One-pot isomerization-cross metathesis-reduction (ICMR) synthesis of lipophilic tetrapeptides

Article abstract of DOI:10.1021/co500020a

An efficient, versatile and rapid method toward homologue series of lipophilic tetrapeptide derivatives (herein, the opioid peptides H-TIPP-OH and H-DIPP-OH) is reported. High atom economy and a minimal number of synthetic steps resulted from a one-pot ta

Full text of DOI:10.1021/co500020a

Research Article
pubs.acs.org/acscombsci
One-Pot Isomerization−Cross Metathesis−Reduction (ICMR)
Synthesis of Lipophilic Tetrapeptides
Mouhamad Jida,*,† Cecilia Betti,† Peter W. Schiller,‡ Dirk Tourwe,† and Steven Ballet*,†
́
†Department of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
‡Department of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Avenue Des Pins Ouest,
Montreal, Quebec H2W 1R7, Canada
S
* Supporting Information
ABSTRACT: An efficient, versatile and rapid method toward
homologue series of lipophilic tetrapeptide derivatives (herein,
the opioid peptides H-TIPP-OH and H-DIPP-OH) is
reported. High atom economy and a minimal number of
synthetic steps resulted from a one-pot tandem isomerization-
cross metathesis-reduction sequence (ICMR), applicable both
in solution and solid phase methodology. The broadly
applicable synthesis proceeds with short reaction times and
simple work-up, as illustrated in this work for alkylated opioid
tetrapeptides.
KEYWORDS: alkylated tetrapeptides, one-pot tandem reactions, isomerization-cross metathesis-reduction process (ICMR),
Grubbs’ second generation and Umicore M2 catalysts, solution and solid phase peptide synthesis
one-pot procedure following a cross metathesis−olefin
reduction reaction on solid support or in solution.
INTRODUCTION
■
In the last two decades, our laboratory has been particularly
interested in the synthesis of novel potent, and receptor
subtype selective opioid peptides.1−3 The prototype peptide H-
Tyr-Tic-Phe-Phe-OH (TIPP) displayed high δ receptor
selectivity and antagonist activity (Figure 1).2 Interestingly,
the corresponding C-terminal amide TIPP-NH2 as well as the
2′,6′-dimethyl-Tyr1 (Dmt1) bearing analogue, DIPP-NH2 (H-
Dmt-Tic-Phe-Phe-NH2, Figure 1), displayed a promising mixed
μ-agonist/δ-antagonist profile, a pharmacological profile that, in
contrast to the classical opioids, may lead to opioids with a
diminished propensity to induce tolerance and physical
dependence during a long-term treatment of pain by chronic
administration.2b
As chain extension, with concomitant increased lipophilicity,
represents one of the standard modifications available to
medicinal chemists in search for an improved therapeutic
profile of lead structures, our group wanted to verify the effect
of (extended) alkyl side chains on the μ-agonist properties/δ-
antagonist profiles of H-TIPP-OH/NH2 and H-DIPP-OH/
NH2. As such, a large set of lipophilic tetrapeptide derivatives,
based on the TIPP and DIPP sequences, containing an alkyl
substituent on the N-, C-terminus, and side chain positions
were synthesized. These peptide derivatives might diminish the
intrinsic drawbacks of peptides as therapeutic agents and
enhance gut-blood or blood-brain barrier (BBB) permeability.
The latter property is the most cumbersome hurdle left for the
TIPP or DIPP family of ligands.1f
Ruthenium-based olefin metathesis catalysts have provided
routes toward new olefins that appear in a variety of valuable
structures for pharmaceutical and material science.4,5,4d,5b
From the numerous catalysts that are available for metathesis
reactions, catalysts 1 (Umicore M2 s generation) and 2
(Grubbs’ second generation), depicted in Figure 2, were
selected in this study. The Umicore M2 metathesis catalyst
yields high E/Z selectivities, it is more resistant to harsh
reaction conditions, as compared to the Grubbs II catalyst,
shows increased activity upon temperature increase, and
excellent stability to air and moisture.6 However, when the
catalysts are stressed by high temperatures, high dilution, and
forced high turnovers, olefin isomerization has been reported in
a few papers (vide infra).5a,15 This isomerization could be
turned to our advantage in the current study. As a consequence
and driven by our interest in the synthesis of more lipophilic
tetrapeptides bearing alkyl chains of variable length, we
discovered a facile and rapid synthesis of novel TIPP- and
DIPP-alkyl derivatives, in which the alkyl chain length varies,
via a one-pot tandem isomerization-cross-metathesis-reduction
sequence. A large set of N-terminus, C-terminus, and side-
chain-derivatized lipopeptides was prepared in good overall
yields and high purity using both a solution and solid phase
parallel synthesis approach.
Received: February 7, 2014
Revised: April 3, 2014
Published: June 6, 2014
To access the desired lipophilic tetrapeptides (TIPP and
DIPP derivatives) in an efficient manner, we aimed to use a
© 2014 American Chemical Society
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Research Article
Figure 1. General structures of the tetrapeptides TIPP-OH and DIPP-NH2 (μ-agonist/δ-antagonist opioid analgesics).
Figure 2. Cross-metathesis: Alkylidene swap between two acyclic olefins.
Figure 3. Structures of modified TIPP and DIPP tetrapeptides as precursors for the tandem ICMR reactions (isomerization−cross metathesis−
reduction).
couplings take place with high yields (See Supporting
Information for more detail).
RESULT AND DISCUSSION
■
To achieve the synthesis of all desired peptide compounds, the
tandem ICMR (isomerization/cross-metathesis/reduction re-
actions) was attempted in solution or on solid phase. First,
precursors 3 and 4 (Figure 3) were prepared stepwise by
solution phase peptide synthesis as outlined in Scheme 1 by use
of propylphosphonic anhydride (T3P). This reagent, com-
monly used as a coupling agent, is a highly reactive water
scavenger that offers several advantages over traditional
reagents, such as low toxicity, commercial availability, a low
price, low epimerization tendency and broad functional group
tolerance. Moreover, it gives way to excellent purity and easy
work up procedures thanks to the formation of water-soluble
byproducts.7 As can be noticed in Scheme 1, all peptide
Next, precursors 5, 6, and 7 (Figure 3) were synthesized on
solid-phase. In general, solid phase organic synthesis (SPOS) is
a rapidly expanding field which is being widely exploited in
search for new peptide derivatization techniques to be used in
medicinal chemistry and compatible with combinatorial
technology.8,9 This has permitted an extraordinary increase in
the speed of synthesis through both work-up simplification and
automation. Although solid-phase peptide synthesis (SPPS) is a
well-established procedure, microwave-assisted SPPS has
received attention since microwave irradiation can accelerate
reaction rates, allow cumbersome couplings (e.g., NMe-amino
acid couplings), and improve the purities and yields in SPPS.10
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Scheme 1. Solution-Phase Synthesis of the CM Precursors H-Tyr-Tic-Phe-Phe-OAll (3) and 3-Butenoyl-Tyr-Tic-Phe-Phe-OH
(4)
Hence, microwave irradiation was also used for the solid phase
synthesis of the peptides in this work.
to give finally the corresponding tetrapeptide 4. This
tetrapeptide 4 was again purified by preparative HPLC.
Solution-Phase Synthesis of Tetrapeptide 3 (Scheme
1). Boc-Phe-OH was condensed with HCl.H-Phe-OBn in the
presence of DIPEA and T3P as a coupling reagent. After N-
terminal Boc-deprotection with in a TFA/CH2Cl2 mixture,
TFA-H-Phe-Phe-OBn was condensed with Boc-Tic-OH via a
DIPEA/T3P coupling procedure (see Supporting Information).
Subsequently, Boc-deprotection allowed the condensation of
Boc-Tyr(tBu)-OH with H-Tic-Phe-Phe-OBn via the same
DIPEA/T3P-mediated coupling procedure. After C-terminal
benzyl hydrolysis with LiOH (THF/H2O), allyl bromide was
stirred with Boc-Tyr(tBu)-Tic-Phe-Phe-OH to present the final
tetrapeptide 3, containing the C-terminal olefin (Boc-Tyr(tBu)-
Tic-Phe-Phe-OAll). This compound was purified by preparative
HPLC.
Microwave-Assisted Solid-Phase Synthesis of Tetra-
peptides 5 and 6 Linked to Wang Resin. The short chain
tetrapeptides 5 and 7 were synthesized manually by Nα-Fmoc
methodology on Wang resin, preloaded with a Phe residue,
under microwave irradiation (Biotage SP Wave, temperature
was set at 75 °C). A 3-fold excess of the building blocks fmoc-
protected amino acid (Fmoc-Xaa−OH), O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and
6-fold excess N,N-diisopropylethylamine (DIEA) in DMF was
added to the swollen resin. Under microwave irradiation, the
mixture was shaken for 5 min with Fmoc-Phe-OH, 5 min with
Fmoc-Tic-OH, and 5 min with Boc-Tyr(tBu)-OH or Boc-
Tyr(All)-OH. The Fmoc protecting group was removed with
20% 4-methyl-piperidine in DMF for 20 min at room
temperature without microwave irradiation. Next, the resin
was filtered and respectively washed by vortexing for 1 min with
DMF, MeOH, and CH2Cl2 to afford the tetrapeptides 5 and 7
linked to Wang resin. For structure confirmation, a micro-
cleavage of the peptide from the resin followed by LC-MS
Solution-Phase Synthesis of Tetrapeptide 4 (Scheme
1). Using the common precursor H-Tic-Phe-Phe-OBn as the
amine coupling partner, Boc-Tyr(Bn)-OH was linked to the N-
terminus of this peptide. After N-terminal Boc deprotection, 3-
butenoic acid was condensed with H-Tyr(Bn)Tic-Phe-Phe-OH
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Table 1. One-pot solution-Phase Synthesis of C-terminal Modified TIPP Via the Tandem Isomerization/Cross-Metathesis/
a
Reduction/Deprotection Sequences
HRMS [M + H]+
b
c
d
n
peptide (3a−h)
rt after HPLC purification (min)
yield after HPLC purification (%)
clogP
calcd
found
1
H-Tyr-Tic-Phe-Phe-O-propyl (3a)
H-Tyr-Tic-Phe-Phe-O-butyl (3b)
H-Tyr-Tic-Phe-Phe-O-pentyl (3c)
H-Tyr-Tic-Phe-Phe-O-hexyl (3d)
H-Tyr-Tic-Phe-Phe-O-heptyl (3e)
H-Tyr-Tic-Phe-Phe-O-octyl (3f)
H-Tyr-Tic-Phe-Phe-O-nonyl (3g)
H-Tyr-Tic-Phe-Phe-O-decyl (3h)
15.62
16.18
16.86
17.52
17.78
18.89
19.84
21.23
13
13
5.162
5.691
6.220
6.749
7.278
7.807
8.336
8.865
677.3334
691.3940
705.3647
719.3803
733.3959
747.4116
761.4272
775.3
677.3311
691.3502
705.3643
719.3604
733.3970
747.4130
761.4272
775.4
2
3
4
5
6
9
11
12
7
7
3
e
8
traces
a
Reaction conditions: (1) 3 (0.1 mmol), 1-hexene (0.4 mmol), Umicore M2 (0.01 mmol); CH2CI2 (3 mL), 24 h, 50 °C; (2) Umicore M2 (0.01
b
mmol), Et3SiH (1 mmol), 48 h, 50 °C; (3) TFA/CH2CI2:50/50, 2 h, room temperature. Retention time was determined by HPLC (standard
gradient) with UV detection at 215 nm. Yields refer to isolated peptides. Estimated by ChemBioOffice 2010. The peptide was not isolated and the
c
d
e
rt refer to the HPLC and the mass refer to the LCMS of the crude mixture.
analysis, confirmed that the desired supported peptides 5 and 7
were obtained with excellent purity.
the peptide was accomplished by treatment with TFA/CH2Cl2
50:50 for 2 h. The peptides were isolated by filtration and
purified by preparative RP-HPLC on a Supelco Discovery BIO
wide pore preparative C18 column in good overall yield.
Surprisingly, this one-pot tandem reaction resulted in the
formation of eight peptides 3a−h (Table 1). To our delight, the
compounds could be separated to high purity in a 30 to 100%
CH3CN in water gradient. The structure of the pure
compounds was confirmed by high-resolution electrospray
ionization (ESP) mass spectrometry. Although moderate yields
are logically obtained for each compound, the sum of the
isolated compounds represents 70%. (See Supporting In-
formation for more details and HPLC chromatogram
examples.) All results are summarized in Table 1.
Indeed, the isomerization/migration of 1-hexene is catalyzed
by Umicore M2 complexes and leads to the thermodynamically
more stable products: 2-hexene and 3-hexene, compounds
which are of significant industrial interest.11 The fast isomer-
ization of 1-hexene to 2-hexene or 3-hexene is in accordance
with previous observations described in literature.11 In 1975,
Porri et al. reported the first observation of a double bond
isomerization/cross-metathesis process, after the observation of
olefin mixture formation, with mixed iridium/silver systems,
rather than the expected polyolefins by olefin polymerization.12
Thereafter, Grubbs et al. demonstrated that the conversion of
1-octadecene into an olefin mixture with a broad chain length
distribution can be achieved by using the same catalyst
system.13 Such isomerization reactions offer the possibility of
forming multiple products in a single step.14 More recently,
Consorti and Dupont reported the formation of a mixture of
olefins with up to 17 methylene units via the isomerizing
metathesis of (E)-3-hexene with a combination of 0.5 mol %
modified Hoveyda−Grubbs metathesis catalyst and 1 mol %
ruthenium hydride isomerization catalyst in an ionic liquid.15
Many Ru-hydrides are known to effect alkene isomerization in
Solid-Phase Synthesis of Tetrapeptide 7 Linked to
Wang Resin. The linear solid supported tetrapeptide 7 was
synthesized manually by Nα-Fmoc methodology on Wang resin,
preloaded with a Phe residue, using O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)/N-
methylmorpholine (NMM) as the coupling mixtures. A 3-fold
excess of the building blocks (Fmoc-Phe-OH, Fmoc-Tic-OH,
and Boc-Dmt(All)-OH] and activating agents were applied in
the presence of 3 mL of NMM (0.4 M in DMF). Fmoc
deprotections were carried out by treating the resin with 20% 4-
methyl-piperidine in DMF. After completion of the coupling,
the resin was filtered and respectively washed with DMF,
MeOH, and CH2Cl2 to give the tetrapeptide 7 linked to Wang
resin. A microcleavage and subsequent LC-MS analysis
confirmed the presence of pure solid-supported peptide
precursor.
Synthesis of Alkylated Tetrapeptides through Isomer-
ization-Cross-Metathesis Reduction (ICMR). Having ole-
fin-bearing tetrapeptide building blocks 3 to 7 in hand, the next
step consisted of studying the tandem cross-metathesis-
reduction reactions, to obtain the desired set of alkylated
tetrapeptides. Toward this end, it was decided to first
investigate the cross metathesis (CM) approach on the C-
terminal allyl ester 3 as the peptide substrate with 1-hexene in
the presence of Umicore M2 catalyst 1, followed by reduction
of double bonds by use of Et3SiH in the presence of Umicore
M2 via one-pot process (Table 1). The reaction was performed
in a dried and sealed vial containing peptide in dry CH2Cl2, 1-
hexene (4 equiv) and Umicore M2 catalyst 1 (0.1 equiv). The
solution was heated at 50 °C in a sealed vial for 24 h. Next,
additional Umicore M2 catalyst (0.1 equiv) and added Et3SiH
(10 equiv) resulted in quantitative reduction of the olefin at 50
°C in CH2Cl2 for 48 h. Final Boc and t-butyl deprotection of
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Figure 4. Proposed mechanism for isomerizing self-metathesis between the olefin-bearing peptide and the isomers of 1-hexene.
Table 2. One-Pot Solution-Phase Synthesis of N-Terminal-Modified TIPP via the Isomerization/Cross-Metathesis/Reduction
a
Sequences
HRMS [M + H]+
b
c
d
n
peptide (4a−h)
rt after HPLC purification (min) yield after HPLC purification (%) clogP
calcd
795.3
found
795.4
e
1
propyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4a)
butyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4b)
pentyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4c)
hexyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4d)
heptyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4e)
octyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4f)
nonyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4g)
decyl-CO-Tyr(Bn)-Tic-Phe-Phe-OH (4h)
19.97
20.45
21.00
21.58
22.12
22.61
23.22
23.57
traces
17
10
12
9
7.357
7.886
2
3
4
5
6
7
8
809.3909
845.3883
837.4241
851.4379
887.4330
901.4492
893.4896
809.3922
845.3885
837.4222
851.4378
877.4354
901.4510
893.4848
8.415
8.944
9.473
9
10.002
10.531
11.060
4
3
a
Reaction conditions: (1) 4 (0.1 mmol), 1-hexene (0.4 mmol), Umicore M2 (0.01 mmol); CH2CI2 (3 mL), 24 h, 50 °C; (2) Umicore M2 (0.01
b
mmol), Et3SiH (1 mmol), 48 h, 50 °C; (3) Pd/C/H2, MeOH, atmospheric pressure, 2 h, room temperature. Retention time was determined by
HPLC (standard gradient) with UV detection at 215 nm. Yields refer to isolated pure peptides. Estimated by ChemBioOffice 2010. The peptide
c
d
e
was not isolated and the rt refer to the HPLC and the mass refer to the LCMS of the crude mixture.
general, as demonstrated for the second generation Grubbs
catalyst 2.16 The double bond migration is thought to be the
result of the presence of metal-hydride intermediates formed in
solution under metathesis condition.17
metathesis, occurs. The fact that no chains longer than in 3g′
were observed, indicates that such an isomerization−metathesis
pathway is not occurring to an appreciable extent.
Additionally, we now report a new method for in situ olefin
reduction under mild “hydrogen-free” (i.e., no added H2)
conditions, using Et3SiH and the ruthenium-based olefin
metathesis catalysts, through a one-pot process ICMR. More
recently, an efficient solution and SPOS reduction of α,β-
unsaturated alkenes was published by Mata and co-workers
employing Grubbs’ II catalyst in a nonmetathetic role and
Et3SiH under microwave irradiation conditions.19 However, our
procedure is interesting as it shows that microwave heating is
not always necessary to reach high conversions and excellent
yields. Moreover, in this study the methodology is applied on
peptide sequences that are of higher molecular complexity
when compared to previous substrates.
The CM reactions are more complex due to competition
between the desired CM reaction, undesired self-metathesis
(which leads to homodimers), and CM consuming the isomers
formed in the solution. According to the literature,18 by-
products formed by alkene-isomerization (AI) and subsequent
self-metathesis can be formed in situ. In our case, isomerization
and self-metathesis products of 1-hexene underwent CM with
olefin-derivatized peptide 3 to afford the unexpected peptides
3a′−g′ (prime numbering corresponds to unsaturated and
intermediate peptide analogues). The chain lengths of alkene
isomerization/CM products observed by LC-MS were generally
1, 2, 3, or 4 CH2 units longer or shorter, as compared to the
nonisomerized olefin CM product 3d′ (Figure 4). It cannot be
excluded that under the reactions conditions, also isomerization
of the alkenes in 3a′−g′, with potentially further cross
To expand the reaction scope and to study the feasibility of
the one pot isomerization self-metathesis/cross-metathesis/
reduction sequence in solution (Table 1), we decided to repeat
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Table 3. One-Pot Solid-Phase Synthesis of N-Terminal-Modified TIPP Via the Tandem Isomerization/Cross-Metathesis/
a
Reduction/Cleavage Reactions
HRMS [M + H]+
b
c
d
n
peptide (5a−h)
rt after HPLC purification (min) yield after HPLC purification (%) clogP
calcd
found
1
propyl-CO-Tyr-Tic-Phe-Phe-OH (5a)
butyl-CO-Tyr-Tic-Phe-Phe-OH (5b)
pentyl-CO-Tyr-Tic-Phe-Phe-OH (5c)
hexyl-CO-Tyr-Tic-Phe-Phe-OH (5d)
heptyl-CO-Tyr-Tic-Phe-Phe-OH (5e)
octyl-CO-Tyr-Tic-Phe-Phe-OH (5f)
Nonyl-CO-Tyr-Tic-Phe-Phe-OH (5g)
Decyl-CO-Tyr-Tic-Phe-Phe-OH (5h)
16.10
17.09
17.39
18.08
19.10
18.87
20.66
21.21
20
16
5.003
705.3282
719.3439
733.3596
747.3752
761.3909
775.4066
789.4257
803.4
705.3298
719.3406
733.3608
747.3763
761.3925
775.4070
789.4222
803.3
2
3
4
5
6
5.532
6.061
6.590
7.119
7.648
8.177
8.706
12
11
8
5
7
3
e
8
traces
a
Reaction conditions: (1) 5 (0.1 mmol), 1-hexene (0.4 mmol), Umicore M2 (0.01 mmol); CH2CI2 (3 mL), 24 h, 50 °C; (b) Umicore M2 (0.01
b
mmol), Et3SiH (1 mmol), 48 h, 50 °C; (c) TFA/CH2CI2 = 50/50, 2 h, room temperature. Retention time was determined by HPLC (standard
gradient) with UV detection at 215 nm. Yields refer to isolated pure peptides. Estimated by ChemBioOffice 2010. The peptide was not isolated
c
d
e
and the rt refer to the HPLC and the mass refer to the LCMS of the crude mixture.
Table 4. One-Pot Solid-Phase Synthesis of Side-Chain-Modified TIPP Via the Tandem Isomerization/Cross-Metathesis/
a
Reduction/Cleavage Reactions
HRMS [M + H]+
b
c
d
n
peptide (6a−h)
rt after HPLC purification (min) yield after HPLC purification (%) clogP
calcd
found
1
H-Tyr(O-propyl)-Tic-Phe-Phe-OH (6a)
H-Tyr(O-butyl)-Tic-Phe-Phe-OH (6b)
H-Tyr(O-pentyl)-Tic-Phe-Phe-OH (6c)
H-Tyr(O-hexyl)-Tic-Phe-Phe-OH (6d)
H-Tyr(O-heptyl)-Tic-Phe-Phe-OH (6e)
H-Tyr(O-octyl)-Tic-Phe-Phe-OH (6f)
H-Tyr(O-nonyl)-Tic-Phe-Phe-OH (6g)
H-Tyr(O-decyl)-Tic-Phe-Phe-OH (6h)
15.41
16.05
16.70
17.45
18.21
18.99
9.84
18
14
15
9
3.359
677.3334
691.3940
705.3647
719.3803
733.3959
747.4116
761.4272
775.4421
677.3331
691.3466
705.3622
719.3822
733.3943
747.4127
761.4295
775.4429
2
3
4
5
3.888
4.417
4.946
5.475
6.004
6.533
7.062
7
6
5
e
7
5
e
8
10.88
3
a
Reaction conditions: (1) 6 (0.1 mmol), 1-hexene (0.4 mmol), Umicore M2 (0.01 mmol); CH2Cl2 (3 mL), 24 h, 50 °C; (2) Umicore M2 (0.01
b
mmol), Et3SiH (1 mmol), 48 h, 50 °C. 3) TFA/CH2Cl2 = 50/50, 2 h, room temperature. Retention time was determined by HPLC (standard
gradient) with UV detection at 215 nm. Yields refer to isolated pure peptides. Estimated by ChemBioOffice 2010. The rt refer to the HPLC (60−
100% of CH3CN as gradient).
c
d
e
the IMCR procedure for N-terminal-modified tetrapeptide 4 by
application of the above-described reaction conditions. As
expected, eight peptides were obtained (six of which could be
isolated after prep-HPLC). Again a good global yield of 64%
(taking into consideration the sum of all components) and high
purity was observed after preparative RP-HPLC (see
Supporting Information). The apparent low to moderate yields
of the individual components are not considered as problematic
since our primary interest is the rapid synthesis of a variety of
peptide derivatives for initial biological evaluation, rather than
high individual yields, commonly aimed for in methodological
studies. As a result of the reduction reaction and subsequent
acidolysis, only the benzyl ester was removed, while the benzyl
ether group remained on the peptide. This observation could
potentially be of interest as one derivatization handle remains
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Table 5. One-Pot Solid-Phase Synthesis of Side-Chain-Modified DIPP Via the Tandem Isomerization/Cross-Metathesis/
a
Reduction/Cleavage Reactions
HRMS[M + H]+
b
c
d
n
peptide (7a−h)
rt after HPLC purification (min) yield after HPLC purification (%) clogP
found
calcd
1
H-Dmt(OHJ-Tic-Phe-Phe-OH (7a)
13.74
15.04
16.43
17.11
17.78
18.49
19.22
20.00
21.08
17
2.613
663.3177
705.3647
719.3803
733.3959
747.4116
761.4235
775.4429
789.5
663.3166
705.3657
719.3795
733.3958
747.4092
761.4235
775.4414
789.6
2
3
4
5
6
H-Dmt(O-propyl)-Tic-Phe-Phe-OH (7b)
H-Dmt(O-butyl)-Tic-Phe-Phe-OH (7c)
H-Dmt(O-pentyl)-Tic-Phe-Phe-OH (7d)
H-Dmt(O-hexyl)-Tic-Phe-Phe-OH (7e)
H-Dmt(O-heptyl)-Tic-Phe-Phe-OH (7f)
H-Dmt(O-octyl)-Tic-Phe-Phe-OH (7g)
H-Dmt(O-nonyl)-Tic-Phe-Phe-OH (7h)
H-Dmt(O-decyl)-Tic-Phe-Phe-OH (7i)
12
4.257
4.786
5.315
5.844
6.373
6.902
7.431
7.960
11
9
9
7
7
4
e
8
traces
traces
e
9
803.5
803.6
a
Reaction conditions: (1) 7 (0.1 mmol), 1-hexene (0.4 mmol), Grubbs’ second generation (0.01 mmol); CH2Cl2 (3 mL), 24 h, 50 °C; (2) Grubbs’
b
second generation (0.01 mmol), Et3SiH (1 mmol), 48 h, 50 °C; (3) TFA/CH2Cl2 = 50/50, 2 h, room temperature. Retention time was determined
by HPLC (standard gradient) with UV detection at 215 nm. Yields refer to isolated pure peptides. Estimated by ChemBioOffice 2010. The
c
d
e
peptide was not isolated and the rt refer to the HPLC and the mass refer to the LCMS of the crude mixture.
Scheme 2. Preparation of Tetrapeptide H-Tyr(O-heptyl)-Tic-Phe-Phe-OH (6e) Under Classical Cross-Metathesis Reaction
Conditions in a Roundbottom Flask
on the peptide analogue. Detailed results are collected in Table
2.
one of the olefin substrates makes its homodimerization less
favored due to site isolation (pseudodilution), while the olefin
that remains in solution can be added in excess to push the
reaction to total conversion. The dimer or byproducts can
easily be eliminated by filtration. Additionally, solid-phase
organic synthesis permits automation and parallel synthesis,
In an attempt to broaden the applicability of the ICMR
conditions, compatibility with solid supports was verified.
Generally, olefin cross metathesis on solid phase has several
potential advantages as compared to its solution phase
counterpart. Under SPOS conditions, the immobilization of
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ACS Combinatorial Science
Research Article
opening a gateway for the rapid generation of combinatorial
libraries.20
Of the 40 lipophilic tetrapeptides, 32 were obtained with
purity of ≥95% in 3−13 mg quantity. The calculated clogP
(3.359−11.060) values of the peptides in this library matched
very well with those obtained for previously reported bioactive
opioid peptides.1c−e,2c,22 The clogP values for our isolated
lipophilic tetrapeptides 3a−f, 5a−f, 6a−f, and 7a−f (modified
DIPP and TIPP derivatives) are situated between 3.359 and
7.807, which make them hydrophobic peptide candidates for
further membrane transport and, in this case, opioid bioactivity
studies.
Exposure of tetrapeptides 5 (N-terminal-modified TIPP) and
6 (side chain modified TIPP), which are still linked to Wang
resin, to the one-pot tandem ICMR conditions, afforded
directly the desired and expected products 5a−h and 6a−h
with overall yields of 75% and 76%, respectively. Interestingly,
in the case of the resin-bound tetrapeptide 5, the overall yield
was 11% higher than was observed in the reaction performed
with the corresponding peptide (4) in solution.
In summary, we were able to establish a new and efficient fast
protocol to generate wide sets of lipophilic tetrapeptide TIPP
and DIPP derivatives by one-pot tandem isomerization/cross-
metathesis/reduction reactions (ICMR) on solid phase and/or
in solution. The ICMR reactions were achieved by application
of Grubbs’ second-generation or Umicore M2 precatalyst to the
olefin cross-metathesis of N-terminally, C-terminally or side
chain allylated tetrapeptides, followed by reduction of the
alkenes in the presence of triethylsilane on a solid supports or/
and in solution. The solid phase experimental procedure did
not require any air exclusion precautions. The desired products
were isolated in good global yields and with excellent purity, as
determined by RP-HPLC and LC-MS. The biological activity
and the potential of the new lipophilic tetrapeptides are under
investigation and will be reported in due course.
Furthermore, the N-propyl-containing peptide 5a was the
most abundant component, whereas it was produced in traces
only in the solution phase synthesis carried out with 4. The
peptides were obtained in moderate yields and were purified by
RP-HPLC. The results are summarized in Tables 3 and 4. This
method is of value since only a few protocols (based on diimide
reduction) of double bonds on solid phase are available.21 In
addition, the diimide protocol appeared to be potentially
problematic when attempting reproduction in solution.19 It can
be noted that cleavage of the peptides from the Wang resin,
directly after the cross-metathesis reaction on 6, and before
reduction, resulted only in O-dealkylated peptide, due to the
well-known acid sensitivity of O-allyl type protecting groups.
This is an indirect argument for the fact that double bond
isomerization (such as discussed for 3a′−g′, Figure 4) in the
final compounds is not, or in a very minor amount, occurring.
Finally, to show that the ICMR protocol is not restricted to
the Umicore M2 catalyst 1, we switched to the more commonly
used Grubbs’ II 2 ruthenium catalyst. The one-pot tandem
ICMR reactions of Wang resin-linked tetrapeptides of type 7
(i.e., side chain modified DIPP) was accomplished by use of
catalyst 2, to give the expected eight tetrapeptides (7 of which
isolated by purification, see Table 5) in a global yield 52%. In
this case the conversion was not as complete, compared to the
equivalent ICMR conditions using Umicore M2 catalyst (Table
5 versus Table 4). The observed phenol-deprotected
tetrapeptide 7a was isolated in 17% yields because of the acid
sensitivity of the O-allyl-type protecting group presented in
building block 7.
To confirm the actual identity of all homologues obtained
through the ICMR method, tetrapeptides 6a−h were prepared
individually in solution or on solid-phase (Scheme 2). At this
point, it should be noted that the cross-metathesis under
classical heating conditions (i.e., with round-bottom flask and
cooler), affords only a single peptide (see Supporting
Information for more details). This in contrast to the described
conditions in a sealed vial (vide supra), for which a mixture of
derivatives are observed and isolated. As depicted in Scheme 2,
the use of Wang resin turned out to be problematic when
attempting the isolation of the alkenyl-derived peptide. During
the acidic cleavage from the solid support (pathway A) removal
of the alkenyl substituent was observed. Tetrapeptide 6e was
however prepared in good yield via two pathways: (B) using
the 2-chlorotrityl resin as solid support (milder acidic
conditions are required for peptide cleavage), followed by
double bound hydrogenation and subsequent removal of the
Boc protecting group and (C) performing the CMR reaction
on the Boc-Tyr(All)-OMe and alkenyl reduction before the
peptide couplings. The peptide products that were obtained in
this way were purified by preparative RP-HPLC and
subsequently coinjected in HPLC with the purified ICMR
products. As such, the structures of 6a−h were confirmed by
identical retention times.
EXPERIMENTAL PROCEDURES
■
General Procedure for the Direct Solid-Phase Syn-
thesis of a Series of Lipophilic Tetrapeptides (7a−h) Via
the Cascade Coupling/Isomerization/Cross-Metathesis/
Reduction/Cleavage Reactions. The linear tetrapeptides
were synthesized manually by Nα-Fmoc methodology on Phe-
Wang resin (0.1 mmol scale) using O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)/N-
methylmorpholine (NMM) as the coupling reagents/mixtures.
A 3-fold excess of the building blocks [Fmoc-Phe-OH, Fmoc-
Tic-OH, and Boc-Dmt(OAll)-OH] and activating agents was
applied in the presence of 3 mL of NMM (0.4 M in DMF).
Fmoc-AA-OH and NMM in DMF were added to the swollen
solid support, and the reaction mixture was shaken for 3 h. The
resin was washed three times with DMF, three times with i-
PrOH and three times with CH2Cl2. Completion of the
coupling was tested by means of the Kaiser or chloranil test.
Coupling of Boc-Dmt(OAll)-OH was repeated twice to give a
negative chloranil test. The next amino-acid was consecutively
coupled using the procedure described above. Fmoc depro-
tections were carried out by treating the resin twice (5 and 30
min) with 20% 4-methyl-piperidine in DMF. After completion
of the coupling, the resin was transferred into a sealed dry vial
containing 3 mL of dry CH2Cl2 then, 1-hexene (0.4 mmol) and
Grubbs’ second generation catalyst 2 (0.01 mmol) were added
consecutively. The solution was heated at 50 °C in a sealed vial
for 24 h. Rh(I)-catalyzed homogeneous hydrogenation using
Grubbs catalyst (0.01 mmol) in the presence of Et3SiH (1
mmol) effected quantitative reduction of the resin-attached
alkene at 50 °C in CH2Cl2 for 48 h. The resin was washed three
times with DMF, three times with i-PrOH, and three times with
CH2Cl2. Final cleavage of the peptide from the resin as well as
the Boc side chain protection group removal was accomplished
by treatment with TFA/CH2Cl2/Et3SiH/H2O 60:35:2.5/2.5
for 2 h. The peptides were isolated by filtration and purified by
RP-HPLC (30% to 100% of CH3CN as gradient) on a Supelco
Discovery BIO wide pore preparative C18 column in good
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ACS Combinatorial Science
Research Article
overall yield and was >95% pure as determined by analytical
RP-HPLC and. The structure of pure compounds was
confirmed by high-resolution electrospray ionization (ESP)
mass spectrometry. This reaction conducted to the formation
of eight peptides: H-Dmt-Tic-Phe-Phe-OH (17%, 12.9 mg), H-
Dmt(propyl)-Tic-Phe-Phe-OH (12%, 9.6 mg), H-Dmt(butyl)-
Tic-Phe-Phe-OH (11%, 10 mg), H-Dmt(pentyl)-Tic-Phe-Phe-
OH (9%, 7.5 mg), H-Dmt(hexyl)-Tic-Phe-Phe-OH (9%, 7.6
mg), H-Dmt(heptyl)-Tic-Phe-Phe-OH (7%, 6 mg), H-Dmt-
(octyl)-Tic-Phe-Phe-OH (4%, 3.5 mg), H-Dmt(nonyl)-Tic-
Phe-Phe-OH (traces), H-Dmt(decyl)-Tic-Phe-Phe-OH
(traces).
Peptide Characterization. H-Dmt-Tic-Phe-Phe-OH (7a).
HPLC (standard gradient): tret = 13.74 min. ESI-HRMS [M +
H+]: m/z = 663.3166 (calcd for C39H42H+N4O6 663.3177).
H-Dmt(propyl)-Tic-Phe-Phe-OH (7b). HPLC (standard
gradient): tret = 15.04 min. TLC Rf (EBAW), 0.79. ESI-
HRMS [M + H+]: m/z = 705.3657 (calcd for C42H48H+N4O6
705.3647).
H-Dmt(butyl)-Tic-Phe-Phe-OH (7c). HPLC (standard gra-
dient): tret = 16.43 min. TLC Rf (EBAW), 0.80. ESI-HRMS [M
+ H+]: m/z = 719.3795 (calcd for C43H50H+N4O6 719.3803).
H-Dmt(pentyl)-Tic-Phe-Phe-OH (7d). HPLC (standard
gradient): tret = 17.11 min. TLC Rf (EBAW), 0.81. ESI-
HRMS [M + H+]: m/z = 733.3958 (calcd for C44H52H+N4O6
733.3959).
H-Dmt(hexyl)-Tic-Phe-Phe-OH (7e). HPLC (standard gra-
dient): tret = 17.78 min. TLC Rf (EBAW), 0.82. ESI-HRMS [M
+ H+]: m/z = 747.4092 (calcd for C45H54H+N4O6 747.4116).
H-Dmt(heptyl)-Tic-Phe-Phe-OH (7f). HPLC (standard
gradient): tret = 18.49 min. TLC Rf (EBAW), 0.83. ESI-
HRMS [M + H+]: m/z = 761.4235 (calcd for C46H56H+N4O6
761.4272).
ABBREVIATIONS
■
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylfor-
mamide; Dmt, 2′,6′-dimethyl-(S)-tyrosine; TBTU, O-(benzo-
triazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate;
NMM, N-methylmorpholine; BBB, blood−brain barrier;
ICMR, isomerization/cross-metathesis/reduction
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and full spectroscopic data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: mouhamad.jida@vub.ac.be.
*E-mail: sballet@vub.ac.be.
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Author Contributions
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Research Foundation
Flanders (FWO), the Minister
̀
e du Dev
́
eloppement Econo-
mique, de l’Innovation et de l’Exportation (MDEIE) du
Quebec, and the U.S. National Institutes of Health (Grant
DA004443).
́
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