On-resin cyclization of a head-to-tail cyclopeptide using an allyldimethylsilyl polystyrene resin pre-loaded by metathesis

Article abstract of DOI:10.1016/j.tet.2005.05.078

Here, we report the solid-phase synthesis of a 17-mer cyclopeptide which is expected to have anti-angiogenic properties. The peptidic synthesis is performed on an allyldimethylsilyl polystyrene support loaded by metathesis with a conveniently functionalized D-Tyrosine amino acid. The linear peptide was assembled by standard Fmoc chemistry and on-resin cyclization was enabled after selective deprotection of the C-terminal group with 2% hydrazine/DMF at room temperature. Final cleavage was realized under mild acidic conditions allowing to obtain a cyclopeptide under partially protected form.

Full text of DOI:10.1016/j.tet.2005.05.078

Tetrahedron 61 (2005) 7789–7795
On-resin cyclization of a head-to-tail cyclopeptide using an
allyldimethylsilyl polystyrene resin pre-loaded by metathesis
b
b
a
Mario Gonc¸alves,a,b Karine Estieu-Gionnet,b,c Georges Laın, Mireille Bayle, Natacha Betz
¨
b,
*
´ ´ ´
and Gerard Deleris
aCEA Saclay, DSM/DRECAM/LSI/LPI, Bat. 466, 91191 Gif-sur-Yvette Cedex, France
´ ´
CNRS UMR 5084 CNAB, Groupe de Chimie Bioorganique, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, F-33076 Bordeaux
b
c
Cedex, France
´ ` ´ ´
INSERM E0113, Laboratoire des mechanismes moleculaires de l’angiogenese, Avenue des Facultes-Universite Bordeaux 1, 33405 Talence,
France
Received 22 December 2004; revised 19 May 2005; accepted 23 May 2005
Available online 15 June 2005
Abstract—Here, we report the solid-phase synthesis of a 17-mer cyclopeptide which is expected to have anti-angiogenic properties. The
peptidic synthesis is performed on an allyldimethylsilyl polystyrene support loaded by metathesis with a conveniently functionalized D-
Tyrosine amino acid. The linear peptide was assembled by standard Fmoc chemistry and on-resin cyclization was enabled after selective
deprotection of the C-terminal group with 2% hydrazine/DMF at room temperature. Final cleavage was realized under mild acidic conditions
allowing to obtain a cyclopeptide under partially protected form.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
to a considerable loss of product. An attractive alternative
includes solid support linkage to an amino acid side-chain
and solid-phase chain assembly of the linear sequence,
followed by cyclization while the peptide still remains
anchored to the resin. The pseudo-dilution attributed to the
solid-phase favours intramolecular reactions over inter-
molecular reactions.10 So far, this method has been applied
to aspartic and glutamic acid,11,12 lysine,13 tyrosine14 and
recently to serine and threonine15 in the Fmoc strategy with
different C-terminal protecting groups. To date, general and
commercial synthesis of head-to-tail cyclic peptides has
used only Allyl group (All) for temporary protection of the
a-COOH group of resin-bound amino acid.16 The selective
removal of the Allyl ester requires complex mixtures such
as Pd(PPh3)4–AcOH–CHCl3-N-methylmorpholine (NMM)
over an extended period of 2 h. Moreover, the use of
tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) requires
inert atmosphere and degazed solvents. To overcome these
drawbacks, a novel 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene)-3-methylbutyl]-amino} benzyl alcohol protect-
ing group called Dmab has been described and used in the
synthesis of a model cyclic heptapeptide17 using standard
Fmoc/tBu procedures.18 Recently, the use of this Dmab
group as a temporary a-COOH protecting group has been
reported for automated solid-phase synthesis of an expanded
(29-mer) cyclic peptide.19
Synthesis of head-to-tail cyclopeptides has attracted a
considerable interest since the antibiotic gramicidin S was
found to be a cyclic decapeptide.1 Many antibiotics and
toxins are also known to be cyclic peptides. Cyclization of
amino acid sequences results in increased metabolic
stability, potency, receptor selectivity and bioavaila-
bility.2–4 Thus, cyclic peptides present suitable properties
for investigating ligand–receptor interactions and structure–
activity relationships5,6 as well as for developing drugs with
increased metabolic stability and receptor selectivity.7–9
Classical methods used to prepare cyclic peptides involve
the synthesis of partially protected linear precursors either in
solution or on solid-phase, their subsequent cleavage, and
cyclization in solution; this one has to be performed under
high dilution to minimize the formation of cyclodimers and
oligomers. This synthetic procedure present some dis-
advantages, such as the necessity to isolate the desired
peptide from the excess reagents, which leads in some cases
Keywords: Solid-phase peptidic synthesis; Cyclic peptides; Metathesis;
On-resin cyclization; Mitsunobu reaction.
Corresponding author. Tel./fax: C33 557 571002;
e-mail: gerard.deleris@u-bordeaux2.fr
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.05.078

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M. Gonc¸alves et al. / Tetrahedron 61 (2005) 7789–7795
2. Results and discussion
the expected compound 2. To protect the a-COOH of D-
Tyrosine, Dmab group was preferred to allyl group, due to
the metathesis step needed for resin loading. Indeed, allyl
group could interfere with the terminal olefin of compound
2 during the loading step.
In a previous work, we have reported the structural and the
biological properties of a 17-mer cyclopeptide.20 This 17-
amino acid molecule, described as cyclic vascular endo-
thelial growth inhibitor (cyclo-VEGI), revealed to be an
attractive candidate for the development of novel angiogen-
esis inhibitor molecules useful for the treatment of cancer
and other angiogenesis-related diseases, now under pre-
clinical development. Angiogenesis allows the establish-
ment of a vascular supply, which is fundamental not only for
organ development but also for important processes in the
adults such as wound healing and reproductive functions.21,22
Angiogenesis is also implicated in the pathogenesis of a
variety of disorders: proliferative retinopathies, age-related
Compound 2 was then involved in our 17-mer cyclic peptide
synthesis. Solid-phase peptide synthesis (Scheme 2) was
performed on an allyldimethylsilyl polystyrene resin which
has a silicon content of 1.3 mmol/g. Loading of the resin
was achieved by cross-metathesis in refluxing CH2Cl2 using
10 mol% of Grubb’s catalyst (first generation) and 1 mmol
of 2 per gram of resin. After 18 h, the resin was filtered off
and washed with DMF, CH2Cl2, MeOH and Et2O. Residual
diethyl ether was removed under vacuum, and a substitution
level of 0.14 mmol/g was determined for this new pre-
loaded resin. 0.1 mmol of this resin was then used for the
linear peptide synthesis on a ABI-433A continuous-flow
automated peptide synthesizer. Standard Fmoc chemistry
was used throughout. Couplings were made with 10 M
excess of the acylating amino acid by activation with a
0.45 M 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate/hydroxybenzotriazole (HBTU/
HOBt) in DMF solution as recommended in the automated
synthesizer manual for 0.1 mmol scale peptide synthesis.
After completion of the peptide assembly, the resin was
treated with 2% hydrazine/DMF at room temperature for
3 min. The treatment was repeated two more times, and the
partially protected resin was thoroughly washed with DMF.
For the intramolecular cyclization 3 M equiv of each
benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexa-
fluorophosphate(PyBOP)andHOBtwereusedinthepresence
of 6 M equiv of diisopropylethylamine (DIEA) for 72 h.
¨
macular degeneration, tumors, rheumatoıd arthritis and
psioriasis. A number of angiogenesis regulators such as
Vascular Endothelial Growth Factors (VEGFs) and Fibro-
blast Growth Factors (FGFs) have been identified.23–26
Cyclo-VEGI encompasses residues 79–93 of VEGF which
are involved in VEGF–VEGF type2 receptor interactions.
Efforts have been made to conveniently functionalize one of
the cyclo-VEGI side-chain’s group in order to broaden its
biological properties.27 Indeed, introduction of a terminal
olefin could allow different ways of functionalization on
cyclo-VEGI with the aim to enhance its anti-angiogenic
properties. However, cyclo-VEGI basic side-chains (i.e.,
Arg, Lys and His) should be kept free due to their key role in
VEGF–receptor interaction.
In this work, we describe a novel solid-phase peptide synthesis
for such compounds which allows high functionalization
potential. It takes advantage of an allyldimethylsilyl poly-
styrene support which could be loaded by cross metathesis
with functionalized terminal olefins.28 Then cyclo-VEGI was
modified by substitution of D-Phenylalanine by D-Tyrosine,
due to their structural homology. Hence, it affords a new side-
chain group from which a terminal olefin could be introduced.
Scheme 1 shows a synthetic way to convenientlyfunctionalize
the commercially available Fmoc-DTyr(OtBu)-OH (Novabio-
chem) to realize solid-phase peptide synthesis and on-resin
cyclization. The esterification of the D-Tyrosine derivative
with Dmab-OH was accomplished by activation with
diisopropylcarbodiimide (DIPCDI) to yield Fmoc-DTyr-
(OtBu)–ODmab 1, which on treatment with trifluoroacetic
acid (TFA) in CH2Cl2 gave the required Fmoc-DTyr-ODmab.
Etherification has been attempted using K2CO3 and 4-bromo-
1-butene in acetone, but it was unsuccessful. Then etherifica-
tion with an alkenyl group was realized under Mitsunobu
reaction conditions, that is, with triphenylphosphine/diethyl
azodicarboxylate (PPh3/DEAD) and but-3-en-1-ol, to yield
Peptide was cleaved from the resin with a solution of 1%
TFA in CH2Cl2 affording a protected modified cyclo-VEGI.
A portion of this peptide was taken for final deprotection
step employing a mixture of trifluoroacetic acid (TFA) in the
presence of suitable scavengers to obtain the functionalized free
cyclic peptide. Peptides were purified by reverse-phase high
performance liquid chromatography (RP-HPLC) and MALDI
mass spectrometry analysis gave the expected mass results.
3. Conclusion
To sum up, we have presented here, a new way to synthesize
a cyclic peptide with one side-chain selectively functiona-
lized which was obtained directly after cleavage from the
resin. To our knowledge this is the first time that a peptide
synthesis was performed on allyldimethylsilyl polystyrene
resin. Thus this 17 amino-acid anticancer cyclic peptide
Scheme 1. Functionalization of Fmoc-DTyr(OtBu)-OH. Reagents and conditions: (i) 1.5 equiv DIPCDI and HOBt, 3 equiv DIEA, CH2Cl2, rt, 18 h, 80%;
(ii) TFA/CH2Cl2 (95/5 v/v), rt, 3 h, 92%; iii) 1.5 equiv of each PPh3-DEAD-but-3-en-1-ol, CH2Cl2, rt, 48 h, 52%.

M. Gonc¸alves et al. / Tetrahedron 61 (2005) 7789–7795
7791
Scheme 2. Solid phase synthesis of the 17-mer cyclopeptide. Reagents and conditions: (i) 2, 10% mol Cl2(PPh3)2RuZCHPh, CH2Cl2; (ii) 16 cycles of Fmoc/
tBu solid-phase peptide synthesis a—20% piperidine/N-methylpyrrolidone (NMP), b—Fmoc-AA-OH, HBTU/HOBt/NMP, DIEA with final deprotection;
(iii) 2% hydrazine/DMF; (iv) PyBOP, HOBt, DIEA, NMP; (v) TFA/triisopropylsilane (Tis)/thioanisole/water/phenol; (vi) 3% TFA/CH2Cl2.
could be obtained either under protected or unprotected form.
Our synthetic approach opens the door to subsequent
functionalizations such as radio-labelling or dimerization of
cyclopeptide 3 and study in order to investigate the structural
and biological properties of these new compounds.
III Bruker apparatus, and HRMS were run on a LCT premier
from Waters. For the NMR spectra, a Bruker Avance 300
was used. Chemicals shifts are reported in parts per million
relative to tetramethylsilane as an internal standard (in
NMR description sZsinglet, dZdoublet, tZtriplet, qZ
quartet, mZmultiplet, brZbroad peak). UV measurements
were taken on a Genesys 5 Spectronic Instruments
spectrophotometer. For column chromatography, 63–200
mesh silica gel 60 (VWR International) was used as the
stationary phase.
4. Experimental
4.1. General information
MALDI mass spectra were run using a MALDI-TOF Reflex
The N-(9-Fluorenylmethoxycarbonyl, Fmoc) protected

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M. Gonc¸alves et al. / Tetrahedron 61 (2005) 7789–7795
natural aminoacids and allyldimethylsilyl polystyrene resin
were purchased from Advanced Chemtech. All trifunctional
aminoacids were suitably protected. The a-carboxyl group
of D-Tyrosine was protected with the 4-{N-[1-(4,4-
dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}
benzyl alcohol (Dmab) group. The 3-amino group of lysine
was protected with the (tert-butoxy)carbonyl (Boc) group.
The histidine imidazole group and the amide group of
glutamine were protected with the triphenylmethyl (Trityl,
Trt) group. The guanidino function of arginine was
protected with the 2,2,4,6,7-pentamethyldihydrobenzo-
furan-5-sulfonyl (Pbf) group and the u-carboxyl group of
glutamic acid was protected with the tert-butyl (tBu) group.
Solution of 0.45 M 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HBTU) in N-hydroxy-
benzotriazole (HOBt) was purchased from Applied
Biosystems. Diethyl azodicarboxylate (DEAD), diisopro-
pylethylamine (DIEA), dimethylformamide (DMF), phenol,
thioanisole, trifluoroacetic acid (TFA), triisopropylsilane
and triphenylphosphine were purchased from Aldrich.
Diethyl ether, piperidine and potassium hydroxide were
purchased from Avocado. Hydrazine was purchased from
Acros and Grubb’s 1st generation catalyst was purchased
from Strem chemicals. Absolute ethanol, dichloromethane,
ethyl acetate, n-hexane and methanol were purchased from
J. T. Baker.
using a 4-fold excess of aminoacids activated as HOBt ester
by means of a 0.45 M HBTU/HOBt solution. Removal of
the Dmab protecting group was performed after N-terminal
Fmoc deprotection. The peptidyl resin was weighed (1.12 g,
0.11 mmol) and placed on a frit in a syringe barrel and
allowed to equilibrate for 5 min in DMF (20 mL/g of resin).
The solvent was removed from the resin by applying a
nitrogen pressure and the residue was resuspended in a
solution of 2% hydrazine monohydrate in DMF (20 mL/g of
resin). Reaction was allowed to proceed for 3 min with
gentle manual agitation and the hydrazine treatment was
repeated a further 2 times to ensure complete reaction. The
peptide-resin was washed with DMF (5!20 mL/g of resin)
and resuspended in a solution of DIEA in DMF (1/9 v/v;
20 mL/g of resin) for 10 min. Finally, the peptidyl resin was
washed successively with DMF, MeOH, Et2O and dried in
vaccuo over KOH. On-resin cyclization was performed by
mixing peptidyl resin (890 mg, 0.09 mmol) with a solution
of PyBOP (140.5 mg, 0.27 mmol), HOBt (36.5 mg,
0.27 mmol) and DIEA (93.5 mL, 0.54 mmol) in 20 mL of
NMP. The mixture was swelled at room temperature for
72 h. The peptidyl resin was washed with 50 mL of each
NMP, CH2Cl2, MeOH and was dried under high vacuum.
Final cleavage of cyclo(DYFPQIMRIKPHQGQHIGE)
from the resin without loss of any side-chain protecting
group was performed with a solution of 1% TFA in CH2Cl2
(10 mL/g of resin). Cyclopeptidyl resin was mixed with
dilute TFA and shake for 4 min. Then the solution was
filtered and filtrate was collected in a flask containing a
solution of 10% pyridine in MeOH (2 mL/10 mL of 1%
TFA). It was repeated 5 times and resin was washed with
3!30 mL of CH2Cl2, 3!30 mL of MeOH, 2!30 mL of
CH2Cl2 and 3!30 mL of MeOH. Then filtrate was
evaporated under reduced pressure to 5% of the volume.
Thereafter, 40 mL of cold water were added to the residue to
aid precipitation of the product which was isolated by
filtration through a sintered glass funnel. Product was
washed three times with fresh water, dissolved in a solution
of CH3CN/H20 (70/30) with 0.1% TFA (eluant B) and then
loaded onto a preparative Hibar Purosphere column C18.
The elution was achieved using the following conditions:
eluant A, 0.1% TFA in water; eluant B, 0.1% TFA in
CH3CN/H2O (70/30); gradient: 10% of B at 0 min, 20% of
B at 5 min, 100% of B at 7 min and 100% of B at 30 min;
4.2. Anchoring of Fmoc-DTyr(O–(CH2)2–CH]CH2)–
ODmab to the allyldimethyl silyl polystyrene resin
Fmoc-DTyr(O–(CH2)2–CH]CH2)–ODmab
(540 mg,
0.7 mmol) was dissolved in degazed CH2Cl2 (25 mL).
Then allyldimethylsilyl polystyrene resin (702 mg,
1.3 mmol gK1) and Grubb’s first generation catalyst
(28.9 mg, 35.11 mmol) were successively added. The
resulting suspension was refluxed under argon atmosphere
overnight. Then Grubb’s catalyst (28.9 mg, 35.11 mmol)
was added again and the reaction mixture was maintained
under refluxed for 12 h. The resin was filtered off and
successively washed with DMF, CH2Cl2, MeOH and Et2O
(100 mL of each). The residual diethyl ether was removed
under high vacuum. The substitution level was determined
spectrophotometrically by Fmoc cleavage. Fmoc-DTyr-
(resin)–ODmab (5.4 mg) was introduced into a test tube
and a solution of 20% piperidine in DMF was added
(0.5 mL). 20% piperidine in DMF (0.5 mL) was also added
to an empty test tube to serve as a blank. Over the next
15 min, the test tube with the resin was swirled two or three
times to make sure all the resin has come in contact with the
piperidine solution. DMF was added to both tubes to bring a
volume of 50 mL. The blank was used to zero the UV
spectrophotometer at 301 nm. The absorbance of the
solution is 0.126. The substitution level was calculated
from the formula: Abs301!Vol (mL)/(7800!m (g)) and
was determined to be 0.15 mmol/g.
4.3. Peptide synthesis
Cyclo(DYFPQIMRIKPHQGQHIGE) was synthesized by
Fmoc/t-Bu batch solid phase synthesis on an Applied
Biosystems 433A automated peptide synthesizer. Pre-
loaded Fmoc-DTyr(resin)–ODmab was used for the linear
chain assembly. Subsequent Fmoc aminoacids were coupled
Scheme 3. Protected peptide analytical HPLC performed on Hibar
Purosphere C18 column with an isochratic of 1% TFA in CH2CL2 over a
period of 25 min.

M. Gonc¸alves et al. / Tetrahedron 61 (2005) 7789–7795
7793
flow rate: 4 mL minK1; detector: 214 nm. Then 130 mg
(35%) of protected cyclo(DYFPQIMRIKPHQGQHIGE)
were obtained. MALDI mass spectrometry analysis gave
the expected result (theoretical value: 3716.64 Da; experi-
mental value: 3716.94 Da) and analytical HPLC on Hibar
Purosphere column C18, eluted with an isochratic gradient
of CH2Cl2 with 0.1% TFA, gave the following profile:
(Scheme 3)
2H, CH2 Fmoc), 4.65 (m, 1H, CH DTyr), 5.12 (s, 2H, CH2
3
Bzl), 5.27 (d, 1H, NH DTyr, JH–HZ8.07 Hz), 6.86 (d,
3
2H, CHAr DTyr, JH–HZ7.86 Hz), 6.94 (d, 2H, CHAr DTyr,
3
3JH–HZ7.86 Hz), 7.09 (d, 2H, CHAr Bzl, JH–HZ7.78 Hz),
7.27 (d, 2H, CHAr Bzl, 3JH–HZ7.78 Hz), 7.24–7.40 (m, 4H,
3
CHAr Fmoc), 7.54 (d, 2H, CHAr Fmoc, JH–HZ7.13 Hz),
7.74 (d, 2H, CHAr Fmoc, 3JH–HZ7.13 Hz), 15.3 (s, 1H, NH
Dmab).
A portion of the product was treated with 0.75 g of phenol in
a TIS/thioanisole/H20/TFA solution (1:2:2:40) for 3 h at
room temperature. The product was precipitated from cold
diethyl ether and filtered. Preparative Hibar Purosphere
column C18 was achieved using the same conditions as
below. Then MALDI mass spectrometry analysis gave the
expected result (theoretical value: 2082 Da; experimental
value: 2081.69 Da) and analytical HPLC on Hibar Puro-
sphere column C18, eluted with a gradient between eluant A
and B (see below), gave the following profile: (Scheme 4).
13C NMR (CDCl3) d ppm: 14.09, 22.56, 28.77, 29.54, 30.01,
37.63, 38.33, 47.08, 52.26, 53.75, 54.86, 66.24, 66.97,
78.45, 107.75, 119.96, 124.17, 125.01, 126.67, 127.01,
129.13, 129.70, 130.17, 134.59, 137.03, 141.28, 143.67,
154.56, 155.53, 171.44, 176.37, 196.37, 200.21.
IR (cmK1): 3307; 2957; 2869; 1725; 1644; 1557; 1507;
1451; 1414; 1387; 1366; 1325; 1163; 1051; 898; 759; 741.
MALDI: expected 770.97; found 769.32.
HRMS (ESC): calculated for C48H55N2O7 771.4009; found
771.3978.
[a]2D0 C23.148, c 1.08 in CH2Cl2.
4.5. Deprotection of compound 1: N-a-Fmoc-D-Tyrosine-
4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-
methylbutyl]-amino} benzyl ester
To a solution of compound 1 (6.13 g, 7.95 mmol) in CH2Cl2
(80 mL) was added TFA (80 mL). The reaction mixture was
stirred at room temperature for 3 h. The solution was
concentrated over reduced pressure to 5% of the volume.
Cold water was added to the residue and a white precipitate
appears. The solid was isolated by filtration and washed 3
times with cold water. Then it was dried in dessicator under
high vacuum over KOH to give 5.22 g (92%) of deprotected
compound 1 as a white solid (mp 92–94 8C).
Scheme 4. Deprotected peptide analytical HPLC performed on Hibar
Purosphere C18 column with a gradient of eluant A and B over a period of
25 min (eluant A: 1% TFA in water; eluant B: 1% TFA in CH3CN/H2O
(70:30)).
1H Nmr (CDCl3) d ppm: 0.76 (d, 6H, (CH3)2–CH, 3JH–H
6.63 Hz), 1.06 (s, 6H, 2 CH3), 1.84 (m, 1H, (CH3)2–CH),
Z
4.4. Compound 1: N-a-Fmoc-O-tertbutyl-D-Tyrosine-4-
{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-
methylbutyl]-amino} benzyl ester
2.42 (br s, 4H, 2 CH2), 2.99 (m, 4H, CH2–CH and CH2
3
DTyr), 4.18 (t, 1H, CH Fmoc, JH–HZ6.59 Hz), 4.34–4.43
(m, 2H, CH2 Fmoc), 4.65 (br s, 1H, CH DTyr), 5.13 (d, 1H,
To a solution of Fmoc-Dtyr(tBu)-OH (5 g; 10.88 mmol) in
CH2Cl2 was added successively DIEA (5.65 mL;
32.64 mmol), DIC (2.5 mL; 16.32 mmol) and HOBt
(2.21 g; 16.32 mmol). After complete dissolution, Dmab-
OH (5 g; 15.23 mmol) was added. Thereafter, the solution
was stirred at room temperature for 18 h. Then the reaction
mixture was filtered and washed with water (4!60 mL).
The organic layer was dried over MgSO4 and filtrated. The
solvent is evaporated under reduced pressure to give a
greenish oil which was purified by silica gel chromatog-
raphy (n-hexane/ethyl acetate, 60:40, 70:30). Then 6.68 g
(80%) of compound 1 were obtained as a yellowish oil.
NH DTyr, 3JH–HZ8.07 Hz), 5.27 (s, 2H, CH2 Bzl), 6.66 (d,
3
2H, CHAr DTyr, JH–HZ7.86 Hz), 6.83 (d, 2H, CHAr DTyr,
3
3JH–HZ7.86 Hz), 7.07 (d, 2H, CHAr Bzl, JH–HZ7.89 Hz),
7.26 (d, 2H, CHAr Bzl, 3JH–HZ7.89 Hz), 7.24–7.39 (m, 4H,
3
CHAr Fmoc), 7.53 (d, 2H, CHAr Fmoc, JH–HZ7.32 Hz),
7.74 (d, 2H, CHAr Fmoc, 3JH–HZ7.32 Hz), 15.1 (s, 1H, NH
Dmab).
13C NMR (CDCl3) d ppm: 22.59, 28.25, 29.53, 30.04, 37.44,
38.42, 47.19, 52.25, 54.93, 66.28, 67.03, 107.91, 115.47,
120.01, 125.02, 126.63, 127.75, 129.54, 130.44, 134.76,
141.34, 143.69, 155.11, 155.59, 171.44, 176.48, 196.61.
1H NMR (CDCl3) d ppm: 0.75 (d, 6H, (CH3)2–CH, 3JH–H
Z
IR (cmK1): 3340; 2958; 2868; 1704; 1616; 1553; 1515;
1450; 1414; 1326; 1248; 1170; 1103; 1051; 827; 759; 740.
6.65 Hz), 1.06 (s, 6H, 2 CH3), 1.29 (s, 9H, 3 CH3), 1.82 (m,
1H, (CH3)2–CH), 2.38 (s, 2H, CH2), 2.48 (s, 2H, CH2), 2.99
3
(d, 2H, CH2–CH, JH–HZ6.80 Hz), 3.06 (br s, 2H, CH2
3
DTyr), 4.23 (t, 1H, CH Fmoc, JH–HZ6.69 Hz), 4.36 (br s,
MALDI: expected 714.86; found 715.30.

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M. Gonc¸alves et al. / Tetrahedron 61 (2005) 7789–7795
´
the “Ligue Nationale contre le Cancer, Comite de la
Gironde” is greatly acknowledged.
HRMS (ESC): calculated for C44H47N2O7 715.3383; found
715.3384.
[a]2D0 C45.808, c 1.31 in CH2Cl2.
4.6. Compound 2: N-a-Fmoc-O-butenyl-D-Tyrosine-4-
{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-
methylbutyl]-amino} benzyl ester
References and notes
1. Consden, R. J.; Gordon, A. H.; Martin, A. J. P.; Synge,
R. D. M. Biochem. J. 1947, 28, 596–602.
To a solution of deprotected compound 1 (4.5 g, 6.29 mmol)
in CH2Cl2 (150 mL) was added triphenylphosphine (2.47 g,
9.44 mmol). After complete dissolution the solution was
cooled at 0 8C and DEAD (1.48 mL, 9.44 mmol) was added.
Thereafter, the reaction mixture was warmed to room
temperature and then but-3-en-1-ol (0.81 mL, 9.44 mmol)
was added. The reaction mixture was stirred at room
temperature for 48 h. Then the solvent was evaporated
under reduced pressure. The residue was purified by silica
gel chromatography (n-hexane/ethyl acetate, 70:30) to give
2.54 g (52%) of compound 2 as a yellowish solid (mp: 104–
106 8C).
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1H NMR (CDCl3) d ppm: 0.75 (d, 6H, (CH3)2–CH, 3JH–H
6.56 Hz), 1.06 (s, 6H, 2 CH3), 1.26–1.28 (m, 2H, ]CH2),
1.82 (m, 1H, (CH3)2–CH), 2.38 (s, 2H, CH2), 2.51 (s, 2H,
Z
7. Hruby, V. J.; Bartosz-Bechowski, H.; Davis, P.; Slaninova, J.;
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3
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CH2), 2.98 (d, 2H, CH2–CH, JH–HZ6.86 Hz), 3.04 (br s,
3
2H, CH2 DTyr), 3.95 (t, 1H, CH Fmoc, JH–HZ6.57 Hz),
4.15–4.22 (m, 2H, O–CH2 DTyr), 4.41 (m, 2H, CH2 Fmoc),
9. Satoh, T.; Li, S.; Friedman, T. M.; Wiaderkiewicz, R.;
Korngold, R.; Huang, Z. Biochem. Biophys. Res. Commun.
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4.76 (br s, 1H, CH DTyr), 5.06–5.16 (m, 4H, CH2 Bzl and
CH2–CH]), 5.21 (d, 1H, NH DTyr, 3JH–HZ8.18 Hz), 5.85
3
(m, 1H, CH]CH2), 6.76 (d, 2H, CHAr DTyr, JH–H
Z
7.99 Hz), 6.94 (d, 2H, CHAr DTyr, 3JH–HZ7.99 Hz), 7.08 (d,
10. Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1978, 101,
677–683.
3
2H, CHAr Bzl, JH–HZ8.03 Hz), 7.26 (d, 2H, CHAr Bzl,
3JH–HZ8.03 Hz), 7.24–7.41 (m, 4H, CHAr Fmoc), 7.53 (d,
2H, CHAr Fmoc, 3JH–HZ7.09 Hz), 7.74 (d, 2H, CHAr Fmoc,
3JH–HZ7.09 Hz), 15.3 (s, 1H, NH Dmab).
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37.37, 38.33, 46.93, 52.26, 53.75, 54.88, 66.22, 66.96,
67.15, 107.74, 115.35, 117.06, 119.80, 124.92, 126.46,
126.87, 127.54, 129.01, 130.12, 134.23, 137.03, 141.27,
143.67, 155.52, 158.19, 171.46, 176.37, 196.36, 200.21.
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IR (cmK1): 3307; 2958; 1722; 1641; 1555; 1513; 1415;
1242; 1064; 761; 742.
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HRMS (ESC): calculated for C48H53N2O7 769.3853; found
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[a]2D0 C7.698, c 1.30 in CH2Cl2.
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