On the synthesis of conformationally modified peptides through isonitrile chemistry: Implications for dealing with polypeptide aggregation

Article abstract of DOI:10.1021/ja2023898

A method for introducing a dimethyleneoxy constraint joining the N atoms of two consecutive amino acids in the context of a polypeptide has been developed. This constraint can profoundly affect the tendency of a polypeptide to suffer aggregation and desolubilization, and it can be readily removed under mild conditions.

Full text of DOI:10.1021/ja2023898

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pubs.acs.org/JACS
On the Synthesis of Conformationally Modified Peptides through
Isonitrile Chemistry: Implications for Dealing with Polypeptide
Aggregation
Xiangyang Wu,† Peter K. Park,† and Samuel J. Danishefsky*,†,‡
†Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, New York 10065,
United States
‡Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States
S Supporting Information
b
For these purposes, we drew from the growing body of
chemistry that emerged from our recent studies of isonitriles.9
ABSTRACT: A method for introducing a dimethyleneoxy
constraint joining the N atoms of two consecutive amino
acids in the context of a polypeptide has been developed.
This constraint can profoundly affect the tendency of a
polypeptide to suffer aggregation and desolubilization, and it
can be readily removed under mild conditions.
We revisited a key reaction that we discovered, namely, the
coupling of a carboxylic acid (a) with an isonitrile (b) to afford an
N-formylamide (c), presumably via the intermediacy of the
formimidate carboxylate mixed anhydride (FCMA),9a as shown.
This line of conjecture led to the proposal shown in Scheme 1.
Thus, an acid (1) would combine with an amino acid-derived
isonitrile (2).9d The coupling reaction described above and
subsequent discharge of the protecting group (P) would give
acid 3. Reiteration of this reaction with amino acid-derived isonitrile
4 would in principle give rise to 5, which contains N-formyl groups on
contiguous amidic nitrogens. It was hoped that these proximal,
imide-like formyl groups could be exploited to generate con-
strained tripeptide 6. Elongation in both the N- and C-terminal
directions would lead to site-specific constrained polypeptide 7.
Cleavage of the molecular constraint would then generate
polypeptide 8 in a natural motif. While the concept is applicable
in principle to many versions of “X,” in our initial explorations
described below, we examined the case where X = oxygen.
Our studies commenced with coupling of valine-related
azide 9 with leucine-derived isonitrile 109f to afford 11 in 82%
yield (Scheme 2). Cleavage of the ester generated acid 12. We
found it convenient to house the eventual N-terminii of our
constructs in terminal azido linkages. Azides hold up well
under the initial 1 þ 1 acid þ isonitrile coupling conditions,
including the thermolytic O f N acyl migration step, and
thereafter in the 2 þ 1 coupling step, which sets the stage
for building the constrained tripeptide motifs. As will be seen
(see below), the reductive cleavage of the azido group serves
as an effective means of exposing the N-terminus (NH2) of the
abbreviated constrained tripeptide to be elongated. In Table 1,
we report three additional examples of this type of sequence.
The next phase of the plan involved building tripeptidyl
constructs containing contiguous N-formyl groups. The key
reaction for accomplishing this end is illustrated in the 2 þ 1
coupling of acid 12 with isonitrile ester 16 (Scheme 3). This
reaction was conducted under our conditions of hindered
thiophenol mediation.9g In this case, a 50% yield of the target
construct 17 was obtained. Curiously, this reaction also gave
rise to a 5% yield of dipeptide 18. An assortment of related
eptides built from proteogenic L-amino acids often possess
P
high affinity and selectivity in binding to various biotargets.
However, the value of peptides bearing strictly natural motifs in
clinical medicine tends to be compromised by poor in vivo
pharmacoproperties, particularly bioavailability.1 A widely con-
sidered strategy to upgrade the value of polypeptides envisions
the incorporation of strategically placed conformational con-
straints into such molecules. Experience has shown that systems
with designed motifs can have useful properties, including
increased resistance to protease-induced degradation2 and de-
creased flexibility relative to native linear peptides. Well-designed
constraints in peptidic structures may result in enhanced affinity
of the polypeptide to target receptors.3 For instance, Kim and co-
workers designed peptidomimetic inhibitors of HIV-1 infection
that outperform analogous linear peptides lacking these con-
formational constraints.4 Such successes have provided incentive
for the development of improved methods for the synthesis of
constrained peptides. In a particularly elegant example, Grubbs
and co-workers5 demonstrated that ring-closing metathesis can
enable the construction of constrained peptide-like structures by
exploiting enantiohomogeneous tertiary amides bearing appro-
priate unsaturation projecting from the amidic nitrogens.
In the research described below, we undertook the development
of a logic wherein R-amino acids in a regular polypeptide motif
would serve to anchor the constraint. In principle, our chemistry
could encompass inclusion of either natural or “unnatural” amino
acid building blocks. In the first instance, we chose to focus on strictly
proteogenic amino acids with the additional proviso that the restraining
device should be detachable, with restoration of the natural motif. The
feature of “reversibility” could be of particular value in peptide
synthesis. Thus, a molecular constraint might serve to attenuate the
aggregation tendencies of prepolypeptide building blocks that are
otherwise difficult to manage.6ꢀ8 The constraint could be dis-
mantled when a more stable (foldable) polypeptide is in hand.
Received: March 16, 2011
Published: May 03, 2011
r
2011 American Chemical Society
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Scheme 1. Synthetic Strategy
Table 2. Synthesis of Tripeptidesa
a Conditions: microwave, 120 °C, CHCl3, 2,6-dimethylthiophenol.
Scheme 2. Synthesis of Azido Precursors of Dipeptide Acidsa
reaction of 12 and 16 was conducted in the absence of
hindered thiophenol (requiring thermolysis at 150 °C rather
than 120 °C), the ratio of the unexpected product 18 to the
target 17 was as high as 5:1.
The N-formyl group within 12 plays a key role in the critical
second coupling reaction leading to bis(N-formyl) tripeptide 17
in that it stabilizes the intermediate FCMA arising from the
reaction of 12 þ 16 against oxazolone formation, thereby
allowing the required O f N acyl tranfer to occur. In Table 2,
we summarize five additional examples of this chemistry.11
With the bis(N-formyl) tripeptides in hand, we turned to the
synthesis of the constrained systems. Initial studies centered on
the reduction of the two N-formyl functions of the mixed imides,
with the goal of generating the corresponding diol derivatives
(cf. 26). These efforts were complicated somewhat by the
sensitivity of the imide-like N-formyl group to bases and nucleo-
philes. Fortunately, after considerable experimentation, it was
found that treatment of bis(N-formyl) tripeptides 17, 21, 23, and
24 with lithium borohydride in a mixed solvent system
(20:1 CH2Cl2/n-PrOH) at ꢀ60 °C in the presence of acetic
anhydride furnished the desired methylols (Table 3).9l The
presence of acetic anhydride apparently served to suppress the
formation of side products arising from deformylation, possibly
by quenching the n-PrOLi generated during the course of the
reaction.12 Moreover, careful tuning of the workup conditions was
crucial to ensure liberation of the diols from their presumed boronate
esters [see the Supporting Information (SI)]. Happily, treatment of
the crude diols with trifluoroacetic acid (TFA) in CH2Cl2 furnished
the corresponding constrained tripeptides (28ꢀ31) in unoptimized
but still manageable overall yields (Table 3).
a Key: (a) microwave, 150 °C, CHCl3, 82%; (b) HCO2H, 100%.
Table 1. Reaction of Azido Acids with Isonitrilesa
a Conditions: (a) microwave, 150 °C, CHCl3; (b) HCO2H.
Scheme 3. ArSH-Mediated Synthesis of Bis(N-formyl)
Tripeptidesa
Armed with a reliable method for obtaining these novel
constrained tripeptides, we turned to the task of synthesizing
larger polypeptides with site-specific constraining motifs. In this
way, we hoped to probe the effect of the constraint on the peptide
conformation and the resulting properties (Scheme 4). In a typical
example, reductive cleavage of tripeptide 28 with triphenylphosphine
(Ph3P) followed by coupling with BocꢀLysꢀGlyꢀCOOH employ-
ing HATU and DIPEA as condensing agents provided the desired
pentapeptide in 75% yield over the two steps. Hydrogenolytic
a Key: (a) microwave, 120 °C, CHCl3, 2,6-dimethylthiophenol.
mechanisms to rationalize formation of this “deletion” pro-
duct can be proposed, but the actual pathway has not been
pinned down.10 In this connection, we note that when the
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Table 3. Synthesis of Constrained Tripeptidesa
Scheme 5. Deprotection of Constrained Peptides 34 and 35a
a Key: (a) 0.1 M HCl, CF3CH2OH, HSCH2CH2CH2SH.
analysis), but most intriguingly, no product could be isolated
because of the formation of an intractable precipitate, presum-
ably as a result of aggregation of linearized 35.14 Efforts to
solubilize the residue in H2O, DMSO, DMF, CH3CN, MeOH,
and CH2Cl2 proved fruitless. The apparent insolubility of 35 in
aqueous solution is especially striking considering that its con-
strained counterpart 33 is perfectly soluble in H2O at concentra-
tions of at least 10 mg/mL. This dramatic contrast suggests that the
concept of using constraints to enforce intramolecular proximities,
thereby attenuating proclivities to aggregation, may well have merit.
As noted above, such a capability could also be of advantage in synthesis.
A particularly important application would be that of stabilizing
aggregation-prone sequences of β-amyloids.15
Extensive NMR studies provided deeper insights into the con-
formational consequences of our constrained heptamers 32 and 33.
Systematic inspection of the NOESY and ROESY data for each
constrained peptide in pure D2O and 9:1 H2O/D2O at 4 °C did not
reveal suggestive through-space interactions between any of the
backbone RH or amide NH protons. However, in both cases, an
NOE correlation was observed between one of the R-protons of
Gly-2 and a side-chain γ-methyl of Val-5 (shown for 32 in
Figure 1a). In contrast, the deconstrained linear system 34 did
not exhibit any discernible NOEs between protons of noncontig-
uous residues, as would be expected (Figure 1b). Taken together,
these data indicate that the CH2ꢀOꢀCH2 constraint induces a
conformational bias wherein the peptide backbone is somehow
folded such a way that residues 2 and 5 are brought into proximity. It
could well be that this effect is responsible for the abatement of
aggregation in 33. The constraint may promote solubility by
providing a scaffold that juxtaposes the hydrophobic surfaces of
the molecule, enabling them to achieve energetically favorable
noncovalent interactions at the intramolecular level.16 Linear pep-
tide 35, lacking the constraint, achieves its noncovalent interactions
by intermolecular means, namely, aggregation.
a Key: (a) LiBH4, CH2Cl2, n-PrOH, Ac2O, ꢀ60 °C; (b) TFA, CH2Cl2.
Scheme 4. Synthesis of Large Constrained Peptidesa
a Key: (a) PPh3, THF, H2O; (b) BocꢀLysꢀGlyꢀCO2H, HATU,
DIPEA, DMF; 75% over two steps; (c) Pd/C, MeOH, H2; (d) HCl
3
H2NꢀGlyꢀAlaꢀOBn, HATU, DIPEA, DMF; 80% over two steps;
(e) 10% Pd/C, MeOH, H2; (f) TFA, CH2Cl2; 80% over two steps.
In summary, we have developed a method for assembling
conformationally biased polypeptides that is enabled by our recently
developed isonitrile coupling technology. Consecutive two-compo-
nent coupling reactions of isonitriles exploiting a strategic N-terminal
azide linkage in the first union afforded a novel tripeptide containing
contiguous N-formyl amidic linkages. These N-formyl groups were
used to establish a tripeptide bearing a CH2ꢀOꢀCH2 bridge
spanning contiguous amides. This system was further elaborated
to construct heptameric peptides containing this bridge, which could
then be dismantled under mild treatment. Studies of the constrained
and linear systems so produced have underscored the possibility of
modulating protein conformations that influence proclivities to
aggregation. The broader implications of these findings are the
subject of considerable current interest in our laboratory.
cleavage of the derived benzyl ester followed by coupling of the
resultant C-terminal acid with dipeptide H2NꢀGlyꢀAlaꢀOBn
afforded the heptapeptide in 80% yield over two steps. Finally, global
deprotection furnished the desired heptapepeptide 32 in 80% yield
over two steps. Following this general protocol, the same precursor
28 was converted to constrained system 33.
In line with our original plan (see above), we hoped to remove
the methylene bridging constraint to generate the native peptide.
Indeed, treatment of 32 with 0.1 M HCl and 1,3-propanedithiol
in trifluoroethanol at room temperature furnished the corre-
sponding water-soluble linear heptapeptide 34 in 95% yield
(Scheme 5).13 When 33 was subjected to the same conditions,
the reaction proceeded to ∼90% conversion (by LCꢀMS
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(10) See the SI for further mechanistic discussion.
Figure 1. Selected 600 MHz NOESY data for (a) constrained peptide
32 and (b) linear peptide 34. The data were obtained for 4.7 mM peptide
samples at 4 °C in D2O (pH 3.6ꢀ3.8, not corrected for deuterium) with
a 250 ms mixing time.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
analytical data for new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
s-danishefsky@ski.mskcc.org
(11) See the SI for further discussion of substrate scope.
’ ACKNOWLEDGMENT
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(13) Corey, E. J.; Reichard, G. A. Tetrahedron Lett. 1993, 34, 6973.
(14) Since the identity of the deprotected material could not be
confirmed by further structural characterization, we synthesized 35
independently by iterative solution-phase couplings. Although the linear
heptapeptide remained in solution during HPLC purification, the
lyophilized material was insoluble in aqueous and organic solvents.
(15) Tickler, A. K.; Clippingdale, A. B.; Wade, J. D. Protein Pept. Lett.
2004, 11, 377.
(16) For a conceptually related approach thought to disrupt inter-
molecular forces by promoting “internal solubilization” using polyethy-
lene glycol-based protecting groups, see: (a) Mutter, M.; Mutter, H.;
Uhmann, R.; Bayer, E. Biopolymers 1976, 15, 917. (b) Zier, A.; Ryan, D.;
Mutter, M. Tetrahedron Lett. 1994, 35, 1039. (c) Zinieris, N.; Zikos, C.;
Ferderigos, N. Tetrahedron Lett. 2006, 47, 6861. (d) Kocsis, L.;
Bruckdorfer, T.; Orosz, G. Tetrahedron Lett. 2008, 49, 7015.
This work was supported by the NIH (CA28824 to S.J.D.).
P.K.P. was supported by a postdoctoral fellowship (PF-11-014-
01-CDD) from the American Cancer Society. The authors thank
Dr. George Sukenick, Ms. Hui Fang, and Ms. Sylvi Rusli of the NMR
Core Facility at SKI for MS and NMR assistance. Dr. John Decatur of
the NMR facility at Columbia University is gratefully acknowledged
for NMR assistance. We also thank Professors Ann McDermott,
Tom Muir, and Pavel Nagorny for advice and helpful discussions. We
thank Ms. Rebecca Wilson for valuable advice and suggestions.
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