Oxyma-B, an excellent racemization suppressor for peptide synthesis

Article abstract of DOI:10.1039/c4ob01612b

Peptide-bond formation is a key process in the synthesis of peptide oligomers. Among the many coupling techniques reported, carbodiimides combine strong acylation potency and smooth reaction conditions and are commonly used in the presence of additives. Recently, ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure) has emerged as a highly reactive alternative to the classic and explosion-prone benzotriazolic additives, namely 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Here we report on a new oxime additive 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (Oxyma-B). This new additive showed satisfactory solubility in various solvents (DMF, ACN, and THF). It was also more effective in the control of optical purity during the synthesis of Z-Phg-Pro-NH₂, Z-Phe-Val-Pro-NH₂, H-Gly-Ser-Phe-NH₂, H-Gly-Cys-Phe-NH₂, H-Gly-Cys(Acm)-Phe-NH₂ and H-Gly-His-Phe-NH₂ than related Oxyma- and benzotriazole-based reagents. Oxyma-B also proved to be advantageous compared to the related HONM, because the latter cannot be used with the carbodiimide. Furthermore, Oxyma-B showed satisfactory performance in assembling demanding sequences such as the Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH₂). This journal is

Full text of DOI:10.1039/c4ob01612b

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Oxyma-B, an excellent racemization suppressor
for peptide synthesis†
Cite this: DOI: 10.1039/c4ob01612b
Received 29th July 2014,
Accepted 27th August 2014
a
b
a,c
a
Yahya E. Jad, Sherine N. Khattab, Beatriz G. de la Torre, Thavendran Govender,
a
b,d
a,c,e,f,g
Hendrik G. Kruger, Ayman El-Faham* and Fernando Albericio*
DOI: 10.1039/c4ob01612b
www.rsc.org/obc
Peptide-bond formation is a key process in the synthesis of peptide This step is commonly carried out through an active ester,
oligomers. Among the many coupling techniques reported, carbo- which can be previously prepared, isolated, purified, and
diimides combine strong acylation potency and smooth reaction characterized, or alternatively prepared in situ by the use of
conditions and are commonly used in the presence of additives. carbodiimides or onium salts. In all cases, the cornerstone of
Recently, ethyl 2-cyano-2-(hydroxyimino)acetate (OxymaPure) has the process is the leaving group. The most used groups are
1
–7
emerged as
a highly reactive alternative to the classic and those derived from phenols and N-hydroxy derivatives.
8
explosion-prone benzotriazolic additives, namely 1-hydroxybenzo- These include 1-hydroxybenzotriazole (HOBt, 1), 7-aza-1-
triazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Here we hydroxybenzotriazole (HOAt, 2) (which are reported as the
report on a new oxime additive 5-(hydroxyimino)-1,3-dimethyl- most potent and expensive analogs), and 6-chloro-1-hydroxy-
9
pyrimidine-2,4,6(1H,3H,5H)-trione (Oxyma-B). This new additive benzotriazole (6-Cl-HOBt, 3) (an analog of medium reactivity
1
0
showed satisfactory solubility in various solvents (DMF, ACN, and with a more accessible potency-to-cost ratio). HOBt deriva-
THF). It was also more effective in the control of optical purity tives are therefore either used in combination with a carbodii-
during the synthesis of Z-Phg-Pro-NH
Ser-Phe-NH , H-Gly-Cys-Phe-NH , H-Gly-Cys(Acm)-Phe-NH
H-Gly-His-Phe-NH than related Oxyma- and benzotriazole-based HCTU (6) ]. However, the explosive nature of HOBt and its
2 2
, Z-Phe-Val-Pro-NH , H-Gly- mide or another coupling agent or are built into a stand-alone
1
1,12
13
14
2
2
2
and reagent such as an iminium salt
[HBTU (4), HATU (5),
1
5
2
1
6
reagents. Oxyma-B also proved to be advantageous compared to related additives has recently been reported. This obser-
the related HONM, because the latter cannot be used with the vation has led to the reclassification of these compounds
carbodiimide. Furthermore, Oxyma-B showed satisfactory perform- under the Class 1 explosive category and has consequently
ance in assembling demanding sequences such as the Aib-enkeph- increased transportation difficulties.
alin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH
2
).
Recently, our group reevaluated OxymaPure (ethyl 2-cyano-
2
-(hydroxyimino)acetate) 7, which was first reported a few
1
7
decades ago. OxymaPure 7 displayed remarkable efficiency in
inhibiting racemization and showed an impressive coupling
efficiency in both automated and manual synthesis. These
effects are superior to those shown by HOBt 1 and comparable
Introduction
The reaction of a carboxylic acid with an amine to render
amide derivatives requires activation of the carboxylic acid.
1
8–20
to those of HOAt 2 (Fig. 1).
Later, we devised a third generation of uronium salt, COMU
8, which carries a morpholonium-based uronium moiety as
the proton acceptor and OxymaPure 7 as the leaving group.
COMU 8 shows coupling efficiency superior to that of HBTU 4
a
Catalysis and Peptide Research Unit, School of Health Sciences, University of
Kwazulu-Natal, Durban 4001, South Africa. E-mail: albericio@irbbarcelona.org
b
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426,
Ibrahimia, Alexandria 21321, Egypt. E-mail: aymanel_faham@hotmail.com
20–23
and comparable to that of HATU 5.
c
School of Chemistry, Yachay Tech, Yachay City of Knowledge, 100119-Urcuqui,
More recently, a new family of uronium salts (HTMU, 10;
HMMU, 11; and HDmPyMU, 12) based on isonitroso
Meldrum’s acid (HONM, 9) was reported as stand-alone coup-
Ecuador
d
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh11451, Saudi Arabia
e
24
School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001,
ling reagents. HONM 9 shows structural similarities to 7,
South Africa
except for the presence of two carbonyl groups as electron-
withdrawing substituents located in the six-membered cyclic
structure. This modification should enhance the reactivity of
the oxime-based additive as a result of its more powerful
f
Institute for Research in Biomedicine and CIBER-BBN, Barcelona 08028, Spain
g
Department of Organic Chemistry, University of Barcelona, Barcelona 08028, Spain
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob01612b
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synthesis. Oxyma-B 14, which has been developed from 1,3-
dimethylbarbituric acid 13, shows a special orientation of the
carbonyl moiety, similar to HONM 9. This orientation can play
an assisted basic catalytic role by enhancing the nucleo-
philicity of the amino function during coupling. In addition,
14 does not hold an ester moiety in its structure and, there-
fore, there is no risk of a side-reaction.
Results and discussion
Synthesis
Fig. 1 Structure of benzotriazole additives 1–3 and their coupling
reagents 4–5, OxymaPure 7 and COMU 8.
Oxyma-B 14 was readily prepared by reaction of 1,3-dimethyl-
2
barbituric acid 13 with NaOH, NaNO , and AcOH in a mixture
of water and methanol for 1 h at 0 °C, followed by acidification
by 35% HCl to afford the desired compound as a white solid
in 84% yield (Scheme 1).
electron-withdrawing effect compared with 7. The cyclic struc-
ture of 9 may also be beneficial because the hydroxy function
becomes more accessible (Fig. 2).
HONM 9 is highly reactive, even reacting with carbodi-
imides. Such reactivity translates into a consumption of coup-
Solubility
ling reagents and a decrease in the yield. However, uronium Solubility is a key feature in determining the suitability of
salts (10, 11, and 12) show greater reactivity when compared coupling reagents, especially in the automatic mode. Oxyma-B
2
4
with classical coupling reagents. In parallel, some of our col- 14 showed acceptable solubility in DMF (the most widely used
leagues have reported the inconvenience of a side reaction solvent in peptide bond formation), ACN (which was reported
2
6
when removing the ethyl ester moiety from the structure under to be a suitable alternative to DMF in SPPS) and THF.
1
8,25
very specific conditions.
Here we describe a new additive Furthermore, 14 showed partial solubility in DCM.
that has been designed, prepared, and screened for peptide
Racemization
α
The undesired loss of configuration at C is dramatic in more
sensitive building blocks, such as phenylglycine or Cys. In
order to check the coupling efficiency and impact of epimeri-
2
7,28
12,18,21,24,29–33
zation,
stepwise models
(1 + 1) (Z-Phg-Pro-
16) Oxyma-
NH 15) and (2 + 1) segment (Z-Phe-Val-Pro-NH
2
2
B-based couplings in solution-phase synthesis were carried
out. In the first model of 15, the α-phenyl moiety in Phg
1
8
ensured high sensitivity towards epimerization (Table 1).
In this simple model of stepwise coupling of 15, both
OxymaPure 7 and Oxyma-B 14 showed excellent results com-
pared to HOBt 1 and even to HOAt 2 in terms of reducing race-
mization (entries 3, 4 vs. 1, 2).
Fig. 2 Structure of HONM 9 and its uronium salts.
Scheme 1 Synthesis of Oxyma-B from 1,3-dimethylbarbituric acid.
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Table 1 Yield and epimerization during the formation of Z-Phg-Pro-
Oxyma-B 14 showed the best performance in reducing epi-
a
NH
2
15 using additives (solution-phase synthesis)
merization, as demonstrated by further epimerization exper-
3
5
iments. We selected the tripeptides H-Gly-Ser-Phe-NH
2
17,
b
c
Entry
Coupling reagent
Yield (%)
DL/LL (%)
1
8,36
H-Gly-Cys-Phe-NH
2
18,
H-Gly-Cys(Acm)-Phe-NH 19 and
H-Gly-His-Phe-NH 20 to study the loss of configuration of
2
3
7
1
2
3
4
DIC/HOBt (1)
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B (14)
94.3
91.5
94.4
90.0
11
2
3.9
0.9
1.0
the most tricky residues, namely Ser, Cys and His. These
amino acids (Ser, Cys, His) gave unusual epimerization during
3
7–41
SPPS.
The tripeptides 17–20 were assembled by stepwise
a
Couplings were performed without preactivation in DMF at room
b
solid-phase synthesis with 5 min preactivation and Ser(tBu),
Cys(Trt), Cys(Acm) and His(Trt) as protecting groups,
temperature. Conversion yield was calculated by HPLC. Retention
2
times of Z-Phg-OH and Z-Phg-Pro-NH were identified by injection of a
c
pure sample. Retention times for each epimer were identified after respectively.
co-injection with pure LL and DL samples onto reverse-phase HPLC
On the basis of the results shown in Table 3, we conclude
that Oxyma-B 14 stands out as one of the most promising addi-
tives in peptide synthesis. It showed better results than Oxyma-
Pure 7 when used as a racemization suppressor and exceeded
those achieved with HOAt 2 in both stepwise and segment
coupling in solid- and solution-phase peptide synthesis.
Furthermore, an additional feature of Oxyma-B 14 is that
the reaction can be monitored through the change in the color
of the solution. The solution is blue at the start of the reaction
and after completion becomes a yellowish green. This
phenomenon also occurs when OxymaPure 7 is used.
using a linear gradient of 25 to 50% 0.1% TFA in CH
O over 15 min, detection at 220 nm and a Phenomex C18 (3 μm,
.6 × 50 mm) column, t (LL) = 6.4 min, t (DL) = 6.8 min.
3
CN/0.1% TFA in
H
4
2
R
R
Having evaluated the stepwise system, we tackled the most
demanding [2 + 1] coupling for the assembly of dipeptide
Z-Phe-Val-OH onto H-Pro-NH affording tripeptide 16
Table 2). The activation of a dipeptide acid is a very interest-
2
,
3
4
(
ing example to test the performance of an additive, since
oxazolone formation is promoted as a result of the electron-
2
1,31
donating effect of the N-aminoacyl substitution.
The per-
centages of LDL epimers were considerably higher than in the
previous stepwise system (Table 2 and Fig. 3).
Peptide synthesis
In this tripeptide 16 model, Oxyma-B 14 showed better
results than OxymaPure 7 and HOAt 2 (entry 4 vs. entries 2
and 3), while the worst result was obtained with HOBt 1
The acylation potency of 14 in hindered couplings was evalu-
ated during SPPS. For this purpose, solid-phase assembly of
the Aib-enkephalin pentapeptide 21 was used as a model for
SPPS (Table 4).
(entry 1). These results are important because in our previous
work involving OxymaPure 7, we also detected that it caused
less racemization than HOBt 1, but sometimes slightly more
than that produced when using HOAt 2.
The design of 21 consisted of introducing the hindered
α,α-disubstituted amino acid (Aib) to replace Gly residues (Leu-
enkephaline), thereby achieving differences in the coupling
performance of each of the additives tested. 21 was stepwise
assembled manually on a Fmoc-RinkAmide-AM-PS-resin by
means of 1 h coupling times (except Aib–Aib where 1 hour
double coupling was applied) with the use of an excess of
Table 2 Yield and epimerization during the formation of Z-Phe-Val-
a
Pro-NH
2
16 using additives (solution-phase synthesis)
b
c
Entry
Coupling reagent
Yield (%)
LDL/LLL (%)
3
equiv. of Fmoc-amino acid/additive/carbodiimides.
1
2
3
4
DIC/HOBt (1)
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B (14)
96.3
97.6
91.9
90.7
14.8
5.9
7.7
5.1
Table 3 Racemization studies on the solid-phase assembling of H-Gly-
AA-Phe-NH (where AA = Ser, Cys, Cys(Acm) or His)
2
a
Couplings were performed without preactivation in DMF at room
b
DL/LL
(%)
temperature. Conversion yield was calculated by HPLC. Retention
Entry
Coupling model
Coupling reagent
times of Z-Phe-Val-OH and Z-Phe-Val-Pro-NH
2
were identified by
c
injection of a pure sample. Retention times for each epimer were
identified after co-injection with pure LLL and LDL samples onto
reverse-phase HPLC using a linear gradient of 30 to 60% 0.1% TFA in
1
2
3
4
5
6
7
8
9
H-Gly-Ser-Phe-NH
2
(17)
(18)
DIC/HOBt (1)
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B (14)
DIC/HOBt (1)
3.3
0.4
0.4
0.3
0.5
0.4
0.3
0.3
0.4
0.3
0.3
0.3
1.1
1.9
3.0
1.0
CH
Phenomex C18 (3 μm, 4.6 × 50 mm) column, t
6.9 min.
3
CN–0.1% TFA in H
2
O over 15 min, detection at 220 nm and a
R
(LLL) = 5.8 min, t (LDL)
R
=
H-Gly-Cys-Phe-NH
2
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B (14)
DIC/HOBt (1)
2
H-Gly-Cys(Acm)-Phe-NH (19)
10
11
12
13
14
15
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B (14)
DIC/HOBt (1)
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B (14)
2
H-Gly-His-Phe-NH (20)
Fig. 3 Peptide models used in the racemization test.
16
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Table 4 Percentage of tetrapeptide des-Aib (H-Tyr-Aib-Phe-Leu-NH
2
)
DIC (Fluka, lot number BCBK8348 V); HOBt (Gl Biochem
during solid-phase assembly of pentapeptide 21 (H-Tyr-Aib-Aib-Phe-
(
Shanghai) Ltd, Lot number GLS110604-00602); HOAt (Gl
Biochem (Shanghai) Ltd, lot number GLS121115-00601); and
OxymaPure (Luxembourg Biotech., Batch number
a
2
Leu-NH )
b
Preactivation
time
Penta
(%)
des-Aib
(%)
Entry
Coupling reagent
1301117008). Melting points were determined with a Barn-
stead IA9300 system (Electrothermal, UK) and are uncorrected.
1
2
3
4
5
6
7
DIC/HOBt (1)
DIC/HOAt (2)
DIC/OxymaPure (7)
DIC/Oxyma-B(14)
DIC/Oxyma-B(14)
DIC/Oxyma-B(14)
DIC/Oxyma-B(14)
3 min
3 min
3 min
No preactivation
8.4
37.5
42.8
19.4
26.4
10.5
9.4
83.1
60.2
50.4
72.6
61.1
79.7
82.8
1
13
NMR spectra ( H NMR and C NMR) were recorded on a
Bruker AVANCE III 400 MHz spectrometer. Chemical shift
values are expressed in ppm downfield from TMS as an
internal standard. IR was recorded using a Bruker-ALPHA
spectrophotometer. Follow-up of the reactions and checks of
the purity of the compounds was performed by TLC on silica
gel-protected aluminium sheets (Type 60 GF254, Merck Milli-
3 minc
3 min
7 min
a
One-hour coupling times were generally applied, except for Aib–Aib
b
(one-hour double coupling). Deletion tetrapeptide (des-Aib) was
identified by peak overlap in HPLC with an authentic sample obtained pore, Bedford, MA, USA), and the spots were detected by
in the solid phase. The crude H-Tyr-Aib-Aib-Phe-Leu-NH
by reverse-phase HPLC using linear gradient of 30 to 60% 0.1% TFA in
CH CN–0.1% TFA in H O over 15 min, detection at 220 nm and a
Phenomex C18 (3 μm, 4.6 × 50 mm) column, t
2
was analyzed
exposure to an UV-lamp at λ 254 nm for a few seconds. Analyti-
cal HPLC was performed on an Agilent 1100 system, and the
3
2
R
= 6.68 (pentapeptide), Chemstation software was used for data processing. LC-MS
was performed on Shimadiz 2020 UFLC-MS using an YMC-
Triart C18 (5 μm, 4.6 × 150 mm) column, and data processing
was carried out by the LabSolution software. Buffer A: 0.1%
c
6
.78 (des-Aib) min. 0.1% DIEA was used.
Our first idea was to apply a range of preactivation times
2 3
formic acid in H O; and buffer B: 0.1% formic acid in CH CN.
with the use of 14 and DIC. A 3 min preactivation gave the best
result (entry 5 vs. entries 4 and 7). In order to enhance the
coupling potency of 14, 0.1 equiv. of DIEA was used. However,
it showed less reactivity than expected (entry 5 vs. 6). In com-
parison with other additives, 14 showed a better performance
than HOBt 1 (entry 5 vs. 1) and a worse performance than
HOAt 2 and OxymaPure 7 (entry 5 vs. entries 2 and 3,
respectively).
High-resolution mass spectrometric data were obtained using
a Bruker micrOTOF-Q II instrument operating at room temp-
erature and a sample concentration of approximately 1 ppm.
Synthesis of 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6
(
1H,3H,5H)-trione (Oxyma-B 14)
15.6 g (0.1 mol) of 1,3-dimethylbarbituric acid 14 was dis-
solved in 60 mL of water containing 6.0 g (0.15 mol) NaOH.
Next, 10 mL of methanol was added to the transparent solu-
tion obtained, then a solution of 7.6 g (0.11 mol) of sodium
nitrite in 30 mL of water was poured into the mixture, which
was then stirred for 2 to 3 min. The mixture was cooled to
Conclusions
Our results indicate that Oxyma-B 14 is a superior racemiza-
tion suppressor than OxymaPure7. Moreover, 14 performs
better than HOAt 2, which to date was considered to be the
most outstanding in that regard. Also, we envisaged that an
additional effect, namely the presence of the carbonyl groups
oriented in the same direction as the N-OH group in this mole-
cule, would increase the reactivity of Oxyma-B 14. Hence, these
groups enhanced basic catalysis; thereby improving the
nucleophilicity of the amine function during the coupling,
1
0 °C, and then 18 g (0.3 mol) of acetic acid was added drop-
wise. The mixture was kept at 25 °C for 1 h. Next, 25 mL of
0% hydrochloric acid was added to the mixture and stirred
for 10 min. The precipitate was filtered, and washed first with
0% methanol and then with water. The product was recrystal-
3
5
lized from aqueous methanol 2 : 1 to obtain Oxyma-B 14 as a
white crystal in 84.0% yield (15.56 g); m.p. 140–142 °C. IR
(
(
(
ATR): 3200–2300 (br, OH), 1667 (s, CO, amide) cm− . H NMR
1
1
2
4
400 MHz, CDCl
br, 1H, OH, D O exchangeable). C NMR (CD
7.82, 135.66, 149.99, 157.45. HRMS (ESI+) m/z calcd for
3
) δ 3.43 (s, 3H, CH
3
3
), 3.46 (s, 3H, CH
3
), 15.94
and occurs when HOAt 2 and HONM 9 are used. Further-
more, Oxyma-B does not contain any ethyl ester, which can
lead to side reactions.
1
2
3
CN): δ 26.93,
2
−
−
6 7 3 4
C H N O : [M − H] 184.0358; found [M − H] 184.0369.
The preparation and study of the applications of the corres-
ponding uronium salt are underway and will be published
elsewhere.
1
8,33,34,42–44
General method for the racemization experiments
0
0
.125 mmol of an acid (Z-Phg-OH or Z-Phe-Val-OH),
.125 mmol of H-Pro-NH , and 0.125 mmol of the corres-
2
ponding additive were dissolved in 2 mL of DMF, and the solu-
tion was cooled in an ice bath and treated with 0.125 mmol of
DIC. The mixture was stirred at 0 °C for 1 h and at room temp-
Experimental
Materials
The solvents used were of HPLC reagent grade. Chemicals and erature overnight. An aliquot (10 μL) of the solution was then
2
amino acid derivatives were purchased from Sigma-Aldrich, taken and diluted to 1 mL with a mixture of CH CN–H O
3
Fluka, Gl Biochem (Shanghai) Ltd, Iris Biotech GmbH, or (1 : 2). Next, 5 μL of this solution was injected into a reverse-
Merck Millipore. The following coupling reagents were used: phase HPLC apparatus.
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Z-Phg-Pro-NH
2
tail were concentrated under nitrogen. The crude peptide was
precipitated with cold Et O (3 × 5 mL) and lyophilized. It was
then analyzed by reversed-phase HPLC, with the use of a Phe-
nomex C18 (3 μm, 4.6 × 50 mm) column, linear gradient 0 to
2
3
A linear gradient of 25–50% 0.1% TFA in CH CN/0.1% TFA in
O over 15 min was applied, with a flow rate of 1.0 mL min−
1
H
2
and detection at 220 nm using a Phenomex C18 (3 μm, 4.6 ×
0 mm) column, t (LL) = 6.4 min, t (DL) = 6.8 min,
(Z-Phg-OH) = 9.1 min.
4
0% of 0.1% TFA in CH CN/0.1% TFA in H O over 15 min,
3
2
5
R
R
with detection at 220 nm. The t
R
values of the LL and DL
t
R
epimers were 6.79 and 7.95 min, respectively. LC–MS showed
the expected mass for the tripeptide at m/z = 325.0.
Z-Phe-Val-Pro-NH2
A linear gradient of 30–60% 0.1% TFA in CH
H O over 15 min was applied, with a flow rate of 1.0 mL min
3
CN/0.1% TFA in Study of cysteine racemization during assembly of H-Gly-
−
1
2
Cys(Acm)-Phe-NH on the solid phase
2
and detection at 220 nm using a Phenomex C18 (3 μm, 4.6 ×
0 mm) column, t (LLL) = 5.8 min, t (LDL) = 6.9 min,
tR(Z-Phe-Val OH) = 8.1 min.
Experiments consisted of the study of the stepwise coupling of
Cys and Gly residues onto previously formed H-Phe-Rink-
Amide-AM-PS-resin (0.6 mmol g , 100 mg), with the use of
5
R
R
−1
the Fmoc/tBu and Cys(Acm) protection strategy. Glycine was
introduced in order to achieve better separation of LL and DL
isomers than of des-Gly dipeptides. Coupling times of 1 h were
used after 5 min preactivation of a solution of Fmoc-amino
acids (3 equiv., excess), the corresponding additive (3 equiv.),
and DIC (3 equiv.) in DMF (0.5 mL) at room temperature.
Fmoc was removed with 20% piperidine in DMF for 7 min.
The peptide chain was released from the resin by treatment
Study of serine racemization during assembly of H-Gly-Ser-
Phe-NH on the solid phase
2
3
5,37
Experiments consisted of the study of the stepwise coupling of
Ser and Gly residues onto previously formed H-Phe-Rink-
−
1
Amide-AM-PS-resin (0.6 mmol g , 100 mg), with the use of
t
the Fmoc/tBu and Ser( Bu) protection strategy. Glycine was
introduced in order to achieve better separation of LL and DL
isomers than des-Gly dipeptides. Coupling times of 1 h were
used after 5 min preactivation of a solution of Fmoc-amino
acids (3 equiv.), the corresponding additive (3 equiv.), and DIC
with TFA–H O–TIS (95 : 2.5 : 2.5) for 1 h at room temperature.
2
The colorless solution was filtered, and the resin was washed
with CH
cleavage cocktail were concentrated under nitrogen. The crude
peptide was precipitated with cold Et O (3 × 5 mL) and lyo-
2 2
Cl (3 × 0.5 mL). The solvent and residues from the
(
3 equiv.) in DMF (0.5 mL) at room temperature. Fmoc was
removed with 20% piperidine in DMF for 7 min. The peptide
chain was released from the resin by treatment with TFA–H O–
TIS (95 : 2.5 : 2.5) for 1 h at room temperature. The colorless
solution was filtered, and the resin was washed with CH Cl
3 × 0.5 mL). The solvent and residues from the cleavage cock-
tail were concentrated under nitrogen. The crude peptide was
precipitated with cold Et O (3 × 5 mL) and then lyophilized. It
was then analyzed by reversed-phase HPLC, with the use of a
Phenomex C18 (3 μm, 4.6 × 50 mm) column, linear gradient Study of histidine racemization during assembly of H-Gly-His-
2
2
philized. It was then analyzed by reversed-phase HPLC, with
the use of a Phenomex C18 (3 μm, 4.6 × 50 mm) column, linear
2
2
gradient 0 to 40% of 0.1% TFA in CH
CN/0.1% TFA in H
2
O
3
(
over 15 min, with detection at 220 nm. The t
R
values of the LL
and DL epimers were 6.70 and 7.54 min, respectively. LC–MS
showed the expected mass for the tripeptide at m/z = 396.0.
2
3
7
0
1
to 30% of 0.1% TFA in CH
5 min, with detection at 220 nm. The t
3
CN/0.1% TFA in H
values of the LL and
2
O over Phe-NH on the solid phase
2
R
Experiments consisted of the study of the stepwise coupling of
Ser and Gly residues onto previously formed H-Phe-RinkA-
DL epimers were 5.55 min and 6.06 min, respectively. LC–MS
showed the expected mass for the tripeptide at m/z = 309.0.
−1
mide-AM-PS-resin (0.6 mmol g , 100 mg), with the use of the
Fmoc/tBu and the His(Trt) protection strategy. Glycine was
introduced in order to achieve better separation of LL and DL
isomers than des-Gly dipeptides. Coupling times of 1 h were
Study of cysteine racemization during assembly of H-Gly-Cys-
1
8,36–38,45
Phe-NH on the solid phase
2
Experiments consisted of the study of the stepwise coupling of used after 5 min preactivation of a solution of Fmoc-amino
Cys and Gly residues onto previously formed H-Phe-RinkA- acids (3 equiv., excess), the corresponding additive (3 equiv.),
−
1
mide-AM-PS-resin (0.6 mmol g , 100 mg), with the use of the and DIC (3 equiv.) in DMF (0.5 mL) at room temperature.
Fmoc/tBu and the Cys(Trt) protection strategy. Glycine was Fmoc was removed with 20% piperidine in DMF for 7 min.
introduced in order to achieve better separation of LL and DL The peptide chain was released from the resin by treatment
2
isomers than des-Gly dipeptides. Coupling times of 1 h were with TFA–H O–TIS (95 : 2.5 : 2.5) for 1 h at room temperature.
used after 5 min preactivation of a solution of Fmoc-amino The colorless solution was filtered, and the resin was washed
acids (3 equiv.), the corresponding additive (3 equiv.), and DIC with CH Cl (0.5 mL × 3). The solvent and residues from the
2
2
(3 equiv.) in DMF (0.5 mL) at room temperature. Fmoc was cleavage cocktail were concentrated under nitrogen. The crude
removed with 20% piperidine in DMF for 7 min. The peptide peptide was precipitated with cold Et O (3 × 5 mL) and lyo-
2
chain was released from the resin by treatment with TFA–H O– philized. It was then analyzed by reversed-phase HPLC, with
2
TIS (95 : 2.5 : 2.5) for 1 h at room temperature. The colorless the use of a Phenomex C18 (3 μm, 4.6 × 50 mm) column, linear
solution was filtered, and the resin was washed with CH
2
Cl
2
3 2
gradient 5 to 20% of 0.1% TFA in CH CN/0.1% TFA in H O
(
3 × 0.5 mL). The solvent and residues from the cleavage cock- over 15 min, with detection at 220 nm. The t values of the LL
R
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and DL epimers were 4.01 min and 4.65 min, respectively. LC–
MS showed the expected mass for the tripeptide at m/z = 359.0.
5 C. A. G. N. Montalbetti and V. Falque, Tetrahedron, 2005,
61, 10827–10852.
6
E. Valeur and M. Bradley, Chem. Soc. Rev., 2009, 38,
06–631.
7 A. El-Faham and F. Albericio, Chem. Rev., 2011, 111,
557–6602.
6
Solid-phase synthesis of H-Tyr-Aib-Aib-Phe-Leu-
1
2,18,21–24,29–33
NH2
6
The synthesis was carried out in a plastic syringe attached to a
8
9
W. König and R. Geiger, Chem. Ber., 1970, 103, 788–798.
L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397–4398.
vacuum manifold to achieve the rapid removal of reagents and
solvent. The Fmoc-RinkAmide-AM-PS resin (0.6 mmol g−
1
,
1
0 G. Sabatino, B. Mulinacci, M. C. Alcaro, M. Chelli,
P. Rovero and A. M. Papini, Peptides 2002: Proceedings of
the Twenty-Seventh European Peptide Symposium, Aug. 31–
Sept. 6, Sorrento, Italy, 2002.
1
00 mg), was washed with DMF, DCM, and DMF (2 × 10 mL
each) and then treated with 20% piperidine in DMF (10 mL)
for 10 min. The resin was then washed with DMF, DCM, and
DMF (2 × 10 mL each) and acylated with a solution of Fmoc-
Leu-OH (3 equiv.), the corresponding additive (3 equiv.) and
DIC (3 equiv.) in DMF (0.5 mL, previously preactivated). After
peptide coupling, the resin was washed with DMF and then
de-blocked by treatment with 20% piperidine in DMF for
1
1
1
1
1
1
1 F. Albericio, J. M. Bofill, A. El-Faham and S. A. Kates, J. Org.
Chem., 1998, 63, 9678–9683.
2 A. El-Faham, S. N. Khattab, M. Abdul-Ghani and
F. Albericio, Eur. J. Org. Chem., 2006, 1563–1573.
3 R. Knorr, A. Trzeciak, W. Bannwarth and D. Gillessen,
Tetrahedron Lett., 1989, 30, 1927.
4 L. A. Carpino, A. El-Faham and F. Albericio, Tetrahedron
Lett., 1994, 35, 2279.
7
1
min. The resin was washed with DMF, DCM, and DMF (2 ×
0 mL each). Then, coupling with the next amino acid, as
explained previously, and de-blocking were repeated to obtain
the pentapeptide. The peptide was cleaved from the resin with
5 O. Marder, Y. Shvo and F. Albericio, Chim. Oggi, 2002, 20,
TFA–H
under nitrogen, and the crude peptide was purified with cold
Et O (3 × 10 mL) and lyophilized. The ratio of the penta- and
2
O (9 : 1) at room temperature for 2 h. TFA was removed
37–41.
6 K. D. Wehrstedt, P. A. Wandrey and D. Heitkamp,
J. Hazard. Mater., 2005, A126, 1–7.
2
tetra-peptide was determined by HPLC analysis using a Pheno-
mex C18 (3 μm, 4.6 × 50 mm) column, with a linear gradient of
1
1
7 M. Itoh, Bull. Chem. Soc. Jpn., 1973, 46, 2219–2221.
8 R. Subirós-Funosas, R. Prohens, R. Barbas, A. El-Faham
and F. Albericio, Chem. – Eur. J., 2009, 15, 9394–9403.
9 R. Subirós-Funosas, A. El-Faham and F. Albericio, Tetra-
hedron, 2011, 67, 8595–8606.
2
0 to 40% of 0.1% TFA in CH
3
CN/0.1% TFA in H
2
O over
−
1
1
5 min, flow rate = 1.0 mL min , detection at 220 nm. The t
R
1
2
values for pentapeptide and des-Aib were 6.68 min and
.78 min, respectively. LC–MS showed the expected mass for
the pentapeptide at m/z = 611.0, and also for des-Aib at m/z =
26.
6
0 R. Subiros-Funosas, S. N. Khattab, L. Nieto-Rodriguez,
A. El-Faham and F. Albericio, Aldrichimica Acta, 2013, 46,
5
21–40.
21 A. El-Faham, R. S. Funosas, R. Prohens and F. Albericio,
Chem. – Eur. J., 2009, 15, 9404–9416.
Acknowledgements
22 R. Subiros-Funosas, G. A. Acosta, A. El-Faham and
F. Albericio, Tetrahedron Lett., 2009, 50, 6200.
We thank Yoav Luxembourg (Luxembourg Ind.,) for his con-
tinuous support during this study. This work was funded in
part by the CICYT (CTQ2012-30930), the Generalitat de Catalu-
nya (2009SGR1024), and the Institute for Research in Biomedi-
cine Barcelona (IRB Barcelona) (Spain); the National Research
Foundation, the University of KwaZulu Natal; AsphenPharma
care for their financial support (South Africa); and SENESCYT
2
2
3 A. El-Faham and F. Albericio, J. Pept. Sci., 2010, 16, 6–9.
4 A. El-Faham, R. Subirós-Funosas and F. Albericio,
Eur. J. Org. Chem., 2010, 3641–3649.
2
5 Although, we have forced the side reaction involving the
ethyl ester moiety: mixing of the amino component in the
presence of OxymaPure (10 equiv.) for 12 h or microwave
heating of the amino component with OxymaPure
(Ecuador).
(
10 equiv.) at 80 °C for 10 min in DMF or NMP, we did not
appreciate the side reaction at all. For more experimental
details, see ref. 18.
2
6 G. A. Acosta, M. del Fresno, M. Paradis-Bas, M. Rigau-
DeLlobet, S. Cote, M. Royo and F. Albericio, J. Pept. Sci.,
2009, 15, 629–633.
Notes and references
1
F. Albericio and L. A. Carpino, in Methods in Enzymology,
ed. B. F. Gregg, Academic Press, 1997, vol. 289, pp. 27 J. S. Davies and A. K. Mohammed, J. Chem. Soc., Perkin
04–126. Trans. 1, 1981, 2982–2990.
J. M. Humphrey and A. R. Chamberlin, Chem. Rev., 1997, 28 Y. Zhang, S. M. Muthana, D. Farnsworth, O. Ludek,
1
2
3
4
9
7, 2243–2266.
K. Adams, J. J. Barchi and J. C. Gildersleeve, J. Am. Chem.
Soc., 2012, 134, 6316–6325.
29 R. Subiros-Funosas, A. El-Faham and F. Albericio, Org.
Biomol. Chem., 2010, 8, 3665–3673.
F. Albericio, R. Chinchilla, D. J. Dodsworth and C. Najera,
Org. Prep. Proced. Int., 2001, 33, 203.
S.-Y. Han and Y.-A. Kim, Tetrahedron, 2004, 60, 2447–2467.
Org. Biomol. Chem.
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Communication
3
3
3
3
3
3
3
3
0 A. El-Faham, S. N. Khattab, R. Subiros-Funosas and 38 T. Kaiser, G. J. Nicholson, H. J. Kohlbau and W. Voelter,
F. Albericio, J. Pept. Sci., 2014, 20, 1–6. Tetrahedron Lett., 1996, 37, 1187–1190.
1 A. El-Faham and F. Albericio, J. Org. Chem., 2008, 73, 2731– 39 N. Robertson, L. Jiang and R. Ramage, Tetrahedron, 1999,
737. 55, 2713–2720.
2 A. El-Faham and F. Albericio, Org. Lett., 2007, 9, 4475– 40 H. Hibino and Y. Nishiuchi, Tetrahedron Lett., 2011, 52,
477. 4947–4949.
3 S. N. Khattab, R. Subiros-Funosas, A. El-Faham and 41 H. Hibino, Y. Miki and Y. Nishiuchi, J. Pept. Sci., 2012, 18,
F. Albericio, ChemistryOpen, 2012, 1, 147–152. 763–769.
4 L. A. Carpino, A. El-Faham and F. Albericio, J. Org. Chem., 42 A. El-Faham and F. Albericio, Eur. J. Org. Chem., 2009,
995, 50, 3561. 1499–1501.
5 A. Di Fenza, M. Tancredi, C. Galoppini and P. Rovero, Tetra- 43 L. A. Carpino, J. Xia and A. El-Faham, J. Org. Chem., 2004,
hedron Lett., 1998, 39, 8529–8532. 69, 54–61.
6 Y. Han, F. Albericio and G. Barany, J. Org. Chem., 1997, 62, 44 L. A. Carpino, H. Imazumi, B. M. Foxman, M. J. Vela,
2
4
1
4
307.
7 W. Van Den Nest, S. Yuval and F. Albericio, J. Pept. Sci.,
001, 7, 115–120.
P. Henklein, A. El-Faham, J. Klose and M. Bienert, Org.
Lett., 2000, 2, 2253–2256.
2
45 H. Hibino and Y. Nishiuchi, Org. Lett., 2012, 14, 1926–1929.
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