Side chain anchoring of tryptophan to solid supports using a dihydropyranyl handle: Synthesis of brevianamide F

Article abstract of DOI:10.1007/s10989-011-9274-8

The multifunctional character of tryptophan has made it a target for the development of new molecules with therapeutic applications. In this sense the design of alternative solid phase routes would allow the widening of synthetic possibilities to access these molecules through conventional or combinatorial strategies. The present work describes a new strategy for side-chain anchoring of tryptophan to dihydropyranyl-functionalized polystyrene resins and its application to the synthesis of the natural diketopiperazine Brevianamide F. For this study a new handle (4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid) was prepared in order to functionalize aminomethyl or methylbenzhydrylamine resins. A preliminary study in solution using Fmoc-Trp-OR (R = Allyl or Me) and suitable resin models showed that the formation of an hemiaminal linkage with the indole system could be brought about by either conventional or microwave heating in 1,2-dichloroethane and in the presence of pyridine p-toluenesulfonate in yields of 70-95% practically without the formation of subproducts. On the other hand the amino acid could be liberated from the resin at room temperature in yields of up to 90% using trifluoroacetic acid in dichloromethane in the presence of 1,3-dimethoxybenzene as a cation scavenger. The conditions found in solution for the reversible formation of the hemiaminal were only reproducible in solid-phase work using conventional heating. These conditions were used in the synthesis of Brevianamide F, furnishing the diketopiperazine in an overall yield of 56%. These results demonstrate the potential of this strategy for the preparation of new molecules based upon tryptophan as a synthetic precursor. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s10989-011-9274-8

Int J Pept Res Ther (2012) 18:7–19
DOI 10.1007/s10989-011-9274-8
Side Chain Anchoring of Tryptophan to Solid Supports Using
a Dihydropyranyl Handle: Synthesis of Brevianamide F
•
Carolina Torres-Garcıa Mireia Dıaz Daniel Blasi Immaculada Farras
•
•
•
´
Irene Fernandez Xavier Ariza Jaume Farras Paul Lloyd-Williams
´
`
•
•
•
•
´
Miriam Royo Ernesto Nicolas
`
•
´
Accepted: 15 September 2011 / Published online: 4 October 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The multifunctional character of tryptophan has
made it a target for the development of new molecules with
therapeutic applications. In this sense the design of alter-
native solid phase routes would allow the widening of
synthetic possibilities to access these molecules through
conventional or combinatorial strategies. The present work
describes a new strategy for side-chain anchoring of tryp-
tophan to dihydropyranyl-functionalized polystyrene resins
and its application to the synthesis of the natural diketo-
piperazine Brevianamide F. For this study a new handle
(4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid) was
prepared in order to functionalize aminomethyl or
methylbenzhydrylamine resins. A preliminary study in solu-
tion using Fmoc-Trp-OR (R = Allyl or Me) and suitable
resin models showed that the formation of an hemiaminal
linkage with the indole system could be brought about by
either conventional or microwave heating in 1,2-dichloro-
ethane and in the presence of pyridine p-toluenesulfonate in
yields of 70–95% practically without the formation of sub-
products. On the other hand the amino acid could be liberated
from the resin at room temperature in yields of up to 90%
using trifluoroacetic acid in dichloromethane in the presence
of 1,3-dimethoxybenzene as a cation scavenger. The condi-
tions found in solution for the reversible formation of the
hemiaminal were only reproducible in solid-phase work using
conventional heating. These conditions were used in the
synthesis of Brevianamide F, furnishing the diketopiperazine
in an overall yield of 56%. These results demonstrate the
potential of this strategy for the preparation of new molecules
based upon tryptophan as a synthetic precursor.
´
`
`
C. Torres-Garcıa ꢀ I. Farras ꢀ X. Ariza ꢀ J. Farras ꢀ
´
P. Lloyd-Williams ꢀ E. Nicolas (&)
Department of Organic Chemistry, University of Barcelona,
Diagonal 647, 08028 Barcelona, Spain
e-mail: enicolas@ub.edu
Keywords Dihydropyranyl handle ꢀ DKPs ꢀ
Side chain anchoring ꢀ Solid phase ꢀ Tryptophan
´
M. Dıaz
Institut de Recerca Biomedica (IRB Barcelona), Parc Cientıfic de
Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain
`
`
D. Blasi
Plataforma Tecnologica Drug Discovery, Parc Cientıfic de
Introduction
`
Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain
´
The amino acid tryptophan has become a focus of attention
in the area of Bioorganic Chemistry on account of the
structural and chemical properties (the multifunctional
chemical nature) of its indole side chain. Thus tryptophan
plays a crucial role in the function of proteins on account of
indole system giving rise to different types of interactions
such as hydrophobic (Mant et al. 2009; Samanta et al.
2000), p-cation (Ma and Dougherty 1997), packing inter-
actions with other aromatic residues (ring-stacking or edge-
to-face interactions) (Samanta et al. 1999) or the formation
´
I. Fernandez
Serveis Cientificotecnics de la Universitat de Barcelona,
Diagonal 647, 08028 Barcelona, Spain
`
`
X. Ariza ꢀ J. Farras
Institut de Biomedicina de la Universitat de Barcelona (IBUB),
Barcelona, Spain
M. Royo
Unitat de Quımica Combinatoria, Parc Cientıfic de Barcelona,
´
Baldiri Reixac 10, 08028 Barcelona, Spain
`
´
123

8
Int J Pept Res Ther (2012) 18:7–19
of hydrogen bonds (Black et al. 2001). On the other hand
tryptophan is a synthetic and biosynthetic precursor to
more complex heterocyclic systems with important bio-
logical properties (Rodrigues de Sa Alves et al. 2009; Ruiz-
Sanchis et al. 2011).
molecular sieves and bubbled with N2 for 30 min. Et2O
was stored over sodium under N2.
Organic solutions were dried over anhydrous magne-
sium or sodium sulfate and solvent removal was performed
by rotatory evaporation at reduced pressure (water pump).
Melting points are uncorrected and were measured with a
microscope equipped with a hot stage and a temperature
controller. Microwave irradiation was carried out in sealed
reaction vessels and reaction temperatures were monitored
using an external surface sensor within the cavity of the
reactor.
The multifunctional character of tryptophan makes it a
target compound for carrying out synthetic transforma-
tions in the search for lead compounds and building
blocks in therapeutic applications (Gordon and Kerwin
1998; Sawyer 1997). In this sense the development of
synthetic strategies involving the union of the amino acid
to solid supports through its side chain are potentially
attractive since this would allow chemical modifications
in different parts of the carbon skeleton to be carried out
in order to, in this way, access new peptide derivatives
such as cyclic peptides or peptides modified at the car-
boxyl terminal, or non-peptide structures such as hetero-
cyclic derivatives from modifications of the indole
system. The use of an amino acid side-chain for anchor-
ing to a solid support has proved to be useful for the
preparation of peptide derivatives with Asp, Glu, Ser or
Lys, trifunctional amino acids that allow union of the
peptide chain to the resin through their side-chain func-
tional groups (Spatola and Romanovskis 2000). The side-
chain attachment of tryptophan to solid supports, on the
other hand, has not been studied to any great extent. In
fact, as far as we are aware, the only previous report of
such an application is that of Bernhardt et al. (1997), who
described the synthesis of a tetrapeptide anilide on a
chlorotrityl resin. With the objective of widening the
application of the side-chain anchorage of tryptophan to
solid supports, we decided to explore the use of the tet-
rahydropyranyl function as a protecting group for indole
nitrogen that would allow its linkage to the polymer
through the formation of an hemiaminal. We were par-
ticularly interested in adapting this approach to the syn-
thesis of tryptophan containing peptides for further
on-resin cyclization (Spatola and Romanovskis 2000).
This would then allow cyclic peptides to be cleaved
directly from the solid support and would constitute a
novel approach to such molecules. Furthermore, the
anchorage of other indole derivatives could, in principle,
provide a method for the solid-phase synthesis of libraries
of small molecules.
Thin-layer chromatography (TLC), was carried out on
silica gel plates (20 9 20 cm, 0.2 mm) and were visualized
using UV light at 254 nm. The plates were also developed
using a phosphomolybdic acid solution. Column chroma-
tography was carried out using silica gel (70–230 mesh,
particle size 35–70 lm) neutralized by elution with a 1%
(v/v) solution of TEA in DCM. Columns were packed as a
suspension of silica gel in the eluent and crude products
were loaded as concentrated solutions in the eluent or
adsorbed on silica gel. Column elution was performed with
the assistance of compressed air. Trial reactions of the
reversible union between dihydropyran (DHP) and trypto-
phan that were performed in solution were monitored using
reversed-phase HPLC on an analytical C-18 Nucleosil
˚
column (4 9 250 mm, 10 mm, 120 A) using mixtures of
0.045% trifluoroacetic acid (TFA) in H2O (eluent A) and
0.036% TFA in MeCN (eluent B). Reversed-phase reten-
tion times in the Experimental Sections are given along
with the elution gradient expressed as an initial percentage
of eluent B in eluent A and a final percentage of eluent B in
eluent A over the duration of the gradient. The flow rate
was 1 ml min-1 and detection was carried out at 220 and
280 nm. HPLC–MS was performed using mixtures of H2O
(containing 0.1% HCOOH) and MeCN (containing 0.07%
HCOOH) as eluents and a diode-array detector. In all cases
elution was performed for 30 min. HPLC on a chiral sta-
tionary phase was carried out using a Chiralpak AS-H
column (4.6 9 250 mm, 10 mm) and a mixture of hexanes
and i-PrOH (85:5) as eluent. Elution was carried out iso-
cratically for 30 min at 1 ml min-1 and detection was
performed at 220 nm.
IR spectroscopy was performed using KBr discs or as
thin films of the pure products. NMR spectroscopy was
carried out on 300, 400, 500 and 600 MHz machines, either
using TMS as internal standard or the signals of the deu-
terated solvent as secondary references. Complete spec-
troscopic characterization was achieved using COSY,
HSQC and HMBC NMR spectroscopy. Signal multi-
plicities are designated as s (singlet), bs (broad singlet),
d (doublet), bd (broad doublet), t (triplet), bt (broad
triplet), m (multiplet). Multiplicities designated with an
asterisk indicate that duplicate signals corresponding to
Materials and Methods
All reagents were synthesis grade or better and were used
without purification. Before use DCM was passed through
an alumina column, tetrahydrofuran (THF) was distilled
˚
over sodium under N2 and DMF was placed over 4 A
123

Int J Pept Res Ther (2012) 18:7–19
9
diastereomers or rotamers were observed. High-resolution
mass spectrometry (HRMS) was performed on an electro-
spray coupled to quadrupole time-of-flight apparatus.
J = 5.3 Hz, 1H), 6.42 (bs, 1H), 6.94 (d, J = 9.2 Hz, 2H),
7.30, (m, 5H), 7.75 (d, J = 9.2 Hz, 2H); 13C-NMR
(100 MHz,CDCl3)d19.7, 24.9, 44.6, 70.7, 73.5,101.2,114.9,
127.4, 128.0, 128.4, 129.2, 129.3, 138.9, 143.9, 161.8, 167.3;
HRMS calcd for C20H22NO3 [M ? H]? 324.1594, found,
324.1597; Reversed-phase HPLC tR 16.2 min, from 40 to
100% eluent B in eluent A over 30 min.
4-[(3,4-Dihydro-2H-pyran-2-yl)methoxy]benzoic
acid (3)
Methyl 4-hydroxybenzoate (2.51 g, 24.2 mmol) and tri-
phenylphosphine (7.68 g, 29.0 mmol) were dissolved in
anhydrous THF (40 ml) under Ar. After stirring for
30 min, (3,4-dihydro-2H-pyran-2-yl)methanol (2.51 ml,
24.2 mmol) and diisopropyl azodicarboxylate (DIPAD)
(6.25 ml, 29.0 mmol) were added and the mixture was
stirred at rt under Ar for 48 h. The solvent was removed
and LiOH (2.66 g, 109 mmol) in H2O/MeOH [(2:1),
75 ml] was added with stirring and the mixture was
brought to reflux and was maintained at this temperature
for 48 h. On cooling to rt a white precipitate appeared
(triphenylphosphine) and the mixture was extracted with
DCM (5 9 60 ml). The aqueous phase was cooled in an
ice-water bath and aqueous HCl (2 M) was added to pH 2
(pH paper). The white solid formed was recovered by fil-
tration, and was dried in vacuo, furnishing the product,
(4.06 g, 72%). Mp = 141–143°C; Rf 0.02 (DCM); IR
(KBr) mmax 3420–2554, 1683, 1606, 1428, 1257,
(3,4-Dihydro-2H-pyran-2-yl)methyl
N-benzylcarbamate (5)
(3,4-Dihydro-2H-pyran-2-yl)methanol 2 (441 ll, 4.14 mmol)
was dissolved in DCM (8 ml) and stirred under Ar. Solu-
tions of DSC (957 mg, 3.73 mmol) in MeCN (10 ml)
under Ar and DMAP (52 mg, 0.42 mmol) in DCM (1 ml)
under Ar were then added via canula and the mixture was
stirred for 4 h at rt. Benzylamine (102 ll, 0.93 mmol) and
DMAP (22 mg, 0.18 mmol) were added and the mixture
was stirred at rt for a further 20 h. After washing with 10%
HCl (3 9 10 ml), brine (3 9 10 ml) and H2O (3 9 5 ml),
the organic phase was dried. Filtration and solvent removal
gave a thick, colorless oil that was purified by chroma-
tography [neutralized silica gel, hexanes/EtOAc (1:3)]
affording the product as a white solid (185 mg, 79%).
Mp = 44–45°C; Rf 0.30 [hexanes/EtOAc (1:3)]; IR(KBr)
mmax 3306, 3059, 2885, 1681, 1651, 1532, 1238, 1051,
1
1170 cm-1; H-NMR (300 MHz, DMSO-d6) d 1.70 (m,
1
1H), 1.93 (m, 1H), 1.98 (m, 1H), 2.08 (m, 1H), 4.13 (m,
3H), 4.70 (m, 1H), 6.39 (d, J = 6 Hz, 1H), 7.02 (d,
J = 9 Hz, 2H), 7.87 (d, J = 9 Hz, 2H); 13C-NMR
(75 MHz, DMSO-d6) d 21.5, 26.3, 72.7, 75.5, 103.3, 145.9,
116.9, 126.1, 134.0, 164.8, 169.9; HRMS calcd for
C13H15O4 [M ? H]? 235.0964, found 235.0969.
694 cm-1; H-NMR (400 MHz, CDCl3) d 1.68 (m, 1H),
1.85 (m, 1H), 1.99 (m, 1H), 2.10 (m, 1H), 4.04 (m, 1H),
4.16 (dd, J1 = 11.4 Hz, J2 = 7 Hz, 1H), 4.26 (dd,
J1 = 11.4 Hz, J2 = 3.6 Hz, 1H), 4.38 (d, J = 6 Hz, 2H),
4.70 (bs, 1H), 5.12 (bs, 1H), 6.38 (d, J = 6.2 Hz, 1H), 7.30
(m, 5H); 13C-NMR (100 MHz, CDCl3) d 19.3, 24.3, 45.4,
67.1, 73.3, 100.9, 127.7 (92), 129.0, 138.6, 143.5, 156.6;
HRMS calcd for C14H18NO3 [M ? H]? 248.1281, found,
248.1289.
N-Benzyl-4-[(3,4-dihydro-2H-pyran-2-
yl)methoxy]benzamide (4)
4-[(3,4-Dihydro-2H-pyran-2-yl)methoxy]benzoic acid
3
Fmoc-(S)-Trp-OAllyl (6)
(234 mg, 1 mmol) was dissolved in DCM (7 ml) and
EDCꢀHCl (232 mg, 1.2 mmol), N-hydroxybenzotriazole
(HOBt) (186 mg, 1.2 mmol) and benzylamine (134 ll,
1.2 mmol) were added and the mixture was stirred at rt for
22 h. After washing with (0.1 N HCl 3 9 5 ml), 10%
NaHCO3 (3 9 5 ml), brine (3 9 5 ml) and H2O (3 9
5 ml), the organic phase was dried. Filtration and solvent
removal gave a pale yellow oil that was purified by chro-
matography [neutralized silica gel, DCM/MeOH (995:5)],
affording the product as a pale yellow oil (143 mg, 44%).
Mp = 173–175°C; Rf 0.32 [DCM/MeOH (9.9:1)]; IR
H-(S)-Trp-OAllylꢀHCl (3.00 g, 10.68 mmol) was dissolved
in a mixture of 10% aqueous Na2CO3/THF (2:1, 75 ml)
and the mixture was cooled to 0°C. A solution of Fmoc-Cl
(3.03 g, 11.73 mmol) in THF (24 ml) was added dropwise
with vigorous stirring which, after addition was complete,
was continued for a further 30 min at 0°C and then for a
further 7 h at rt. The mixture was extracted with EtOAc
(3 9 30 ml) and the organic phase was dried. After filtra-
tion and solvent removal the yellow solid obtained was
purified by chromatography [silica gel, hexanes/EtOAc
(3:1)], furnishing the product as a foamy white solid
(3.30 g, 86%). Mp = 134–136°C; Rf 0.18 [hexanes/EtOAc
(3:1)]; [a]D = ?11.42 (CHCl3, c 1.15); IR (KBr) mmax
3317, 3051, 2912, 1736, 1699, 1522, 1221 cm-1; 1H-NMR
1
(KBr) mmax 3310, 3052, 2923, 1634, 1607 cm-1; H-NMR
(400 MHz, CDCl3) d 1.83 (m, 1H), 1.98 (m, 1H), 2.06 (m,
1H), 2.16 (m, 1H), 4.04 (dd, J1 = 9.8 Hz, J2 = 4.4 Hz,
1H), 4.13 (dd, J1 = 9.8 Hz, J2 = 6.1 Hz, 1H), 4.21 (m,
1H), 4.62 (d, J = 5.7 Hz, 2H), 4.74 (m, 1H), 6.41 (d,
(400 MHz, DMSO-d6)
d
3.08 (dd, J1 = 14.7 Hz,
123

10
Int J Pept Res Ther (2012) 18:7–19
J2 = 9.4 Hz, 1H), 3.21 (dd, J1 = 14.7 Hz, J2 = 5.4 Hz,
1H), 4.17 (m, 1H), 4.22 (m, 2H), 4.33 (m, 1H), 4.54 (m, 2H),
5.15 (bd, J = 10.8 Hz, 1H), 5.24 (bd, J = 17.6 Hz, 1H),
5.81 (m, 1H), 6.98 (td, J1 = 7.4 Hz, J2 = 0.7 Hz, 1H), 7.06
(td, J1 = 7.6 Hz, J2 = 0.8 Hz, 1H), 7.17 (bd, J = 2.2 Hz,
1H), 7.29 (m, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.40 (m, 2H),
7.53 (d, J = 7.8 Hz, 1H), 7.65 (m, 2H), 7.87 (d, J = 7.7 Hz,
2H), 7.89 (d, J = 8.2 Hz, 1H), 10.9 (bs, 1H); 13C-NMR
(100 MHz, CDCl3) d 27.1, 46.7, 55.2, 65.0, 65.9, 109.8,
111.6, 117.8, 118.1, 118.6, 120.3, 121.1, 124.0, 125.4, 127.2
(92), 127.8, 132.5, 136.3, 140.9, 143.9, 156.2, 172.2; HRMS
calcd for C29H27N2O4 [M ? H]? 467.1965, found,
467.1962; Reversed-phase HPLC tR 22.0 min, from 40 to
100% eluent B in eluent A over 30 min.
removal gave a brown oil that was purified by chroma-
tography [neutralized silica gel, hexanes/EtOAc (2:1)]
furnishing the product as a white foamy solid (190 mg,
80%). Mp = 58–60°C; Rf 0.30 [hexanes/EtOAc (3:1)];
IR(KBr) mmax 3341, 2943, 1721, 1510, 1461, 1450, 1337,
1232, 1202, 1078, 1036, 758, 739 cm-1
;
1H-NMR
(400 MHz, DMSO-d6) d 1.51 (m, 2H), 1.71 (m, 1H), 1.82
(m, 1H), 1.91 (m, 1H), 1.99 (m, 1H), 3.05 (m, 1H), 3.17
(dd, J1 = 14.6 Hz, J2 = 5.1 Hz, 1H), 3.69 (m, 1H), 3.88
(m, 1H), 4.17 (m, 1H), 4.22 (m, 2H), 4.32 (m, 1H), 4.54 (m,
2H), 5.16 (dd, J1 = 10.2 Hz, J2 = 4.2 Hz, 1H), 5.25 (dd,
J1 = 17.3 Hz, J2 = 11 Hz, 1H), 5.53 (d, J = 10.3 Hz,
1H), 5.82 (m, 1H), 7.05 (t, J1 = 7.3 Hz, J2 = 7.5 Hz, 1H),
7.14 (t, J1 = 7.7 Hz, J2 = 7.6 Hz, 1H), 7.28 (m, 2H), 7.34
(d, J = 6.8 Hz, 1H), 7.40 (m, 2H), 7.50 (d, J = 8.2 Hz,
1H), 7.54 (d, J = 7.9 Hz, 1H), 7.67 (m, 2H), 7.87 (d,
J = 7.6 Hz, 2H), 7.94 (d*, J = 5.6 Hz, 1H); 13C-NMR
(100 MHz, CDCl3) d 22.9, 25.0, 26.9, 30.4, 46.8, 52.4,
55.1, 65.9, 67.2, 82.5, 110.9, 111.0, 117.9, 118.6, 119.8,
120.4, 121.8, 124.1, 125.7, 127.5, 128.0, 128.2, 132.5,
136.3, 141.1, 144.1, 156.2, 172.0; HRMS calcd for
C34H35N2O5 [M ? H]? 551.2540, found 551.2534;
Reversed-phase HPLC tR 25.3 min, from 40 to 100% elu-
ent B in eluent A over 30 min.
Fmoc-(S)-Trp-OMe (7)
H-(S)-Trp-OMeꢀHCl (0.99 g, 3.90 mmol) was dissolved in
a mixture of 10% aqueous Na2CO3/THF (2:1, 45 ml) and
the mixture was cooled to 0°C. A solution of Fmoc-OSu
(1.45 g, 4.30 mmol) in THF (15 ml) was added dropwise
with vigorous stirring which, after addition was complete,
was continued for a further 30 min at 0°C and then for a
further 7 h at rt. The mixture was extracted with EtOAc
(3 9 30 ml) and the organic phase was dried. After filtra-
tion and solvent removal the yellow solid obtained was
purified by chromatography [silica gel, hexanes/EtOAc
(3:1)], furnishing the product as a foamy white solid
(1.64 g, 96%). Mp = 118–120°C; Rf 0.12 [hexanes/EtOAc
(3:1)]; [a]D = ?27.75 (CHCl3, c 1.10); IR (KBr) mmax
3385, 3057, 2951, 1741, 1688, 1535, 1218 cm-1; 1H-NMR
(400 MHz, DMSO-d6) d 3.05 (dd, J1 = 14.6 Hz, J2 =
5.4 Hz, 1H), 3.17 (dd, J1 = 14.3 Hz, J2 = 9.4 Hz, 1H),
3.60 (s, 3H), 4.17 (m, 1H), 4.20 (m, 2H), 4.30 (m, 1H), 6.98
(td, J1 = 7.5 Hz, J2 = 0.8 Hz, 1H), 7.06 (td, J1 = 7.5 Hz,
J2 = 0.7 Hz, 1H), 7.17 (d, J = 2.2, 1H), 7.28 (m, 2H), 7.34
(d, J = 8 Hz, 1H), 7.39 (m, 2H), 7.52 (d, J = 7.7, 1H),
7.64 (t, J = 8 Hz, 2H), 7.86 (d, J = 7.7 Hz, 2H), 7.90 (d,
J = 7.6 Hz, 1H), 10.90 (bs, 1H); 13C-NMR (100 MHz,
CDCl3) d 28.7, 48.4, 53.7, 56.8, 67.5, 111.5, 113.3, 119.8,
120.2, 121.9, 122.8, 125.6, 127.0, 128.8 (92), 129.4, 137.9,
142.5, 145.5, 157.7, 174.5; HRMS calcd for C27H25N2O4
[M ? H]? 441.1809, found, 441.1807; Reversed-phase
HPLC tR 20.3 min, from 40 to 100% eluent B in eluent A
over 30 min.
Fmoc-Trp(2-THP)-OMe (9)
Fmoc-Trp-OMe (100 mg, 0.22 mmol) was dissolved in
DCE (4 ml) and DHP (14 ll, 0.16 mmol) and PPTS
(91 mg, 0.36 mmol) were added. This mixture was sub-
mitted to microwave irradiation at a temperature of 120°C
for 14 min with stirring. After cooling the solution was
washed with H2O (3 9 5 ml) and then dried. Filtration and
solvent removal gave a brown oil that was purified by
chromatography [neutralized silica gel, hexanes/EtOAc
(2:1)] furnishing the product as a white foamy solid
(80 mg, 69%). Mp = 62.6–65.3°C; Rf 0.20 [hexanes/
EtOAc (2:1)]; IR (KBr) mmax 3321, 2921, 2850, 1715, 1511,
1462, 1203, 1033, 736 cm-1; 1H-NMR (400 MHz, DMSO-
d6) d 1.51 (m, 2H), 1.70 (m, 1H), 1.82 (m, 1H), 1.90 (m,
1H), 1.99 (m, 1H), 3.03 (dd, J1 = 14.4, J2 = 9.4 Hz, 1H),
3.16 (dd, J1 = 14.7 Hz, J2 = 5 Hz, 1H), 3.61 (s, 3H), 3.68
(m, 1H), 3.88 (m, 1H), 4.17 (m, 1H), 4.21 (m, 2H), 4.28 (m,
1H), 5.53 (d, J = 10.2 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H),
7.14 (t, J1 = 7.7 Hz, J2 = 7.2 Hz, 1H), 7.29 (m, 2H), 7.33
(d, J = 6.5 Hz, 1H), 7.40 (m, 2H), 7.50 (d, J = 8.4 Hz,
1H), 7.54 (d, J = 8.2 Hz, 1H), 7.67 (m, 2H), 7.87 (d,
J = 7.3 Hz, 2H), 7.91 (d*, J = 5.6 Hz, 1H); 13C-NMR
(100 MHz, CDCl3) d 23.1, 27.1, 25.4, 30.7, 47.1, 52.4,
55.5, 66.1, 67.4, 82.9, 110.9, 111.0, 118.7, 119.8, 120.4,
121.8, 123.9, 125.6, 127.4, 128.0, 128.1, 136.2, 144.0,
144.1, 157.2, 173.8; HRMS calcd for C32H33N2O5
[M ? H]? 525.2384, found, 525.2381; Reversed-phase
Fmoc-Trp(2-THP)-OAllyl (8)
Fmoc-Trp-OAllyl (200 mg, 0.43 mmol) was dissolved in
DCE (5 ml) and DHP (40 ll, 0.43 mmol) and PPTS
(181 mg, 0.68 mmol) were added. The mixture was stirred
at 70°C for 8 h. After cooling the solution was washed with
H2O (3 9 5 ml) and then dried. Filtration and solvent
123

Int J Pept Res Ther (2012) 18:7–19
11
HPLC tR 23.7 min, from 40 to 100% eluent B in eluent A
over 30 min.
HRMS: calcd for C47H45N3O7 [M ? H]? 764.3330, found
764.3337. HPLC tR 25.8 min, from 40 to 100% eluent B in
eluent A over 30 min.
Nin-{6-[4-(N-Benzylcarbamoyl)benzoxymethyl]
tetrahydropyran-2-yl}-N-(9-fluorenyl-
methoxycarbonyl)-(S)-tryptophan allyl ester (10)
Fmoc-(S)-Trp(Boc)-OMe (12)
Fmoc-Trp-OMe (143 mg 0.32 mmol) was dissolved in
anhydrous MeCN (3 ml) under N2 and a solution of DMAP
(4.0 mg, 0.033 mmol) and Boc2O (87.0 mg, 0.39 mmol) in
anhydrous MeCN (1 ml) was added via canula. The mix-
ture was stirred for 6 h at rt. The mixture was then diluted
with H2O (5 ml) and extracted with Et2O (2 9 20 ml). The
organic phase was washed with 1 M KHSO4 (3 9 20 ml),
saturated NaHCO3 (3 9 20 ml) and brine (3 9 20 ml) and
then dried. After filtration and solvent removal the pale
yellow crude was purified by chromatography [silica gel,
hexanes/EtOAc (4:1)] furnishing the product as a white
foamy solid (96 mg, 55%). Mp = 51.3–53.5°C; Rf 0.29
[hexanes/EtOAc (4/1)]; IR (KBr) mmax 3369, 3066, 2951,
Fmoc-Trp-OAllyl (150 mg, 0.32 mmol) was dissolved in
DCE (4 ml) and N-benzyl-4-[(3,4-dihydro-2H-pyran-
2-yl)methoxy]benzamide (74 mg, 0.23 mmol) and PPTS
(132 mg, 0.51 mmol) were added. The mixture was sub-
mitted to microwave irradiation at a temperature of 120°C
for 3 h with stirring. After cooling the solution was washed
with H2O (3 9 5 ml) and then dried. Filtration and solvent
removal gave a brown oil that was purified by chroma-
tography [neutralized silica gel, hexanes/EtOAc (1:1)]
furnishing the product as a white foamy solid (90 mg,
1
50%). H-NMR (400 MHz, DMSO-d6) d 1.20 (m, 1H), 1.
38 (m, 1H), 1.72 (m, 1H), 1.81 (m, 1H), 1.86 (m, 1H), 1.98
(m, 1H), 3.07 (dd, J1 = 14.7 Hz, J2 = 5.2 Hz, 1H), 3.19
(dd, J1 = 14.1 Hz, J2 = 9.5 Hz, 1H), 3.93 (m, 1H), 4.02
(m, 1H), 4.14 (m, 1H), 4.17 (m, 1H), 4.22 (m, 2H), 4.33 (m,
1H), 4.45 (d, J = 5.9 Hz, 2H), 4.55 (m, 2H), 5.17 (m, 1H),
5.26 (m, 1H), 5.70 (bt, J = 9.8 Hz, 1H), 5.83 (m, 1H),
6.92–6.97 (d*, J = 8.7 Hz, 2H), 7.08 (m, 1H), 7.17 (m,
1H), 7.22 (t, J = 6.7 Hz, J = 6.4 Hz, 1H), 7.30 (m, 2H),
7.31 (m, 4H), 7.36 (s*, 1H), 7.41 (m, 2H), 7.56 (m, 2H),
7.68 (m, 2H), 7.83–7.84 (d*, J = 8.7 Hz, 2H), 7.89 (d,
7.4 Hz, 2H), 7.97 (m*, 1H), 8.88 (m, 1H); 13C-NMR
(100 MHz, CDCl3) d 22.2, 26.5, 26.8, 29.8, 42.5, 46.6,
54.9, 64.9, 65.7, 70.5, 75.3, 82.8, 110.6, 110.8, 114.0,
117.7, 118.4, 119.5, 120.1, 121.5, 123.5, 125.2, 126.6,
127.6, 127.0, 127.1 (92), 127.8, 128.2, 132.3 (92), 135.8,
139.9, 140.7, 143.7, 156.0, 160.7, 165.6, 171.8; HRMS
calcd for C49H48N3O7 [M ? H]? 790.3485, found
790.3486. HPLC tR 26.9 min, from 40 to 100% eluent B in
eluent A over 30 min.
1
1729, 1256 cm-1; H-NMR (400 MHz, CDCl3) d 1.65 (s,
9H), 3.27 (m, 2H), 3.71 (s, 3H), 4.21 (t, J = 7 Hz, 1H),
4.38 (m, 2H), 4.76 (m, 1H), 5.4 (d, J = 8 Hz, 1H), 7.22 (td,
J1 = 7.4 Hz, J2 = 1 Hz, 1H), 7.29 (m, 2H), 7.31 (td,
J1 = 7.7 Hz, J2 = 1.1 Hz, 1H), 7.39 (m, 2H), 7.43 (m,
1H), 7.51 (m, 1H), 7.55 (m, 2H), 7.76 (m, 2H), 8.12, (bd,
J = 7.4 Hz, 1H); 13C-NMR (100 MHz, CDCl3) d 28.1,
28.4, 47.4, 52.7, 54.2, 67.5, 84.1, 115.0, 115.5, 119.0,
120.2, 122.9, 124.4, 124.8, 125.3, 127.3, 127.9, 130.7,
135.5, 141.5, 143.9, 149.7, 155.9, 172.2; HRMS calcd for
C32H33N2O6 [M ? H]? 541.2333, found, 541.2338; HPLC
(chiral stationary phase) tR 18.2 min.
Fmoc-Trp(Boc)-OMe (Racemate)
As above for the preparation of Fmoc-(S)-Trp(Boc)-OMe
but using racemic Fmoc-Trp-OMe (300 mg, 0.68 mmol).
The product was obtained as yellow oil (154 mg, 41%).
HPLC (chiral stationary phase) tR 13.8 min (R enantiomer),
18.2 min (S enantiomer).
Nin-{6-[4-(N-Benzylcarbamoyl)benzoxymethyl]
tetrahydropyran-2-yl}-N-(9-fluorenyl-
methoxycarbonyl)-(S)-tryptophan methyl ester (11)
N-Benzyl-4-[(6-(2,4-dimethoxyphenyl)tetrahydro-2H-
pyran-2-yl)methoxy]benzamide (13)
This product was prepared using similar conditions that those
used for the analogue 10. 0.5 g (1.07 mmol) of 7 and 0.25 g
(0.77 mmol) of 4 afforded 43 mg of 11 (7%) as a white solid.
1H-NMR (400 MHz, CDCl3) d 1.56 (m, 2H), 1.84 (m, 1H),
2.02(m, 2H), 2.12(m, 1H), 3.29(d, J = 5.4, 2H), 3.67(s, 3H),
4.0(m, 1H), 4.07(m, 1H), 4.09(m, 1H), 4.19(m, 1H), 4.36(m,
2H), 4.74 (m, 1H), 4.62 (d, J = 5.3 Hz, 2H), 5.36 (m, 1H),
5.54 (dd, J1 = 2.9 Hz, J2 = 2.3 Hz, 1H), 6.27 (bs, 1H), 6.87
(d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 7.13 (m, 1H), 7.22 (m, 1H),
7.26 (m, 3H), 7.34 (m, 4H), 7.37 (m, 2H), 7.42 (m, 1H), 7.52
(m, 1H), 7.53(m, 2H), 7.75(m, 2H), 7.86(d, J = 8.5 Hz, 2H);
N-Benzyl-4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benz-
amide 8 (40 mg, 0.124 mmol) was dissolved in TFA/
mDMB (9:1, 1 ml) and the solution was allowed to stand
for 60 min at rt. After solvent removal the reddish oil was
purified by chromatography [silica gel, DCM/EtOAc (1:1)]
affording the product (34.2 mg, 60%) as a foamy white
solid. Mp = 55–59°C; Rf 0.62 [DCM/EtOAc (1:1)]; IR
(KBr) mmax 3320, 2928, 2834, 1605, 1500, 1288, 1251,
1
1205; H-NMR (400 MHz, CDCl3) d 1.37 (m, 1H), 1.46
(m, 1H), 1.60 (m, 2H), 1.94 (m, 2H), 3.73 (s*, 3H), 3.75
123

12
Int J Pept Res Ther (2012) 18:7–19
(s*, 3H), 3.81 (dd, J1 = 9.3 Hz, J2 = 7.3 Hz, 1H), 3.93
(dd, J1 = 9.5 Hz, J2 = 3.2 Hz, 1H), 3.96 (m, 1H), 4.59 (t,
J = 7.8 Hz, 1H), 4.61 (d, J = 5.6 Hz, 2H), 6.40 (m, 1H),
6.41 (m, 2H), 6.87 (d, J = 8.8 Hz, 2H), 7.05 (m, 1H), 7.29
(m, 1H), 7.34 (m, 4H), 7.73 (d, J = 8.8 Hz, 2H); 13C-NMR
(100 MHz, CDCl3) d 23.9, 33.1, 34.5, 35.4, 44.2, 55.4,
55.7, 70.1, 72.5, 99.0, 104.0, 114.5, 126.1, 127.1, 127.6,
128.0, 128.6, 128.9 (92), 138.5, 158.3, 158.8, 161.5, 167.0;
HRMS for C28H32NO5 [M ? H]? calcd, 462.2275, found,
462.2273; HPLC Rt 19.8 min, from 40 to 100% eluent B in
eluent A over 30 min.
fitted with a porous polystyrene frit and was washed suc-
cessively with DCM (10 9 30 s), TFA (40% v/v) in DCM
(1 9 1 min and 2 9 10 min), DCM (5 9 30 s), DIEA
(5% v/v) in DCM (6 9 2 min), DCM (5 9 30 s),
DMF (5 9 30 s) and DCM (5 9 30 s). 3 (65 mg, 0.28
mmol), N,N0-diisopropylcarbodiimide (DIPCDI) (43 ll,
0.28 mmol) and ethyl cyanoglyoxyl-2-oxime (39 mg,
0.28 mmol) in DCM (1 ml) were then added and the
mixture was allowed to stand for 1 h at rt with occasional
manual stirring. The resin was then washed with DCM
(5 9 30 s). 6 (64 mg, 0.14 mmol) and PPTS (53 mg,
0.21 mmol) in DCE (0.5 ml) were then added to the
handle-resin and the suspension was shaken at 80°C for
16 h in an Advanced Chemtech PLS 4 9 4 organic syn-
thesizer. After cooling to rt the aminoacyl-resin was
washed successively with DCM (5 9 30 s), DMF (5 9
30 s) and MeOH (5 9 30 s). Spectrophotometric quanti-
fication of the dibenzofulvene-piperidine adduct from a
portion of the resin indicated a 76% yield for amino acid
coupling. After washing with DMF (5 9 30 s) and DCM
(5 9 30 s) this resin was placed under Ar and Pd(PPh3)4
(43 mg, 0.037 mmol) and PhSiH3 (544 ll, 4.4 mmol) in
DCM (2 ml) were added. The mixture was shaken for
30 min at rt, filtered and washed with DCM (8 9 30 s). A
second treatment with Pd(PPh3)4 (43 mg, 0.037 mmol)
and PhSiH3 (544 ll, 4.4 mmol) in DCM (2 ml) was then
carried out. After filtration the resin was washed succes-
sively with DCM (8 9 30 s), diethyl dithiocarbamate (5%
v/v) in DMF (2 9 5 min), DMF (5 9 1 min), DCM
(5 9 30 s) and DMF (5 9 30 s). The resin (140 mg) was
then treated with H-Pro-OMeꢀHCl (19 mg, 0.28 mmol),
PyBOP (76 mg, 0.28 mmol) and DIEA (77 ll, 0.83
mmol) in DMF (0.5 ml) for 60 min with occasional
manual stirring. The resin was washed with DCM
(5 9 30 s) and DMF (5 9 30 s). This coupling reaction
and washing cycle was then repeated twice using the
same quantities of reagents and solvents. The resulting
resin was treated with piperidine (20% v/v) in DMF
(2 9 10 min), was washed with DMF (5 9 30 s) and
DCM (5 9 30 s) and dried. Cleavage of the product from
the resin was brought about by treatment with TFA/
mDMB/DCM (5:5:90 v/v) (3 9 10 min) and the collected
washings were submitted to solvent removal. The crude
product was precipitated with hexanes and the remaining
solid was centrifuged (10 min at 6000 rpm) and filtered,
affording 14 mg of a foamy white solid that co-eluted
with an authentic sample of cTrpPro. HPLC analysis
indicated a cleavage yield of 74% for this process. HRMS
calcd for C16H18N3O2 [M ? H]? 284.1399, found
284.1394.
Synthesis of Brevianamide F (1) in Solution
Z-Trp-OH (0.51 g, 1.48 mmol), H-Pro-OMeꢀHCl (257 mg,
1.48 mmol), HOBt (231 mg, 1.48 mmol) and EDCꢀHCl
(284 mg, 1.48 mmol) were suspended in MeCN (20 ml).
DIEA (383 ll, 2.22 mmol) was added and the mixture was
stirred overnight at rt under N2. The solvent was removed
and EtOAc (20 ml) was added. The organic phase was
washed successively with H2O (2 9 20 ml), aqueous 10%
Na2CO3 (2 9 20 ml), H2O (2 9 20 ml), 0.1 M HCl
(2 9 20 ml) and brine (2 9 20 ml) and the resulting
organic solution was dried. After filtration and solvent
removal the residue was dissolved in anhydrous MeOH
(50 ml) and was added to 10% Pd/C (54 mg, 10 mol%).
This mixture was then stirred vigorously under H2 at stp
overnight. After filtration through Celite the solvent was
removed affording a foamy white solid that was purified
by chromatography [neutralized silica gel, hexanes/Et2O
(2:8)] to yield the desired product as a foamy white solid
(285 mg, 68%). Mp = 166–168°C; [a]D = -69.5 (MeOH,
1
c 0.44); Rf 0.30 [DCM/EtOAc (2:1)]; H-NMR (400 MHz,
DMSO-d6) d 1.37 (m, 1H), 1.64 (m, 2H), 1.95 (m, 1H),
3.08 (dd, J1 = 14.9, J2 = 5.7 Hz, 1H), 3.26 (m, 2H), 3.39
(m, 1H), 4.04 (bt, J = 8.3 Hz, 1H), 4.29 (bt, J = 4.9 Hz,
1H), 6.95 (bt, J = 7.5 Hz, 1H), 7.05 (bt, J = 7.5 Hz, 1H),
7.18 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.56
(d, J = 7.9 Hz, 1H), 7.72 (s, 1H), 10.9 (s, 1H); 13C-NMR
(100 MHz, DMSO-d6) d 21.9, 25.8, 27.7, 44.6, 55.3, 58.4,
109.3, 111.2, 118.2, 118.7, 120.9, 124.4, 127.4, 136.0,
165.5, 169.1; HRMS calcd for C16H18N3O2 [M ? H]?
284.1399, found 284.1391; HPLC: tR 15.8 min, from 5 to
100% eluent B in eluent A over 30 min.
Solid-Phase Synthesis of Brevianamide F (1)
Aminomethyl-polystyrene resin (0.37 mmol g-1, 249 mg,
0.092 mmol) was introduced into a polypropylene syringe
123

Int J Pept Res Ther (2012) 18:7–19
13
Assays of Conventional and Microwave Heating
in the Reaction of Fmoc-Trp-OR (6 for R = Allyl or 7
for R = Me) with 3,4-Dihydro-2H-pyran and with
N-Benzyl-4-[(3,4-dihydro-2H-pyran-2-
were added. The tubes were closed and heated to the
desired temperature with mechanical stirring in an
Advanced Chemtech PLS 4 9 4 organic synthesizer.
Optimum heating of the vials was achieved using suitably-
sized aluminium adaptors.
yl)methoxy]benzamide (4)
The reaction conditions assayed were: 1.5 and 3 eq for
6; 1, 2 and 6 eq for PPTS; 100, 200 and 300 ll for DCE
and 70, 80 and 90°C (the reaction time was 16 h in all
cases). The yields of amino acid anchoring were between
67 and 90% for aminomethylpolystyrene-resin, and
between 64 and 84% for MBHA-resin. The final func-
tionalization levels and reaction yields were determined by
spectrophotometric quantification of the dibenzofulvene
adduct formed on elimination of the Fmoc group.
Conventionally heated reactions were carried out in 2 ml
Wheaton sealable vials with magnetic stirring. In order to
ensure homogeneous heating of the vials they were inserted
into an aluminium block that incorporated a heating sensor.
Reaction time was 16 h and the temperature was 70°C in
all cases. Product yields were determined by measuring the
peak areas in reversed-phase chromatography (see
‘‘Materials and Methods’’).
Microwave heated reactions were carried out with
magnetic stirring using 5 ml vials (see ‘‘Materials and
Methods’’). Reaction times were 30 min when DHP was
used, and 30 and 150 min for reactions involving 4. The
temperature was 120°C in all cases.
For reactions involving DHP (9.5 ll, 0.104 mmol,
1 eq), the relevant amino acid derivative (76.2 mg of 6 and
72.3 mg of 7, 0.156 mmol, 1.5 eq) and PPTS (60.5 mg,
0.239 mmol, 2.3 eq) were dissolved in DCE (1.5 ml). For
those reactions involving 4 (20.0 mg, 61.7 mmol, 1 eq),
the relevant amino acid derivative (45.3 mg of 6 and
42.9 mg of 7, 92.6 mmol, 1.5 eq) and PPTS (35.7 mg,
0.142 mmol, 2.3 eq), were dissolved in DCE (1 ml). Yields
were 68–70% with conventional heating and 63–67%
under microwave heating for DHP, and 95–96% with
conventional heating and 97–99% under microwave heat-
ing in the case of 4.
Assays of Anchoring Fmoc-Trp-OAllyl (6) to the Solid
Support Using Microwave Heating
Standard protocol: aminomethylpolystyrene resin (59.6 mg,
0.37 mmol g-1, 0.022 mmol) was introduced into a poly-
styrene syringe fitted with a porous polystyrene frit and was
washed with, successively, DCM (10 9 30 s), 40% TFA in
DCM (1 9 1 min and 2 9 10 min), DCM (5 9 30 s), 5%
DIEA in DCM (6 9 2 min), DCM (5 9 30 s), DMF
(5 9 30 s) and DCM (5 9 30 s). The handle 3 (15 mg,
3 mmol) in DCM (1 ml) was added together with DIPCDI
(10.22 ll, 3 mmol) and ethyl cyanoglyoxyl-2-oxime (9 mg,
3 mmol) in DCM (1 ml) and the mixture was allowed to
stand for 3 h at rt with occasional manual stirring. The
resulting resin was then washed with DCM (5 9 30 s), DMF
(5 9 30 s) and DCE (5 9 30 s) and added to a solution of 6
(15 mg, 1.5 mmol) and PPTS (13 mg, 2.3 mmol) in anhy-
drous DCE (0.3 ml). The suspension was then subjected to
microwave irradiation for 2 h at 120°C. After cooling it was
washed with DMF (5 9 30 s), DCM (5 9 30 s), MeOH
(5 9 30 s) and DCM (5 9 30 s). The yield of amino acid
anchoring was judged to be 47% as determined by spectro-
photometric quantification of the dibenzofulvene adduct
formed on elimination of the Fmoc group. Other trial reac-
tions carried out with an aminomethylpolystyrene resin with a
functionalization level of 0.55 mmol g-1 gave yields between
30 and 58%.
Assays of Anchoring Fmoc-Trp-OAllyl (6) to the Solid
Support Using Conventional Heating
Aminomethylpolystyrene-resin (batches of 59–65 mg) and
MBHA-resin (batches of 61–68 mg), with functionaliza-
tions 0.37 and 1.00 mmol g-1, respectively were used. The
resin batches were introduced into polypropylene syringes
fitted with porous polystyrene frits and were treated with,
successively, DCM (10 9 30 s), 40% TFA in DCM (1 9 1
and 2 9 10 min), DCM (5 9 30 s), 5% DIEA in DCM
(6 9 2 min), DCM (5 9 30 s), DMF (5 9 30 s) and DCM
(5 9 30 s).
Assays of Stability of Nim-{6-[4-(N-Benzylcarbamoyl)
phenoxymethyl]tetrahydropyran-2-yl}-
The aminomethylpolystyrene-resin and MBHA-resin
batches were then subjected to treatment with the handle 3
(3 eq), DIPCDI (3 eq) and ethyl cyanoglyoxyl-2-oxime (3
eq) in DCM for 3 h. (After treatment the ninhydrin test was
negative in all cases). Excess reagents were filtered and the
resulting resins were washed with DCM (5 9 30 s), DMF
(5 9 30 s) and DCE (3 9 30 s).
N-(9-fluorenylmethoxycarbonyl)-(S)-tryptophan
Allyl Ester (10) Under Acidic Conditions
Reactions were carried out at rt in polypropylene vials
equipped with a magnetic stirrer bar. Between 4.8 and
5.5 mg of tryptophan derivative (6.1–11.2 mmol) was
dissolved in 1 ml of acidolytic mixture. Product yields
shown in Table 1 were determined by measuring the peak
Batches of both resins in DCE were then introduced into
2 ml screw-cap vials (Wheaton) and 6, PPTS and DCE
123

14
Int J Pept Res Ther (2012) 18:7–19
Table 1 Stability of Nim-{6-[4-(N-benzylcarbamoyl)phenoxymethyl]-
tetrahydropyran-2-yl}-N-(9-fluorenylmethoxycarbonyl)tryptophan
allyl ester (10) under different acidolytic conditions
the side-chain anchoring of suitably protected trifunctional
amino acids such as Thr (Graham et al. 2002), Ser (Vil-
lorbina et al. 2007) and 4-hydroxyproline (Hyp) (Anderson
et al. 2005). However, the solid-phase protection of nitro-
gen-based functional groups as their tetrahydropyranyl
derivatives has not been extensively studied. The reversible
union of purines (Nugiel et al. 1997; Chang et al. 2005) and
benzimidazoles (Wang et al. 2002) through one of the
imidazole nitrogens has been reported. We were interested
by Smith et al. (1998) report describing a similar approach
for indoles using a tetrahydropyranyl-functionalized resin.
Typical anchoring conditions involved submitting the
indole derivative (1.4 eq with respect to resin functionali-
zation) and a sulfonic acid as catalyst in 1,2-dichloroethane
(DCE) as solvent to moderately high temperatures
(60–70°C) and long reaction times (12–30 h) in the pres-
ence of the solid support. On the other hand, cleavage of
the hemiaminal linkage between the heterocycle and the
resin is usually carried out using TFA in DCM at rt and
requires only short reaction times.
Entry Acidolytic mixture
Time 10 (%)a 6 (%)b
(min)
1
TFA/DCM (9:1)
TFA/DCM (1:9)
TFA/TIS/DCM (1:1:8)
TFA/H2O (9:1)
30
30
60
60
50
14
2
65 (41)c 21 (16)c
3
–
13
4
34
75
–
8
5
TFA/mDMB/DCM (0.2:1:8.8) 60
TFA/mDMB/DCM (0.5:1:8.5) 30
TFA/mDMB/DCM (1:0.2:8.8) 15
17
6
90d
68 (56)e
72
7
–
8
TFA/mDMB/DCM (1:1:8)
TFA/mDMB/DCM (4:1:5)
TFA/mDMB (9:1)
60
60
60
60
–
9
–
40
10
11
a
–
17
TFA/mDMB/THF (4:1:5)
34 (25)f 21 (7)f
Percentage of unreacted starting material
Based on integration of chromatographic peaks
After 1 h
b
c
d
e
f
A similar result was obtained after 1 h reaction time
These results encouraged us to study the reversible
linkage of suitably protected tryptophan derivatives to a
tetrahydropyranyl-functionalized resin with a view to
developing new synthetic strategies based on tryptophan as
the synthetic precursor. As a first approximation, in this
communication we report on our in-depth study of the
anchoring of tryptophan to tetrahydropyranyl-functiona-
lized resins and on its application to the synthesis of the
naturally occurring diketopiperazine Brevianamide F 1.
Cyclic peptides such as this have interesting biological
properties and potential applications in a range of fields
(Milne and Kilian 2010). Our study demonstrates the via-
bility of our strategy and its potential use in the preparation
of larger cyclic peptides or other tryptophan containing
derivatives.
After 30 min
After 3 h
areas in reversed-phase chromatography (see ‘‘Materials
and Methods’’).
Assays of Cleavage of Fmoc-Trp-OAllyl (6)
from the Resin
The aminoacyl resins obtained by conventional heating and
by microwave irradiation were introduced into polystyrene
syringes fitted with porous polystyrene frits and were
washed with DCM (5 9 30 s). They were then treated with
a mixture of TFA/mDMB/DCM (5:10:85) for 1 h at rt and
were washed with DCM (5 9 30 s). Cleavage yields of
80% for the resin subjected to conventional heating and
33% for that subjected to microwave heating were obtained
as judged by spectrophotometric quantification of the dib-
enzofulvene adduct formed on elimination of the Fmoc
group.
Commercial polystyrene-based resins functionalized
with tetrahydropyranyl groups are available. Nevertheless,
greater versatility would be achieved by using a handle that
could be incorporated into different aminomethylated res-
ins (polyestyrene, polyethyleneglycol, etc.) in this way
extending application to other types of solid support. Basso
and Ernst (2001) have described the use of (3,4-dihydro-
2H-pyran-2-yl)methoxyacetic acid in the functionalization
of aminomethylated ‘Syn-PhaseTM-lanterns’ for the syn-
thesis of cyclic peptides in which the peptide was attached
to the solid support via the hydroxyl group of Hyp. In our
laboratory this particular handle was obtained as an
hygroscopic, low melting-point and difficult to manipulate
solid. Consequently, in the present study we used 4-[(3,
4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid as an
alternative, which is a white solid with mp 141–143°C.
This product 3 (see Scheme 1) was obtained by Mitsunobu
reaction between (3,4-dihydro-2H-pyran-2-yl)methanol 2
Results and Discussion
The functionalization of a resin with tetrahydropyranyl
groups was first described by Thompson and Ellman (1994)
for the anchoring of molecules to solid supports through
their hydroxyl groups. Such supports have been shown to
be useful for a range of organic molecules (Kitade et al.
2006; Liu et al. 2005; Nam et al. 2003; Tanaka et al. 2006;
Wallace 1997). More specifically, they have been used for
123

Int J Pept Res Ther (2012) 18:7–19
15
O
CO2H
O
NH
Bz
O
O
Bz
O
O
O
N
c
OH
O
O
a
b
H
5
2
3
4
Scheme 1 Reagents and conditions: (a) (i) methyl p-hydroxybenzo-
ate (1 eq), PPh3 (0.8 eq), THF, Ar (30 min); then 2 (1 eq) and DIPAD
(0.8 eq), 48 h, (ii) LiOH (4.5 eq), MeOH (2)/H2O (1) (72%, two
steps); (b) benzylamine (1.2 eq), EDCꢀHCl (1.2 eq), HOBt (1.2 eq),
DCM, 22 h (44%); (c) (i) DSC (0.9 eq), DMAP (0.1 eq), DCM (10)/
MeCN (1), Ar, 4 h, (ii) benzylamine (0.22 eq), DMAP (0.04 eq), 20 h
(79%, two steps)
and methyl 4-hydroxybenzoate followed by ester hydro-
lysis. Isolation of the intermediate ester led to low overall
yields (25%) but preparation of the acid directly without
isolating the ester gave the desired handle in 72% yield.
As a prior step to the solid phase, it was considered
desirable to find suitable conditions for the formation of the
hemiaminal using amide 4 as a soluble model of an ami-
nomethyl polystyrene functionalized with tetrahydropyra-
nyl groups (Scheme 1). Thus, this product was easily
obtained from handle 3 and benzylamine under similar
conditions to those used in solid phase work. On the other
hand, alcohol 2 was used in order to obtain carbamate
model 5 with the objective of exploring an alternative
functionalization of the resin. This product was obtained
from 2 and benzylamine using N,N0-disuccinimidyl carba-
mate (DSC) in the presence of 4-dimethylaminopyridine
(DMAP).
The reaction conditions necessary for the reversible
union of tryptophan via its side-chain to the model com-
pound 4, were first explored using DHP as the simplest
hemiaminal precursor. A comparison was made between
results obtained with conventional heating and with
microwave radiation as an alternative to the conditions
described by Smith et al. (1998), in order to reduce reaction
times. Thus, when mixtures of DHP (1 eq) and 6 or 7
(1.5 eq) were heated (70°C for 16 h) or irradiated (120°C
for 30 min) in the presence of pyridinium 4-toluenesulfo-
nate (PPTS) (2.3 eq) in DCE (Scheme 2), diastereomeric
Fmoc-Trp(THP)-OAllyl 8 or Fmoc-Trp(THP)-OMe 9 were
obtained, respectively. These diastereomers were practi-
cally indistinguishable by NMR spectroscopy and HPLC.
Conventional heating and microwave irradiation afforded
similar yields of 65–70% and no other products were
detected, indicating that a-amino and carboxyl protecting
groups were stable to the conditions employed.
In order to attach tryptophan to a tetrahydropyran-
functionalized resin via its indole ring, the a-amino and
carboxyl functions must be protected with groups that are
compatible with the conditions necessary for the forma-
tion of the hemiaminal linkage and with the elongation of
the peptide chain itself. We considered that the three-
dimensional orthogonal strategy based on the 9-fluore-
nylmethoxycarbonyl (Fmoc), t-Bu and allyl groups (Kates
et al. 1993; Trzeciak and Bannwarth 1992) had much to
recommend it, since the reversible union of tryptophan to
the resin requires acidic conditions and the resulting
hemiaminal linkage is stable to bases such as piperidine,
used for removal of the Fmoc group, or the nucleophiles
used to remove the allyl group under neutral conditions
(Guibe 1998). Nevertheless, we also considered protecting
the carboxyl group as a methyl ester since this could be
cleanly removed if required by basic treatment (LiOH) in
the presence of the hemiaminal linkage (Cabrele et al.
1999; Gu and Silverman 2003; Liu et al. 2005). Therefore
the amino acid derivatives used in this study were Fmoc-
Trp-OAllyl 6 and Fmoc-Trp-OMe 7, that were prepared
using slightly modified classical methods starting from
the commercially available products H-Trp-OAllyl and
H-Trp-OMe (Kuriyama and Kitahara 2001; Richard et al.
2004).
When monomeric analog of dihydropyranyl function-
alized aminomethylated resin 4 was reacted with trypto-
phan derivatives 6 and 7 (70°C, 16 h), HPLC monitoring
showed the disappearance of 4 and the appearance of two
new products 10 and 11, respectively. Both of these were
formed as diastereomeric mixtures but, like 8 and 9, only
one chromatographic peak was detected by HPLC. On the
other hand a precise analysis of derivative 10 by NMR
revealed a single set of signals, indicating very probably,
the presence of one very major diastereomer. The purities
of 10 and 11, as determined by integration of the HPLC
peak areas were around 95% and no other products were
observed. Similar results were obtained when the same
reactions were subjected to microwave radiation (120°C,
3 h).
We also addressed the issue of the possible racemization
of these tryptophan derivatives using HPLC on a chiral
stationary phase. This required conversion of a racemic
mixture of 7 into its Nin-t-butoxycarbonyl (Boc) derivative
12 by treatment with Boc2O/DMAP, in order to achieve
optimal peak separation (Scheme 2). Optically pure amino
acid 7 was then subjected to the conditions necessary for
the anchorage of tryptophan, and was further transformed
to the corresponding Nin-Boc derivative. HPLC analysis on
123

16
Int J Pept Res Ther (2012) 18:7–19
Scheme 2 Reagents and
conditions: (a) from 6: DHP
(1 eq), PPTS (1.6 eq), DCE,
70°C, 8 h; from 7: DHP
(0.7 eq), PPTS (1.6 eq), DCE,
120°C (MW), 14 min (69%);
(b) 4 (0.72 eq), PPTS (1.6 eq),
DCE, 120°C (MW), 3 h (10:
50%, 11: 7%); (c) from 7:
Boc2O (1.22 eq), DMAP
(0.1 eq), MeCN, N2, 6 h (55) %
(similar conditions were used
for a racemic mixture of 7,
40%); (d) TFA (0.5)/mDMB
(1)/DCM (8.5), 30 min (90%)
a chiral stationary phase showed that chiral integrity had
been maintained.
The use of TFA in the absence of cation scavengers
furnished tryptophan derivative 6 in low yields and dif-
ferent reaction sub products (entries 1 and 2). These
results were not improved by adding TIS (entry 3) or H2O
(entry 4) to the mixture as cation scavengers. How-
ever, 3,5-dimethoxybenzene (mDMB) (Stathopoulos et al.
2006) was found to be an efficient scavenger since it
notably improved the yield of 6 using similar conditions
of TFA (compare entry 8 with entries 2 and 3). Higher
concentrations of acid (entries 9 and 10) or other solvents
(compare entries 9 and 11) did not lead to improvements
in the yields of 6; nevertheless, the reduction of the TFA
concentration to 5% furnished the desired tryptophan
derivative in excellent yield (entry 6). HPLC analysis of
this reaction showed the protected amino acid 6, an
unidentified by-product (10%) and product 13 resulting
from electrophilic aromatic substitution of the scavenger
by the pyranyl carbocation derived from monomeric
analog 4 under acidic conditions (Scheme 2). The identity
of 13 was established by mass spectrometry and by
chromatographic comparison with a pure sample of this
product obtained by reaction of 4 with mDMB in TFA.
Finally, treatment of 10 with lower concentrations of TFA
(entry 5), lower concentrations of mDMB (compare
entries 7 and 8) or longer reaction times (entry 7) led to
lower cleavage yields of 6.
The coupling of tryptophan to an aminomethyl resin
functionalized by direct union of DHP derivative 2 to the
polymer via a carbamate linkage was also assayed using
the dihydropyranyl model 5 (Scheme 1). Unfortunately,
HPLC showed that product 5 was unstable to the coupling
conditions and this route was not pursued further.
After completion of our studies in solution, we next
turned to the union of the amino acid derivative to a
polymeric solid support. Thus, handle 3 was attached to
aminomethyl-polystyrene and benzhydrylamino (MBHA)-
polystyrene resins using benzotriazol-1-yl-oxytrispyrroli-
dinophosphonium hexafluorophosphate (PyBOP) with
N,N-diisopropylethylamine (DIEA) in N,N0-dimethylform-
amide (DMF). Microwave irradiation of these resins in the
presence of 3 eq of protected amino acid 6 and 1.6 eq of
PPTS in DCE gave variable results with yields in the range
30–60%. On the other hand, conventional heating repro-
ducibly gave yields above 80% making it the method of
choice for the anchorage of tryptophan to these resins.
Final amino acid functionalization levels were determined
by spectrophotometric quantification of the Fmoc group
(Ma and Sonveaux 1989).
As with the anchoring of the amino acid to the solid
support, the cleavage of the amino acid from the resin
was first studied in solution with monomeric analogs and
was monitored by HPLC. Table 1 summarizes the results
obtained with 10 under different acidolytic conditions.
The application of these optimized cleavage conditions
to tryptophanyl-polystyrene resins obtained from anchoring
reactions carried out using conventional heating gave good
123

Int J Pept Res Ther (2012) 18:7–19
17
O
O
we chose to apply it to Brevianamide F 1, a natural dike-
topiperazine with antibiotic properties isolated from certain
bacteria (Mehdi et al. 2009, and references therein)
(Scheme 3). However, in order to facilitate the identifica-
tion of the product synthesized by solid phase methods, we
elected to synthesize the molecule in solution first in order
to provide material for HPLC comparison. From among the
different methods described in the literature for the syn-
thesis of Brevianamide F (Ashnagar et al. 2007; Caballero
et al. 2003; Jhaumeer-Laulloo et al. 2003; Steyn 1973), that
of Ashnagar et al. (2007) was used for various reasons: the
product is obtained in very good yields, the strategy
developed employs protecting groups that are removed
under mild conditions and that allows it to be adapted to a
solid phase synthesis, and the synthetic protocol is based on
coupling proline to the a-carboxyl of tryptophan, an
approximation that is compatible with the union of this
amino acid to the polymer through its side chain. Thus,
Z-Trp-OH and H-Pro-OMe were coupled using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) in DCM and
hydrogenolysis of the Z group at rt using 10% Pd/C then
led to spontaneous cyclization (68% yield). NMR spec-
troscopic data of the crude product obtained in this way
indicated no significant impurities and further purification
was not required.
Fmoc
N
H
a
H2N
N
O
b
O
N
O
N
N
O
N
H
O
H
N
c
N
H
O
Brevianamide F
1
Scheme 3 Reagents and conditions: (a) (i) 3 (3 eq), DIPCDI (3 eq),
OxymeTM (3 eq), DCM, 1 h, (ii) 6 (1.5 eq), PPTS (2.3 eq), DCE,
80°C, 16 h (76%, two steps); (b) (i) Pd(PPh3)4 (0.4 eq), PhSiH3
(48 eq), DCM, 30 min (two treatments), (ii) H-Pro-OMeꢀHCl (3 eq),
PyBOP (3 eq), DIEA (9 eq), DCM, 1 h (three treatments), (iii) 20%
of piperidine in DMF, 10 min (two treatments); (c) TFA (0.5)/mDMB
(0.5)/DCM (9), 10 min (three treatments) (74% from amino-acyl
resin)
yields although longer reaction times were required (1 h,
80%). However with tryptophanyl-polystyrene resins pre-
pared using microwave irradiation cleavage yields were
much lower (around 30%) although they could be
improved to around 50% by adding TIS (3%) and H2O
(2%) to the cleavage cocktails. We believe that this dif-
ference in behavior may be explained by non-specific
union of the amino acid to the resins or by structural
changes produced in the polymers at the high temperatures
generated on microwave irradiation.
Solid-phase synthesis of 1 was then undertaken starting
from an aminomethyl-polystyrene resin to which handle 3
had been attached (Scheme 3). Tryptophan derivative 6
was anchored (76% yield) as described above using con-
ventional heating (80°C, 16 h). The allyl group was
removed using Pd(PPh3)4 in the presence of PhSiH3 and
H-Pro-OMeꢀHCl was coupled using PyBOP/DIEA.
Removal of the Fmoc group with piperidine then led to
spontaneous cyclization as demonstrated by a negative
ninhydrin test on the resin at this point. Cleavage from the
resin was effected using TFA/mDMB/DCM (5:5:90 v/v),
In order to demonstrate the usefulness of this new
approach to the solid-phase synthesis of peptide derivatives
Fig. 1 Solid-phase synthesis of
Brevianamide F: a HPLC of the
acidolytic crude; b HPLC of the
same crude after washing with
hexanes (see experimental
details for HPLC conditions)
123

18
Int J Pept Res Ther (2012) 18:7–19
through catalytic palladium pi-allyl methodology. Tetrahedron
54:2967–3042
giving the essentially pure cyclic peptide in an overall yield
of 56%, as determined by HPLC analysis (Fig. 1). Cycli-
zation of the dipeptide resulting from coupling of Fmoc-
Pro-OH to the a-amino group of tryptophan required longer
reaction times.
Jhaumeer-Laulloo S, Khodabocus A, Jugoo A, Jheengut D, Sobha S
(2003) Synthesis of diketopiperazines containing prolinyl unit—
cyclo(L-prolinyl-L-leucine), cyclo(L-prolinyl-L-isoleucine) and
cyclo(L-tryptophyl-L-proline). J Indian Chem Soc 80:765–768
Kates SA, Sole NA, Johnson CR, Hudson D, Barany G, Albericio F
(1993)
A novel, convenient, three-dimensional orthogonal
strategy for solid-phase synthesis of cyclic peptides. Tetrahedron
Lett 34:1549–1552
Kitade M, Tanaka H, Oe S, Iwashima M, Iguchi K, Takahashi T
Conclusions
(2006) Solid-phase synthesis and biological activity of
combinatorial cross-conjugated dienone library. Chem Eur J
12:1368–1376
a
With the synthesis of Brevianamide F, this work demon-
strates the promise of anchoring tryptophan through its side
chain to a DHP functionalized solid support as an approach
to the synthesis of peptide derivatives containing trypto-
phan such as cyclic peptides. In principle, it would be also
applicable to the solid-phase synthesis of other tryptophan-
containing molecules with potential pharmacologic interest
and libraries of such compounds. On the other hand,
4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid has
proved to be a suitable handle to functionalize amino-
methyl- or MBHA-polystyrene resins with dihydropyranyl
groups.
Kuriyama W, Kitahara T (2001) Synthesis of apicidin. Heterocycles
55:1–4
Liu S, Gu W, Lo D et al (2005) N-Methylsansalvamide and peptide
analogues. Potent new antitumor agents. J Med Chem 48:
3630–3638
Ma JC, Dougherty DA (1997) The cation-p interaction. Chem Rev
97:1303–1324
Ma Y, Sonveaux E (1989) The 9-fluorenylmethyloxycarbonyl group
as a 50-OH protection in oligonucleotide synthesis. Biopolymers
28:965–973
Mant CT, Kovacs JM, Kim HM, Pollock DD, Hodges RS (2009)
Intrinsic amino acid side-chain hydrophilicity/hydrophobicity
coefficients determined by reversed-phase high-performance
liquid chromatography of model peptides: comparison with
other hydrophilicity/hydrophobicity scales. Biopolymers 92:
573–595
Acknowledgment We are grateful for financial support from Min-
´
isterio de Ciencia e Innovacion (CTQ2006-12460).
Mehdi RBA, Shaaban KA, Rebai IK, Smaoui S, Bejar S, Mellouli L
(2009) Five naturally bioactive molecules including two
rhamnopyranoside derivatives isolated from the Streptomyces
sp. strain TN58. Nat Prod Res B 23:1095–1107
References
Milne PJ, Kilian G (2010) The properties, formation, and biological
activity of 2,5-diketopiperazines. In: Mander L, Liu HW (eds)
Comprehensive natural products II: chemistry and biology, vol 5.
Elsevier Science, Amsterdam, pp 657–698
Nam N-H, Sardari S, Parang K (2003) Reactions of solid-supported
reagents and solid supports with alcohols and phenols through
their hydroxyl functional group. J Comb Chem 5:479–546
Nugiel DA, Cornelius LAM, Corbett JW (1997) Facile preparation of
2,6-disubstituted purines using solid-phase chemistry. J Org
Chem 62:201–203
Richard DJ, Schiavi B, Joullie MM (2004) Synthetic studies of
roquefortine C: synthesis of isoroquefortine C and a heterocycle.
Proc Natl Acad Sci USA 101:11971–11976
Rodrigues de Sa Alves F, Barreiro EJ, Fraga CAM (2009) From
nature to drug discovery: the indole scaffold as a ‘Privileged
Structure’. Mini Rev Med Chem 9:782–793
Ruiz-Sanchis P, Savina Svetlana A, Albericio F, Alvarez M (2011)
Structure, bioactivity and synthesis of natural products with
hexahydropyrrolo[2,3-b]indole. Chemistry 17:1388–1408
Samanta U, Pal D, Chakrabarti P (1999) Packing of aromatic rings
against tryptophan residues in proteins. Acta Crystallogr D
D55:1421–1427
Samanta U, Pal D, Chakrabarti P (2000) Environment of tryptophan
side chains in proteins. Proteins Struct Funct Genet 38:288–300
Sawyer TK (1997) Peptidomimetic and nonpeptide drug discovery:
impact structure-based drug design. In: Veerapandian P (ed)
Structure based drug design: disease, targets, techniques and
development. Marcel Dekker, New York, pp 559–634
Smith AL, Stevenson GI, Swain CJ, Castro JL (1998) Traceless solid
phase synthesis of 2,3-disubstituted indoles. Tetrahedron Lett
39:8317–8320
Anderson MO, Shelat AA, Guy RK (2005) Solid-phase approach to
the phallotoxins: total synthesis of [Ala7]-phalloidin. J Org
Chem 70:4578–4584
Ashnagar A, Bailey PD, Cochrane PJ, Mills TJ, Price RA (2007)
Unusual rearrangements and cyclizations involving polycyclic
indolic systems. Arkivoc 11:161–171
Basso A, Ernst B (2001) Solid-phase synthesis of hydroxyproline-
based cyclic hexapeptides. Tetrahedron Lett 42:6687–6690
Bernhardt A, Drewello M, Schutkowski M (1997) The solid-phase
synthesis of side-chain-phosphorylated peptide-4-nitroanilides.
J Pept Res 50:143–152
Black KM, Clark-Lewis I, Wallace CJA (2001) Conserved tryptophan
in cytochrome c: importance of the unique side-chain features of
the indole moiety. Biochem J 359:715–720
´
Caballero E, Avendan˜o C, Menendez JC (2003) Brief total synthesis
of the cell cycle inhibitor tryprostatin B and related preparation
of its alanine analogue. J Org Chem 68:6944–6951
Cabrele C, Langer M, Beck-Sickinger AG (1999) Amino acid side
chain attachment approach and its application to the synthesis of
tyrosine-containing cyclic peptides. J Org Chem 64:4353–4361
Chang J, Dong C, Guo X et al (2005) A solid-phase approach to novel
purine and nucleoside analogs. Bioorg Med Chem 13:4760–4766
Gordon EM, Kerwin JF (1998) Combinatorial chemistry and molec-
ular diversity in drug discovery. Wiley-Liss, New York
Graham KAN, Wang Q, Eisenhut M, Haberkorn U, Mier W (2002) A
general method for functionalising both the C- and N-terminals
of Tyr3-octreotate. Tetrahedron Lett 43:5021–5024
Gu W, Silverman RB (2003) Solid-phase total synthesis of scytal-
idamide A. J Org Chem 68:8774–8779
Guibe F (1998) Allylic protecting groups and their use in a complex
environment. Part II: allylic protecting groups and their removal
123

Int J Pept Res Ther (2012) 18:7–19
19
Spatola AF, Romanovskis P (2000) Head-to-tail cyclic peptides and
cyclic peptide libraries. In: Greenberg A, Breneman CM,
Liebman JF (eds) The amide linkage: selected structural aspects
in chemistry, biochemistry and materials science. Wiley, New
York, pp 519–564
Stathopoulos P, Papas S, Tsikaris V (2006) C-terminal N-alkylated
peptide amides resulting from the linker decomposition of the
Rink amide resin. J Pept Sci 12:227–232
Steyn PS (1973) Structure of five dioxopiperazines from Aspergillus
ustus. Tetrahedron 29:107–120
Tanaka H, Ishida T, Matoba N, Tsukamoto H, Yamada H,
Takahashi T (2006) Efficient polymer-assisted strategy for the
deprotection of protected oligosaccharides. Angew Chem Int
Ed 45:6349–6352
Thompson LA, Ellman JA (1994) Straightforward and general
method for coupling alcohols to solid supports. Tetrahedron
Lett 35:9333–9336
Trzeciak A, Bannwarth W (1992) Synthesis of head-to-tail cyclized
peptides on solid support by Fmoc [9-fluorenylmethoxycarbo-
nyl] chemistry. Tetrahedron Lett 33:4557–4560
Villorbina G, Canals D, Carde L et al (2007) Solid-phase synthesis of a
combinatorial library of dihydroceramide analogues and its activity
in human alveolar epithelial cells. Bioorg Med Chem 15:50–62
Wallace OB (1997) Solid phase synthesis of ketones from esters.
Tetrahedron Lett 38:4939–4942
Wang X, Choe Y, Craik CS, Ellman JA (2002) Design and synthesis
of novel inhibitors of gelatinase B. Bioorg Med Chem Lett
12:2201–2204
123