Solid-phase peptide synthesis and solid-phase fragment coupling mediated by isonitriles

Article abstract of DOI:10.1073/pnas.1310431110

The synthesis of polypeptides on solid phase via mediation by isonitriles is described. The acyl donor is a thioacid, which presumably reacts with the isonitrile to generate a thio-formimidate carboxylate mixed anhydride intermediate. Applications of this

Full text of DOI:10.1073/pnas.1310431110

Solid-phase peptide synthesis and solid-phase
fragment coupling mediated by isonitriles
Ting Wanga and Samuel J. Danishefskya,b,1
aLaboratory of Bioorganic Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and bChemistry Department, Columbia University,
New York, NY 10027
Contributed by Samuel J. Danishefsky, June 4, 2013 (sent for review May 20, 2013)
The synthesis of polypeptides on solid phase via mediation by
isonitriles is described. The acyl donor is a thioacid, which pre-
sumably reacts with the isonitrile to generate a thio-formimidate
carboxylate mixed anhydride intermediate. Applications of this
chemistry to reiterative solid-phase peptide synthesis as well as
solid-phase fragment coupling are described.
of SPPS, so ably pioneered by Merrifield (5), clearly heralded
a paradigm shift in the synthesis of peptides and even proteins by
chemical means. Our program was launched by mixing solid-
support bound glutamine 8, and asparagine-derived thioacid 9,
with tert-butyl isocyanide (t-BuNC) in dimethylformamide. After
cleavage from resin, dipeptide 10 was obtained in 95% yield
(Table 1, entry 1). The experiments summarized in Table 1 serve
to clarify and integrate the findings herein, conducted under
SPPS conditions, with the previously described (20, 27) solution-
based amidation of thioacids. Previously, we had demonstrated
a highly exploitable oxidatively activable pathway for enhancing
the acyl-donating properties of thioacids (18). In solution phase,
oxidative amidation is operative even in the absence of isonitrile
mediation. In the solution phase experiments, particularly with
simple, unhindered thioacids, it proved to be very difficult to avoid
significant levels of amide formation, even following attempted
avoidance of oxidation and in the absence of isonitrile mediation.
This “negative” result could be interpreted as reflecting our in-
ability to fully prevent low levels of oxidation. Alternatively, some
amidation may have been triggered through the presence of traces
of chain-carrying oxidation impurities (such as diacyldisulfides)
with high acyl-donating potential in the substrate samples of thio
acid (27). Although these questions cannot yet be definitively
answered in the solution phase experiments, the situation under
SPPS conditions were more revealing (Table 1). Thus, entry 1
demonstrates the viability of the isonitrile (thio-FCMA) pathway.
Although slightly improved yields are obtained with the inclusion
of HOBt (entry 2), no meaningful rate increase is observed. Ac-
cordingly, it seems that the small improvement in yield between
entries 1 and 2 does not suggest a significant change of mecha-
nism. Indeed, entry 3 establishes the dominance of the thio-FCMA
pathway, indicating a possible low-yielding acyl activation pathway
by HOBt itself, independent of isonitrile mediation or adventitious
oxidative activation. Entry 4 demonstrates that, at least under the
more discriminating SPPS conditions, we are able to effectively
avoid oxidative acylation and there are no discernable acyl-
donating properties by thioacids, themselves, with respect to
amine nucleophile.
mide bond formations are arguably among the most im-
A
portant constructions in organic chemistry (1, 2). The cen-
trality of the amide linkage, as found in polypeptides and proteins,
in the maintenance of life hardly needs restatement. Numerous
strategies, resulting in a vast array of protocols to synthesize bi-
ologically active polypeptides and proteins, have been demon-
strated (3, 4). Central to reiterative polypeptide bond formations
was the discovery and remarkable development of solid-phase
peptide synthesis (SPPS) (5, 6). The extraordinary impact of SPPS
in fostering enhanced access to homogeneous polypeptides is clear
to everyone in the field.
As we have described elsewhere, by classical, mechanistic
reasoning, we were led to conjecture about some hitherto-
unexplored possibilities relevant to the chemistry of isonitriles
(7–14). It was anticipated that isonitriles might be able to me-
diate the acylation of amines, thus giving rise to amides (15).
Early experiments focused on free carboxylic acids as the acyl-
ating agents. As our studies progressed, it was found that the
combination of thioacids, amines, and isonitriles leads to the
efficient formation of amide bonds under stoichiometric or near-
stoichiometric conditions (7–13, 16, 17). Although there remain
unresolved issues of detail and nuance, the governing mechanism
for amide formation under these conditions involves reaction of
the thioacid, 1, with an isonitrile, 2, to generate a thio-formimidate
carboxylate mixed anhydride (thio-FCMA), 3, which is intercepted
by the “acyl-accepting” amine to generate amide, 5, and thio-
formamide, 6 (Fig. 1). The efficiency of the amidation was fur-
ther improved through the use of hydroxybenzotriazole (HOBt)
(18), which could well give rise to HOBt ester 7, although this
pathway has not been mechanistically proven.
The potentialities of the isonitrile-mediated amidation method
were foreshadowed via its application to the synthesis of cyclo-
sporine (19). The power of the method was particularly well
demonstrated in the context of our recent total synthesis of oxy-
tocin (OT) (20), wherein isonitrile mediation was used in each of
the peptide bond constructions, leading to the synthesis of the
hormone in high yield and excellent purity. This nonapeptide is
involved in a range of biological functions including parturition
and lactation (21, 22). Signaling of OT to its receptor (OTR) is
apparently an important factor in quality maintenance of various
CNS functions (23). The ability to synthesize such modestly sized,
but bio-impactful peptides in both native (wild-type) form, and as
strategically modified variants, is one of the current missions of
our laboratory, with the objective of possible applications to the
very serious problem of autism (24–26).
We continued our exploration in this method of amide for-
mation using varying amines and thioacids (19, 28, 29). Coupling
of solid-support glutamine 8 and leucine-derived thioacid 11 (19)
under mediation by t-BuNC and HOBt, followed by cleavage
from resin, provided dipeptide 12 in 94% yield (Fig. 2, Eq. 1).
The solid-phase isonitrile-mediated peptide bond formation was
also shown to be highly efficient by coupling solid-support serine
derivative 13 with arginine derivative 14, to afford a 92% yield of
15 after cleavage from supporting polymer (Fig. 2, Eq. 2). In
a similar fashion, a glycine-leucine dipeptide 17 was obtained in
Author contributions: T.W. and S.J.D. designed research; T.W. performed research; T.W.
and S.J.D. analyzed data; and T.W. and S.J.D. wrote the paper.
The authors declare no conflict of interest.
Results and Discussion
1To whom correspondence should be addressed. E-mail: s-danishefsky@ski.mskcc.org.
We wondered whether isonitrile-mediated amide bond forma-
tion could be used in the context of SPPS. The enormous impact
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1310431110/-/DCSupplemental.
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O
R2
R2
O
H
S
1.t-BuNC, HOBt, DMF
R2
H
O
O
N
HS
R3NH2
4
NH2
O
N
+
R2
+
RT
HX
NHFmoc
NHFmoc
X
2. TFA/TIPS/H2O
+
C
N
R2
N
H
R1
NHR3
R1
SH
R1
O
O
R1
R1
S
H
X= O or NH
2
5
6
1
thio-FCMA 3
HOBt
O
O
O
H
NH2
O
N
+
HO
NHFmoc
HS
(1)
NHFmoc
11
O
R3NH2
4
O
O
O
8
R1
OBt
R1
NHR3
TrtHN
12, 94% yield
O
H2N
HOBt ester 7
5
HN
NHPbf
NH
HN
NH2
NH
OH
O
O
O
H
NH2
HS
H2N
O
H
Me
Me
N
O
+
(2)
(3)
NHFmoc
N
H
HO
HO
NHFmoc
O
HN
HN
O
Ot-Bu
13
14
S
S
15, 92% yield
Me
O
O
O
H
N
O
N
O
O
Me
N
H
O
O
H
H
NH2
HS
+
N
O
O
O
N
O
NHFmoc
HO
NHFmoc
H2N
N
O
16
H
O
NH2
11
NH2
17, 95% yield
oxytocin (OT)
PbfHN
HN
NH
HN
NH2
SSt-Bu
Fig. 1. Isonitrile-mediated amidation; structure of OT.
SSt-Bu
HN
N
O
+
(4)
NH
O
N
O
H
NH
O
Fmoc
N
H
N
95% (Fig. 2, Eq. 3). Also encouraging was the success of the
method in producing tetrapeptide 20 through a miniligation.
Solid-support–bound 18 underwent amidation with cysteine-
proline–derived thioacid 19, providing ligation product 20 in
87% yield (Fig. 2, Eq. 4).
HN
NH3
HS
O
Fmoc
N
O
H
19
18
O
H2N
O
20, 87% yield
Fig. 2. Isonitrile-mediated SPPS: substrate scope. Entry 1, 2, 3: Novasyn
tentagel 4-carboxytrityl (TGT) resin; entry 4: rink amide polyethylene glycol-
dimethylacrylamide copolymer (PEGA) resin.
Having demonstrated the efficiency of isonitrile-mediated thi-
oacid amidation on solid support, we next investigated the
applicability of this chemistry to enable an iterative “peptide
homologation” process. The isonitrile-mediated reiterative SPPS
sequence started with cleavage of the fluorenylmethyloxycarbonyl
(Fmoc) group. The resulting N terminus served to amidate amino
acid-derived thioacid to afford solid-support dipeptide. The se-
quence was reiterated through several cycles (vide infra) through
the same two-step sequence of Fmoc deprotection followed by
isonitrile (and HOBt)-mediated acylation. Following cleavage
from the resin, the polypeptide is obtained. Tripeptide 21 was
elaborated after two deprotection/isonitrile-mediated coupling
cycles in excellent yield (85%; Fig. 3). Happily, isonitrile-mediated
SPPS proved to be extendable to a C-terminal histidine initiated
tetrapeptide, thereby providing pentapeptide 22 in 72% yield.
The program was further extended to a C-terminal glutamate-
bound pentapeptide to afford hexapeptide 23 in 68% yield. It is
worth noting that only 1.2–1.5 eq of both thioacids and reagents
were used in all of the cases shown in Fig. 3, thus demonstrating
a high level of efficiency in the isonitrile/HOBt-mediated
SPPS method.
Having established the feasibility of the core isonitrile-medi-
ated SPPS strategy, we turned to the synthesis of the historically
important mammalian hormone vasopressin, 25 (30, 31). Vaso-
pressin is a well-known neurohypophysial hormone found in
most mammals. Much of the hormone is maintained in vesicles
at the posterior pituitary, awaiting release into the bloodstream
to control the reabsorption of molecules in the tubules of the
O
O
R2
...
AA3 AA4
then
1. 20% piperidine
DMF
H
NHFmoc
N
Polypeptide
O
O
NH
O
Fmoc
R1
R1
TFA/TIPS/H2O
2. Fmoc-AA-SH,
t-BuNC,
HOBt, DMF
Ph
O
NH2
O
O
N
N
NHFmoc
O
O
H
N
N
O
Table 1. Application of thio-FCMA chemistry to SPPS
H
H
O
HO
N
H
HO
N
N
O
O
N
H
NH2
Fmoc
O
21, 85% yield
22, 72% yield
HN
H2N
HN
NH
HO
O
NH2
O
H
O
O
H
H
Entry
Additive
Yield, %
N
N
N
HO
O
N
N
NHFmoc
H
O
H
1
2
3
4
t-BuNC
t-BuNC, HOBt
HOBt
95
99
20
O
O
OH
O
NH2
23, 68% yield
None
ND
ND, not detected; TGT, tentagel 4-carboxytrityl.
Wang and Danishefsky
Fig. 3. Reiterative isonitrile-mediated SPPS.
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OH
OH
O
O
H2N
O
H2N
HS
O
N
H
N
H
HN
HN
HN
HN
HN
HN
NH2
HN
HN
NH2
S
S
Ph
O
Ph
O
SH
O
O
O
O
H
N
H
N
N
O
N
O
N
H
O
N
H
O
H
N
H
N
O
O
O
O
N
N
H
H
NH2
NH2
O
O
NH2
vasopressin 25
NH2
dihydro-vasopressin 24
H2N
O
H2N
O
Fig. 4. Structure of vasopressin.
kidneys, by affecting the tissue permeability. However, vasopressin Cleavage of the Fmoc protecting group in the resulting dipeptide
is also released directly into the brain, where it also plays an im- 28, followed by ligation with cysteine-proline–derived thioacid
portant role in social behavior and bonding. As seen, vasopressin
is structurally similar to the nonapeptide oxytocin (Fig. 4, 25 venture then progressed through the same two-step deprotection-
vs. 8) (32, 33). Accordingly, the interactivity of these two hor- coupling homologation process using thioacids 31–35, derived
mones has been studied extensively. We undertook to revisit from asparagine, glutamine, phenylalanine, tyrosine, and cyste-
29, efficiently provided solid-support tetrapeptide 30. This ad-
the synthesis of vasopressin in the context of isonitrile-
mediated SPPS.
The synthesis of vasopressin 25 began with coupling solid-
supported glycine 26 and arginine-derived thioacid 27 (Fig. 5).
ine, respectively, to afford the vasopressin backbone, 36, on solid
support. Happily, cleavage of the nonapeptide from the resin,
using TFA/triisopropylsilane (TIPS)/H2O, provided dihydro-va-
sopressin in 43% overall yield, and enabled us to characterize 24
O
O
H
H
HN
HN
N
O
N
O
HN
HN
S
S
O
O
STrt
Fmoc-Arg(pbf)-SH (27),
t-BuNC, HOBt, DMF
N
O
1. 20% piperidine in DMF
NHFmoc
O
O
O
H
H
O
NH2
N
N
2. Fmoc-Cys(Trt)-Pro-SH (29),
t-BuNC, HOBt, DMF
N
N
NHFmoc
N
N
H
H
H
H
O
O
26
28
30
Ot-Bu
OH
Boc
HN
O
O
O
O
H2N
HS
O
N
H
N
H
H
HN
HN
HN
HN
N
HN
HN
HN
HN
NH2
TrtS
STrt
Ph
S
Ph
O
O
O
SH
Asn Gln Phe Tyr Cys
Isonitrile mediated SPPS
TFA/TIPS/H2O
O
O
O
O
H
N
H
N
N
N
N
H
O
O
N
H
O
O
H
H
O
O
O
N
O
O
N
N
N
O
H2N
N
O
H
H
H
NHTrt
NH2
O
NHTrt
O
NH2
36
dihydro-vasopressin 24
43% over 14 steps
OH
O
H2N
O
N
H
HN
HN
HN
HN
NH2
S
S
Ph
O
air, pH=7
71%
O
O
H
N
N
N
H
O
O
H
O
O
N
H2N
N
O
H
NH2
O
NH2
Vasopressin 25
Fig. 5. Synthesis of vasopressin. Reagents in the isonitrile-mediated SPPS: Asn = Fmoc-Asn(Trt)-SH (31); Gln = Fmoc-Gln(Trt)-SH (32); Phe = Fmoc-Phe-SH (33);
Tyr = Fmoc-Tyr(t-Bu)-SH (34); Cys = Boc-Cys(Trt)-SH (35).
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Wang and Danishefsky

...
Isonitrile mediated ligation
Fmoc-peptide 2-SH
AA3 AA4
then
peptide 1
peptide 1 peptide 2
Polypeptide
TFA/TIPS/H2O
H2N
HN
NH
O
O
NHTrt
O
O
O
H
O
H
H
H
N
N
+
N
N
N
N
H
O
NH2
HS
N
H
NHFmoc
H
O
O
O
O
O
38
NHTrt
37
= Novasyn TGT resin
H2N
O
t-BuNC, HOBt,
DMF
H2N
HN
NH
TrtHN
O
O
O
O
O
H
O
H
N
H
N
H
N
N
N
N
N
N
H
NHFmoc
H
H
H
O
O
O
O
O
O
NHTrt
H2N
O
39
1. 20% piperidine in DMF
2. 38, t-BuNC, HOBt, DMF
3. TFA/TIPS/H2O
78% over 4 steps
H2N
NH
H2N
HN
NH
H2N
HO
O
HN
O
O
O
O
O
O
H
H
N
H
H
H
H
N
N
N
N
N
N
N
H
O
N
N
H
N
N
H
NHFmoc
H
H
H
O
O
O
O
O
O
O
NH2
H2N
O
H2N
O
40
Fig. 6. Isonitrile-mediated SPFC.
as a homogeneous entity. Finally, aeration of 24 in pH 7 aqueous
solution provided vasopressin 25 in 71% isolated yield. As in
the recently reported case of oxytocin (20), the high field pro-
ton spectra of 24 and 25 were quite similar, thus suggesting a
high order of preorganization, even in the absence of disulfide
bond formation.
for lysine), thus simplifying purification by HPLC. Ideally, a two-
step deprotection–fragment condensation process would allow for
assembly of a large peptide or even a protein in an efficient way
without the need for postligation purification (Fig. 6).
To evaluate this concept, solid-support–based pentapeptide 37
and unprotected tetrapeptide-derived thioacid 38* were assem-
bled (Fig. 6). Isonitrile-mediated chemoselective SPFC served
to smoothly amidate the C-terminus thioacid, affording solid-
support nonapeptide 39. A deprotection–ligation homologation
process followed by cleavage from resin using TFA/TIPS/H2O
furnished polypeptide 40 in 78% yield.
We next addressed the question of C-terminal epimerization
during the course of this isonitrile-mediated SPFC. In the simplest
case, solid-supported glycine 41 underwent SPFC with peptide-
derived thioacid 42* to provide 43 in 83% yield with no detectable
loss of stereointegrity (Fig. 7, Eq. 1). Similarly, the more complex
resin-bound peptide, 37, readily coupled with 42 to afford
decapeptide 44 in 82% yield with no epimerization (Fig. 7, Eq. 2).
However, when the C-terminal phenylalanine-derived thioacid 45*
served as the ligation partner, a significant level of epimerization
product (21%) was observed, presumably because Phe is partic-
ularly prone to C-terminal epimerization (Fig. 7, Eq. 3) (41). More
detailed studies examining a wide range of C-terminal thioacids
and their application in peptide synthesis are underway.
We next evaluated the feasibility of a more challenging prop-
osition, namely, the possibility of an iterative chemoselective solid-
phase fragment coupling (SPFC) via isonitrile-mediated amida-
tion. Due to the high levels of convergence, SPFC is, in principle,
an attractive strategy for the synthesis of large peptides and/or
peptides with “difficult” sequences (34–40). Moreover, SPFC may
offer considerable advantages in allowing for interim purification,
which should simplify obtaining homogeneous end product.
However, ligations in the SPPS mode have often been plagued by
limitations. Protected peptide fragments, which are used in tra-
ditional SPFC, are difficult to purify by HPLC due to their poor
solubility in aqueous solvents (34, 35). To solve this problem, it
would be ideal if minimally protected peptide fragments could be
used in the ligation step. It was in the context of this problem that
we could envision a major advantage of the chemistry developed
above. Thus, we have shown that a suitably rendered isonitrile
could selectively activate a thioacid functional group at room
temperature in the presence of carboxylic acid, amide, guanidine,
or phenol (18). The resulting thio-FCMA (or its corresponding
HOBt ester) acylates primary amine more readily than the alco-
hol, imidazole, indole, or phenol-based putative acyl acceptors.
Given this unique aspect of the method, it seemed possible that
one could execute SPFC by using unprotected fragments (except
*Peptide-derived thioacids were prepared following the procedure documented in refs.
18 and 20.
Wang and Danishefsky
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HO
O
OH
O
O
O
O
O
O
H
H
H
H
SPFC
AcHN
N
N
HO
N
N
NHAc
(1)
NH2
+
N
N
SH
N
N
H
N
O
H
H
H
H
O
O
O
O
O
41
43
42
NH
HN
83 % yield
no epimerization
HO
H2N
O
NH
H2N
NH
OH
O
H2N
O
O
H
N
H
O
O
O
O
H
O
SPFC
+
H
H
H
N
AcHN
N
37
(2)
HO
N
N
N
N
N
SH
N
H
N
H
O
N
H
N
H
N
H
H
H
O
O
O
O
O
O
O
NHAc
42
NH
NH
NH2
44
HN
82% yield
no epimerization H2N
H2N
NH
HO
H2N
HO
O
OH
O
O
O
O
O
O
O
O
H
H
H
H
H
H
N
SPFC
+
N
N
N
N
AcHN
N
37
(3)
N
H
N
H
O
N
H
N
H
N
N
H
N
H
SH
H
O
O
O
O
O
NHAc
O
O
Ph
Ph
NH2
45
NH
HN
46
75% yield
21% epimerization
H2N
NH
H2N
NH
Fig. 7. Isonitrile-mediated SPFC: substrate scope.
Conclusion
our isonitrile-based protocols permit the use of near-stoichiometric
levels of amino acid coupling partners, thereby providing a valuable
economic advantage over prevailing SPPS methods. Presumably,
this method is also applicable to substrates bearing unnatural
side chains and extended backbones. Furthermore, SPFC could
conceivably accommodate a recombinant coupling partner (al-
beit one lacking a lysine residue). Given these attributes, the
methods described herein are likely to be quite useful in poly-
peptide synthesis. More detailed studies and further application
to the synthesis of large peptides and proteins will be disclosed in
due course. Full experimental details and spectra are included in
the SI Appendix.
In summary, isonitrile/HOBt-mediated solid-phase amidation has
been demonstrated. In an iterative fashion, this highly efficient
methodology was used in SPPS, requiring consumption of only
small excesses of reagents. Adoption of this central strategy
enabled the total synthesis of homogeneous dihydrovasopressin
24, which was then oxidatively cyclized to furnish vasopressin,
25. In addition to single amino acid homologation, an iterative
chemoselective SPFC concept, using unprotected peptide-derived
thioacids, has been reduced to practice. This ligation method, al-
though not free of C-terminal epimerization issues, already offers
enough ligation sites so as to provide the basis for a widely general
method for ligating in the SPPS mode. We call this method SPFC.
As practiced herein, SPFC offers the advantage of minimal pro-
tection of side chains. For the moment, only lysine residues appear
to require protection. Moreover, although standard approaches
generally require multiple equivalents of costly amino acid precursors,
ACKNOWLEDGMENTS. We thank Dr. George Sukenick, Hui Fang, and Sylvi
Rusli of Sloan-Kettering Institute’s NMR core facility for assistance. We thank
Prof. Phil Dawson for sharing valuable insights in the field of SPFC. Support
for this research was provided by National Institutes of Health Grant
HL25848 (to S.J.D.).
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