Synthesis of nucleotidylated poliovirus VPg proteins

Article abstract of DOI:10.1021/jo100757t

Phosphitylation of the side chain hydroxyl function of Fmoc protected tyrosine with 5′-phosphoramidites of suitably protected cytidine, adenosine, and guanosine, followed by oxidation gave three novel nucleotidylated amino acid building blocks. After protective group manipulation, these building blocks were used in a solid phase peptide synthesis to afford the nucleotidylated poliovirus proteins VPgpC, VPgpA, and VPgpG.

Full text of DOI:10.1021/jo100757t

pubs.acs.org/joc
clarified completely extensive research on for instance po-
Synthesis of Nucleotidylated Poliovirus VPg Proteins
liovirus has shown that VPg and its uridinylylated forms,
VPgpU and VPgpUpU, act as primers in both negative- and
positive-strand RNA synthesis.10-14 In poliovirus VPgpU
the O4 of tyrosine-3 of the 22 amino acid long VPg protein is
modified with a 50-uridinylyl moiety, whereas the elongated
version VPgpUpU is modified with a 50-(uridinylyl-(50f30))-
uridinylyl moiety. These poliovirus nucleoproteins are
formed under influence of a cis-acting replication element
(CRE) within the viral RNA.15-19 It has been shown that the
introduction of point mutations in this CRE motif led to
reaction of CTP, ATP, or GTP with O4 tyrosine of VPg to
produce VPgpC, VPgpA, and VPgpG, respectively.15
Gerbrand J. van der Heden van Noort,
Herman S. Overkleeft, Gijsbert A. van der Marel, and
Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden University,
P.O. Box 9502, 2300 RA Leiden, The Netherlands.
filippov@chem.leidenuniv.nl
Received April 29, 2010
As part of a program20 to develop synthetic routes to
naturally occurring nucleotidylated proteins and derivatives
thereof we set out to develop a solid phase synthesis of
nucleotidylated poliovirus VPg’s. We demonstrate the via-
bility of a generic, solid-phase approach toward the nucleo-
tidylated polypeptide containing all four naturally occurring
ribonucleotides. In earlier reports VPgpU and VPgpUpU of
certain picornaviruses were assembled either by block cou-
pling or by solid-phase peptide synthesis.21-23 These nucleo-
peptides prove to be valuable tools in the ongoing research
on viral replication of picornaviruses.24-26 For the synthesis
of poliovirus derived VPgpC, VPgpA, and VPgpG a solid-
phase synthesis with prenucleotidylated Fmoc-tyrosine
building blocks appeared most convenient.23 Key to the
success of such a strategy is the choice of the protecting
groups for the exocyclic amino functions of the nucleobases.
Such protecting groups should be stable during the solid-
phase peptide synthesis and leave the peptide intact during
their deprotection procedure at the end of the synthesis.
Phosphitylation of the side chain hydroxyl function of
Fmoc protected tyrosine with 50-phosphoramidites of
suitably protected cytidine, adenosine, and guanosine,
followed by oxidation gave three novel nucleotidylated
amino acid building blocks. After protective group ma-
nipulation, these building blocks were used in a solid
phase peptide synthesis to afford the nucleotidylated
poliovirus proteins VPgpC, VPgpA, and VPgpG.
(10) Steil, B. P.; Barton, D. J. J. Virol. 2009, 83, 12660–12670.
(11) Van Ooij, M. J. M.; Vogt, D. A.; Paul, A.; Castro, C.; Kuijpers, J.;
van Kuppeveld, F.; J., M.; Cameron, C. E.; Wimmer, E.; Andino, R.;
Melchers, W. J. G. J. Gen. Virol. 2006, 87, 103–113.
(12) Murray, K.; Barton, D. J. Virol. 2003, 77, 4739–4750.
(13) Pathak, H. B.; Arnold, J. J.; Wiegand, P. N.; Hargittai, M. R. S.;
Cameron, C. E. J. Biol. Chem. 2007, 282, 16202–16213.
(14) Goodfellow, I. G.; Kerrigan, D.; Evans, D. J. RNA 2003, 9, 124–137.
(15) Paul, A. V.; Yin, J.; Mugavero, J.; Rieder, E.; Liu, Y.; Wimmer, E. J.
Biol. Chem. 2003, 278, 43951–43960.
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Funct., Bioinf. 2006, 63, 719–726.
Nucleotidylation of proteins is a widespread posttransla-
tional modification.1-4 For example, adenylylation of tyr-
osine has been recognized as an important regulatory event
both in bacteria and in higher organisms.5,6 A number of
RNA viruses use the uridinylylated or guanylylated form of
so-called VPg proteins to initiate the replication of the
genome (VPg = Viral Protein genome-linked).7 Perhaps
the most investigated protein nucleotidylation process takes
place in Picornaviridae, a family of positive strand RNA
viruses that causes illnesses such as poliomyelitis, common
cold, hepatitis A, and foot and mouth disease.8 All these
viruses share the presence of a small VPg, which is covalently
linked to the 50-end of the viral genome.9 Although the
mechanism of replication of Picornaviridae has not been
(19) Rieder, E.; Paul, A. V.; Kim, D. W.; van Boom, J. H.; Wimmer, E. J.
Virol. 2000, 74, 10371–10380.
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H. S.; van der Marel, G. A.; Filippov, D. V. J. Am. Chem. Soc. 2010, 132,
5236–5240.
(21) Filippov, D.; Kuyl-Yeheskiely, E.; van der Marel, G. A.; Tesser,
G. I.; van Boom, J. H. Tetrahedron Lett. 1998, 39, 3597–3600.
(22) Kriek, N. M. A.; Meeuwenoord, N. J.; van den Elst, H.; Heus, H. A.;
van der Marel, G. A.; Filippov, D. V. Org. Biomol. Chem. 2006, 4, 3576–3586.
(23) Kriek, N. M. A.; 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.
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DOI: 10.1021/jo100757t
r
Published on Web 07/22/2010
J. Org. Chem. 2010, 75, 5733–5736 5733
2010 American Chemical Society

van der Heden van Noort et al.
JOCNote
SCHEME 1. Synthesis of Fmoc-Tyrosine Derivatives
SCHEME 2. Solid-Phase Synthesis of VPgpC, VPgpA, and
VPgpG
We selected the acyl-based protective groups as those are
well established in the field of nucleopeptide synthesis.27,28
Because the stability of acyl protecting groups depends on
the nature of the nucleobase, the benzoyl group was selected
to protect the exocyclic amino functions of both cytidine and
adenosine while guanosine was equipped with the more
labile phenoxyacetyl group.29
The solid-phase synthesis of the target nucleopeptides
starts with the solution-phase synthesis of suitably protected
nucleotidylated tyrosine building blocks 13-15 (Scheme 1).
Known 4-N-benzoylcytidine30 (1), 6-N-benzoyladenosine31
(2), and 2-N-phenoxyacetylguanosine32 (3) were silylated
with TBDMSCl in pyridine, treated with isobutyric anhy-
dride, and subsequently desilylated with acid to give partially
protected nucleosides 4, 5, and 6, respectively.
Treatment of 4, 5, and 6 with 2-cyanoethoxy-N,N-diiso-
propylaminochlorophosphine in the presence of triethylamine
as base led to 50-phosphoramidites 7, 8, and 9. Tetrazole-
mediated phosphitylation of the free hydroxyl function on the
side chain of Fmoc-Tyr-OAll with amidites 7, 8, and 9
followed by oxidation of the intermediate phosphite triesters
with mCPBA gave 10, 11, and 12. Subsequent liberation of the
acid by palladium-catalyzed removal of the allyl protective
group gave the respective nucleotidylated amino acid building
blocks 13, 14, and 15.
Next attention was directed to the synthesis of the protected
and immobilized 19-mer peptide 16, a common intermediate
en route to target VPgpC, VPgpA, and VPgpG (Scheme 2).
Tentagel S Ram resin was functionalized with the first amino
acid by attachment of Fmoc-Glu-OtBu via its side chain.23
Automated solid-phase peptide synthesis based on Fmoc chem-
istry with use of commercially available amino acid building
blocksandHCTUascondensingagentproceededuneventfully.
Immobilized 19-mer 16 was manually elongated by the
sequential incorporation of citydinylated tyrosine 13, using a
BOP/HOBt based coupling protocol. Treatment with 2%
DBU in DMF resulted in cleavage of the Fmoc and cya-
noethyl protecting groups, after which the phosphodiester
containing peptide was elongated with Fmoc-Ala-OH and
Fmoc-Gly-OH to give the partially protected VPgpC (17,
Scheme 2). TFA-mediated cleavage of the nucleopeptide
from the resin and concomitant unmasking of the amino
acid side chains, deprotection of all acyl protection groups
with 25% aq ammonia, and final purification with RP-
HPLC afforded homogeneous VPgpC 20 in 45% yield based
on initial loading of the resin (Figure 1).
A similar sequence of reactions as described for the
formation of 17, using in this case adenylylated tyrosine
14, gave immobilized VPgpA 18. In the first instance,
cleavage from the solid support, removal of the remaining
protecting groups, and purification did not result in homo-
geneous VPgpA. It turned out that despite the relative acid
stability of the glycosidic linkages of ribonucleosides, TFA
treatment of partially protected VPgpA 18 was accompanied
by depurination of the adenosine moiety, which was not
separable from nucleoprotein 21.33 This was unexpected
because the 50-phosphorylated adenosine residue was re-
ported to be stable to TFA treatment34 and 50-adenylyl O-
tyrosine, specifically, is known to withstand a treatment with
strong acid (0.3 N HCl, 37 °C, 3 h).35 Homogeneous VPgpA
(27) Robles, J.; Beltran, M.; Marchan, V.; Perez, Y.; Travesset, I.;
Pedroso, E.; Grandas, A. Tetrahedron 1999, 55, 13251–13264.
(28) Dreef-Tromp, C. M.; van Dam, E. M. A.; van den Elst, H.; van der
Marel, G. A.; van Boom, J. H. Nucleic Acids Res. 1990, 18, 6491–6495.
(29) Schulhof, J. C.; Molko, D.; Teoule, R. Nucleic Acids Res. 1987, 15,
397–416.
(30) Sung, W. L.; Narang, S. A. Can. J. Chem. 1982, 60, 111–120.
(31) Cui, Z.; Zhang, L.; Zhang, B Tetrahedron Lett. 2001, 42, 561–563.
(32) Wu, T.; Ogilvie, K. K. Tetrahedron Lett. 1988, 29, 4249–4252.
(33) See the Supporting Information for a crude LCMS trace of 18.
(34) Al-Eryani, R. A.; Ball, H. L. Tetrahedron Lett. 2010, 51, 1730–1731.
(35) Medzihradszky, D.; Chen, S. L.; Kenyon, G. L.; Gibson, B. W. J.
Am. Chem. Soc. 1994, 116, 9413–9419.
5734 J. Org. Chem. Vol. 75, No. 16, 2010

van der Heden van Noort et al.
JOCNote
The residue was taken up in 4:1 MeCN/H2O (30 mL, v/v) and p-
TsOH(1 equiv, 0.55 g) was added after which thereaction mixture
was stirred overnight at room temperature. The reaction mixture
was concentrated and purified with silica gel column chromatog-
raphy DCM/MeOH (100/0 to 98/2) to afford the title compound
as a white foam (0.88 g, 1.8 mmol, 63%). 13C NMR (100 MHz,
CDCl3) δ 176.1, 175.6, 162.8, 155.4, 145.7 (C6 Cyt.), 133.1, 133.0
(Arom. Bz), 128.8 (Arom. Bz), 127.8 (Arom. Bz), 97.7 (C5 Cyt.),
88.9 (C10), 84.1 (C40), 74.2 (C20), 71.0 (C30), 61.3 (C50), 33.8, 33.7
(CH iBu), 18.8, 18.7 (CH3 iBu). 1H NMR (400 MHz, CDCl3) δ
8.42, 8.40 (d, 1H, H6 Cyt.), 7.83, 7.81 (d, 2H, Bz), 7.52-7.49 (m,
2H, Arom. Bz, H5 Cyt.), 7.41-7.37 (m, 2H, Arom. Bz), 6.19, 6.18
(d, 1H, H10), 5.58-5.53 (m, 2H, H20, H30), 4.40 (br s, 1H),
4.22-4.21 (m, 1H, H40), 3.98-3.83 (dd, 2H, H50), 2.58-2.49
(m, 2H, CH iBu), 1.13-1.08 (m, 12H, CH3 iBu). IR 1743, 1653,
1624, 1558, 1481, 1311, 1244, 1099. LCMS (10-90% MeCN in 15
min), Rt= 6.90. ESI-MS m/z487.9 [M]þ. HRMS[C24H29N3O8 þ
H]þ calcd 488.2027, found 488.2026.
2-Cyanoethoxy-N,N0-diisopropylamino(4-N-benzoy-20,30-di-
O-isobutyrylcytidinyl-50-yl)phosphine (7). To a stirred solution of
4-N-benzoyl-20,30-di-O-isobutyryl-β-D-cytidine (4) (0.89 g, 1.8
mmol), coevaporated with MeCN, in DCM (5 mL), containing
TEA (0.84 mL, 5.9 mmol) was added 2-cyanoethoxy-N,N-
diisopropylaminochlorophosphine (0.50 mL, 2.25 mmol) under
an argon atmosphere. The reaction mixture was stirred for 45
min at room temperature. After 31P NMR showed complete
consumption of the 2-cyanoethoxy-N,N-diisopropylamino-
chlorophosphine (200 MHz, CDCl3: δ 180.4) and formation
of the product (200 MHz, CDCl3: δ 150.3, 149.6), 30 mL of
DCM was added and the reaction mixture was washed with
water and 5% NaHCO3. The organic phase was dried (MgSO4)
and concentrated. The residue was applied on a silica gel column
and eluted with a gradient of EtOAc in PE (50/50 to 100/0) to
yield the title compound as an off-white foam (0.9 g, 1.3 mmol,
73%). 13C NMR (100 MHz, CDCl3) δ 175.2, 175.0, 174.8, 174.7,
162.2, 154.2, 144.0 (C6 Cyt.), 143.9 (C6 Cyt.), 132.7, 132.5
(Arom. Bz), 128.3 (Arom. Bz), 127.4 (Arom. Bz), 117.3, 117.2,
97.0, 96.9 (C5 Cyt.), 87.1, 86.7 (C10), 82.4, 82.3, 82.0, 81.9 (C40),
73.6, 73.5 (C20), 70.3, 70.1 (C30), 62.4, 62.3, 62.2, 62.0 (C50), 58.4,
58.1, 57.9 (CH2 CNEO), 42.79, 42.76, 42.7, 42.6 (CH iPr), 33.3,
33.2, 33.1 (CH iBu), 24.22, 24.15, 24.10, 24.08 (CH3 iPr), 19.96,
19.88, 19.86, 19.79 (CH2 CNEO), 18.31, 18.29, 18.27, 18.13
FIGURE 1. LCMS traces of purified VPgpG, VPgpC, and VPgpA.
21, however, could be obtained in 9% yield based on initial
loading of the resin by acidolysis, RP-HPLC purification of
the intermediate nucleopeptide, ammonia treatment, and
finally a second RP-HPLC purification (Figure 1).
Adoption of the procedure of VPgpC for the synthesis of
VPgpG and LCMS analysis of the crude product showed the
presence of truncated sequences while depurination products
were lacking. Guided by the work of Bae and Lakshman36,37
reporting that BOP and PyBOP mediated condensations
may lead to side reactions at the C-6 of guanosine we
introduced the last three amino acids in the synthesis of
VPgpG using the HATU coupling reagent.38 Treatment with
TFA, 25% aq ammonia, and RP-HPLC purification af-
forded VPgpG in 29% yield based on initial loading of the
resin (Figure 1).
In conclusion, three unprecedented nucleotidylated amino
acid building blocks were prepared and applied in a concise
solid-phase procedure to the synthesis of poliovirus derived
proteins VPgpC, VPgpA, and VPgpG. This methodology
opens the way to the synthesis of more elaborated nucleotidy-
lated proteins, such as VPgpG of norovirus. The synthetic, well-
defined constructs of this type may prove to be invaluable in the
structural and biological studies of viral replication.
1
(CH3 iBu). H NMR (400 MHz, CDCl3) δ 8.20-8.16 (m, 1H,
H6 Cyt.), 7.84-7.82 (m, 2H, Bz), 7.46-7.42 (m, 1H, Bz),
7.36-7.32 (m, 3H, Bz, H5 Cyt.), 6.25, 6.24, 6.18, 6.17 (2 ꢀ d,
1H, H10), 5.39-5.28 (m, 1H, H20), 4.23 (m, 1H, H30), 4.08-3.80
(m, 5H, H40, H50, CH2 CNEO), 3.69-3.62 (m, 2H, CH iPr),
2.76-2.69 (m, 2H, CH2 CNEO), 2.63-2.55 (m, 2H, CH iBu),
1.27-1.14 (m, 24H, CH3 iBu, CH3 iPr). 31P NMR (161 MHz,
CDCl3) δ 150.3, 149.6. IR 2980, 1746, 1666, 1624, 1556, 1481,
1310, 1245, 1153. LCMS (10-90% B in 15 min), Rt = 6.80, ESI-
MS m/z 605.1 [M þ H]þ of the corresponding H-Phosphonate.
TLCMS 688.5 [M þ H]þ. HRMS [C33H46N5O9P þ H]þ calcd
688.3106, found 688.3118.
Experimental Section
The experimental data for compounds 4, 7, 10, 13, and 20 are
given as typical representatives of VPgpN synthesis.
4-N-Benzoyl-20,30-di-O-isobutyryl-β-D-cytidine (4). 4-N-ben-
zoyl-β-D-cytidine (1) (1.0 g, 2.9 mmol) was dissolved in pyridine
(5 mL) after coevaporation with pyridine. TBDMS-Cl (5.8 mmol,
0.87 g) was added and the reaction mixture was stirred until it
became a clear solution. After this isobutyric anhydride (1.6
mmol, 1.93 mL) was added and the reaction mixture was stirred
at room temperature for 16 h, upon which TLC analysis (10%
MeOH/DCM) indicated complete conversion of the starting
material. The reaction mixture was concentrated and taken up
in EtOAc. After washing with sat. aq NaHCO3, 5% citric acid,
and H2O the organic phase was dried (MgSO4) and concentrated.
2-Cyanoethoxy(Nr- Fmoc-tyrosin-4-yl allyl ester)(4-N-ben-
zoyl- 20,30-di-O-isobutyrylcytidinyl-50-yl) Phosphate (10). NR-
Fmoc-tyrosine allyl ester [Fmoc-Tyr-OAll] (0.33 g, 0.75 mmol)
was added to compound 7 (0.50 g, 0.72 mmol) and the two
compounds were coevaporated with MeCN (5 mL) and DCE
(5 mL) and then dissolved in 1:1 MeCN/DCM (5 mL, v/v).
Tetrazole (200 mg, 2.88 mmol) was added and the reaction
mixture was stirred for 45 min until 31P NMR (200 MHz: δ
135.4, 134.9) showed complete consumption of the phosphor-
amidite. The reaction mixture was cooled to 0 °C and m-CPBA
(5 equiv) was added in portions until 31P NMR (200 MHz: δ
-6.2, -6.3) showed complete consumption of the phosphite.
The reaction mixture was diluted in 20 mL of DCM and washed
with 5% Na2S2O3 and water. The organic phase was dried
(36) Bae, S.; Lakshman, M. K. J. Am. Chem. Soc. 2007, 129, 782–789.
(37) Bae, S.; Lakshman, M. K. Org. Lett. 2008, 10, 2203–2206.
(38) Pritz, S.; Wolf, Y.; Klemm, C.; Bienert, M. Tetrahedron Lett. 2006,
47, 5893–5896.
J. Org. Chem. Vol. 75, No. 16, 2010 5735

van der Heden van Noort et al.
JOCNote
(MgSO4) and concentrated. The residue was applied to a silica
gel column and eluted with a gradient of EtOAc in PE (50/50 to
100/0) to yield the title compound as a colorless gel in 74% over
two steps (0.55 g, 0.53 mmol). 13C NMR (100 MHz, CDCl3) δ
175.7, 175.6, 175.5, 171.0, 170.9, 162.5, 155.7, 155.6, 149.03,
148.96, 143.8 (C6 Cyt.), 143.7 (C6 Cyt.), 141.2, 133.8, 133.7,
133.2, 132.8, 131.3, 131.04, 131.00, 128.9, 127.7, 127.6, 127.01,
126.98, 125.0, 119.9, 119.2, 119.1 (CH2 Allyl), 116.1 (CN), 89.0,
88.4 (C10), 81.01, 80.96, 80.9 (C40), 73.5 (C20), 69.7, 69.6 (C30),
67.1 (C50), 66.9, 66.8, 66.11, 66.08 (CH2 Fmoc, CH2 Allyl),
63.02, 62.97 (CH2 CNEO), 54.7 (CR), 47.1 (CH Fmoc), 37.3,
37.2 (Cβ), 33.70, 33.68, 33.64 (CH iBu), 19.63, 19.55 (CH2
CNEO), 18.75, 18.72, 18.6 (CH3 iBu). 1H NMR (400 MHz,
2.75-2.63 (m, 2H, CH2 CNEO), 2.62-2.58 (m, 2H, CH iBu), 1.16
(m, 12H, CH3 iBu). 31P NMR (161 MHz, CDCl3) δ -6.4, -6.8. IR
1699, 1668, 1624, 1558, 1481, 1450, 1249. LCMS (10-90% B in 15
min) Rt = 9.06. ESI-MS m/z 1006.7 [M þ H]þ. HRMS [C51H52-
N5O15P þ H] þ calcd 1006.3270, found 1006.3281.
VPgpC (20). The protected peptide 16 was prepared via solid-
phase peptide synthesis starting from Tentagel S RAM resin (200
mg, 50 μmol) to which Fmoc-Glu-OtBu was attached via the side
chain carboxylic acid functionality by using an automated peptide
synthesizer. As repetitive cycle was used: (1) Fmoc cleavage with
20% piperidine in NMP, (2) coupling of the appropriate amino
acid applying a 5-fold excess, activation by 5 equiv of HCTU in
NMP (0.25M) and 12.5 equiv of DiPEA in NMP (1.25 M) for 1 h,
and (3) capping with 0.5 M acetic anhydride, 0.125 M DipEA, and
0.015 M HOBt in NMP for 1 min.
CDCl3)
δ 7.89-7.76, 7.74-7.73, 7.61-7.45, 7.41-7.36,
7.31-7.24, 7.19-7.12 (6 ꢀ m, 19H), 6.25, 6.24, 6.18, 6.17 (2 ꢀ
d, 1H, H10), 5.91-5.82 (m, 1H, CH Allyl), 5.65, 5.63 (d, 1H,
NH), 5.49-5.44 (m, 1H, H30), 5.39-5.22 (m, 4H, CH2 Allyl),
5.22-5.18 (m, 1H, H20), 4.75-4.73 (m, 2H, CH2 β), 4.65-4.62
(m, 5H, CH R, CH2 CNEO, CH2 Fmoc), 4.40-4.33 (m, 2H,
H50), 4.27-4.26 (m, 1H, H40), 4.20-4.14 (m, 1H, CH Fmoc),
3.20-3.08 (m, 2H, CH2 β), 2.81-2.70 (m, 2H, CH iBu),
2.63-2.55 (m, 2H, CH2 CNEO), 1.21-1.14 (m, 12H, CH3 iBu).
31P NMR (161 MHz, CDCl3) δ -5.7, -5.9. IR 2980, 1740, 1700,
1668, 1626, 1556, 1506, 1481, 1250, 1188, 1036. LCMS (50-90%
B in 15 min), Rt = 6.26. ESI-MS m/z 1046.5 [M þ H]þ. HRMS
[C54H56N5O15P þ H]þ calcd 1046.3583, found 1046.3593.
2-Cyanoethoxy(Nr-Fmoc-tyrosin-4-yl)(4-N-benzoyl-20,30-di-
O-isobutyrylcytidinyl-50-yl) Phosphate (13). Compound 10
(0.55 g, 0.53 mmol) was coevaporated with MeCN and dissolved
in 1:1 THF/DCM (10 mL, v/v). After addition of AcOH (2.6 mmol,
0.15 mL), Bu3SnH (0.32 mL), and Pd(PPh3)4 (20 mg) the reaction
was stirred for 2 h. The reaction mixture was concentrated and the
residue was applied to a silica gel column and eluted with a gradient
of MeOH in DCM (0/100 to 5/95) to yield the title compound as a
white foam (0.30 g, 0.29 mmol, 56%). 13C NMR (100 MHz, CDCl3)
δ 175.50, 175.46, 175.4, 174.3, 167.2, 163.2, 163.1, 155.6, 154.3,
148.8, 148.7, 145.0 (C6 Cyt.), 143.7, 143.6, 141.1, 134.0, 133.0, 132.5,
131.0, 128.5, 128.0, 127.5, 126.9, 125.0, 124.9, 119.8, 119.6, 116.3,
97.7 (C5 Cyt.), 89.8, 89.3 (C10), 80.7, 80.6 (C40), 73.4 (C20), 69.4,
69.3 (C30), 66.9, 66.9, 66.7 (CH2 Fmoc, C50), 63.0, 63.0 (CH2
CNEO), 54.6 (CR), 46.9 (CH Fmoc), 37.0 (Cβ), 33.5 (CH iBu),
The last three amino acids were introduced manually on 12.5
μmol of 16 by a double coupling protocol: (1) Fmoc cleavage
with 2% DBU in DMF, (2) coupling of the appropriate amino
acid applying a 5-fold excess, activation by 5 equiv of BOP/
HOBt in NMP (0.25M) and 12.5 equiv of DiPEA in NMP (1.25
M) for 1 h, and (3) capping with 0.5 M acetic anhydride, 0.125 M
DiPEA, and 0.015 M HOBt in NMP for 1 min. The crude
partially protected nucleopeptide was cleaved off the resin with
TFA/TIS/H2O (95/5/5, v/v/v, 5 mL) by shaking for 2.5 h. After
filtration into cold Et2O the precipitate was coevaporated with
toluene (2 ꢀ 5 mL) and then treated with 25% aq ammonia (2.5
mL) in 1,4-dioxane (2.5 mL). After stirring for 4 h, concentra-
tion of the reaction mixture afforded the crude title compound.
RP-HPLC purification was conducted on an automated HPLC
system supplied with a semipreparative C18 column (250 ꢀ 10.00
mm, 5 μ, flow: 4 mL/min). The applied eluent system was (A)
H2O, (B) MeCN, and (C) 0.5% TFA in H2O with detection at
280 nm. Gradient: 10-25% B (10% C as stationary phase) in 3
CV. The collected fractions were lyophilized, yielding 14.89 mg,
5.6 μmol (45% based on original loading of resin) of the
nucleopeptide. 31P NMR (161 MHz, D2O) δ - 4.24. LCMS
(10-90% B in 15 min.), Rt = 4.18 min. ESI-MS m/z 2659.2
[M]þ, 1330.4 [M]2þ, 887.6 [M]3þ. HRMS [C114H189N34O37P þ
H]2þ calcd 1329.6918, found 1329.6934.
Acknowledgment. This work has been funded by the
Dutch Organisation for Scientific Research (NWO)
1
19.4, 19.3 (CH2 CNEO), 18.64, 18.62 (CH3 iBu). H NMR (400
MHz, CDCl3) δ 7.92-7.87 (m, 3H), 7.75, 7.73 (m, 2H), 7.54-7.47
(m, 4H), 7.39-7.35 (m, 5H), 7.29-7.25 (m, 2H), 7.21-7.14 (m,
4H), 6.10, 6.06 (d, 1H, C10), 5.76, 5.74, 5.70, 5.68 (dd, 1H, NH),
5.46-5.42 (m, 2H, C30, C20), 4.70 (m, 1H, CH R), 4.48-4.43 (m,
2H, CH2 Fmoc), 4.33-4.31 (m, 3H, C40, C50), 4.30-4.23 (m, 2H,
CH2 CNEO), 4.13 (m, 1H, CH Fmoc), 3.18-3.11 (m, 2H, CH2 β),
Supporting Information Available:
Experimental proce-
dures and spectroscopic data for all compounds, as well as
copies of 1H, 13C, and 31P NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
5736 J. Org. Chem. Vol. 75, No. 16, 2010