Solid-phase synthesis of phenylalanine containing peptides using a traceless triazene linker

Article abstract of DOI:10.1021/jo301630h

The use of a triazene function to anchor phenylalanine to a polymeric support through its side chain is reported. To prove the usefulness of this strategy in solid-phase peptide synthesis, several bioactive peptides have been prepared including cyclic, C-modified, and protected peptides. The triazene linkage is formed by coupling the diazonium salt of Fmoc-Phe(pNH ₂)-OAllyl to a MBHA-polystyrene resin previously functionalized with isonipecotic acid (90%). Further assembly of the peptide chain, cleavage from the resin using 2-5% TFA in DCM, and reduction of the resulting diazonium salt of the peptide with FeSO₄-7H₂O in DMF afforded the desired products in high purities (73-94%).

Full text of DOI:10.1021/jo301630h

Note
pubs.acs.org/joc
Solid-Phase Synthesis of Phenylalanine Containing Peptides Using a
Traceless Triazene Linker
Carolina Torres-García,†,‡ Daniel Pulido,‡,§ Magdalena Carceller,† Ivan Ramos,† Miriam Royo,*,‡,§
́
and Ernesto Nicolas*,†
́
†Department of Organic Chemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Spain
‡Unitat de Química Combinator
̀
ia, Parc Científic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain
§Biomedical Research Networking in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Baldiri Reixac 10, 08028
Barcelona, Spain
S
* Supporting Information
ABSTRACT: The use of a triazene function to anchor phenylalanine to a
polymeric support through its side chain is reported. To prove the
usefulness of this strategy in solid-phase peptide synthesis, several bioactive
peptides have been prepared including cyclic, C-modified, and protected
peptides. The triazene linkage is formed by coupling the diazonium salt of
Fmoc-Phe(pNH2)-OAllyl to a MBHA-polystyrene resin previously
functionalized with isonipecotic acid (90%). Further assembly of the
peptide chain, cleavage from the resin using 2−5% TFA in DCM, and
reduction of the resulting diazonium salt of the peptide with FeSO4·7H2O
in DMF afforded the desired products in high purities (73−94%).
eptide research on drug design and drug discovery is one
polymeric support through a triazene linkage. The potential of
Pof the most promising fields in the development of new this approach has been proven by the synthesis of some
biologically active peptide derivatives.
drugs. However, linear peptides are generally not suitable for
use as drugs because of their poor bioavailability. For this
reason, much attention is being paid to the search for molecules
with potential therapeutic properties together with improved
structural and enzymatic stability. In this sense, cyclic peptides,
either naturally occurring or synthetically constructed, and C-
modified peptides have proven to be excellent molecular
scaffolds for design purposes.1 In this field of research,
phenylalanine has become an important pharmacophore in
SAR studies because of the key role that plays eliciting intrinsic
activity in compounds that contain this amino acid.2 Thus, the
nonpolar nature and steric bulkiness of its side chain is
responsible for crucial hydrophobic interactions in molecular
recognition processes.3 Moreover, a large number of natural
peptides covering a wide range of biological properties contain
at least one phenylalanine residue1 and are, therefore, potential
targets for the design of new drugs.
From a synthetic point of view, connection of the amino acid
to the resin via its side chain as the starting building block is
one of the most suitable approaches to the synthesis of cyclic4
and C-modified5 peptides because both extremes of the peptide
chain are left free for further chemical transformations. As a
part of our ongoing studies devoted to explore novel synthetic
strategies based on side-chain anchoring of aromatic amino
acids to solid supports,6 we have developed a new synthetic
strategy for the side-chain anchoring of phenylalanine to a
In general, side-chain anchoring solid-phase strategies have
taken advantage of Fmoc chemistry because they can be easily
adapted to protection schemes such as Fmoc/tBu/Allyl,7 and
most of the proteinogenic trifunctional amino acids have been
used in such strategies as starting building blocks with this
purpose.4,5 However, scarce literature can be found about
phenylalanine because of its side-chain nature. In this sense,
Silverman et al. have reported an elegant silicon-based linking
strategy to attach the aromatic side chain of phenylalanine that
has proven to be efficient in the synthesis of small peptides8
and cyclic peptides.9−11 Thus, peptides can be obtained in a
traceless way upon cleavage from the resin under acidic
conditions or halogen ipso-substituted phenylalanine containing
peptides can be formed (Br or I) by cleavage of the peptide in
the presence of suitable electrophile precursors (Br2 or ICl).12
Inspired by this concept, we focused our attention on the
possibility of tethering phenylalanine to the polymeric support
through a triazene linker. This linker group was originally
introduced by Brase et al.13 for the solid-phase synthesis of
̈
aromatic and heteroaromatic compounds.14 This system is
easily accessible by coupling the target aromatic amine through
its diazonium salt to a disubstituted amino-functionalized
Received: August 2, 2012
Published: September 28, 2012
© 2012 American Chemical Society
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Note
a
Scheme 1. Synthesis of the Peptides Reported in This Work
a
Key: (a) (i) In solution diazonium salt of 1: BF3Et2/tBuNO2; DCM; −10 °C; (ii) solid-phase coupling: pyridine, DCM, rt; (b) (i) SPPS; (ii) 5%
TFA in DCM, 5 min; (iii) FcSO4−7H2O/DMF; (c) (i) SPPS; (ii) 2% TFA in DCM, 3 × 2 min; (iii) FeSO4−7H2O/DMF.
polymeric support. A broad spectrum of synthetic approaches
can be applied to the aromatic compound attached to the resin
due to the robustness of the linker (stable toward daylight,
oxygen, moisture, bases, reducing agents, or oxidizing agents).
However, acidic cleavage of the triazene resin under mild
conditions yields the amine resin, which can be recycled, and
the modified aryldiazonium salt, which can be further reduced
after the cleavage step. In this sense, the triazene linker can be
envisaged as a multifunctional linker13 because of the wide
range of chemical transformations that diazonium salts can
provide,14a which could be used in a combinatorial fashion for
SAR studies.
this function to basic and neutral conditions. With the aim of
coupling phenylalanine to the polymeric support, the p-amino-
substituted amino acid was required (Fmoc-Phe(pNH2)-OAllyl,
1. This derivative was prepared following two synthetic
pathways using slightly modified classical methods starting
from the commercially available Fmoc-Phe(pNO2)-OH by C-
terminal protection with allyl bromide (98%)21 followed by
reduction of the nitro group with Zn dust in acidic conditions
(69% yield).22 We decided to use a piperazinyl-type resin14a to
anchor 1 through its side chain. The reaction conditions to
carry out this reaction were optimized from those described in
the literature with a commercial piperazinylmethylpolystyrene
resin, but we further moved to a MBHA-polystyrene resin
modified with Boc-isonipecotic acid (DIC/HOBt in DMF) to
have the possibility to control the functionalization of the
polymeric support and to perform on-resin quantitative amino
acid analysis by the introduction of a standard amino acid. The
best coupling results were achieved (up to 91%) when the resin
was treated under Ar atmosphere with 2.5 equiv of the
diazonium salt of 1 (preformed with BF3·Et2O/tBuNO2 in
DCM at −10 °C, 1 h) and 30 equiv of pyridine in DCM at rt
during 3 h. Final amino acid functionalization levels were
determined by spectrophotometric quantification of Fmoc
group.23
To prove the feasibility of this synthetic approach with
phenylalanine, several peptides were chosen as models (Scheme
1): endomorphin 2 (2),15 an endogenous opioid tetrapeptide
carboxamide with high selectivity and specificity for the μ-
opioid receptor; the Ac-AAPF p-nitroanilide peptide (3),16
a
chromogenic substrate for protease activity studies; the same
peptide but biotinylated at the C-terminal (4)17 as an example
of a potential molecular probe; cPhePro (5),18 a DKP with
antifungal and antimicrobial properties found in a wide range of
organisms; and unguisin A (6),19 a highly hydrophobic cyclic
heptapeptide isolated from the marine fungus Emericella unguis
and with antibacterial activity against staphylococus aureus.
The three-dimensional orthogonal strategy based on
Fmoc/tBu/allyl groups20 was chosen to attach phenylalanine
to a solid support via triazene linkage because of the stability of
The peptides that are shown in Scheme 1 were synthesized
as follows. The peptide chain of 2 was assembled from the N-
terminal of the phenylalanine attached to the resin and was
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Note
completed with the coupling of the C-terminal phenylalanine
carboxamide. C-Terminal-modified Ac-AAPF peptides 3 and 4
were synthesized using different synthetic approaches. Thus, p-
nitroaniline was coupled to the N-urethane-protected phenyl-
alanine before peptide chain elongation to avoid racemization
of this amino acid under the experimental conditions used in
this reaction,24 while biotin was attached at the end of the
assembly of the peptide chain. DKP 5 was prepared by coupling
proline to the C-terminal of phenylalanine in order to perform
on-resin cyclization through the primary N-terminal of
phenylalanine. Finally, the peptide sequence of cyclopeptide 6
was grown from the N-terminal of phenylalanine and cyclized
before cleavage from the resin.
with 5% TFA in DCM. Three 2 min treatments with this
solution were carried out to drive the reactions to completion.
In the case of peptide 2, the tyrosine side chain remained
protected under these conditions and required a treatment of 3
t
h with TFA/TIS/H2O (95:2.5:2.5) in order to remove the Bu
group. However, further treatment with THF to reduce the
resulting diazonium salts gave unreproducible results and
complex HPLC profiles. That moved us to explore other
strategies for reduction. To our surprise, the hydrodediazonia-
tion process mediated by FeSO4·7H2O in the presence of DMF
as hydrogen donor36 proved to be very efficient, giving clean
reaction crudes with the absence of byproducts. In general,
reactions went to completion at room temperature after a few
minutes, and products could be easily isolated performing a
simple workup that included ion-exchange chromatography to
remove any trace of iron, which was evidenced by a qualitative
assay using the colorimetric reagent ammonium thiocyanate.
Peptides crudes 2−6 were obtained with chromatographic
purities of 73−94%.
In the case of peptide 2, we decided to evaluate potential
epimerization during the C-terminal coupling of H-Phe-
NH2·HCl to the peptide backbone despite not detecting
diastereoisomers by HPLC. This reaction was performed using
DIC/oxyme as coupling reagents, conditions which are
reported to give rise to low degree of C-terminal
racemization.32 However, in order to determine the epimeriza-
tion more precisely, some experiments using Ac-Phe-Phe-NH2
(9) as model peptide were carried out. The dipeptide was
prepared in solid phase under conditions similar to those used
in the synthesis of 2. Thus, the protected amino acid 1 was
anchored to the resin, the N-terminal was acetylated after
removing the Fmoc group, and C-terminal coupling of H-Phe-
NH2·HCl was carried out. The analysis of the acidolytic crude
showed an 8−12% of racemization when it was compared with
the corresponding standards Ac-L-Phe-L-Phe-NH2 (9) and Ac-
D-Phe-L-Phe-NH2 (10), previously prepared on a Rink amide
resin following standard solid-phase protocols. It is interesting
to note that this drawback was overcome when C-terminal
coupling was performed on an Fmoc-protected Phe residue
attached to the resin, followed by Fmoc group elimination and
further N-terminal acetylation.24
The cleavage of the peptide under mild acidic conditions
offers the possibility of using this methodology for the synthesis
of protected peptides if required for convergent strategies. In
this sense, side-chain anchoring of the peptide chain allows one
to obtain the protected peptide with one of the extremes
unprotected if suitable orthogonal protections for the N-
terminal or the C-terminal are used. This approach is
exemplified by the synthesis of the side-chain protected peptide
hemorphine-7, an endogenous peptide belonging to the family
of opioid peptides released from hydrolyzed hemoglobin.37
Thus, this peptide was solid-phase assembled using DIC/HOBt
in DMF as N-terminal- or C-terminal-protected chains, and
both were cleaved from the resin with 2% TFA in DCM (5
treatments of 2 min) to afford the side-chain-protected peptides
7 or 8, respectively, in high yields (more than 90%) and purity
(more that 93%, as determined by integration of the
chromatographic peak areas).
N-Terminal peptide chain elongation was performed by
anchoring stepwise Na-Fmoc-protected amino acids with DIC/
HOBt25 and final acetylation with Ac2O/DIEA in the case of 3
and 4. Reactions were monitored until completeness using the
Kaiser test for primary amines26 or the chloranil test for
proline.27 Removal of the Fmoc protecting group was carried
out with 20% of piperidine in DMF, except for 6 where 3% of
the non-nucleophilic base DBU28 in DMF was used to remove
Fmoc prior to on-resin cyclization in order to avoid C-terminal
piperidylamide formation as a byproduct during this step.7,29
Furthermore, stronger basic conditions were necessary for 6 to
carry out Na deprotection after the coupling of GABA and a
mixture of 10% of DBU and 10% of piperidine in DMF was
required for complete removal.30 The C-terminal allyl
protection of phenylalanine was removed using Pd(PPh3)4 in
the presence of PhSiH331 to further attach H-Phe-NH2 (2), N-
Fmoc-diethylenediamine hydrochloride (4) and H-Pro-
OMe·HCl (5) with DIC/ethyl cyanoglyoxyl-2-oxime
(Oxyma)32 following a double-coupling protocol to ensure
complete reaction. In the case of peptide 3, five treatments with
p-nitroaniline and PyAOP/HOAt/DIEA in DMF33 were
needed to anchor the amine, as revealed by HPLC−MS
analysis. This coupling system was also used to attach biotin to
complete the assembly of 4, but only one treatment was
required in this particular case, as shown by the Kaiser test. On-
resin cyclizations of peptides 5 and 6 were carried out following
different strategies. Thus, removal of the Fmoc group from the
linear dipeptide methyl ester using a mixture of 10% of DBU
and 10% of piperidine in DMF took place with concomitant
intramolecular cyclization to afford the desired DKP skeleton.
In this sense, it is interesting to note that cyclization of the
dipeptide resulting from coupling of Na-Fmoc-Pro-OH to N-
terminal of phenylalanine gave complex crudes under the same
experimental conditions. Concerning 6, after removal of C-
terminal allyl ester and the N-terminal Fmoc group, the peptide
backbone was head-to-tail cyclized upon activation with
PyOAP/HOAt/DIEA in DMF. Both cyclization processes
went to completion as monitored by the Kaiser test.
Once the linear precursors were built on the resin, peptide-
resins were subjected to an acidolytic cleavage treatment.
Several acids under or followed by reducing conditions have
been applied to cleave small nonpeptide aromatic molecules
linked to a solid support via triazene. Thus, HCl in the presence
of THF,13 H2PO3,13 HSiCl3,34 or TFA followed by THF/
DMF36 have been used to obtain the traceless product. In view
of these precedents, we decided to use conditions similar to
those reported by Schunk et al.35 because we believed those
conditions would be more amenable to peptide chemistry
protocols in terms of compatibility with amino acid side-chain
functionalities. Thus, peptides 2−6 were cleaved from the resin
In summary, we have developed a new versatile and efficient
methodology for the solid-phase synthesis of peptides
containing phenylalanine, based on the anchorage of this
amino acid to the solid support through its side chain. Key
aspects of this strategy include the use of Fmoc-Phe(pNH2)-
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Note
2H), 5.89 (m, 1H), 5.30 (m, 3H), 4.61 (m, 3H), 4.41 (dd, J1 = 10.5
Hz, J2 = 7.2 Hz, 1H), 4.32 (dd, J1 = 10.7 Hz, J2 = 7.0 Hz, 1H), 4.20 (t, J
= 7.2 Hz, 1H), 3.51 (bs, 2H), 3.02 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 171.4, 155.6, 145.4, 143.8, 141.3, 131.5, 130.2, 127.7, 127.0,
125.3, 125.2, 120.0, 119.0, 115.3, 67.0, 66.0, 55.0, 47.2, 37.4; HRMS
calcd for C27H27N2O4 [M + H]+443.1965, found 443.1962.
OAllyl as the starting building block, coupling of this amino
acid via triazene to an MBHA-polystyrene resin modified by the
spacer Boc-isonipecotic acid, the use of low concentrated TFA
for peptide cleavage from the resin, and mild removal of the
resulting peptide diazonium salt with FeSO4·7H2O. This
strategy allows the modification of both ends of the peptide
chain, as well as the synthesis of protected peptides if required
for a convergent approach. Alternatively, the scope of
application could be broadened by the introduction of diversity
through the chemical transformation of the diazonium salt to
obtain peptide analogues potentially useful for SAR studies.
Investigations in this direction are currently underway.
Isonipecotic Acid Loaded MBHA Resin. MBHA resin (1.57 g,
0.63 mmol/g, 0.99 mmol) was introduced into a polypropylene
syringe fitted with a porous polystyrene frit and was washed
successively with DCM (10 × 30 s), TFA (40% v/v) in DCM (1 ×
1 min and 2 × 10 min), DCM (5 × 30 s), DIEA (5% v/v) in DCM (5
× 2 min), DCM (5 × 30 s), and DMF (5 × 30 s). Then, Fmoc-Gly-
OH (internal reference) (0.88 g, 2.97 mmol), HOBt (0.40 g, 2.97
mmol), and DIC (460 μL, 2.97 mmol) in DMF (4 mL) were added.
The suspension was left for 1 h at rt with occasional manual stirring
and was washed with DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 ×
30 s), and DCM (5 × 30 s). Then, Fmoc aminoacyl protection was
removed with 20% piperidine in DMF (1 × 1 min and 2 × 10 min)
and the Boc-isonipecotic acid (0.68 g, 2.97 mmol), HOBt (0.40 g, 2.97
mmol) and DIC (460 μL, 2.97 mmol) added in DMF (4 mL). After 1
h at rt with occasional manual stirring, the mixture was washed with
DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 ×
30 s). Finally, the resin was treated with 40% of TFA in DCM to
remove Boc group (2 × 10 min) and washed with DCM (5 × 30 s),
DIEA (5% v/v) in DCM (5 × 2 min), DCM (5 × 30 s), MeOH (5 ×
30 s), DCM (5 × 30 s), and DMF (5 × 30 s).
General Procedure A for the Coupling of Phenylalanine to
the Resin via Triazene. Compound 1 (1.24 g, 2.80 mmol) was
dissolved under Ar in anhydrous DCM (35 mL), and the resulting
solution was cooled to −10 °C, and then BF3·Et2O (1.80 mL, 14.20
mmol) and tBuNO2 (1.85 mL, 14.00 mmol) were added. The mixture
was stirred while maintaining this temperature for 1 h under Ar and
was added via cannula to a preformed mixture of the piperidine linker
resin (1.57 g, 0.99 mmol) and anhydrous pyr (18 mL) at −10 °C. The
resulting suspension was shaken at rt for 3 h under Ar, and then after
filtration, the resin was washed with DCM (5 × 30 s), MeOH (5 × 30
s), DCM (5 × 30 s), and DMF (5 × 30 s). Spectrophotometric
quantification of Fmoc groups afforded an amino acid coupling of 91%
yield.
Capping of Unreacted Piperidino Groups. The aminoacyl resin
was washed with DMF (5 × 30 s) and treated with Ac2O (1.9 mL,
20.03 mmol) and DIEA (3.5 mL, 20.09 mmol) in DMF (4 mL) for 30
min. Then, the mixture was washed with DMF (5 × 30 s), DCM (5 ×
30 s), MeOH (5 × 30 s), and DCM (5 × 30 s).
General Procedure B: Amino Acid Coupling. To the resin were
added the amino acid (3 equiv), HOBt (3 equiv), and DIC (3 equiv)
in DMF (4 mL) and the mixture reacted for 1 h at rt with occasional
manual stirring. Then, the resin was washed with DMF (5 × 30 s),
DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 × 30 s).
General Procedure C: Removal of the Fmoc Group. The resin
was treated with piperidine (20% v/v) in DMF (8 mL) (1 × 1 min and
2 × 10 min) or DBU (3% v/v) in DMF (1 × 1 min and 2 × 10 min)
in the case of the N-terminal Fmoc group, and then it was washed with
DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 ×
30 s).
EXPERIMENTAL SECTION
■
Materials and Methods. Solid-phase peptide synthesis was
carried out in polypropylene syringes fitted with porous polystyrene
frits. Reactions were performed at rt using occasional manual stirring,
and the resins were washed, unless stated otherwise, with DMF (5 ×
30 s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 × 30 s).
Couplings were monitored until completeness using the Kaiser test for
primary amines27 or the chloranil test for proline.28 Solvents and
excess of reagents were removed by filtration under reduced pressure.
On-resin quantifications were carried out by hydrolysis and amino acid
analysis as follows: the peptidyl resin samples (7.5−9.5 mg) were
washed with DMF (5 × 30 s), DCM (5 × 30 s), and MeOH (5 × 30
s) and then dried under reduced pressure. Hydrolyses were performed
using 300 μL of aqueous HCl (37% v/v) and 300 μL of propionic acid.
The acidolytic crudes were dissolved in aqueous HCl (20 mM) to
achieve a suitable concentration for amino acid analysis. Flash
chromatography was carried out in an automated flash system.
Semipreparative HPLC was carried out with a Sunfire C18, 19 × 100
mm column. XBridge C18, 4.6 × 50 mm and XBridge BEH 130 C18
columns were used for analytical HPLC−MS. NMR spectra were
collected in CDCl3 at 25 °C. Mass spectra were acquired with
quadrupole detection and an electrospray ion source in positive-ion
mode.
(S)-Fmoc-Phe(pNO2)-OAllyl. (S)-Fmoc-Phe(pNO2)-OH (3.07 g,
7.63 mmol) was dissolved in DMF (60 mL), and NaHCO3 (2.69 g,
32.02 mmol) and allyl bromide (1.5 mL, 17.20 mmol) were added.
The mixture was stirred at rt for 16 h, and then the solvent was
removed. The pale yellow solid obtained was dissolved in EtOAc (60
mL) and was washed with H2O (3 × 60 mL). The organic phase was
dried, and the solvent was removed under vacuum, furnishing the
product as a white solid that was used in the next step without further
chromatography purification (3.52 g, 98%): mp = 143−146 °C; Rf
0.47 [tBuOMe/hexanes (1:1)]; [α]20 = +15.6 (c 1, CHCl3,); IR
D
1
(ATR) 3329, 1749, 1687, 1516, 1338, 1263, 1213 cm−1; H NMR
(400 MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.6 Hz,
2H), 7.56 (m, 2H), 7.41 (m, 2H), 7.30 (m, 2H), 7.22 (d, J = 8.3 Hz,
2H), 5.86 (m, 1H), 5.31 (m, 3H), 4.70 (m, 1H), 4.62 (d, J = 5.7 Hz,
2H), 4.51 (dd, J1 = 10.9 Hz, J2 = 6.9 Hz, 1H), 4.40 (dd, J1 = 10.7 Hz, J2
= 6.4 Hz, 1H), 4.19 (t, J = 6.4 Hz, 1H), 3.27 (dd, J1 = 13.9 Hz, J2 = 5.8
Hz, 1H), 3.16 (dd, J1 = 13.8 Hz, J2 = 5.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 170.5, 155.4, 147.2, 143.6, 143.5, 141.4, 131.0, 130.3,
127.8, 127.1, 124.9, 123.7, 120.0, 119.7, 66.8, 66.5, 54.4, 47.2, 38.1;
HRMS calcd for C27H24N2O6Na [M + Na]+ 495.1526, found
495.1527.
(S)-Fmoc-Phe(pNH2)-OAllyl (1). (S)-Fmoc-Phe(pNO2)-OAllyl
(1.91 g, 4.04 mmol) and Zn dust (1.29 g, 19.73 mmol) were
suspended in absolute EtOH (50 mL). Glacial AcOH (50 mL) was
added to the mixture, and the resulting suspension was stirred at 60 °C
for 1 h. Solvent was evaporated, and the product was purified by flash
chomatography using DCM/EtOAc (9:1) as eluent, affording the
product as a white solid (1.24 g, 69%): mp = 124−127 °C; Rf 0.41
[DCM/EtOAc (9:1)]; [α]20 = +15.2 (c 1, CHCl3); IR (ATR) 3389,
2931, 1740, 1692, 1260 cm−1D; 1H NMR (400 MHz, CDCl3) δ 7.76 (d,
J = 7.7 Hz, 2H), 7.56 (t, J = 6.4 Hz, 2H), 7.39 (m, 2H), 7.30 (tt, J1 =
4.4 Hz, J2 = 3.7 Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 6.59 (d, J = 8.3 Hz,
General Procedure D: Removal of Allyl Group. The resin was
washed with DMF (5 × 30 s) and DCM (5 × 30 s). Then the resin
was suspended in DCM and degassed by bubbling Ar for 5 min, when
Pd(PPh3)4 (0.4 equiv) and PhSiH3 (48 equiv) in DCM (8 mL) were
added. The mixture was shaken for 30 min at rt, filtered, and washed
with DCM (8 × 30 s). This treatment was carried out twice under the
same conditions. After filtration, the resin was washed with DCM (8 ×
30 s), a solution of sodium diethyl dithiocarbamate (5% v/v) in DMF
(2 × 5 min), DMF (5 × 1 min), and DCM (5 × 30 s).
General Procedure E: Cleavage of the Peptide from the
Resin and Diazonium Salt Reduction. Cleavage of the peptide
from the resin was brought about by treatment with TFA in DCM (5/
95 v/v) (3 × 2 min) (peptides 2−6) and (2/98 v/v) (5 × 2 min)
(peptides 7 and 8), and the collected washings were evaporated under
vacuum to dryness (the diazonium salt was protected from light).
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Note
Then the diazonium salt was dissolved in DMF (15 mL), and
FeSO4·7H2O (1.5 equiv) was added. The mixture was stirred at rt for
5 min, and the solvent was removed under vacuum. The presence of
iron impurities after chromatographic purifications was evaluated as
follows: a drop of a solution of peptide samples in a mixture of
aqueous HCl (5% v/v) (4 mL) and CH3CN (4 mL) was mixed with a
drop of saturated aqueous ammonium thiocyanate. Samples that
showed a red color after 15 min were treated with Amberlite IRC748
until a colorless test was obtained.
semipreparative reversed-phase HPLC (5% → 20% B in 1 min and
20% → 33% B in 5 min with a flow rate of 16 mL/min over 7 min and
214 nm UV detection, tR = 4.35 min) to be obtained, yielding 26.9 mg
of 4 (global yield of 32%). ESI-HRMS: calcd for C34H51N8O7S [M +
H]+ 715.3601, found, 715.3587. HPLC analysis was performed with
220 nm UV detection on a C18 analytical column (4.6 × 100 mm)
eluting with a gradient of 5% → 100% B over 11 min with a flow rate
of 2 mL/min. tR = 4.45 min.
Schizandrochin B (5). From 270 mg (0.170 mmol) of aminoacyl
resin prepared according to procedure A. After removal of the allyl
group (general procedure D), H-Pro-OMe·HCl (0.07 g, 0.54 mmol),
PyAOP (0.26 g, 0.50 mmol), HOAt (0.07 g, 0.51 mmol), and DIEA
(265 μL, 1.52 mmol) in DMF (8 mL) were added. The mixture was
left for 1 h at rt when the Fmoc group was removed following the
general procedure C (four times). Then, the mixture was washed with
DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 ×
30 s). Peptide cleavage from the resin (>99% yield) and diazonium salt
reduction (general procedure E) afforded the title peptide (79% of
purity by HPLC) that was purified by semipreparative reversed-phase
HPLC (5% → 20% B in 1 min and 20% → 33% B in 5 min with a flow
rate of 16 mL/min and 214 nm UV detection, tR = 4.62 min), yielding
17.1 mg of 5 (global yield of 67%). ESI-HRMS calcd for C14H17N2O2
[M + H]+ 245.1290, found 245.1287. HPLC analysis was performed
with 220 nm UV detection on a C18 analytical column (4.6 × 100
mm) eluting with a gradient of 5% → 100% B over 11 min with a flow
rate of 2 mL/min. tR = 4.45 min.
Unguisin A (6). From 320 mg (0.202 mmol) of aminoacyl resin
prepared according to procedure A. After removal of the Fmoc group
(general procedure C), the peptide chain was assembled following the
general procedure B except for removal of the Fmoc group after
anchoring GABA and further amino acids, where a mixture of DBU
(10% v/v) and piperidine (10% v/v) in DMF (1 × 1 min and 2 × 10
min) was used. Then the C-terminal allyl group and N-terminal Fmoc
group were removed following the general procedure D and using
DBU (3% v/v) in DMF, respectively. Cyclization was carried out with
two treatments of PyAOP (0.32 g, 0.61 mmol), HOAt (0.08 g, 0.59
mmol), and DIEA (212 μL, 1.22 mmol) in DMF (8 mL) for 1 h at rt
with occasional manual stirring. Then, the resin was washed with DMF
(5 × 30 s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 × 30 s).
Peptide cleavage from the resin (76% yield) and diazonium salt
reduction (general procedure E) gave the desired product (84.2% of
purity by HPLC) that was purified by semipreparative reversed-phase
HPLC (5% → 35% B in 1 min and 35% → 48% B in 5 min with a flow
rate of 16 mL/min and 214 nm UV detection, tR = 4.90 min), yielding
28.7 mg of 6 (global yield of 30%). ESI-HRMS calcd for C40H55N8O7
[M + H]+, 759,4194; found, 759,4180. HPLC analysis was performed
with 220 nm UV detection on a C18 analytical column (4.6 × 100
mm) eluting with a gradient of 5% → 100% B over 13 min with a flow
rate of 2 mL/min. tR = 6.23 min.
Protected Hemorphine-7 Derivatives (7 and 8). From 800 mg
(0.504 mmol) of aminoacyl resin prepared according to procedure A.
After removal of Fmoc group of phenylalanine (general procedure C),
the peptide chain was assembled following the general procedure B
obtaining the desired peptidyl resin fully protected (peptidyl resin A).
Protected Hemorphine-7 Derivative 7. Peptidyl resin A (250 mg,
0.158 mmol) was used in the synthesis. The C-terminal allyl group was
removed following the general procedure D, and the resin was washed
with DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM
(5 × 30 s). Peptide cleavage from the resin (98% yield) and diazonium
salt reduction (general procedure E) gave the desired product (93.4%
of purity by HPLC) that was purified by semipreparative reversed-
phase HPLC (5% → 95% B in 1 min, 95% → 100% B in 5 min and
100% B in 5 min with a flow rate of 16 mL/min and 214 nm UV
detection, tR = 6.60 min), yielding 106.4 mg of 7 (global yield of
34.5%). ESI-HRMS: calcd for C109H129N12O18S [M + H]+ 1925,9269,
found 1925.9248. HPLC analysis was performed with 220 nm UV
detection on a C18 analytical column (4.6 × 100 mm) eluting with a
gradient of 5% → 100% B over 13 min with a flow rate of 2 mL/min.
tR = 10.57 min.
Endomorphin-2 (2). From 308 mg (0.194 mmol) of aminoacyl
resin prepared according to procedure A. Peptide chain was elongated
until the N-terminal end following general procedures B and C. The C-
terminal allyl group was then removed following general procedure D,
and H-Phe-NH2 (0.10 g, 0.61 mmol), DIC (91 μL, 0.59 mmol),
oxyme (0.08, 0.58 mmol) in DMF (8 mL) were added. The mixture
was reacted for 1 h at rt with occasional manual stirring (this
procedure was repeated twice to ensure the complete coupling), and
the N-terminal Fmoc group was removed according to general
procedure C. Subsequent peptide cleavage from the resin (98% yield)
and diazonium salt reduction (general procedure E) afforded a crude
material (73.1% of purity by HPLC) that was treated with the mixture
TFA/TIS/H2O (95/2.5/2.5 v/v) for 3 h at rt. Volatiles were removed
under vacuum, the product was precipitated with Et2O, and the solid
was centrifuged (10 min at 3500 rpm) and filtered. Endomorphin-2
was purified by semipreparative reversed-phase HPLC (5% → 15% B
in 1 min and 15% → 23% in 5 min with a flow rate of 16 mL/min and
214 nm UV detection, tR = 4.90 min), yielding 26.1 mg of 2 (global
yield of 38%). ESI-HRMS calcd for C32H38N5O5 [M + H]+, 572,2873;
found 572,2861. HPLC analysis was performed with 220 nm UV
detection on a C18 analytical column (4.6 × 100 mm) eluting with a
gradient of 5% → 100% B over 11 min with a flow rate of 2 mL/min.
tR = 4.65 min.
Ac-AAPF-pNA (3). From 310 mg (0.195 mmol) of aminoacyl resin
prepared according to procedure A. The general procedure D was
followed to remove the allyl group and the resin was treated five times
with p-nitroaniline (0.3 g, 2.17 mmol), PyAOP (0.3 g, 0.58 mmol),
HOBt (0.08 g, 0.59 mmol), and DIEA (200 μL, 1.15 mmol) in DMF
(8 mL) for 1 h at rt. Then, the mixture was washed with DMF (5 × 30
s), DCM (5 × 30 s), MeOH (5 × 30 s), and DCM (5 × 30 s). The
peptide chain was further assembled (general procedure B), the N-
terminal Fmoc group was removed (general procedure C), and finally,
acetylation was performed (general procedure A). Peptide cleavage
from the resin (96% yield) and diazonium salt reduction (general
procedure E) afforded a peptide crude (86.4% of purity by HPLC)
that was purified by semipreparative reversed-phase HPLC (5% →
35% B in 1 min and 35% → 53% in 5 min with a flow rate of 16 mL/
min and 214 nm UV detection, tR = 4.92 min), yielding 12.8 mg of 3
(global yield of 19%). ESI-HRMS calcd for C28H35N6O7 [M + H]+,
567,2567; found, 567,2586. HPLC analysis was performed with 220
nm UV detection on a C18 analytical column (4.6 × 100 mm) eluting
with a gradient of 5% → 100% B over 11 min with a flow rate of 2
mL/min. tR = 6.35 min.
Biotynilated Ac-AAPF (4). From 301 mg (0.190 mmol) of
aminoacyl resin prepared according to procedure A. The peptide
chain was grown according to general procedures B and C when
acetylation was carried out as described in general procedure A, and
the C-terminal allyl group was removed following the general
procedure D. Then, mono-Fmoc-ethylene diamine hydrochloride
(0.30 g, 0.95 mmol), DIC (90 μL, 0.58 mmol), oxyme (0.08 g, 0.57
mmol), and DIEA (165 μL, 0.95 mmol) in DMF/DCM (1/1 v/v) (8
mL) were added. The mixture was left for 1 h with occasional manual
stirring. The N-terminal Fmoc group was then removed using the
general procedure C, and biotin (0.14 g, 0.57 mmol) was added
together with PyAOP (0.3 g, 0.58 mmol), HOAt (0.08 g, 0.59 mmol),
and DIEA (200 μL, 1.15 mmol) in DMF/DCM (1/1 v/v) (8 mL).
The mixture was left for 1 h with occasional manual stirring, and the
resin was washed with DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 ×
30 s), and DCM (5 × 30 s). Peptide cleavage from the resin (97%
yield) and diazonium salt reduction (general procedure E) allowed the
desired product (93.4% of purity by HPLC) that was purified by
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The Journal of Organic Chemistry
Note
Protected Hemorphine-7 Derivative 8. Peptidyl resin A (250 mg,
0.158 mmol) was used in the synthesis. After the peptidyl resin was
washed with DMF (5 × 30 s), N-terminal Fmoc was removed by
treatment with DBU (3% v/v) in DMF (1 × 1 min and 2 × 10 min)
and then the resin washed with DMF (5 × 30 s), DCM (5 × 30 s),
MeOH (5 × 30 s) ,and DCM (5 × 30 s). Peptide cleavage from the
resin (98% yield) and diazonium salt reduction (general procedure E)
gave the desired product (94% of purity by HPLC) that was purified
by semipreparative reversed-phase HPLC (5% → 10% B in 1 min and
10% → 25% B in 5 min with a flow rate of 16 mL/min and 214 nm
UV detection, tR = 4.30 min), yielding 88.1 mg of 8 (global yield of
32%). ESI-HRMS: calcd for C97H123N12O16S [M + H]+ 1743.8901,
found 1743.8860. HPLC analysis was performed with 220 nm UV
detection on a C18 analytical column (4.6 × 100 mm) eluting with a
gradient of 5% → 100% B over 13 min with a flow rate of 2 mL/min.
tR = 9.87 min.
Racemization Study. Synthesis of Model Peptides Ac-L-Phe-L-
Phe-NH2 and Ac-D-Phe-L-Phe-NH2 (9 and 10). Fmoc-Rink-amide
MBHA resin (0.73 mmol/g, 0.73 mmol) was introduced into a
polypropylene syringe fitted with a porous polystyrene frit and was
washed successively with DCM (10 × 30 s) and DMF (10 × 30 s).
Then, the Fmoc group was removed following general procedure C,
and Fmoc-L-Phe-OH (0.85 g, 2.19 mmol), DIC (0.34 mL, 2.20
mmol), and oxyme (0.28 g, 2.19 mmol) in DMF (15 mL) were added.
The mixture was reacted for 1 h at room temperature with occasional
manual stirring. After removal of the Fmoc group of phenylalanine
(general procedure C), the peptidyl resin was divided in two batches
(A and B). Then, Fmoc-L-Phe-OH (batch A) and Fmoc-D-Phe-OH
(batch B) were coupled using DIC (3 equiv) and oxyme (3 equiv) in
DMF. After 1 h at room temperature with occasional manual stirring,
the Fmoc group was removed following the general procedure C and
acetylation performed following general procedure A. Diastereoiso-
meric peptides were cleaved from the corresponding resins using the
acidolytic mixture TFA/TIS:DCM (95:2.5:2.5) for 2h. The crude
products were precipitated with hexanes and the remaining solids were
centrifuged (5 min at 3500 rpm) and filtered, yielding white solids that
were analyzed by HPLC: C18 analytical column (4.6 × 100 mm)
eluting at 220 nm with a gradient of 20% → 50% B over 4.5 min with a
flow rate of 2 mL/min; tR = 1.87 min (Ac-L-Phe-L-Phe-NH2, 9) and tR
= 2.23 min (Ac-D-Phe-L-Phe-NH2, 10).
Synthesis of Model Peptide Ac-L-Phe-L-Phe-NH2 (9) Using a
Triazene Linkage. Synthetic Route 1. From 142 mg (0.09 mmol) of
aminoacyl resin prepared according to general procedure A. After
removal of Fmoc group (general procedure C), acetylation was
performed following general procedure A. Then, C-terminal allyl
group was removed following the general procedure D, and the resin
was washed with DMF (5 × 30 s), DCM (5 × 30 s), MeOH (5 × 30
s) and DCM (5 × 30 s). Then H-Phe-NH2·HCl (0.05 g, 0.27 mmol),
DIC (40 μL, 0.26 mmol), and oxyme (0.03 g, 0.27 mmol) in DMF (8
mL) were added. The mixture was reacted for 1 h at rt with occasional
manual stirring (this procedure was repeated twice to ensure the
complete coupling). Finally, peptide cleavage from the resin and
diazonium salt reduction (general procedure E) afforded peptide crude
that was analyzed by HPLC.
Synthetic Route 2. From 153 mg (0.1 mmol) of aminoacyl resin
prepared according to general procedure A. The C-terminal allyl group
was removed following general procedure D, and H-Phe-NH2·HCl
(0.06 g,0.30 mmol), DIC (46 μL, 0.30 mmol), and oxyme (0.04 g, 0.32
mmol) in DMF (8 mL) were added. The mixture was reacted for 1 h
at rt with occasional manual stirring (this procedure was repeated
twice to ensure the complete coupling), and the N-terminal Fmoc
group was removed according to general procedure C. Finally,
acetylation was performed following the general procedure D. Peptide
cleavage from the resin and diazonium salt reduction (general
procedure E) afforded peptide crude that was analyzed by HPLC.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of phenylalanine derivatives, HPLC-
MS data of crude peptides, HPLC and HRMS of pure peptides.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: enicolas@ub.edu, mroyo@pcb.ub.es.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work has been partially financed by Ministerio de Ciencia
́
e Innovacion (CTQ2006-12460 and CTQ2008-00177.
REFERENCES
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