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REAGENTS
H
C
C
COOH
PG
G1 NH
COO
C
NH2
PG2
C
PG1 NH
C
CO
NH
COO
PG2
Coupling Reagents
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.
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 pentauorophenyl (OPfp) and hydroxysuccinimido (OSu or
NHS) esters are reactive amino acid derivatives nding 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,
Coupling Reagents
Fig. 2.
Mechanism of
base-catalyzed
racemization during
activation.
Fig. 3.
Carbodiimidemediated amide
bond formation.
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
Coupling Reagents
Fig. 4.
Mechanism of the
DCC/HOBt-mediated
peptide coupling.
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. Dprez, 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.
Knig 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. Knig and R. Geiger, Chem. Ber. 103, 788
(1970)
W. Knig 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 efcient polymeric auxiliary for the synthesis of amides.
Simple ltration allows the separation of
the product from the polymer.
Coupling Reagents
HOAt
(1-Hydroxy-7-aza-1H-benzotriazole)
DMAP
(4-(N,N-Dimethylamino)pyridine)
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. Subirs-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. Subirs-Funosas, A. El-Faham, F. Albericio, Peptide Science 98, 89 (2012)
BOP (Q-1980)
(Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexauorophosphate)
BOP was the rst 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
PyBrOP
(Bromo-tripyrrolidino-phosphonium hexauorophosphate)
Coupling Reagents
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. Boll, 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. Mayower Worldwide (2002)
F. Dettner, A. Hnchen, D. Schols, L. Toti, A.
Nusser, R. D. Sssmuth, Angew. Chem. Int.
Ed. Engl. 48, 1856 (2009)
PyOxim (Q-2760)
(Ethyl cyano(hydroxyimino)acetato-O2)tri-(1-pyrrolidinyl)-phosphonium hexauorophosphate)
4. Aminium/Uronium-Imonium Reagents
TBTU (BF4-) (Q-1665)/ HBTU (PF6-)
(2-(1H-Benzotriazol-1-yl)-N,N,N,Ntetramethylaminium tetrauoroborate/
hexauorophosphate)
Fig. 5.
Coupling with TBTU
in the presence of
DIPEA.
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
hexauorophosphate N-oxide)
The HOAt analog to HBTU is a highly efcient coupling reagent for solid- and
solution-phase reactions. Contrary to HOBt-
12
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 tetrauoroborate)
TFFH (Q-2280)
(Tetramethyluoroformamidinium hexauorophosphate)
Literature:
M. A. Bailn, R. Chinchilla, D. J. Dodsworth,
C. Njera, J. Org. Chem. 64, 8936 (1999)
13
Coupling Reagents
Fig. 8.
Mechanism
of DMTMMmediated
amide bond
formation.
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.
14
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 eld 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)
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
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 hexauorophosphate)
Q-1980
COMU
(1-[1-(Cyano-2-ethoxy2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium hexauorophosphate)
Q-2735
PyBOP
(Benzotriazol-1-yloxy-tripyrrolidinophosphonium hexauorophosphate)
Q-2715
PyOxim
((Ethyl cyano(hydroxyimino)acetatoO2)-tri-(1-pyrrolidinyl)-phosphonium
hexauorophosphate)
Q-2760
DEPBT
(3-(Diethoxy-phosphoryloxy)-1,2,3benzo[d]triazin-4(3H)-one)
Q-2565
TBTU
(N-[(1H-Benzotriazol-1-yl)
(dimethylamino)-methylene]-N-methylmethanaminium tetrauoroborate
N-oxide)
Q-1665
TFFH
(Tetramethyluoroformamidinium
hexauorophosphate)
Q-2280
EEDQ
(N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline)
Q-1735
TOTT
(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethylisothiouronium tetrauoroborate)
Q-2600
HDMC
(N-[(5-Chloro-1H-benzotriazol-1-yl)dimethylamino-morpholino]-uronium
hexauorophosphate 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
DIABETES
PEPTIDES
ENDOTHELINS
FRET
SUBSTRATES
GHRELIN,
LEPTIN AND
OBESTATIN
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
2011123
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