Novabiochem®

Innovations 3/10

Critical evaluation of in situ

coupling reagents for SPPS
With the plethora of coupling reagents available for mediating amide
bond formation in peptide synthesis, making a rational decision as to
the optimal reagent for a given application can be difficult. In this
Novabiochem® Innovations, we compare a wide range of novel and
commercially available coupling reagents (Figure 1) for efficiency,
solution stability and solubility, with a view to simplifying the selection
of coupling reagents.

HBTU1 HATU2 HCTU

PF6 PF6 PF6
N N N
N N N

N N N
N N N
N N N
N Cl
O O O

3
HDMC COMU4 TOTU

PF6

PF6 O PF6
N O
N
N N N N

N
O O
N
N N
Cl N
O O
O CN CN

O O

PyOxim5 PyBOP6 Ezracom

O

CN N
N PF6 N PF6 N CN
N N PF6
N P O N P O N N
OC2H5 O N P O
OC2H5
N N
O N
O

Fig. 1: Coupling reagents examined in this study.

 Solubility Table 3: Closed-vial stabilities of coupling reagents [7].

Coupling reagent 2 days 7 days 14 days

Solubility of coupling reagents was tested by placing 2.5 COMU 67 46 36
mmol of coupling reagent in a measuring cylinder. DMF was HDMC 99 99 98
added in 10 ml aliquots and the mixture stirred for 5 minutes
HATU 99 99 98
between additions of DMF (Table 1). Solubilities above 1.5 M
HBTU 100 98 98
were not tested.
HCTU 99 99 98
Table 1: Solubilities of coupling reagents.
TOTU 89 85 74
Coupling reagent Molarity (lit) Molarity [7] PyBOP 88 85 81
HATU 0.433 0.45 PyOxim 90 88 86
HBTU 0.463 0.5 Ezracom 90 85 79
HCTU 0.503 0.8
The colors of the solutions were also determined by
HDMC 1.003 0.75
measuring the OD of the solutions at 400 nm (Figure 2). In
COMU 1.444 1.5
general the color of the solution correlates with stability.
TOTU 0.8
PyBOP 1.55 1.5
PyOxim 2.55 1.5
Ezracom 0.4

Stability
Open- and closed-vial stability of 0.25 M solutions of
coupling reagents in DMF was determined by 1H nmr time
course studies in d7-DMF at room temperature (Tables 2 & 3)
[7]. These results indicate that solutions of standard uronium
coupling reagents have excellent long term stability in open
vials, whereas those of phosphonium coupling reagents
should not be stored in open vials for longer than 2 days.
Uronium coupling reagents appear to be a good choice for
Figure 2: Solution stability of coupling reagents determined by solution
use with synthesizers based on an open XY platform. OD at 400 nm [7].
Closed vial stability closely mimics the conditions

Coupling efficiency
employed on synthesizers such as the ABI 433 or PTI
Symphony. As expected solutions of uronium coupling
reagents are extremely stable under those conditions.
Phosphonium reagent solutions can be safely used up to 7 Three peptides selected from the literature [2, 3] (Table 4)
days if stored under these conditions. Surprisingly, and in were used as models to evaluate the efficiency of the
contradiction of published results, COMU appeared to be coupling reagents.
rather unstable in solution.
Table 2: Open-vial stabilities of coupling reagents [7]. Peptide sequence

Coupling reagent 0 days 1 day 2 days 1 H-Tyr-Sar-Sar-Phe-Leu-NH2

COMU 96 47 14
COMU (dried) 98 58 16 2 H-Tyr-Aib-Aib-Phe-Leu-NH2
HDMC 99 98 93
HATU 99 98 97 3 H-Tyr-MeLeu-MeLeu-Phe-Leu-NH2
HBTU 99 99 99
HCTU 99 99 98 Table 4: Peptides prepared in this study.
TOTU 98 72 42
PyBOP 99 73 34
PyOxim 99 82 47
Ezracom 98 86 59

2
Peptide synthesis conditions Table 6 summarizes the results obtained using peptide 3
as the model. These data reveal remarkedly consistent trends.
The peptides were assembled under the conditions shown in The nature of the coupling reagent leaving group appears to
Table 5. In every case the C-terminal three residues were be the most important factor influencing efficiency. The
coupled for 1 h. In the case of peptides 1 and 3 the final two activating moiety appears to have little influence. HATU was
residues were coupled for 5 mins to exaggerate the the most efficient reagent for amide bond formation. All four
differences in efficiencies between the coupling reagents Oxyma-based reagents, COMU, TOTU, PyOxim and Ezracom,
under test. With peptide 2, a 1 h coupling time was used for gave very similar results and were more effective than Cl-
all residues. HOBt and HOBt-based reagents. HOBt-based reagents were
Table 5: Conditions used for synthesis of test peptides. the least effective.
Table 6: Composition of products obtained using various coupling
Conditions reagents in the synthesis of peptide 3 [7].

Resin Rink amide AM resin

% Compostion of products by HPLC
Instrument ABI 433A Coupling
MeLFL YMeLFL MeLMeLFL YMeLMeLFL
reagents
Coupling Fmoc-Aaa-OH:Coupling reagent:DIPEA (4:4:8)
PyBOP 60 31 5 4
Deblock 20% Piperidine (3 or 4 times 3 min)
PyOxim 10 45 5 40
Cleavage TFA/water/TIS (95:2.5:2.5) for 3 h HBTU 57 31 8 4

HCTU 31 35 9 25

Results HATU 0 16 7 77

TOTU 5 58 2 35
In our hands, only peptide 3 proved useful as a tool for
COMU 9 47 4 40
evaluating these coupling reagents: peptide 1 was efficiently
assembled by all reagents tested; with peptide 2, the des-Aib Ezracom 9 50 5 36
peptide could not be baseline resolved from the desired
pentapeptide using three different HPLC columns; whereas HDMC 24 46 7 21
with peptide 3, all the by-products from synthesis are fully

Conclusions
resolved and the peptide appears to be highly discriminating
between coupling reagents (Figure 3).

PyOxim appears to combine high reactivity and solubility

with moderate stability, making it an excellent choice of
coupling reagent for synthesizers employing closed-bottle
reagent storage. Furthermore, unlike uronium-based coupling
reagents, it will not give rise to guanidinylated by-products,
making it ideal for coupling of slow-to-activate amino acids,
cyclizations and fragment condensations. HATU was the
most effective coupling reagent of those tested, but its high
cost limits its use to special applications. COMU should be
dissolved and used immediately. For synthesizers employed
open-vials, uronium-based reagents should be employed
Fig. 3: HPLC profile of H-Tyr-MeLeu-MeLeu-Phe-Leu-NH2 using
owing to their high stability.
COMU.

3
 Ordering information 851008 TBTU 5g
25 g
100 g
Novabiochem®’s coupling reagents
851004 BOP 5g 851090 TFFH 1g
25 g 5g
100 g 25 g
851085 COMU 5g 851088 TOTU 5g
25 g 25 g
100 g 100 g
851091 DEPBT 5g 851007 WSC°HCl 5g
25 g 25 g

References
100 g
851013 HATU 5g
25 g
1. R. Knorr, et al. (1989) Tetrahedron Lett., 30, 1927.
851006 HBTU 5g
2. L. A. Carpino, et al. (1993) J. Am. Chem. Soc., 115, 4397.
25 g
100 g 3. A. El-Faham & F. Albericio (2008) J. Org. Chem., 73, 2731.
4. A. El-Faham, et al. (2009) Chem. Eur. J., 15, 9404.
851012 HCTU 5g
25 g 5. R. Subirós-Funosas, et al. (2010) Org. Biomol. Chem., 8, 3665.
100 g 6. J. Coste, et al. (1990) Tetrahedron Lett., 31, 205.
7. R. Behrendt, et al., Poster 83, Presented at the 31st European peptide Symposium,
851011 MSNT 1g
Copenhagen, Sept 2010.
5g
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25 g
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100 g
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25 g
100 g
851095 PyOxim 5g
NEW 25 g
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