Ethyl 2-Cyano-2-(hydroxyimino)acetate (Oxyma): An efficient and convenient additive used with tetramethylfluoroformamidinium hexafluorophosphate (TFFH) to replace 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) during peptide synthesis

Article abstract of DOI:10.1246/bcsj.20100075

The appropriateness of ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) as a substitute for benzotriazole-based additives, for use in the TFFH approach for peptide synthesis, is discussed in terms of its capacity to control racemization, its coupling efficiency in difficult couplings either for stepwise or segment coupling in solution- and solid-phase coupling. In addition, Boc-based solution-phase peptide synthesis and its stability in the presence of growing peptide chains were studied. Oxyma displayed remarkable results in terms of racemization depression together with impressive coupling efficiency in both solution- and solid-phase synthesis. Furthermore, Oxyma suggests a lower risk of explosion than HOBt and HOAt.

Full text of DOI:10.1246/bcsj.20100075

1374 Bull. Chem. Soc. Jpn. Vol. 83, No. 11, 1374–1379 (2010)
© 2010 The Chemical Society of Japan
Ethyl 2-Cyano-2-(hydroxyimino)acetate (Oxyma): An Efficient and
Convenient Additive Used with Tetramethylfluoroformamidinium
Hexafluorophosphate (TFFH) to Replace 1-Hydroxybenzotriazole
(HOBt) and 1-Hydroxy-7-azabenzotriazole (HOAt)
during Peptide Synthesis
Sherine N. Khattab
Department of Chemistry, Faculty of Science, University of Alexandria, Alexandria 21321, Egypt
Received March 15, 2010; E-mail: ShKh2@link.net
The appropriateness of ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) as a substitute for benzotriazole-based
additives, for use in the TFFH approach for peptide synthesis, is discussed in terms of its capacity to control racemization,
its coupling efficiency in difficult couplings either for stepwise or segment coupling in solution- and solid-phase coupling.
In addition, Boc-based solution-phase peptide synthesis and its stability in the presence of growing peptide chains were
studied. Oxyma displayed remarkable results in terms of racemization depression together with impressive coupling
efficiency in both solution- and solid-phase synthesis. Furthermore, Oxyma suggests a lower risk of explosion than HOBt
and HOAt.
Me
For many years acid chlorides were used for activating the
carboxyl group of an amino acid for amide bond formation.1-6
Among peptide coupling reagents, long ago, gained the
reputation of being “over activated” and therefore prone to
numerous side reactions including loss of configuration.7 Acid
fluorides, on the other hand, are more stable to hydrolysis than
acid chlorides and are not subject to the limitation mentioned
with regard to tert-butyl-based side chain protection. Thus
Fmoc-based solid-phase peptide synthesis can be easily carried
out via Fmoc amino acid fluorides.8,9 The conversion of acids
into acid fluorides was presented by several methods often
involving toxic reagents.9,10 A remarkable progress was the
development of TFFH (tetramethylfluoroformamidinium hexa-
fluorophosphate) (1) (Figure 1). TFFH is a non-hygroscopic
salt stable to handling under ordinary conditions and acts as an
in situ reagent for the preparation of amino acid fluoride during
peptide synthesis.11 TFFH appears to be an ideal coupling
reagent for peptide synthesis in solid and solution phase, as
well as organic synthesis.11-16
For some amino acids, e.g., Fmoc-Aib-OH (Abbreviations
are in the last section), it was found that the use of TFFH alone
gave results that were less satisfactory than those obtained with
isolated amino acid fluorides. The deficiency was traced to
inefficient conversion to the acid fluoride, which under the
conditions used (2 equivalents of diisopropylethylamine
(DIEA)) was accompanied by the corresponding symmetric
anhydride and oxazolone.12,17 On the other hand, it is now
shown that if a fluoride additive such as benzyltriphenylphos-
phonium dihydrogen trifluoride (PTF, 2) or pyridine-hydrogen
fluoride 318 is present during the activation step, the latter two
products are avoided and a maximum yield of acid fluoride is
obtained. Generation of the amino acid fluoride via TFFH (1) is
Me
Me
N
N
H2F3
F
P(Ph)3
PF6
Ph
Me
TFFH, 1
PTF, 2
N
(HF)
n
3
Figure 1. Structures of fluorinating reagents.
more efficient if PTF is present as shown by model solid-phase
syntheses.18 Presumably this technique can also be used to
improve conversion to the isolable acid fluorides.19
The use of an additive to support different coupling reagents
is common in the peptide coupling reactions. A common
method for minimizing loss of configuration during stepwise
coupling in solution phase is to add in the coupling mixture an
additive such as 1-hydroxybenzotriazole (HOBt, 4).20 Recently,
1-hydroxy-7-azabenzotriazole (HOAt, 5) has been described as
a more favorable coupling additive for both solution-21 and
solid-phase synthesis.22 The presence of the latter additives in
the coupling medium induces the formation of an active ester,
which subsequently undergoes aminolysis to afford the desired
peptide bond. It was reported that generation of the amino acid
fluoride using TFFH is more efficient in the presence of HOAt
Published on the web October 21, 2010; doi:10.1246/bcsj.20100075

Sh. N. Khattab
Bull. Chem. Soc. Jpn. Vol. 83, No. 11 (2010) 1375
Z-Phg-Pro-NH2 (7)
Z-Gly-Phe-Pro-NH2 (9)
Z-Phe-Val-Pro-NH2 (8)
Z-Gly-Phe-Val-OMe (10)
N
N
NC
COOEt
OH
N
N
Figure 3. Models of peptides.
OH
Table 1. Yield and Racemization during the Formation of
Z-Phg-Pro-NH2 (7) in DMF (Solution-Phase Synthesis)a)
Oxyma (6)
HOBt (4)
Figure 2. Structure of Oxyma (6) and HOBt (4).
Entry Coupling reagentb) Base (equiv) Yield/% DL/%
1
2
3
4
5
6
7
8
9
TFFH-HOAt
TFFH-HOBt
TFFH-Oxyma
TFFH
DIC-HOAt25a
DIC-HOBt25a
DIC-Oxyma25a
HOTU25b
HATU25b
HBTU25b
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
®
78
80
85
80
81.4
81.9
89.9
78.9
78.4
80.2
1.7
8.2
0.54
7.4
3.3
9.3
1.0
0.17
3.1
8.2
as an additive. The reactivity of the active esters formed is
expected to be directly related to the stability of the leaving
group anions (OBt and OAt ), and accordingly related to their
pKa values. The pKa values of the additives HOBt 4 and HOAt
5 are 4.60 and 3.47 respectively.23
¹
¹
®
®
Recently the explosive properties of HOBt derivatives were
reported.24 This led to their reclassification under a class 1
explosive category and has accordingly increased their trans-
portation difficulties. In a recent paper of our research group,
it was reported that ethyl 2-cyano-2-(hydroxyimino)acetate
(Oxyma, 6) is an excellent replacement for HOBt and its
analogs.25 Compound 6 (Figure 2) was first reported in 1970
with an acidity similar to that of the HOBt (pKa value 4.60).26
Here the appropriateness of 6 as a substitute for the
benzotriazole-based additives is discussed in terms of its
capacity to control racemization, its coupling efficiency in
difficult couplings either for stepwise or segment coupling in
solution- and solid-phase coupling. In addition, Boc-based
solution-phase peptide synthesis and its stability in the presence
of growing peptide chains were studied.
DIEA (2)
DIEA (2)
DIEA (2)
10
a) LL- and DL-Forms of the test dipeptide have been described
elsewhere.27 The tR values of LL and DL were identified by
co-injection with authentic and pure samples of LL. HPLC
system: Linear gradient of 20 to 80% CH3CN/H2O, 0.1% TFA
over 30 min, detection at 200 nm Water Symmetry C18 5 ¯m
4.6 © 150 mm, tR LL = 12.54 min., tR DL = 13.12 min. b)
2 min preactivation time was used.
A
DIEA as base, the performance of Oxyma exceeded that of
HOBt and HOAt in terms of yield and optical purity. The yield
of the dipeptide model 7 using TFFH/Oxyma was 85% with
only 0.54% DL-racemization, while in case of TFFH alone
80% yield was obtained with 7.4% DL-racemization (Table 1,
Entries 3 and 4). On the other hand, the use of TFFH/HOAt
and TFFH/HOBt as coupling reagents gave 78% and 80%
yield with 1.7% and 8.2% DL-racemization respectively. The
TFFH/Oxyma combination and the previously reported oxime-
based HOTU25b gave better conservation of chirality than the
lately studied benzotriazole-based coupling reagents (HATU,
HBTU, HMDA, and HMDB),25b,27 DIC in presence of several
additives (Table 1, Entries 5-7),25a,27 and the fluoroamidinuim
salts (BTFFH and DFIH).12b
Results and Discussion
In our search for a class of safe and efficient additives, we
came across a family of strongly acidic oximes reported earlier
by Itoh.26 Compound 6 one of the oximes studied, has been
tested by our research group as an additive for use in the
carbodiimide approach for the formation of peptide bonds.
Oxyma displayed a remarkable capacity to inhibit racemiza-
tion, together with impressive coupling efficiency in both
automated and manual synthesis, superior to those of HOBt and
HOAt.25 Calorimetry assays (DSC and ARC) suggested a lower
risk of explosion in the case of Oxyma.25
In the case of the more racemization-prone well-studied
[2 + 1] segment coupling (Z-Phe-Val-OH onto H-Pro-NH2),
2 min preactivation was used leading to the tripeptide 8. The
segment coupling was performed in the presence of either DIEA
or TMP as a base. The yield obtained using TFFH as coupling
reagent in the presence of Oxyma in both cases (Table 2, Entries
7 and 8) was very good. Table 2 shows a comparison of the
performance of a number of coupling reagents lately used for
the segment coupling of the tripeptide 8.12b,25,27 In general, it is
observed that the degree of racemization in the presence of
TMP as base is lower than in case of using DIEA. In addition,
the presence of an additive improved the performance of most
of the coupling reagents. The use of HOAt as an additive, when
using TFFH, BTFFH, DFIH, DIC, HATU, and HAPyU as
coupling reagents in the presence of TMP, gave the least degree
of racemization, 2.7%, 2.8%, 3.2%, 2.1%, 2.4%, and 1.6%
respectively. In the TFFH coupling approach the use of Oxyma
as an additive gave comparable optical purity (2.8%) (Table 2,
Our goal is to test Oxyma as an additive to support the TFFH
coupling methodology. Investigations of Oxyma as an additive
in the coupling medium, during the generation of the amino
acid fluoride using TFFH was carried first by infrared
examination. Activation of the protected amino acid Z-Val-
OH by means of TFFH in absence of an additive gives the acid
fluoride (IR: 1842 cm¹1), whereas in case of using Oxyma as an
additive, after 5 min activation, only the Oxyma esters (1720
¹1
(active ester) and 1710 cm (Oxyma ethyl ester)), and a peak
¹1
at 2251 cm corresponding to the CN group were observed.
Further investigation of racemization and the compound’s
effectiveness of coupling yields were carried out. For the study
of racemization, peptide models27,28 were used for comparison
of HOAt, HOBt, and Oxyma. These coupling models are more
reliable than those chosen in the previous papers (Figure 3).
With regard to sensitive stepwise coupling (Z-Phg-OH onto
H-Pro-NH2), using 2 min preactivation in the presence of

1376 Bull. Chem. Soc. Jpn. Vol. 83, No. 11 (2010)
Oxyma: An Efficient Additive Used with TFFH
Table 2. Yield and Racemization during the Formation of
Z-Phe-Val-Pro-NH2 (8) (2 + 1) in DMF (Solution-Phase
Synthesis)a)
Table 4. Yield and Racemization during the Formation of
Z-Gly-Phe-Val-OMe (10) (2 + 1) in DMF (Solution-
Phase Synthesis)a)
Entry Coupling reagent Base (equiv) Yield/% LDL/%
Entry Coupling reagent Base (equiv) Yield/% LDL/%
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
TFFH-HOAt
TFFH-HOAt
TFFH-HOBt
TFFH-HOBt
TFFH
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
®
86
78
90
81
77
75
90
86
86.1
78.8
89.9
85.8
83.2
75.6
72.1
89.7
81.2
91.2
88.7
15.4
2.7
1
2
3
4
5
6
7
8
TFFH-HOAt
TFFH-HOAt
TFFH-HOBt
TFFH-HOBt
TFFH
TFFH
TFFH-Oxyma
TFFH-Oxyma
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
90
87
89
85
88
86
90
89
1.7
0.8
5.7
3.9
6.2
5.6
1.8
1.1
28.4
15.3
30.9
23.6
22.1
2.8
2.1
8.9
3.8
13.9
5.3
10.9
2.4
27.4
14.2
23.6
7.4
TFFH
TFFH-Oxyma
TFFH-Oxyma
DIC-HOAt25a
DIC-HOBt25a
DIC-Oxyma25a
HATU25b
a) LLL- and LDL-Forms of the test tripeptide have been
described elsewhere28 and were co-injected with authentic and
pure samples. HPLC system: Linear gradient 20 to 80%
CH3CN/H2O, 0.1% TFA over 30 min, detection at 200 nm
Water Symmetry C18 5 ¯m 4.6 © 150 mm, tR LLL = 18.06 min,
tR LDL = 18.62 min).
®
®
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
HATU25b
HATU-HOAt25b
HATU-HOAt25b
HBTU25b
HBTU25b
were confirmed. The coupling reaction was performed in
the presence of DIEA or TMP as base showing comparable
results of Oxyma and HOAt clearly better than that seen with
HOBt (Table 4). In addition, an improvement in the coupling
efficiency of TFFH in the presence of an additive was
observed.
Further racemization experiments were carried out for
the stepwise coupling of the four previously studied model
systems, Z-Val-Val-OCH3 (11), Z-Val-Ala-OCH3 (12), Z-
Phe-Val-OCH3 (13), and Z-Phe-Ala-OCH3 (14),28 using
TFFH as coupling reagent in the presence and in absence of
Oxyma. In the presence of Oxyma as an additive an improve-
ment in yield was observed but the optical purity of the studied
models was almost the same (less than 1%) as observed from
NMR analysis. The OCH3 units of the esters 11, 12, 13, and 14
were monitored at 3.72, 3.74, 3.68, and 3.70 ppm respectively.
These peaks correspond to the LL-enantiomers. The slightly
down shielded peaks at 3.87, 3.88, 3.83, and 3.85 ppm
correspond to the DL-enantiomers respectively.
The performance of Oxyma was further tested in the manual
synthesis of Leu-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-NH2,
15) in solution phase using TFFH-Oxyma. Boc chemistry was
used in the solution phase except for the last amino acid
coupling. Fmoc-Tyr(Ot-Bu)OH was used and the Fmoc group
was deblocked by using 30% diethylamine in acetonitrile for
1 h and then the crude peptide was treated with TFA-DCM
(1:1) for two hours at room temperature.
HOTU25b
HOTU25b
a) LLL- and LDL-Forms of the test tripeptide have been
described elsewhere27 and were co-injected with authentic and
pure samples. HPLC system: Linear gradient 20 to 80%
CH3CN/H2O, 0.1% TFA over 30 min, detection at 200 nm
Water Symmetry C18 5 ¯m 4.6 © 150 mm, tR LLL = 6.96 min,
tR LDL = 7.44 min.
Table 3. Yield and Racemization during the Formation of
Z-Gly-Phe-Pro-NH2 (9) (2 + 1) in DMF (Solution-Phase
Synthesis)a)
Entry Coupling reagent Base (equiv) Yield/% LDL/%
1
2
3
4
5
6
7
TFFH-HOAt
TFFH-HOAt
TFFH
TFFH-HOBt
TFFH-HOBt
TFFH-Oxyma
TFFH-Oxyma
DIEA (2)
TMP (2)
TMP (2)
DIEA (2)
TMP (2)
DIEA (2)
TMP (2)
90.3
85.9
80.1
89.2
83.9
89.9
86.9
2.1
1.1
10.6
6.4
4.2
2.9
1.4
a) LLL- and LDL-Forms of the test tripeptide have been
described elsewhere28 and were co-injected with authentic and
pure samples. HPLC system: Linear gradient 20 to 80%
CH3CN/H2O, 0.1% TFA over 30 min, detection at 200 nm
Water Symmetry C18 5 ¯m 4.6 © 150 mm, tR LLL = 11.81 min,
tR LDL = 12.83 min.
For testing the efficiency of TFFH/Oxyma in Boc chemistry,
the pentapeptide H-Tyr-Gly-Gly-Phe-Leu-NH2 was stepwise
elongated using Boc-amino acids. Boc-Phe-OH was preacti-
vated using TFFH/Oxyma/DIEA (1:1:2) for 2 min in DMF and
then coupled to H-Leu-NH2 for 30 min to afford the dipeptide
Boc-Phe-Leu-NH2. After deblocking of the Boc protecting
group, using TFA-DCM (1:1) for 2 h at room temperature, the
dipeptide TFA salt is further coupled to Boc-Gly-OH in the
presence of TFFH/Oxyma using the same strategy to give the
tripeptide. The crude tripeptide was treated directly with TFA-
DCM followed by subsequent coupling with Boc-Gly-OH.
The crude tetrapeptide was treated directly with TFA-DCM.
Entry 8) to that of the HOAt additive (2.7%) (Table 2,
Entry 2). In addition, TFFH/Oxyma gave higher optical purity
than the oxime-based HOTU25b (Table 2, Entry 19) and the
DIC/Oxyma25a coupling approach (Table 2, Entry 11).
For the rather non-sensitive case of segment coupling of
Z-Gly-Phe-OH to H-Pro-NH2, which leads to tripeptide 9,
Oxyma and HOAt additives again perform better than HOBt in
term of optical purity and yield (Table 3).
In the case of formation of the tripeptide Z-Gly-Phe-Val-
OMe (10) which is a less sensitive case the previous results

Sh. N. Khattab
Bull. Chem. Soc. Jpn. Vol. 83, No. 11 (2010) 1377
when compared to HOAt/TFFH or TFFH alone. The percent-
age of the desired pentapeptide was being 98%, where Oxyma
gave a better result than HOBt.
Conclusion
Oxyma was used as an additive in the TFFH approach for
peptide synthesis. It displayed remarkable results in terms
of racemization depression during stepwise and segment
coupling. The TFFH/Oxyma combination showed superiority
to a number of coupling reagents previously reported. As
would be expected, the use of HOAt as an additive with all
coupling reagents confirmed its superiority to HOBt in terms of
both coupling yields and retention of configuration. Remark-
ably, Oxyma often gave results comparable or even better than
those obtained with HOAt. In addition, Oxyma can be handled
with a considerably lower risk than the explosive benzotriazole
and its derivatives, which is an extra advantage.
tR/min
Figure 4. HPLC analysis of the crude sample of TFA¢
Tyr-Gly-Gly-Phe-Leu-NH2 (15). TFFH-Oxyma is used
as coupling reagent. Conditions: HPLC system: Linear
gradient 20 to 80% CH3CN/H2O, 0.1% TFA over 30 min,
detection at 200 nm Water Symmetry C18 5 ¯m 4.6 ©
150 mm. tR = 9.619 min (98.3%).
Experimental
General. All peptides were identified by HPLC analysis of
the crude sample. Conditions: a Waters Symmetry C18 column
(4.6 © 150 mm, 5 ¯m), linear gradient over 30 min of 20 to
Table 5. The Percentages of Des-Aib (H-Tyr-Aib-Phe-
Leu-NH2) Obtained during Solid-Phase Assembly of the
Pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH2, 16)a),b)
¹1
80% CH3CN in H2O/0.1% TFA, flow rate 1.0 mL min
.
Dipeptides were identified by 1H NMR. NMR spectra were
recorded on a JEOL 500 MHz spectrometer at room temper-
ature. Tetramethylsilane (TMS) was used as reference for all
NMR spectra with chemical shifts reported as ¤ units (part per
million, ppm) relative to TMS. HPLC-MS electrospray mass
spectroscopy was used for identification of peptides.
Coupling reagent Base (equiv) Pentapeptide/% Des-Aib/%
TFFH-HOAt
TFFH-HOBt
TFFH
DIEA (2)
DIEA (2)
DIEA (2)
DIEA (2)
95
64
95
98
5
36
5
TFFH-Oxyma
2
Racemization Tests with Model Peptides 7-10 in Solution
a) Tetrapeptide (des-Aib) was confirmed by peak overlap in the
presence of an authentic sample. The crude H-Tyr-Aib-Aib-
Phe-Leu-NH2 was analyzed by HPLC [Symmetry Waters
Phase.
Test couplings were carried out as previously
described for Z-Phg-Pro-NH2,27 Z-Phe-Val-Pro-NH2,27 Z-
Gly-Phe-Pro-NH2,28 and Z-Gly-Phe-Val-OMe.28
C18 (4.6 © 150 mm, 4 ¯m), linear gradient over 30 min of 20
Formation of Z-Phg-Pro-NH2 (7): 0.125 mmol of Z-Phg-
OH, 0.125 mmol coupling reagent, and 0.25 mmol of DIEA as
base were dissolved in 1 mL DMF at 0 °C. The reaction mixture
was preactivated for 2 min. 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 M HCl
(2 © 5 mL), 1 M NaHCO3 (2 © 5 mL), and saturated NaCl (2 ©
5 mL), dried over anhydrous MgSO4, the solvent was removed,
and the crude peptide was directly analyzed by HPLC.
Formation of Z-Phe-Val-Pro-NH2 (8): 0.125 mmol of Z-
Phe-Val-OH, 0.125 mmol coupling reagent, and 0.25 mmol of
base (DIEA or TMP) were dissolved in 1 mL DMF at 0 °C. The
reaction mixture was preactivated for 2 min. 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, followed by the
workup described above.
Formation of Z-Gly-Phe-Pro-NH2 (9) (2 + 1) and Z-Gly-
Phe-Val-OMe (10) (2 + 1) were performed by the same
procedure mentioned previously for the segment coupling.
Crude products were analyzed by reverse-phase HPLC (with
a waters Symmetry C18, 5 ¯m, 4.6 © 150 mm column), linear
gradient over 30 min of 20 to 80% CH3CN in H2O/0.1% TFA,
flow rate 1.0 mL min¹1, detection at 220 nm. In the Z-Phg-Pro-
NH2 model, the tR values of the LL- and DL-epimers were 12.54
and 13.12 min, respectively, whereas in the Z-Phe-Val-Pro-
¹1
to 80% CH3CN in H2O/0.1% TFA, flow rate 1.0 mL min
,
tR penta = 11.3 min, tR des-Aib = 11.56 min]. b) HPLC-MS
showed the right mass for the pentapeptide at 612.0.
The TFA salt of the tetrapeptide was used directly without
purification to couple with Fmoc-Tyr(OBu)-OH under the
same condition used before. Then the Fmoc group was
removed by treatment with 30% diethylamine in acetonitrile
at room temperature for 1 h. The crude product was then treated
with a mixture of TFA-DCM (1:1) at room temperature for
2 h to remove the butyl group. The crude pentapeptide was
obtained in yield (68.9%). The purity by HPLC was 98.3 (at
tR = 9.62 min) (Figure 4). The HPLC-MS showed the right
mass for the pentapeptide at 555.3.
Hindered Amino Acids. In a more demanding example,
the Leu-enkaphlin derivative H-Tyr-Aib-Aib-Phe-Leu-
29
NH2 (16) was manually assembled on Fmoc-Rink Amide
AM-resin using amino acid/activator (3 equiv), DIEA (6
equiv), using 30 min coupling time, except for the case of Aib-
Aib, which required 1 h. Percentages of incorporation for the
coupling of Fmoc-Aib-OH onto the Aib-containing resin were
determined by reverse-phase HPLC analysis, after cleavage of
the peptide from the resin by treatment with TFA/H2O (9:1) for
2 h at room temperature (Table 5). Little improvement was
observed with TFFH in the presence of Oxyma as an additive

1378 Bull. Chem. Soc. Jpn. Vol. 83, No. 11 (2010)
Oxyma: An Efficient Additive Used with TFFH
NH2 case, the tR values of the LLL- and LDL-epimers were 6.96
and 7.44 min, respectively. In the Z-Gly-Phe-Pro-NH2 model,
the tR values of the LLL- and LDL-epimers were 11.81 and
12.83 min, respectively, whereas in the Z-Gly-Phe-Val-OMe
case, the tR values of the LLL- and LDL-epimers were 18.06 and
18.62 min, respectively.
9.62 min). The HPLC-MS showed the right mass for the
pentapeptide at 555.3.
Synthesis of H-Tyr-Aib-Aib-Phe-Leu-NH2 (16) in Solid
Phase.
100 mg of Fmoc-RA-PS (0.7 mmol g¹1) were
deblocked by 10 mL of 20% piperidine in DMF for 10 min,
washed with DMF (2 © 10 mL), DCM (2 © 10 mL), and then
DMF (2 © 10 mL). Fmoc-Leu-OH (3 equiv), TFFH/additive
(3 equiv), and DIEA (6 equiv) were mixed in 0.5 mL DMF and
activated for 1-2 min and then added to the resin and stirred
slowly for 1 min and let to couple for 30 min (1 h double
coupling only for the Aib-Aib), (ninhydrin test was negative).
The resin was washed with DMF, and then deblocked by 20%
piperidine in DMF for 7 min. The resin was washed with DMF,
DCM, and DMF, then coupled with the next amino acid.
Coupling and deblocking steps were repeated to provide the
penta peptide. The percentage of des-Aib (4-mer) (Tyr-Aib-
Phe-Leu-NH2) during solid-phase assembly of the pentapep-
tide (Tyr-Aib-Aib-Phe-Leu-NH2) was confirmed by peak
overlap in the presence of an authentic sample. The crude
H-Tyr-Aib-Aib-Phe-Leu-NH2 was analyzed by HPLC
[Symmetry Waters C18 (4.6 © 150 mm, 4 ¯m), linear gradient
over 30 min of 20 to 80% CH3CN in H2O/0.1% TFA, flow rate
1.0 mL min¹1, tR penta = 11.3 min, tR des-Aib = 11.56 min].
The HPLC-MS analysis showed the right mass for the
pentapeptide at 612.0.
Racemization Tests with Model Dipeptides 11-14 in
Solution Phase. Test couplings were carried out as previously
described for Z-Val-Val-OCH3 (11), Z-Val-Ala-OCH3 (12),
Z-Phe-Val-OCH3 (13), and Z-Phe-Ala-OCH3 (14).28
To a solution of 0.1 mmol of Z-Val-OH or Z-Phe-OH and
0.2 mmol of DIEA in 2 mL of DMF was added 0.1 mmol of the
appropriate coupling reagent at 0 °C. The reaction mixture was
preactivated for 2 min, followed by the addition of a solution
of 0.1 mmol of Val-OMe¢HCl or Ala-OMe¢HCl and 0.1 mmol
of DIEA in 1 mL of DMF. The reaction mixture was stirred at
0 °C for 1 h and at room temperature overnight. After dilution
with 25 mL of ethyl acetate, the organic phase was washed with
1 M HCl (3 © 10 mL), saturated NaHCO3 (3 © 10 mL) and
saturated NaCl (3 © 10 mL) and dried over MgSO4 anhydrous.
After removal of the solvent with a rotary evaporator, the
residue was recrystallized from CH2Cl2/hexane to give the
dipeptide.
The extent of racemization during the preparation of the
model systems was monitored by proton NMR analysis. The
OCH3 units of esters 11, 12, 13, and 14 were monitored at 3.73,
3.74, 3.68, and 3.70 ppm respectively. These correspond to the
LL-enantiomers. The slightly downshielded peaks at 3.87, 3.88,
3.83, and 3.85 ppm correspond to the DL-enantiomers respec-
tively.
Abbreviations
Aib: ¡-aminoisobutyric acid, Boc: t-butyloxycarbonyl,
BTFFH: bis(tetramethylene)fluoroformamidinium hexafluoro-
phosphate, DFIH: 1,3-dimethyl-2-fluoro-4,5-dihydro-1H-
imidazolium hexafluorophosphate, DCM: dichloromethane,
DIC: diisopropylcarbodiimide, DIEA: diisopropylethylamine,
DMF: dimethylformamide, HOBt: 1-hydroxybenzotriazole,
HOAt: 7-aza-1-hydroxybenzotriazole, Oxyma: ethyl 2-cyano-
2-(hydroxyimino)acetate, HAPyU: 1-(1-pyrrolidinyl-1H-1,2,3-
triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmethanaminium
hexafluorophosphate N-oxide, HATU: N-[(dimethylamino)-1H-
1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmethan-
aminium hexafluorophosphate N-oxide, HBTU: N-[(1H-benzo-
triazol-1-yl)(dimethylamino)methylene]-N-methylmethanami-
nium hexafluorophosphate N-oxide, HDTU: O-(3,4-dihydro-4-
oxo-1,2,3-benzotriazin-3-yl)-1,2,3,3-tetramethyluronium hexa-
fluorophosphate, HMDA: 1-[(dimethylimino)(morpholino)-
methyl]-3H-[1,2,3]triazolo[4,5-b]pyridine-1-ium-3-olate hexa-
fluorophosphate, HMDB: 1-[(dimethylimino)(morpholino)-
Solution-Phase Synthesis of Leu-Enkephalin (H-Tyr-
Gly-Gly-Phe-Leu-NH2, 15) Using TFFH-Oxyma.
The
pentapeptide was manually elongated. TFFH/Oxyma (0.05
mmol each) was added to a solution of H-Leu-NH2
(0.05 mmol), DIEA (0.1 mmol), Boc-Phe-OH (0.05 mmol) in
DMF (2 mL) at 0 °C. The reaction mixture was stirred at 0 °C
for 1 h and at rt for another 1 h, then ethyl acetate was added
(40 mL). The organic layer was washed with saturated Na2CO3
(2 © 10 mL), 10% citric acid (2 © 10 mL), saturated NaCl
(2 © 10), dried (MgSO4), filtered and the solvent was removed
under vacuum. The crude dipeptide was treated with TFA-
DCM (1:1, 10 mL) at room temperature for 2 h, then the solvent
and TFA were removed under vacuum and then washed with
ether. The crude TFA salt was used for the next step for the
coupling with Boc-Gly-OH using the same previous coupling
techniques and then workup as usual. The crude tripeptide was
treated directly with TFA-DCM followed by subsequent
coupling with Boc-Gly-OH as mentioned before. The TFA
salt of the tetrapeptide was used directly without purification to
couple with Fmoc-Tyr(OBu)-OH under the same condition
used before. Then the Fmoc group was removed by treatment
with 30% diethylamine in acetonitrile (10 mL) at room temper-
ature for 1 h. The solvent was removed under vacuum and then
the crude product was treated with 10 mL of a mixture of TFA-
DCM (1:1) at room temperature for 2 h. The solvent and TFA
were removed under vacuum and then the crude pentapeptide
was washed with ether to give slight brown solid in yield
(68.9%). The purity by HPLC using linear gradient over 30 min
of 20 to 80% CH3CN in H2O/0.1% TFA, was 98.3% (at tR
methyl]-3H-benzo[1,2,3]triazolo-1-ium-3-olate
phosphate, HOTU: O-[cyano(ethoxycarbonyl)methylidene)-
amino]-1,1,3,3-tetramethyluronium hexafluorophosphate,
hexafluoro-
TFFH: tetramethylfluoroformamidinium hexafluorophosphate,
TFA: trifluoroacetic acid, TMP: 2,4,6-trimethylpyridine (colli-
dine), PTF: benzyltriphenylphosphonium dihydrogen trifluo-
ride, Z: benzyloxycarbonyl. Amino acids and peptides are
abbreviated and designated following the rules of the IUPAC-
IUB Commission of Biochemical Nomenclature (J. Biol.
Chem. 1972, 247, 977).
Prof. Ayman El-Faham, University of Alexandria, College
of Science, Chemistry Department, Alexandria, Egypt and
Prof. Fernando Albericio, University of Barcelona, Spain are

Sh. N. Khattab
Bull. Chem. Soc. Jpn. Vol. 83, No. 11 (2010) 1379
Carpino, A. El-Faham, J. Am. Chem. Soc. 1995, 117, 5401.
12 a) A. El-Faham, Chem. Lett. 1998, 671. b) A. El-Faham,
Org. Prep. Proced. Int. 1998, 30, 477. c) K. Akaji, N. Kuriyama, Y.
Kiso, Tetrahedron Lett. 1994, 35, 3315.
thanked for their advice and support. The Egyptian Academy of
Science is thanked for its partial support through the Joint
Research Project in collaboration with the Spanish University
of Barcelona, Park Scientific (A/9846/07).
13 D. Hudson, J. Org. Chem. 1988, 53, 617.
14 J. Izdebski, J. Bondaruk, S. W. Gumułka, P. Krzaścik, Int. J.
Pept. Protein Res. 1989, 33, 77.
15 R. I. Carey, L. W. Bordas, R. A. Slaughter, B. C. Meadows,
J. L. Wadsworth, H. Huang, J. J. Smith, E. Furusjö, J. Pept. Res.
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Supporting Information
HPLC analysis of 7 (Table 1, Entry 3), HPLC analysis of 8
(Table 2, Entry 8). This material is available free of charge on
the web at http://www.csj.jp/journals/bcsj/.
16 H. Wenschuh, M. Beyermann, E. Krause, M. Brudel, R.
Winter, M. Schuemann, L. A. Carpino, M. Bienert, J. Org. Chem.
1994, 59, 3275.
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