A general synthetic method toward uridylylated picornavirus VPg proteins

Article abstract of DOI:10.1002/psc.2508

Small proteins called viral protein genome-linked (VPg), attached to the 5′-end of the viral RNA genome are found as common structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block in an on-line solid phase-based approach. Copyright 2013 European Peptide Society and John Wiley & Sons, Ltd. The replication of picornaviruses is primed by a small so-called VPg protein linked to a uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block in an on-line solid phase based approach.

Full text of DOI:10.1002/psc.2508

Protocol
Received: 16 January 2013
Revised: 27 February 2013
Accepted: 27 February 2013
Published online in Wiley Online Library: 19 April 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2508
A general synthetic method toward
uridylylated picornavirus VPg proteins
Gerbrand J. van der Heden van Noort,a Catherine H. Schein,b
Herman S. Overkleeft,a Gijsbert A. van der Marela and Dmitri V. Filippova*
Small proteins called viral protein genome-linked (VPg), attached to the 50-end of the viral RNA genome are found as common
structure in the large family of picornaviruses. The replication of these viruses is primed by this VPg protein linked to a single
uridylyl residue. We report a general procedure to obtain such nucleoproteins employing a pre-uridylylated tyrosine building block
in an on-line solid phase-based approach. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: viral nucleoprotein; uridylylation; post-translational modification; on-line peptide synthesis
Scope and Comments
*
Correspondence to: Dmitri V. Filippov, Leiden Institute of Chemistry, Leiden Uni-
versity, P. O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: filippov@chem.
leidenuniv.nl
The replication of many eukaryotic and plant viruses requires a
‘viral protein genome-linked’ (VPg) protein to prime polymerase
mediated RNA synthesis [1–3]. For example, picornaviruses is a
family of (+)-stranded RNA viruses that includes foot and mouth
disease virus and human enteroviruses (HEV), a large group of
pathogens that include poliovirus (PV), coxsackievirus and
human rhinovirus [4]. All picornaviruses share the common
a Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, RA, 2300
Leiden, The Netherlands
b Department of Microbiology and Immunology, University of Texas Medical
Branch, Galveston, TX 77555-0857, USA
J. Pept. Sci. 2013; 19: 333–336
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

VAN DER HEDEN VAN NOORT ET AL.
feature of a VPg covalently linked to the 50-end of the viral genome
[5,6]. This specific protein is required in the priming mechanism of
viral replication, although its exact mode of action remains unclear
[7–17]. Understanding the crucial role of this RNA linked protein in
viral replication may lead to the development of new vaccines and
drugs against diseases caused by these viruses. Initial reports by our
group described the synthesis of uridylylated VPg’s of PV using a
block condensation strategy [18] and an online SPPS synthesis
[19,20] as well as the preparation of other nucleotidylated VPg’s
of PV [21]. These synthetically made nucleoproteins indeed proved
to be useful tools in research on PV replication [22–24]. Because the
sequence of VPg’s in HEV is diverse and those from the more dis-
tantly related picornaviruses such as foot and mouth disease virus
even more so, we set out to demonstrate that our synthetic
methods could be extended from the well-documented synthesis
of PV VPg’s to cover many different related peptide sequences be-
longing to other picornaviral pathogens. We now demonstrate a
general sequential on-line SPPS method to obtain the VPgpU nu-
cleoprotein from two coxsackie strands (A24 and B3W), HEV 71, a
consensus sequence for HEV [25] and PV 1 (see Figure 1). We envi-
sion this method, which employs a pre-uridylylated tyrosine build-
ing block, to be universal for the synthesis of all O4-Tyr linked
VPgpU’s. Guided by these results, we anticipate that this synthetic
method will also be applicable for the preparation of intermediates
that are involved in RNA polymerase priming by other VPg
proteins, such as those of plant pathogens [26,27]. The synthetic
nucleotidylated peptides described in this paper may prove useful
in studying the interaction with polymerases of picornaviruses to
provide further details of the replication mechanism. This should
aid in the design of novel viral reproduction inhibitors.
To avoid this unwanted side reaction, silver carbonate was used
as a base and 7 was obtained in quantitative yield. Subsequent
removal of the t-butyl ether using TFA provided phenol 8 quantita-
tively. Phosphitylation of 8 with uridine-50-phosphoramidite 9
under the agency of 4,5-dicyanoimidazole and subsequent
oxidation of the intermediate phosphite using t-BuOOH yielded
fully protected tyrosine 10 in excellent yield. Liberation of the acid
functionality by palladium catalyzed removal of the allyl protecting
group using N, N-dimethylbarbituric acid as scavenger, gave target
tyrosine derivative 11, which was used in the solid phase peptide
synthesis towards the target nucleoproteins.
Solid Phase Peptide Synthesis
The solid phase synthesis of VPgpU’s 1b–5b is exemplified by the
preparation of coxsackie A24 VPgpU 1b outlined in Scheme 2
and was started with HATU-mediated coupling of Fmoc-Glu-OtBu
to immobilize glutamic acid residue via the side chain acid moiety
to Rink amide resin (Tentagel RAM). This procedure ensures that
during the final acidic cleavage of the VPgpU from the resin, a
carboxamide is generated, converting the initially attached glutamic
acid into the desired C-terminal glutamine residue (see Scheme 2).
Subsequent removal of the Fmoc protection and repetitive
elongation of the resin bound peptide using standard automated
Fmoc solid-phase peptide synthesis and HATU as coupling reagent,
gave the immobilized 19-mer peptide 12. The immobilized peptide
12 was elongated by performing a manual double coupling protocol
using Fmoc-Tyr(pU)-OH (11) and BOP/HOBt as coupling reagent.
Subsequent piperidine treatment liberated the terminal amine and
concomitantly cleaved the cyanoethyl group resulting in the forma-
tion of phosphodiester. The presence of the phosphodiester gave no
problems in the subsequent attachment of the last two N-terminal
amino acids, as no side reactions were detected. Final removal of
the Fmoc group followed by treatment with a mixture of 95% TFA,
2.5 % TIS, and 2.5% H2O, to effectuate removal of acid labile protec-
tive groups and release of the product from the solid support yielded
crude partially protected VPgpU. Treatment with NH4OH in dioxane
to remove 20-O and 30-O acetyl groups from the ribose residue
yielded the completely deprotected crude nucleopeptide, which
was purified using RP-HPLC to afford homogeneous coxsackie A24
VPgpU nucleoprotein 1b. The parent genome linked protein (1a)
could be obtained straightforwardly by elongation of resin bound
peptide (12) with the non-uridylylated tyrosine derivative Fmoc-Tyr
(tBu)-OH. After a final Fmoc-deprotection and acid treatment, the
crude viral protein could be purified using RP-HPLC to give
Experimental Procedure
Synthesis of Uridylylated Tyrosine Building Block
Uridylylated tyrosine building block 11 was selected for the solid
phase synthesis of the target VPgpU’s (see Scheme 1). The synthetic
pathway toward this key intermediate was optimized by the
adaptation of the phosphitylation/oxidation and deprotection
conditions used in prior preparations of analogues compounds
[18,19,21]. A modified route of synthesis to 11 was undertaken that
started with the protection of commercially available Fmoc-Tyr
(tBu)-OH 6 as allyl ester (see Scheme 1). When cesium carbonate
and allyl bromide were used to introduce the allyl ester as initially
reported, the Fmoc group in 6 was found to be partially cleaved.
Figure 1. Coxsackievirus A24, coxsackievirus B3W, human enterovirus 71, consensus human enterovirus, and poliovirus 1 VPg(pU)’s.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 333–336

SYNTHETIC METHOD TOWARD URIDYLYLATED PICORNAVIRUS VPg PROTEINS
Scheme 1. Synthesis of uridylylated tyrosine building block 11. Reagents and conditions: (i) Ag2CO3, DMF, r.t., 15 min; then All-Br, 2.5 h, r.t., (quantitative), (ii) 70%
TFA in DCM, 30 min, r.t., (quantitative), (iii) 4,5-dicyanoimidazole, MeCN, 30 min, r.t., (iv) t-BuOOH, 45 min, rt, (90% based on 8), v. Pd(PPh3)4, N,N-dimethylbarbituric
acid, THF, 2 h, r.t., (92%).
Scheme 2. SPPS for Coxsackie A24 VPg 1a and VPgpU 1b. Solid phase peptide synthesis cycle: (1) 20% piperidine in DMF, (2) Fmoc-AA-OH, HATU, DiPEA in
NMP, (3) Ac2O, HOBt, DiPEA in NMP, (i) 20% piperidine in DMF, (ii) 95% TFA, TIS, H2O, (iii) NH3 in dioxane (only for nucleotidylated peptides).
homogeneous VPg 1a. In a similar fashion, VPgU’s (2b–5b) and
VPg’s (2a–5a) of different species were prepared conveniently
and in a moderate to good yield.
to be more difficult to synthesize using this approach. In the final
alkaline treatment b-elimination could occur, causing the formation
of dehydroalanine and cleavage of the phosphodiester linkage. This
limitation might also apply to nucleopeptides containing unusually
base labile peptide bonds.
Viral protein genome linked to longer RNA fragments cannot
be accessed using this method, even if the corresponding
oligonucleotidylated amino acid building block is crafted, because
the cleavage conditions used to release the final construct from
the resin will almost certainly lead to isomerization of the
internucleosidic phosphodiesters in the oligoribonucleotide and pos-
sibly to partial depurination as well.
Limitations
The method described in this paper is developed for the synthe-
sis of picornaviral VPgpU’s, which are uridylylated at the tyrosine
residue. Nucleopeptides of some other viruses contain nucleo-
tides connected via a phosphodiester to a serine or threonine
residue in the peptide chain. Such nucleopeptides might prove
J. Pept. Sci. 2013; 19: 333–336 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

VAN DER HEDEN VAN NOORT ET AL.
13 Shen M, Reitman ZJ, Zhao Y, Moustafa I, Wang Q, Arnold JJ, Pathak HB,
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Supporting Information
Supporting information may be found in the online version of
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Experimental procedures and spectroscopic data for all com-
pounds, copies of 1H and 31P NMR spectra for (nucleo)proteins 1–5
are provided. This material is available free of charge via the Internet.
Acknowledgement
This work has been funded by the Dutch Organisation for Scientific
Research (NWO).
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