Solid phase synthesis of hydrogen bond surrogate derived α-helices: Resolving the case of a difficult amide coupling

Article abstract of DOI:10.1039/c000905a

Solid phase synthesis of HBS helices involving the Fukuyama-Mitsunobu reaction and triphosgene coupling is described.

Full text of DOI:10.1039/c000905a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Solid phase synthesis of hydrogen bond surrogate derived a-helices: resolving
the case of a difficult amide coupling†
Anupam Patgiri, Michael R. Witten and Paramjit S. Arora*
Received 15th January 2010, Accepted 22nd February 2010
First published as an Advance Article on the web 5th March 2010
DOI: 10.1039/c000905a
Solid phase synthesis of HBS helices involving the
Fukuyama–Mitsunobu reaction and triphosgene coupling is
described.
access to high-yielding protocols for the difficult RCM reaction, we
have been unable to prepare diverse HBS sequences. The synthesis
strategy we typically utilize for the preparation of these artificial
helices from bis-olefins is depicted in Scheme 1. We prepare
bis-olefin 1 by standard Fmoc solid phase synthesis protocols,
introduce the N-allyl functionality as a dimer, and cap the peptide
chain with 4-pentenoic acid. The N-allyl dimer 4 is prepared in so-
lution using the Fukuyama nitrobenzenesulfonamide chemistry,6
as described in a previous report.7 We prepare the N-allyl
containing dimer in solution because of the inherent unreactivity
of the N-allylamino acids toward activated carboxylic acids.8
Coupling to N-allyl amino acids is significantly more challenging
than coupling to N-methyl amino acids. The solution phase
synthesis allows us to isolate purified dimer for further elaboration
of the peptide on solid phase. The analogous synthesis of the
bis-olefin entirely on solid phase leads to inseparable mixtures.
However, the requirement to prepare the dimer in solution limits
our choice of amino acid residues to those that do not require
protecting groups for the side chain functionality. Although, we
have prepared such dimers as needed,4,5 the manipulation of
orthogonal protecting groups for the amine, carboxylic acid and
a-Helices constitute the largest class of protein secondary struc-
tures, and play a major role in mediating protein–protein interac-
tions. The organic chemistry community has focused significant
attention on studying different approaches to either stabilize this
conformation in peptides or mimic it with nonnatural scaffolds.1
We have developed a new strategy for constraining short peptides
in stable a-helical conformations.2 This strategy, termed the
hydrogen bond surrogate approach, involves replacement of one
of the main chain hydrogen bonds with a carbon–carbon bond.
In an a-helix, a hydrogen bond between the C O of the ith amino
=
acid residue and the NH of the i + 4th amino acid residue nucleates
=
and stabilizes the helical structure (Fig. 1). To mimic the C O–H–
N hydrogen bond with a carbon–carbon bond, we utilized a ring
closing metathesis (RCM) reaction between appropriately placed
olefins on the peptide chain. A crucial feature of this helix design is
that all side-chains are available for solvent exposed contacts with
biomolecular targets. The crystal structure of a short HBS a-helix
shows that the RCM-based macrocycle faithfully reproduces the
conformation of a canonical a-helix.3 We have also demonstrated
that hydrogen bond surrogate (HBS) a-helices can target chosen
protein receptors in cell-free and cell-culture assays.4
Fig. 1 An HBS a-helix features a carbon–carbon bond in place of an
N-terminal hydrogen bond. R = amino acid side chains.
The ring-closing metathesis reaction is the key step in our
synthesis of HBS peptides, and we have reported optimized
procedures for this step on solid-phase under oil-bath heating
and microwave irradiation conditions.5 However, despite gaining
Department of Chemistry, New York University, New York, NY 10003,
USA. E-mail: arora@nyu.edu
† Electronic supplementary information (ESI) available: Synthesis and
characterization of HBS-helices. See DOI: 10.1039/c000905a
Scheme 1 Reported synthesis of HBS a-helices.
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Table 1 Bis-olefin peptides prepared by the optimized method
Table 2 Optimized Fukuyama–Mitsunobu reaction conditions for N-
allylation of amino acid residues on Rink amide Resina
bisolefin
sequencea
% yieldb
9
XFEA*IYRLELLKAEEAN-NH2
XFEG*IYRLELLKAEEAN-NH2
XEFL*LRLWLKAibEEAibN-NH2
XRKA*YKRLAMK-NH2
84
91
55
60
10
11
12
a X denotes pentenoic acid residue, and A*, G* and L* refer to N-allyl
A, G and L, respectively. b Refers to the amount of the desired peptide
in crude mixture, after cleavage, as judged by HPLC (see Supplementary
Information).
the side chain functionality restricts the sequence of dimers that
can be synthesized.
entry
T/◦C time/h % conversionb
N-Allylamino acids are significantly less nucleophilic than N-
methyl amino acids for coupling with Fmoc amino acids. We
found most standard coupling reagents to provide low yields of
the desired products even with excess reagents, multiple coupling
cycles, and microwave irradiation. After extensive investigations
with common and exotic coupling reagents, we discovered that
triphosgene (bis-(trichloromethyl)carbonate) delivers the coupled
product in acceptable yields. The optimized solid-phase method-
ology is compatible with all standard side chain protecting groups
and offers the option to drive the difficult coupling reaction to
completion through the use of excess reagents.
o-nitrobenzenesulfonyl protection
1
25
12
0.25
quantitative
quantitative
2
100c
Mitsunobu reaction
3
4
25
25
6
12
0.16
70%
90%
quantitative
5
100c
o-nitrobenzenesulfonyl deprotection
6
7
25
2
0.08
quantitative
quantitative
50c
a Optimization studies were performed with peptide 9 sequence; results of
N-allylation of alanine are shown. b Determined from analytical HPLC
areas. c With microwave irradiation.
Scheme 2 illustrates the new method, which has proven to
be highly effective for the preparation of a diverse set of HBS
sequences on solid phase (Table 1). We utilized microwave assisted
Fukuyama–Mitsunobu chemistry to prepare the N-allylamino
peptide on standard Fmoc resins for the subsequent amide bond
formation reaction with triphosgene. The optimized approach
bypasses the need for preparation of the N-allyl dimer. The
optimization studies for each step were initially performed with
peptide 9, and the best conditions were then tested with the
other sequences. We chose bisolefin 9 for the optimization studies
because it has been a difficult sequence to access with our
previous approach. Coupling of the t-butyl protected glutamic
acid residue to an allylamine, in the solution phase synthesis of
FmocGlu(OtBu)-N-allylAla-OH, is difficult because of the bulky
side chain and the troublesome protecting group manipulations.
The optimized conditions described below provide high yields
of the bis-olefin peptides consisting of six to sixteen residues
independent of the sequence.
The Fukuyama–Mitsunobu reaction has emerged as the best
method for N-alkylation of amino acids and peptides.9 We utilized
this approach to prepare N-allylpeptide 7 on solid-phase under
microwave irradiation. Table 2 shows the results of the Fukuyama
and the Mitsunobu steps for allylation of the alanine residue
needed for the synthesis of bis-olefin 9. Both reactions provide
the desired products in high yields at room temperature and with
microwave irradiation.10 To the best of our knowledge, this is the
first report of a microwave-assisted Fukuyama–Mitsunobu reac-
tion. Palladium catalyzed allylation of nitrobenzenesulfonamides
also proceeds in high yields.8
Fig. 2 Representative crude HPLC traces for conversion of N-allylamine
7 to amide 8 with different coupling agents as described in Table 3.
Peptide was deprotected and cleaved from Rink amide resin with TFA,
and monitored at 220 nm. (a) N-allyl peptide, (b) PyBrOP/DMAP, (c)
TFFH/HOAt, (d) HATU/HOAt, (e) DIC/HOAt, (f) triphosgene.
Scheme 2 Optimized synthesis of bis-olefin 1.
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Table 3 Effectiveness of different coupling reagentsa
the amine in situ; whereas in the thionyl chloride procedure, the
acid chloride must be isolated prior to the coupling step.
We find triphosgene to be a superior coupling reagent compared
to every other combination tested for a variety of sequences
(Table 1), except when the allyl bearing amino acid is a glycine.
N-Allylglycines may be coupled to other Fmoc amino acids
in high yields with the standard DIC/HOAt combination. N-
Allylglycines containing sequences can be prepared either with the
outlined Fukuyama–Mitsunobu method or through substitution
of bromoacetamide with allylamine (Scheme 3). This strategy is
analogous to that used in the synthesis of peptoids (N-alkylglycine
oligomers),16 and begins with coupling of bromoacetic acid to a
peptide chain followed by addition of allylamine to afford 15.
Coupling of unhindered allylglycines proceeds in high yields with
standard coupling agents. This simplified strategy is attractive for
N-allylglycine containing HBS helices.
entry
coupling reagentb,c
T/◦C time/h
% conversiond,e
1
2
3
4
5
6
7
8
9
triphosgene
DIC/HOAt
HATU/HOAt
TFFH/HOAt
PyBrOP/DMAP
HBTU
45
45
45
45
45
25
25
25
25
25
0.5
0.5
0.5
0.5
0.5
94%
34%
11%
9%
trace
trace
trace
trace
trace
trace
12
TBTU
12
12
2
PyBOP
amino acid chloridef
DAST
10
2
a Optimization studies were performed with peptide 9 sequence; re-
sults of amide bond formation between an N-allylalanine residue and
FmocGlu(tBu)-OH on Rink amide resin are shown. b With microwave
irradiation; see the experimental section for details. c DMF was used
as a solvent with all coupling agents except for THF with triphosgene.
d Determined from analytical HPLC. e Results after three different treat-
ments with the Fmoc amino acid and coupling agent. f Pre-synthesized
nosyl amino acid chloride.8
The key reason limiting our access to diverse sets of HBS
helices has been the inefficient coupling of an N-allylamine to
the next Fmoc amino acid.8 Over the past several years, we have
tested a variety of coupling agents for this reaction. Table 3
describes the effectiveness of different coupling reagents for the
amide bond formation between of FmocGlu(tBu)-OH to an
N(allyl)Ala peptide (for the synthesis of peptide 9) on Rink amide
resin. Coupling of the t-butyl protected glutamic acid residue
to an allylamine is difficult because of the bulky side chain.
Traditional reagents such as HBTU, PyBOP and TBTU give
only traces of the amide product.11 Coupling with pre-synthesized
nosyl-amino acid chlorides8 yields a complex mixture with traces
of desired product. In situ generated amino acid fluoride with
DAST12 failed to provide any product. Reagents recommended
for difficult couplings, such as, HATU/HOAt13 TFFH/HOAt12
and PyBrOP/DMAP14 provided low yields. DIC/HOAt afforded
a 34% conversion. Significantly, bis-(trichloromethyl)carbonate,15
commonly known as triphosgene, provided greater than 90%
conversion of the allylamine to the desired amide. No racemization
was observed from the triphosgene mediated coupling as detected
by HPLC, which agrees with the observation by Falb et al.15c
Fig. 2 depicts crude HPLC traces for the amide bond formation
between FmocGlu(tBu)-OH to N-allylalanine residue in 9, after
cleavage of the peptide from resin. The effectiveness of all coupling
reagents was tested at room temperature (data not shown) and
with microwave heating. Table 3 shows optimized conditions
for triphosgene mediated coupling reaction. Percent conversions
reported in Table 3 are results after three different treatments
(“triple coupling”) with the Fmoc amino acid and coupling agent.
It is interesting to note that amino acid chlorides generated by
SOCl2 are less effective than those generated with triphosgene.
This result likely reflects the stability of acid chlorides. In the
triphosgene method, the acid chloride is generated and coupled to
Scheme 3 Synthesis of N-allylglycine containing peptides.
In conclusion, we have described a highly effective approach
to solid phase synthesis of hydrogen bond surrogate helices. The
modified approach utilizes triphosgene as the coupling agent for
the amide bondforming reaction between unreactive N-allylamino
acid residues and Fmoc amino acids. Triphosgene emerged as the
most efficient coupling agent from a variety of traditional and
contemporary reagents. The optimized solid phase microwave-
based protocols utilize the Fukuyama–Mitsunobu synthesis of
N-allylamines and provide an efficient method for synthesizing
HBS sequences.
Experimental section
Representative optimized synthesis of HBS a-helices (Tables 2
and 3)
The desired free amine peptide 6 is synthesized on Rink
amide resin using standard procedures. A solution of 2-
nitrobenzenesulfonylchloride (3 eq) in DCM and 2,4,6-collidine
(5 eq) are added to the pre-swelled resin, and the mixture is
irradiated under microwaves (CEM discover) for 15 min at 100 ◦C.
The resin is washed with DMF (¥3) and DCM (¥3), and dried
under vacuum. Anhydrous THF and triphenylphosphine (5 eq) are
added to the resin (containing 13) under nitrogen atmosphere, and
the mixture is cooled in an ice bath. Diisopropyl azodicarboxylate
(10 eq) and allyl alcohol (10 eq) are added and the mixture is
subjected to microwave irradiation at 100 ◦C for 10 min. The
resin is washed with DMF (¥3) and DCM (¥3), and dried under
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vacuum. DBU (5 eq) and 2-mercaptoethanol (10 eq) are added
to pre-swelled resin (containing 14) in DMF under nitrogen
atmosphere. The reaction mixture is then subjected to microwave
irradiation at 50 ◦C for 5 min. The resin (containing 7) is washed
with DMF (¥3) and DCM (¥3), dried under vacuum. In a separate
flask Fmoc amino acid (10 eq), triphosgene (3.3 eq) and enough
THF are mixed to maintain a triphosgene concentration of 0.15 M;
2,4,6-collidine (28 eq) is added, and the resulting solution is
introduced to the resin (containing 7). The mixture is irradiated
under microwaves at 45 ◦C for 30 min. The coupling step is
repeated twice. The resin (containing 8) is washed with DCM (¥5),
and the Fmoc group is removed with 20% piperidine in DMF. The
desired Fmoc amino acid residue and 4-pentenoic acid are coupled
using standard peptide synthesis methodology as described in
the Supplementary Information. Ring-closing metathesis on bis-
olefin is performed with Hoveyda–Grubbs II catalyst (20 mol%) in
dichloroethane under microwave irradiation at 120 ◦C for 10 min
as described.5
CHE-0234863, and the NCRR/NIH for Research Facilities
Improvement Grant C06 RR-16572.
References
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Representative synthesis of N-allylglycine peptides (Scheme 3)
A solution of bromoacetic acid (20 eq) with DIC (20 eq) and HOAt
(10 eq) in DMF is added to pre-swelled resin, and shaken for 2 h.
The resin is washed sequentially with DMF (¥3), DCM (¥3) and
DMF (¥3). Resin is re-suspended in 1 M allylamine (20 eq) in
DMF and shaken for 20 min. The resin (containing 15) is washed
with DMF (¥3), methanol (¥3) and DCM (¥3), and treated with
the desired Fmoc amino acid (20 eq), DIC (20 eq) and HOAt (10
eq) in DMF under microwave irradiation for 30 min at 60 ◦C.
The resin is washed with DMF (¥3), DCM (¥3), DMF (¥3), and
coupled with the desired Fmoc amino acid residue and 4-pentenoic
acid using standard peptide synthesis methodology as described
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bis-olefin 10 is performed with Hoveyda–Grubbs II catalyst (20
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diisopropylcarbodiimide; HBTU, (2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate); HOAt, N-hydroxy-7-
azabenzotriazole; PyBOP, Benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate; PyBrOP, bromotripyrrolidino-
phosphonium hexafluorophosphate; TBTU, 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFFH, tetra-
methylfluoroformamidinium hexafluorophosphate.
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Acknowledgements
We are grateful for financial support from the NIH (GM073943).
We thank the NSF for equipment Grants MRI-0116222 and
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