PyOxP and PyOxB: The Oxyma-based novel family of phosphonium salts

Article abstract of DOI:10.1039/c003719b

Recent studies described the great impact of a non-benzotriazolic family of coupling reagents based on ethyl 2-cyano-2-(hydroxyimino)acetate, Oxyma, as a powerful coupling methodology for peptide synthesis. Here we present the synthesis and evaluation of the derived phosphonium salts O-[(1-cyano-2-ethoxy- 2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyOxP) and tetrafluoroborate (PyOxB). Both coupling reagents exhibited higher capacity to suppress racemization in various peptide models and enhanced solubility in DMF and DCM than benzotriazole-based reagents. In addition, the hexafluorophosphate analog PyOxP, combined excellent stability with outstanding efficiency in the assembly of demanding penta and decapeptides that include consecutive Aib residues. Cyclization models revealed the advantages of PyOxP, which rendered a higher percentage of cyclic material than other known potent phosphonium salts.

Full text of DOI:10.1039/c003719b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
PyOxP and PyOxB: the Oxyma-based novel family of phosphonium salts†
Ramon Subiro´s-Funosas,a,b Ayman El-Faham*a,c,d and Fernando Albericio*a,b,e
Received 12th March 2010, Accepted 19th May 2010
First published as an Advance Article on the web 17th June 2010
DOI: 10.1039/c003719b
Recent studies described the great impact of a non-benzotriazolic family of coupling reagents based on
ethyl 2-cyano-2-(hydroxyimino)acetate, Oxyma, as a powerful coupling methodology for peptide
synthesis. Here we present the synthesis and evaluation of the derived phosphonium salts
O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl) phosphonium
hexafluorophosphate (PyOxP) and tetrafluoroborate (PyOxB). Both coupling reagents exhibited higher
capacity to suppress racemization in various peptide models and enhanced solubility in DMF and
DCM than benzotriazole-based reagents. In addition, the hexafluorophosphate analog PyOxP,
combined excellent stability with outstanding efficiency in the assembly of demanding penta and
decapeptides that include consecutive Aib residues. Cyclization models revealed the advantages of
PyOxP, which rendered a higher percentage of cyclic material than other known potent phosphonium
salts.
side reactions, usually during slow couplings, such as cyclizations
Introduction
or in the introduction of hindered residues. In these situations, an
Success in peptide synthesis is highly dependent on the coupling
excess of such a coupling reagent usually causes the attachment of
strategy used to form the peptide bond.1,2 Traditionally, carbodi-
a guanidine moiety to the free N-terminus of the growing peptide
chain, thereby blocking any further reaction.11 Moreover, these
reagents are costly for peptide synthesis purposes. Phosphonium
salts differ from the former in the nature of the electrophilic
core as they have a positively charged phosphorus center and
they do not undergo the above mentioned termination of the
peptide chain when added in excess. Thus phosphonium salts
show similar potency to their aminium/uronium counterparts.12
These types of onium salts were first introduced by Kenner
and coworkers in 1969, although they did not gain general
acceptance until the 1990s, when many became commercially
available.13 The most predominant phosphonium salts display a
tris(pyrrolidino)phosphonium-type electrophilic core (PyAOP 12,
PyBOP 13, PyClock, 14; Fig. 2)14–16 while their tris(dimethylamino)
analogs, of which BOP is the most representative, are slightly less
reactive and also originate HMPA as a byproduct, which is a
carcinogenic and toxic phosphoramide.17–19
In both classes of onium salts, aminium/uronium and phos-
phonium, the same trend is observed: HOAt (2) analogs showed
superiority to HOBt (1) and 6-Cl–HOBt (3) ones in activation
and coupling steps and also in reducing the loss of optical
purity.12 In addition, derivatives of 6-Cl–HOBt (3) are generally
less hazardous and more reactive than HOBt (1) counterparts.9
This relative performance is connected to the acidity of the
additive, which is linked to the reactivity of the corresponding
active ester. Therefore, PyAOP (12) is considered the most reactive
phosphonium salt (pKa of HOAt=3.28), whereas PyClock (14) is
a good alternative to PyBOP (13), because the presence of Cl at
position 6 of the benzotriazole ring results in a better leaving group
(pKa of 6-Cl–HOBt=3.35 vs. pKa of HOBt=4.60).20 However,
additional factors contribute to the great difference in reactivity.
Thus, it has been proposed that the nitrogen at the 7-position
of HOAt creates a neighboring group effect, which results in
enhanced reactivity and solubility of its analogs.
imides were considered an efficient approach to assemble low
to moderate sequences, in consumption with additives (HOBt 1,
HOAt 2 and 6-Cl–HOBt 3; Fig. 1 left), which lower the likelihood
of racemization and other side reactions.3–5 However, the need for
stronger activating reagents led to the introduction of stand-alone
onium salts (mainly aminium/uronium and phosphonium ones),
which have become the preferred coupling choice in solution- and
solid-phase peptide synthesis.6
The most powerful onium salts are the aminium/uronium
ones. Their structure comprises a carbocation skeleton and an
additive, usually benzotriazole-based (HATU 4, HBTU 5/TBTU
6, HCTU 7/TCTU 8; Fig. 1 center).7–9 The tetramethylamino
group included in the carbocation skeleton of these classical
coupling reagents has recently evolved to a dimethylmorpholino
moiety (HDMA 9, HDMB 10, 6-Cl–HDMB 11; Fig. 1 right),10
thereby resulting in improved stability and solubility in common
solvents used in peptide synthesis, as well as an increase in
optical purity and reactivity. The high reactivity of both types
of aminium/uronium salts may also lead to the appearance of
aInstitute for Research in Biomedicine, Barcelona Science Park,
Baldiri Reixac 10, 08028, Barcelona, Spain. E-mail: aymanel_faham@
hotmail.com, albericio@irbbarcelona.org; Fax: +34 93-403-71-26; Tel: +34
93-403-70-88
bCIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine Barcelona Science Park, Baldiri Reixac 10, 08028, Barcelona,
Spain
cDepartment of Chemistry, College of Science, King Saud University,
P.O. Box 2455, Riyadh, 11451, Kingdom of Saudi Arabia
dDepartment of Chemistry, Faculty of Science, Alexandria University,
Ibrahimia, 21321, Alexandria, Egypt
eDepartment of Organic Chemistry, University of Barcelona, Mart´ı i
Franque´s 1-11, 08028, Barcelona, Spain
† Electronic supplementary information (ESI) available: 1H and 13C-NMR
spectra for PyOxP and PyOxB, and HPLC characterization of racemiza-
tion and coupling efficiency experiments. See DOI: 10.1039/c003719b
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Fig. 1 Benzotriazole-based additives (left), tetramethyl (center) and dimethylmorpholino (right) aminium salts.
was not tested.26 In the present study, we compared the
capacity of O-[(cyano-(ethoxycarbonyl)methyliden)-amino]-
yloxytripyrrolidinophosphonium hexafluorophosphate
and
tetrafluoroborate (PyOxP 17 and PyOxB 18, Fig. 4) to lower
racemization and enhance coupling extension in linear and cyclic
peptide models with the abovementioned benzotriazole-based
phosphonium salts PyAOP (12), PyBOP (13) and PyClock (14).
Fig. 2 Benzotriazole-based phosphonium salts.
Recently, we reported ethyl 2-cyano-2-(hydroxyimino)acetate
(Oxyma 15, Fig. 3) and its derived uronium salt 1-[(1-
(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-
morpholinomethylene)] methanaminium hexafluorophosphate
(COMU 16, Fig. 3) as efficient additive and coupling reagent
for peptide synthesis.21,22 The active esters formed by these
reagents are extremely reactive due to the low hindrance and
acidity of Oxyma (pKa = 4.60), which results in an excellent
leaving group.23 Oxyma (15) and COMU (16) show superiority to
HOBt (1) derivatives and a similar or even higher potency than
HOAt (2) analogs in suppressing racemization and assembling
demanding peptide models. In terms of safety, 15 and 16 also have
a considerably lower risk of explosion than benzotriazole-based
reagents and are compatible with microwave irradiation.24,25
Fig. 4 Structure of the hexafluorophosphate and tetrafluoroborate
tris(pyrrolidino)phosphonium salts derived from Oxyma.
Results and discussion
The Oxyma-based phosphonium salts PyOxP (17) and PyOxB
(18) were successfully synthesized in one-pot fashion from Oxyma
(15) (Scheme 1). The first step consisted of the rapid formation
of the intermediate potassium salt of the oxime with potassium
carbonate (K2CO3) after 10 min of gently stirring. Subsequent
addition of PyBroP or the analogous tetrafluoroborate salt to the
reaction mixture afforded, after 4 h, the desired phosphonium salts
17 and 18, in 76% yield after recrystallization from DCM–hexane.
Previous formation of the potassium salt is preferred over the
addition of a tertiary base to Oxyma (15), like Et3N, to generate
the anion, since this method yields Et3N+Br-, which is difficult to
separate from the desired phosphonium salt.
Fig. 3 Oxyma and its derived uronium salt, COMU.
The stability of phosphonium salts is a basic feature for their
optimal performance in peptide synthesis, since they are predom-
inantly used in slow-rate couplings, specially cyclizations, and
therefore a rapid hydrolysis of the reagent dramatically decreases
their efficiency. In order to check and compare the stability of the
proposed oxime-based phosphonium salts with their benzotriazole
counterparts, we conducted hydrolysis experiments in an NMR
tube (3 days in acetone) and in an open vial (5 days in DMF)
In view of these outstanding results, the next step was to
include Oxyma (15) in a tris(pyrrolidino)-type phosphonium
salt, in both hexafluorophosphate and tetrafluoroborate
forms. The synthesis and structure determination of the
hexafluorophosphate version was accomplished by Hoffmann
et al. in 2003, although its suitability as a coupling reagent
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Scheme 1
Table 1 Hydrolytic stability (%) of phosphonium salts during NMR and
HPLC assays
Table 2 Solubility of phosphonium salts in DMF and DCM
DMF
DCM
Method of analysis
NMR, 3 days (%)a
Entry Coupling Reagent Wt/g mL-1 Molarity Wt/g mL-1 Molarity
Entry
Coupling Reagent
HPLC, 5 h (%)b
1
2
3
4
5
PyAOP (12)
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
1.04
0.80
0.80
1.36
1.52
1.99
1.54
1.44
2.58
3.24
0.92
0.64
0.40
1.04
1.32
1.79
1.23
0.72
1.97
2.82
1
2
3
4
5
PyAOP (12)
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
10
20
50
60
30
29
39
35
40
25
a The samples were dissolved in acetone-d6 and placed in an NMR tube
(closed vial). b A solution of the phosphonium salt in DMF was kept at rt
in an open vial.
(Table 1). As envisaged, stability in the closed vial was higher
than in the open vessel. The hexafluorophosphonium derivative
17 showed slower hydrolysis than its tetrafluoroborate analog
18 in both conditions (entries 4 vs. 5). The lower stability of
PyOxB (18) was evident during the NMR assays, when a freshly
prepared solution rapidly changed color (from colorless to yellow
in DMF, from yellow to red in DCM), showing 6–7% hydrolysis
in NMR. These observations were consistent with the melting
points observed for the two Oxyma-based phosphonium salts
(131–132 ◦C for PyOxB vs. 185–186 ◦C for PyOxP). Not only
did PyOxP (17) show greater stability than PyOxB (18), but it
was also the most stable phosphonium salt tested, including the
benzotriazole analogs PyAOP (12), PyBOP (13) and PyClock (14)
(entry 4 vs. 1, 2, 3).12 Amongst these classical reagents, 14 exhibited
slower hydrolysis than 12 and a similar rate to 13, as previously
reported (entry 3 vs. 1, 2).16 Finally, in the DMF/DIEA mixture
PyOxB (18) hydrolyzed the most quickly.
Solubility is also a key feature in determining the suitability
of a coupling reagent for peptide synthesis. The solubility of
the various phosphonium salts in DMF (generally the solvent
of choice in peptide bond formation) and DCM was evaluated
(Table 2). All reagents exhibited higher solubility in the more
polar solvent (DMF), as expected. The Oxyma derivatives 17 and
18 were considerably more soluble than the benzotriazole ones
(12, 13, 14) in both solvents (entries 4, 5 vs. 1, 2, 3). Although
displaying lower stability than PyOxP (17), PyOxB (18) was more
soluble, affording up to 3.24 M solutions in DMF (entry 5 vs. 4).
Efficient peptide assembly is highly dependent on extension of
coupling and yield, but also on the control of optical purity. In
order to compare the retention of configuration induced by PyOxP
(17) and PyOxB (18) with that achieved by the benzotriazole-
based derivatives 12, 13 and 14, three previously studied peptide
Fig. 5
models were used, which involve stepwise and segment couplings
(Fig. 5).27–29
The [1+1] stepwise coupling of Z-Phg-OH onto H-Pro-NH2 to
afford Z-Phg-Pro-NH2 19 was performed in the presence of DIEA,
in solution and solid phase (Table 3). The Oxyma-based phospho-
nium salts showed better performance than the benzotriazole-
based derivatives in both approaches. The superiority of these
Table 3 Racemization during the [1+1] formation of Z-Phg-Pro-NH2 in
DMF (solution and solid-phase synthesis)a,b.c
Entry
Coupling approach
Solution-phase
Coupling Reagent
DL (%)
1
2
3
4
5
6
7
8
PyAOP (12)
2.2
5.8
1.6
0.3
0.6
2.6
5.7
1.4
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
PyAOP (12)/HOAt (2)
PyBOP (13)/HOBt (1)
PyClock (14)/6-Cl-HOBt (3)
PyOxP (17)/Oxyma (15)
PyOxB (18)/Oxyma (15)
PyAOP (12)
9
0.6
0.6
10
11
12
13
14
15
Solid-Phase
41.4d
44.2d
50.2d
7.4d
10.8d
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
a DIEA (2 equiv.) was used as base (for detailed procedure and analysis, see
Experimental section).27 b LL and DL forms of the model dipeptide were
identified after co-injection with a pure sample of LL. c Yields obtained
were in the range 88–92%. d The percentage in the table is the average of
two runs.
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Table 4 Racemization during the [2+1] formation of Z-Phe-Val-Pro-NH2
Table 5 Yield and racemization during the [3+3] formation of Z-Gly-
in DMF (solution and solid-phase synthesis) a,b,c
Gly-Val-Pro-Gly-Gly-NH2 in DMF (solid-phase synthesis)a,b,c
Entry Coupling approach Coupling Reagent
LDL (%)
Entry
Coupling Reagent
Base (equiv.)
DIEA (2)
DL (%)
1
2
3
4
5
6
7
8
Solution-phase
PyAOP (12)
3.0
12.5
8.6
5.7
5.3
1.0
8.8
1
2
3
4
5
6
7
8
9
PyAOP (12)
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
PyAOP (12)
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
33.7
51.0
43.1
22.2
22.2
4.4
10.2
11.5
4.1
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
PyAOP (12)/HOAt (2)
PyBOP (13)/HOBt (1)
TMP (2)
PyClock (14)/6-Cl-HOBt (3) 7.9
9
PyOxP (17)/Oxyma (15)
PyAOP (12)
3.4
10
11
12
13
14
15
16
17
18
Solid-Phase
17.3
29.9
28.6
23.0
27.8
15.0
29.5
10
6.9
PyBOP (13)
a For detailed procedure and analysis see Experimental section. b LL and
DL forms of the model hexapeptide were identified after co-injection with
pure samples. c Yields obtained were in the range 88–92%.
PyClock (14)
PyOxP (17)
PyOxB (18)
PyAOP (12)/HOAt (2)
PyBOP (13)/HOBt (1)
PyClock (14)/6-Cl-HOBt (3) 25.2
PyOxP (17)/Oxyma (15) 22.2
the percentage of racemization detected with PyOxP (17), which
was even lower than that recorded for the HOAt analog 12 (entry
4, 9 vs. 1, 6).
a TMP (2 equiv.) was used as base (for detailed procedure and analysis, see
Experimental section).27,29 b LLL and LDL forms of the model tripeptide
were identified after co-injection with a pure sample of LLL. c Yields
obtained were in the range 88–92%.
After addressing loss of optical purity, the coupling efficiency
of PyOxP (17), the most stable and strongest racemization-
suppressing analog of the Oxyma-based phosphonium salts,
was tested in the assembly of complex peptide models, based
on Leu-enkephalin pentapeptide (H-Tyr-Gly-Gly-Phe-Leu-NH2),
its linear dimer (H-Tyr-Gly-Gly-Phe-Leu-Tyr-Gly-Gly-Phe-Leu-
NH2) and ACP (65–74) decapeptide (H-Val-Gln-Ala-Ala-Ile-Asp-
Tyr-Ile-Asn-Gly-NH2). The design of the analogs consisted of
introducing the hindered a,a-disubstituted amino acid Aib to
replace consecutive Gly (Leu-enkephalin and dimer derivatives 22,
23 and 24) or Ala (ACP derivative 25) residues, in order to reduce
coupling extension, thereby improving our capacity to distinguish
differences between coupling reagents (Fig. 6).
salts was greater in the solid-phase synthesis, where racemization
with PyOxP (17) and PyOxB (18) was 4–5 times lower than with
PyAOP (12), PyBOP (13) and PyClock (14) (entries 14, 15 vs.
11, 12, 13). Among these novel phosphonium salts, 17 afforded
the best results regarding optical purity (entries 4, 14 vs. 5, 15).
In solution, 14 gave lower racemization than 12, the most potent
phosphonium salt known to date (entry 3 vs. 1).16 Addition of the
corresponding additive to the onium salt did not have a siginificant
effect on the percentage of racemization (entries 6–10 vs. 1–5).
The performance of the phosphonium salts in stepwise synthesis
was also tested in the Z-Phe-Ser-Pro-NH2 model, although this
system was not sensitive enough to allow a comparative study
(racemization was < 0.1% in all experiments).
The loss of optical purity during the [2+1] segment coupling
of dipeptide Z-Phe-Val-OH onto H-Pro-NH2 to yield Z-Phe-Val-
Pro-NH2 20 was higher than in the previous stepwise model. This
result is attributed to the greater sensitivity of peptide fragments
than single amino acids (Table 4). Given that TMP reduces
racemization in segment couplings more efficiently than other
bases, its use is recommended in these experiments.29 Oxyma-based
PyOxP (17) and PyOxB (18) showed a similar performance, giving
lower racemizations than HOBt and 6-Cl–HOBt analogs (entries
4, 5 vs. 2, 3 and entries 13, 14 vs. 11, 12). The addition of additives
in peptide model 20 produced a clear increase in the control of
optical purity, in contrast to the less sensitive segment coupling 19
(entries 6–9 vs. 1–5 and 15–18 vs. 10–14).
The retention of configuration in fragment couplings was
further tested in the [3+3] formation of the model hexapeptide Z-
Gly-Gly-Val-Pro-Gly-Gly-NH2 21, a modified version of the well-
known system Z-Gly-Gly-Val-Ala-Gly-Gly-NH2, in solid-phase
fashion (Table 5).29 The results indicate the suitability of using the
highly hindered base TMP instead of DIEA for segment couplings.
Again, Oxyma-based phosphonium salts showed an outstanding
performance, regardless of the base used. Of particular note was
Fig. 6
Coupling efficiency was first evaluated in the synthesis of the
highly demanding Aib-enkephalin analog 22 (H-Tyr-Aib-Aib-
Phe-Leu-NH2).7a Coupling times of 30 min, except for the Aib-Aib
assembly (30 min double coupling), were applied during solid-
phase elongation. The pentapeptide/deletion tetrapeptide ratios
obtained with the reagents are given in Table 6. Following the
trend observed in the previous experiments, PyOxP (17) provided
higher yield and extent of coupling than PyAOP (12), which
was the benzotriazole derivative that best performed, followed
by PyClock (14) (entry 4 vs. 1, 2, 3). The desired pentapeptide
was obtained with PyBOP (13) in a poor 48.6% yield, in
contrast to its Oxyma analog 17, which afforded near quantitative
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To conclude the coupling efficiency assays, the Aib67-Aib68
modified ACP (64–75) decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-
Tyr-Ile-Asn-Gly-NH2) 25 was assembled by means of 5 min
couplings, except in the formation of Aib-Ile and Aib-Aib peptide
bonds (30 min and 30 min double coupling respectively).31
Misincorporation of one residue (des-Aib, des-Asn, des-Gln and
des- Ile69) or two (des-Ile69,Ile72) occurred (Table 8). PyOxP (17)
allowed the introduction of Aib, Asn and Ile69 to a greater extent
than PyAOP (12) and PyBOP (13), thereby affording 81% of the
Aib-decapeptide 25. In contrast, the benzotriazole counterparts
provided only 64% and 48% yield (entry 3 vs. 1,2). Higher yield
was also obtained with the Oxyma-based phosphonium salt 17
(83.2%).
Table 6 Percentage of Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-
Phe-Leu-NH2) and des-Aib deletion tetrapeptide (H-Tyr-Aib-Phe-Leu-
NH2) during solid-phase assembly a,b
Entry
Coupling Reagent
Yield (%)
Penta (%)
des-Aib(%)
1
2
3
4
PyAOP (12)
PyBOP (13)
PyClock (14)
PyOxP (17)
89.0
80.2
85.0
89.2
84.8
48.6
76.7
98.5
14.2
47.3
23.3
1.5
a Couplings were performed with 2 equiv. of DIEA b Tetrapeptide (des-
Aib) was confirmed by peak overlap in the presence of a true sample (for
detailed procedure and analysis, see Experimental section).7a
Finally, the suitability of PyOxP (17) and PyOxB (18) as
coupling reagents in cyclization steps was tested in the synthesis of
cyclic Ala-pentapeptide (H-Ala-Ala-NMeAla-Ala-Ala-OH).32,33
The main risk associated with this step is the capping of the
N-terminus amino group. This effect has been reported when
aminium/uronium salts are used and it leads to guanidylation
and termination of the peptide chain (Scheme 2).
Aib-enkephaline 22. Thus, 17 showed better performance than its
benzotriazole counterparts.
The phosphonium salts were further examined in the elongation
of the sterically hindered 8,9- and 3,4-Aib analogs of Leu-
enkephaline dimer (23 and 24), which displays difficult Leu and Tyr
couplings.30 After short couplings, many nonapeptides (des-Phe,
des-Leu, des-Tyr, des-Aib and des-Gly) and octapeptides (des-
Aib,Leu, des-Aib,Tyr, des-Leu,Tyr, des-Gly,Tyr and des-Gly,Aib)
were produced. The introduction of Aib and the adjacent Tyr
was the most demanding coupling (Table 7). The experiments
with PyAOP (12) resulted in an excellent degree of coupling,
although PyOxP (17) performed better, affording the target
decapeptides in >95% yield (entries 4, 8 vs. 1, 5) in solid-
phase syntheses. In the benzotriazole series, PyClock (14) and
PyBOP (13) obtained similar percentages of the 8,9-analog 23
(entry 3 vs. 2); however, in the synthesis of the 3,4-derivative 24,
PyClock (14) showed higher potency than its HOBt counterpart 13
(entry 7 vs. 6).
Scheme 2
Once the linear pentapeptide was successfully assembled in
solid-phase and cleaved from the resin, a 0.01M solution in
DMF was mixed with the corresponding coupling reagent and
DIEA. After 24 h, samples were taken and their content was
analyzed by reverse-phase HPLC-PDA and HPLC-MS (Table 9).
Table 7 Percentage of 8,9-(H-Tyr-Aib-Aib-Phe-Leu -Tyr-Gly-Gly-Phe-Leu-NH2) and 3,4-(H-Tyr-Gly-Gly-Phe-Leu-Tyr-Aib-Aib-Phe-Leu-NH2) Aib-
enkephalin decapeptides and deletion peptides during solid-phase assemblya,b
Coupling
Entry Reagent
des-
des-Aib- des-Aib, des-
des- Leu, des-Gly, des
des-
des-
des-Gly,
Analog
deca (%) Phe (%) Leu (%) Tyr (%)
Leu (%) Tyr (%) Tyr (%) Tyr (%) Aib (%) Gly (%) Aib (%)
c
c
c
c
c
c
c
c
c
8,9-Aib 23
1
2
3
4
5
6
7
8
PyAOP (12)
PyBOP (13)
93.3
56.1
0.1
0.1
0.1
0.1
0.3
0.1
1.0
0.1
—
0.2
0.7
0.1
0.5
0.1
0.3
0.1
0.1
0.4
0.3
0.2
0.4
0.3
0.1
0.1
0.3
0.2
0.2
0.2
0.2
0.3
0.7
0.5
0.7
—
—
—
—
0.9
3.4
2.4
0.1
1.2
0.5
1.0
1.1
3.6
1.3
1.2
1.3
1.7
1.6
1.2
0.8
1.5
—
—
—
—
0.3
0.4
37.7
39.1
0.5
PyClock (14) 56.2
c
PyOxP (17)
PyAOP (12)
PyBOP (13)
PyClock (14) 71.5
PyOxP (17) 95.4
96.5
85.5
51.5
—
3,4-Aib 24
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
9.9
0.6
0.6
0.3
0.1
44.8
24.6
0.5
a Couplings were performed with 2 equiv. of DIEA (for detailed procedure and analysis, see Experimental section). b Apart from the detailed deletion
peptides, unknown peaks were observed in all cases at 5.2 and 5.8 min in the 8,9 analog and at 4.6 and 6.6 min in the 3,4-analog (in the range 0.5–3.0%).
c No misincorporation was detected.
Table 8 Percentage of Aib67-Aib68 analog of ACP (64–75) decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-Tyr-Ile-Asn-Gly-NH2) and deletion peptides during
solid-phase assembly a,b
Entry
Coupling Reagent
Yield (%)
Aib-deca (%)
des-Aib (%)
des-Asn (%)
des-Gln (%)
des-Ile69, Ile72 (%)
des-Ile69 (%)
1
2
3
PyAOP (12)
PyBOP (13)
PyOxP (17)
81.0
78.0
83.2
64.0
48.0
81.0
12.0
32.0
2.2
3.8
2.0
2.2
1.0
0.8
1.0
1.0
5.0
1.7
10.4
12.0
10.1
a Fmoc-amino acids were preactivated with coupling reagents in the presence of 1 equiv. of DIEA and then, after addition to the resin, a second equiv.
of DIEA was added (for detailed procedure and analysis, see Experimental section). b Other unknown peaks were observed, but these were not related to
the peptide.
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Table 9 Cyclization of H-Ala-Ala-NMeAla-Ala-Ala-OH in DMF after
is advantageous and highly recommended in automated peptide
synthesizers.
24 h (10-2 M).a,b
Coupling
Reagent
Cyclic
penta (%)
Linear
penta (%)
Linear
dimer (%)
Experimental
Entry
Materials
1
2
3
4
5
PyAOP (12)
PyBOP (13)
PyClock (14)
PyOxP (17)
PyOxB (18)
54
43
61
70
47
10
28
15
10
15
36
29
24
20
38
The solvents used were of HPLC reagent grade. Oxyma Pure
was obtained from Luxembourg Biotechnologies. Melting points
were measured in open capillary tubes with a Buchi B-540
melting point apparatus and were uncorrected. 1H NMR and
13C NMR magnetic resonance spectra were recorded on a Varian
Mercury 400 MHz spectrometer at rt with chemical shift values
reported in d units (ppm) relative to tetramethylsilane (TMS) as
internal standard. For analytical separation and characterization,
a reverse-phase Waters 2695 HPLC separation module, coupled
to a Waters 2998 PDA UV detector, was used. Chromatograms
were processed with Empower software. Peptide mass was detected
by means of an HPLC-PDA system as described above, coupled
to a Waters Micromass ZQ mass detector, using the MassLynx
4.1 software. Elemental analyses were performed on a Perkin-
Elmer 2400 elemental analyzer, and the values found were within
0.3% of the theoretical values. Follow-up of the synthesis of
the phosphonium salts was done by TLC on silica gel-protected
aluminium sheets (Type 60 GF254, Merck) and the spots were
detected by exposure to UV-lamp at l 254 nm for a few seconds.
The compounds were named using ChemBioDraw Ultra version
12.0, Cambridge soft Corporation. Samples were introduced into
a Waters Synapt HDMS mass spectrometer by direct infusion and
exact masses were determined (ESI positive polarity, W analyzer,
3000 V capillary voltage, 150 ◦C and 100 ◦C desolvation and source
temperature, 40 V sample cone, 100–1500 m/z).
a The linear pentapeptide was elongated on a 2-chlorotrityl resin by means
of DIC/HOAt (2)-mediated couplings. b 4 equiv. excess of DIEA was
used (for detailed procedure and analysis, see Experimental section). c In
analogous cyclizations performed with HDMA (9) and COMU (16) in
DCM, a high amount of guanidylated material was found (90% and
60% respectively), thus confirming the unsuitability of uronium salts in
cyclization steps.
In all experiments, the cyclic product was detected along with
the starting linear pentapeptide and the linear dimer. PyOxP
(17) afforded the highest percentage of cyclized material, closely
followed by PyClock (14), which showed stronger performance
than its HOAt derivative PyAOP (12) (entry 4 vs. 1, 3). PyBOP
(13) and PyOxB (18) were the least efficient in providing the cyclic
pentapeptide, thus confirming the superiority of the hexafluo-
rophosphate version of the Oxyma-based phosphonium salt over
the tetrafluoroborate counterpart (entry 4 vs. 5).
Conclusions
In summary, PyOxP (17) and PyOxB (18), the hexaflurophosphate
and tetrafluoroborate phosphonium salts derived from Oxyma
(15), have been successfully obtained in a one-pot procedure
from ethyl 2-cyano-2-(hydroxyimino)acetate. This was achieved
through reaction of the intermediate potassium salt and PyBroP or
its analogous tetrafluoroborate salt to afford the reagents in good
yields and high purities. The presence of Oxyma (15) conferred the
reagents higher solubility in standard solvents for peptide synthesis
than PyAOP (12), PyBOP (13) and PyClock (14). Consistently, the
analysis of racemization models, involving either [1+1] stepwise
or [2+1] and [3+3] segment couplings, showed a higher control of
optical purity of PyOxP (17) and PyOxB (18) over their HOBt and
6-Cl–HOBt counterparts, and similar performance to that of the
HOAt derivative.
One-pot synthesis of PyOxP (17) and PyOxB (18)
Oxyma (20 mmol; 2.84 g) was suspended in ACN (50 mL) and
potassium carbonate (10 mmol; 1.38 g) was added. The reaction
mixture was stirred at rt for 10 min and then 20 mmol of the
bromophosphonium salt (PyBroP or its tetrafluoroborate analog)
was added under nitrogen atmosphere. The reaction mixture was
stirred at rt for 4 h, filtered from the precipitate and washed with
ACN (10 mL). The filtrate was concentrated under vacuum and
diethylether was added with stirring to provide the phosphonium
salts in 76% yield (7.9 g), after◦ recrystallization from DCM–
1
hexane. PyOxP: mp = 185–186 C; H NMR (CDCl3): d 1.41
Although PyOxP (17) and PyOxB (18) allow the preparation of
more concentrated solutions, the use of the former is preferred in
view of its higher stability and coupling efficiency, both in the as-
sembly of linear and cyclic peptide models. Apart from being more
stable in solution than PyAOP (12), PyBOP (13) and PyClock (14),
PyOxP (17) also showed greater coupling performance during the
elongation of the sterically demanding Leu-enkephalin and ACP
(64–75) derivatives that present consecutive Aib residues. With
respect to the cyclization of H-Ala-Ala-NMeAla-Ala-Ala-OH,
PyOxP (17) afforded the cyclized product in the highest percentage,
superior to that achieved in the synthesis with PyAOP and with a
lower percentage of the linear by-products.
(t, 3H, CH3), 2.01–2.04 (m, 12H, 6 C-CH2), 3.37–3.41 (m, 12H,
6 N-CH2), 4.47 (q, 2H, CH2) ppm; 13C NMR (CDCl3):d 14.00,
26.39, 48.29, 65.07, 106.31, 135.80, 135.95, 155.87 ppm.; elemental
analysis (%) calculated for C17H29F6N5O3P2 (527.38): C, 38.72;
H, 5.54; F, 21.61; N, 13.28. Found: C, 38.59; H, 5.45; N, 13.38.
PyOxB: mp = 131–132 ◦C; H1 NMR (acetone-d6): d 1.36 (t, 3H,
CH3), 2.04–2.05 (m, 12H, 6 C-CH2), 3.51–3.53 (m, 12H, 6 N-CH2),
4.45 (q, 2H, CH2) ppm,13C NMR (acetone-d6):d 13.47, 26.21,
47.97, 64.28, 106.97, 136.69, 136.84, 156.36 ppm. A sample for
exact mass determination was prepared, dissolving in CH3CN and
diluting 1 to 100 in H2O–CH3CN (1 : 1): [M]+=382.2009. PyOxP
and PyOxB were also characterized by HPLC, using a Sun fire
C18 (3.5 mm, 4.6 ¥ 100 mm) column, linear gradient 5 to 100%
of 0.036% TFA in CH3CN/0.045%TFA in H2O over 8 min; flow
rate = 1.0 ml min-1; detection at 220 nm; tR (PyOxP)= 5.19 min,
tR (PyOxB) = 5.17 min.
In conclusion, PyOxP (17) showed superiority not only over
PyBOP (13) and PyClock (14), but also over PyAOP (12), the
most potent phosphonium salt known to date. Moreover, given
its high stability in open and closed vials, the use of PyOxP
3670 | Org. Biomol. Chem., 2010, 8, 3665–3673
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Racemization tests using model peptides in solution and solid
phase27,29
Solid-Phase Synthesis of Aib-ACP (65–74) decapeptide
(H-Val-Gln-Aib67-Aib68-Ile69-Asp-Tyr-Ile72-Asn-Gly-NH2)31
Solution-phase tests were conducted adding the coupling reagent
(0.125 mmol, 1 equiv.) to a solution of the amino acids (1 equiv.
each) in DMF (1 mL) in the presence of base (2 equiv.) at 0 ◦C. The
resulting mixture was stirred for 3–4 h in an ice bath. The work-up
of the reaction mixture was as previously described for Z-Phg-
Pro-NH2 and Z-Phe-Val-Pro-NH2.27 For solid-phase assays, the
coupling was performed with H-Pro-RinkAmide-PS-resin or H-
Pro-Gly-Gly-RinkAmide-PS-resin, 3 equiv. of the corresponding
acid (Z-Phg-OH, Z-Phe-Val-OH or Z-Gly-Gly-Val-OH) and 3
equiv. of the corresponding coupling reagent in DMF at rt, with
preactivation of 10–30 s with 6 equiv. of base (DIEA or TMP).22
The reaction mixture was left 3–4 h at rt. The peptide was recovered
after cleavage from the resin with 50% TFA in DCM for 1 h at rt.
TFA and DCM were removed in vacuo and the crude peptide was
precipitated with ether or dissolved in ACN and injected directly
into the HPLC apparatus. Crude products from the solution-
and solid-phase tests were analyzed by reverse-phase HPLC-PDA,
using a Sun fire C18 (3.5 mm, 4.6 ¥ 100 mm) column, linear gradient
of 20 to 50% (Z-Phg-Pro-NH2) over 20 min, linear gradient of 20
to 70% (Z-Phe-Val-Pro-NH2) over 8 min or linear gradient of 10%
to 60% (Z-Gly-Gly-Val-Pro-Gly-Gly-NH2) CH3CN/0.036% TFA
in H2O/0.045% TFA over 8 min; flow = 1.0 mL min-1 detection
at 220 nm. tR (Z-L-Phg-Pro-NH2) = 12.51 min, tR (Z-D-Phg-Pro-
NH2) = 13.07 min, tR (Z-Phe-L-Val-Pro-NH2) = 6.95 min, tR (Z-
Phe-D-Val-Pro-NH2) = 7.43 min, tR (Z-Gly-Gly-L-Val-Pro-Gly-
Gly-NH2) = 5.53 min, tR (Z-Gly-Gly-D-Val-Pro-Gly-Gly-NH2) =
5.91 min.
The synthesis was conducted in a plastic syringe attached to
a vacuum manifold so as to effect rapid removal of reagents
and solvent. The Fmoc-RinkAmide-MBHA-PS resin (loading =
0.45 mmol g-1, 100 mg) was swelled in DCM and conditioned in
DMF (2 ¥ 10 mL each). The Fmoc-protecting group was removed
by treatment with 20% piperidine in DMF (10 mL) for 10 min,
and the resin was washed with DMF, DCM and DMF (2 ¥ 10 mL
each). Acylation took place by mixing the resin with a solution
of Fmoc-Gly-OH (3 equiv.), and coupling reagent (2 equiv.) in
0.4 mL DMF, previously preactivated with DIEA (2 equiv.) for
1–2 min, followed by addition of extra 2 equiv. of DIEA. The
reaction mixture was stirred slowly for 1 min and allowed to
couple for 5 min (30 min coupling for Aib-Ile and 30 min double
coupling in case of Aib-Aib). The resin was washed with DMF,
and then deblocked with a solution of 20% piperidine in DMF for
7 min. The resin was washed with DMF, DCM, DMF (2 ¥ 10 mL
each) and then coupled with the next amino acid to obtain the
pentapeptide. The peptide was cleaved from the resin by treatment
with TFA–H2O (9 : 1) at rt for 2 h. TFA was removed in vacuo and
the crude peptide was precipitated with ether. The precipitate was
then redissolved in H2O and lyophilized. The weight of the crude
product was recorded and the purity was determined by HPLC
analysis, using a Sun fire C18 (3.5 mm, 4.6 ¥ 100 mm) column,
linear gradient 10 to 35% of 0.036% TFA in CH3CN/0.045%TFA
in H2O over 8 min; flow rate = 1.0 ml min-1; detection at 220 nm; tR
(Aib-ACP) = 6.41 min, [M+H]+=1091.2; tR (des-Aib) = 6.57 min,
[M+H]+ = 1005.1; tR (des-Asn) = 6.67 min, [M+H]+ = 997.2;
tR (des-Gln) = 6.95 min, [M+H]+ = 963.4; tR (des-Ile69, Ile72) =
5.12 min, [M+H]+= 863.9; tR (des-Ile69) = 6.22 min, [M+H]+=
976.5.
Solid-phase synthesis of Aib-enkephalin pentapeptide
(H-Tyr-Aib-Aib-Phe-Leu-NH2)7a
The synthesis was carried out in a plastic syringe attached to
a vacuum manifold so as to effect rapid removal of reagents and
solvent. The Fmoc-RinkAmide-AM-PS (loading = 0.63 mmol g-1,
100 mg) resin was swelled in DCM and conditioned in DMF (2 ¥
10 mL each). The resin was deblocked by treatment with 20%
piperidine in DMF (10 mL) for 10 min. The resin was washed
with DMF, DCM and DMF (2 ¥ 10 mL each) and then acylated
with a solution of Fmoc-Leu-OH (3 equiv.) and coupling reagent
(3 equiv.) in 0.4 mL of DMF, previously preactivated with DIEA
(6 equiv.) for 1–2 min before addition to the resin. The reaction
mixture was stirred slowly for 1 min and allowed to couple
for 30 min (30 min double coupling for Aib-Aib peptide bond
formation). The resin was washed with DMF, and then deblocked
by treatment with 20% piperidine in DMF for 7 min. The resin
was washed with DMF, DCM and DMF (2 ¥ 10 mL each) and
then coupled with the next amino acid to obtain the pentapeptide.
The peptide was cleaved from the resin after treatment with TFA–
H2O (9 : 1) at rt for 2 h. TFA was removed in vacuo and the crude
peptide was precipitated with diethyl ether. The weight of the crude
product was recorded and purity was determined by reverse-phase
HPLC-PDA, using a Sun fire C18 (3.5 mm, 4.6 ¥ 100 mm) column,
linear gradient 0 to 100% of 0.036% TFA in CH3CN/0.045%TFA
in H2O over 15 min; flow rate = 1.0 ml min-1; detection at 220 nm;
tR (penta) = 6.68 min, [M+H]+=611.0; tR (des-Aib) = 6.78 min,
[M+H]+=526.4. Neither des-Tyr (448.3) nor tripeptide (363.5)
were observed.
Solid-phase synthesis of 8,9-Aib enkephalin decapeptide
(H-Tyr-Aib-Aib-Phe-Leu-Tyr-Gly-Gly-Phe-Leu-NH2)30
The synthesis was carried out in a plastic syringe attached to
a vacuum manifold so as to effect rapid removal of reagents
and solvent. The resin Fmoc-Rink-amide-AM-PS (loading =
0.63 mmol g-1, 100 mg) resin was swelled in DCM and conditioned
in DMF (2 ¥ 10 mL each). The resin was deblocked by treatment
with 20% piperidine in DMF (10 mL) for 10 min. The resin was
washed with DMF, DCM and DMF (2 ¥ 10 mL each) and then
acylated with a solution of Fmoc-Leu-OH (3 equiv.) and coupling
reagent (3 equiv in 0.4 mL of DMF, previously preactivated with
DIEA (6 equiv.) for 1–2 min before addition to the resin. The
reaction mixture was stirred slowly for 1 min and allowed to
couple for 30 min (30 min double coupling for Aib-Aib coupling).
The resin was washed with DMF and then deblocked by treat-
ment with 20% piperidine in DMF for 7 min. The resin was washed
with DMF, DCM and DMF (2 ¥ 10 mL each) and then coupled
with the next amino acid to obtain the pentapeptide. The peptide
was cleaved from the resin after treatment with TFA–H2O (9 : 1)
at rt for 2 h. TFA was removed in vacuo and the crude peptide was
precipitated with diethyl ether. The weight of the crude product
was recorded and purity was determined by reverse-phase HPLC-
PDA, using a XBridge C18 (2.5 mm, 4.6 ¥ 75 mm) column, linear
gradient 30 to 37% of 0.036% TFA in CH3CN/0.045%TFA in H2O
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over 8 min; flow rate = 1.0 ml min-1; detection at 220 nm; tR (des-
Phe) = 3.31 min, [M+H]+ = 1001.5; tR (des-Aib,Tyr) = 3.65 min,
[M+H]+ = 900.6; tR (des-Leu) = 3.74 min, [M+H]+ = 1035.5; tR
(des-Leu,Tyr) = 4.26 min, [M+H]+ = 872.5; tR (des-Aib,Leu) =
4.40 min, [M+H]+ = 950.5; tR (des-Tyr) = 4.82 min, [M+H]+ =
985.7; tR (decapeptide) = 6.17 min, [M+H]+ = 1148.7; tR (des-
Aib) = 6.73 min, [M+H]+ = 1063.6; tR (des-Gly) = 8.11 min,
[M+H]+ = 1091.6.
resin was washed with DMF, DCM, and DMF (2 ¥ 50 mL each)
and then coupled with the next Fmoc-Ala-OH (3 mmol) by means
of DIC (3 mmol), and HOAt (3 mmol) for 1 h (1 h double
coupling for the coupling of Fmoc-Ala-OH with H-NMeAla-Ala-
Ala-Resin). The coupling and deblocking cycles were repeated to
obtain the pentapeptide. The peptide was cleaved from the resin
by treatment with 50 mL of TFA/DCM (2%) at rt for 5 min. The
resin was filtered and then washed with an extra solution of 2%
in TFA/DCM (20 mL). TFA and DCM were removed in vacuo
and the crude peptide was precipitated with cold diethyl ether.
The precipitate was then collected and washed two extra times.
After drying the peptide under vacuum (1.0 g, 66.5% yield), purity
(99.0%) was determined by reverse-phase HPLC analysis using a
Sun fire C18 (3.5 mm, 4.6 ¥ 100 mm) column, linear gradient 5 to
25% of 0.036% TFA in CH3CN/0.045%TFA in H2O over 8 min;
flow rate = 1.0 ml min-1; detection at 220 nm; tR (penta Ala) =
2.99 min, [M+H]+ = 388.2.
B: Cyclization. The cyclization step was performed as reported
in a previous study.32 The linear pentapeptide (0.1 mmol) was
cyclized by treatment with the corresponding coupling reagent
(0.11 mmol) and 0.44 mmol of DIEA in DMF (10 mL, 0.01 M).
After removing DMF under reduced pressure, the crude product
was extracted with methanol and precipitated with ether. The
sample was dissolved with 100 ml of 1%TFA/H2O, completing
the volume to 1 ml with ACN. 10 ml of the resulting solution was
injected into a reverse-phase HPLC apparatus using a Sun fire
C18 (3.5 mm, 4.6 ¥ 100 mm) column, linear gradient 5 to 55%
of 0.036% TFA in CH3CN/0.045%TFA in H2O over 8 min; flow
rate = 1.0 ml min-1; detection at 220 nm; tR (linear pentapeptide) =
2.65 min, [M+H]+ = 388.2; tR (cyclic pentapeptide)= 7.12 min,
[M+H]+ = 370.7; tR (linear dimer) = 8.67 min, [M+H]+ = 739.5.
Solid-phase synthesis of 3,4-Aib enkephalin decapeptide
(H-Tyr-Gly-Gly-Phe-Leu-Tyr-Aib-Aib-Phe-Leu-NH2)30
The synthesis was carried out in a plastic syringe attached to
a vacuum manifold so as to effect rapid removal of reagents
and solvent. The resin Fmoc-Rink-amide-AM-PS (loading =
0.63 mmol g-1, 100 mg) resin was swelled in DCM and conditioned
in DMF (2 ¥ 10 mL each). The resin was deblocked by treatment
with 20% piperidine in DMF (10 mL) for 10 min. The resin was
washed with DMF, DCM and DMF (2 ¥ 10 mL each) and then
acylated with a solution of Fmoc-Leu-OH (3 equiv.) and coupling
reagent (3 equiv.) in 0.4 mL of DMF, previously preactivated with
DIEA (6 equiv.) for 1–2 min before addition to the resin. The
reaction mixture was stirred slowly for 1 min and allowed to couple
for 30 min (only 10 min for the last five residues). The resin was
washed with DMF and then deblocked by treatment with 20%
piperidine in DMF for 7 min. The resin was washed with DMF,
DCM, DMF (2 ¥ 10 mL each) and the coupling and deblocking
cycles were repeated to obtain the decapeptide. The peptide was
cleaved from the resin after treatment with TFA–H2O (9 : 1) at rt
for 2 h. TFA was removed in vacuo and the crude peptide was
precipitated with diethyl ether. The weight of the crude product
was recorded and purity was determined by reverse-phase HPLC-
PDA, using a XBridge C18 (2.5 mm, 4.6 ¥ 75 mm) column, linear
gradient 32 to 35% of 0.036% TFA in CH3CN/0.045%TFA in
H2O over 8 min; flow rate = 1.0 ml min-1; detection at 220 nm;
tR (des-Phe) = 3.43 min, [M+H]+ = 1001.4; tR (des-Aib,Leu) =
3.71 min, [M+H]+ = 950.6; tR (des-Leu) = 4.27 min, [M+H]+ =
1035.5; tR (des-Leu,Tyr) = 4.81 min, [M+H]+ = 872.5; tR (des-
Gly,Tyr) = 5.69 min, [M+H]+ = 928.7; tR (des-Tyr) = 5.90 min,
[M+H]+ = 985.8; tR (des-Aib,Tyr) = 6.24 min, [M+H]+ = 950.5;
tR (decapeptide) = 7.54 min, [M+H]+ = 1148.7; tR (des-Aib) =
8.21 min, [M+H]+ = 1063.7; tR (des-Gly) = 8.53 min, [M+H]+ =
1091.8; tR (des-Gly,Aib) = 9.04 min, [M+H]+ = 1006.8
Acknowledgements
This work was partially supported by CICYT (CTQ2006-
03794/BQU), Luxembourg Bio Technologies, Ltd. (Rehovot),
the Generalitat de Catalunya (2005SGR 00662), the Institute for
Research in Biomedicine, and the Barcelona Science Park. RS-
F thanks the Ministerio de Educacio´n y Ciencia for a FPU PhD
fellowship.
Notes and references
1 Abbreviations not defined in text: Aib, a-aminoisobutyric acid; ACN,
CH3CN, ACP, acyl carrier protein decapeptide (65–74); AM-PS,
aminomethyl-polystyrene resin; DIC, N,N¢-diisopropylcarbodiimide;
BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluo-
rophosphate; COMU, 1-[(1-(cyano-2-ethoxy-2-oxoethylideneami-
nooxy)-dimethylamino-morpholinomethylene)] methanaminium hexa-
fluorophosphate DCM, dicloromethane; DIEA, N,N-diisopropy-
lethylamine; DMF, N,N-dimethylformamide; HOBt, 1-hydroxyben-
zotriazole; 6-Cl-HOBt, 6-chloro-1-hydroxybenzotriazole; HOAt,
7-aza-1-hydroxybenzotriazole; HATU, N-[(dimethylamino)-1H-1,2,3-
triazolo[4,5-b]pyridin-1-yl-methylene)-N-methylmethanaminium he-
xafluorophosphate N-oxide; HBTU, N-[(1H-benzotriazol-1-yl)-
(dimethylamino)methylene]N-methylmethanaminium hexafluoropho-
sphate
(dimethylamino)methylene]N-methylmethanaminium hexafluoropho-
sphate N-oxide; HDMA, 1-((dimethylimino)(morpholino)-
methyl)3-H-[1,2,3]triazolo[4,5-b]pyridine-1-3-olate hexafluorophos-
phate; HDMB, 1-((dimethylimino)(morpholino)methyl)3-H-benzo-
[1,2,3]triazolo-1-ium-3-olate hexafluorophosphate; 6-Cl-HDMB,
Synthesis and cyclization of H-Ala-Ala-NMeAla-Ala-Ala-OH.32,33
A: Solid-phase synthesis.32. The synthesis was conducted in a
plastic syringe attached to a vacuum manifold in order to effect
rapid removal of reagents and solvent. The 2-chlorotritylchloride
resin (loading = 1.55 mmol g-1; 1 g) was swelled in DCM (3 ¥
50 mL), and then acylated with Fmoc-Ala-OH (2 mmol) and
DIEA (14 mmol) in 10 mL of DCM. The reaction mixture was
stirred slowly for 1 min and allowed to couple for 15 min. Then,
an extra 6 mmol of DIEA was added and the resin was left to
stand with stirring from time to time for 45 min. 1 mL of MeOH
was added to the resin, stirred for 5 min and allowed to stand for
an additional 5 min. The resin was filtered and then washed with
DCM and DMF (2 ¥ 50 mL each). Deblocking of the N-terminus
was accomplished with 20% piperidine in DMF for 7 min. The
N-oxide;
HCTU,
N-[(6-chloro-1H-benzotriazol-1-yl)-
3672 | Org. Biomol. Chem., 2010, 8, 3665–3673
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1-((dimethylimino)(morpholino)methyl)3-H-6-chlorobenzo[1,2,3]tri-
azolo-1-ium-3-olate hexafluorophosphate; MBHA-PS, 4-methyl-
benzhydrylamine resin; Oxyma, ethyl 2-cyano-2-(hydroxyimino)
acetate; Phg, a-phenylglycine; PyAOP, azabenzotriazol-1-yl-N-
oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; PyBOP,
benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluoro-
phosphate; PyClock, 6-chloro-benzotriazol-1-yl-N-oxy-tris-pyrro-
lidino-phosphonium hexafluorophosphate; PyOxB, O-[(1-cyano-2-
12 F. Albericio, J. M. Bofill, A. El-Faham and S. A. Kates, J. Org. Chem.,
1998, 63, 9678–9683.
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ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl)
ium tetrafluoroborate; PyOxP, O-[(1-cyano-2-ethoxy-2-oxoethy-
lidene)amino]-oxytri(pyrrolidin-1-yl) phosphonium hexafluoro-
phosphon-
phosphate TBTU, N-[(1H-benzotriazol-1-yl)-(dimethylamino)methy-
lene]N-methylmethanaminium tetrafluoroborate N-oxide; TCTU, N-
[(6-chloro-1H-benzotriazol-1-yl)-(dimethylamino)methylene]N-meth-
ylmethanaminium tetrafluoroborate N-oxide; TFA, trifluoroacetic
acid; TMP, 2,4,6-trimethylpyridine (collidine); Z, benzyloxycarbonyl;
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