CITU: A Peptide and Decarboxylative Coupling Reagent

Article abstract of DOI:10.1021/acs.orglett.7b03121

Tetrachloro-N-hydroxyphthalimide tetramethyluronium hexafluorophosphate (CITU) is disclosed as a convenient and economical reagent for both acylation and decarboxylative cross-coupling chemistries. Within the former set of reactions, CITU displays reactiv

Full text of DOI:10.1021/acs.orglett.7b03121

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX-XXX
CITU: A Peptide and Decarboxylative Coupling Reagent
Justine N. deGruyter,†,⊥ Lara R. Malins,†,⊥ Laurin Wimmer,† Khalyd J. Clay,† Javier Lopez-Ogalla,†
Tian Qin,† Josep Cornella,† Zhiqing Liu,‡ Guanda Che,‡ Denghui Bao,‡ Jason M. Stevens,§
Jennifer X. Qiao,∥ Martin P. Allen,∥ Michael A. Poss,∥ and Phil S. Baran*,†
†Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡Asymchem Laboratories, (Tianjin) Co., Ltd., TEDA, Tianjin 200457, P. R. China
§Chemistry and Synthetic Development, Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, New Jersey 08901, United States
∥Department of Discovery Chemistry, Bristol-Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543, United States
S
* Supporting Information
ABSTRACT: Tetrachloro-N-hydroxyphthalimide tetramethyluronium hexa-
fluorophosphate (CITU) is disclosed as a convenient and economical reagent
for both acylation and decarboxylative cross-coupling chemistries. Within the
former set of reactions, CITU displays reactivity similar to that of common
coupling reagents, but with increased safety and reduced cost. Within the latter,
increased yields, more rapid conversion, and a simplified procedure are possible
across a range of reported decarboxylative transformations.
catalyst. Tetrachloro-N-hydroxyphthalimide (TCNHPI) esters,
forged through activation of carboxylic acids with TCNHPI and
N,N-diisopropylcarbodiimide (DIC), are preferred RAE sub-
strates in several of the cross-coupling protocols.2 One limitation
of this chemistry is the long activation periods and large excesses
of activating agents that are required for the efficient conversion
of peptide-based substrates. Inspired by historical advances in
peptide coupling reagents,3 the promising TCNHPI motif was
C−C bond formation via cross-coupling and C−N bond
formation via amide bond construction remain the most useful
reactions in the modern organic chemist’s arsenal.1 The objective
of this work was to develop an inexpensive and safe multipurpose
reagent that would conveniently enable both acylation of
heteroatomswith particular interest in solid- and solution-
phase peptide synthesisand decarboxylative cross-coupling
reactions. Herein, we report the realization of this goal with the
invention of CITU (1, Figure 1), a reagent that successfully
blends high reactivity in both reaction manifolds with a safety
profile that is particularly attractive for industrial applications.
Over the past year and a half, a variety of decarboxylative cross-
coupling reactions have emerged which exploit simple activating
agents for the formation of redox-active esters (RAEs)so
named for their ability to accept an electron from a suitable metal
modified to incorporate a tetramethyluronium moiety,4
a
cationic core commonly found in similar reagents. Thus,
tetrachloro-N-hydroxyphthalimide tetramethyluronium hexa-
fluorophosphate (CITU) was invented. Synthesized from the
reaction of tetramethyluronium chloride and TCNHPI (itself
derived from a nontoxic flame retardant) in the presence of mild
base, CITU can be safely made on multikilogram scale (Figure
1). The reagent features great versatility in reaction scope,
performing well in peptide coupling reactions as compared to
industry standards. Extensive epimerization studies (vide infra)
indicate a negligible degree of epimerization at the α-stereo-
center in solution, while epimerization on solid phase is both
minimal and comparable with that of similar reagents (e.g.,
OxymaPure/DIC, PyAOP, HATU). Additionally, CITU has
proven to be a powerful activator in the suite of recently reported
nickel-catalyzed decarboxylative cross-coupling methods, form-
ing adducts that readily engage with various zinc reagents in
alkylation,2a arylation,5 alkenylation,2c and alkynylation2d re-
actions. Formation of the requisite redox-active ester is efficient,
mild, and can be similarly achieved in either solid- or solution-
phase reactions. When applied to peptide substrates, the CITU-
mediated cross-coupling approach enables direct introduction of
Received: October 6, 2017
Figure 1. CITU: a dual-purpose activating reagent.
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03121
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Scheme 1. CITU Activation and Decarboxylative Cross-Coupling
non-natural side chains onto native sequences, a challenge that
has captivated synthetic chemists for decades.6
solution-phase side-chain decarboxylative cross-couplings pro-
ceeded smoothly at room temperature, with alkylation (9 and
10), alkenylation (11), and alkynylation (12) all readily
accessible pathways. Of note, formation of the TCNHPI ester
of 13 did not proceed after 24 h with TCNHPI/DIC, while the
NHPI ester required 16 h before complete consumption of the
starting material was observed. In contrast, conversion to the
TCNHPI active ester is complete after 2 h at ambient
temperature when using CITU. Finally, tetrapeptide 13 was
activated under standard CITU conditions and then subjected to
nickel-catalyzed cross-coupling with the chiral zinc reagent 14.
Complete ester hydrolysis was observed under the reaction
conditions to afford the free acid 15, with the chiral center intact.
A second CITU-mediated activation−alkylation sequence of the
resulting carboxylic acid provided 16 in 32% yield. One drawback
to the use of CITU, however, is that decarboxylative Suzuki
cross-couplings7 do not proceed, presumably due to inhibition
by the PF6− counterion.
To evaluate efficiency as an acylating agent (Scheme 2A),
CITU was next used to assemble select endogenous opioid
peptides, manually in the case of hemorphin-4 (17) and
spinorphin (18), and both manually and via automated SPPS
for β-neoendorphin (19) and Leu enkephalin (20) (see the
Supporting Information for details). Rubiscolin-6 (22), a δ
opioid peptide, was similarly synthesized, albeit in lower yield;
premature capping of the resin-bound amine with CITU
hydrolysis products accounts for the mass balance in most
cases. Further, CITU performed well in the synthesis of 21, a
hexapeptide recognized for its notoriously “difficult” to
Investigations into the reactivity of CITU began with the
construction of tri- and tetrapeptides assembled under standard
Fmoc-solid-phase peptide synthesis (Fmoc-SPPS) conditions
(Scheme 1A). Solid-phase cross-couplings necessitated orthog-
onal protection of the embedded glutamic (Glu) or aspartic
(Asp) acids, and incorporation of allyl esters allowed for selective
deprotection of the acid moieties while retaining the resin linkage
and other acid-labile protecting groups. Once unmasked, a
solution of CITU and N-methylmorpholine (NMM) in DMF
was added to the resin-bound acid, which was then agitated at
room temperature for 2 h. Following the activation period, the
newly formed RAE was treated with a solution of the nickel−
ligand complex and the desired zinc reagent for 16−24 h. While
care was taken to exclude water from the SPPS vessel, no
specialized equipment or skill is required for successful cross-
coupling, a testament to the robust nature of the TCNHPI ester.
Of note, one-pot, tandem activation (8) of Glu and Asp side-
chains within a single peptide was accomplished in moderate
yield (7% over 13 steps); judicious choice of orthogonal acid
protecting groups should allow for sequential introduction of
distinct groups when greater diversity is desired.
For solution-phase substrates (Scheme 1B), Glu was
incorporated with the standard acid-labile tert-butyl ester
protecting group, which was readily removed under the acidic
conditions employed for resin-cleavage. Subsequent HPLC
purification afforded the tetrapeptide 13 (shown in Scheme
1C) in excellent yield (79%). As with the solid-phase variant,
B
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Scheme 2. Peptide Acylation and Small Molecule Functionalization
synthesize sequence.8 Activation of small molecules (Scheme
2B) to form TCNHPI ester (23), thioester (24), and racemic
amide (25) proceeded with similar facility, with yields ranging
from fair to excellent. Notably, only 50% of the TCNHPI ester 23
was formed when using TCNHPI/DIC, a sharp contrast to the
84% conversion observed with CITU. CITU-mediated decar-
boxylative cross-coupling of several small molecules was also
achieved (Scheme 2C), providing access to a variety of intriguing
alkynyl substitutions, including that of bis-protected Glu (26),2d
biotin (27), and the N-tosyl azetidine (29). Interestingly,
alkylation of indole-3-propionic acid proceeded in good yield
(28, 49%), with no protection of the indole core required.
Comprehensive solution-phase epimerization studies were
undertaken (Figure 2A, see the SI for detailed analysis) for the
synthesis of dipeptide Fmoc-AlaPhe-NH2 (30) using a variety of
solvents, bases, and additives. Of the 48 experiments conducted,
only two instances of epimerization were identified, both of
which used N-methylpiperidine as the base (max of 1.33%
epimerization). Comparison of solid-phase degrees of epimeriza-
tion with CITU to that of industry-standard coupling reagents
utilized a fragment coupling approach for tripeptide Phth-
AlaValPhe-NH2 (31) and the iterative assembly of tripeptide H-
GlyCysPhe-NH2 (32), a substrate commonly employed in
similar studies (Figure 2B, see the SI for experimental
protocols).9 For the latter, studies indicate that CITU performs
better than comparable “all-in-one” reagents, but slightly worse
than several that require additives (e.g., DIC). However, when
cost and convenience are considered, the slight decrease in dr
should not deter widespread process- and laboratory-scale
implementation.
Figure 2. Epimerization studies. (a) Percent epimerization determined
by analytical HPLC (UV trace, see the SI for details). (b) Average of
values obtained in triplicate experiments as determined by analytical
HPLC (UV trace, see the SI for details). (c) Percent epimerization
determined by analytical HPLC (mass integration, see the SI for details).
Given the explosive properties of benzotriazole-based
coupling reagents,10 differential scanning calorimetry (DSC)
data were obtained (see the SI for experimental details) to assess
the safety profile of CITU. The temperature of onset was
established at 170 °C, with a decomposition heat of −509 J/g.
C
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352, 801−805. (b) Qin, T.; Malins, L. R.; Edwards, J. T.; Merchant, R.
R.; Novak, A. J. E.; Zhong, J. Z.; Mills, R. B.; Yan, M.; Yuan, C.; Eastgate,
M. D.; Baran, P. S. Angew. Chem. 2017, 129, 266−271. (c) Edwards, J.
T.; Merchant, R. R.; McClymont, K. S.; Knouse, K. W.; Qin, T.; Malins,
L. R.; Vokits, B.; Shaw, S. A.; Bao, D.-H.; Wei, F.-L.; Zhou, T.; Eastgate,
M. D.; Baran, P. S. Nature 2017, 545, 213−218. (d) Smith, J. M.; Qin, T.;
Merchant, R. R.; Edwards, J. T.; Malins, L. R.; Liu, Z.; Che, G.; Shen, Z.;
Shaw, S. A.; Eastgate, M. D.; Baran, P. S. Angew. Chem., Int. Ed. 2017, 56,
11906−11910.
(3) (a) El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557−6602.
(b) For a graphical guide to amide bond formation, see: Ishihara, Y.;
Montero, A.; Baran, P. S. The Portable Chemist’s Consultant, version
2.7.1; Apple Publishing Group: New York, 2016; pp 835−895.
(4) For discussion of representative tetramethyluronium-containing
reagents, see the following. (a) HBTU, HATU, and TATU: Abdelmoty,
I.; Albericio, F.; Carpino, L. A.; Foxman, B. M.; Kates, S. A. Lett. Pept. Sci.
1994, 1, 57−67. (b) HBTU: Dourtoglou, V.; Ziegler, J.-C.; Gross, B.
Tetrahedron Lett. 1978, 19, 1269−1272. (c) TDTU and HDTU:
Carpino, L. A.; El-Faham, A.; Albericio, F. J. Org. Chem. 1995, 60, 3561−
3564. (d) HOTT: Garner, P.; Anderson, J. T.; Dey, S.; Youngs, W. J.;
Galat, K. J. Org. Chem. 1998, 63, 5732−5733.
(5) (a) Cornella, J.; Edwards, J. T.; Qin, T.; Kawamura, S.; Wang, J.;
Pan, C.-M.; Gianatassio, R.; Schmidt, M.; Eastgate, M. D.; Baran, P. S. J.
Am. Chem. Soc. 2016, 138, 2174−2177. (b) Sandfort, F.; O’Neill, M. J.;
Cornella, J.; Wimmer, L.; Baran, P. S. Angew. Chem., Int. Ed. 2017, 56,
3319−3323.
(6) For a representative review, see: Sletten, E. M.; Bertozzi, C. R.
Angew. Chem., Int. Ed. 2009, 48, 6974−6998.
(7) Wang, J.; Qin, T.; Chen, T.-G.; Wimmer, L.; Edwards, J. T.;
Cornella, J.; Vokits, B.; Shaw, S. A.; Baran, P. S. Angew. Chem., Int. Ed.
2016, 55, 9676−9679.
The data revealed no correlation when run through the Yoshida
explosivity prediction model, signifying that the compound is
unlikely to be shock sensitive and/or explosion propagating. For
comparison, TBTUone of the most structurally similar of the
common coupling reagentsis classified as an explosive with an
energy release of 401 kJ/mol, a value nearly 1.5 times higher than
that of CITU (277 kJ/mol).10a Importantly, the reagent has been
prepared in kilogram batches from tetrachlorophthalic anhy-
dride, a factor which all but eliminates both cost and safety
concerns.10c CITU is bench-stable when stored as a solid at
ambient temperature; however, as with many comparable
reagents, stock solutions (including those frequently employed
in automated peptide synthesizers) should be freshly prepared
every 2 days to avoid degradation and hydrolysis.
CITU, now commercially available,11 is an inexpensive, dual-
purpose reagent that enables both solid- and solution-phase
acylation and decarboxylative cross-coupling reactions. It is best
suited for practitioners seeking a safe alternative to the standard
array of coupling reagents, particularly on large scale. While
CITU is not recommended for use in the previously disclosed
decarboxylative Suzuki cross-coupling reaction,7 it can be readily
employed for the analogous Ni-catalyzed Negishi alkylation,
arylation, alkenylation, and alkynylation reactions, as well as for
solution- and solid-phase acylations.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b03121.
(8) Katritzky, A. R.; Haase, D. N.; Johnson, J. V.; Chung, A. J. Org.
Chem. 2009, 74, 2028−2032.
(9) Han, Y.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307−
4312.
Detailed experimental procedures and analytical data for
linear peptides and small molecules (PDF)
(10) (a) Wehrstedt, K. D.; Wandrey, P. A.; Heitkamp, D. J. Hazard.
́
Mater. 2005, 126, 1−7. (b) Subiros-Funosas, R.; Prohens, R.; Barbas, R.;
AUTHOR INFORMATION
■
El-Faham, A.; Albericio, F. Chem. - Eur. J. 2009, 15, 9394−9403. (c)
Personal communication (poster session, Pfizer, Inc.): Sperry, J. B.;
Minteer, C.; Tao, L.; Johnson, R.; Duzguner, R.; Hawksworth, M.; Oke,
S.; Eisenbeis, H.; Barnhart, R.; Bill, D. R.; Giusto, R.; Weaver, J. Thermal
Stability of Common Peptide Couping Reagents, Oct 2017.
(11) CITU is now available in the MilliporeSigma catalog
(ALD00598).
Corresponding Author
*E-mail: pbaran@scripps.edu.
ORCID
Lara R. Malins: 0000-0002-7691-6432
Phil S. Baran: 0000-0001-9193-9053
Author Contributions
⊥J.N.D. and L.R.M. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support for this work was provided by Bristol-Myers
Squibb, NSF GRFP (J.N.D.), NIH (F32GM117816 postdoctoral
■
fellowship to L.R.M. and GM-118176), Schrodinger Fellowship
̈
of the Austrian Science Fund (L.W.), Marie Skłodowska-Curie
Fellowship (H2020-MSCA-IF-2014 to J.L.-O.), and Catalan
Government (postdoctoral fellowship to J.C.). We are grateful to
Drs. D.-H. Huang and L. Pasternack (TSRI) for assistance with
nuclear magnetic resonance (NMR) spectroscopy, Dr. M.
Schmidt (BMS) for help with DSC data, Dr. J. Chen (TSRI)
for purification and analysis assistance, and Dr. Y. Shao (TSRI)
for providing Phth-AV-OH.
REFERENCES
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̈
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