IntroductionWith the plethora of coupling reagents available for mediating amide bondformation in peptide synthesis, making a rational decision as to the optimalreagent for a given application can be difficult. In this poster, we compare awide range of novel and commercially available coupling reagents (Figure 1) forefficiency, solution stability, and solubility, with a view to simplifying theselection of coupling reagents.

Fig.1: Coupling reagents examined in this study.

SolubilitySolubility of coupling reagents was tested by placing 2.5 mmol of couplingreagent in a measuring cylinder. DMF was added in 10 ml aliquots and themixture stirred for 5 minutes between additions of DMF (Table 1). Solubilitiesabove 1.5 M were not tested.

Table 1: Solubility of coupling reagents.

StabilityOpen and closed vial stabilities of various coupling reagents were determinedby 1H nmr time course studies in d7-DMF (Tables 2 & 3). Closed vial stabilityclosely mimics the conditions employed on synthesizers such as the ABI 433 orPTI Symphony. As expected solutions of uronium coupling reagents areextremely stable under those conditions. Phosphonium reagent solutions canbe safely used up to 7 days if stored under these conditions. Surprisingly, andin contradiction of published results, COMU appeared to be highly unstable insolution. Uronium coupling reagents appear to be a good choice for use withsynthesizers based on an open XY platform.

Table 2: Open vial stability of coupling reagents.

Table 3: Closed vial stability of coupling reagents.

The stabilities of solutions of coupling reagents in DMF containing DIPEA werealso tested by the NMR method (Table 4). HBTU/DIPEA and PyBOP/DIPEAmixtures were found to be sufficiently stable to to be usable after 2 days.

Table 4: Closed vial stability of coupling reagents.

Coupling efficiencyThree peptides selected from the literature [2, 3] (Table 5) were used as modelsto evaluate the efficiency of the coupling reagents.

Table 5: Peptides prepared in this study.

Peptide synthesis conditionsThe peptides were assembled under the conditions shown in Table 6. In everycase the C-terminal three residues were coupled for 1 h. In the case of peptides11 and 33 the final two residues were coupled for 5 mins to exaggerate thedifferences in efficiencies between the coupling reagents under test. Withpeptide 22, a 1 h coupling time was used for all residues.

Table 6: Conditions used for synthesis of test peptides.

ResultsIn our hands, only peptide 33 proved useful as a tool for evaluating thesecoupling reagents: peptide 11 was efficiently assembled by all reagents tested;with peptide 22, the des-Aib peptide could not be baseline resolved from thedesired pentapeptide using three different HPLC columns; whereas withpeptide 33, all the by-products from synthesis are fully resolved and the peptideappears to be highly discriminating between coupling reagents (Figure 4).

Table 7 summarizes the results obtained using peptide 33 as the model. Thesedata reveal remarkably consistent trends. The nature of the coupling reagentleaving group appears to be the most important factor influencing efficiency.The activating moiety appears to have little influence. HATU was the mostefficient reagent for amide bond formation. All three Oxyma-based reagents,COMU, PyOxim and Ezracom, gave very similar results and were more effectivethan Cl-HOBt and HOBt-based reagents. HOBt-based reagents were the leasteffective.

Table 7: Composition of products obtained using various coupling reagents in the synthesis of peptide 3.

References1. R. Knorr, et al. (1989) Tetrahedron Lett., 30, 1927.

2. L. A. Carpino, et al. (1993) J. Am. Chem. Soc., 115, 4397.

3. A. El-Faham & F. Albericio (2008) J. Org. Chem., 73, 2731.

4. A. El-Faham, et al. (2009) Chem. Eur. J., 15, 9404.

5. R. Subiros-Funosas, et al. (2010) Org. Biomol. Chem., 8, 3665.

6. J. Coste, et al. (1990) Tetrahedron Lett., 31, 205.

Novabiochem, NovaSyn, NovaTag, and PyBOP are trademarks of Merck KGaA.

Critical evaluation of in situ coupling reagents for SPPSR. Behrendt1 , A. Bruenner1 & P. White2

1Novabiochem, Merck & Cie, Im Laternenacker 5, CH-8200 Schaffhausen, Switzerland2Novabiochem, Merck Chemicals Ltd., Beeston, Notts, NG9 2JR, U.K.

Conclusions PyOxim appeared to combine high reactivity and solubility with moderate

stability, making it an excellent choice of coupling reagent forsynthesizers employing closed-bottle reagent storage. Furthermore,unlike uronium-based coupling reagents, it will not give rise toguanidinylated 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 itshigh cost limits its use to special applications.

For synthesizers employed open-vials, uronium-based reagents should beemployed owing to their high stability.

Peptide sequence

1 H-Tyr-Sar-Sar-Phe-Leu-NH2

2 H-Tyr-Aib-Aib-Phe-Leu-NH2

3 H-Tyr-MeLeu-MeLeu-Phe-Leu-NH2

Fig. 4: HPLC profile of H-Tyr-MeLeu-MeLeu-Phe-Leu-NH2 using COMU.

Novabiochem

Coupling reagent Molarity (lit) MolarityHATU 0.433 0.45HBTU 0.463 0.5HCTU 0.503 0.8HDMC 1.003 0.75COMU 1.444 1.5PyBOP 1.55 1.5PyOxim 2.55 1.5Ezracom 0.4

N

N

N

N

N

N

O

PF

6

N

N

N

N

N

O

PF

6

N

N

N

N

N

O

PF

6

Cl

N

N

N

N

N

O

PF

6

Cl

O

O

CN

N

O

NN

O

PF

6

O

P O

PF

6

N

N

N

N

N

N

P O

N

OC

2

H

5

O

PF

6

CN

N

N

N

P O

N

OC

2

H

5

O

PF

6

CN

N

N

N

O

O

O

HBTU

1

HATU

2

HCTU

HDMC

3

COMU

4

PyOxim

5

PyBOP

6

Ezracom

O

O

CN

N

O

NN

PF

6

TOTU

Conditions

Resin Rink amide AM resin

Instrument ABI 433A

Coupling Fmoc-Aaa-OH:Coupling reagent:DIPEA (4:4:8)

Deblock 20% Piperidine (3 or 4 times 3 min)

Cleavage TFA/water/TIS (95:2.5:2.5) for 3 h

Coupling reagent

Sequence PyBOP PyOxim HBTU TOTU HCTU HATU COMU Ezracom HDMC

MeLFL 60 10 57 5 31 0 9 9 24

YMeLFL 31 45 31 58 35 16 47 50 46

MeLMeLFL 5 5 8 2 9 7 4 5 7

YMeLMeLFL

4 40 4 35 25 77 40 36 21

Coupling reagent 2d 7d 14d

HDMC 99 99 98

COMU 67 46 36

PyOxim 90 88 86

Ezracom 90 85 79

PyBOP 88 85 81

HATU 99 99 98

HBTU 100 98 98

HCTU 99 99 98

TOTU 89 85 81

Coupling reagent 0 d 1 d 2d

HDMC 99 98 93

COMU 96 47 14

PyOxim 99 73 34

Ezracom 98 86 59

PyBOP 99 73 34

HATU 99 98 97

HBTU 99 99 99

HCTU 99 99 98

TOTU 98 72 42

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

Coupling reagent 1d 2d 5d

HBTU/DIPEA (1:2) 90 86 77

HCTU/DIPEA (1:2) 84 77 49

HATU/DIPEA (1:2) 79 69 58

PyBOP/DIPEA (1:2) 80 77 63