Stepwise solid phase synthesis of uridylylated viral genome-linked peptides using uridylylated amino acid building blocks

Article abstract of DOI:10.1016/S0040-4020(03)00042-5

The uridylylated amino acid building blocks 2-cyanoethyl-(N^α-9-fluorenylmethoxy-carbonyl-tyrosin-4-yl)- (2′,3′-di-O-acetyluridin-5′-yl) phosphate and 2-chlorophenyl-(N^α-fluorenyl-methoxycarbonyl-serin-3-yl)- (2′,3′-di-O-acetyluridin-5′-yl) phosphate have been used successfully in an on-line SPPS of the VPgpU from the polio, coxsackie and cowpea mosaic virus.

Full text of DOI:10.1016/S0040-4020(03)00042-5

Tetrahedron 59 (2003) 1589–1597
Stepwise solid phase synthesis of uridylylated viral genome-linked
peptides using uridylylated amino acid building blocks
Nicole M. A. J. Kriek,a Dmitri V. Filippov,a Hans van den Elst,a Nico J. Meeuwenoord,a
Godefridus I. Tesser,b Jacques H. van Booma and Gijs A. van der Marela
,
*
aGorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
bLaboratory of Organic Chemistry, Catholic University Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands
Received 9 September 2002; revised 9 December 2002; accepted 2 January 2003
Abstract—The uridylylated amino acid building blocks 2-cyanoethyl-(N a-9-fluorenylmethoxy-carbonyl-tyrosin-4-yl)-(20,30-di-O-acetyl-
uridin-50-yl) phosphate and 2-chlorophenyl-(N a-fluorenyl-methoxycarbonyl-serin-3-yl)-(20,30-di-O-acetyluridin-50-yl) phosphate have been
used successfully in an on-line SPPS of the VPgpU from the polio, coxsackie and cowpea mosaic virus. q 2003 Elsevier Science Ltd. All
rights reserved.
1. Introduction
Virus replication in the infected host is a two-step process,
carried out primarily by the RNA polymerase in conjunction
with other viral and possibly also cellular proteins. The
incoming viral RNA is transcribed into complementary
minus strands, which are then used as templates for the
synthesis of the progeny plus strands. Although the basic
steps are well known, very little is understood about the
details of these processes and particularly about the exact
function of the cis-acting RNA structures contained within
picornaviral RNAs.3 Thus, one of the important unanswered
Poliovirus is perhaps the best known member of the
Picornaviridae, a family of plus-stranded RNA viruses
including a large number of pathogens with widely different
host range and disease symptoms.1 An unusual but common
feature of the picornaviruses genome is the presence of a
small protein, VPg (viral protein genome-linked), cova-
lently attached to the 50-end of the plus-stranded viral
genome which in turn is terminated with a poly(A)-tail.2
Figure 1. VPgpUs from poliovirus (1b), coxsackie virus (2b) and cowpea mosaic virus (3b).
Keywords: genome-linked peptides; uridylylated amino acid building blocks; solid phase peptide synthesis; Rink Amide resin.
*
Corresponding author. Tel.: þ31-71 5274280; fax: þ31-71-5274307; e-mail: g.marel@chem.leidenuniv.nl
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00042-5

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Chart 1. Synthesis of poliovirus VPgpU by fragment condensation.5
questions about minus-strand synthesis is how the viral
RNA polymerase recognizes and selects only its own RNA
as template. It has been shown4 that the postulated first step
in the replication of the plus-stranded poliovirus RNA could
be initiated using both synthetic VPg (1a, Fig. 1) and
VPgpU5 (1b) as primers. For example, uridylylation of 1a
with uridine triphosphate, Mg2þ ions, purified poliovirus
RNA polymerase 3Dpol and using poly(A) as a template led
to the formation of VPg-poly(U), the 50-end of the minus
strand RNA.4 It was envisaged that the poly(A)-dependent
process would confer sufficient specificity to poliovirus
RNA replication. However, recent studies6 clearly indicated
that a small RNA hairpin in the coding region of the protein
2C in poliovirus RNA serves as the primary template in the
in vitro uridylylation of VPg (1a).
transcripts VPgpU (see Figure 1) from polio (1b),
coxsackie7 (2b) as well as cowpea mosaic8 (3b) virus.
2. Results and discussion
The synthetic route to VPgpU (1b) devised earlier in our
laboratory5 comprised, as denoted in Chart 1, a fragment
condensation of the N-terminal allyloxycarbonyl (Alloc)
protected peptide 4 with the Alloc side-chain protected
C-terminal allyl (All) ester peptide 5, and subsequent
removal under neutral conditions of the protecting groups.
Both fragments were readily accessible by a stepwise solid
phase peptide synthesis (SPPS) based on Fmoc-chemistry.
Thus, sequential elongation of the acid-labile 4-hydroxy-
methyl-3-methoxyphenoxybutyric acid (HMPB)-Tentagel
resin with the appropriately protected amino acids and
the uridylylated tyrosine unit 6 gave, after cleavage and
With the objective of studying the latter process in more
detail, we here report an online SPPS of the initially formed
Scheme 1. Synthesis of VPgpU 1b from poliovirus. Reagents and conditions: (i) (a) Cs2CO3 (0.98 equiv.), DMF, 08C!room temperature, 20 min;
(b) 2-chlorotrityl chloride (1.1 equiv.), DMF, room temperature, 30 min. (ii) 9 (1 equiv.), o-NPT (2 equiv.), CH2Cl2, room temperature, 10 min. (iii) t-BuOOH,
CH2Cl2, room temperature, 30 min. (iv) TFA/CH2Cl2 (2/98, v/v), TES, room temperature, 1 h, 38% yield based on 7.

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1591
Scheme 2. Synthesis of VPgpU 1b from poliovirus. Reagents and conditions: (i) repeat (21£) the following three-step cycle: (a) 2% DBU in DMF, room
temperature, 5 min; (b) Fmoc-AA-OH (5 equiv.), BOP (5 equiv.), HOBt (5 equiv.), DiPEA (10 equiv.), NMP, room temperature, 1 h; (c) Ac2O/
DiPEA/NMP/HOBt, room temperature, 1 min. (ii) 2% DBU in DMF, room temperature, 5 min. (iii) TFA/TIS/H2O (95/2.5/2.5, v/v/v), room temperature, 2 h.
(iv) 25% conc. NH3·H2O/1,4-dioxane (1/1, v/v), room temperature, 3 h. (iv) RP-HPLC purification.
partial unmasking, the uridylylated fragment 4. Similarly,
the partially protected0 sequence 5 was obtained by
elongation of the 4-(20,4 -dimethoxyphenyl-aminomethyl)-
phenoxyacetamido-L-norleucyl-MBHA resin (Rink Amide
MBHA resin) followed by cleavage and concomitant
removal of the acid-labile side-chain protecting groups.
This rather elaborate and time-consuming approach was an
incentive to explore the feasibility of constructing VPgpU
1b and 2b via a continuous solid phase synthesis. In this
respect, it was essential to find out whether both target
compounds would be unscathed by the harsh acidic
conditions required for the deprotection of the acid-labile
side-chain protecting groups (cf. Chart 1) of the amino
acids introduced in the on-line SPPS of 1b and 2b based on
Fmoc-chemistry. To this end, the uridylylated tyrosine
building unit 6 (see Chart 1) was subjected to neat
trifluoroacetic acid (TFA). Work-up of the mixture, after
24 h at 208C, led to a near quantitative recovery of 6,
indicating that the phosphodiester in both target peptides
will be compatible with the unblocking of the side-chain
protecting groups in the final stage of the synthesis of
VPgpU 1b and 2b. It also turned out that the number of steps
in the synthesis of the earlier reported5 uridylylated tyrosine
building block 6 could be reduced following the procedure
depicted in Scheme 1. Thus, tritylation of the cesium-salt
of Fmoc-Tyr (7) with 2-chlorotrityl chloride led, after
work-up, to the isolation of 8. Phosphitylation of the
phenolic hydroxyl in crude 8 with known5 2-cyanoethyl-
(20,30-di-O-acetyl-uridin-50-yl)-phosphoramidite (9) in the
presence of 5-(ortho-nitrophenyl)tetrazole (o-NPT),9
followed by in situ oxidation with tert-butylhydroperoxide,
resulted in the isolation of 10. Acidolysis of the 2-chloro-
trityl ester in 10 using triethylsilane (TES) as a trityl cation
scavenger gave after purification the homogeneous tyrosine
building block 6 in 38% overall yield.
VPgpU (1b) is depicted in Scheme 2. Accordingly, the
Fmoc-group in the commercially available glutamic acid
derivative 11, attached via the side-chain to Rink Amide
MBHA resin, was removed with 2% 1,8-diazabicyclo-
[5,4,0]-undec-7-ene (DBU) in N,N-dimethylformamide
(DMF).10 Subsequently, the free amine was condensed
with the suitably protected amino acids under the agency of
benzotriazole-1-yloxy-tris-(dimethylamino)phosphonium
hexafluorophosphate (BOP) and 1-hydroxybenzotriazole
(HOBt) in the presence of N,N-diisopropylethylamine
(DiPEA). The b-branched amino acids, Fmoc-Asn(Trt)-
OH, Fmoc-Arg(Pbf)-OH as well as the key uridylylated
tyrosine building block 6 were incorporated by executing
a double condensation step. Although it is known5 that
removal of the Fmoc group at this stage of the synthesis is
accompanied by b-elimination of the cyanoethyl group, the
resulting phosphodiester is not expected11 to give rise to
the formation of unwanted side-products in the ensuing two
elongation steps. The synthesis of the fully protected
immobilized sequence 12 was completed by double
coupling of the last two amino acids i.e. Fmoc-Ala-OH
and Fmoc-Gly-OH. The nucleopeptide 12 was deprotected
and cleaved from the resin by the following three-step
procedure. First, the N-terminal Fmoc-group was removed
by treatment with 2% DBU in DMF. Secondly, the peptide
was cleaved from the resin with concomitant removal of the
side-chain protecting groups by acidolysis with 95% aq.
TFA in the presence of triisopropylsilane (TIS) as cation
scavenger. Deblocking of the acetyl groups in the uridine
moiety with concentrated aqueous ammonia in 1,4-dioxane
led to the isolation of the crude nucleopeptide 1b. RP-HPLC
purification afforded homogeneous poliovirus VPgpU 1b
(see Figure 2a), the identity of which was ascertained
by 31P NMR (D2O, d 24.1 ppm) and MALDI-TOF-MS
(m/z¼2659.6 [MþH]þ) analysis. The steps involved in the
transformation of 12 into target VPgpU 1b could be reduced
by taking into consideration that the Fmoc group can be
The sequence of events in the stepwise SPPS of poliovirus

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N. M. A. J. Kriek et al. / Tetrahedron 59 (2003) 1589–1597
Figure 2. RP-HPLC trace of (a) poliovirus VPgpU 1b and (b) coxsackie virus VPgpU 2b.
readily removed12 under the same basic conditions as used
in the deacetylation step. Indeed, acidolysis of 12 followed
by treatment with concentrated ammonia led to the isolation
of 1b, which was in every aspect identical to the same
compound obtained via deblocking of 12 by the three-step
protocol.
two-step protocol, as described for the unmasking of 12,
gave crude 2b. Purification by RP-HPLC yielded homo-
geneous coxsackie virus VPgpU 2b (see Figure 2b), the
structure of which was ascertained by 31P NMR (D2O, d
24.2 ppm) and MALDI-TOF-MS (m/z¼2715.9 [MþH]þ).
The uneventful and smooth on-line SPPS of both VPgpU’s
1b and 2b urged us to adopt the same approach to the
preparation of the VPgpU 3b of cowpea mosaic virus
containing an N-terminal serine linked via a phosphodiester
bond to the 50-hydroxyl function of uridine. It is well
established13 that the serine uridylylated moiety in the target
VPgpU 3b will be rather prone, especially so under the
influence of a strong base, to b-elimination leading to the
unwanted dehydroalanine derivative of VPgpU 3b and
The successful outcome of the on-line SPPS of VPgpU (1b)
of poliovirus was an incentive for constructing VPgpU (2b)
from coxsackie virus by the same methodology. Accord-
ingly, elongation of the immobilised glutamic acid deriva-
tive 11, as outlined in Scheme 3, with suitably protected
amino acids proceeded uneventfully to give the immobi-
lized and fully protected uridylylated peptide 13. Deprotec-
tion and cleavage of 13 from the resin following the
Scheme 3. Synthesis of VPgpU 2b from coxsackie virus. Reagents and conditions: (i) repeat (21£) the following three-step cycle: (a) 2% DBU in DMF, room
temperature, 5 min; (b) Fmoc-AA-OH (5 equiv.), BOP (5 equiv.), HOBt (5 equiv.), DiPEA (10 equiv.), NMP, room temperature, 1 h; (c) Ac2O/
DiPEA/NMP/HOBt, room temperature, 1 min. (ii) TFA/TIS/H2O (95/2.5/2.5, v/v/v), room temperature, 2 h. (iii) 25% conc. NH3·H2O/1,4-dioxane (1/1, v/v),
room temperature, 3 h. (iv) RP-HPLC purification.

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1593
Scheme 4. Synthesis of the uridylylated serine derivative 19. Reagents and conditions: (i) Ag2CO3 (1.3 equiv.), AllBr (4 equiv.), DMF, 08C!room
temperature, 3 h, 89%. (ii) 20,30-Di-O-acetyluridine (1 equiv.), 1,4-dioxane. (iii) 15, pyridine/1,4-dioxane, room temperature, 2 h, 87%. (iv) AcOH (4.5 equiv.),
Bu3SnH (2 equiv.), Pd(PPh3)4 (4 mol%), CH2Cl2/THF (1/1, v/v), room temperature, 1 h, 91%.
uridine-50-phosphate. The latter undesired scission can,
however, be decreased substantially using a less strong base.
For example, the integrity of the phosphodiester linkage
can be preserved for a relatively long period of time in
concentrated ammonia.14 Moreover, the same basic con-
ditions can also be used successfully, as mentioned before,
in the unmasking of an Fmoc protecting group.12 It may
therefore be expected that the unblocking of the N-terminal
Fmoc group from the serine uridylylated moiety, introduced
in the final stage of the SPPS, will be compatible with the
presence of the phosphodiester linkage in the target
compound 3b. Previous studies from our laboratory on the
synthesis of serine uridylylated containing peptides
revealed15 that a 2-chlorophenyl protecting group could be
removed under mild conditions16 with fluoride ions (i.e.
TBAF/pyridine/DMF/H2O). The latter operation generates
Scheme 5. Synthesis of VPgpU 3b of cowpea mosaic virus. Reagents and conditions: (i) repeat (26£) the following three-step cycle: (a) 20% piperidine in
DMF, room temperature, 5 min; (b) Fmoc-AA-OH (5 equiv.), BOP (5 equiv.), HOBt (5 equiv.), DiPEA (10 equiv.), NMP, room temperature, 1 h;
(c) Ac2O/DiPEA/NMP/HOBt, room temperature, 1 min; then 20% piperidine in DMF, room temperature, 5 min. (ii) 19 (10 equiv.), DIC (10 equiv.), HOBt
(10 equiv.), CH2Cl2/DMF, (95/5, v/v), room temperature, 1 h. (iii) TBAF (0.25 M) in pyridine/DMF/H2O (3/4/1, v/v/v), room temperature, 2 h.
(iv) TFA/TIS/H2O/PhOH/EDT (90/0.12/0.8/0.12/0.8, v/v/v/w/v), room temperature, 4 h. (v) aq. conc. NH3·H2O (25%)/1,4-dioxane (1/1, v/v), room
temperature, 3 h. (vi) 20% piperidine in DMF, room temperature, 5 min.

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N. M. A. J. Kriek et al. / Tetrahedron 59 (2003) 1589–1597
a phosphodiester function which will survive, as required,
ensuing deblocking steps involving base and acid treat-
ments. On the basis of the foregoing information it was
decided17 to use the serine uridylylated derivative 19 (see
Scheme 4), the phosphodiester function of which is
temporarily protected with the 2-chlorophenyl group, as
the building unit in the on-line SPPS of target VPgpU 3b.
occurrence of b-elimination, the introduction of building
block 19 was executed under slightly acidic conditions
using N,N0-diisopropylcarbodiimide (DIC) and HOBt to
furnish the immobilised and fully protected nucleopeptide
21. Transformation of 21 into VPgpU 3b was performed in
a three-step sequence starting with the removal of the
2-chlorophenyl group by treatment with aq. TBAF. The
peptide was then subjected to TFA-mediated cleavage from
the resin with concomitant removal of all acid-labile
protecting groups. Unmasking of both the N-terminal
amine function as well as the hydroxyl groups with
concentrated aqueous ammonia in 1,4-dioxane yielded
crude VPgpU 3b not contaminated, as evidenced by LC/
MS analysis, with the undesired dehydroalanine derivative
22. Purification by cation exchange followed by desalting,
gave homogeneous (see Figure 3) 3b, the identity of which
was corroborated by ESI-MS (m/z¼1923.4 [Mþ2H]2þ) and
31P NMR (D2O, d 20.1 ppm).
The synthesis of 19 is depicted in Scheme 4 and commences
with the selective allylation of the silver-salt of Fmoc-serine
14 to give allyl ester 15. The uridine phosphate moiety was
then introduced by the following one-pot-two-step pro-
cedure. Thus, phosphorylation of 20,30-di-O-acetyluridine
with the known18 bifunctional phosphorylating agent 16,
and in situ condensation of intermediate 17 with the
protected serine derivative 15, gave the 2-chlorophenyl
phosphotriester derivative 18. Pd(0)-catalyzed hydro-
stannolysis19 of the allyl ester in 18 furnished the serine
building block 19 in 70% overall yield.
On the other hand, subjection of 21 to the following
sequential four-step deblocking process (i.e. fluoride
ions, 20% piperidine, TFA/TIS/H2O/PhOH/EDT and then
ammonolysis) revealed, as gauged by LC/MS analysis, the
presence of the dehydroalanine derivative 22 of VPgpU 3b
as the major product. The latter observation also nicely
confirms the incompatibility of the serine phosphodiester
function, resulting from the fluoride treatment of 21, with
the removal of the Fmoc-group using piperidine as the base.
The synthesis of 3b started, as outlined in Scheme 5, with
the assembly of the immobilized peptide 20. Initially, a
stepwise SPPS of 20 was performed under the same
conditions as previously described for the assembly of
nucleopeptide 1b. However, deprotection and cleavage
from the resin followed by extensive LC/MS analysis
showed the presence of truncated fragments due to
incomplete couplings of the amino acids: [Gln10], [Gln11],
[Tyr12], [Tyr14], [Pro18], [Leu19] and [Lys20]. The latter
could be circumvented by executing double couplings
for these amino acids. The resulting immobilized peptide 20
was then elongated by condensation with 2-chlorophenyl-
N a-(9-fluorenylmethoxycarbonyl)-serin-3-yl-(20,30-di-O-
acetyluridin-50-yl) phosphate 19. To prevent the possible
In conclusion, the results presented in this paper clearly
show that biologically important uridylylated VPg’s can be
readily prepared via an on-line SPPS approach.
3. Experimental
3.1. General methods and materials
Pyridine (Acros Organics), N,N-dimethylformamide
(Baker, p.a.), 1,4-dioxane (Baker, p.a.) and 1,2-dichloro-
ethane (Baker, p.a.) were stored over molecular sieves
˚
(4 A). Acetonitrile (extra dry, DNA synthesis grade) was
purchased from Biosolve. All reagents were obtained from
Acros Chemicals and used as received unless otherwise
stated. Pd(PPh3)2Cl2 was bought from Aldrich. Solvents
used in the automated peptide synthesis, DiPEA and TFA
were all of peptide synthesis grade (Biosolve) and used as
received. BOP was obtained from Senn Chemicals and
anhydrous HOBt from Neosystem. Fmoc-Glu (Rink Amide
MBHA resin)-Ot-Bu, 2-chlorotrityl chloride and the pro-
tected amino acids were bought at NovaBiochem. All the
amino acids applied in the synthesis were the naturally
occurring L-amino acids. TLC analysis was performed on
Merck 25DC Plastikfolien Kieselgel 60 F254. Detection by
UV absorption (254 nm) and spraying with one of the
following solutions: (a) 20% H2SO4 in EtOH followed by
charring; (b) ammonium molybdate (25 g L21)/ceric
ammonium sulfate (10 g L21) in 10% aq. H2SO4 followed
by charring; (c) KMnO4 (10 g L21 in 2% aq. Na2CO3).
Fluka silica gel (230–400 mesh) was used for column
chromatography. The solvents for chromatography were of
technical grade and distilled before use. 1H NMR, 13C NMR
and 31P NMR spectra were recorded with a Bruker AC200
Figure 3. RP-HPLC trace of cowpea mosaic virus VPgpU 2. The purified
product is contaminated with a small quantity (see arrow) of a product
having a mass of 16 m.u. higher than the target VPgpU 3b. The latter
finding may be ascribed to oxidation of the methionine unit in 3b.

N. M. A. J. Kriek et al. / Tetrahedron 59 (2003) 1589–1597
1595
instrument at 200, 50.1 and 80.7 MHz, respectively.
Chemical shifts (d) are given in ppm, relative to tetra-
chloride (1.3 g, 4.4 mmol) was added resulting in a clear
solution followed by rapid formation of a white precipitate.
After stirring for 2 h the reaction was quenched by addition
of methanol (5 mL) and the solvents were evaporated.
The residue was dissolved in EtOAc (100 mL), washed
with water (50 mL) and aqueous NaHCO3 (5%, 50 mL).
The organic phase was dried (MgSO4), filtered and
concentrated in vacuo. Obtained crude title compound 8
was used without further purification in the phosphitylation
reaction.
1
methylsilane as an internal standard for H NMR and 13C
NMR and 85% H3PO4 as an external standard for 31P NMR.
LC/MS analysis was performed on a Jacso HPLC system
(detection simultaneously at 214 and 254 nm) coupled to a
Perkin–Elmer Sciex API 165 mass instrument equipped
with a custom-made Electrospray Interface (ESI). An ana-
lytical Alltima C18 column (Alltech, 4.6 mmD£250 mmL,
5 mm particle size) was used. Buffers: A: H2O, B: CH3CN
and C: 0.5% aq. TFA. For RP-HPLC purification of the
uridylylated peptides a BioCAD ‘Vision’ automated HPLC
system (PerSeptive Biosystems, inc.), supplied with a semi-
preparative Alltima C18 column (Alltech, 10.0 mmD£250
mmL, 5 mm particle size, running at 4 mL min21) was used.
The applied buffers were A: H2O, B: CH3CN and C: 1% aq.
TFA. Detection was performed by UV, simultaneous at
214 and 254 nm. Cation exchange chromatography was
executed on a Porosw 10S column running at 5 mL min21
(PerSeptive Biosystems, inc., 4.6 mm£100 mm) using A:
H2O, B: CH3CN, C: NaCl (3 M) and D: Hepes (0.05 M) in
NaOAc (0.05 M, pH 7.5) as buffer-solutions and detection
at 280 nm. Subsequent desalting was achieved on a Porosw
R2 column (4.60 mm£100 mm, 5 mL min21, PerSeptive
Biosystems, inc.) applying buffers: A: H2O, B: CH3CN and
C: 1% aq. TFA. Detection was performed at 280 nm.
MALDI-TOF-MS spectra were recorded on a Voyager-DE
PRO mass spectrometer (PerSeptive Biosystems, inc.).
3.1.2. 20,30-Di-O-acetyl-50-O-(2-cyanoethyl)-N,N-diiso-
propylaminophosphoramidite (9). 20,30-Di-O-acetyluri-
dine20 (1.3 g, 4.0 mmol) was dried by coevaporation with
1,4-dioxane (2£15 mL) and dissolved in CH2Cl2 (20 mL).
Under a blanket of nitrogen triethylamine (2.7 mL,
19 mmol) was added followed by slow addition of a
solution of 2-cyanoethoxy-N,N-diispropylamino chlorophos-
phine21 (1.1 mL, 4.8 mmol) in CH2Cl2 (10 mL). The
reaction mixture was stirred for 30 min, diluted with
CH2Cl2 (50 mL) and washed with water (40 mL) and aq.
NaHCO3 (5%, 20 mL). The organic phase was dried
(MgSO4), filtered and concentrated. The residue was
dissolved in CH2Cl2 (10 mL) and precipitated by addition
to light petroleum (75 mL). After centrifugation, the
supernatant was carefully decanted and 9 was obtained
as a slightly yellow coloured oil, which was used in the
next step without further purification. 31P NMR (CH2Cl2,
acetone capillary): d 150.2, 149.5.
General procedure for solid phase peptide synthesis
(SPPS). The nucleopeptides 1b, 2b and 3b were prepared
on an ABI 433A (Applied Biosystems, division of Perkin–
Elmer) automatic peptide synthesiser using the FastMocw
peptide synthesis. The peptides0were prepared on a 50 mmol
scale starting from 4-(20,4 -dimethoxyphenyl-amino-
methyl)-phenoxyacetamido-L-norleucyl-MBHA resin (Rink
Amide MBHA resin) to which Fmoc-Glu-Ot-Bu is attached
via the side-chain carboxylic acid functionality (initial
loading: 0.34 mmol g21).
3.1.3. 2-Cyanoethyl-N a-(9-fluorenylmethoxycarbonyl)-
tyrosin-4-yl-(20,30-di-O-acetyluridin-50-yl)phosphate (6).
N a-Fmoc-tyrosine 2-chlorotrityl ester 8 (crude, 4 mmol)
and 20,30-di-O-acetyluridine phosphoramidite 9 (crude,
4 mmol) were coevaporated with CH3CN (2£20 mL) and
dissolved in CH3CN (20 mL). Under nitrogen atmosphere
dry o-NPT (1.5 g, 8 mmol) was added and the reaction
mixture was stirred for 1 h after which 31P NMR analysis
(acetone capillary, d 135.3 ppm) showed complete con-
sumption of the amidite. Subsequently, t-BuOOH (1.9 mL,
16 mmol) was added and stirring was continued for 30 min.
The mixture was poured into EtOAc/H2O (2/1, v/v, 60 mL)
and the layers were separated. The organic layer was
washed with aq. NaHCO3 (5%, 25 mL), dried (MgSO4),
filtered and concentrated to give crude 10. 31P NMR
(CDCl3): d 26.1; 26.2. Crude 10 was dissolved in
TFA/CH2Cl2 (2/98, v/v, 10 mL) and stirred for 10 min
followed by addition of TES (0.5 mL). After 10 min the
addition of TFA/CH2Cl2 and TES with intermediates of 10
and 5 min respectively, was repeated twice. The reaction
mixture was concentrated and purified (silica gel, light
petroleum/EtOAc/MeOH, 1/1/0!0/95/5, v/v/v). Concen-
tration of the appropriate fractions furnished title compound
6 (1.3 g, 1.5 mmol, 38% based on Fmoc-tyrosine) as an oil.
13C NMR (CDCl3/MeOD/D2O, 45/50/5, v/v/v): d 173.4
(CvO Tyr), 169.8 (2£CvO Ac), 163.9 (C-4 U), 155.8
(CvO Fmoc), 150.4 (C-2 U), 144.9 (Cq Tyr), 143.4, 140.9
(Cq Fmoc), 140.1 (C-6 U), 134.2 (Cq Tyr), 130.7, 127.3,
126.7, 124.7, 119.5 (Carom Fmoc, Tyr), 116.4 0(CN), 102.7
(C-5 U), 87.6 (C-10), 72.2, 69.3, 66.4 (C-20, C-3 , C-40), 66.4
(CH2 Fmoc), 63.0 (C-50), 54.7 (aCH Tyr), 46.7 (CH Fmoc),
36.7 (bCH2 Tyr), 19.7, 19.1 (2£CH3 Ac). ESI-MS: m/z¼
847.5 [MþH]þ, 869.4 [MþNa]þ.
The consecutive steps performed in each cycle were:
1. Deprotection of the Fmoc-group with 2% DBU in DMF
for 5£1 min, unless stated otherwise.
2. Coupling of the appropriate amino acid applying a five-
fold excess. Generally, the amino acid (0.25 mmol) was
dissolved in NMP (0.5 mL) and subsequently 0.25 mmol
of BOP/HOBt (0.5 M BOP/0.5 M HOBt in DMF/NMP
1/1, v/v) and 0.63 mmol of DiPEA (1.25 M in NMP)
were added. The resulting solution was transferred to
the reaction vessel, which was then shaken for 1 h.
3. The unreacted amino functions were capped by acetyl-
ation with 0.5 M acetic anhydride, 0.125 M DiPEA and
0.015 M HOBt in NMP. The resulting suspension was
shaken for 1 min.
3.1.1. N a-(9-Fluorenylmethoxycarbonyl)-tyrosine
2-chlorotrityl ester (8). To a cooled solution (08C) of
N a-Fmoc-tyrosine 7 (1.6 g, 4.0 mmol) in DMF (50 mL)
was added a solution of Cs2CO3 (0.64 g, 1.9 mmol) in water
(1 mL). The reaction mixture was stirred at room tempera-
ture for 20 min and concentrated in high vacuum. The
residue was suspended in DMF (50 mL) and 2-chlorotrityl

1596
N. M. A. J. Kriek et al. / Tetrahedron 59 (2003) 1589–1597
3.1.4. Poliovirus VPgpU (1b). Protected peptide 12 was
synthesised as described in the general procedure for solid
phase peptide synthesis starting from Fmoc-Glu (Rink
Amide MBHA resin)-Ot-Bu 11 (147 mg, 50 mmol,
0.34 mmol g21). The following amino acid derivatives
1H, vCH All), 5.70 (d, 1H, J¼6.58 Hz, NH), 5.31 (t, 2H,
J¼17.55 Hz, J¼11.69 Hz, vCH2 All), 4.70 (d, 2H, J¼
5.12 Hz, OCH2 All), 4.44 (d, 2H, J¼6.58 Hz, OCH2 Fmoc),
4.24 (t, 1H, J¼ 6.58 Hz, J¼7.31 Hz, CH Fmoc), 4.02 (m,
3H, aCH, bCH2 Ser). ESI-MS: m/z¼368.2 [MþH]þ, 390.3
[MþNa]þ.
were
applied:
Fmoc-Ala-OH,
Fmoc-Asn(Trt)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-
Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Thr
(t-Bu)-OH and Fmoc-Val-OH. Amino acid derivative 6
was coupled twice by a standard coupling cycle. A small
portion of the fully protected immobilised peptide 12
(100 mg, approximately 12 mmol) was transferred into a
round bottom flask, a solution of 2% DBU in DMF (5 mL)
was added and the suspension was shaken for 5 min. The
resin was placed on a glass filter and washed with DMF
(5£5 mL). Next, the resin was suspended in a mixture of
TFA/TIS/H2O (95/2.5/2.5, v/v/v, 10 mL) and shaken for
4 h. The resin was filtered, washed with TFA and the filtrate
was concentrated to dryness. The residue was coevaporated
with toluene (2£10 mL) and subsequently dissolved in a
mixture of 25% conc. aq. ammonia and 1,4-dioxane (1/1,
v/v, 10 mL). After 5 h, careful evaporation of the solvents
yielded crude 1b. RP-HPLC purification (linear gradient of
15!20% B in 17 min; 3.5 CV) followed by lyophilisation
furnished nucleopeptide 1b (6.8 mg, 19%, TFA-salt) as a
fluffy solid. 31P NMR (D2O): d 24.14. MALDI-TOF-MS:
m/z¼2659.56 [MþH]þ, 2681.38 [MþNa]þ, 2697.64
[MþK]þ, calculated mass 2659.4. LC/MS analysis: Rt
8.35 min (linear gradient of 5!50% B in 20 min), ESI-MS:
m/z¼2660.0 [MþH]þ, 1330.8 [Mþ2H]2þ, 887.4 [Mþ
3.1.7. O-(2-Chlorophenyl)-N a-(9-fluorenylmethoxycarbo-
nyl-serin-3-yl allyl ester)-(20,30-di-O-acetyl-uridin-50-yl)
phosphate (18). To a stirred solution of 20,30-di-O-
acetyluridine (1.6 g, 5.0 mmol, coevaporated with pyridine
(2£20 mL) in 1,4-dioxane (20 mL) was added O-(2-chloro-
phenyl)-O,O-bis(1-benzotriazolyl) phosphate 1715 (0.2 M
in 1,4-dioxane, 25 mL, 5 mmol). After 1 h, TLC analysis
(MeOH/CH2Cl2, 1/9, v/v) indicated complete conversion of
the starting material into the lower running compound 18.
Then a solution of N a-Fmoc-serine allyl ester 15 (1.8 g,
5.0 mmol) in pyridine (15 mL) was added and stirring
was continued for 2.5 h. The reaction mixture was diluted
with CH2Cl2 (100 mL), washed with aq. NaHCO3 (2%,
2£50 mL), water (2£75 mL) and aq. AcOH (2%,
2£50 mL), dried (MgSO4), filtered and concentrated.
Purification by silica gel column chromatography (EtOAc/
light petroleum, 1/1!100/0, v/v) furnished0 18 as a colour-
less oil (3.8 g, 4.4 mmol, 87%, based on 2 ,30-di-O-acetyl-
uridine). 31P NMR (CDCl3): d 26.4, 26.9. 13C NMR
(CDCl3): d 169.4, 169.5 (2£CvO Ac), 168.1 (CvO ester),
163.0 (C-4 U), 155.7 (CvO Fmoc), 150.3 (C-2 U), 143.4,
141.0 (Cq Fmoc), 139.6 (C-6 U), 130.5 (vCH All), 130.9,
127.9, 127.4, 126.8, 126.3, 124.9, 121.2, 119.7 (Carom Fmoc,
Ph), 118.8 (vCH2 All), 103.2 (C-5 U), 86.9. 86.5 (C-10 U),
80.2, 72.2, 69.4 (C-20,0C-30, C-40 U), 68.5, 67.1, 66.4, 60.1
(OCH2 All, Fmoc; C-5 U; bCH2 Ser), 54.2 (aCH Ser), 46.7
(CH Fmoc), 20.2 (2£CH3 Ac). 1H NMR (CDCl3): d 9.77 (br
d, 1H, NH), 7.76 (m, 2H, Harom Fmoc), 7.61 (m, 2H, Harom
Fmoc), 7.43-7.10 (m, 9H, Harom Fmoc, Ph; H-6 U), 6.08 (m,
2H, H-1 U, NH Ser), 5.89 (m, 1H, ¼CH All), 5.69 (d, 1H,
J¼8.04 Hz, H-5 U), 5.40 (m, 2H, H-20, H-30), 5.26 (m, 2H,
vCH2 All), 4.70-4.22 (m, 11H, OCH2 All, Fmoc; CH
Fmoc; H-40, H-50a, H-50b U; aCH, bCH2 Ser), 2.11 (s, 6H,
2£CH3 Ac). ESI-MS: m/z¼868.3 [MþH]þ, 890.2
[MþNa]þ.
3H]3þ, 665.6 [Mþ4H]4þ
.
3.1.5. Coxsackie virus VPgpU (2b). The nucleopeptide
13 was synthesised starting from 11 and deprotected as
described for 1b. The crude nucleopeptide 2b (50 mmol)
was purified by RP-HPLC (linear gradient of 15!23% B in
12.3 min, 2.5 CV). Lyophilisation yielded 22.5 mg (14%,
TFA-salt) of pure coxsackie virus VPgpU 2b as a fluffy
solid. 31P NMR (D2O): d 24.15. MALDI-TOF-MS: m/z¼
2715.85 [MþH]þ, 2738.01 [MþNa]þ, calculated mass
2715.7. LC/MS analysis: Rt 8.01 min (linear gradient of
5!50% B in 20 min), ESI-MS: m/z¼2716.0 [MþH]þ,
1358.4 [Mþ2H]2þ, 906.0 [Mþ3H]3þ, 679.8 [Mþ4H]4þ
.
3.1.8. O-(2-Chlorophenyl)-N a-(9-fluorenylmethoxycarbo-
nyl)-serin-3-yl-(20,30-di-O-acetyluridin-50-yl) phosphate
(19). Allyl ester 18 (3.68 g, 4.2 mmol) was dissolved in
a mixture of THF/CH2Cl2 (1/1, v/v, 20 mL). Acetic acid
(1.1 mL, 19 mmol), Bu3SnH (2.2 mL, 8.4 mmol) and
Pd(PPh3)2Cl2 (0.12 g, 0.17 mmol) were added, resulting in
a yellow reaction mixture that immediately turned dark red.
After stirring at room temperature for 30 min, the solvents
were evaporated. The remaining oil was redissolved in
CH2Cl2 and applied onto a silica gel column. Elution with
CH2Cl2/MeOH (100/0!95/5, v/v) yielded uridylylated
building block 19 as slightly yellow coloured oil (3.2 g,
3.8 mmol, 90%). 31P NMR (CDCl3): d 26.52, 26.62. 13C
NMR (CDCl3/MeOD, 3/1, v/v): d 171.0 (CvO acid), 169.6
(2£CvO Ac), 165.1 (C-4 U), 157.2 (CvO Fmoc), 151.3
(C-2 U), 144.4, 141.9 (Cq Fmoc), 141.4 (C-6 U), 131.5,
129.0, 128.4, 127.7, 127.5, 125.8, 122.1, 120.5 (Carom Fmoc,
Ph), 103.6 (C-5 U), 88.7 (C-10 U), 80.8, 73.4, 70.3 (C-20,
C-30, C-40 U), 69.6, 67.9, 62.1 (OCH2 Fmoc, bCH2 Ser, C-50
U), 46.9 (CH Fmoc), 20.4 (2£CH3 Ac). 1H NMR (CDCl3): d
3.1.6. N a-(9-Fluorenylmethoxycarbonyl)-serine allyl
ester (15). To a cooled solution (08C) of Fmoc-Ser-OH 14
(3.7 g, 10 mmol) in DMF (40 mL) silver carbonate
(3.6 g, 13 mmol) was added and the mixture was stirred
for 15 min at room temperature. After addition of
allylbromide (4.0 mL, 46 mmol), stirring was continued
for 2.5 h. The mixture was filtered, diluted with ethyl acetate
(200 mL) and washed with 10% aq. KHSO4 (2£50 mL) and
water (2£75 mL). The organic layer was dried (MgSO4),
filtered and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography (light petro-
leum/EtOAc, 100/0!1/3, v/v) afforded compound 15 as a
white solid (3.7 g, 8.9 mmol, 89%). 13C NMR (CDCl3): d
169.5 (CvO ester), 155.5 (CvO Fmoc), 143.0, 142.8,
140.1 (Cq Fmoc), 130.9 (vCH All), 126.7, 126.1, 124.3,
118.9 (Carom Fmoc), 116.9 (vCH2 All), 65.9, 64.6, 61.5
(OCH2 All, Fmoc; bCH2 Ser), 55.7 (aCH Ser), 46.0 (CH
Fmoc). 1H NMR (CDCl3): d 7.77 (m, 2H, Harom Fmoc), 7.61
(m, 2H, Harom Fmoc), 7.37 (m, 4H, Harom Fmoc), 5.92 (br m,

N. M. A. J. Kriek et al. / Tetrahedron 59 (2003) 1589–1597
1597
USA 1978, 75, 4868–4872. (c) Kitamura, N.; Semler, B. L.;
Rothberg, P. G.; Larsen, G. R.; Adler, C. J.; Dorner, A. J.;
Emini, E. A.; Hanecak, R.; Lee, J. J.; van der Werf, S.;
Anderson, C. W.; Wimmer, E. Nature 1981, 291, 547–553.
3. Agol, V. I.; Paul, A. V.; Wimmer, E. Virus Res. 1996, 62,
129–147.
7.75 (m, 2H, Harom Fmoc), 7.61 (m, 2H, Harom Fmoc), 7.48-
7.07 (br m, 9H, Harom Fmoc, Ph; H-6 U), 6.11 (m, 2H, H-10
U, NH Ser), 5.73 (d0, 1H, J¼8.04 Hz, H-5 U), 5.40, 5.29
(2£m, 2H, H-20, H-3 U), 4.65-3.88 (br m, 9H, CH, OCH2
Fmoc; H-40, H-50a, H-50b U; aCH, bCH2 Ser), 2.08 (s,
6H, 2£CH3 Ac). ESI-MS: m/z¼828.3 [MþH]þ, 850.1
[MþNa]þ.
4. Paul, A. V.; van Boom, J. H.; Filippov, D.; Wimmer, E. Nature
1998, 393, 280–284.
3.1.9. Cowpea mosaic virus VPgpU (3b). Immobilised
peptide 20 was synthesised according to the general
procedure for solid phase peptide synthesis. In every
Fmoc deprotection cycle piperidine/DMF (1/4, v/v) was
used. Additional double couplings were introduced for
[Gln10], [Gln11], [Tyr12], [Tyr14], [Pro18], [Leu19] and
[Lys20]. A small amount of peptide-resin 20 (50 mg,
^13 mmol) was removed from the synthesiser, transferred
into a round bottom flask and suspended in CH2Cl2 (2 mL).
Subsequently, uridylylated serine building block 19
(107 mg, 0.13 mmol) and HOBt (18 mg, 0.13 mmol) were
added, and the reaction mixture was gently stirred for
15 min. Then DIC (20 mL, 0.13 mmol) was added and
stirring was continued for 1 h. The solvent and reagents
were removed by filtration and the resin was repeatedly
washed with CH2Cl2 (2£5 mL), MeOH (5 mL) and CH2Cl2
(2£5 mL). After the coupling-procedure was repeated
once more, a negative Kaiser-test showed complete
coupling of the uridylylated building block. The fully
protected peptide-resin 21 was suspended in a solution of
0.2 M TBAF (1 M in THF, 1.25 mL) in DMF/H2O/pyridine
(4/1/3, v/v/v, 4 mL) and stirred for 2 h. The resin was
filtered and washed with CH2Cl2 (2£5 mL), MeOH (5 mL)
and CH2Cl2 (2£5 mL). Next, the resin was suspended in a
mixture of TFA/TIS/H2O/PhOH/EDT (90/1.2/0.8/0.8/1.2,
v/v/v/w/v, 10 mL) and gently stirred under a blanket of
argon for 4 h. After filtration, the resin was washed with
TFA (3£5 mL) and the filtrate was concentrated. The
remaining oil was dried in high vacuum (0.5 mm Hg,
15 min), redissolved in TFA (1 mL) and precipitated by
addition to diethyl ether (15 mL). After washing with ether
(3£5 mL), the white solid was taken up in NH3·H2O
(25%)/1,4-dioxane (1/1, v/v, 10 mL) and stirred for 5 h at
room temperature. Careful evaporation of the solvents
afforded crude 3b. The obtained product 3b was purified by
cation exchange chromatography (linear gradient (A/B/C/D,
60/20/0/20!52.5/20/7.5/20, v/v/v/v in 12 CV). Next, the
obtained product was desalted (linear gradient of (10!50%
B in 10 CV). After lyophilisation nucleopeptide 3b was
obtained as a white fluffy solid (2.7 mg, 5%, TFA-salt). 31P
NMR (D2O): d 20.10. LC/MS analysis: Rt 18.54 min
(linear gradient of 10!50% B in 20 min). ESI-MS: m/z¼
5. Filippov, D.; Kuyl-Yeheskiely, E.; van der Marel, G. A.;
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G.; Shanks, M.; Beyreuter, K. EMBO J. 1984, 3, 1629–1634.
9. Pon, R. T. Tetrahedron Lett. 1987, 28, 3643–3646.
10. Meldal, M.; Bielfeldt, T.; Peters, S.; Jensen, K. J.; Paulsen, H.;
Bock, K. Int. J. Pept. Protein Res. 1994, 43, 529–536.
11. Wakamiya, T.; Saruta, K.; Yasuoka, J.; Kusumoto, S. Chem.
Lett. 1994, 1099–1102.
12. (a) Chang, C.-D.; Waki, M.; Ahmad, M.; Meienhofer, J.;
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´
59–66. (b) Robles, J.; Beltran, V.; Perez, Y.; Travesset, I.;
Pedroso, E.; Grandas, A. Tetrahedron 1999, 55,
13251–13264.
13. (a) Shabarova, L. A. Progress in Nucleic Acids Research and
Molecular Biology; Davidson, N. J., Cohn, W. E., Eds.;
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1995, 23, 4151–4161. (b) Zubin, E. M.; Romanova, E. A.;
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Marel, G. A.; van Boom, J. H. Nucleic Acids Res. 1989, 17,
2897–2905. (b) Kuyl-Yeheskiely, E.; van der Klein, P. A. M.;
Visser, G. M.; van der Marel, G. A.; van Boom, J. H. Recl.
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van Boom, J. H. Tetrahedron 1988, 20, 6515–6523.
16. It has also been reported that the Fmoc group can be rapidly
removed by fluoride ions (i.e. dry TBAF in DMF): Ueki, M.;
Amemiya, M. Tetrahedron Lett. 1987, 28, 6617–6620.
17. It is also of interest to note that the 2-cyanoethyl group, which
can also be removed with fluoride ions, has been used earlier to
serve the same purpose. See in this respect: (a) Dreef-Tromp,
C. M.; van den Elst, H.; van der Marel, G. A.; van Boom, J. H.
Nucleic Acids Res. 1992, 20, 4015–4020. Robles, J.; Pedroso,
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18. Van der Marel, G. A.; van Boeckel, C. C. A.; Wille, G.; van
Boom, J. H. Tetrahedron Lett. 1981, 22, 3887–3890.
1923.0 [Mþ2H]2þ, 1282.1 [Mþ3H]3þ, 962.0 [Mþ4H]4þ
770.1 [Mþ5H]5þ, calculated mass 3842.9.
,
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