A practical large-scale synthesis of cyclic RGD pentapeptides suitable for further functionalization through click' chemistry

Article abstract of DOI:10.1055/s-0030-1258396

A multigram batch of the cyclo[Arg-Gly-Asp-d-Phe-Lys] and its N - azido derivative was accomplished via solution-phase synthesis using an epimerization-free fragment condensation. The C-terminus of d-Phe was protected as its tert-butyl ester. Fmoc (Arg, Gly, Asp, d-Phe) and Boc (Lys) groups were used to protect all N - termini. The Ts and NO² groups, respectively were chosen to protect the guanidine group. The macrocyclization step (between d-Phe and l-Lys) was carried out under TBTU/HOBt or DPPA condensation conditions. Finally, the -amino group of the lysine residue was selectively converted into the azido group by a diazo-transfer reaction. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0030-1258396

PAPER
653
A Practical Large-Scale Synthesis of Cyclic RGD Pentapeptides Suitable for
Further Functionalization through ‘Click’ Chemistry
Synthesis
i
of Cyc
ř
lic
R
GD
í
Pentapeptide
P
s
aleček, Gerald Dräger, Andreas Kirschning*
Institut für Organische Chemie and Biomolekulares Wirkstoffzentrum (BMWZ), Leibniz Universität Hannover, Schneiderberg 1b,
30167 Hannover, Germany
E-mail: andreas.kirschning@oci.uni-hannover.de
Received 18 November 2010
triazole formation (tandem cycloaddition–retro-Diels–
Abstract: A multigram batch of the cyclo[Arg-Gly-Asp-D-Phe-
Alder reaction).11
Lys] and its N-e-azido derivative was accomplished via solution-
phase synthesis using an epimerization-free fragment condensation.
The C-terminus of D-Phe was protected as its tert-butyl ester. Fmoc
(Arg, Gly, Asp, D-Phe) and Boc (Lys) groups were used to protect
all N-a-termini. The Ts and NO2 groups, respectively were chosen
to protect the guanidine group. The macrocyclization step (between
D-Phe and L-Lys) was carried out under TBTU/HOBt or DPPA con-
densation conditions. Finally, the e-amino group of the lysine resi-
due was selectively converted into the azido group by a diazo-
transfer reaction.
In view of the importance of cyclo-RGDfK, there is a
quest to develop a synthesis which can easily be upscaled,
particularly as solid-phase peptide synthesis is of reduced
practicability if gram amounts of a target peptide are re-
quired. In the present publication we, therefore, report the
first approach towards cyclo[Arg-Gly-Asp-D-Phe-Lys]
peptide (1) and its N-e-azido derivative 2 solely based on
solution-phase synthesis (Figure 1).
Key words: RGD-peptides, solution-phase synthesis, amino acids,
cyclizations, diazo compounds
H2N
NH
Arg
R
HN
Cyclic pentapeptides containing the RGD (Arg-Gly-Asp)
motif were developed as highly active and selective antag-
onists for the avb3 integrin receptor.1 A class of this het-
erodimeric transmembrane protein2 exerts an important
role in cell signaling and cell–cell and cell–matrix interac-
tions and recognitions.3 The cyclo-RGDfK,4 its methylat-
ed analogues5 and other related cyclic RGD-peptides6
were designed, synthesized and frequently tested for their
crucial location in tumor angiogenesis and metastasis,3,7
as well as for the stimulation of cell adhesion.6b,8
O
NH
Gly
HN
G
O
O
Lys
K
R = NH2, 1
N3, 2
NH
HN
R
N
CO2H
O
O
f
H
D
Asp
Ph
D-Phe
Figure 1 cyclo-RGDfK peptide (1) and N-e-azido cyclo-RGDfK
peptide (2)
Cyclic RGD pentapeptides mentioned above have typical-
ly been prepared by solid-phase synthesis.4–6 Commonly,
the linear-protected pentapeptide was prepared first, fol-
lowed by cleavage from the polymeric resin. Cyclization
and removal of the protecting groups finalized the synthe-
sis according to the original or improved protocols of
Kessler et al.4a
In planning the synthesis a large variety of alternative
coupling strategies for the various protected R-G-f-K ami-
no acid fragments could be envisaged.12 Retrosynthetical-
ly, the synthesis of title cyclic pentapeptide 1 and 2
(Scheme 1) should by achieved by macrolactamization of
Importantly, the e-amino lysine (K) moiety of the cRGD-
fK peptide can be readily modified and used for further
functionalization by means of a 1,3-dipolar cycloaddition
(‘click’ chemistry)9 between alkynes and organic azides
to afford the corresponding 1,4-disubstituted 1,2,3-triaz-
oles. In many examples the method was applied in the
synthesis of glycoconjugates, oligosaccharides, and gly-
copeptides.10
N-e-Azido derivative of cyclic RGD peptides4e,6e,11 were
recently employed in the ‘click’ reaction with dendrimeric
alkynes4e,11 or under metal-free conditions to afford CF3-
the
linear
pentapeptide
[Lys(PG)-Arg(PG)-Gly-
Asp(OPG)-D-Phe-OH] via a one-pot acidic deprotection
of the terminal N-a-Boc-lysine and tert-butyl ester of D-
phenylalanine [Boc-Lys(PG)-Arg(PG)-Gly-Asp(OPG)-
D-Phe-Ot-Bu]. The 4-toluenesulfonyl (Ts) group and al-
ternatively the nitro (NO2) group were chosen to protect
the guanidine unit of arginine. The Fmoc group was cho-
sen as the protecting group for the R-G-f amino acids at
the N-a-termini. The e-amino group of lysine should be
selectively converted into the azido group by a diazo-
transfer reaction in the last step.
Our solution-phase RGD protocol started with D-phenyl-
alanine (Scheme 2), which was converted into its tert-bu-
tyl ester 3 by reaction with isobutene in a mixture of
dioxane and sulfuric acid (82%).13
SYNTHESIS 2011, No. 4, pp 0653–0661
Advanced online publication: 11.01.2011
DOI: 10.1055/s-0030-1258396; Art ID: Z28210SS
© Georg Thieme Verlag Stuttgart · New York
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x
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x
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1
1

654
J. Paleček et al.
PAPER
Fmoc-Asp(OBn)-OH,
EDC, HOBt, DIPEA,
CH2Cl2, r.t., overnight
PGHN
NH
COOBn
O
R = N3
H
N
HN
D-Phe-OR
R = H
t-BuO
Ph
NHFmoc
95%
O
R = PGHN
O
2-methylpropene, dioxane,
H2SO4 (3 equiv), r.t., 2 d
82%
4
NH
R = Ot-Bu, 3
HN
O
O
a) Et2NH (30 equiv), CH2Cl2, r.t.,
overnight, full conv.
b) Fmoc-Gly-OH, EDC, HOBt,
DIPEA, CH2Cl2, r.t., overnight
NH
79%
HN
O
R
N
CO2PG
O
H
COOBn
O
Ph
cyclization
O
H
N
NHFmoc
t-BuO
Ph
N
H
O
5
PGHN
NH
NH
a) Et2NH (30 equiv), CH2Cl2, r.t.,
overnight, full conv.
b) Fmoc-Arg(Ts)-OH, EDC, HOBt, b') Fmoc-Arg(NO2)-OH, PyAOP,
COOPG
DIPEA, CH2Cl2, r.t., overnight
2,4,6-collidine, DMF, 0 °C, 5 h
then 4 °C, 2 d
O
O
O
H
H
PG = Ts, 6; 70%
N
N
NH2
PG = NO2, 11; 75%
N
N
H
HO
Ph
H
O
O
PGHN
NH
NH
HN
PG
COOBn
O
Lys(PG)-Arg(PG)-Gly-Asp(OPG)-D-Phe-Ot-Bu
O
H
H
N
N
N
H
NHFmoc
t-BuO
Scheme 1 Retrosynthetic analysis of the cyclo-RGDfK peptide (1
and 2)
O
O
Ph
6, 11
The linear peptide fragments were synthesized using the
standard epimerization-free condensation conditions
(EDC and HOBt) according to Ley’s protocol.12b Firstly,
D-Phe-Ot-Bu (3) was reacted with Fmoc-Asp(OBn)-OH
to afford the corresponding dipeptide Fmoc-Asp(OBn)-D-
Phe-Ot-Bu (4) (95%). Next, the Fmoc group was
removed12c,h,14 from dipeptide 4. The reaction was carried
out with diethylamine in dichloromethane and subsequent
coupling with Fmoc-Gly-OH gave the protected tripep-
tide Fmoc-Gly-Asp(OBn)-D-Phe-Ot-Bu (5) in 79% yield
over two steps.
a) Et2NH (30 equiv), CH2Cl2, r.t., PG = Ts, 7, 80%
overnight, full conv.
NO2, 12, 78%
b) Boc-Lys(Z)-OH, EDC, HOBt,
DIPEA, CH2Cl2, r.t., overnight
PGHN
NH
NH
O
COOBn
O
O
H
H
N
N
NHBoc
t-BuO
Ph
N
N
H
H
O
O
Then, the synthesis of peptides 1 and 2 progressed via the
tripeptide 5 following two alternative routes that are based
on two different protecting group strategies for the guani-
dine moiety, namely the 4-toluenesulfonyl and nitro group
protection, respectively.
7, 12
NHZ
Scheme 2 The ‘tosyl’ and ‘nitro’ routes: Synthesis of the linear
pentapeptides: Boc-Lys(Z)-Arg(Ts)-Gly-Asp(OBn)-D-Phe-Ot-Bu (7)
and Boc-Lys(Z)-Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu (12).
Via the ‘tosyl’ route, the linear tetrapeptide Fmoc-
Arg(Ts)-Gly-Asp(OBn)-D-Phe-Ot-Bu (6) was formed af-
ter removal of the Fmoc group from the tripeptide 5 and
the subsequent reaction with Fmoc-Arg(Ts)-OH (70%
yield over two steps). Instead of the Fmoc functionalized
lysine, Boc-protected lysine was selected for the last lin-
ear coupling step. The linear pentapeptide Boc-Lys(Z)-
Arg(Ts)-Gly-Asp(OBn)-D-Phe-Ot-Bu (7) was prepared
Treatment of the pentapeptide 7 with a mixture of trifluo-
roacetic acid/dichloromethane4b,12g,h,15 led to quantitative
deprotection of its terminal carboxylate and amino groups
(Scheme 3) to give the ammonium salt of pentapeptide
Lys(Z)-Arg(Ts)-Gly-Asp(OBn)-D-Phe-OH·TFA (8). The
crucial macrolactamization step was performed with
again via a two step sequence: (a) Fmoc deprotection of TBTU/HOBt12b,16 conditions and afforded the cyclic pen-
tetrapeptide 6 and (b) condensation with Boc-Lys(Z)-OH tapeptide cyclo[Arg(Ts)-Gly-Asp(OBn)-D-Phe-Lys(Z)]
(80% over two steps).
(9) in 78% yield over two steps.
Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York

PAPER
Synthesis of Cyclic RGD Pentapeptides
655
7
12
O2NHN
N
NHFmoc
O
a) TFA, CH2Cl2, r.t.,
a) TFA, CH2Cl2, r.t.,
NH
overnight
overnight
b) 8, TBTU, HOBt,
DIPEA, CH2Cl2,
r.t., overnight
PG = Ts
b) 13, DPPA, NaHCO3
DMF, r.t., overnight
PG = NO2
78%
85%
Figure 2 The structure of intramolecular d-lactam formation of
Fmoc-arginine(NO2)
For the cyclization, the ammonium salt of pentapeptide
Lys(Z)-Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu·TFA (13)
(Scheme 3) was quantitatively formed from peptide 12 in
the presence of trifluoroacetic acid in dichloromethane.
Then, the cyclization of pentapeptide 13 was repeated
with TBTU/HOBt leading to full conversion of peptide 13
into cyclic peptide 14 as judged by thin layer chromatog-
raphy (CH2Cl2–MeOH, 9:1). Purification of peptide 14 by
column chromatography on silica gel turned out to be very
problematic as elution was difficult due to its low solubil-
ity in organic solvents.
PGHN
NH
HN
O
NH
HN
O
O
PG = Ts, 9
NO2, 14
NH
HN
O
HN
Z
N
CO2Bn
O
H
Therefore, we added liquid reagent DPPA/NaHCO3 in
N,N-dimethylformamide to the cyclization mixture.18 The
pure cyclic peptide 14 was obtained (85%) after filtration
of solid sodium hydrogen carbonate, extraction (side
products of coupling reagents were removed), and crystal-
lization from methanol (traces of DPPA were removed).
Ph
1. H2, Pd/C
CH2Cl2–MeOH (1:1)
overnight
2. 10, HF/anisole,
0 °C, 2 h
95%
86%
H2, Pd/C
AcOH–MeOH (1:1)
overnight
99%
cRGDfK•AcOH (1)
The synthesis of cRGDfK peptide (1) was achieved via
both routes after removal of the three remaining protect-
ing groups (Scheme 3). For the ‘tosyl’ route, peptide 9
was transformed to the RGD peptide 1 in two steps. First,
the benzyl and benzyloxycarbonyl groups were cleaved
by hydrogenation19 in methanol (95%). The final depro-
tection step required detosylation of the N-tosyl guanidine
group in 10 with an excess of anhydrous hydrogen fluo-
ride in the presence of anisole as an electrophilic scaven-
ger using a Teflon flask.12d,e,20 After treatment of the
reaction mixture, the residue was dissolved in a 5% aque-
ous acetic acid solution (peptide 1 was transferred from
the Teflon flask into a glass flask) and lyophilized, and the
pure title peptide (monoacetate salt) 1 was obtained by
crystallization from a mixture of diethyl ether–methanol
(1:1) (yield 86%, purity >95% according to NMR spec-
troscopy).
a) t-BuOH–H2O (1:1), pH adjusted
to 10 (1 N NaOH), CuSO4•5H2O
b) TfN3 in CH2Cl2, r.t., overnight
90%
N-ε-azido cRGDfK (2)
Scheme 3 ‘Cyclization via the ‘tosyl’ and ‘nitro’ routes, respective-
ly, deprotection steps: cyclo[Arg-Gly-Asp-D-Phe-Lys] (1) and ‘diazo-
transfer’: N-e-azido cyclo[Arg-Gly-Asp-D-Phe-Lys] (2)
For the alternative ‘nitro’ route, the protected cyclic pen-
tapeptide cyclo[Arg(NO2)-Gly-Asp(OBn)-D-Phe-Lys(Z)]
(14) (Scheme 3) was synthesized again from tripeptide 5
via the nitro-guanidine-protected linear tetrapeptide 11
and pentapeptide 12 (Scheme 2) under similar conditions
described above for the ‘tosyl’ route.
The alternate ‘nitro’ route (Scheme 3), allowed all three
protecting groups (Bn, Z and N-nitro guanidine21) in 14 to
be cleaved simultaneously by catalytic hydrogenation in a
mixture of acetic acid–methanol. We had noted that the
presence of acetic acid plays an important role for accel-
erating the hydrogenation. Pure monoacetic acid salt of
cRGDfK (1) was quantitatively isolated after simple fil-
tration through a short pad of Celite (purity was >95% ac-
cording to NMR analysis).
Coupling of Fmoc-deprotected tripeptide 5 with Fmoc-
Arg(NO2)-OH using EDC/HOBt unexpectedly gave a low
yield (30%) of the corresponding tetrapeptide Fmoc-
Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu (11). Therefore,
we had to employ different conditions for the coupling
with the protected arginine.17 In fact, minimization of the
intramolecular d-lactam formation of Fmoc-arginine had
to be achieved.17f–h After substantial optimization, in order
to suppress d-lactam formation (Figure 2) peptide 11 was
obtained in good yield (75%) using the reagent system
PyAOP in N,N-dimethylformamide and 2,4,6-collidine as
base. Then, the pentapeptide Boc-Lys(Z)-Arg(NO2)-Gly-
Asp(OBn)-D-Phe-Ot-Bu (12) was again synthesized un-
der the typical EDC/HOBt condition (78% over two
steps).
Finally, the e-amino group of lysine was selectively con-
verted into the corresponding azide by a diazo-transfer re-
action (Scheme 3).4e,6e,22 Pure azido-RGD peptide 2 was
obtained after column chromatography on Sephadex G-
2512e,f,23 (yield 90% for 0.3 mmol scale, >95% purity ac-
cording to NMR analysis). Noteworthy, the synthesis can
be carried out on a multigram scale.
Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York

656
J. Paleček et al.
PAPER
The spectroscopic data of D-Phe-Ot-Bu (3) were in full agreement
In summary, we describe a practical and scalable solution-
phase synthesis of cyclo-RGDfK (1) and N-e-azido cyclo-
RGDfK (2) peptides. The e-amino or e-azido group in the
lysine moiety of RGD peptides 1 and 2 are suitable mate-
rials for further elaboration in various field of application.
For example the fusion of peptide 2 with biomedical ma-
terials through ‘click’ cycloaddition has great potential for
applications in the field of tissue engineering. These lines
of research are currently pursued in our laboratories.
with those reported in the literature.24
Fmoc-Asp(OBn)-D-Phe-Ot-Bu (4)
DIPEA (5.9 mL, 33.9 mmol, 1.5 equiv) and EDC (5.42 g, 28.3
mmol, 1.25 equiv) were added successively to a mixture of D-Phe-
Ot-Bu (3, 5 g, 22.6 mmol, 1 equiv), Fmoc-Asp(OBn)-OH (10.6 g,
23.7 mmol, 1.05 equiv), and HOBt (4.58 g, 33.9 mmol, 1.5 equiv)
in CH2Cl2 (500 mL) at 0 °C. The mixture was warmed to r.t. and
stirred overnight (checked by TLC, CH2Cl2–MeOH, 99:1). Then,
the mixture was concentrated under reduced pressure and purified
by column chromatography (silica gel, CH2Cl2–MeOH, 99:1;
Rf = 0.47) to afford 4 (13.9 g, 21.4 mmol; 95%) as colorless crystals;
mp 42–44 °C.
All solvents were dried by conventional methods. Starting materials
and reagents were purchased from commercial suppliers and used
without further purification. Preparative column chromatography
was performed using silica gel 60, particle size 0.040–0.063 mm
(230–240 mesh, flash). The azido RGD-peptide 2 was purified us-
ing Sephadex G-25. Analytical TLC was carried out employing sil-
ica gel 60 F254 plates from Macherey&Nagel. Visualization of the
chromatograms was achieved by UV detection (254 nm), by color-
ation with a phosphomolybdic acid soln in EtOH or ninhydrin soln
in EtOH. NMR spectra were recorded on Bruker ARX-400 or 500
spectrometers (1H, 400 MHz or 500 MHz; 13C, 100 MHz or 125
MHz). All spectra were measured using standard Bruker pulse se-
quences. 2D NMR spectroscopy (COSY, HSQC and HMBC) was
used for the assignment of signals in the 1H and 13C NMR spectra.
Mass spectra and HRMS data were recorded on a QTof Premier
equipped with an Acquity UPLC (both Waters). Melting points
were measured on a SRS OptiMelt apparatus and are uncorrected.
The optical rotation of D-Phe-Ot-Bu (3) was measured with a Perkin
Elmer 341 polarimeter. The IR spectrum of azido peptide 2 was re-
corded with a Bruker Vektor 22 FT-IR spectrophotometer (Golden-
Gate ATR unit).
1H NMR (400 MHz, CDCl3): d = 1.43 [s, 9 H, C(CH3)3], 2.66 (dd,
3J = 6.5 Hz, 2J = 17.1 Hz, 1 H, bCH2 Asp), 3.05 (m, 1 H, bCH2 Asp, 1
3
2
H, bCH2 Phe), 3.11 (dd, J = 6.1 Hz, J = 13.7 Hz, 1 H, bCH2 Phe),
4.22 (t, 3J = 7.2 Hz, 1 H, OCH2CHFmoc), 4.40 (m, 2 H,
OCH2CHFmoc), 4.63 (m, 1 H, aCHAsp), 4.73 (m, 1 H, aCHPhe), 5.15
3
(s, 2 H, COOCH2Ph), 5.95 (d, J = 8.2 Hz, 1 H, aNHAsp), 6.99 (d,
3J = 7.5 Hz, 1 H, aNHPhe), 7.11–7.44 (m, 14 H, ArH, Fmoc), 7.59 (t,
3J = 6.8 Hz, 2 H, Fmoc), 7.79 (d, 3J = 7.5 Hz, 2 H, Fmoc).
13C NMR (100 MHz, CDCl3): d = 27.9 [C(CH3)3], 36.3 (bCH2 Asp),
38.0 (bCH2 Phe), 47.1 (OCH2CHFmoc), 50.9 (aCHAsp), 53.8 (aCHPhe),
66.9 (COOCH2Ph), 67.4 (OCH2CHFmoc), 82.5 [C(CH3)3], 120.0
(CHFmoc), 125.1 (CHFmoc), 126.9 (CHAr), 127.1 (CHAr and CHFmoc),
127.7 (CHFmoc), 128.3 (CHAr), 128.4 (CHAr), 128.6 (CHAr), 129.4
(CHAr), 135.3 (CAr), 136.0 (CAr), 141.3 (CFmoc), 143.6 (CFmoc), 155.9
(NHCOOFmoc), 169.5 (NHCO), 170.0 (COOt-Bu), 171.5
(COOCH2Ph).
HRMS (ESI+): m/z [M + Na]+ calcd for: C39H40N2O7Na: 671.2733;
found: 671.2733.
Abbreviations: Arg (R): arginine; Asp (D): aspartate; D-Phe (f): D-
phenylalanine; DPPA: diphenylphosphoryl azide; EDC: 1-(3-di-
methylaminopropyl)-3-ethylcarbodiimide hydrochloride; Gdn:
guanidine; Gly (G): glycine; HOBt: 1-hydroxybenzotriazole; Lys
(K): lysine; PyAOP: (7-azabenzotriazol-1-yloxy)tripyrrolidino-
phosphonium hexafluorophosphate; TBTU: 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate.
Fmoc-Gly-Asp(OBn)-D-Phe-Ot-Bu (5)
Et2NH (31 mL, 296 mmol, 30 equiv) was added dropwise to a
stirred mixture of 4 (6.4 g, 9.87 mmol, 1 equiv) in CH2Cl2 (400 mL)
at r.t. and stirred overnight (checked by TLC, CH2Cl2–MeOH, 99:1
and 95:5). Then, the mixture was evaporated and dried overnight
under vacuum. The nonpolar fluorenyl side product was removed
by flash chromatography (silica gel, CH2Cl2–MeOH, 95:5 to 9:1).
Then the crude Asp(OBn)-D-Phe-Ot-Bu was directly employed in
the second peptide coupling step.
D-Phe-Ot-Bu (3)
A mixture of D-phenylalanine (5 g, 30.3 mmol, 1 equiv) and concd
H2SO4 (8.9 g, 90.8 mmol, 3 equiv) in anhyd 1,4-dioxane (60 mL)
was cooled to –78 °C in a 2-neck round-bottomed flask (500 mL).
2-Methylpropene (50 g, 891 mmol, 29 equiv) was slowly bubbled
and condensed into the flask. The mixture was warmed to r.t. and
stirred for 2 d. Then the mixture was diluted with 1 M NaOH soln
(500 mL) and Et2O (200 mL). The phases were separated and the
aqueous phase was extracted with Et2O (3 × 200 mL). The com-
bined organic layers were dried (MgSO4), filtered and concentrated
under reduced pressure. The crude product was purified by flash
column chromatography (silica gel, EtOAc; Rf = 0.36) to afford 3
(5.5 g, 24.9 mmol; 82%) as a yellow oil.
DIPEA (2.58 mL, 14.8 mmol, 1.5 equiv) and EDC (2.37 g, 12.3
mmol, 1.25 equiv) were added successively to a mixture of
Asp(OBn)-D-Phe-Ot-Bu (approx. 9.87 mmol; calculated as quanti-
tative yield after the first step; Fmoc-deprotection as described
above), Fmoc-Gly-OH (3.23 g, 10.9 mmol, 1.1 equiv) and HOBt
(2.0 g, 14.8 mmol, 1.5 equiv) in CH2Cl2 (500 mL) at 0 °C. The mix-
ture was warmed to r.t. and stirred overnight (checked by TLC,
CH2Cl2–MeOH, 99:1). Then, the mixture was concentrated under
reduced pressure and purified by column chromatography (silica
gel, CH2Cl2–MeOH, 99:1; Rf = 0.25) to afford 5 (5.5 g, 7.79 mmol;
79%) as colorless crystals; mp 49.5–50 °C.
20
24
[a]D –24.4 (c 1.12, EtOAc) [Lit.24a [a]D –22.4 (c 10.0 EtOH);
1H NMR (400 MHz, CDCl3): d = 1.42 [s, 9 H, C(CH3)3], 2.62 (dd,
Lit.24b [a]D20 +25.1 (c 2.8, EtOAc) for L-Phe-Ot-Bu].
3J = 6.5 Hz, 2J = 17.1 Hz, 1 H, bCH2 Asp), 3.01 (m, 1 H, bCH2 Asp, 1
3
2
1H NMR (400 MHz, CDCl3): d = 1.46 [s, 9 H, C(CH3)3], 1.48 (s, 2
H, NH2), 2.87 (dd, 3J = 7.8 Hz, 2J = 13.6 Hz, 1 H, CH2), 3.07 (dd,
3J = 5.7 Hz, 2J = 13.6 Hz, 1 H, CH2), 3.64 (dd, 3J = 5.7 Hz, 3J = 7.8
Hz, 1 H, CH), 7.21–7.38 (m, 5 H, ArH).
13C NMR (100 MHz, CDCl3): d = 27.9 [C(CH3)3], 41.2 (CH2), 56.3
(CH), 81.1 [C(CH3)3], 126.6 (CHAr), 128.3 (CHAr), 129.3 (CHAr),
137.5 (CAr), 174.3 (COOt-Bu).
H, bCH2 Phe), 3.11 (dd, J = 6.1 Hz, J = 14.0 Hz, 1 H, bCH2 Phe),
3
3
3.87 (d, J = 5.1 Hz, 2 H, aCH2 Gly), 4.26 (t, J = 6.8 Hz, 1 H,
OCH2CHFmoc), 4.44 (d, 3J = 6.5 Hz, 2 H, OCH2CHFmoc), 4.70 (m, 1
H, aCHPhe), 4.86 (m, 1 H, aCHAsp), 5.10 (s, 2 H, COOCH2Ph), 5.43
(br s, 1 H, aNHGly), 7.06 (br d, 3J = 6.5 Hz, 1 H, aNHPhe), 7.14 (d,
3J = 6.8 Hz, 2 H, ArH), 7.22 (br d, 3J = 6.8 Hz, 1 H, aNHAsp), 7.18–
7.37 (m, 10 H, ArH, Fmoc), 7.43 (t, 3J = 7.5 Hz, 2 H, Fmoc), 7.62
(d, 3J = 7.2 Hz, 2 H, Fmoc), 7.78 (d, 3J = 7.5 Hz, 2 H, Fmoc).
HRMS (ESI+): m/z [M + H]+ calcd for C13H20NO2: 222.1494;
found: 222.1493.
13C NMR (100 MHz, CDCl3): d = 27.9 [C(CH3)3], 35.8 (bCH2 Asp),
37.9 (bCH2 Phe), 44.6 (aCH2 Gly), 47.1 (OCH2CHFmoc), 49.0
Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York

PAPER
Synthesis of Cyclic RGD Pentapeptides
657
(aCHAsp), 53.9 (aCHPhe), 66.9 (COOCH2Ph), 67.4 (OCH2CHFmoc),
82.4 [C(CH3)3], 120.0 (CHFmoc), 125.1 (CHFmoc), 126.9 (CHAr),
127.1 (CHAr and CHFmoc), 127.7 (CHFmoc), 128.3 (CHAr), 128.4
(CHAr), 128.6 (CHAr), 129.4 (CHAr), 135.3 (CAr), 136.1 (CAr), 141.3
(CFmoc), 143.7 (CFmoc), 156.6 (NHCOOFmoc), 168.8 (NHCO), 169.3
(NHCO), 170.1 (COOt-Bu), 171.5 (COOCH2Ph).
flash chromatography (silica gel, CH2Cl2–MeOH, 95:5 to 9:1).
Then, the crude Arg(Ts)-Gly-Asp(OBn)-D-Phe-Ot-Bu was directly
used in the second peptide coupling step.
DIPEA (0.9 mL, 5.16 mmol, 1.5 equiv) and EDC (0.82 g, 4.3 mmol,
1.25 equiv) were added successively to a mixture of Arg(Ts)-Gly-
Asp(OBn)-D-Phe-Ot-Bu (approx. 3.44 mmol; calculated as quanti-
tative yield after the first step; Fmoc-deprotection as described
above), Boc-Lys(Z)-OH (1.44 g, 3.78 mmol, 1.1 equiv) and HOBt
(0.7 g, 5.16 mmol, 1.5 equiv) in CH2Cl2 (400 mL) at 0 °C. The mix-
ture was warmed to r.t. and stirred overnight (checked by TLC,
CH2Cl2–MeOH, 95:5). Then, the mixture was concentrated under
reduced pressure and purified by column chromatography (silica
gel, CH2Cl2–MeOH, 95:5; Rf = 0.43) to afford 7 (3.19 g, 2.76 mmol;
80%) as yellowish crystals; mp 82–99 °C (dec.).
HRMS (ESI+): m/z [M + Na]+ calcd for C41H43N3O8Na: 728.2948;
found: 728.2968.
Fmoc-Arg(Ts)-Gly-Asp(OBn)-D-Phe-Ot-Bu (6)
Et2NH (22.1 mL, 212.5 mmol, 30 equiv) was added dropwise to a
stirred mixture of 5 (5.0 g, 7.08 mmol, 1 equiv) in CH2Cl2 (350 mL)
at r.t. and stirred overnight (checked by TLC, CH2Cl2–MeOH, 99:1
and 95:5). Then, the mixture was evaporated and dried overnight
under vacuum. The nonpolar fluorenyl side product was removed
by flash chromatography (silica gel, CH2Cl2–MeOH, 95:5 to 9:1).
Then, the crude D-Phe-Asp(OBn)-Gly was directly used in the sec-
ond peptide coupling step.
1H NMR (400 MHz, CDCl3): d = 1.35 (m, 2 H, gCH2 Lys), 1.38 [s, 9
H, C(CH3)3], 1.41 [s, 9 H, C(CH3)3], 1.48 (m, 4 H, bCH2 Lys, dCH2 Lys),
1.57 (m, 2 H, gCH2 Arg), 1.73 (m, 1 H, bCH2 Arg), 1.88 (m, 1 H,
bCH2 Arg), 2.36 (s, 3 H, CH3 Ts), 2.74 (dd, 3J = 5.8 Hz, 2J = 17.5 Hz,
1 H, bCH2 Asp), 2.85 (dd, 3J = 5.5 Hz, 2J = 17.5 Hz, 1 H, bCH2 Asp),
2.96 (dd, 3J = 7.2 Hz, 2J = 13.9 Hz, 1 H, bCH2 Phe), 3.03 (dd, 3J = 6.4
Hz, 2J = 13.9 Hz, 1 H, bCH2 Phe), 3.12 (m, 2 H, eCH2 Lys), 3.27 (m,
2 H, dCH2 Arg), 3.81 (m, 1 H, aCH2 Gly), 3.96 (m, 1 H, aCH2 Gly), 4.13
(m, 1 H, aCHLys), 4.47 (m, 1 H, aCHArg), 4.60 (m, 1 H, aCHPhe),
4.85 (m, 1 H, aCHAsp), 5.04 (s, 2 H, COOCH2Ph), 5.06 (s, 2 H,
COOCH2Ph), 5.39 (br s, 1 H, eNHLys), 5.44 (br d, 3J = 7.2 Hz, 1 H,
aNHLys), 6.45 (br s, 2 H, NHGdn), 7.11–7.40 (m, 19 H, 17 ArH, aN-
DIPEA (1.85 mL, 10.6 mmol, 1.5 equiv) and EDC (1.76 g, 9.20
mmol, 1.3 equiv) were added successively to a mixture of Gly-
Asp(OBn)-D-Phe-Ot-Bu (approx. 7.08 mmol; calculated as quanti-
tative yield after the first step; Fmoc-deprotection as described
above), Fmoc-Arg(Ts)-OH (5.07 g, 9.21 mmol, 1.3 equiv) and
HOBt (1.43 g, 10.6 mmol, 1.5 equiv) in CH2Cl2 (500 mL) at 0 °C.
The mixture was stirred at 0 °C for 2 h, then warmed to r.t. and
stirred overnight (checked by TLC, CH2Cl2–MeOH, 97:3). Then,
the mixture was concentrated under reduced pressure and purified
by column chromatography (silica gel, CH2Cl2–MeOH, 97:3;
Rf = 0.25) to afford 6 (5.05 g, 4.97 mmol; 70%) as yellowish crys-
tals; mp 89–93 °C.
1H NMR (400 MHz, CDCl3): d = 1.35 [s, 9 H, C(CH3)3], 1.58 (m, 2
H, gCH2 Arg), 1.72 (m, 1 H, bCH2 Arg), 1.88 (m, 1 H, bCH2 Arg), 2.33
(s, 3 H, CH3 Ts), 2.66 (dd, 3J = 6.2 Hz, 2J = 17.1 Hz, 1 H, bCH2 Asp),
2.86 (dd, 3J = 4.8 Hz, 2J = 17.1 Hz, 1 H, bCH2 Asp), 2.96 (dd, 3J = 7.2
Hz, 2J = 14.0 Hz, 1 H, bCH2 Phe), 3.04 (dd, 3J = 6.2 Hz, 2J = 14.0 Hz,
1 H, bCH2 Phe), 3.20 (m, 1 H, dCH2 Arg), 3.35 (m, 1 H, dCH2 Arg), 3.83
(dd, 3J = 4.8 Hz, 2J = 16.4 Hz, 1 H, aCH2 Gly), 3.96 (dd, 3J = 4.1 Hz,
2J = 16.4 Hz, 1 H, aCH2 Gly), 4.15 (t, 3J = 7.2 Hz, 1 H,
OCH2CHFmoc), 4.36 (m, 3 H, OCH2CHFmoc, aCHArg), 4.61 (m, 1 H,
aCHPhe), 4.82 (m, 1 H, aCHAsp), 5.02 (s, 2 H, COOCH2Ph), 6.13 (d,
3J = 6.2 Hz, 1 H, aNHArg), 6.46 (br s, 2 H, NHGdn), 7.09–7.39 (m, 18
H, 12 ArH, 4 Fmoc, aNHPhe, NHGdn), 7.45 (br d, 3J = 6.2 Hz, 1 H,
aNHAsp), 7.57 (t, 3J = 6.8 Hz, 2 H, Fmoc), 7.75 (m, 5 H, 2 ArH, 2
Fmoc, aNHGly).
H
Phe, aNHArg, NHGdn), 7.48 (br d, 3J = 6.2 Hz, 1 H, aNHAsp), 7.76 (d,
3J = 7.1 Hz, 2 H, ArH), 7.84 (br s, 1 H, aNHGly).
13C NMR (100 MHz, CDCl3): d = 21.4 (CH3PhSO2), 24.3
(gCH2 Lys), 27.8 [C(CH3)3], 28.2 [C(CH3)3], 29.3 (gCH2 Arg), 29.4
(bCH2 Arg), 30.6 (dCH2 Lys), 32.4 (bCH2 Lys), 36.1 (bCH2 Asp), 37.9
(bCH2 Phe), 40.5 (eCH2 Lys), 41.9 (dCH2 Arg), 43.7 (aCH2 Gly), 49.2
(aCHAsp), 53.1 (aCHArg), 54.2 (aCHPhe), 54.9 (aCHLys), 66.4
(COOCH2Ph), 66.7 (COOCH2Ph), 80.2 [C(CH3)3], 82.1 [C(CH3)3],
125.9 (CHAr), 126.8 (CHAr), 128.0 (CHAr), 128.1 (CHAr), 128.3
(CHAr), 128.4 (CHAr), 128.5 (CHAr), 128.6 (CHAr), 129.1 (CHAr),
129.2 (CHAr), 129.3 (CHAr), 135.4 (CAr), 136.3 (CAr), 136.6 (CAr),
140.5 (CAr), 142.1 (CAr), 156.3 (NHCOOBoc), 156.8 (NHCOOZ),
156.9 (CGdn), 169.3 (NHCO), 169.8 (COOCH2Ph), 170.4 (COOt-
Bu), 170.5 (NHCO), 172.7 (NHCO), 173.6 (NHCO).
HRMS (ESI+): m/z [M + Na]+ calcd for C58H77N9O14SNa:
1178.5190; found: 1178.5208.
Lys(Z)-Arg(Ts)-Gly-Asp(OBn)-D-Phe-OH·TFA (8)
13C NMR (100 MHz, CDCl3): d = 21.4 (CH3PhSO2), 27.8
[C(CH3)3], 29.4 (gCH2 Arg), 29.5 (bCH2 Arg), 35.9 (bCH2 Asp), 37.9
(bCH2 Phe), 40.0 (dCH2 Arg), 43.3 (aCH2 Gly), 47.1 (OCH2CHFmoc),
49.1 (aCHAsp), 54.3 (aCHPhe and aCHArg), 66.8 (COOCH2Ph), 67.1
(OCH2CHFmoc), 82.3 [C(CH3)3], 119.9 (CHFmoc), 125.1 (CHFmoc),
125.9 (CHAr), 126.9 (CHAr), 127.1 (CHAr and CHFmoc), 127.7
(CHFmoc), 128.2 (CHAr), 128.3 (CHAr), 128.6 (CHAr), 129.2 (CHAr),
129.3 (CHAr), 135.3 (CAr), 136.2 (CAr), 140.5 (CAr), 141.2 (CFmoc),
142.1 (CAr), 143.7 (CFmoc), 156.5 (NHCOOFmoc), 156.6 (CGdn), 169.3
(NHCO), 169.8 (NHCO), 170.5 (COOt-Bu), 171.2 (NHCO), 171.3
(COOCH2Ph).
TFA (10 mL, 131 mmol, 150 equiv) was added dropwise to a stirred
mixture of 7 (1 g, 0.87 mmol, 1 equiv) in CH2Cl2 (200 mL) at 0 °C.
Then, the mixture was warmed to r.t. and stirred overnight (checked
by MS and TLC, CH2Cl2–MeOH, 95:5 and 9:1), evaporated and
dried overnight under vacuum. The resulting ammonium salt of lin-
ear pentapeptide Lys(Z)-Arg(Ts)-Gly-Asp(OBn)-D-Phe-OH (8)
was directly used for the second cyclization step.
HRMS (ESI+): m/z [M + H]+ calcd for C49H62N9O12S: 1000.4239;
found: 1000.4243.
cyclo[Arg(Ts)-Gly-Asp(OBn)-D-Phe-Lys(Z)] (9)
HRMS (ESI+): m/z [M + Na]+ calcd for C54H61N7O14SNa:
DIPEA (1.52 mL, 8.7 mmol, 10 equiv) and TBTU (0.56 g, 1.74
mmol, 2 equiv) were added successively to a mixture of Lys(Z)-
Arg(Ts)-Gly-Asp(OBn)-D-Phe-OH (8, approx. 0.87 mmol; calcu-
lated as quantitative yield after the first step: Boc- and tert-butyl es-
ter deprotection) and HOBt (2.35 g, 1.74 mmol, 2 equiv) in CH2Cl2
(1 L) at r.t. The mixture was stirred overnight (checked by TLC,
CH2Cl2–MeOH, 9:1). Then, the mixture was diluted with H2O (300
mL). The phases were separated and the aqueous phase was extract-
ed with CH2Cl2 (3 × 200 mL). The combined organic layers were
dried (MgSO4), filtered, and concentrated under reduced pressure.
1038.4047; found: 1038.3857; m/z [M
+
H]+ calcd for
C54H62N7O14S: 1016.4248; found: 1016.4247.
Boc-Lys(Z)-Arg(Ts)-Gly-Asp(OBn)-D-Phe-Ot-Bu (7)
Et2NH (10.7 mL, 103.2 mmol, 30 equiv) was added dropwise to a
stirred mixture of 6 (3.5 g, 3.44 mmol, 1 equiv) in CH2Cl2 (300 mL)
at r.t. and stirred overnight (checked by TLC, CH2Cl2–MeOH, 95:5
and 9:1). Then, the mixture was evaporated and dried overnight un-
der vacuum. The nonpolar fluorenyl side product was removed by
Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York

658
J. Paleček et al.
PAPER
The crude product was purified by column chromatography (silica
gel, CH2Cl2–MeOH, 9:1; Rf = 0.38) to afford 9 (0.67 g, 0.68 mmol;
78%) as yellowish crystals; mp 99–121 °C (dec.).
(CHAr), 128.4 (CHAr), 128.6 (CHAr), 129.4 (CHAr), 135.5 (CAr),
136.3 (CAr), 141.4 (CFmoc), 143.7 (CFmoc), 157.0 (NHCOOFmoc),
159.2 (CGdn), 169.3 (NHCO), 170.1 (COOt-Bu), 170.8 (NHCO),
171.0 (COOCH2Ph), 173.3 (NHCO).
HRMS (ESI+): m/z [M + H]+ calcd for C47H54N8O11: 907.3990;
found: 907.3962.
1H NMR (400 MHz, CDCl3–CD3OD, 9:1): d = 1.06 (m, 2 H,
gCH2 Lys), 1.28–1.68 (m, 7 H, 2 bCH2 Lys, 2 dCH2 Lys, 2 gCH2 Arg, 1
bCH2 Arg), 1.84 (m, 1 H, bCH2 Arg), 2.37 (s, 3 H, CH3 Ts), 2.66 (dd,
2
3J = 6.1 Hz, J = 16.4 Hz, 1 H, bCH2 Asp), 2.85–3.08 (m, 5 H, 1
bCH2 Asp, 2 bCH2 Phe, 2 eCH2 Lys), 3.15 (m, 2 H, dCH2 Arg), 3.32 (m,
2 H, aCH2 Gly), 3.93 (m, 1 H, aCHLys), 4.21 (m, 1 H, aCHArg), 4.51
(m, 1 H, aCHPhe), 4.80 (dd, 1 H, 3J = 6.5 Hz, 3J = 7.9 Hz, aCHAsp),
5.04 (s, 2 H, COOCH2Ph), 5.06 (s, 2 H, COOCH2Ph), 7.12–7.35 (m,
17 H, ArH), 7.70 (d, 3J = 8.2 Hz, 2 H, ArH). Signals NH were not
detected in the spectrum.
13C NMR (100 MHz, CDCl3–CD3OD, 9:1): d = 22.3 (CH3PhSO2),
23.9 (gCH2 Lys), 30.1 (bCH2 Arg), 30.2 (dCH2 Lys), 30.8 (bCH2 Lys),
32.4 (gCH2 Arg), 36.2 (bCH2 Asp), 38.2 (bCH2 Phe), 41.2 (eCH2 Lys),
41.4 (dCH2 Arg), 44.8 (aCH2 Gly), 50.5 (aCHAsp), 56.1 (aCHArg), 56.2
(aCHPhe), 56.3 (aCHLys), 67.7 (COOCH2Ph), 67.9 (COOCH2Ph),
127.0 (CHAr), 128.1 (CHAr), 128.9 (CHAr), 129.2 (CHAr), 129.3
(CHAr), 129.5 (CHAr), 129.6 (CHAr), 129.7 (CHAr), 129.8 (CHAr),
130.2 (CHAr), 130.4 (CHAr), 136.6 (CAr), 137.4 (CAr), 137.8 (CAr),
141.7 (CAr), 143.4 (CAr), 158.1 (NHCOOZ), 158.5 (CGdn), 171.7
(NHCO), 171.8 (COOCH2Ph), 172.1 (NHCO), 173.4 (NHCO),
173.6 (NHCO), 174.3 (NHCO).
Boc-Lys(Z)-Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu (12)
Et2NH (11.2 mL, 108.2 mmol, 30 equiv) was added dropwise to a
stirred mixture of 11 (3.27 g, 3.61 mmol, 1 equiv) in CH2Cl2 (300
mL) at r.t. and stirred overnight (checked by TLC, CH2Cl2–MeOH,
95:5 and 9:1). Then, the mixture was evaporated and dried over-
night under vacuum. The nonpolar fluorenyl byproduct was re-
moved by flash chromatography (silica gel, CH2Cl2–MeOH, 95:5 to
9:1). Then, the crude Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu was
directly used in the second peptide coupling step.
DIPEA (0.94 mL, 5.42 mmol, 1.5 equiv) and EDC (0.87 g, 4.51
mmol, 1.25 equiv) were added successively to a mixture of
Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu (approx. 3.61 mmol; cal-
culated as quantitative yield after the first step; Fmoc-deprotection
as described above), Boc-Lys(Z)-OH (1.51 g, 3.97 mmol, 1.1 equiv)
and HOBt (0.73 g, 5.42 mmol, 1.5 equiv) in CH2Cl2 (400 mL) at 0
°C. The mixture was warmed to r.t. and stirred overnight (checked
by TLC, CH2Cl2–MeOH, 95:5). Then, the mixture was concentrat-
ed under reduced pressure and purified by column chromatography
(silica gel, CH2Cl2–MeOH, 95:5; Rf = 0.14) to afford 12 (2.95 g,
2.82 mmol; 78%) as colorless crystals; mp 139–140 °C.
HRMS (ESI+): m/z [M + Na]+ calcd for C49H59N9O11SNa:
1004.3952; found: 1004.3948.
Fmoc-Arg(NO2)-Gly-Asp(OBn)-D-Phe-Ot-Bu (11)
1H NMR (400 MHz, CD3OD): d = 1.33 (m, 2 H, gCH2 Lys), 1.39 [s,
Et2NH (17.8 mL, 175.5 mmol, 30 equiv) was added dropwise to a
stirred mixture of 5 (4.05 g, 5.75 mmol, 1 equiv) in CH2Cl2 (350
mL) at r.t. and stirred overnight (checked by TLC, CH2Cl2–MeOH,
99:1 and 95:5). Then, the mixture was evaporated and dried over-
night under vacuum. The non-polar fluorenyl side product was re-
moved by flash chromatography (silica gel, CH2Cl2–MeOH, 95:5 to
9:1). Then, the crude Gly-Asp(OBn)-D-Phe-Ot-Bu was directly
used in the second peptide coupling step.
9 H, C(CH3)3], 1.42 [s, 9 H, C(CH3)3], 1.47 (m, 4 H, bCH2 Lys,
dCH2 Lys), 1.61 (m, 2 H, gCH2 Arg), 1.71 (m, 2 H, bCH2 Arg), 2.70 (dd,
2
3
3J = 7.5 Hz, J = 16.4 Hz, 1 H, bCH2 Asp), 2.79 (dd, J = 6.1 Hz,
2J = 16.4 Hz, 1 H, bCH2 Asp), 2.98 (dd, 3J = 8.1 Hz, 2J = 14.0 Hz, 1
3
2
H, bCH2 Phe), 3.07 (dd, J = 6.6 Hz, J = 14.0 Hz, 1 H, bCH2 Phe),
3.09 (m, 2 H, eCH2 Lys), 3.25 (m, 2 H, dCH2 Arg), 3.84 (m, 2 H,
aCH2 Gly), 3.98 (dd, 3J = 5.6 Hz, 3J = 8.5 Hz, 1 H, aCHLys), 4.36 (m,
1 H, aCHArg), 4.52 (dd, 3J = 6.6 Hz, 3J = 8.0 Hz, 1 H, aCHPhe), 4.82
(dd, 3J = 6.1 Hz, 3J = 7.5 Hz, 1 H, aCHAsp), 5.05 (s, 2 H,
COOCH2Ph), 5.09 (s, 2 H, COOCH2Ph), 7.18–7.33 (m, 15 H, ArH).
Signals NH were not detected in the spectrum.
2,4,6-Collidine (0.76 mL, 5.75 mmol, 1.0 equiv) was added to a
mixture of Gly-Asp(OBn)-D-Phe-Ot-Bu (approx. 5.75 mmol; cal-
culated as quantitative yield after the first step; Fmoc-deprotection
as described above), Fmoc-Arg(NO2)-OH (3.55 g, 8.05 mmol, 1.4
equiv) and PyAOP (4.20 g, 8.05 mmol, 1.4 equiv) in DMF (30 mL)
at 0 °C. The mixture was stirred for 5 h at the same temperature and
then stored in a refrigerator at 4 °C for 2 d (checked by TLC,
CH2Cl2–MeOH, 95:5). Then, the mixture was concentrated under
reduced pressure and purified by column chromatography (silica
gel, CH2Cl2–MeOH, 95:5; Rf = 0.22) to afford 11 (3.90 g, 4.30
mmol; 75%) as colorless crystals; mp 110–111 °C.
13C NMR (100 MHz, CD3OD): d = 24.0 (gCH2 Lys), 28.2 [C(CH3)3],
28.8 [C(CH3)3], 29.8 (gCH2 Arg), 29.9 (bCH2 Arg), 30.5 (dCH2 Lys),
32.5 (bCH2 Lys), 37.1 (bCH2 Asp), 38.5 (bCH2 Phe), 41.4 (eCH2 Lys),
41.7 (dCH2 Arg), 43.7 (aCH2 Gly), 50.9 (aCHAsp), 54.4 (aCHArg), 56.0
(aCHPhe), 56.2 (aCHLys), 67.3 (COOCH2Ph), 67.7 (COOCH2Ph),
80.8 [C(CH3)3], 83.1 [C(CH3)3], 127.9 (CHAr), 128.8 (CHAr), 128.9
(CHAr), 129.2 (CHAr), 129.3 (CHAr), 129.4 (CHAr), 129.5 (CHAr),
129.6 (CHAr), 130.5 (CHAr), 137.3 (CAr), 138.1 (CAr), 138.4 (CAr),
158.2 (NHCOOBoc), 158.9 (NHCOOZ), 160.9 (CGdn), 171.2 (NH-
CO), 171.7 (COOCH2Ph), 171.9 (COOt-Bu), 172.1 (NHCO), 174.5
(NHCO), 175.7 (NHCO).
1H NMR (400 MHz, CDCl3–CD3OD, 9:1): d = 1.35 [s, 9 H,
C(CH3)3], 1.61 (m, 2 H, gCH2 Arg), 1.67 (m, 1 H, bCH2 Arg), 1.83 (m,
3
1 H, bCH2 Arg), 2.74 (m, 2 H, bCH2 Asp), 2.97 (dd, J = 7.5 Hz,
2J = 14.0 Hz, 1 H, bCH2 Phe), 3.04 (dd, 3J = 6.5 Hz, 2J = 14.0 Hz, 1
H, bCH2 Phe), 3.22 (m, 1 H, dCH2 Arg), 3.83 (m, 2 H, aCH2 Gly), 4.15
(m, 2 H, OCH2CHFmoc, aCHArg), 4.35 (dd, 3J = 6.5 Hz, 2J = 10.6 Hz,
1 H, OCH2CHFmoc), 4.42 (dd, 3J = 6.8 Hz, 2J = 10.6 Hz, 1 H,
HRMS (ESI+): m/z [M + H]+ calcd for C51H71N10O14: 1047.5151;
found: 1047.5133.
Lys(Z)-Arg(NO2)-Gly-Asp(OBn)-D-Phe-OH·TFA (13)
3
3
OCH2CHFmoc), 4.59 (t, J = 6.8 Hz, 1 H, aCHPhe), 4.80 (t, J = 6.5
Hz, 1 H, aCHAsp), 5.04 (s, 2 H, COOCH2Ph), 7.10–7.39 (m, 14 H,
10 ArH, 4 Fmoc), 7.57 (t, 3J = 6.8 Hz, 2 H, Fmoc), 7.73 (d, 3J = 7.5
Hz, 2 H, Fmoc). Signals NH were not detected in the spectrum.
TFA (10 mL, 131 mmol, 137 equiv) was added dropwise to a stirred
mixture of 12 (1 g, 0.96 mmol, 1 equiv) in CH2Cl2 (200 mL) at 0 °C.
Then, the mixture was warmed to r.t. and stirred overnight (checked
by MS and TLC, CH2Cl2–MeOH, 95:5 and 9:1), evaporated and
dried overnight under vacuum. The resulting ammonium salt of lin-
ear pentapeptide Lys(Z)-Arg(NO2)-Gly-Asp(OBn)-D-Phe-OH (13)
was directly used in the second cyclization step.
13C NMR (100 MHz, CDCl3–CD3OD, 9:1): d = 27.8 [C(CH3)3],
29.0 (gCH2 Arg), 29.1 (bCH2 Arg), 36.0 (bCH2 Asp), 37.8 (bCH2 Phe),
40.6 (dCH2 Arg), 42.8 (aCH2 Gly), 47.2 (OCH2CHFmoc), 49.3
(aCHAsp), 54.3 (aCHPhe and aCHArg), 66.9 (COOCH2Ph), 67.0
(OCH2CHFmoc), 82.7 [C(CH3)3], 120.0 (CHFmoc), 125.0 (CHFmoc),
125.1 (CHAr), 126.9 (CHFmoc), 127.2 (CHFmoc), 127.8 (CHAr), 128.3
HRMS (ESI+): m/z [M + H]+ calcd for C42H55N10O12: 891.4001;
found: 891.3987.
Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York

PAPER
Synthesis of Cyclic RGD Pentapeptides
659
cyclo[Arg(NO2)-Gly-Asp(OBn)-D-Phe-Lys(Z)] (14)
philized and purified by crystallization (Et2O–MeOH, 1:1) to afford
cyclo[Arg-Gly-Asp-D-Phe-Lys]·AcOH (1) (1.05 g, 1.74 mmol;
86%; the purity >95% according to NMR) as colorless crystals.
DPPA (0.79 g, 2.88 mmol, 3 equiv) was added successively to a
mixture of Lys(Z)-Arg(NO2)-Gly-Asp(OBn)-D-Phe-OH (13, ap-
prox. 0.96 mmol; calculated as quantitative yield after the first step:
Boc and tert-butyl ester deprotection) and NaHCO3 (0.4 g, 4.8
mmol, 5 equiv) in DMF (1 L) at r.t. The mixture was stirred over-
night (checked by TLC, CH2Cl2–MeOH, 9:1). Then, the solid
NaHCO3 was filtered off and DMF evaporated. The residue was
dissolved in a mixture of CH2Cl2–DMF (95:5, 500 mL) and extract-
ed with H2O (3 × 300 mL). The organic layer was evaporated and
dried overnight under vacuum. The crude product was purified by
crystallization (MeOH) to afford 14 (0.71 g, 0.81 mmol; 85%) as
colorless crystals; mp 202 °C (dec.); Rf = 0.42 (CH2Cl2–MeOH,
9:1).
cyclo[Arg-Gly-Asp-D-Phe-Lys]·AcOH (1) via ‘Nitro’ Route
Pd/C (4.5 g, 10% weight) was added to a soln of 14 (1.5 g, 1.72
mmol) in a mixture of AcOH–MeOH (1:1, 200 mL). The mixture
was purged with H2 three times and stirred under H2 atmosphere
overnight at r.t. The suspension was filtered through a short pad of
Celite, washed with a mixture of AcOH–MeOH (1:5), concentrated,
and dried overnight under vacuum to afford colorless crystals of cy-
clo[Arg-Gly-Asp-D-Phe-Lys]·AcOH (1) (1.03 g, 1.71 mmol; 99%);
purity >95% (NMR); mp 191 °C (dec.).
1H NMR (500 MHz, D2O): d = 0.70 (m, 2 H, gCH2 Lys), 1.27–1.57
(m, 7 H, 2 bCH2 Lys, 2 dCH2 Lys, 2 gCH2 Arg, 1 bCH2 Arg), 1.72 (m, 1
H, bCH2 Arg), 1.76 (s, 3 H, CH3CO), 2.39 (dd, 3J = 7.2 Hz, 2J = 15.4
1H NMR (400 MHz, DMSO-d6): d = 1.05 (m, 2 H, gCH2 Lys), 1.28–
1.65 (m, 7 H, 2 bCH2 Lys, 2 dCH2 Lys, 2 gCH2 Arg, 1 bCH2 Arg), 1.74
3
2
3
2
(m, 1 H, bCH2 Arg), 2.59 (dd, J = 6.0 Hz, J = 15.9 Hz, 1 H,
bCH2 Asp), 2.79–3.00 (m, 5 H, 1 bCH2 Asp, 2 bCH2 Phe, 2 eCH2 Lys),
Hz, 1 H, bCH2 Asp), 2.51 (dd, J = 7.0 Hz, J = 15.4 Hz, 1 H,
3
bCH2 Asp), 2.71 (t, J = 7.6 Hz, 2 H, eCH2 Lys), 2.81 (m, 1 H,
2
3
3.15 (m, 2 H, dCH2 Arg), 3.28 (dd, J = 14.9 Hz, J = 4.0 Hz, 1 H,
bCH2 Phe), 2.97 (dd, 3J = 5.3 Hz, 2J = 13.0 Hz, 1 H, bCH2 Phe), 3.05
(m, 2 H, dCH2 Arg), 3.32 (d, 2J = 14.6 Hz, 1 H, aCH2 Gly), 3.73 (m, 1
2
3
aCH2 Gly), 3.95 (m, 1 H, aCHLys), 4.08 (dd, J = 14.9 Hz, J = 7.4
Hz, 1 H, aCH2 Gly), 4.21 (m, 1 H, aCHArg), 4.49 (m, 1 H, aCHPhe),
4.76 (m, 1 H, aCHAsp), 5.05 (s, 2 H, COOCH2Ph), 5.09 (s, 2 H,
COOCH2Ph), 7.14–7.46 (m, 16 H, 15 ArH, eNHLys), 7.57 (d,
3J = 8.0 Hz, 1 H, aNHArg), 8.06 (d, 3J = 7.3 Hz, 1 H, aNHLys), 8.12
(d, J = 7.3 Hz, 1 H, aNHPhe), 8.17 (d, J = 8.4 Hz, 1 H, aNHAsp),
8.44 (dd, 3J = 4.1 Hz, 3J = 7.4 Hz, 1 H, aNHGly). Signals NHGdn were
not observed in the spectrum.
2
H, aCHLys), 4.05 (d, J = 14.6 Hz, 1 H, aCH2 Gly), 4.26 (m, 1 H,
aCHArg), 4.42 (dd, 3J = 5.3 Hz, 3J = 10.5 Hz, 1 H, aCHPhe), 4.56 (m,
1 H, aCHAsp), 7.11–7.25 (m, 5 H, ArH). Signals NH were not de-
tected in the spectrum.
3
3
13C NMR (125 MHz, D2O): d = 22.0 (gCH2 Lys), 23.1 (CH3CO), 24.2
(gCH2 Arg), 25.9 (dCH2 Lys), 27.3 (bCH2 Arg), 29.5 (bCH2 Lys), 36.5
(bCH2 Phe), 37.9 (bCH2 Asp), 38.9 (eCH2 Lys), 40.3 (dCH2 Arg), 43.7
(aCH2 Gly), 50.7 (aCHAsp), 52.3 (aCHArg), 54.7 (aCHPhe), 55.6
(aCHLys), 127.1 (CHAr), 128.7 (CHAr), 129.1 (CHAr), 135.9 (CAr),
156.6 (CGdn), 171.2 (NHCOGly), 172.3 (NHCOAsp), 172.5 (NH-
COArg), 173.5 (NHCOPhe), 174.1 (NHCOLys), 176.6 (CH3CO), 177.8
(COOHAsp).
HRMS (ESI+): m/z [M + H]+ calcd for C27H42N9O7: 604.3207;
found: 604.3207.
HRMS (ESI–): m/z [M – H]+ calcd for C27H40N9O7; 602.3051;
found: 602.3057.
13C NMR (100 MHz, DMSO-d6): d = 23.7 (gCH2 Lys), 29.6
(bCH2 Arg), 29.8 (dCH2 Lys), 31.6 (bCH2 Lys), 31.8 (gCH2 Arg), 36.1
(bCH2 Asp), 38.4 (bCH2 Phe), 40.9 (eCH2 Lys), 41.0 (dCH2 Arg), 44.2
(aCH2 Gly), 49.8 (aCHAsp), 52.8 (aCHArg), 55.3 (aCHPhe), 55.6
(aCHLys), 66.1 (COOCH2Ph), 67.5 (COOCH2Ph), 128.6 (CHAr),
128.7 (CHAr), 128.8 (CHAr), 128.9 (CHAr), 129.1 (CHAr), 129.2
(CHAr), 129.3 (CHAr), 129.4 (CHAr), 130.0 (CHAr), 137.0 (CAr),
138.1 (CAr), 138.2 (CAr), 157.0 (NHCOOZ), 160.2 (CGdn), 170.6
(NHCO), 170.7 (COOCH2Ph), 170.9 (NHCO), 171.5 (NHCO),
172.2 (NHCO), 172.9 (NHCO).
HRMS (ESI+): m/z [M + H]+ calcd for C42H53N10O11: 873.3895;
found: 873.3854.
The spectroscopic data of the cyclo[Arg-Gly-Asp-D-Phe-Lys] (1)
were in full agreement with those reported in the literature.4a
N-e-Azido cyclo[Arg-Gly-Asp-D-Phe-Lys] (2)
cyclo[Arg-Gly-Asp-D-Phe-Lys]·AcOH (1) via ‘Tosyl’ Route
cyclo[Arg(Ts)-Gly-Asp-D-Phe-Lys] (10)
Triflyl azide preparation: NaN3 (0.216 g, 3.32 mmol, 10 equiv) was
dissolved in distilled H2O (8 mL) and CH2Cl2 (20 mL) was added.
The mixture was cooled in an ice bath and Tf2O (0.11 mL, 0.664
mmol, 2 equiv) was added dropwise over 5 min. The mixture was
stirred for 2 h. The phases were separated and the organic phase was
extracted with H2O (3 × 10 mL). The organic phase was directly
used in the next diazo-transfer step.
Pd/C (10 g, 10% weight) was added to a soln of 9 (2.25 g, 2.29
mmol) in a mixture of CH2Cl2–MeOH (1:1, 400 mL). The mixture
was purged with H2 three times and stirred under H2 atmosphere
overnight at r.t. The suspension was filtered through a short pad of
Celite, washed with MeOH, concentrated and dried overnight under
vacuum to afford colorless crystals of the cyclo[Arg(Ts)-Gly-Asp-
D-Phe-Lys] (10, 1.65 g, 2.18 mmol; 95%) which was directly used
in the next N-tosyl deprotection step.
HRMS (ESI+): m/z [M + H]+ calcd for C34H48N9O9S: 758.3296;
found: 758.3294.
HRMS (ESI–): m/z [M – H]+ calcd for C34H46N9O9S: 756.3139;
found: 756.3113.
cyclo[Arg-Gly-Asp-D-Phe-Lys] (1, 0.2 g, 0.332 mmol, 1 equiv) was
dissolved in a mixture of t-BuOH –H2O (1:1, 20 mL) at r.t. Then,
pH was adjusted to 10 by the addition of 1 M NaOH soln. The mix-
ture was treated with CuSO4·5 H2O (8 mg, 0.033 mmol, 0.1 equiv),
a soln of TfN3 in CH2Cl2 (20 mL, prepared as described above) and
stirred overnight at r.t. (checked by MS). Then, the mixture was ex-
tracted with CH2Cl2 (3 × 10 mL) and the aqueous peptide phase lyo-
philized. The crude product was purified by Sephadex G-25 gel
chromatography (H2O as solvent) to afford N-e-azido cyclo[Arg-
Gly-Asp-D-Phe-Lys] (2) (0.188 g, 0.299 mmol; 90%; >95% purity
according to NMR analysis) as colorless crystals; mp 199 °C (dec.).
cyclo[Arg-Gly-Asp-D-Phe-Lys]·AcOH (1)
A mixture of cyclo[Arg(Ts)-Gly-Asp-D-Phe-Lys] (10, 1.53 g, 2.02
mmol, 1 equiv) and anhyd anisole (1 mL) was cooled to –78 °C in
a Teflon flask. The anhyd HF (8 mL) was slowly bubbled and con-
densed from a HF-bomb attached to a Teflon flask via a septum and
a Teflon-cannula. The mixture was then warmed to 0 °C and stirred
for 2 h. The HF was removed at 0 °C under vacuum (a Teflon-flask
with the mixture was connected to vacuum via a safety flask with
silica gel). The residue was dried overnight, dissolved in 5% aq
AcOH soln and overfilled from a Teflon flask into a glass flask, lyo-
IR (film): 2099 cm–1.
1H NMR (500 MHz, D2O): d = 0.75 (m, 2 H, gCH2 Lys), 1.26–1.59
(m, 7 H, 2 bCH2 Lys, 2 dCH2 Lys, 2 gCH2 Arg, 1 bCH2 Arg), 1.71 (m, 1
3
2
H, bCH2 Arg), 2.38 (dd, J = 7.2 Hz, J = 15.7 Hz, 1 H, bCH2 Asp),
3
2
2.53 (dd, J = 7.0 Hz, J = 15.7 Hz, 1 H, bCH2 Asp), 2.80 (m, 1 H,
bCH2 Phe), 3.00 (dd, 3J = 5.4 Hz, 2J = 13.0 Hz, 1 H, bCH2 Phe), 3.05
Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York

660
J. Paleček et al.
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3
(m, 2 H, dCH2 Arg), 3.09 (t, J = 7.0 Hz, 2 H, eCH2 Lys), 3.33 (d,
2J = 15.2 Hz, 1 H, aCH2 Gly), 3.71 (m, 1 H, aCHLys), 4.06 (d,
2J = 15.2 Hz, 1 H, aCH2 Gly), 4.27 (m, 1 H, aCHArg), 4.43 (dd,
G.; Billen, D.; Dager, I.; Kocienski, P. J.; Sliwinski, E.; Tai,
L. R.; Boyle, F. T. Tetrahedron 2008, 64, 4700.
(g) Petersen, S.; Alonso, J. M.; Specht, A.; Doudu, P.;
Goeldner, M.; del Campo, A. Angew. Chem. Int. Ed. 2008,
47, 3192.
3J = 5.5 Hz, J = 10.8 Hz, 1 H, aCHPhe), 4.56 (m, 1 H, aCHAsp),
7.06–7.28 (m, 5 H, ArH). Signals NH were not detected in the spec-
trum.
13C NMR (125 MHz, D2O): d = 22.4 (gCH2 Lys), 24.1 (gCH2 Arg),
27.1 (dCH2 Lys), 27.3 (bCH2 Arg), 29.8 (bCH2 Lys), 36.6 (bCH2 Phe),
37.7 (bCH2 Asp), 40.4 (dCH2 Arg), 43.7 (aCH2 Gly), 50.5 (eCH2 Lys),
50.7 (aCHAsp), 52.2 (aCHArg), 55.2 (aCHLys), 55.3 (aCHPhe), 127.2
(CHAr), 128.8 (CHAr), 129.1 (CHAr), 135.8 (CAr), 156.6 (CGdn),
171.2 (NHCOGly), 172.3 (NHCOAsp), 172.4 (NHCOArg), 173.4 (NH-
COPhe), 174.4 (NHCOLys), 177.9 (COOHAsp).
3
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HRMS (ESI+): m/z [M + H]+ calcd for C27H40N11O7: 630.3112;
found: 630.3101.
HRMS (ESI–): m/z [M – H]+ calcd for C27H38N11O7: 628.2956;
found: 628.2944.
The spectroscopic data of the N-e-azido cyclo[Arg-Gly-Asp-D-Phe-
Lys] (2) were in full agreement with those reported in the litera-
ture.4e
Acknowledgment
This work was funded by the Deutsche Forschungsgemeinschaft
(DFG) for the Cluster of Excellence REBIRTH (EXC 62) and by
the Fonds der Chemischen Industrie. The authors thank Dr. E. Hofer
and Dr. J. Fohrer (Gottfried Wilhelm Leibniz Universität Hannover,
Institut für Organische Chemie, Hannover, Germany) for NMR
measurements.
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Synthesis 2011, No. 4, 653–661 © Thieme Stuttgart · New York