Coupling Reagents

COUPLING
REAGENTS
R
H
Cα
Cα
COOH
PG1 NH
R
H
COO
NH2
PG2
R
H
Cα
PG1 NH
R
H
Cα
CO
NH
COO
PG2
Coupling Reagents
COUPLING REAGENTS AND
ADDITIVES OFFERED BY BACHEM
The coupling reaction i.e. the formation of an amide bond
between amino acids and/or peptides is the crucial step
in peptide synthesis. The reaction consists of two consecutive steps:
1. Activation of the carboxy moiety
2. Acylation of the amino group
During the first step the protected amino acid (or peptide) reacts with a so-called coupling reagent yielding
a reactive intermediate. The chemistry behind and the
most important coupling reagents will be presented in
this brochure.
Activation during Peptide Synthesis
The formation of an amide bond between a
carboxylic acid moiety and an amino function of two amino acids is the core reaction
in peptide synthesis:
PEPTIDE
SYNTHESIS
Our comprehensive offer of coupling
reagents, amino acids derivatives and
further building blocks, and resins
at www.bachem.com will fulfill the
needs of solid-phase and solution
peptide synthesis. If the product you
need is lacking, please let us know.
2
The first step of this condensation reaction,
the activation of the carboxyl moiety
(I. in Fig. 1) is often the critical one. Depending on the type of activating reagent, the
intermediate A is a stable compound which
could be isolated if desired. During the
second step (II. in Fig. 1), A is attacked by a
nucleophile such as the α-amino group of a
carboxy-protected amino acid. Steps I and
II can be performed either consecutively or,
with certain types of coupling reagents, as
a one-pot reaction.
Over the years, numerous activation
methods have been developed, such as the
generation of carboxylic halides (chlorides,
fluorides), carboxylic azides, symmetrical
or mixed anhydrides or the use of carbodiimides (DCC, DIC, EDC · HCl) with or without
additives.
Fig. 1.
Activation and coupling of a protected
amino acid.
PG, PG’: protecting
groups
Act: activating
group
If the amino compound contains other functional groups able to take part in the coupling reaction, use of a stable preactivated
carboxylic compound A is recommended.
Active esters such as the pentafluorophenyl (OPfp) and hydroxysuccinimido (OSu or
NHS) esters are reactive amino acid derivatives finding broad application in peptide
synthesis. For our comprehensive offer of
active esters please see our online shop.
Coupling Reagents
Carbodiimides have been used as activators for decades in solid-phase and solution
peptide synthesis. They still hold their place,
though in recent years two classes of coupling reagents became popular, the phosphonium- and the aminium-(imonium-)
type reagents such as BOP, PyBOP, PyBrOP,
TBTU, HBTU, HATU, COMU, and TFFH. These
compounds achieve high coupling rates accompanied by few undesired side reactions.
In contrast to activation by carbodiimides,
peptide couplings using the latter compounds require the presence of a base. Diisopropylethylamine (DIPEA) and N-methylmorpholine (NMM) are the most frequently
used ones in Fmoc/tBu-based solid-phase
synthesis. In cases of a markedly increased
risk of racemization, the weaker base
sym.-collidine has been recommended for
substituting DIPEA or NMM.
Racemization is one of the main side reactions, when activating carboxyl groups of
amino acids (except for glycine or proline).
Electron-withdrawing groups bound to
the α-amino moiety (e.g. acyl, peptidyl
(i.e.peptide fragments)) increase the
NONE OF THE COUPLING
REAGENTS IS APPLICABLE
TO ALL TYPES OF
COUPLING REACTIONS
3
Coupling Reagents
Fig. 2.
Mechanism of
base-catalyzed
racemization during
activation.
tendency to racemize considerably. Urethane derivatives, which include standard
α-amino protecting groups as Fmoc, Boc,
and Z, of amino acids usually retain their
optical purity upon activation. The mechanism of racemization (Fig. 2) involves the
abstraction of the α-hydrogen from the
α-carbon atom of the activated amino acid,
either by direct formation of an enolic intermediate (direct α-abstraction, path A) or
by formation of a 5-membered oxazolinone
ring (path B), which isomeric aromatic configuration is readily formed in the presence
of bases.
Many other side reactions have been
described so far, depending on the type of
side chain functionalities, the combination
of protecting groups, the reactivity of the
carboxyl group, or the basicity of the amino
function. They will be mentioned later as
special information for the respective coupling reagent.
Every coupling reagent has its individual
advantages and drawbacks. None of them
will render good results for all types of
amino acid derivatives!
This brochure gives information on a large
number of currently available coupling
reagents, their scope and limitations. We
hope that it can help the chemist to make
the appropriate choice.
4
Coupling Reagents and Additives
1. Carbodiimides
DCC
(Dicyclohexylcarbodiimide)
This popular condensation reagent has
been applied for coupling since 1955 and
is still much in use today. DCC is routinely
applied in solution as well as in solid-phase
peptide synthesis, mostly in combination
with additives such as HOBt or HOSu in
order to reduce epimerization in the case
of peptides or racemization in the case of
amino acids. Couplings in solution with
amino acid derivatives provided as salts
(e.g. hydrochlorides) require one equivalent
of a tertiary base (as NMM). Else, the reaction does not require additional base, so
that racemization can be kept minimal.
Application of DCC in solid-phase synthesis
is limited due to the by-product dicyclohexylurea (DCU) with is formed simultaneously
with the coupling reaction (see Fig. 3). This
by-product is sparingly soluble in most
solvents, and therefore DCC should better
be replaced by other carbodiimides e.g. DIC
(diisopropylcarbodiimide).
Fig. 3.
Carbodiimidemediated amide
bond formation.
However, in the manufacture of active
esters, DCC is still one of the reagents of
choice. In solution, the low solubility of DCU
turns into an advantage of this activation
method.
Application of preformed OPfp esters reduces the risk of concomitant racemization
during couplings. Fmoc-AA-OPfp-esters
can be isolated and purified by crystallization. They are stable but highly reactive
building blocks routinely used for couplings
in fully automated SPPS.
Literature:
J. C. Sheehan and G. P. Hess, J. Am. Chem.
Soc. 77, 1067 (1955)
G. W. Anderson and F. M. Callahan, J. Am.
Chem. Soc. 80, 2902 (1958)
I.Schön and L.Kisfaludy, Synthesis 303
(1986)
DIC (or DIPCDI) is a useful reagent for automated SPPS, because the corresponding
urea is soluble in standard solvents such as
isopropanol and can be washed out more
readily than the one obtained from DCC.
If base-free conditions are required as to
minimize racemization, the combination of
DIC and HOBt (or HOAt, Oxyma Pure) is still
one of the best methods e.g. for coupling
Fmoc-Cys(Trt)-OH.
Literature:
A. Williams and I. T. Ibrahim, Chem. Rev. 81,
589 (1981)
L. A. Carpino, A. El-Faham, Tetrahedron 55,
6813 (1999)
EDAC · HCl, EDC · HCl, WSC· HCl (Q-1955)
(N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide · HCl)
DIC
(Diisopropylcarbodiimide)
This water-soluble carbodiimide was especially designed for couplings in aqueous
solution, even though the stability of the
5
Coupling Reagents
Fig. 4.
Mechanism of the
DCC/HOBt-mediated
peptide coupling.
reagent under these conditions is limited.
Normally, EDAC-mediated couplings are
performed in polar solvents such as DMF or
NMP or even in methylene chloride. EDAC
has also been employed in SPPS on highly
polar resins compatible with water-containing solvents. Contrary to DCU, the urea
formed from EDAC is readily soluble.
EDAC is the reagent of choice for the conjugation of peptides, labels, small organic
molecules etc. to proteins.
Literature:
J. C. Sheehan, P. A. Cruickshank, G. L.
Boshart, J. Org. Chem. 26, 2525 (1961)
K. D. Kopple and D.E.Nitecki, J. Am. Chem.
Soc. 84, 4457 (1962)
J. C. Sheehan, J. Preston, P. A. Cruickshank,
J. Am. Chem. Soc. 87, 2492 (1965)
K. Hojo, H. Ichikawa, Y. Fukumori, K. Kawasaki, Int. J. Pept. Res. Ther. 14, 373 (2008)
Y. J. Pu, R. K. Vaid, S. K. Boini, R. W. Towsley,
C. W. Doecke, D. Mitchell, Org. Process Res.
Dev. 13, 310 (2009)
6
In addition to racemization another side
reaction with carbodiimides is an O-Nmigration of the activated carboxyl function forming an N-acyl urea (see Fig. 3).
This stable compound is unable to take
part in further couplings reaction. As this
side reaction depends on the temperature
and coupling behavior of the corresponding amino function, low temperatures are
always recommended in carbodiimidemediated couplings.
Due to the fact, that all carbodiimides are
condensing reagents, side reactions of unprotected amino acid-side chains (Asn, Gln),
such as conversion of amides to nitriles
have been observed. Appropriate side-chain
protecting groups will prevent these side
reactions.
Other side reactions depending on the nature of additives are not a special problem
of carbodiimides and will be described later.
2. Additives
Additives such as HOBt, HOAt or Oxyma
pure® are strongly recommended in all
cases of amide bond formations with carbodiimides, in order to enhance the reactivity and also to reduce formation of epimers
as well as N-acylureas.
Literature:
I.E. Pop, B.P. Déprez, A.L. Tartar, J. Org. Chem.
62, 2594 (1997)
HOOBt (HODhbt)
(Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine)
HOBt
(1-Hydroxybenzotriazole)
N-Hydroxybenzotriazole, developed by W.
König and R. Geiger in 1970 was the most
popular additive during the last decades.
Today, HOBt is still one of the most effective
suppressors of racemization in carbodiimide-mediated reactions.
The most important drawback of the additive lies in its explosive character, especially
in water-free form. Therefore, its availability
is more restricted today and it should be
substituted by more stable additives in the
future.
The mechanism of activation by HOBt used
in combination with DCC is shown in Fig. 4.
Literature:
W. König and R. Geiger, Chem. Ber. 103, 788
(1970)
W. König and R. Geiger, Chem. Ber. 103, 2024
(1970)
HOBt-6-sulfonamidomethyl resin · HCl
(200-400 mesh) (D-2435)
(1-Hydroxybenzotriazole-6-sulfonamidomethyl resin · HCl)
“Polymeric HOBt”, a highly efficient polymeric auxiliary for the synthesis of amides.
Simple filtration allows the separation of
the product from the polymer.
ODhbt esters, generated during couplings
with a carbodiimide and HOOBt or DEPBTmediated couplings, are more reactive than
HOBt esters. The release of the benzotriazine can be monitored spectrophotometrically.
Literature:
E. Atherton, S. Cameron, M. Meldal, R.C.
Sheppard, J. Chem. Soc. Chem. Commun.
1763 (1986)
HOSu (Q-1800)
(N-Hydroxysuccinimide)
In contrast to HOBt and HODhbt, HOSu is
completely shelf-stable. It is also a wellknown additive in carbodiimide-mediated
reactions since many years. On the other
hand HOSu forms stable and isolable active
esters, which are applicable in organic as
well as in aqueous solution.
A drawback of HOSu is due to its hydroxamic acid structure susceptible to the Lossenrearrangement. This side reaction can be
observed under condensation conditions
and leads to introduction of an additional
β-alanine.
Literature:
G. W. Anderson, J. E. Zimmerman, F. M. Callahan, J. Am. Chem. Soc. 86, 1839 (1964)
F. Weygand, D. Hoffmann, E. Wünsch, Z.
Naturforsch. 21b, 426 (1966)
S. J. Davies and A. K. Mohammed, J.Chem.
Soc. Perkin Trans. I, 2982 (1981)
7
Coupling Reagents
HOAt
(1-Hydroxy-7-aza-1H-benzotriazole)
More than HOBt, this additive accelerates
coupling reactions and suppresses racemization. However, the explosive properties of
this compound restrict its applications and
availability.
Literature:
L. A. Carpino, A. El-Faham, Tetrahedron 55,
6813 (1999)
J. Klose, A. El-Faham, P. Henklein, L. A. Carpino, M. Bienert, Tetrahedron Lett. 40, 2045
(1999)
L. A. Carpino, H. Imazumi, B. M. Foxman, M.
J. Vela, P. Henklein, A. El-Faham, J. Klose,
J. Bienert, Org. Lett. 2, 2253 (2000)
Oxyma Pure® (Q-2750)
(Ethyl 2-cyano-2-(hydroximino)acetate)
DMAP
(4-(N,N-Dimethylamino)pyridine)
Esterifications of carboxylic acids with
primary or secondary, aliphatic alcohols
employing carbodiimides proceed smoothly,
when performed in non-polar solvents
(DCM, toluene,…). Nevertheless, a remarkable acceleration is observed, if catalysts as
DMAP are present, forming highly reactive
intermediates. In the case of amino acid
compounds, racemization is often a main
disadvantage of this coupling method,
which could be reduced sometimes by
running the reaction at low temperatures
or applying the alcoholic partner in high
concentrations.
Literature:
S. S. Wang, J. P. Tam, B. S. Wang, R. B. Merrifield, Int. J. Pept. Protein Res. 18, 459 (1981)
J.-P. Gamet, R. Jacquier, J. Verducci, Tetrahedron, 40, 1995 (1984)
K. Takeda, A. Ayabe, M. Suzuki, Y. Konda, Y.
Harigaya, Synthesis 689 (1991)
C. Grondal, Synlett 1568 (2003)
3. Phosphonium Reagents
A trademark of Luxembourg Bio Technologies Ltd, Rechovot, Israel
This more recently developed additive is a
non-explosive alternative to HOBt or HOAt,
and allows high coupling rates at low racemization when applied in combination with
carbodiimides.
In practice, Oxyma Pure can be used in an
identical manner as HOBt in DMF on automated synthesizers.
Literature:
R. Subirós-Funosas, R. Prohens, R. Barbas,
A. El-Faham, F. Albericio, Chem. Eur. J. 15,
9394 (2009)
S. N. Khattab, Bull. Chem. Soc. Jpn. 83, 1374
(2010)
R. Subirós-Funosas, A. El-Faham, F. Albericio, Peptide Science 98, 89 (2012)
8
BOP (Q-1980)
(Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate)
BOP was the first of a broad range of phosphonium type coupling reagents. It was
introduced by Castro et al. already in 1975.
BOP provides excellent coupling behavior
and good solubility in most of the common
solvents, in solid-phase as well as in solution. It converts carboxyl groups into -OBt
esters and has no guanylation-activity to
amino functions as aminium-compounds
like TBTU. It is a useful reagent for lactonization, selective esterification or amidation
of α-amino acids without racemization.
However, its severe drawback is the toxicity
problem due to the carcinogenic HMPA
(hexamethylphosphoramide) formed as byproduct during the reaction.
Literature:
B. Castro, J. R. Dormoy, G. Evin, C. Selve,
Tetrahedron Lett. 14, 1219 (1975)
B. Castro, G. Evin, C. Selve, R. Seyer,
Synthesis, 413 (1977)
J.-A. Fehrentz and B. Castro, Synthesis, 676
(1983)
R. P. McGeary, Tetrahedron Lett. 39, 3319
(1998)
A. Wahhab and J. Leban, Tetrahedron Lett.
40, 235 (1999)
PyBOP® (Q-2715)
(Benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate)
A trademark of Merck KGaA, Darmstadt,
Germany
Introduced as a non-toxic version of BOP,
PyBOP has the same effective coupling
properties in solid phase as BOP.
PyBOP has been used as well for obtaining
peptide thioesters.
Literature:
J. Martinez, J. Laur, B. Castro, J. Med. Chem.
28, 1874 (1985)
J. Coste, D. Le-Nguyen, B. Castro,
Tetrahedron Lett. 31, 205 (1990)
T. Høeg-Jensen M. H. Jakobsen, C. E. Olsen,
A. Holm, Tetrahedron Lett. 32, 7617 (1991)
R. von Eggelkraut-Gottanka, A. Klose, A. G.
Beck-Sickinger, M. Beyermann, Tetrahedron
Lett. 44, 3551 (2002)
PyBrOP®
(Bromo-tripyrrolidino-phosphonium hexafluorophosphate)
A trademark of Merck KGaA, Darmstadt,
Germany
This reagent was developed by J. Coste, to
overcome the lack of PyBOP®, as well as
other HOBt-containing coupling reagents, in
incomplete couplings to N-methyl-aminoacids. Further good results were reported in
coupling of Aib-derivatives.
Additionally, due to its high reactivity,
PyBrOP® is not a standard coupling reagent
for all amino acids. The formation of oxazolones during prolonged couplings accompanied by higher racemization limits the use
of the coupling reagent.
Literature:
J. Coste, M.-N. l. Dufour, A. Pantaloni,
B. Castro, Tetrahedron Lett. 31, 669 (1990)
J. Coste, E. Frérot, P. Jouin, B. Castro,
Tetrahedron Lett. 32, 1967 (1991)
S. Gazal, G. Gellerman, E. Glukhov, C. Gilon,
J. Pept. Res. 58, 527 (2001)
PyAOP
(7-Aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate)
The HOAt-analog to PyBOP®, developed by
L. Carpino gave also remarkable faster coupling rates as PyBOP®, due to the enhanced
electron withdrawing effect of the corresponding formed -OAt active esters during
coupling reaction.
9
Coupling Reagents
Contrary to HATU PyAOP cannot react with
amino groups yielding guanidines.
it is the reagent of choice for coupling the
racemization prone Fmoc-His(Trt)-OH.
Literature:
F. Albericio, M. Cases, J. Alsina, S. A. Triolo,
L. A. Carpino, S. A. Kates, Tetrahedron Lett.
38, 4853 (1997)
F. Albericio, J. M. Bofill, A. El-Faham,
S. A. Kates, J. Org. Chem. 63, 9678 (1998)
Literature:
C.-X. Fan, X.-L. Hao, Y.-H. Ye, Synth. Commun. 26, 1455 (1996)
H. Li, X. Jiang, Y. H. Ye, C. Fan, T. Romoff, M.
Goodman, Org. Lett. 1, 91 (1999)
M. Mergler, F. Dick, T. Vorherr, Innovation and
Perspectives in Solid Phase Synthesis and
Combinatorial Libraries, 7th International
Symposium, Southampton, p. 235, R. Epton,
ed. Mayflower Worldwide (2002)
F. Dettner, A. Hänchen, D. Schols, L. Toti, A.
Nusser, R. D. Süssmuth, Angew. Chem. Int.
Ed. Engl. 48, 1856 (2009)
PyOxim (Q-2760)
(Ethyl cyano(hydroxyimino)acetato-O2)tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate)
This recently developed coupling reagent
contains Oxyma pure as part of the molecule instead of the explosive compounds
HOBt or HOAt. This improvement of safety
in handling is accompanied with an accelerated reactivity in couplings as well as
a minimized allergenic potential, making
PyOxim to one of the best coupling reagents
in solid-phase reactions. The only drawback
is the formation of tris-pyrrolidinophosphamide as side product, which can cause
problems in separation when the reagent is
applied in solution.
Literature:
R. Subiros-Funosas, A. El-Faham,
F. Albericio, Org. Biomol. Chem. 8, 3665
(2010)
DEPBT (Q-2565)
(3-(Diethoxy-phosphoryloxy)-1,2,3-benzo[d]
triazin-4(3H)-one)
Phosphonium-based coupling reagents do
not affect amino groups and are well suited
to be used as cyclization reagents, when
applied in excess.
Two classes of building blocks should be
disclaimed from coupling procedures with
phosphonium reagents:
a. Phosphorylated amino acids (FmocSer(PO,OH,OBzl)-OH,…) can undergo undesired couplings between reagent and the
unprotected phosphoryl-side chain.
b. Compounds containing nucleotides (e.g.
PNA-building blocks) with oxo-functions
like guanine, etc. are prone to be attacked
under structural rearrangement.
Literature:
S. Pritz, Y. Wolf, C. Klemm, M. Bienert, Peptides 2006, Proceedings of the 29th European Peptide Symposium, Gdansk, Poland,
p. 472, edited by K. Rolka, P. Rekowski and J.
Silbering (2006)
J. W. Perich, N. J. Ede, S. Eagle, A. M. Bray,
Lett. Pept. Sci. 6, 91 (1999)
4. Aminium/Uronium-Imonium Reagents
TBTU (BF4-) (Q-1665)/ HBTU (PF6-)
(2-(1H-Benzotriazol-1-yl)-N,N,N’,N’tetramethylaminium tetrafluoroborate/
hexafluorophosphate)
Contrary to the other described phosphonium reagents, DEPBT is a mixed anhydride of
HOOBt and dietylphosphoric acid. It mediates amide bond formations with a remarkable resistance to racemization. Therefore
10
Fig. 5.
Coupling with TBTU
in the presence of
DIPEA.
These two compounds, only differing in
their counterions (BF4-, PF6-) have nearly
identical chemical properties and belong to
the most popular coupling reagents. Their
application is widespread in solid-phase
reactions as well as in solution, because all
resulting by-products are soluble in water
as well as in standard organic solvents
(see Fig. 5). For couplings of phosphorylated amino acids TBTU and HBTU are the
reagents of choice.
Literature:
P. Henklein, C. Mügge, B. Costisella, V. Wray,
T. Domke, A. El-Faham, Y. Lee, S A. Kates, L.
A. Carpino, Innovation and Perspectives in
Solid-Phase Synthesis and Combinatorial
Libraries, 5th International Symposium,
London, p. 309, R. Epton, ed. Mayflower
Scientific (1998)
R. Knorr, A. Trzeciak, W. Bannwarth, D.
Gillessen, Peptides 1988, Proceedings of
the 20th European Peptide Symposium,
Tübingen, p. 37, G. Jung and E. Bayer, eds. W.
de Gruyter, Berlin (1989)
R. Knorr, A. Trzeciak, W. Bannwarth, D. Gillessen, Tetrahedron Lett. 30, 1927 (1989)
I. Abdelmoty, F. Albericio, L. A. Carpino,
B. M. Foxman, S. A. Kates, Lett. Pept. Sci. 1,
57 (1994)
J. M. Matsoukas, D. Panagiotopoulos,
M. Keramida, T. Mavromoustakos, R. Yamdagni, Q. Wu, G. J. Moore, M. Saifeddine,
M. D. Hollenberg, J. Med. Chem. 39, 3585
(1996)
R. F. Poulain, A. L. Tartar, B. P. Déprez, Tetrahedron Lett. 42, 1495 (2001)
A well-known limitation of aminium/
uronium-derivatives is a possible reaction
with free amino groups yielding guanidines,
when the coupling reagent is applied in
excess (Fig. 6). This side reaction is normally
inhibited by application of a slight excess of
carboxyl compound in relation to coupling
reagent and by a short period of preactivation before adding to the amino compound.
HCTU
(2-(6-Chloro-1H-benzotriazol-1-yl)N,N,N’,N’-tetramethylaminium hexafluorophosphate)
The replacement of HOBt by 6-Chloro-HOBt
as part of the molecule, leads to higher
reaction rates and improved results in the
synthesis of difficult peptides.
Literature:
O. Marder, Y. Shvo, F. Albericio, Chimica Oggi
20, 37 (2002)
O. Marder and F. Albericio, Chimica Oggi 21,
6 (2003)
11
Coupling Reagents
Fig. 6.
N-Terminal guanidinylation by the
coupling reagent.
HDMC (Q-2765)
(N-[(5-Chloro-1H-benzotriazol-1-yl)dimethylamino-morpholino]-uronium
hexafluorophosphate N-oxide)
solution-phase reactions. Contrary to
HOBt-based reagents they have been used
successfully in couplings of N-methylamino acids.
Literature:
L. A. Carpino, J. Am. Chem. Soc. 115, 4397
(1993)
F. Albericio, J. M. Bofill, A. El-Faham, S. A.
Kates, J. Org. Chem. 63, 9678 (1998)
When modifying the uronium part of HCTU
by introduction of a morpholine moiety, a
further increase of reactivity can be gained.
Even coupling rates of HATU could be exceeded in some cases by HDMC.
COMU (Q-2735)
(1-[1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]uronium hexafluorophosphate)
Literature:
A. El-Faham and F. Albericio, J. Org. Chem.
73, 2731 (2008)
TATU (BF4-) (Q-2150)/ HATU (PF6-) (Q-2780)
(2-(7-Aza-1H-benzotriazol-1-yl)-N,N,N’,N’tetramethylaminium tetrafluoroborate/
hexafluorophosphate)
The HOAt analogs to HBTU/TBTU are highly
efficient coupling reagents for solid- and
12
COMU is a novel coupling reagent with
coupling efficiencies comparable to HATU.
The incorporation of Oxyma Pure as part
of the molecule in place of the explosive
compounds HOBt or HOAt results in safer
handling in combination with better solubility and a reduced allergenic potential than
HBTU/TBTU or HATU. COMU is especially
suited for microwave-accelerated SPPS.
Recently it has been demonstrated that
COMU can be used to prepare esters of
all types of alcohols at room temperature
under mild conditions in the presence of
organic bases.
Fig. 7.
EEDQ-mediated
generation of mixed
anhydride.
Literature:
A. El-Faham and F. Albericio, J. Org. Chem.
73, 2731 (2008)
A. El-Faham, R. Subiros Funosas, R. Prohens, F. Albericio, Chem. Eur. J. 15, 9404
(2009)
R. Subiros-Funosas, G. A. Acosta, A. ElFaham, F. Albericio, Tetrahedron Lett. 50,
6200 (2009)
A. El-Faham and F. Albericio, J. Pept. Sci. 16,
6 (2010)
J.K. Twibanire and T.B. Grindley et al.Org.
Lett. 13, 2988 (2011)
TOTT (Q-2600)
(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethylisothiouronium tetrafluoroborate)
TOTT is a thiuronium salt of 2-mercaptopyridone-1-oxide which showed good results in
couplings of sterically hindered or methylated amino acids, comparable with HATU.
Racemization levels of those couplings are
reported to be lower as with other reagents.
TFFH (Q-2280)
(Tetramethylfluoroformamidinium hexafluorophosphate)
A highly efficient coupling reagent which
generates amino acid fluorides in situ.
Amino acid fluorides are especially suited
for the coupling of sterically hindered α,αdisubstituted amino acids such as Aib.
Literature:
L.A. Carpino and A. El-Faham, J. Am. Chem.
Soc. 117, 5401 (1995)
A. El-Faham, OPPI Briefs 30, 477 (1998)
A. El-Faham and S.N. Khattab, Synlett 886
(2009)
S. N. Khattab, Bull. Chem. Soc. Jpn. 83, 1374
(2010)
5. Miscellaneous Coupling Reagents
EEDQ (Q-1735)
(N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline)
Literature:
M. A. Bailén, R. Chinchilla, D. J. Dodsworth,
C. Nájera, J. Org. Chem. 64, 8936 (1999)
EEDQ is an “old” reagent (developed in
1967), which still is of interest due to its re-
13
Coupling Reagents
Fig. 8.
Mechanism
of DMTMMmediated
amide bond
formation.
markable properties. It converts the amino
acid derivative into an anhydride EEDQ
does not require the presence of a tertiary
base nor does it affect the amino function
or other reactive groups such as hydroxyls
(Fig. 7). Albeit the formation of the anhydride is slow, it is consumed very rapidly by
the amino-component which minimizes
racemization during product formation.
As an example, EEDQ was the only reagent
amongst the tested ones which mediated
the coupling 3,5-dinitrobenzoyl-Leu-OH to
3-aminopropyl-silica with negligible level of
concomitant racemization.
T3P
(2-Propanephosphonic acid anhydride)
Literature:
B. Belleau and G. Malek, J. Am. Chem. Soc.
90, 1651 (1968)
B. Belleau, R. R. Martel, G. Lacasse, M. Menard, N. L. Weinberg, Y. G. Perron, J. Am. Chem.
Soc. 90, 823 (1968)
R. Pougeois, FEBS Lett. 154, 47 (1983)
H. Kunz, H. Waldmann, C. Unverzagt, Int. J.
Pept. Protein Res. 26, 493 (1985)
M. H. Hyun, M. H. Kang, S. C. Han,
Tetrahedron Lett. 40, 3435 (1999)
A. Yang, A. P. Gehring, T. Li, J. Chromatogr. A
878, 165 (2000)
Merck Index, 14th ed. No. 3518, (2006)
Literature:
J. Klose, M. Bienert, C. Mollenkopf, D. Wehle,
C.-W. Zhang, L. A. Carpino, P. Henklein,
Chem. Commun. 1847 (1999)
P. Koch, C. Vedder, T. Schaffer, Chimica Oggi
26, 6 (2008)
A trademark of Euticals
T3P (or PPA) is a cyclic anhydride of propylphosphonic acid, which is normally used
in combination with tertiary amines for
solution-phase and cyclization reactions.
PPA gives superior results especially for
sterically hindered peptides.
DMTMM and related compounds
(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium salts)
Triazines - a group of coupling reagents,
developed by Kaminski et al. - are condensation products of substituted cyanuric
14
chloride with tertiary amines as NMM or
DABCO, able to mediate peptide couplings
in water or alcoholic solutions. These
reagents proved to be particularly efficient
allowing high yields and low racemization
levels. For the coupling mechanism of
DMTMM see Fig. 8.
Literature:
K. Jastrzabek, B. Kolesinska, G. Sabatino, F.
Rizzolo, A. Papini, Z. Kaminski, Int. J. Pept.
Res .Ther. 13, 229 (2007)
Z. J. Kaminski, B. Kolesinska, J. Kolesinska,
G. Sabatino, M. Chelli, P. Rovero, M. Blaszczyk, M. L. Glowka, A. M. Papini, J. Am. Chem.
Soc. 127, 16912 (2005)
B. Kolesinska, J. Fraczyk, G. Sabatino, A.
Papini, Z. Kaminski, Chimica Oggi 25, 26
(2007).
BTC
(bis-Trichloromethylcarbonate or “Triphosgene”)
The stable trimeric form of phosgene is a
well-known reagent for generating acid
chlorides from carboxylic acids. Its applicability for peptide couplings was evaluated
by C. Gilon et al. for solution- and solidphase couplings. They used collidine as
base and THF or DCM as solvents. Other
solvents such as DMF or NMP have to be
strictly avoided, as they can react with BTC.
Care must be taken when handling the
reagent, because BTC and even more so
phosgene which is formed as intermediate
are highly toxic compounds. Beyond these
restrictions, BTC is one of the most efficient
activation reagents and recommended
especially for difficult couplings and less
reactive amines such as anilines.
Literature:
E. Falb, T. Yechezkel, Y. Salitra, C. Gilon,
J. Pept. Res. 53, 507 (1999)
B. Thern, J. Rudolph, G. Jung, Tetrahedron
Lett. 43, 5013 (2002)
CDI
(1,1’-Carbonyldiimidazole)
CDI, as the related compounds disuccinimidyl carbonate or 4-nitrophenyl-chloroformate, is a phosgene analog, a derivative of
carbonic acid. CDI is not routinely used for
peptide couplings. Its field of application
lies in the synthesis of ureas and urethanes
from amines, alcoholic compounds or resins
and linkers.
Nevertheless CDI has been successfully
used in peptide couplings. The protocol for
couplings has to include a preactivation
step during which the reactive acid imidazolide is formed, which is added to the amino
component.
Literature:
G.W. Anderson and R. Paul, J. Am. Chem.
Soc. 80, 4423 (1958)
H. Ogura, T. Kobayashi, K. Shimizu, K. Kawabe, K. Takeda, Tetrahedron Lett. 20, 4745
(1979)
K. Takeda, Y. Akagi, A. Saiki, T. Tsukahara, H.
Ogura, Tetrahedron Lett. 24, 4569 (1983)
T. Kamijo, H. Harada, K. Iizuka, Chem.
Pharm. Bull. 32, 5044 (1984)
Conclusion
TBTU, HBTU and PyBOP are well suited
reagents for most standard coupling
reactions. They all contain the potentially
explosive HOBt as part of the molecule. The
same problem is encountered with the more
reactive HOAt-based coupling reagents.
Due to safety considerations, all of them
will have to be replaced in the future by
Oxyma Pure-based reagents such as COMU
or PyOxim.
More specialized reagents as HATU, HDMC,
TOTT or DEPBT can be required to succeed
in incorporating amino acid derivatives
prone to side reactions. However, “old” reagents as EEDQ still can be useful in cases
where other reagents rendered poor results.
Bachem offers a large selection of coupling
reagents to meet all requirements of the
synthetic chemist.
15
Coupling Reagents
REFERENCES
For more information as well as many
valuable hints regarding properties and application of coupling reagents the following
review articles are recommended:
F. Albericio and L.A. Carpino
Coupling reagents and activation. Methods
for solid-phase assembly of peptides.
Meth. Enzymol. 289, 104-126 (1997)
F. Albericio, R. Chinchilla, D. J. Dodsworth,
C. Nájera
New trends in peptide coupling reagents.
Org. Prep. Proced. Int. 33, 203-303 (2001)
S.-Y. Han and Y.-A. Kim
Recent development of peptide coupling
reagents in organic synthesis.
Tetrahedron 60, 2447-2467 (2004)
A.R. Katritzky, K. Suzuki, K. Singh
N-Acylation in combinatorial chemistry.
ARKIVOC 12-35 (2004)
C.A.G.N. Montalbetti and V. Falque
Amide bond formation and peptide coupling.
Tetrahedron 61, 10827-10852 (2005)
E. Valeur and M. Bradley
Amide bond formation: beyond the myth of
coupling reagents.
Chem. Soc. Rev. 38, 606-631 (2009)
16
M.M. Joullié and K.M. Lassen
Evolution of amide bond formation.
ARKIVOC 189-250 (2010)
C. Roche, M. Pucheault, M. Vaultier,
A. Commerçon
Onium salt supported peptide synthesis.
Tetrahedron 66, 8325-8334 (2010)
A. El-Faham and F. Albericio
Peptide coupling reagents: more than a
letter soup.
Chem. Rev. 111, 6557-6602 (2011)
V.R. Pattabiraman and J.W. Bode
Rethinking amide bond synthesis.
Nature 480, 471-479 (2011)
T.I. Al-Warhi, H.M.A. Al-Hazimi, A. ElFaham
Recent development in peptide coupling
reagents.
J. Saudi Chem. Soc. 16, 97-116 (2012)
R. Subirós-Funosas, S.N. Khattab, L.
Nieto-Rodríguez, A. El-Faham, F. Albericio
Advances in acylation methodologies enabled by Oxyma-based reagents.
Aldrichim. Acta 46, 21-40 (2013)
J. R. Dunetz, J. Magano, G. A. Weisenburger
Large-Scale Applications of Amide Coupling
Reagents for the Synthesis of Pharmaceuticals.
Org. Proc. Res. Devel.20, 140-177 (2016)
COUPLING
REAGENTS
AND
ADDITIVES
We also offer a comprehensive choice of pre-formed active esters of
Fmoc-, Boc-, and Z-amino acids:
Fmoc-Xaa-OPfp and Fmoc-Xaa-OSu
Boc-Xaa-ONp and Boc-Xaa-OSu
Z-Xaa-ONp and Z-Xaa-OSu
for all strategies of peptide synthesis.
Appropriately protected active esters of the proteinogenic amino acids
as well as of unusual amino acids can be found in our online shop
shop.bachem.com
17
Coupling Reagents
COUPLING
REAGENTS
BOP
(Benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate)
Q-1980
COMU
(1-[1-(Cyano-2-ethoxy2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate)
Q-2735
DEPBT
(3-(Diethoxy-phosphoryloxy)-1,2,3benzo[d]triazin-4(3H)-one)
Q-2565
EDAC · HCl, EDC · HCl, WSC · HCl
(N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide · HCl)
Q-1955
EEDQ
(N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline)
Q-1735
HATU
(N-[(7-Aza-1H-benzotriazol-1-yl)
(dimethylamino)-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide)
Q-2780
PyBOP
(Benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate)
Q-2715
PyOxim
((Ethyl cyano(hydroxyimino)acetatoO2)-tri-(1-pyrrolidinyl)-phosphonium
hexafluorophosphate)
Q-2760
TATU
(N-[(7-Aza-1H-benzotriazol-1-yl)
(dimethylamino)-methylene]-N-methylmethanaminium tetrafluoroborate
N-oxide)
Q-2150
TBTU
(N-[(1H-Benzotriazol-1-yl)
(dimethylamino)-methylene]-N-methylmethanaminium tetrafluoroborate
N-oxide)
Q-1665
TFFH
(Tetramethylfluoroformamidinium
hexafluorophosphate)
Q-2280
TOTT
(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethylisothiouronium tetrafluoroborate)
Q-2600
HDMC
(N-[(5-Chloro-1H-benzotriazol-1-yl)dimethylamino-morpholino]-uronium
hexafluorophosphate N-oxide)
Q-2765
ADDITIVES
HOSu
(N-Hydroxysuccinimide)
Q-1800
Oxyma Pure
(Cyano-hydroxyimino-acetic ethyl
ester)
Q-2750
18
HOBt-6-sulfonamidomethyl resin ·
HCl (200-400 mesh)
(1-Hydroxybenzotriazole-6-sulfonamidomethyl resin · HCl)
D-2435
PRODUCT BROCHURES
AMYLOID
PEPTIDES
ANTIMICROBIAL
PEPTIDES
CALCITONIN
GENE-RELATED
PEPTIDES
CASPASE
SUBSTRATES
INHIBITORS
CYSTEINE
DERIVATIVES
DAP AND DAB
DERIVATIVES
DIABETES
PEPTIDES
ENDOTHELINS
FRET
SUBSTRATES
GHRELIN,
LEPTIN AND
OBESTATIN
1
1
LHRH
AGONISTS AND
ANTAGONISTS
MATRIX
METALLOPROTEINASES
MELANOMA
PEPTIDES
N-METHYLATED
AMINO ACID
DERIVATIVES
NEUROPEPTIDE Y
NON-IONIC
DETERGENTS
ORTHOGONALITY
OF PROTECTING
GROUPS
PAR
ACTIVATING
PEPTIDES
PEPTIDE YY
PEPTIDES
IN COSMETICS
SECRETASE
SUBSTRATES
INHIBITORS
VETERINARY
PEPTIDES
VIP/PACAP



PRION
PEPTIDES
PSEUDOPROLINE
DIPEPTIDES
Europe, Africa, Middle East and Asia Pacific
Bachem AG
Tel. +41 58 595 2020
sales.ch@bachem.com
Americas
Bachem Americas, Inc.
Tel. +1 888 422 2436 (toll free in USA & Canada)
+1 310 539 4171
sales.us@bachem.com
Visit our website
www.bachem.com
or shop online
shop.bachem.com
All information is compiled to the
best of our knowledge. We cannot be
made liable for any possible errors
or misprints. Some products may be
restricted in certain countries.
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2011123 published by Global Marketing, Bachem Group, August 2016
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