Synthesis of 4-thiouracil KPGEPGPK analogues as potential TIIICBP identification tools

Article abstract of DOI:10.1007/s10989-010-9227-7

In this paper, we described the synthesis of three octapeptides analogues which are obtained by the incorporation of photochemical 4-thiouracil (s ⁴U) probes selectively activables. This probe has been incorporated at the 1 and/or 3 positions in KPGEPGPK sequence since it has been demonstrated that those positions could be mutated without major anti-thrombotic activity modifications. These analogues could be used to identify a new receptor for type III collagen named Type III Collagen Binding Protein (TIIICBP).

Full text of DOI:10.1007/s10989-010-9227-7

Int J Pept Res Ther (2010) 16:257–266
DOI 10.1007/s10989-010-9227-7
Synthesis of 4-Thiouracil KPGEPGPK Analogues as Potential
TIIICBP Identification Tools
•
Viviane Silva Pires Sophie da Nascimento
•
Pascal Sonnet
Accepted: 27 July 2010 / Published online: 7 August 2010
Ó Springer Science+Business Media, LLC 2010
Abstract In this paper, we described the synthesis of
three octapeptides analogues which are obtained by the
incorporation of photochemical 4-thiouracil (s4U) probes
selectively activables. This probe has been incorporated at
the 1 and/or 3 positions in KPGEPGPK sequence since it
has been demonstrated that those positions could be
mutated without major anti-thrombotic activity modifica-
tions. These analogues could be used to identify a new
receptor for type III collagen named Type III Collagen
Binding Protein (TIIICBP).
iBuOCOCl Isobutyl chloroformate
FmocOSu
9-Flourenylmethoxycarbonyl-N-
hydroxysuccinimide
Dimethyl sulfoxide
DMSO
Introduction
In a previous work, the identification of a new receptor for
type III collagen named Type III Collagen Binding Protein
`
(TIIICBP) has been reported (Monnet and Fauvel-Lafeve
Keywords 4-Thiouracil probe Á TIIICBP Á Type III
collagen Á KPGEPGPK Á Anti-thrombotic activity
2000). This receptor which is not yet identified is supposed to
interact with the octapeptide sequence KPGEPGPK located
in the a1-chain of CB4 fragment of the type III collagen. This
octapeptide specifically inhibits platelet interaction with
type III collagen in static and flow conditions. This octa-
peptide prevents in vivo, the thrombosis photochemically-
induced in arterioles of mice without affecting bleeding time
(Maurice et al. 2006a, b). Actually, the TIIICBP structure
and the mechanisms of its corresponding interactions with
KPGEPGPK are so far unknown. Our ultimate goal will be to
Abbreviations
TIIICBP
s4U
Type III Collagen Binding Protein
4-Thiouracil probe
PNA
Peptides nucleic acids
ARN
Ribonucleic acid
NMP
DIEA
HBTU
N-Methyl-2-pyrrolidone
N,N-Diisopropylethylamine
2-(1H-benzotriazol-1-yl)-1,1,3,
3-tetramethyluronium hexafluorophosphate
Trifluoroacetic acid
ˆ
identified TIIICBP (Pires et al. 2007; Pecher et al. 2009). For
this, a solution could be provided by using the 4-thiouracil
(s4U) probe which could be able to form covalent photoad-
ducts, under irradiation, with TIIICBP.
TFA
Dde
N-[1-(4,4-Dimethyl-2,
6-dioxocyclohexylidene)ethyl]
4-Methylmorpholine
The thionucleobases were used initially as intrinsic
probes photoactivated for structural analyzes of the ribo-
nucleic acid (ARN) and to identify the probable contacts
between nucleic acids and proteins such as in the complexes
nucleoproteins of various systems (replisome, spliceosome,
transcription and ribosomes). The thioresidues used (4-thi-
ouracil (s4U), 4-thiothymine, 6-mercaptopurine, 6-thiogua-
nine) absorb the light in a wavelength of 360 nm. Many
works have been done concerning the nature of the
NMM
Boc
tert-Butyloxycarbonyl
Fluorenylmethyloxycarbonyl
Fmoc
V. S. Pires Á S. da Nascimento Á P. Sonnet (&)
UMR-CNRS 6219, Laboratoire des Glucides, Faculte de
´
´
Pharmacie, Universite de Picardie Jules Verne,
1 rue des Louvels, 80037 Amiens, France
e-mail: pascal.sonnet@u-picardie.fr
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Int J Pept Res Ther (2010) 16:257–266
4
´
photoadducts formed after s U irradiation (Saintome et al.
1994, 2000; Clivio et al. 1991, 1992a, b). The 4-thiouracil
derivatives are also known for their photochemical inter-
actions with different compounds (Fourrey et al. 1973,
1974; Jouin and Fourrey 1975; Fourrey and Moron 1976).
However, the greatest interest of this molecule is related to
their uses in the synthesis of the oligonucleotides analogues
and an additional sprayer (Lock Spray) for the reference
compound. The source and desolvation temperatures were
kept at 80 and 150°C, respectively. Nitrogen was used as the
drying and nebulizing gas at flow rates of 350 and 50 l/h,
respectively. The capillary voltage was 3 kV, the cone
voltage 100 V and the RF lens1 energy was optimised for
each sample (30–150 V). Lock mass correction, using
appropriate cluster ions of an orthophosphoric acid solution
(0.1% in 50/50 acetonitrile/water) or of a sodium iodide
solution (2 lg/ll in 50/50 propan-2-ol/water ? 0.05 lg/ll
caesium iodide), was applied for accurate mass measure-
ments. The mass range was typically 50–2,050 Da and
spectra were recorded at 2 s/scan in the profile mode at a
resolution of 10,000 (FWMH). Data acquisition and pro-
cessing were performed with MassLynx 4.0 software.
Melting points were determined on a Kofler plate and are
given uncorrected. Infrared spectra (IR) were recorded on a
NICOLET-210 spectrometer using KBr pellets or a Jasco
FT/IR 4200. Nuclear magnetic resonance (1H and 13C
NMR) spectra were recorded on a BRUKER AVANCE 500
spectrometer (500 MHz) and tetramethylsilane (TMS) was
used as an internal standard. 1H NMR analyses were
obtained at 500 MHz (s: singlet, d: doublet, t: triplet, dd:
double doublet, m: multiplet), whereas 13C NMR analyses
were obtained at 125 MHz. The chemical shifts (d) are
given in parts per million relative to TMS (d = 0.00).
´
(Saintome et al. 1994, 2000; Clivio et al. 1991, 1992a, b) and
peptides nucleic acids (PNA) (Clivio et al. 1997, 1998;
Kosynkina et al. 1994). The peptides nucleic acids were
highlighted for the first time by Nielsen and co-workers
(Nielsen et al. 1991; Egholm et al. 1992). Since 1991, s
everal methods of syntheses, in liquid and solid phases, have
been proposed for PNAs (Gasser and Spiccia 2008; Pritz
et al. 2006; Upert et al. 2005; Falkiewicz et al. 2001;
For reviews see Hyrup and Nielsen 1996; Koppelhus and
Nielsen 2003).
In this paper, we described the synthesis of three octa-
peptides analogues that could be able to bind by a covalent
way to TIIICBP via the use of 4-thiouracil (s4U) probe
selectively activable. Those octapeptides analogues are
obtained by the incorporation of s4U, at the 1 and/or 3
positions in KPGEPGPK sequence, since Erickson and
colleagues have been demonstrated that those correspond-
ing positions could be mutated without major anti-throm-
botic activity modifications (Erickson et al. 1992).
Procedure for Preparation of 1a, 1b and 1c
Materials and Methods
Fmoc(9-fluorophenylmethoxy)-aminoacidsandFmoc-amino
acids-SasrinÒ were purchased from Bachem (Germany). The
other chemicals compounds were purchased from Sigma-
Aldrich and used without further purification. The peptides
were synthesized on an Applied Biosystems Model 433A
peptide synthesizer, using standard automated continuous-
flow solid-phase peptide synthesis methods. Both manual
and automated systems (Applied Biosystem 433A Peptide
Synthesizer) were used to prepare the peptides 1a, 1b and 1c.
Ten-fold molar excess of the above amino acids were used in
a typical coupling reaction. Fmoc-deprotection was accom-
plished by treatment with a solution of 20% (v/v) piperidine
in N-methyl-2-pyrrolidone (NMP). The coupling reaction
was achieved by treatment with 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
and N,N-diisopropylethylamine (DIEA) in NMP using a
standard Fast-Moc protocol. The primary amine protecting
group N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]
(Dde) was removed with a solution of 2% hydrazine (v/v) in
DMF at room temperature. The peptides were cleaved and
side-chain deprotected by treatment of the peptide resin with
CH2Cl2/TFA (1:9 v/v) during 3 h at room temperature. The
octapeptides 1 were purified on an RP-HPLC C18 column
The chemicals compounds were purchased from Sigma-
Aldrich and used without further purification. Column
chromatography was performed on Kielselgel 60 (40–63 lm)
ASTM (Merck). Reactions were analyzed on precoated
silica gel 60 F254 plates (Merck) and the compounds were
visualized with a UV lamp (254 nm) and the phosphomo-
lybdic acid reagent and heating. The compounds were
analyzed on an analytical reverse-phase HPLC Shimadzu
instrument with a prosphere 100 C18 5 l column (4.6 9
150 mm) using a linear gradient of: (A) 0.1% (v/v) TFA in
water and (B) 0.1% (v/v) TFA acetonitrile/water mixture
(80/20, v/v), at a flow rate of 1 ml/min with UV detection at
220 nm. Compounds were purified by reverse-phase HPLC
using a Shimadzu semi-preparative HPLC system on an
prosphere 100 C18 5 l column (10 9 250 mm) by elution
with a linear gradient of (A): aqueous 0.1% TFA and (B):
0.1% (v/v) TFA in acetonitrile/water mixture (80/20), at a
flow rate of 3 ml/min with UV detection at 220 nm. The
mass spectra and the high-resolution electrospray mass
spectra in the positive ion mode were obtained on a Q-TOF
Ultima Global hybrid quadrupole/time-of-flight instrument
(Waters-Micromass, Manchester, U.K.), equipped with a
pneumatically assisted electrospray (Z-spray) ion source
(ProsphereÒ C18, 100 A 15 lm, 25 9 100 mm) using a
˚
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Int J Pept Res Ther (2010) 16:257–266
259
mixture of aqueous 0.1% (v/v) TFA (A) and 0.1% (v/v) TFA
in acetonitrile/water mixture (80/20, v/v) (B) as the mobile
phase (flow rate of 3 ml/min) and employing UV detection
at 220 nm. Electrospray mass spectrometric sequence
analysis has been used to confirm the correct sequences
(MS–MS, m/z).
a standard Fast-Moc protocol. The completed octapeptide
was cleaved from the resin and side chain deprotected by
treatment with a mixture of CH2Cl2/TFA (1:9 v/v) for 3 h at
room temperature. The solid support was removed by fil-
tration, the filtrate concentrated under reduced pressure, and
the octapeptide precipitated from diethyl ether to give
15.22 mg of the octapeptide 1b, as a yellow powder, after
reverse-phase chromatography purification (46%). m.p.:
H2N-Lys(s4U)-Pro-Gly-Glu-Pro-Gly-Pro-Lys-OH 1a
1
187°C. H NMR (500 MHz, CD3OD), d (ppm): 1.40 (m,
Synthesis of octapeptide started from commercially avail-
able Fmoc-Lys(Boc)-SasrinÒ 9 (177.0 mg, 0.105 mmol).
Then the octapeptide was cleaved from its resin and side
chain deprotected by treatment with a mixture of CH2Cl2/
TFA (1:9 v/v) for 3 h at room temperature. The solid
support was removed by filtration, the filtrate concentrated
under reduced pressure, and the free octapeptide precipi-
tated from diethyl ether. 27.36 mg of octapeptide 1a was
obtained, as a yellow powder, after reverse-phase chro-
3H, CH2c, Pro), 1.60 (m, 3H, CH2c, Pro), 1.65 (m, 2H,
CH2c, Lys), 1.80 (m, 2H, CH2b, Glu), 1.80 (m, 2H, CH2c,
Lys), 1.80 (m, 3H, CH2d, Lys), 1.90 (m, 2H, CH2c, Glu),
2.01 (m, 6H, CH2b, Lys), 2.07 (m, 3H, CH2d, Lys), 2.05 (m,
2H, CH2c, Lys), 2.49 (m, 6H, CH2b, Pro), 2.99 (m, 4H, CHe2,
Lys), 3.10 (m, 2H, CHe2, Lys), 3.62 (m, 3H, CH2d, Pro), 3.80
(m, 3H, CH2d, Pro), 4.10 (m, 2H, CH2, Gly), 4.22 (m, 3H,
CHa, Lys), 4.49 (m, 2H, CH2, s4U), 4.50 (m, 3H, CHa, Pro),
4.75 (m, 1H, CHa, Glu), 6.36 (d, J = 7.31 Hz, 1H, CH, s4U),
7.30 (d, J = 7.37 Hz, 1H, CH, s4U). 13C NMR (125 MHz,
CD3OD), d (ppm): 20.7 (CH2c, Pro), 22.0 (CH2c, Pro), 22.3
(CH2c, Pro), 24.7 (CH2b, Glu), 26.0 (CH2b, Lys), 26.5
(CH2b, Lys), 26.7 (CH2b, Lys), 29.1 (CH2d, Lys, 3C), 28.3
(CH2c, Glu), 29.2 (CH2b, Pro), 29.4 (CH2b, Pro), 29.5 (CH2b,
Pro), 30.5 (CH2c, Lys), 30.6 (CH2c, Lys), 30.8 (CH2c, Lys),
38.7 (CHe2, Lys), 38.9 (CH2e, Lys), 39.4 (CHe2, Lys), 41.4
(CH2, Gly), 46.5 (CH2d, Pro, 3C), 50.4 (CHa, Glu), 51.2
(CH2, s4U), 51.4 (CHa, Lys), 53.2 (CHa, Lys, 2C), 60.2
(CHa, Pro, 3C), 112.6 (CH, s4U), 141.2 (CH, s4U), 149.2
(CO, s4U), 167.2 (CO, Gly), 167.6 (CO, Lys), 168.2 (CH2-
CO, s4U), 170.9 (CO, Glu), 171.1 (CO, Lys), 172.7 (CO, Pro),
172.9 (CO, Pro), 173.1 (CO, Pro), 173.5 (COOH, Glu), 175.1
(COOH, Lys), 191.3 (CS, s4U). HRMS [MH]? calculated for
C46H74N13O13S: 1048.5250, found: 1048.5275.
1
matography purification (30%). Mp: 187°C. H NMR
(500 MHz, MeOD), d (ppm): 1.55 (m, 3H, CH2c, Pro), 1.75
(m, 3H, CH2c, Pro), 1.79 (m, 2H, CH2b, Lys), 1.80 (m, 4H,
CH2d, Lys), 1.85 (m, 2H, CH2b, Glu), 1.90 (m, 2H, CH2c,
Glu), 2.05 (m, 4H, CH2c, Lys), 2.11 (m, 2H, CH2b, Lys),
3.00 (m, 6H, CH2b, Pro), 3.30 (m, 4H, CHe2, Lys), 3.65 (m,
3H, CH2d, Pro), 3.76 (m, 3H, CH2d, Pro), 4.05 (m, 4H,
CH2, Gly), 4.29 (m, 1H, CHa, Lys), 4.49 (m, 1H, CHa,
Lys), 4.49 (m, 2H, CH2, s4U), 4.50 (m, 3H, CHa, Pro), 4.80
(m, 1H, CHa, Glu), 6.35 (m, 1H, CH, s4U), 7.36 (m, 1H, CH,
s4U). 13C NMR (125 MHz, MeOD), d (ppm): 20.8 (CH2c,
Pro), 22.0 (CH2c, Pro, 2C), 24.4 (CH2b, Lys), 24.9 (CH2b,
Lys), 26.1 (CH2b, Glu), 26.5 (CH2c, Glu), 29.0 (CH2d, Lys),
29.1 (CH2d, Lys), 29.2 (CH2b, Pro), 29.3 (CH2b, Pro, 2C),
29.5 (CH2c, Lys), 30.5 (CH2c, Lys), 38.1 (CH2e, Lys), 39.4
(CHe2, Lys), 41.4 (CH2, Gly), 42.2 (CH2, Gly), 46.6 (CH2d,
Pro, 3C), 50.4 (CHa, Glu), 50.5 (CH2, s4U), 51.3 (CHa,
Lys), 51.7 (CHa, Lys), 60.2 (CHa, Pro), 60.3 (CHa, Pro),
60.9 (CHa, Pro), 112.6 (CH, s4U), 141.1 (CH, s4U), 149.2
(CO, s4U), 167.6 (CO, Gly), 167.7 (CO, Lys), 168.1 (CO,
s4U), 169.9 (CO, Gly), 170.8 (CO, Glu), 172.9 (CO, Pro),
173.0 (CO, Pro), 173.1 (CO, Pro), 173.5 (COOH, Glu),
175.4 (COOH, Lys), 191.4 (CS, s4U). HRMS [MH]? cal-
culated for C42H65N12O13S: 977.4515, found: 977.4525.
H2N-Lys(s4U)-Pro-Lys(s4U)-Glu-Pro-Gly-Pro-Lys-OH
1c
Synthesis of octapeptide started from commercially avail-
able Fmoc-Lys(Boc)-SasrinÒ 9 (182.24 mg, 0.108 mmol).
The octapeptide Boc-Lys(Fmoc)-Pro-Lys(Dde)-Glu(OtBu)-
Pro-Gly-Pro-Lys(Boc)-SasrinÒ 10 was obtained and Fmoc-
deprotection was accomplished by treatment with a solution
of 20% (v/v) piperidine in NMP. The probe 4-thiouracil-1-
ylacetic acid 3 was coupled at the N-terminal of the side
chain of lysine in position 1, using a standard Fast-Moc
protocol. The octapeptide Boc-Lys(s4U)-Pro-Lys(Dde)-
Glu(OtBu)-Pro-Gly-Pro-Lys(Boc)-SasrinÒ was obtained and
Dde protecting group was manually removed with a solution
of 2% hydrazine (v/v) in DMF at room temperature. A
second probe 4-thiouracil-1-ylacetic acid 3 was coupled at
the N-terminal of the side chain of lysine in position 3
automatically, using a standard Fast-Moc protocol. The
completed octapeptide was cleaved from the resin and side
H2N-Lys-Pro-Lys(s4U)-Glu-Pro-Gly-Pro-Lys-OH 1b
Synthesis of octapeptide started from commercially avail-
able Fmoc-Lys(Boc)-SasrinÒ 9 (169.5 mg, 0.101 mmol).
The octapeptide Boc-Lys(Boc)-Pro-Lys(Dde)-Glu(OtBu)-
Pro-Gly-Pro-Lys(Boc)-SasrinÒ was obtained and the Dde
protecting group was manually removed with 2% hydra-
zine (v/v) in DMF at room temperature. Then the probe
4-thiouracil-1-ylacetic acid 3 was coupled at the N-terminal
of the side chain of lysine in position 3 automatically, using
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Int J Pept Res Ther (2010) 16:257–266
chain deprotected by treatment with a mixture of CH2Cl2/
TFA (1:9 v/v) for 3 h at room temperature. The solid support
was removed by filtration, the filtrate concentrated under
reduced pressure. Peptide was purified using reverse-phase
chromatography to afford 20.22 mg of octapeptide 1c as a
yellow powder (26%). m.p.: 188°C. HRMS [MH]? calcu-
lated for C52H77N15O15S2: 1215.5165, found: 1215.5109.
(CS). HRMS [MNa]? calculated for C6H6N2O3S23Na:
208.9997, found: 209.0006.
2-(2,4-Dioxo-5,6-dihydro-3H-pyrimidin-1-yl)-methyl
ethanoate 4
A suspension of uracil (10 g, 89.2 mmol) and K2CO3
(30.82, 223.03 mmol) in water (100 ml) was stirred at
80°C for 15 min. A solution of methyl 2-bromoethanoate
(20.4 g, 133.3 mmol) in water (10 ml) was added and the
solution was stirred at 80°C for 1 h. After cooling, the
precipitate was filtered, washed with cold water and dried
at room temperature in a desiccator, in the presence of
CaCl2 and under vacuum, to give 10 g of 4 as a white
powder (61%). m.p.: 180°C. IR (KBr, cm-1): 3136 (NH),
1700 (CO). 1H NMR (500 MHz, DMSO-d6), d (ppm): 3.68
(s, 3H, OCH3), 4.55 (s, 2H, CH2), 5.56 (d, J = 7.60 Hz,
1H, CH), 7.55 (d, J = 7.60 Hz, 1H, N–CH), 11.40 (s, 1H,
NH). 13C NMR (125 MHz, DMSO-d6), d (ppm): 49.0
(CH2), 52.7 (OCH3), 101.6 (N–CH), 146.3 (CH), 151.4
(CO–N), 164.1 (CO–CH), 169.1 (COO). MS [MNa]?
C7H8N2O4Na found: 207.04 (100%, molecular peak).
Fmoc-Lys(s4U)-OH 2
Sodium carbonate (5.5 mg, 65.47 lmol) and FmocOSu
(6.4 mg, 18.97 lmol) in dioxane (5 ml) were added to a
solution of H2N-Lys(s4U)-OH 8 (5 mg, 15.87 lmol) in
water (5 ml). The reaction mixture was stirred overnight at
room temperature. The reaction was cooled to 0°C and
acidified to pH 3 with HCl 9 N. The product was extracted
with EtOAc (3 9 30 ml). The organic phase was washed
with water, dried (MgSO4) and evaporated. After pre-
parative HPLC purification, 5.13 mg of Fmoc-Lys(s4U)-
OH 2 was obtained as a yellow oil (60%). 1H NMR
(500 MHz, CD3OD), d (ppm): 1.40 (m, 2H, CH2b, Lys),
1.50 (m, 2H, CH2d, Lys), 1.90 (m, 2H, CH2c, Lys), 3.21 (m,
2H, CH2e, Lys), 3.60 (m, 1H, CHa, Lys), 3.80 (m, 2H,
OCH2, Fmoc), 3.80 (m, 1H, CH, Fmoc), 4.40 (s, 2H, CH2,
s4U), 6.35 (d, J = 7.44 Hz, 1H, CH, s4U), 7.20 (m, 2H,
CH, Fmoc), 7.25 (m, 2H, CH, Fmoc), 7.25 (m, 1H, CH,
s4U), 7.60 (m, 2H, CH, Fmoc), 7.80 (m, 2H, CH,
Fmoc). 13C NMR (125 MHz, CD3OD), d (ppm): 22.8
(CH2b, Lys), 25.0 (CH2d, Lys), 28.4 (CH2c, Lys), 36.6
(CHe2, Lys), 49.0 (CH2, s4U), 50.2 (CH, Fmoc), 52.0 (CHa,
Lys), 66.6 (OCH2, Fmoc), 112.9 (CH, s4U), 119.6 (CH,
Fmoc, 2C), 124.8 (CH, Fmoc, 2C), 126.7 (CH, Fmoc, 2C),
127.4 (CH, Fmoc, 2C), 140.3 (CH, s4U), 141.2 (Cq, Fmoc,
2C), 143.9 (Cq, Fmoc, 2C), 148.9 (CO, s4U), 168.1 (CO,
s4U), 169.2 (CO, Fmoc), 173.4 (COOH, Lys), 191.3 (CS,
s4U). MS [MNa]? C27H28N4O6SNa found: 559.23 (100%,
molecular peak).
2-(2-Oxo-4-thioxo-5,6-dihydro-3H-pyrimidin-1-yl)-
methyl ethanoate 5
To a boiling solution of 4 (10 g, 54.3 mmol) in dioxane
(100 ml) was added P2S5 (14.42 g, 65.2 mmol). The
solution was stirred for 2 h and then filtered on CeliteÒ.
The filtrate was then concentrated to dryness. After puri-
fication by silica gel chromatography give, 15 g of 5 were
obtained as a yellow powder (69%). m.p.: 180°C. 1H NMR
(500 MHz, DMSO-d6), d (ppm): 3.68 (s, 3H, OCH3), 4.55
(s, 2H, CH2), 6.32 (d, J = 7.33 Hz, 1H, CH), 7.53 (d,
J = 7.33 Hz, 1H, N–CH), 12.80 (s, 1H, NH). 13C NMR
(125 MHz, DMSO-d6), d (ppm): 49.7 (CH2), 52.9 (OCH3),
112.8 (N–CH), 141.9 (CH), 148.8 (CO), 168.5 (COO),
191.3 (CS). MS [MH]? C7H9N2O3S found: 201.03 (100%,
molecular peak).
2-(2-Oxo-4-thioxo-5,6-dihydro-3H-pyrimidin-1-yl)-
ethanoic acid 3
Boc-Lys(s4U)-OCH3 6
A suspension of 5 (8 g, 40.0 mmol) in 2 N NaOH aqueous
(50 ml) was sonicated for 30 min. The reaction was cooled
to 0°C and acidified to pH 2 with HCl 9 N. The formed
precipitate was filtrated, washed with cold water, and dried
at room temperature in a dessicator over CaCl2 under
vacuum yielding 4.84 g of 3 as a slightly yellow powder
4-Thiouracil-1-ylacetic acid 3 (2.95 g, 15.89 mmol), NMM
(1.74 g, 15.89 mmol), and iBuOCOCl (2.17 g, 15.89 mmol)
were dissolved in CH2Cl2 (25 ml). After few minutes,
triethylamine (1.6 g, 15.89 mmol) was added, followed
by an addition of a solution of Boc-Lys-OCH3 (3.72 g,
14.30 mmol) in CH2Cl2 (25 ml). The reaction was stirred for
30 min at room temperature. The solvent was then evapo-
rated at reduced pressure, and the residue was purified on
silica gel with ethyl acetate (100%) to give 5.22 g of 6 as a
1
(65%). m.p.: 220°C. H NMR (500 MHz, DMSO-d6), d
(ppm): 4.40 (s, 2H, CH2), 6.29 (d, J = 6.85 Hz, 1H, CH),
7.52 (d, J = 6.85 Hz, 1H, N–CH), 12.65 (s, 1H, NH). 13C
NMR (125 MHz, DMSO-d6), d (ppm): 49.7 (CH2), 112.5
(N–CH), 142.1 (CH), 148.9 (CO), 169.4 (COOH), 191.2
1
yellow powder (81%). m.p.: 128°C. H NMR (500 MHz,
CD3OD), d (ppm): 1.35 (s, 9H, CH3, Boc), 1.30 (m, 2H,
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Int J Pept Res Ther (2010) 16:257–266
261
CH2b, Lys), 1.47 (m, 2H, CH2d, Lys), 1.72 (m, 2H, CH2c,
Lys), 3.19 (m, 2H, CHe2, Lys), 3.65 (s, 3H, OCH3), 4.03 (m,
1H, CHa, Lys), 4.41 (s, 2H, CH2, s4U), 6.31 (d, J = 7.32 Hz,
1H, CH, s4U), 7.39 (d, J = 7.32 Hz, 1H, CH, s4U), 8,35 (s,
1H, NH, Lys). 13C NMR (125 MHz, CD3OD), d (ppm): 22.7
(CH2b, Lys), 27.3 (CH3, Boc), 28.4 (CH2d, Lys), 30.8 (CH2c,
Lys), 39.0 (CHe2, Lys), 50.3 (CH2, s4U), 51.2 (OCH3), 53.6
(CHa, Lys), 79.2 (Cq, Boc), 112.5 (CH, s4U), 140.9 (CH,
s4U), 149.1 (CO, s4U), 156.7 (CO, Boc), 167.3 (CO, s4U),
173.6 (COO, Lys), 191.4 (CS, s4U). HRMS [MNa]? calcu-
lated for C18H28N4O6S23Na: 451.1627, found: 451.1631.
temperature. The solvent was then evaporated at reduced
pressure, and the residue was washed with ether to give
1
16.7 mg of 8 as a yellow powder (87%). m.p.: 130°C. H
NMR (500 MHz, CD3OD), d (ppm): 1.37 (m, 2H, CH2b,
Lys), 1.59 (m, 2H, CH2d, Lys), 1.85 (m, 2H, CH2c, Lys),
3.19 (m, 2H, CHe2, Lys), 3.65 (m, 1H, CHa, Lys), 4.33 (s,
2H, CH2, s4U), 6.20 (d, J = 6.81 Hz, 1H, CH, s4U), 7.18
(d, J = 6.81 Hz, 1H, CH, s4U). 13C NMR (125 MHz,
CD3OD), d (ppm): 20.3 (CH2b, Lys), 25.1 (CH2d, Lys),
28.4 (CH2c, Lys), 37.4 (CHe2, Lys), 49.0 (CH2, s4U), 52.1
(CHa, Lys), 111.1 (CH, s4U), 139.4 (CH, s4U), 147.7 (CO,
s4U), 166.0 (CO, s4U), 170.5 (COOH, Lys), 189.9 (CS,
s4U). MS [MH]? C12H19N4O4S found: 315.29 (100%,
molecular peak).
Boc-Lys(s4U)-OH 7
A solution of Boc-Lys(s4U)-OCH3 (1 g, 2.33 mmol) in
methanol (25 ml) was added an aqueous solution of 10%
NaOH (25 ml). The reaction was stirred for 2 h at 0°C. The
organic solvent was then evaporated at reduced pressure,
and the solution was neutralized using excess HCl. The
product was extracted in EtOAc (3 9 30 ml). The organic
phase was washed with water, dried (MgSO4) and evapo-
rated to furnish 724 mg of 7 as a yellow powder (75%).
m.p.: 130°C. 1H NMR (500 MHz, CD3OD), d (ppm): 1.39
(s, 9H, CH3, Boc), 1.40 (m, 2H, CH2b, Lys), 1.45 (m, 2H,
CH2d, Lys), 1.75 (m, 2H, CH2c, Lys), 3.25 (m, 2H, CH2e,
Lys), 4.09 (m, 1H, CHa, Lys), 4.43 (s, 2H, CH2, s4U), 6.35
(d, J = 7.20 Hz, 1H, CH, s4U), 7.38 (d, J = 7.20 Hz, 1H,
CH, s4U). 13C NMR (125 MHz, CD3OD), d (ppm): 22,8
(CH2b, Lys), 27.3 (CH3, Boc), 28.4 (CH2d, Lys), 30.0
(CH2c, Lys), 38.9 (CH2e, Lys), 50.2 (CH2, s4U), 53.4 (CHa,
Lys), 79.1 (Cq, Boc), 112.5 (CH, s4U), 140.9 (CH, s4U),
149.1 (CO, s4U), 156.8 (COO, Boc), 167.3 (CO, s4U),
174.8 (COO, Lys), 191.5 (CS, s4U). HRMS [MH]?
calculated for C17H26N4O6S: 437.1471, found: 437.1462.
Results and Discussion
In order to obtain the peptides 1, the 4-thiouracil probe at
the N-terminal of the side chain of the lysine could be
incorporated, according to a Fmoc chemistry strategy, in
the position 1 and/or 3 into the reference octapeptide
KPGEPGPK sequence. The three corresponding octapep-
tides 1a–c could be obtained from the modified lysine 2
starting from the 4-thiouracil-1-ylacetic acid 3 (s4U)
(Scheme 1).
The synthesis of the simple derivative 4-thiouracil 3 is
based on a methodology initially described by Wheeler and
Liddle (1908) and modified by Egholm et al. (1992). In our
case, the probe 3 was prepared in three steps from the
commercially available uracil (Scheme 2) (Clivio et al.
1998; Egholm et al. 1992).
Treatment of uracil with methyl 2-bromoethanoate and
K2CO3, as a base, furnished 4 in 61% yield (Scheme 2).
13C NMR of 4 indicates a signal at 49.0 ppm corresponding
to the methylenic group and 52.7 ppm to the methyl group.
This compound treated with a phosphorus pentasulfide
solution under boiling solution affords the sulfur compound
5. The spectra 13C NMR confirm the appearance of a signal
H2N-Lys(s4U)-OH 8
Boc-Lys(s4U)-OH 7 (20 mg, 45.75 lmol) was cleaved by
TFA treatment (10%) in CH2Cl2 (5 ml) during 3 h at room
Scheme 1 Retrosynthesis
of octapeptides analogues 1
O
OH
O
Boc-K-OCH3
O
N
H
+
K(R1)PXEPGPK 1
OH
HN
R1 = H or s4U
X = G or K(s4U)
O
O
O
O
K(s4U)PKEPGPK
KPK(s4U)EPGPK
K(s4U)PK(s4U)EPGPK
1a
1b
1c
HN
3 (s4U)
2
HN
S
S
123

262
Int J Pept Res Ther (2010) 16:257–266
S
N
S
N
O
N
O
4-thiouracil must, of course, be realized safe from the light.
13C NMR of the acid 3 confirms the presence of the thio-
amide group represented by the signal at 191.2 ppm and
the liaison CH=CH by the signal at 112.5 and 142.1 ppm
(Fig. 1). We can also check the presence of the acid group
by the signal at 169.4 ppm. The high-resolution electro-
spray mass spectra of 3 show the molecular ion at 209.0006
[MNa]?.
ii
i
iii
HN
HN
HN
HN
65%
61%
69%
O
O
O
O
N
H
5
COOCH3
COOH
COOCH3
4
3
Scheme 2 Synthesis of 4-thiouracil-1-ylacetic acid 3. Reagents: (i)
K2CO3, BrCH2COOCH3, DMF; (ii) P2S5, dioxane, reflux; (iii) NaOH,
water
The probe lysine Fmoc 2 was synthesized by two dif-
ferent methods (Scheme 3). First, the lysine 2 synthesis
was carried out by a direct coupling reaction between the
Fmoc-Lys-OH and the probe s4U 3 in the presence of
at 191.3 ppm corresponding to the C=S group. Finally,
the compound 5 was saponified to yield the corresponding
s4U 3 (total yield 29%). These reactions containing the
1
Fig. 1 13C NMR and H NMR
(DMSO-d6) spectrum of probe 3
123

Int J Pept Res Ther (2010) 16:257–266
263
iBuOCOCl in CH2Cl2. During this reaction, many impu-
rities have been formed and observed by HPLC and the
corresponding lysine 2 was obtained in low yield (33%
HPLC yield). Moreover, the retention times of these dif-
ferent compounds being too close, HPLC purification was
so constraining and unfortunately prevented us from
working with significant amounts.
observed impurities generally formed in the coupling
reaction. This unfortunately prevents us from working with
significant amounts of the probe 2.
Consequently, to limit the formation of secondary
products; we have decided to directly incorporate the
probe 3 into the lysine in position 1 and/or 3 at the end of
the peptide synthesis. For that, the octapeptide Fmoc
synthesis is carried out starting from the amino acid
L-Fmoc-Lys(Boc) grafted with the SasrinÒ resin in
C-terminal. The different amino acids are incorporated by
peptide coupling using HBTU/DIEA. At each step, the
Fmoc group is cleaved using a 20% piperidine solution in
the NMP. The difficulty here consist to incorporate a
lysine into 1 and/or 3 position with a selectively protec-
tive group (Dde, Fmoc, Boc) grafted in the function
amine Ne of the side chain. After synthesis of the octa-
peptide, the side chain amino function of the lysine in
position 1 and/or 3 is selectively cleaved to be coupled
with the probe thiouracil 3 (s4U).
The octapeptide K(s4U)PGEPGPK 1a is synthesized
starting from Fmoc-K(Boc)-SasrinÒ resin 9 (Scheme 4). The
seven amino acids are introduced into a sequential order in
fourteen steps to obtain the correctly protected octapeptide
10 (R1 = Fmoc, X1 = G). The last added amino acid is a
Boc-Lys(Fmoc)-OH. The Fmoc group was cleaved selec-
tively using a 20% piperidine solution in NMP. Conse-
quently, only the function amine Ne of the side chain of lysine
is free. The probe 3 was then coupled to this octapeptide
Consequently, a second new way has been used in which
the probe was incorporated at the beginning of the syn-
thesis. For this, the probe lysine protected 2 was obtained
in four steps starting from the Boc-Lys-OCH3 (Scheme 3).
The coupling reaction with the protected lysine and the
probe s4U 3 was accomplished in the presence of iBuO-
COCl/NMM to afford 6 (81%). The corresponding 1H
NMR spectra shows the presence of the nine protons of
Boc group at 1.35 ppm and two protons ethylene CH=CH
of the probe at 6.31 ppm and 7.39 ppm. In NMR 13C, the
presence of carbons related to these two CH protons were
observed at 112.5 and 140.9 ppm. The compound 6 was
then saponified to give 7 as 75% yield. After Boc depro-
tection of 5, the corresponding lysine 8 was engaged with
FmocOSu to afford compound 2, after preparative HPLC
purification (60% yield). 1H NMR showed the signal
characteristic of Fmoc group at 7.20, 7.25, 7.60 and
7.80 ppm. In this pathway synthesis (31% total yield), the
photoactivable probe remains stable during all the synthe-
sis, in particular, in the acidic conditions used to obtain the
deprotected lysine 8. In the two methods used, we always
Scheme 3 Synthesis of amino
acid 2. Reagents: (i)
O
O
H
N
H
N
O
O
O
4-thiouracil-1-ylacetic acid 3,
iBuOCOCl, NMM, TEA,
CH2Cl2; (ii) NaOH, MeOH,
water; (iii) TFA 10%, CH2Cl2;
(iv) FmocOSu, NaHCO3,
dioxane, water; (v) Fmoc-K-
OH, iBuOCOCl, NMM, TEA,
CH2Cl2
H
OH
OMe
O
N
OMe
NH2
O
O
ii
i
O
75%
81%
S
S
NH
NH
HN
N
HN
N
O
O
7
6
O
O
iii
87%
O
H2N
OH
NH
O
O
O
S
HN
OH
HN
iv
S
HN
v
N
60%
O
N
33%
O
8
2
NH
O
S
3
COOH
HN
N
O
O
123

264
Int J Pept Res Ther (2010) 16:257–266
y3, GPK), 244.2 (ion y2, PK) and 147.1 (ion y1, K). Only
some ions b, corresponding to the loss of amino acids on
the N-terminal side have been observed at 580.2 (ion b4,
K(s4U)PGE), 451.2 (ion b3, K(s4U)PG) and 394.1 (ion b2,
K(s4U)P). Some other fragments (ions b) are also observ-
ables corresponding with peptide fragments in which the
probe is missing on the level of the side chain of lysine:
226.1 (ion KP) and 509.2 (ion KPGEP).
Fmoc-K(Boc)
9
R
R1 = Fmoc or Boc
R2 = s4U 3 or Boc
i
7 steps
Boc-K(R1)PX1E(tBu)PGPK(Boc)
ii
10
R
X1 = G or K(Dde)
11 Boc-K(R2)PX1E(tBu)PGPK(Boc)
R
iii
13C NMR spectra shows the corresponding carbonyl
peaks between 149 and 175 ppm: 149.2 (s4U), 167.6 (G),
167.7 (K), 168.1 (s4U), 169.9 (G), 170.8 (E), 172.9 (P),
173.0 (P), 173.1 (P), 173.5 (E) and 175.4 ppm (K). The
signal at 191.4 ppm confirms the presence of the probe
group C=S. The two probe ethylene carbons are present at
1a K(s4U)PKEPGPK
1b
1c K(s4U)PK(s4U)EPGPK
KPK(s4U)EPGPK
Scheme 4 Synthesis of octapeptide analogues 1a, 1b and 1c.
Reagents: (i) Fmoc-amino acids, HBTU; (ii) R1 = Fmoc; X1 = G:
(a) piperidine 20%, NMP; (b) 4-thiouracil-1-ylacetic acid 7, HBTU;
R1 = Boc; X1 = K(Dde): (a) NH2NH2 2%, DMF; (b) 4-thiouracil-
1-ylacetic acid 3, HBTU; R1 = Fmoc; X1 = K(Dde): (a) piperidine
20%, NMP; (b) 4-thiouracil-1-ylacetic acid 3, HBTU; (c) NH2NH2
2%, DMF; (d) 4-thiouracil-1-ylacetic acid 3, HBTU; (iii) TFA 90%,
CH2Cl2
1
141.1 and 112.6 ppm. H NMR spectra shows the probe
ethylene protons at 6.42 ppm and 7.38 ppm. The a amino
acids protons are present at 4.05 (G), 4.29 (K), 4.49 (K),
4.50 (P) and 4.80 ppm (E).
The second octapeptide modified KPK(s4U)EPGPK 1b
was synthesized according to the same protocol synthesis
such as octapeptide 1a previously described. The lysine
which incorporated the probe possesses a Fmoc a-amino
function protection whereas the side chain amino function
is protected with a Dde group (Scheme 4). In the case of
the lysine in position 1, in which no probe is incorporated,
its two amino functions are Boc protected. After intro-
duction of the seven corresponding amino acids, starting
from the Fmoc-Lys(Boc)-SasrinÒ resin 9, the correctly
protected octapeptide 10 (R1 = Boc, X1 = K(Dde)) is
obtained. The cleavage of the Dde group (side chain of the
lysine located in position 3) is carried out manually (i.e. out
of automatic synthetizer) by the addition of a 2% hydrazine
function using HBTU/DIEA to give the corresponding
octapeptide 11 (R2 = s4U, X1 = G) grafted on the resin and
incorporating the probe. Lastly, after cleavage of the pro-
tective groups and the resin, using a 90% TFA solution in
CH2Cl2, the octapeptide K(s4U)PGEPGPK 1a is obtained,
after purification by preparative HPLC, with a total yield of
30%.
Peptide K(s4U)PGEPGPK 1a was characterized by the
mass and NMR spectroscopic analysis. The molecular ion
[MH]? is represented by the peak at 977.4 on the mass
spectra (Fig. 2). The mass spectra shows peptide fragments
at 681.4 (ion y7, PGEPGPK), 584.3 (ion y6, GEPGPK),
527.3 (ion y5, EPGPK), 398.2 (ion y4, PGPK), 301.2 (ion
Fig. 2 MS spectrum of peptide K(s4U)PGEPGPK 1a
123

Int J Pept Res Ther (2010) 16:257–266
265
Fig. 3 MS spectrum of peptide
KPK(s4U)EPGPK 1b
solution in DMF. The corresponding peptide is then cou-
pled with the probe 3 using HBTU/DIEA to afford the
octapeptide KPK(s4U)EPGPK 1b with a total yield of 46%,
after cleavage of the protective groups (Boc) and the resin
using a solution 90% TFA solution in CH2Cl2 and pre-
parative HPLC purification. The two octapeptides 1a and
1b present similar mass spectra (Fig. 3). The molecular ion
1b is observed at 1048.5 [MH]?. The fragments corre-
sponding to the ions b are represented by the masses at
506.9 (ion b6, KPK(s4U)EPG), 652.7 (ion b4, KPK(s4U)E),
523.6 (ion b3, KPK(s4U)) and 129.1 (ion b1, K). Other
peptide fragments are seen (ions y) at 920.4 (ion y7,
PK(s4U)EPGPK), 822.9 (ion y6, K(s4U)EPGPK), 527.3
(ion y5, EPGPK), 398.2 (ion y4, PGPK), 301.2 (ion y3,
GPK) and 244.1 (ion y2, PK).
positions 1 and 3. The lysine in position 3 has a (-amino
function Fmoc protected and a side chain amino function
Dde protected (Fmoc-Lys(Dde)). The lysine in position 1
possesses a a-amino function Boc and a side chain amino
function Fmoc protected (Boc-Lys(Fmoc)) (Scheme 4).
From L-Fmoc-Lys(Boc) grafted on the resin 9, the
sequential introduction of seven amino acids was carried
out. Then, after the side chain lysine deprotection (Fmoc)
by using piperidine, the probe 3 is coupled. The Dde group
(lysine in position 3) is cleaved manually using a 2%
hydrazine solution in DMF. The second probe 3 was then
incorporated by peptide coupling using HBTU. Lastly,
after final deprotection and resin cleavage, the octapeptide
K(s4U)PK(s4U)EPGPK 1c is obtained with a total yield of
26% after purification on preparative HPLC.
The last octapeptide modified 1c possesses two probes
incorporated in the two lysine side chains located into
The octapeptide K(s4U)PK(s4U)EPGPK 1c was identified
by spectroscopy of mass (Fig. 4). The peak corresponding
Fig. 4 MS spectrum of peptide K(s4U)PK(s4U)EPGPK 1c
123

266
Int J Pept Res Ther (2010) 16:257–266
at the ion multi-charged [M ? 2H]2? and the ion molecular
are found respectively at 608.7633 and 1215.5109. Some
other peptide fragments (ions y) are visible at 920.4 (ion y7,
PK(s4U)EPGPK), 527.3 (ion y5, EPGPK), 398.2 (ion y4,
PGPK) and 244.1 (ion y2, PK). The fragment corresponding
to the lysine appears at 129.1 (ion b1) and to K(s4U)P at 394.1
(ion b2).
Fourrey JL, Moron J (1976) Thiocarbonyl photochemistry VII.
Photochemistry of 1,3-dimethyl 4-thiouracil in presence of
triethylamine. Tetrahedron Lett 17:301–304
Fourrey JL, Jouin P, Moron J (1973) Thiocarbonyl photochemistry II.
The reaction of 4-thiouracil derivatives with unsaturated nitriles.
Tetrahedron Lett 14:3229–3232
Fourrey JL, Jouin P, Moron J (1974) Thiocarbonyl photochemistry
III. Thietanes obtention from 4-thiouracil derivatives. Tetrahe-
dron Lett 15:3005–3006
Gasser G, Spiccia L (2008) Synthesis of a ferrocenyl uracil PNA
monomer for insertion into PNA sequences. J Organomet Chem
693:2478–2482
Hyrup B, Nielsen PE (1996) Peptide nucleic acids (PNA): synthesis,
properties and potential applications. Bioorg Med Chem 4:5–23
Jouin P, Fourrey JL (1975) Thiocarbonyl photochemistry V. Light
induced reactions of 3-methyl 4-thiouracil with methacrylonit-
rile. Tetrahedron Lett 16:1329–1332
Koppelhus U, Nielsen PE (2003) Cellular delivery of peptide nucleic
acid (PNA). Adv Drug Deliv Rev 55:267–280
Kosynkina L, Wang W, Liang TC (1994) A convenient synthesis of
chiral peptide nucleic acid (PNA) monomers. Tetrahedron Lett
35:5173–5176
Maurice P, Waeckel L, Pires V, Sonnet P, Lemesle M, Arbeille B,
Vassy J, Rochette J, Legrand C, Fauvel-Lafeve F (2006a) The
platelet receptor for type III collagen (TIIICBP) is present in
platelet membrane lipid microdomains (rafts). Histochem Cell
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Antithrombotique effect of the type III collagen-related octa-
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Conclusions
In conclusion, we have synthesized three new KPGEPGPK
analogues in which the probe photochemical selectively
activable 4-thiouracil (s4U) is incorporated in position 1
and/or 3 by Fmoc chemistry synthesis with a total yield
superior at 25%. These analogues could be used to identify
the putative new receptor for type III collagen named Type
III Collagen Binding Protein (TIIICBP).
Acknowledgment Viviane Silva Pires was the recipient of a grant
from the CNPq (Brazil).
Conflict of interest The authors have no competing interests that
might be perceived to influence the results and/or discussion reported
in this paper.
`
Monnet E, Fauvel-Lafeve F (2000) A new platelet receptor specific to
type III collagen. Type III collagen-binding protein. J Biol Chem
275:10912–10917
Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-
selective recognition of DNA by strand displacement with a
thymine-substituted polyamide. Science 254:1497–1500
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