Amide and Peptide Bond Formation in Water at Room Temperature

Article abstract of DOI:10.1021/acs.orglett.5b01812

A general and environmentally responsible method for the formation of amide/peptide bonds in an aqueous micellar medium is described. Use of uronium salt (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) as a coupling reagent, 2,6-lutidine, and TPGS-750-M represents mild conditions associated with these valuable types of couplings. The aqueous reaction medium is recyclable leading to low E Factors.

Full text of DOI:10.1021/acs.orglett.5b01812

Letter
pubs.acs.org/OrgLett
Amide and Peptide Bond Formation in Water at Room Temperature
Christopher M. Gabriel,† Megan Keener,† Fabrice Gallou,‡ and Bruce H. Lipshutz*,†
†Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, United States
‡Novartis Pharma AG, Basel, Switzerland
S
* Supporting Information
ABSTRACT: A general and environmentally responsible
method for the formation of amide/peptide bonds in an
aqueous micellar medium is described. Use of uronium salt (1-
cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-
morpholinocarbenium hexafluorophosphate (COMU) as a
coupling reagent, 2,6-lutidine, and TPGS-750-M represents
mild conditions associated with these valuable types of
couplings. The aqueous reaction medium is recyclable leading
to low E Factors.
urrently, the most common strategies for generating
amide bonds involve use of carbodiimide, uronium, or
remain.1 To date, very few methods have been developed which
utilize greener alternatives, such as water, as the reaction
medium.6,7
Our approach toward making a more environmentally sound
process for the formation of amide and peptide bonds was to
utilize TPGS-750-M (3, Figure 2), a surfactant designed by our
C
phosphonium reagents (e.g., EDCI, HATU, BOP, etc.), which
rely on benzotriazole-based activators such as HOBt and
HOAt, in order to increase reaction rates and suppress
racemization.1 Although these methods tend to be highly
effective, especially in peptide bond formation, unpredictable
autocatalytic decomposition makes reagents containing a
benzotriazole scaffold undesirable from the standpoint of
safety.2 More recently, oxime-based activators such as Oxyma
1 (Figure 1) have been put forth as safer choices relative to
Figure 2. Structure of TPGS-750-M.
group8 that, upon dissolution in water, spontaneously
assembles into nanomicelles. These particles can then be
utilized to solvate water-insoluble reagents and act as
“nanoreactors” of the appropriate size (50−60 nm) and
shape (spheres or worms) in an exclusively aqueous medium,
thereby replacing commonly used organic solvents with water.
Herein we report a robust general procedure for amide/peptide
bond formation in water at ambient temperatures that (a) takes
place in high yields; (b) offers considerable scope in terms of
coupling partners; (c) shows high protecting group tolerance;
(d) occurs with negligible racemization; (e) allows for facile
recyclability of the reaction medium; and (f) produces very
little organic waste as manifested by low E Factors.
Optimization focused on the coupling of p-toluic acid with L-
Leu-OEt·HCl. Several tertiary amine bases,9 concentrations,
and surfactants10 were screened. Eventually, the amide product
could be formed in 93% yield after 13 h (Table 1, entry 2)
using N-methylmorpholine as a base in 4 wt % TPGS-750-M/
H2O. Further screening with pyridyl bases (e.g., 2,6-lutidine,
Figure 1. Structures of Oxyma and COMU.
triazole-based reagents.3 The emerging popularity of Oxyma
has led to the development of a highly active Oxyma-derived
uronium salt (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)-
dimethylaminomorpholino carbenium hexafluorophosphate
(COMU), 2, which is an attractive alternative to current
coupling methods, in particular with regard to stereoretention.4
Another approach to activated ester synthesis of amides comes
in the form of boronic acid catalysis (BAC).5 While BAC does
offer the ability to form amide bonds catalytically, high catalyst
cost and modest scope may be viewed as limitations of this
method.
Notwithstanding these advances in amide/peptide bond
constructions (vide supra), which lead to increased efficiency
and, therefore, less organic waste, a “greener” protocol would
likely be welcomed since nearly all coupling reactions are run in
polar aprotic solvents (e.g., DMF, DCM).1 Hence, issues such
as safety, waste disposal, health, and lack of recyclability
Received: June 24, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b01812
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Letter
protecting groups such as Cbz, Boc, and Fmoc, along with
several C-terminus protecting groups such as alkyl, allyl, and
benzyl esters, was noted. Especially sterically hindered amino
acids such as N-Cbz protected α-aminoisobutyric acid (Z-Aib-
OH) coupled exceptionally well under these aqueous
conditions (products 5−9) with no obvious limitations
involving coupling partners such as Phe-OMe HCl, 8, and
Pro-OBn HCl, 9. Similarly, coupling to form Boc-Phg-Phg-
OMe, 15, was achieved in high yield (88%) after 4 h. Lipophilic
product Fmoc-Val-Leu-OEt, 20, was also formed in high yield
(96%) under the optimized conditions, while the same reaction
yielded only 53% product in the absence of surfactant,
indicative of the role of TPGS-750-M in this technology.
Fmoc deprotection, typically occurring under basic condi-
tions,11 was not observed. Successful couplings were also
achieved for several important oligopeptide natural product
intermediates including Chlamydocin precursor 21,12 and
Streptocidin C precursor 2313 with comparable yields and
reaction times analogous to those seen previously.
Table 1. Optimized Conditions for Amide Bond Formation
a
entry
base
time (h)
yield of 4 (%)
1
2
3
4
N-methylmorpholine (2.0 equiv)
N-methylmorpholine (3.0 equiv)
2,6-lutidine (3.0 equiv)
13
13
0.25
4
70
93
>99
>99
2,4,6-collidine (3.0 equiv)
a
Yields of isolated products.
2,4,6-collidine) resulted in quantitative yield of the desired
product, as well as a substantial increase in reaction rate.
The optimized conditions of the model system leading to
amide 4 translated well to the formation of peptide bonds. In
fact, a lower surfactant concentration (2 rather than 4 wt %),
while maintaining a global concentration in water of 0.5 M,
could be employed, affording comparable yields and reaction
times (Figure 3). The scope of this reaction proved to be quite
broad, where tolerance to several common peptide N-terminus
Use of 2,6-lutidine was originally pursued for its known
ability as an effective base for amide couplings, while reducing
the extent of epimerization/racemization due to steric bulk
surrounding its basic site.14 Under our standard conditions
leading to products Z-Aib-Phg-OMe (8) and Z-Aib-Phe-OMe
(23), the chiral integrity of these materials was maintained
(Table 2). Interestingly, no epimerization was observed even
Table 2. Study of Epimerization under Standard Conditions
a
b
entry
product
base
time (h)
yield
ee
1
2
3
24
24
2,6-lutidine
pyridine
0.5
0.5
2.5
93%
94%
90%
>99%
>99%
>99%
8
2,6-lutidine
a
b
Isolated yield. Determined by HPLC analysis.
when pyridine was used in its place. The low occurrence of
epimerization is likely due to the mildly acidic to neutral
conditions (pH = 6−7) involved, which is known to be
insufficient for deprotonation even of the α-proton in residues
such as phenylglycine.15 When stronger bases such as DBU or
DABCO were employed, a more alkaline reaction mixture
resulted (pH = 9), and no detectable coupling product was
observed. Furthermore, it was found that either increasing or
decreasing the pH of a reaction where 2,6-lutidine is used as the
base leads as well to lower overall yields.16
The correlation between pH and reaction yield may simply
be due to amino acid solubility. Most coupling partners will
dissolve readily into the aqueous surfactant solution containing
2,6-lutidine, which may solubilize each component as its
corresponding carboxylate or ammonium salt. The reaction
then could take place in water yielding the now insoluble
carboxamide product, which may be solubilized within the
micelles present in solution. This notion is supported in that
these reactions do occur to varying extents in the absence of
surfactant (i.e., on water), albeit, on occasion, with considerably
lower product yields (e.g., Figure 3, product 20).17
Figure 3. Peptide coupling products in TPGS-750-M/H2O.a a Con-
ditions (by order of addition): carboxylic acid (1.1 equiv), 2 wt %
TPGS-750-M/H2O (0.5 M), 2,6-lutidine (3.1 equiv), ammonium
chloride (1.0 equiv), COMU (1.1 equiv). b Yields of isolated products
are reported. c 5.0 mmol scale. d From ammonium tosylate salt of Leu-
In order to further explore the generality of this method,
products resulting from amide bond formation were generated,
e
OAllyl. Isolated yield in the absence of TPGS-750-M.
B
DOI: 10.1021/acs.orglett.5b01812
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as illustrated in Figure 4. As expected, simple alkanoic acid-
alkylamine-derived amides (products 25−27), including
mixture, thereby leading to an exceptionally low E Factor20 of
2.8. The remaining aqueous mixture can then be recycled at
least four additional times without change in yield (Scheme 1).
a
Scheme 1. Recycling and E Factors
a
b
Yields of isolated products are reported. Recycle performed by
introducing all components to the aqueous reaction mixture from the
c
previous reaction, after extraction with MTBE. 96% isolated yield.
Impurities found in the organic extracts are easily removed
using typical acid/base washes followed by filtration through a
silica plug to yield pure peptides, eliminating the need for
further purification in most cases.21
In summary, we have developed a protocol utilizing peptide
coupling reagent COMU (2) and designer surfactant TPGS-
750-M (3) in water as a general method for generating peptide
and amide bonds. This methodology successfully addresses
several environmental issues, including: replacement of organic
solvent with very small amounts of water as the reaction
medium, avoidance of benzotriazole activators, recycling of the
aqueous surfactant solution, and minimal use of organic
solvents for workup/purification. Further studies on this
coupling approach that feature multiple bond-formations in
aqueous nanomicelles in a single pot will be the subject of a
future report from these laboratories.
Figure 4. Amide products formed in TPGS-750-M/H2O.a−c a Con-
ditions (by order of addition): carboxylic acid (1.1 equiv), 2 wt %
TPGS-750-M/H2O (0.5 M), 2,6-lutidine (3.1 equiv), amine (1.0
equiv), COMU (1.1 equiv). b Yields of isolated products are reported.
c 1.0 M. d From ammonium chloride salt of the amine. e From α-
amino-γ-butyrolactone hydrobromide; reaction temperature: 45 °C.
ASSOCIATED CONTENT
■
S
* Supporting Information
secondary amines such as piperazine in 28, are produced in
high yields without modification to the general procedure.
Amides from conjugated carboxylic acids can be produced as
well under standard conditions including amide (29), as well as
substituted benzamides (30−33). Reaction of p-chlorobenzoic
acid with L-Trp-OMe·HCl afforded Benzotript18 methyl ester
31 in high yield (92%) after only 2 h, while the unprotected
indole nitrogen of tryptophan showed no influence on the
coupling. More electron-rich benzoic acids such as 4-n-
butoxybenzoic acid offered an unexpected challenge en route
to racemic 32, where only a 36% yield was obtained after 16 h,
while the undesired Oxyma-activated ester was isolated in 57%
yield. To circumvent the reduced electrophilicity of this
activated ester, the reaction was heated to 45 °C to afford in
90% yield product 32, with only trace amounts of the activated
intermediate remaining. In addition, adduct 33 was isolated in
85% yield after only 1 h from the condensation of m-
bromobenzoic acid and the N,O-dimethylhydroxylamine hydro-
chloride, this being the first reported synthesis of a Weinreb
amide19 in the absence of organic solvent.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b01812.
1
Experimental procedures, characterization data, H and
13C NMR spectra, and HPLC chromatograms (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: lipshutz@chem.ucsb.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support provided by Novartis is warmly acknowl-
edged with thanks. We acknowledge Sigma-Aldrich (Dr. Subir
Ghorai) for kindly supplying some of the reagents used in this
study.
An added benefit to performing these peptide couplings is
realized given the high solubility of the urea and oxime
byproducts of COMU in water. Extraction with a minimum
volume of organic solvent (e.g., i-PrOAc) is sufficient in most
cases to remove the coupling product from the reaction
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(21) The only observed products of this reaction are the desired
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predominately unreacted SM. Addition of extra COMU is usually not
an option due to the slurry-like property of the final reaction mixture.
D
DOI: 10.1021/acs.orglett.5b01812
Org. Lett. XXXX, XXX, XXX−XXX