
and Glu and more elaborated well-defined synthetic ADP-
ribosylated peptides.
Acknowledgment. This work was funded by the Dutch
Organization for Scientific Research (NWO).
Supporting Information Available: Spectroscopic data and
experimental procedures are available free of charge via the
Internet at http://pubs.acs.org.
Conclusion
JA910940Q
In conclusion we have presented a convenient method for
the synthesis of suitably protected ribosylated asparagine (6)
and glutamine (7) building blocks amenable to straightforward
incorporation in the solution and the solid phase peptide
synthesis schemes. These modified amino acid building blocks
(19) Tao, Z. H.; Gao, P.; Liu, H. W. J. Am. Chem. Soc. 2009, 131, 14258–
14260.
(20) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem., Int. Ed. 2008,
47, 10030–10074.
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