BOP-OXy, BOP-OBt, and BOP-OAt: Novel organophosphinic coupling reagents useful for solution and solid-phase peptide synthesis

Article abstract of DOI:10.1002/psc.2579

Stand-alone coupling reagents derived from bis(2-oxo-3-oxazolidinyl) phosphorodiamidic chloride show efficient performance in solution and SPPS. In particular, the Oxyma Pure (Luxembourg Biotech., Tel Aviv, Israel) derivative shows the additional advantag

Full text of DOI:10.1002/psc.2579

Research Article
Received: 29 September 2013
Revised: 2 October 2013
Accepted: 2 October 2013
Published online in Wiley Online Library: 22 November 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2579
BOP-OXy, BOP-OBt, and BOP-OAt: novel
organophosphinic coupling reagents
useful for solution and solid-phase
peptide synthesis
Ayman El-Faham,a,b,c* Sherine N. Khattab,b Ramon Subirós-Funosasc,d,e
and Fernando Albericioc,d,f,g**
Stand-alone coupling reagents derived from bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride show efficient performance in
solution and SPPS. In particular, the Oxyma Pure (Luxembourg Biotech., Tel Aviv, Israel) derivative shows the additional advantage
of being highly soluble in DMF and even fairly soluble in CH3CN, which can extend its use for the synthesis of complex peptides. These
new stand-alone coupling reagents have the advantage of not bearing any counteranion such as PF6 or BH4, whose presence can jeop-
ardize the purification of final peptides prepared in solution. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: Oxyma Pure; phosphorous reagent; amino acids; solid-phase peptide synthesis; BOP-Cl
that BOP-Cl does not work properly in solid phase. This can be
because activation of the urethane-protected amino acid with
Introduction
Takeuchi and Yamada [1] reported in 1974 DPPA (1) as the first
mixed carboxylic phosphoric anhydride method for peptide
bond formation. DPPA was followed by the development of
various organophosphorous reagents [2–5].
BOP-Cl is converted first to the acid chloride and then to the
oxazolone, which is a rather poor acylating derivative, and in
addition is accompanied by racemization [23].
Herein, the use of the BOP moiety in peptide synthesis in both
solution and solid phase is expanded through the introduction of
the most common additives to carbodiimides: HOBt (9) [24],
HOAt (10) [25–27], and Oxyma Pure (11) [28–30]. HOBt and its
The next generation of organophosphinic reagents was
represented by thiophosphinic-type coupling reagents such as
dimethylphosphinothioyl azide (MPTA, 2) and 3-dimethylpho-
sphinothioyl-2(3H)-oxazolone (MPTO, 3) (Figure 1) [6]. Because
MPTA (2) generates a carbamoyl azide or urea derivative as
by-product, Ueki introduced MPTO (3), in which the azide group
of MTPA is replaced by a 2-oxazolone group. On the basis of the
earlier development of organophosphorous reagents, consider-
able effort has been focused on developing various coupling re-
agents of a similar kind [7–17]. In the last decade [18,19],
phosphoric acid diethyl 2-phenylbenzimidazol-1-yl ester
(DOEPBI, 4), diphenylphosphinic acid 2-phenylbenzimidazol-1-
yl ester (DPPBI, 5), diphenyl 2-phenyl-1H-benzo[d]imidazol-1-yl
phosphate (DOPPBI, 6) [19], and TFMS-DEP (7) [20] (Figure 2)
have been reported as highly efficient coupling reagents. Their
efficiency was evaluated through the synthesis of a range of
amide-containing compounds and peptides, and the extent of
racemization was found to be negligible.
Bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride (BOP-Cl,
8) is the most widely used peptide coupling reagent of the
organophosphinic family (Figure 3) [21]. BOP-Cl (8) [22] is a
well-known excellent dehydrating reagent in peptide synthesis,
featuring enhanced stability, lesser corrosive character, and more
easiness to handle than other activating agents.
However, despite such excellent performance, the use of BOP-
Cl in peptide synthesis has been limited to a few cases involving
N-methyl amino acids [22]. Furthermore, it is commonly regarded
*
Correspondence to: Ayman El-Faham, Department of Chemistry, College of
Science, King Saud University, P.O. Box 2455, 11451 Riyadh, Kingdom of Saudi
Arabia Department of Chemistry. E-mail: Aymanel_faham@hotmail.com
** Correspondence to: Fernando Albericio, Institute for Research in Biomedicine-
Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain. E-mail: albericio@
irbbarcelona.org
a Department of Chemistry, College of Science, King Saud University, P.O.
Box 2455, 11451 Riyadh, Kingdom of Saudi Arabia
b Department of Chemistry, Faculty of Science, Alexandria University, P.O.
Box 426, Ibrahimia, Alexandria 21321, Egypt
c
Institute for Research in Biomedicine-Barcelona, Baldiri Reixac 10, 08028
Barcelona, Spain
d Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-
BBN), Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
e Institut fuer Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2,
D-12489 Berlin
f
School of Chemistry & Physics, University of KwaZulu-Natal, 4001 Durban,
South Africa
g Department of Organic Chemistry, University of Barcelona, Marti i Franques
1-11, 08028 Barcelona, Spain
J. Pept. Sci. 2014; 20: 1–6
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

EL-FAHAM ET AL.
Table 1. Solubility of the organophosphinic coupling reagents in dif-
ferent solvents
Entry Coupling reagent
Solubility (molarity)
DMF
DCM
CH3CN
THF
Figure 1. Organophosphorous reagents: DPPA (1), MPTA (2), and MPTO (3).
1
2
3
4
5
6
7
8
BOP-Cl (8)
BOP-OBt (13)
BOP-OAt (14)
BOP-OXy (15)
BOP-N-OXy (16)
COMU
0.19
0.01
0.06
0.12
Insolublea
Insolublea 0.006 Insolublea
0.12 Insolublea
0.02
Insolublea
0.06
0.03b
—
1.18
0.06
0.72
0.24
1.44
0.43
0.46
Insolublea 0.35b
—
—
—
—
—
—
HATU
—
HBTU
—
aCompounds labeled as ‘insoluble’ refer to 30 mg that could not be
dissolved in 15 ml of solvent (<0.002 M).
bSolutions of BOP-N-Oxyma show a characteristic brown color.
Figure 2. Organophosphorous reagents: DOEPBI (4), DPPBI (5), DOPPBI
(6), and TFMS-DEP (7).
recently of interest, especially CH3CN and THF [42]. In this sense, a
solubility study in different solvents has been carried out.
As it is shown in Table 1, BOP-OXy is almost as soluble as (1-cyano-
2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-
carbenium hexafluorophosphate (COMU) [33] and three times
more soluble than HATU and HBTU. Furthermore and regardless
of solvent used, the Oxyma Pure-based reagent is more soluble
than the OAt/OBt ones, showing a relatively good solubility in
CH3CN (better than HATU/HBTU in DMF). This result could
enlarge the use of BOP-OXy for synthesis of complex peptides.
Figure 3. Bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride (BOP-Cl, 8).
more potent aza derivative HOAt are being discontinued for
safety reasons [31]. Oxyma Pure does not show that problem,
and it has been shown to be superior in all cases to HOBt and
even in many cases with the same performance than HOAt
[28,32–40]. In addition, the derivative of the amide counterpart,
N-Oxyma (12) [41], was also prepared.
Epimerization Control
In order to check the impact of epimerization in solution when
using the novel phosphinic coupling reagents, BOP-Cl (8), BOP-
OBt (13), BOP-OAt (14), BOP-OXy (15), and BOP-NOXy (16), the
model peptides (17–19) were selected (Figure 4). These peptides
are described to give rise to considerable racemization during
stepwise and [2 + 1] fragment couplings [43,44]. After coupling
of equimolar quantities of the amine- and acid-containing coun-
terparts and aqueous work-up, the impact of epimerization for
the three peptides (17–19) was determined by HPLC, using opti-
mized conditions that give clear separation of the two epimers.
The performance of the activating reagents (13–16) in a step-
wise coupling was examined during the assembly of Z-Phg-OH
onto H-Pro-NH2 (17) (Table 2) [43]. The Phg residue shows a high
sensitivity toward epimerization, because of the high stability of
the counteranion. In this model, remarkably, most of the
phosphonic derivatives (13–16) were able to maintain the desired
chirality of the test dipeptide (entries 6–8). In particular, BOP-OXy
(15) was the most efficient in reducing epimerization (entry 7).
The retention of configuration during the [2 + 1] segment
coupling of dipeptide Z-Gly-Phe-OH onto H-Pro-NH2 to yield Z-Gly-
Phe-Pro-NH2 (18) was higher than in the previous stepwise model
as has been also observed in other works for segment couplings
(Table 3) [28,33,45]. The use of 2,4,6-trimethylpyridine (TMP) in these
experiments is recommended because it reduces racemization in seg-
ment couplings more efficiently than other bases such as DIEA [46].
Results and Discussion
Synthesis of Phosphinic Coupling Reagents
The synthetic strategy consists in the reaction of BOP-Cl (8) with
HOBt (9), HOAt (10), Oxyma Pure (11), and N-Oxyma (12) in DCM
in presence of TEA as base to afford the corresponding desired
phosphinic coupling reagents (13–16) (Scheme 1).
Solubility of the Novel Phosphinic Reagents
In similarity to most organic processes, the solubility of the reactants
plays a crucial role in the conversion. Although DMF is the common
solvent of choice for both the solid phase and solution strategies of
peptide synthesis, the use of greener solvent alternatives has been
Figure 4. Peptide models used to test the reduction of epimerization in-
Scheme 1. Synthesis of phosphinic derivatives (13–16).
duced by the phosphinic coupling reagents.
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Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
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BOP DERIVATIVES AS COUPLING REAGENTS
Table 2. Yield and epimerization during the formation of Z-Phg-Pro-
NH2 (17) in DMF at room temperature by using the phosphinic cou-
pling reagents (solution phase synthesis)a
Table 4. Yield and epimerization during the [2 + 1] formation of Z-
Phe-Val-Pro-NH2 (19) in DMF at room temperature by using the
phosphinic coupling reagents (solution phase synthesis)a
Entry
Coupling reagent
Pre-activation Yield [%] DL [%]b
Entry Coupling reagent
Base
Yield [%] DL [%]b
1
2
3
4
5
6
7
8
BOP-Cl (8)
2 min
2 min
2 min
1 min
86.0
83.0
86.0
81.0
78.0
86.0
90.0
81.0
3.47
5.67
2.78
1.17
6.1
1
2
3
4
5
6
7
8
BOP-Cl (8)
DIEA (2 equiv)
DIEA (2 equiv)
DIEA (2 equiv)
TMP (2 equiv)
TMP (2 equiv)
TMP (2 equiv)
DIEA (2 equiv)
TMP (2 equiv)
87.0
86.0
85.0
83.0
80.3
87.0
92.0
87.0
35.2
18.3
6.9
BOP-Cl/HOBt
BOP-Cl/HOAt
BOP-Cl/Oxyma Pure
BOP-OBt (13)
BOP-OAt (14)
BOP-OXy (15)
BOP-N-OXy (16)
BOP-Cl/HOBt
BOP-Cl/HOAt
BOP-Cl/Oxyma Pure
BOP-OBt (13)
BOP-OAt (14)
BOP-OXy (15)
BOP-OXy (15)
12.9
16.1
2.7
1.4
0.16
1.2
20.4
9.2
a2 equiv DIEA was used.
aNo pre-activation was performed.
bThe LL and DL epimers of this peptide model have been
described in the literature [43]. Retention times for each
epimer were identified after co-injection with a pure LL sample.
bThe LLL and LDL epimers of this tripeptide model have been
described in the literature [44]. The retention times for each
epimer were identified after co-injection with a pure sample.
Table 3. Yield and epimerization during the [2 + 1] formation of Z-
Gly-Phe-Pro-NH2 (18) in DMF at room temperature by using the
phosphinic coupling reagents (solution phase synthesis)a
Table 5. Impact of des-Aib (H-Tyr-Aib-Phe-Leu-NH2) tetrapeptide during
solid-phase assembly of the pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH2
(20) by using the phosphinic coupling reagentsa
Entry Coupling reagent
Base
Yield [%] DL [%]b
Entry
Coupling
reagent
Yield Pentapeptide 20
Des-Aib
[%]b
[%]
[%]
1
2
3
4
BOP-Cl (8)
TMP (2 equiv)
TMP (2 equiv)
TMP (2 equiv)
TMP (2 equiv)
83.0
83.0
95.0
80.0
15.1
1.1
1
2
3
4
5
BOP-Clc (8)
75
85
89
89
86
10.9
78.0
66.8
5.6
BOP-OAt (14)
BOP-OXy (15)
BOP-NOXy (16)
BOP-OBt (13)
BOP-OAt (14)d
BOP-OXy (15)
BOP-NOXy (16)
33.3
90.5
93.1
77.5
1.8
6.6
6.9
aCoupling was conducted without pre-activation time.
22.5
bThe LLL and LDL epimers of this tripeptide model have been
described in the literature [44]. Retention times for each
epimer were identified after co-injection with a pure sample.
a1–2 min pre-activation and 30-min coupling times were generally
applied, except for Aib-Aib (30-min double coupling)
bDeletion tetrapeptide des-Aib was identified by peak overlap in
HPLC with an authentic sample obtained in solid phase.
cExtra peaks were observed at 5.38 (4.05%), 5.71 (2.28%), 6.16
(3.44%), and 6.54 min (1.26%).
Best results were obtained with BOP-OAt (14) (entry 2), whereas
BOP-OXy (15) performed closely (entry 3). All BOP-OR phosphonic
reagents superseded in configuration control that of the parent
BOP-Cl (entry 1).
dExtra peak was observed at 6.25 min (3.69%).
Similar tendencies were observed in the [2+ 1] segment cou-
pling (Z-Phe-Val-OH onto H-Pro-NH2, 19) (Table 4). In this model
system, BOP-Cl (8) was the poorest racemization-suppressing re-
agent of the compounds tested, with a percentage of LDL epimer
of nearly 35% (Table 4, entry 1). Best results were obtained with
BOP-OAt (14) (entry 6), whereas BOP-Oxy (15) provided remarkably
low epimerization (entry 8). In all cases (Tables 2–4), the use of a
mixture of BOP-Cl and the N-hydroxy derivative provides poorer
results than when the coupling reagent is used alone.
respectively (entries 1,2 vs 4,3). These results confirm that BOP-Cl
is not suitable at all for SPPS.
H-Tyr-Aib-Aib-Phe-Leu-NH2
20
Conclusions
In conclusion, BOP-based stand-alone coupling reagents can be
used in both solution and solid-phase approaches. BOP-OXy
showed superiority to BOP-OAt in reducing epimerization in step-
wise couplings, whereas in 2 + 1 segment couplings performed
closely to BOP-OAt. Concerning coupling potency, BOP-OXy gave
close results to BOP-OAt in the synthesis of the H-Tyr-Aib-Aib-Phe-
Leu-NH2 pentapeptide, whereas the worst results were obtained
with BOP-Cl. BOP-OXy showed advantageous performance over
BOP-NOXy in all cases, highlighting the ester to amide difference in
reactivity with regard to BOP-OXy. An additional advantage of BOP-
OXy compared with OAt derivative is its superior solubility in DMF
and even in CH3CN, a feature that can expand its use for the prepa-
ration of complex peptides. Finally, these new stand-alone coupling
reagents have the advantage of not bearing any counteranion such
as PF6 or BH4, whose presence can jeopardize the purification of final
peptides prepared in solution.
Coupling Potency
To conclude with the evaluation of the novel organophosphinic
reagents, coupling efficiency assays were designed. Thus, the pen-
tapeptide H-Tyr-Aib-Aib-Phe-Leu-NH2 (20, Table 5) was assembled
in solid phase by means of 30-min couplings, except in the forma-
tion of Aib-Aib peptide bond (30-min double coupling) [47].
Misincorporation of one Aib residue (des-Aib) is the most important
side reaction and therefore gives a clear reference of the quality of
coupling reagent (Table 5). BOP-OXy exhibited closer coupling po-
tency to BOP-OAt with enhanced purity (entries 4 and 3), achieving
an impressive 93.1% and 90.5% yield of 20, respectively. In contrast,
BOP-Cl and BOP-OBt gave the worst results (entries 1 and 2),
providing only 10.9% and 33.3% yield of the pentapeptide 20,
J. Pept. Sci. 2014; 20: 1–6
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EL-FAHAM ET AL.
129.53, 135.30, 149.22, 151.80, 157.12. Anal. Cacld for C11H11N6O6P
(354.22): C, 37.30; H, 3.13; N, 23.73. Found: C, 37.55; H, 3.00; N, 23.91.
Experimental Section
Materials and General Methods
Ethyl 2-(bis(2-oxooxazolidin-3-yl)phosphoryloxyimino)-2-cyanoacetate
(BOP-OXy, 15)
The solvents used were of HPLC reagent grade. Melting points were
determined with a Buchi B-540 apparatus (BUCHI Labortechnick
GmbH, Essen, Germany) and are uncorrected. NMR spectra
(1H NMR and 13C NMR spectra) were recorded on Varian Mercury
400 MHz spectrometer (Vernon Hills, IL, USA) with chemical shift
values reported in δ units (ppm) relative to an internal standard.
Follow-up of the reactions and checks of the purity of the
compounds was performed by TLC on silica gel-protected alumi-
num sheets (Type 60 GF254, Merck Millipore, Bedford, MA, USA),
and the spots were detected by exposure to UV-lamp at λ 254 nm
for a few seconds. For analytical separation and characterization,
a reverse-phase Waters 2695 HPLC separation module, coupled to
a Waters 996 PDA UV detector (Milford, MA, USA), was used.
Chromatograms were processed with Empower software. Peptide
mass was detected by means of an HPLC-PDA system as described
earlier, coupled to a Waters Micromass ZQ mass detector, using the
MassLynx 4.1 software (Milford, MA, USA).
Methods A and B: The product was obtained as a white
powder, mp 152–153 °C, in 2.1 g (73.3%) yield. 1H NMR
(CDCl3): δ 1.45 (t, 3H, CH3), 4.22 (t, 4H, 2CH2), 4.49 (q, 2H,
CH2), 4.58 (t, 4H, 2CH2). 13C NMR (CDCl3): δ 14.14, 45.48, 46.05,
64.77, 65.38, 106.47, 136.43, 155.48, 156.68. Anal. Calcd for
C11H13N4O8P (360.22): C, 36.68; H, 3.64; N, 15.55. Found: C,
36.82; H, 3.61; N, 15.86.
N-(Bis(2-oxooxazolidin-3-yl)phosphoryloxy)-2-(ethyl amino)-2-
oxoacetimidoyl cyanide (BOP-NOXy, 16)
Method A: The product was obtained as a white powder, mp
1
135–137 °C, in 2.98 g (83.0%). H NMR (acetone-d6): δ 1.18 (t, 3H,
CH3), 3.39 (t, 4H, 2CH2), 4.17 (q, 2H, CH2), 4.60 (t, 4H, 2CH2). 13C
NMR (acetone-d6): δ 14.03, 35.09, 45.37, 45.41, 65.23, 65.33,
106.89, 155.48. Anal. Calcd for C11H14N5O7P (359.23): C, 36.78; H,
3.93; N, 19.50. Found: C, 36.59; H, 3.88; N, 19.81.
General Procedure for Synthesis of Phosphonic Derivatives (13–16)
General Method for the Racemization Experiments Using the
New Phosphonic Derivatives (BOP-Cl, BOP-OBt, BOP-OAt,
BOP-OXy, and BOP-NOXy derivatives) [28,33,35,36,45]
Method A
To a solution of N-hydroxy derivatives (10 mmol) and TEA
(10 mmol) in dry DCM (20 ml) was added the BOP-Cl (10 mmol) at
0 °C. The reaction mixture was stirred at this temperature for 1 h
and cooled to room temperature. Then, the reaction mixture was
stirred for 6 h under N2 at room temperature, filtered from the
precipitate, and washed with 10ml of DCM to afford a white solid
in pure state as indicated from the NMR. To the filtrate, 50 ml of
DCM was added and washed with 10% NaHCO3 (2× 10 ml), 1 N
HCl (2× 10 ml), and saturated NaCl (2× 10 ml) and dried. The
solvent was removed under vacuum, and the residue was
crystallized from DCM/hexane to afford extra 10% yield.
Some 0.125 mmol of an acid (Z-Phg-OH, Z-Gly-Phe-OH, or Z-Phe-
Val-OH) and (20 μl) 0.125 mmol BOP-Cl, BOP-OBt, BOP-OAt, BOP-
OXy, and BOP-NOXy were dissolved in 2 ml DMF at 0 °C. Then,
0.125 mmol of H-Pro-NH2 was added. The reaction mixture was
stirred at 0 °C for 1 h and at room temperature overnight. The
reaction mixture was diluted with 25 ml of ethyl acetate and
extracted with 1 N HCl (2 × 5 ml), 1 N NaHCO3 (2 × 5 ml), and satu-
rated NaCl (2 × 5 ml). It was then dried over anhydrous MgSO4.
The solvent was then removed, and the crude peptide was
directly analyzed by HPLC.
Method B
Z-Phg-Pro-NH2 17
BOP-Cl (10 mmol) was added to a suspension of the potassium
salt of N-hydroxy derivatives (10 mmol) in CH3CN at 0 °C. The re-
action mixture was stirred at this temperature for 1 h and cooled
to room temperature with stirring for 6 h, under N2. It was filtered
from the precipitate and washed with 20 ml CH3CN, and then the
solvent was removed under vacuum. The residue was
recrystallized from DCM/hexane or CH3CN/ether to give a white
solid in pure state as indicated from the NMR.
A linear gradient of 25–50% CH3CN/H2O and 0.1% TFA over 20min
was applied, with a flow rate of 1.0 ml/min and detection at 220 nm
using a Waters 996 PDA detector and a Sunfire C18 column 3.5 μm,
4.6 × 100 mm (Milford, MA, USA), tR LL = 9.89 min, tR DL = 10.50 min.
Z-Gly-Phe-Pro-NH2 18
A linear gradient of 20–70% CH3CN/H2O and 0.1% TFA over 8 min
was applied, with detection at 220 nm using a Waters 996 PDA
1H-Benzo[d][1,2,3]triazol-1-yl bis(2-oxooxazolidin-3-yl)phosphinate
(BOP-OBt, 13)
detector and a Sunfire C18 column 3.5 μm, 4.6 × 100 mm, tR LLL
5.61 min, tR LDL = 5.94 min.
=
Method A: The product was obtained as a white solid, mp 178–181 °C,
in 2.3 g (65.0%) yield. H NMR (acetone-d6): δ 3.83 (t, 4H, 2CH2),
Z-Phe-Val-Pro-NH2 19
1
A linear gradient of 20–70% CH3CN/H2O and 0.1% TFA over 8 min
was applied, with a flow rate of 1.0 ml/min and detection at
220 nm using a Waters 996 PDA detector and a Sunfire C18
column 3.5 μm, 4.6 × 100 mm, tR LLL = 6.81 min, tR LDL = 7.24 min.
4.29 (t, 4H, 2CH2), 7.41 (t, 1H, CH), 7.54 (t, 1H, CH), 7.73 (d, 1H, CH),
7.95 (d, 1H, CH). 13C NMR (acetone-d6): δ 46.05, 64.33, 121.40,
129.53, 149.22, 157.11. Anal. Calcd for C12H12N5O6P (353.23): C,
40.80; H, 3.42; N, 19.83. Found: C, 40.57; H, 3.20; N, 20.05.
3H-[1,2,3]Triazolo[4,5-b]pyridin-3-yl bis(2-oxooxazolidin-3-yl)phosphinate
(BOP-OAt, 14)
Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH2 (20) in solid phase
[28,33,35,36,45]
Method A: The product was obtained as a white solid, mp 184–185 °C,
in 2.7 g (76.3%) yield. H NMR (acetone-d6): δ 3.81 (t, 4H, 2CH2),
The pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH2 (20) was prepared
following a published procedure. The synthesis was carried out
in a plastic syringe that was attached to a vacuum manifold so
as to effect rapid removal of reagents and solvent. The resin
1
4.26 (t, 4H, 2CH2), 7.50 (dd, 1H, CH), 8.50 (d, 1H, CH), 8.75 (dd, 1H,
CH). 13C NMR (acetone-d6): δ 46.01, 46.05, 64.24, 64.33, 121.40,
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J. Pept. Sci. 2014; 20: 1–6

BOP DERIVATIVES AS COUPLING REAGENTS
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carboxyl groups: diphenyl 2-oxo-3-oxazolinylphosphonate. Tetrahe-
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Fmoc-Rink-amide-AM-PS with loading 0.75 mmol/g (100 mg) was
washed with DCM and DMF (2 × 10 ml each), deblocked with
20% piperidine in DMF (10 ml) for 10 min, and washed with
DMF (2 × 10 ml), DCM (2 × 10 ml), and then with DMF
(2 × 10 ml). Fmoc-Leu-OH (3 equiv), coupling reagent (3 equiv),
and DIEA (6 equiv) were mixed in DMF (0.4 ml), pre-activated
for 1–2 min, and then added to the resin. The resulting slurry
was stirred slowly for 30–60 min and allowed to couple for
30 min (30-min double coupling for Aib-Aib). The ninhydrin test
was performed during the introduction of the first residue (neg-
ative). The resin was subsequently washed with DMF and then
deblocked by 20% piperidine in DMF for 7 min. Next, the peptide
resin was washed with DMF, DCM, and DMF, and coupled with
the next amino acid. Coupling and deblocking steps were re-
peated to provide the pentapeptide. The peptide was cleaved
from the resin by TFA/H2O (9 : 1) at room temperature for 2 h.
TFA was removed in vacuum, and the crude peptide was precip-
itated with ether. The crude weight was recorded, and the per-
centage of des-Aib (Tyr-Aib-Phe-Leu-NH2) during solid-phase
assembly of the pentapeptide (Tyr-Aib-Aib-Phe-Leu-NH2) was
confirmed by peak overlap in the presence of an authentic sam-
ple. The crude H-Tyr-Aib-Aib-Phe-Leu-NH2 (20) was analyzed by
HPLC analysis using a Sunfire C18 column (3.5 μm, 4.6 × 100 mm)
with a 996 PDA detector at 220 nm, flow rate 1 ml/min, using a
linear gradient of 10/900 in 20 min. CH3CN/H2O/0.1TFA. tR (pen-
tapeptide) = 7.73 min, [M + H]+ = 611.35; tR (tetrapeptide, des-
Aib) = 7.98 min, [M + H]+ = 526.30, none of the des-Tyr (448) or
the tripeptide (363) was observed.
16 Li H, Jian X, Ye Y-H, Fan C, Romoff T, Goodman M. 3-
(Diethoxyphosphoryl oxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT):
a
new coupling reagent with remarkable resistance to racemization.
Org. Lett. 1999; 1: 91–94.
17 Xie H-B, Tian G-L, Ye Y-H. Application of an organophosphorus re-
agent depbt for synthesis of cycloheptapeptide. Synth. Commun.
2000; 30: 4233–4240.
18 Gardiner JM, Procter J. Synthesis of N-alkoxybenzimidazoles with differ-
entiated C2 and O-substituents. Tetrahedron Lett. 2001; 42: 5109–5111.
19 Kokare ND, Nagawade RR, Rane VP, Shinde DB. Organophosphorus
esters of 1-hydroxy-2-phenylbenzimidazole: synthesis and utilization
as novel peptide coupling reagents. Synthesis 2007; 5: 766–772.
20 Yasuhara T, Nagaoka YT, Tomioka K. An activated phosphate for an ef-
ficient amide and peptide coupling reagent. J. Chem. Soc., Perkin
Trans. 1 2000, 17: 2901–2902.
Acknowledgements
In Spain, this work was partially supported by CICYT (CTQ2012-
30930), the Generalitat de Catalunya (2009SGR 1024), and the
Institut for Research in Biomedicine (IRB-Barcelona). The authors
extend their appreciation to the Deanship of Scientific Research
at King Saud University for funding this work through research
group no RGP-VPP-234.
21 Diago-Messeguer J, Palomo-Coll AL, Fernandez-Lizarbe JR, Zugaza-Bil-
bao A. A new reagent for activating carboxyl groups; preparation and
reactions of N,N-bis[2-oxo-3-ox-azolidinyl]phosphorodiamidic chlo-
ride. Synthesis 1980; 7: 547–551.
22 Van der Auwera C, Anteunis MJO. N,N′-bis(2-oxo-3-oxazolidinyl)
phosphinic chloride (BOP-Cl) a superb reagent for coupling at and
with iminoacid residues. Int. J. Pept. Protein. 1987; 29: 574–588.
23 Carpino LA, Ionesco D, El-Faham A, Henklein P, Wenschuh H, Bienert M,
Beyermann M Protected amino acid chlorides vs protected amino acid
fluorides: reactivity comparisons. Tetrahedron Lett. 1998; 39: 241–244.
24 Sheehan JC, Hess GP. A new method of forming peptide bonds. J. Am.
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Additional supporting information may be found in the online
version of this article at the publisher’s web site.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2014; 20: 1–6