COUPLING

REAGENTS

H R H R

Cα Cα

PG1 NH COOH NH2 COO PG2

H R H R

Cα Cα

PG1 NH 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 consecu-
tive 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

in peptide synthesis:
The formation of an amide bond between a The first step of this condensation reaction,
carboxylic acid moiety and an amino func- the activation of the carboxyl moiety
tion of two amino acids is the core reaction (I. in Fig. 1) is often the critical one. Depend-

ing on the type of activating reagent, the

intermediate A is a stable compound which
PEPTIDE could be isolated if desired. During the
second step (II. in Fig. 1), A is attacked by a
SYNTHESIS nucleophile such as the α-amino group of a
carboxy-protected amino acid. Steps I and
Our comprehensive offer of coupling II can be performed either consecutively or,
reagents, amino acids derivatives and with certain types of coupling reagents, as
further building blocks, and resins a one-pot reaction.
at www.bachem.com will fulfill the
needs of solid-phase and solution Over the years, numerous activation
peptide synthesis. If the product you methods have been developed, such as the
need is lacking, please let us know. generation of carboxylic halides (chlorides,
fluorides), carboxylic azides, symmetrical
or mixed anhydrides or the use of carbodi-

2
 Fig. 1.
Activation and cou-
pling of a protected
amino acid.
PG, PG’: protecting
groups
Act: activating
group

imides (DCC, DIC, EDC · HCl) with or without type reagents such as BOP, PyBOP, PyBrOP,
additives. TBTU, HBTU, HATU, COMU, and TFFH. These
If the amino compound contains other func- compounds achieve high coupling rates ac-
tional groups able to take part in the coupl- companied by few undesired side reactions.
ing reaction, use of a stable preactivated In contrast to activation by carbodiimides,
carboxylic compound A is recommended. peptide couplings using the latter com-
Active esters such as the pentafluorophe- pounds require the presence of a base. Di-
nyl (OPfp) and hydroxysuccinimido (OSu or isopropylethylamine (DIPEA) and N-methyl-
NHS) esters are reactive amino acid deriva- morpholine (NMM) are the most frequently
tives finding broad application in peptide used ones in Fmoc/tBu-based solid-phase
synthesis. For our comprehensive offer of synthesis. In cases of a markedly increased
active esters please see our online shop. risk of racemization, the weaker base
sym.-collidine has been recommended for
Coupling Reagents substituting DIPEA or NMM.
Carbodiimides have been used as activa- Racemization is one of the main side reac-
tors for decades in solid-phase and solution tions, when activating carboxyl groups of
peptide synthesis. They still hold their place, amino acids (except for glycine or proline).
though in recent years two classes of cou- Electron-withdrawing groups bound to
pling reagents became popular, the phos- the α-amino moiety (e.g. acyl, peptidyl
phonium- and the aminium-(imonium-) (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. Ure- Coupling Reagents and Additives

thane derivatives, which include standard
α-amino protecting groups as Fmoc, Boc, 1. Carbodiimides
and Z, of amino acids usually retain their
optical purity upon activation. The mecha- DCC
nism of racemization (Fig. 2) involves the (Dicyclohexylcarbodiimide)
abstraction of the α-hydrogen from the
α-carbon atom of the activated amino acid,
either by direct formation of an enolic in-
termediate (direct α-abstraction, path A) or
by formation of a 5-membered oxazolinone
ring (path B), which isomeric aromatic con- This popular condensation reagent has
figuration is readily formed in the presence been applied for coupling since 1955 and
of bases. is still much in use today. DCC is routinely
Many other side reactions have been applied in solution as well as in solid-phase
described so far, depending on the type of peptide synthesis, mostly in combination
side chain functionalities, the combination with additives such as HOBt or HOSu in
of protecting groups, the reactivity of the order to reduce epimerization in the case
carboxyl group, or the basicity of the amino of peptides or racemization in the case of
function. They will be mentioned later as amino acids. Couplings in solution with
special information for the respective cou- amino acid derivatives provided as salts
pling reagent. (e.g. hydrochlorides) require one equivalent
Every coupling reagent has its individual of a tertiary base (as NMM). Else, the reac-
advantages and drawbacks. None of them tion does not require additional base, so
will render good results for all types of that racemization can be kept minimal.
amino acid derivatives! Application of DCC in solid-phase synthesis
This brochure gives information on a large is limited due to the by-product dicyclohex-
number of currently available coupling ylurea (DCU) with is formed simultaneously
reagents, their scope and limitations. We with the coupling reaction (see Fig. 3). This
hope that it can help the chemist to make by-product is sparingly soluble in most
the appropriate choice. solvents, and therefore DCC should better
be replaced by other carbodiimides e.g. DIC
(diisopropylcarbodiimide).

4
 Fig. 3.
Carbodiimide-
mediated amide bond
formation.

However, in the manufacture of active DIC (or DIPCDI) is a useful reagent for au-
esters, DCC is still one of the reagents of tomated SPPS, because the corresponding
choice. In solution, the low solubility of DCU urea is soluble in standard solvents such as
turns into an advantage of this activation isopropanol and can be washed out more
method. readily than the one obtained from DCC.
Application of preformed OPfp esters re- If base-free conditions are required as to
duces the risk of concomitant racemization minimize racemization, the combination of
during couplings. Fmoc-AA-OPfp-esters DIC and HOBt (or HOAt, Oxyma Pure) is still
can be isolated and purified by crystal- one of the best methods e.g. for coupling
lization. They are stable but highly reactive Fmoc-Cys(Trt)-OH.
building blocks routinely used for couplings
in fully automated SPPS. Literature:
A. Williams and I. T. Ibrahim, Chem. Rev. 81,
Literature: 589 (1981)
J. C. Sheehan and G. P. Hess, J. Am. Chem. L. A. Carpino, A. El-Faham, Tetrahedron 55,
Soc. 77, 1067 (1955) 6813 (1999)
G. W. Anderson and F. M. Callahan, J. Am.
Chem. Soc. 80, 2902 (1958) EDAC · HCl, EDC · HCl, WSC· HCl (4031236)
I.Schön and L.Kisfaludy, Synthesis 303 (N-(3-Dimethylaminopropyl)-N’-ethylcar-
(1986) bodiimide · HCl)

DIC
(Diisopropylcarbodiimide)

This water-soluble carbodiimide was es-

pecially designed for couplings in aqueous
solution, even though the stability of the

5
Coupling Reagents

Fig. 4.
Mechanism of the DCC/
HOBt-mediated pep-
tide coupling.

reagent under these conditions is limited. In addition to racemization another side

Normally, EDAC-mediated couplings are reaction with carbodiimides is an O-N-
performed in polar solvents such as DMF or migration of the activated carboxyl func-
NMP or even in methylene chloride. EDAC tion forming an N-acyl urea (see Fig. 3).
has also been employed in SPPS on highly This stable compound is unable to take
polar resins compatible with water-con- part in further couplings reaction. As this
taining solvents. Contrary to DCU, the urea side reaction depends on the temperature
formed from EDAC is readily soluble. and coupling behavior of the correspond-
EDAC is the reagent of choice for the con- ing amino function, low temperatures are
jugation of peptides, labels, small organic always recommended in carbodiimide-
molecules etc. to proteins. mediated couplings.
Due to the fact, that all carbodiimides are
Literature: condensing reagents, side reactions of un-
J. C. Sheehan, P. A. Cruickshank, G. L. protected amino acid-side chains (Asn, Gln),
Boshart, J. Org. Chem. 26, 2525 (1961) such as conversion of amides to nitriles
K. D. Kopple and D.E.Nitecki, J. Am. Chem. have been observed. Appropriate side-chain
Soc. 84, 4457 (1962) protecting groups will prevent these side
J. C. Sheehan, J. Preston, P. A. Cruickshank, reactions.
J. Am. Chem. Soc. 87, 2492 (1965) Other side reactions depending on the na-
K. Hojo, H. Ichikawa, Y. Fukumori, K. Kawa- ture of additives are not a special problem
saki, Int. J. Pept. Res. Ther. 14, 373 (2008) of carbodiimides and will be described later.
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
2. Additives Literature:
I.E. Pop, B.P. Déprez, A.L. Tartar, J. Org. Chem.
Additives such as HOBt, HOAt or Oxyma 62, 2594 (1997)
pure® are strongly recommended in all
cases of amide bond formations with car- HOOBt (HODhbt)
bodiimides, in order to enhance the reactiv- (Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzo-
ity and also to reduce formation of epimers triazine)
as well as N-acylureas.

HOBt
(1-Hydroxybenzotriazole)

ODhbt esters, generated during couplings

with a carbodiimide and HOOBt or DEPBT-
mediated couplings, are more reactive than
HOBt esters. The release of the benzotri-
N-Hydroxybenzotriazole, developed by W. azine can be monitored spectrophotometri-
König and R. Geiger in 1970 was the most cally.
popular additive during the last decades.
Today, HOBt is still one of the most effective Literature:
suppressors of racemization in carbodi- E. Atherton, S. Cameron, M. Meldal, R.C.
imide-mediated reactions. Sheppard, J. Chem. Soc. Chem. Commun.
The most important drawback of the addi- 1763 (1986)
tive lies in its explosive character, especially
in water-free form. Therefore, its availability HOSu (4025878)
is more restricted today and it should be (N-Hydroxysuccinimide)
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: In contrast to HOBt and HODhbt, HOSu is

W. König and R. Geiger, Chem. Ber. 103, 788 completely shelf-stable. It is also a well-
(1970) known additive in carbodiimide-mediated
W. König and R. Geiger, Chem. Ber. 103, 2024 reactions since many years. On the other
(1970) hand HOSu forms stable and isolable active
esters, which are applicable in organic as
HOBt-6-sulfonamidomethyl resin · HCl well as in aqueous solution.
(200-400 mesh) (4027860) A drawback of HOSu is due to its hydroxam-
(1-Hydroxybenzotriazole-6-sulfonamido- ic acid structure susceptible to the Lossen-
methyl resin · HCl) rearrangement. 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. Cal-
“Polymeric HOBt”, a highly efficient poly- lahan, J. Am. Chem. Soc. 86, 1839 (1964)
meric auxiliary for the synthesis of amides. F. Weygand, D. Hoffmann, E. Wünsch, Z.
Simple filtration allows the separation of Naturforsch. 21b, 426 (1966)
the product from the polymer. S. J. Davies and A. K. Mohammed, J.Chem.
Soc. Perkin Trans. I, 2982 (1981)

7
Coupling Reagents

HOAt DMAP
(1-Hydroxy-7-aza-1H-benzotriazole) (4-(N,N-Dimethylamino)pyridine)

Esterifications of carboxylic acids with

More than HOBt, this additive accelerates primary or secondary, aliphatic alcohols
coupling reactions and suppresses racemi- employing carbodiimides proceed smoothly,
zation. However, the explosive properties of when performed in non-polar solvents
this compound restrict its applications and (DCM, toluene,…). Nevertheless, a remark-
availability. able acceleration is observed, if catalysts as
DMAP are present, forming highly reactive
Literature: intermediates. In the case of amino acid
L. A. Carpino, A. El-Faham, Tetrahedron 55, compounds, racemization is often a main
6813 (1999) disadvantage of this coupling method,
J. Klose, A. El-Faham, P. Henklein, L. A. Car- which could be reduced sometimes by
pino, M. Bienert, Tetrahedron Lett. 40, 2045 running the reaction at low temperatures
(1999) or applying the alcoholic partner in high
L. A. Carpino, H. Imazumi, B. M. Foxman, M. concentrations.
J. Vela, P. Henklein, A. El-Faham, J. Klose, Literature:
J. Bienert, Org. Lett. 2, 2253 (2000) S. S. Wang, J. P. Tam, B. S. Wang, R. B. Merri-
field, Int. J. Pept. Protein Res. 18, 459 (1981)
Oxyma Pure® (4019721) J.-P. Gamet, R. Jacquier, J. Verducci, Tetra-
(Ethyl 2-cyano-2-(hydroximino)acetate) hedron, 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 Technolo-
gies Ltd, Rechovot, Israel BOP (4031234)
This more recently developed additive is a (Benzotriazol-1-yloxy-tris(dimethylamino)-
non-explosive alternative to HOBt or HOAt, phosphonium hexafluorophosphate)
and allows high coupling rates at low race-
mization when applied in combination with
carbodiimides.
In practice, Oxyma Pure can be used in an
identical manner as HOBt in DMF on auto-
mated synthesizers.

Literature:
R. Subirós-Funosas, R. Prohens, R. Barbas,
A. El-Faham, F. Albericio, Chem. Eur. J. 15, BOP was the first of a broad range of phos-
9394 (2009) phonium type coupling reagents. It was
S. N. Khattab, Bull. Chem. Soc. Jpn. 83, 1374 introduced by Castro et al. already in 1975.
(2010) BOP provides excellent coupling behavior
R. Subirós-Funosas, A. El-Faham, F. Alberi- and good solubility in most of the common
cio, Peptide Science 98, 89 (2012) solvents, in solid-phase as well as in solu-
tion. It converts carboxyl groups into -OBt
esters and has no guanylation-activity to
amino functions as aminium-compounds

8
like TBTU. It is a useful reagent for lactoni- PyBrOP®
zation, selective esterification or amidation (Bromo-tripyrrolidino-phosphonium hexa-
of α-amino acids without racemization. fluorophosphate)
However, its severe drawback is the toxicity
problem due to the carcinogenic HMPA
(hexamethylphosphoramide) formed as by-
product 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, A trademark of Merck KGaA, Darmstadt,
Synthesis, 413 (1977) Germany
J.-A. Fehrentz and B. Castro, Synthesis, 676 This reagent was developed by J. Coste, to
(1983) overcome the lack of PyBOP®, as well as
R. P. McGeary, Tetrahedron Lett. 39, 3319 other HOBt-containing coupling reagents, in
(1998) incomplete couplings to N-methyl-amino-
A. Wahhab and J. Leban, Tetrahedron Lett. acids. Further good results were reported in
40, 235 (1999) coupling of Aib-derivatives.
Additionally, due to its high reactivity,
PyBOP® (4031604) PyBrOP® is not a standard coupling reagent
(Benzotriazol-1-yloxy-tripyrrolidino-phos- for all amino acids. The formation of oxazo-
phonium hexafluorophosphate) lones during prolonged couplings accompa-
nied 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,
A trademark of Merck KGaA, Darmstadt, J. Pept. Res. 58, 527 (2001)
Germany
Introduced as a non-toxic version of BOP, PyAOP
PyBOP has the same effective coupling (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-
properties in solid phase as BOP. phosphonium hexafluorophosphate)
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) The HOAt-analog to PyBOP®, developed by
R. von Eggelkraut-Gottanka, A. Klose, A. G. L. Carpino gave also remarkable faster cou-
Beck-Sickinger, M. Beyermann, Tetrahedron pling rates as PyBOP®, due to the enhanced
Lett. 44, 3551 (2002) electron withdrawing effect of the corre-
sponding formed -OAt active esters during
coupling reaction.

9
Coupling Reagents

Contrary to HATU PyAOP cannot react with it is the reagent of choice for coupling the
amino groups yielding guanidines. racemization prone Fmoc-His(Trt)-OH.

Literature: Literature:
F. Albericio, M. Cases, J. Alsina, S. A. Triolo, C.-X. Fan, X.-L. Hao, Y.-H. Ye, Synth. Com-
L. A. Carpino, S. A. Kates, Tetrahedron Lett. mun. 26, 1455 (1996)
38, 4853 (1997) H. Li, X. Jiang, Y. H. Ye, C. Fan, T. Romoff, M.
F. Albericio, J. M. Bofill, A. El-Faham, Goodman, Org. Lett. 1, 91 (1999)
S. A. Kates, J. Org. Chem. 63, 9678 (1998) M. Mergler, F. Dick, T. Vorherr, Innovation and
Perspectives in Solid Phase Synthesis and
PyOxim (4071639) Combinatorial Libraries, 7th International
(Ethyl cyano(hydroxyimino)acetato-O2)- Symposium, Southampton, p. 235, R. Epton,
tri-(1-pyrrolidinyl)-phosphonium hexafluo- ed. Mayflower Worldwide (2002)
rophosphate) F. Dettner, A. Hänchen, D. Schols, L. Toti, A.
Nusser, R. D. Süssmuth, Angew. Chem. Int.
Ed. Engl. 48, 1856 (2009)

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
This recently developed coupling reagent phosphonium reagents:
contains Oxyma pure as part of the mol- a. Phosphorylated amino acids (Fmoc-
ecule instead of the explosive compounds Ser(PO,OH,OBzl)-OH,…) can undergo unde-
HOBt or HOAt. This improvement of safety sired couplings between reagent and the
in handling is accompanied with an ac- unprotected phosphoryl-side chain.
celerated reactivity in couplings as well as b. Compounds containing nucleotides (e.g.
a minimized allergenic potential, making PNA-building blocks) with oxo-functions
PyOxim to one of the best coupling reagents like guanine, etc. are prone to be attacked
in solid-phase reactions. The only drawback under structural rearrangement.
is the formation of tris-pyrrolidinophos-
phamide as side product, which can cause Literature:
problems in separation when the reagent is S. Pritz, Y. Wolf, C. Klemm, M. Bienert, Pep-
applied in solution. tides 2006, Proceedings of the 29th Euro-
pean Peptide Symposium, Gdansk, Poland,
Literature: p. 472, edited by K. Rolka, P. Rekowski and J.
R. Subiros-Funosas, A. El-Faham, Silbering (2006)
F. Albericio, Org. Biomol. Chem. 8, 3665 J. W. Perich, N. J. Ede, S. Eagle, A. M. Bray,
(2010) Lett. Pept. Sci. 6, 91 (1999)

DEPBT (4029249)
(3-(Diethoxy-phosphoryloxy)-1,2,3-benzo[d] 4. Aminium/Uronium-Imonium Reagents
triazin-4(3H)-one)
TBTU (BF4-) (4013268)/ HBTU (PF6-)
(2-(1H-Benzotriazol-1-yl)-N,N,N’,N’-
tetramethylaminium tetrafluoroborate/
hexafluorophosphate)

Contrary to the other described phosphoni-

um reagents, DEPBT is a mixed anhydride of
HOOBt and dietylphosphoric acid. It medi-
ates amide bond formations with a remark-
able resistance to racemization. Therefore

10
 Fig. 5.
Coupling with TBTU
in the presence of
DIPEA.

These two compounds, only differing in A well-known limitation of aminium/

their counterions (BF4-, PF6-) have nearly uronium-derivatives is a possible reaction
identical chemical properties and belong to with free amino groups yielding guanidines,
the most popular coupling reagents. Their when the coupling reagent is applied in
application is widespread in solid-phase excess (Fig. 6). This side reaction is normally
reactions as well as in solution, because all inhibited by application of a slight excess of
resulting by-products are soluble in water carboxyl compound in relation to coupling
as well as in standard organic solvents reagent and by a short period of preactiva-
(see Fig. 5). For couplings of phosphory- tion before adding to the amino compound.
lated amino acids TBTU and HBTU are the
reagents of choice. HCTU
(2-(6-Chloro-1H-benzotriazol-1-yl)-
Literature: N,N,N’,N’-tetramethylaminium hexafluoro-
P. Henklein, C. Mügge, B. Costisella, V. Wray, phosphate)
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. The replacement of HOBt by 6-Chloro-HOBt
de Gruyter, Berlin (1989) as part of the molecule, leads to higher
R. Knorr, A. Trzeciak, W. Bannwarth, D. Gil- reaction rates and improved results in the
lessen, Tetrahedron Lett. 30, 1927 (1989) synthesis of difficult peptides.
I. Abdelmoty, F. Albericio, L. A. Carpino,
B. M. Foxman, S. A. Kates, Lett. Pept. Sci. 1, Literature:
57 (1994) O. Marder, Y. Shvo, F. Albericio, Chimica Oggi
J. M. Matsoukas, D. Panagiotopoulos, 20, 37 (2002)
M. Keramida, T. Mavromoustakos, R. Yam- O. Marder and F. Albericio, Chimica Oggi 21,
dagni, Q. Wu, G. J. Moore, M. Saifeddine, 6 (2003)
M. D. Hollenberg, J. Med. Chem. 39, 3585
(1996)
R. F. Poulain, A. L. Tartar, B. P. Déprez, Tetra-
hedron Lett. 42, 1495 (2001)

11
Coupling Reagents

Fig. 6.
N-Terminal gua-
nidinylation by the
coupling reagent.

HDMC (4069891) efficient coupling reagents for solid- and

(N-[(5-Chloro-1H-benzotriazol-1-yl)- solution-phase reactions. Contrary to
dimethylamino-morpholino]-uronium HOBt-based reagents they have been used
hexafluorophosphate N-oxide) successfully in couplings of N-methyl-
amino 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)

COMU (4062545)
When modifying the uronium part of HCTU (1-[1-(Cyano-2-ethoxy-2-oxoethylidene-
by introduction of a morpholine moiety, a aminooxy)-dimethylamino-morpholino]-
further increase of reactivity can be gained. uronium hexafluorophosphate)
Even coupling rates of HATU could be ex-
ceeded in some cases by HDMC.

Literature:
A. El-Faham and F. Albericio, J. Org. Chem.
73, 2731 (2008)

TATU (BF4-) (4026098)/ HATU (PF6-)

(4032172) COMU is a novel coupling reagent with
(2-(7-Aza-1H-benzotriazol-1-yl)-N,N,N’,N’- coupling efficiencies comparable to HATU.
tetramethylaminium tetrafluoroborate/ The incorporation of Oxyma Pure as part
hexafluorophosphate) of the molecule in place of the explosive
compounds HOBt or HOAt results in safer
handling in combination with better solubil-
ity 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
The HOAt analogs to HBTU/TBTU are highly organic bases.

12
 Fig. 7.
EEDQ-mediated
generation of mixed
anhydride.

Literature: TFFH (4062158)

A. El-Faham and F. Albericio, J. Org. Chem. (Tetramethylfluoroformamidinium hexa-
73, 2731 (2008) fluorophosphate)
A. El-Faham, R. Subiros Funosas, R. Pro-
hens, F. Albericio, Chem. Eur. J. 15, 9404
(2009)
R. Subiros-Funosas, G. A. Acosta, A. El-
Faham, F. Albericio, Tetrahedron Lett. 50,
6200 (2009)
A. El-Faham and F. Albericio, J. Pept. Sci. 16, A highly efficient coupling reagent which
6 (2010) generates amino acid fluorides in situ.
J.K. Twibanire and T.B. Grindley et al.Org. Amino acid fluorides are especially suited
Lett. 13, 2988 (2011) for the coupling of sterically hindered α,α-
disubstituted amino acids such as Aib.
TOTT (4033112)
(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl- Literature:
isothiouronium tetrafluoroborate) 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)

TOTT is a thiuronium salt of 2-mercaptopyr-

idone-1-oxide which showed good results in 5. Miscellaneous Coupling Reagents
couplings of sterically hindered or methyl-
ated amino acids, comparable with HATU. EEDQ (4015690)
Racemization levels of those couplings are (N-Ethoxycarbonyl-2-ethoxy-1,2-dihydro-
reported to be lower as with other reagents. quinoline)

Literature:
M. A. Bailén, R. Chinchilla, D. J. Dodsworth,
C. Nájera, J. Org. Chem. 64, 8936 (1999)

EEDQ is an “old” reagent (developed in

13
Coupling Reagents

Fig. 8.
Mechanism
of DMTMM-
mediated
amide bond
formation.

1967), which still is of interest due to its re-

markable properties. It converts the amino T3P
acid derivative into an anhydride EEDQ (2-Propanephosphonic acid anhydride)
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 anhy-
dride is slow, it is consumed very rapidly by
the amino-component which minimizes A trademark of Euticals
racemization during product formation. T3P (or PPA) is a cyclic anhydride of pro-
As an example, EEDQ was the only reagent pylphosphonic acid, which is normally used
amongst the tested ones which mediated in combination with tertiary amines for
the coupling 3,5-dinitrobenzoyl-Leu-OH to solution-phase and cyclization reactions.
3-aminopropyl-silica with negligible level of PPA gives superior results especially for
concomitant racemization. sterically hindered peptides.

Literature: Literature:
B. Belleau and G. Malek, J. Am. Chem. Soc. J. Klose, M. Bienert, C. Mollenkopf, D. Wehle,
90, 1651 (1968) C.-W. Zhang, L. A. Carpino, P. Henklein,
B. Belleau, R. R. Martel, G. Lacasse, M. Men- Chem. Commun. 1847 (1999)
ard, N. L. Weinberg, Y. G. Perron, J. Am. Chem. P. Koch, C. Vedder, T. Schaffer, Chimica Oggi
Soc. 90, 823 (1968) 26, 6 (2008)
R. Pougeois, FEBS Lett. 154, 47 (1983)
H. Kunz, H. Waldmann, C. Unverzagt, Int. J. DMTMM and related compounds
Pept. Protein Res. 26, 493 (1985) (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-
M. H. Hyun, M. H. Kang, S. C. Han, 4-methylmorpholinium salts)
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)

Triazines - a group of coupling reagents,

developed by Kaminski et al. - are conden-

14
sation products of substituted cyanuric
chloride with tertiary amines as NMM or CDI
DABCO, able to mediate peptide couplings (1,1’-Carbonyldiimidazole)
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.
CDI, as the related compounds disuccinimi-
Literature: dyl carbonate or 4-nitrophenyl-chlorofor-
K. Jastrzabek, B. Kolesinska, G. Sabatino, F. mate, is a phosgene analog, a derivative of
Rizzolo, A. Papini, Z. Kaminski, Int. J. Pept. carbonic acid. CDI is not routinely used for
Res .Ther. 13, 229 (2007) peptide couplings. Its field of application
Z. J. Kaminski, B. Kolesinska, J. Kolesinska, lies in the synthesis of ureas and urethanes
G. Sabatino, M. Chelli, P. Rovero, M. Blaszc- from amines, alcoholic compounds or resins
zyk, M. L. Glowka, A. M. Papini, J. Am. Chem. and linkers.
Soc. 127, 16912 (2005) Nevertheless CDI has been successfully
B. Kolesinska, J. Fraczyk, G. Sabatino, A. used in peptide couplings. The protocol for
Papini, Z. Kaminski, Chimica Oggi 25, 26 couplings has to include a preactivation
(2007). step during which the reactive acid imidaz-
olide is formed, which is added to the amino
BTC component.
(bis-Trichloromethylcarbonate or “Triphos-
gene”) Literature:
G.W. Anderson and R. Paul, J. Am. Chem.
Soc. 80, 4423 (1958)
H. Ogura, T. Kobayashi, K. Shimizu, K. Kaw-
abe, K. Takeda, Tetrahedron Lett. 20, 4745
The stable trimeric form of phosgene is a (1979)
well-known reagent for generating acid K. Takeda, Y. Akagi, A. Saiki, T. Tsukahara, H.
chlorides from carboxylic acids. Its applica- Ogura, Tetrahedron Lett. 24, 4569 (1983)
bility for peptide couplings was evaluated T. Kamijo, H. Harada, K. Iizuka, Chem.
by C. Gilon et al. for solution- and solid- Pharm. Bull. 32, 5044 (1984)
phase couplings. They used collidine as
base and THF or DCM as solvents. Other
solvents such as DMF or NMP have to be Conclusion
strictly avoided, as they can react with BTC.
Care must be taken when handling the TBTU, HBTU and PyBOP are well suited
reagent, because BTC and even more so reagents for most standard coupling
phosgene which is formed as intermediate reactions. They all contain the potentially
are highly toxic compounds. Beyond these explosive HOBt as part of the molecule. The
restrictions, BTC is one of the most efficient same problem is encountered with the more
activation reagents and recommended reactive HOAt-based coupling reagents.
especially for difficult couplings and less Due to safety considerations, all of them
reactive amines such as anilines. will have to be replaced in the future by
Oxyma Pure-based reagents such as COMU
Literature: or PyOxim.
E. Falb, T. Yechezkel, Y. Salitra, C. Gilon, More specialized reagents as HATU, HDMC,
J. Pept. Res. 53, 507 (1999) TOTT or DEPBT can be required to succeed
B. Thern, J. Rudolph, G. Jung, Tetrahedron in incorporating amino acid derivatives
Lett. 43, 5013 (2002) prone to side reactions. However, “old” re-
agents 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 M.M. Joullié and K.M. Lassen
valuable hints regarding properties and ap- Evolution of amide bond formation.
plication of coupling reagents the following ARKIVOC 189-250 (2010)
review articles are recommended: C. Roche, M. Pucheault, M. Vaultier,
A. Commerçon
F. Albericio and L.A. Carpino Onium salt supported peptide synthesis.
Coupling reagents and activation. Methods Tetrahedron 66, 8325-8334 (2010)
for solid-phase assembly of peptides. A. El-Faham and F. Albericio
Meth. Enzymol. 289, 104-126 (1997) Peptide coupling reagents: more than a
F. Albericio, R. Chinchilla, D. J. Dodsworth, letter soup.
C. Nájera Chem. Rev. 111, 6557-6602 (2011)
New trends in peptide coupling reagents. V.R. Pattabiraman and J.W. Bode
Org. Prep. Proced. Int. 33, 203-303 (2001) Rethinking amide bond synthesis.
S.-Y. Han and Y.-A. Kim Nature 480, 471-479 (2011)
Recent development of peptide coupling T.I. Al-Warhi, H.M.A. Al-Hazimi, A. El-
reagents in organic synthesis. Faham
Tetrahedron 60, 2447-2467 (2004) Recent development in peptide coupling
A.R. Katritzky, K. Suzuki, K. Singh reagents.
N-Acylation in combinatorial chemistry. J. Saudi Chem. Soc. 16, 97-116 (2012)
ARKIVOC 12-35 (2004) R. Subirós-Funosas, S.N. Khattab, L.
C.A.G.N. Montalbetti and V. Falque Nieto-Rodríguez, A. El-Faham, F. Albericio
Amide bond formation and peptide cou- Advances in acylation methodologies en-
pling. abled by Oxyma-based reagents.
Tetrahedron 61, 10827-10852 (2005) Aldrichim. Acta 46, 21-40 (2013)
E. Valeur and M. Bradley J. R. Dunetz, J. Magano, G. A. Weisenburger
Amide bond formation: beyond the myth of Large-Scale Applications of Amide Coupling
coupling reagents. Reagents for the Synthesis of Pharmaceu-
Chem. Soc. Rev. 38, 606-631 (2009) ticals.
Org. Proc. Res. Devel.20, 140-177 (2016)

16
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 BOP
(Benzotriazole-1-yl-oxy-tris-
PyBOP
(Benzotriazol-1-yloxy-tripyrrolidino-

REAGENTS (dimethylamino)-phosphonium
hexafluorophosphate)
phosphonium hexafluorophosphate)
4031604
4031234
PyOxim
COMU ((Ethyl cyano(hydroxyimino)acetato-
(1-[1-(Cyano-2-ethoxy- O2)-tri-(1-pyrrolidinyl)-phosphoni-
2-oxoethylideneaminooxy)-dimeth- um hexafluorophosphate)
ylamino-morpholino]-uronium 4071639
hexafluorophosphate)
4062545 TATU
(N-[(7-Aza-1H-benzotriazol-1-yl)
DEPBT (dimethylamino)-methylene]-N-
(3-(Diethoxy-phosphoryloxy)-1,2,3- methylmethanaminium tetrafluo-
benzo[d]triazin-4(3H)-one) roborate N-oxide)
4029249 4026098

EDAC · HCl, EDC · HCl, WSC · HCl TBTU

(N-(3-Dimethylaminopropyl)-N’- (N-[(1H-Benzotriazol-1-yl)
ethylcarbodiimide · HCl) (dimethylamino)-methylene]-N-
4031236 methylmethanaminium tetrafluo-
roborate N-oxide)
EEDQ 4013268
(N-Ethoxycarbonyl-2-ethoxy-1,2-
dihydroquinoline) TFFH
4015690 (Tetramethylfluoroformamidinium
hexafluorophosphate)
HATU 4062158
(N-[(7-Aza-1H-benzotriazol-1-yl)
(dimethylamino)-methylene]-N- TOTT
methylmethanaminium hexafluoro- (2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tet-
phosphate N-oxide) ramethylisothiouronium tetrafluo-
4032172 roborate)
4033112
HDMC
(N-[(5-Chloro-1H-benzotriazol-1-yl)-
dimethylamino-morpholino]-uroni-
um hexafluorophosphate N-oxide)
4069891

ADDITIVES HOSu
(N-Hydroxysuccinimide)
HOBt-6-sulfonamidomethyl resin ·
HCl (200-400 mesh)
4025878 (1-Hydroxybenzotriazole-6-sulfon-
amidomethyl resin · HCl)
Oxyma Pure 4027860
(Cyano-hydroxyimino-acetic ethyl
ester)
4019721

18
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