4-(4,6-Di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate. Triazine-based coupling reagents designed for coupling sterically hindered substrates

Article abstract of DOI:10.1021/jo2002038

4-(4,6-Di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (DFET/NMM/BF₄) was prepared and used as a reagent for coupling sterically hindered substrates. The formation of the appropriate triazine "superactive" ester in a reaction of DFET/NMM/BF₄ with carboxylic acids was confirmed. The efficiency of the reagent has been studied in the synthesis of Leu-enkephaline pentapeptide carried out on a Fmoc-RinkAmide-AM-PS resin, by systematically modifying the -Gly-Gly- fragment for N-methyl or α,α-disubstituted residues and compared with the efficiency of classic aminium salt 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU) under a variety of reaction conditions. In syntheses of Aib-Aib (Aib: α-aminoisobutyric acid), MeVal-MeVal, and MeLeu-MeLeu, the considerably superior performance of enkephaline analogues was obtained for DFET/NMM/BF₄ relative to TBTU, regardless of reaction conditions. Analysis of the couplings involving triazine reagent suggests that factors controlling efficiency of coupling sterically hindered substrates are the structure of the leaving group permitting formation of the cyclic intermediate or cyclic transition state and the absence of strongly solvating solvents. It has to be considered as highly probable that the absence of strongly solvating milieu favors cyclic intermediates or the cyclic transition state. Arrangement of both components into the cyclic intermediate or cyclic transition state by accumulation of the geminal (vicinal) substituents effect (known as the Thorpe-Ingold effect) would compensate retardation of the coupling process caused by steric hindrance.

Full text of DOI:10.1021/jo2002038

ARTICLE
pubs.acs.org/joc
4-(4,6-Di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomor-
pholinium Tetrafluoroborate. Triazine-Based Coupling Reagents
Designed for Coupling Sterically Hindered Substrates
Konrad G. Jastrzabek,†,‡ Ramon Subiros-Funosas,‡,§ Fernando Albericio,*,‡,§,|| Beata Kolesinska,† and
Zbigniew J. Kaminski*,†
†Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
‡Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, 08028-Barcelona, Spain
§CIBER-BBN, Networking Centre on Bioengineering Biomaterials and Nanomedicine, Barcelona Science Park, 08028-Barcelona, Spain
Department of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain
S Supporting Information
b
ABSTRACT: 4-(4,6-Di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-
yl)-4-methylomorpholinium tetrafluoroborate (DFET/NMM/
BF4) was prepared and used as a reagent for coupling sterically
hindered substrates. The formation of the appropriate triazine
“superactive” ester in a reaction of DFET/NMM/BF4 with
carboxylic acids was confirmed. The efficiency of the reagent
has been studied in the synthesis of Leu-enkephaline pentapeptide carried out on a Fmoc-RinkAmide-AM-PS resin, by systematically
modifying the -Gly-Gly- fragment for N-methyl or R,R-disubstituted residues and compared with the efficiency of classic aminium salt
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) under a variety of reaction conditions. In syntheses of
AibꢀAib (Aib: R-aminoisobutyric acid), MeVal-MeVal, and MeLeu-MeLeu, the considerably superior performance of enkephaline
analogues was obtained for DFET/NMM/BF4 relative to TBTU, regardless of reaction conditions. Analysis of the couplings involving
triazine reagent suggests that factors controlling efficiency of coupling sterically hindered substrates are the structure of the leaving group
permitting formation of the cyclic intermediate or cyclic transition state and the absence of strongly solvating solvents. It has to be
considered as highly probable that the absence of strongly solvating milieu favors cyclic intermediates or the cyclic transition state.
Arrangement of both components into the cyclic intermediate or cyclic transition state by accumulation of the geminal (vicinal)
substituents effect (known as the ThorpeꢀIngold effect) would compensate retardation of the coupling process caused by steric
hindrance.
’ INTRODUCTION
activation and prolonged coupling time may severely deteriorate
the yield and purity of the required peptide.
In recent years, most of the peptides used in combating various
diseases as well as peptides showing activity as insecticides,
contraceptives, growth promoters, metabolism regulators, and
many other uses have been prepared using unnatural building
blocks such as noncoded amino acids. The main reason for
practicing this approach is the expected increase of the resistance
of unnatural structures toward metabolic degradation, prolonged
activity, reduced dosage, and more convenient application. In
most cases, these advantageous effects of incorporation of an
unnatural moiety into the peptide chain exclude application of
biological approaches for their preparation, and therefore that
depends only on a successful chemical synthetic strategy.
Currently, the arsenals of available reagents and strategies for
the coupling are adequate for successful assembly of peptides
from the 20 coded amino acids in solution and on a solid support,
but often they are found insufficient or even fail in the case of
their unnatural analogues or the so-called difficult sequences.1ꢀ10
The most frustrating syntheses involve the concourse of poor
reactive and sterically hindered building blocks because stronger
The acidity of the additive, either used in combination with
carbodiimide or contained into a stand-alone coupling reagent, is
a critical factor in the efficiency of peptide bond formation.
However, sterical effects are also involved in this process, which
become more influential as the size of the peptide fragment
increases. In most of the cases, an approach to improve the
efficiency of the coupling reagent by expansion of its structure
remains not fruitful because introduction of any additional
structural fragments enlarges the size of the reagent, spoiling
the effect of increased reactivity. Therefore, most of the attempts
were made to shrink the reagent fragment participating at the
stage most sensitive to the steric effects, which for the classic
mechanism of peptide bond synthesis is the formation of the
tetrahedral intermediate during the aminolysis step. Nowadays,
only few reagents were designed in accord with this inspirational
Received: February 16, 2011
Published: April 15, 2011
r
2011 American Chemical Society
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ARTICLE
Scheme 1. Intramolecular Cyclic Transition State Postulated for Coupling Involving Triazine “Superactive Esters”
strategy. All of them are based on good leaving groups of size
smaller than standard HOBt, HOAt, or 6-Cl-HOBt. The addi-
tional goal of the process of declining the size of the leaving group
was elimination of the explosive property caused by instability of
the triazole fragment.11 One of the first and most promising
responses to this problem was coupling reagents based on the
ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) oxime frame,
which contains high electron-withdrawing substituents, increas-
ing the acidity of the additive and consequently the reactivity.12
Its parent uronium salt, COMU, incorporating a hydrogen bond
acceptor in the iminium part resulted in performances superior to
reagents described previously.13ꢀ15
Scheme 2. Synthesis of 2-Chloro-4,6-di
(2,2,2,-trifluoroethoxy)-1,3,5-triazine (CDFET, 2b)
and 4-Di(4,6-[2,2,2-trifluoroethoxy]-1,3,
5-triazyn-2-yl)-4-methylomorpholinium
Tetrafluoroborate (DFET/NMM/BF4, 3b)
The alternative process of size diminution gave coupling
reagents based on the use of pyridine,16,17 pyrimidine,18 or
triazine19ꢀ22 as leaving group. In all cases, an additional mechan-
istic benefit originating from structural features of azines predis-
posed for the aminolysis proceeding via a cyclic, concerted
transition state, similar to the one envisaged for 7-HOAt23
(Scheme 1), has been anticipated. However, for relatively less
reactive derivatives of pyridine and pyrimidine, this benefit has
been less significant than in the case of much more electron-
deficient triazines.24,25
intermediate in a coupling reaction, substantial intensification
of coupling sterically hindered substrates has to be observed.
To increase the reactivity of classic triazine-based coupling
reagents 2a, methoxy groups at positions 2 and 4 in the triazine
ring were substituted with much more strongly electron-with-
drawing 2,2,2-trifluoroethoxy substituents. Synthesis of 2-chloro-
4,6-di(2,2,2-trifluoroethoxy)-1,3,5-triazine (CDFET, 2b) was
accomplished in the two-phase system by treatment of cyanuric
chloride with an equivalent amount of 2,2,2-trifluoroethanol in
the presence of sodium bicarbonate with 52% yield (Scheme 2).
Contrary to the well-known rule of thumb declaring that
substitution of chlorine atoms in cyanuric chloride with other
groups proceeds under gradually more vigorous conditions,
introduction of the trifluoroethoxy group enhanced the reactivity
of triazine. This strongly favored substitution of the next reactive
group in the 1,3,5-triazine ring and promoted formation of
persubstituted derivatives. Therefore, isolation of 2b from the
mixture of products required fractional distillation affording all
three possible triazines substituted with mono-, di-, and tri-(2,2,2-
trifluoroethoxy) groups.
Stable solid coupling reagent 3b was prepared according to a
classic procedure33 by treatment of 2b with N-methylmorpho-
linium tetrafluoroborate in the presence of sodium bicarbonate
in 43% yield. This procedure, reducing the danger of partial
hydrolysis, was found much more convenient for the preparation
of highly reactive 3b than the recently proposed method20,34
involving transformation in aqueous medium, considered as
being less suitable for the highly reactive substrate.
Bearing in mind the ThorpeꢀIngold effect,26 we hypothesized
that increased substitution in substrates could favor formation
of the cyclic intermediate or transition state (at least compared to
the linear transition state or linear intermediate) by tethering the
two reacting centers and finally support acylation of sterically
hindered substrates.27ꢀ30 In general, intramolecular reactions
occur more rapidly than their intermolecular counterparts owing
to their more favorable entropy change on passing to the
transition state.31 When five- and six-membered rings are formed,
like the one proposed for 4-(4,6-di[2,2,2-trifluoroethoxy]-1,3,5-
triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (DFET),
this entropic contribution produces a favorable ΔG and equilibrium
constant. This would compensate the well-known effect of retarda-
tion of synthetic processes by increased steric hindrance of sub-
strates. To verify this assumption, we undertake systematic studies
on incorporation of N-alkylated amino acids and on 2,2-disubsti-
tuted amino acids comparing different coupling reagents.
’ RESULTS AND DISCUSSION
The measurements of nitrogen kinetic isotope effects32 in the
reaction model for difficult coupling involving sterically hindered
trimethylacetic acid and aniline used as a less nucleophilic amino
component provided data compatible with fast release of the
leaving group but not enough characteristics to confirm unequi-
vocally the mechanism via cyclic transition state. One could
expect that decreasing electron density in the triazine ring would
favor attack of the nucleophilic amino component on the
activated carboxylic function, simultaneously sustaining the
character of triazine as an excellent leaving group. If this
modification would be sufficient for promoting the cyclic
Coupling experiments in solution confirmed the expectations
and demonstrated the synthetic usability of 3b. The activation of
carboxylic function proceeded substantially faster than in the case
of analogue 3a (R = CH3)20 (Scheme 3).
Even in the case of sterically hindered carboxylic components
4b, monitoring of the activation process shows the complete
consumption of an equimolar amount of reagent within 15 min.
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Scheme 3. Activation of 4-Methoxybenzoic Acid (4a) and
Trimethylacetic Acid (4b) and with 3b
carried out on a Fmoc-RinkAmide-AM-PS resin, studying the
introduction of the last three residues onto H-Phe-Leu-resin. The
composition of products was determined by LC-MS, and the amount
of incomplete sequences was determined based on UV absorbance at
220 nm.
In the synthesis of the “less” sterically hindered sarcosine-
containing analogue 7 under conditions optimized for uronium
coupling reagents, the productivity of TBTU was com-
parable with DFET/NMM/BF4 (3b) (see Table 2, entries 1ꢀ4
and 5ꢀ7).
However, in the case of Aib analogue 8 under more demand-
ing conditions with short, 1 min preactivation time, significantly
different results were obtained for both compared coupling
reagents (Table 3). Application of TBTU for Aib-Aib coupling
prolonged to 240 min gave the expected enkephaline analogue in
yields not exceeding 24%, with 72ꢀ77% des-Aib side products
present as the predominant impurity. Considerably superior
performance was obtained for DFET/NMM/BF4 (3b) relative
to TBTU, regardless of reaction conditions. The crucial factor
controlling the coupling with TBTU and especially DFET/
NMM/BF4 (3b) was found to be the character of the solvent
used in the synthesis. In the case of strongly solvating DMF
solution (Table 3, entries 5ꢀ7), the yields of the final product
were poor. However, decreasing the solvating ability of solvent
by gradually increasing the amount of DCM from 0 to 100%
(entries 7ꢀ10), the most suitable solvent during activation of the
carboxylic substrate35 significantly promoted the coupling, rais-
ing the yield of the final product from 8 to 71% even for shorter
30 min coupling time. It is worthy to notice that dilution of DCM
with strongly solvating 1,1,3,3-tetramethylurea (TMU) de-
pressed the efficiency of coupling, yielding final products in only
19% yield (Table 3, entry 10 vs 12), whereas an opposite
tendency was observed in DMF (entry 7 vs 11). Further
improvements of results were obtained by prolongation of
difficult coupling of Aib residues to 120 min. The best, 82%
yield again was obtained in the composition of solvents with the
dominating fraction of less polar DCM.
It has been found that also the presence of additives, such as
HOAt, HOBt, Oxyma, or HODMT, in the reaction mixture
could substantially modify the coupling results. On one hand,
addition of classic HOBt severely deteriorated coupling process
(entry 15), increasing the amount of des-Aib peptide to 86%,
whereas on the other hand HOAt, HODMT, and especially
Oxyma increased the efficiency of peptide bond formation. The
performance of HODMT was slightly superior to that of HOAt.
The most beneficial was addition of Oxyma36 (Table 3, entry
17) affording pentapeptide 9 in 94% yield, the highest percen-
tage achieved in this peptide system. Again, this propitious
result of Oxyma was purged away by addition of DMF to
reacting mixture (entry 20).
Table 1. Synthesis of Peptides in Solution Using 3b
Pg-Aaa-Bbb-OMeb
entry
peptide
yield % puritya % Aaa L/D % Bbb L/D %c
1
2
3
4
5
Z-Aib-Aib-OMed
Fmoc-Phe-Ala-OMe
Boc-Leu-Val-OMe
Boc-Pro-Phe-OMe
Z-Aib-Leu-OMed
89
98
93
94
91
94.2
97.1
82.4
96.4
96.2
-
-
99.5/0.5
98.2/1.8
100/0
-
98.9/1.1
99.7/0.3
99.8/0.2
99.2/0.8
a HPLC method. b Determined by GC on a ChromasilVal column after
hydrolysis to amino acids. c Unspecific partial racemization accompany-
ing hydrolytic degradation. d Z: benzoxycarbonyl. Aib: R-
aminoisobutyric acid.
The formation of appropriate triazine “superactive” ester was
confirmed by the presence of a characteristic band at 1757 and
1786 cmꢀ1 in the IR spectrum of both 2-acyloxy-4,6-di(2,2,2-
trifluoroethoxy)-1,3,5-triazines (5a,b) obtained in 73ꢀ81%
yield, respectively (Scheme 3).
In the case of procedures involving 3a, the isolation of neutral
product obtained in the coupling reaction was less convenient
because less soluble appropriate hydroxy-1,3,5-triazines are
formed as side product. Therefore, to completely remove this
side product, it was necessary to prolong the washing procedure
of crude preparation. The coupling results obtained for the
selected peptides are compiled below in Table 1.
The obtained yields were spanned in the range from 89 to 98.
Thus, even the most hindered sequences were obtained in reason-
able good yield. All preparative inconveniences caused by the lower
solubility of 2-hydroxy-4,6-di(2,2,2,-trifluoroethoxy)-1,3,5-triazine
were eliminated when 3b was applied in SPPS; therefore, this
procedure involving 3b was considered as more precise for compar-
ison of reactivity of coupling reagents. The model peptides selected
for the comparative studies were based on the Leu-enkephaline
pentapeptide, by systematically modifying the -Gly-Gly- fragment for
N-methyl or R,R-disubstituted residues. In the performed experi-
ments, the efficiency of classic aminium salt 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) was com-
pared with triazine reagent DFET/NMM/BF4 (3b) under a variety
of reaction conditions. The syntheses of enkephaline analogues were
Considerable diversification of coupling results was found also
in the case of incorporation of consecutive N-methylleucine
residues into the peptide chain. For TBTU, the yield of expected
enkephalin analogue was in the range 1ꢀ11% (Table 4, entries 1
and 2), whereas in experiments with DFET/NMM/BF4 (3b),
even in DMF solution the final product was obtained in 66ꢀ79%
yield (entries 3 and 4). For both reagents, a minimum preactiva-
tion time of 1 min is recommended to enhance active ester
formation (entries 2, 4 vs 1, 3).
Generally recognized as one of the most extremely difficult
couplings, introduction of N-MeVal into N-MeVal-Phe-
Leu-resin was studied during assembly of enkephalin analogue
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Table 2. Synthesis of Tyr-Sar-Sar-Phe-Leu Enkephaline Analogue (7), Preactivation 1 min
entry
coupling reagent
base
coupling time [min]
solvent
pentapeptide [%]
des-Sar [%]
1
2
3
4
5
TBTU
TBTU
TBTU
3b
DIPEA
NMM
-
5
5
5
5
5
DMF
DMF
99.06
99.10
98.39
96.94
96.84
0.58
0.58
0.10
2.49
2.48
DMF:DCM (1:1)
DMF
NMM
NMM
3b
DMF:DCM (1:1)
Table 3. Synthesis of Tyr-Aib-Aib-Phe-Leu Enkephaline Analogue (8), Preactivation 1 min
entry coupling reagent
base
additive
coupling time [min]
solvent
penta peptide [%] des-Aib des-Tyr des-2Aib des-AibTyr
1
2
TBTU
TBTU
TBTU
TBTU
3b
NMM
NMM
NMM
NMM
NMM
NMMa
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
NMM
5 min each
DMF
0.67
18.41
21.16
23.02
11.16
8.02
96.12
77.78
74.79
72.32
85.03
90.35
77.02
59.68
28.21
86.73
80.27
65.40
44.29
17.32
86.24
34.17
4.86
0.12
2.64
2.89
3.51
0.26
--
0.2
2.88
0.53
0.59
0.58
3.36
0.87
0.32
0.12
0.06
0.07
0.06
0.20
0.19
0.19
0.26
0.21
0.15
0.09
--
-
-
-
-
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/2 ꢁ 120/30
5 min each
“
0.65
0.57
0.57
0.19
0.76
0.94
0.76
0.83
0.81
0.72
0.74
0.16
0.11
0.63
0.81
0.85
0.13
0.51
0.32
3
DMF:DCM (1:1)
DMF:DCM (1:8)
DMF
4
5
6
3b
30 each
“
7
3b
30 each
DMF:DCM (1:1)
DMF:DCM (1:8)
DCM
21.71
39.41
70.90
12.38
18.93
33.65
55.37
82.32
12.86
64.81
94.15
66.75
63.35
48.79
--
8
3b
-
-
-
-
30 each
--
9
3b
30 each
--
10
11
12
13
14
15
16
17
18
19
20
3b
30 each
TMU:DMF (1:4)
TMU:DCM (1:4)
DMF
--
3b
30 each
--
3b
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/2 ꢁ 120/30
30/60/30
--
3b
DMF:DCM (1:1)
DMF:DCM (1:8)
DMF:DCM (1:1)
DMF:DCM (1:1)
DMF:DCM (1:1)
DMF:DCM (1:1)
DCMb
--
3b
0.05
--
3b
HOBt
HOAt
Oxyma
HODMT
-
3b
--
3b
--
3b
33.03
36.09
50.70
--
3b
--
3b
Oxyma
30/2 ꢁ 120/30
DCMb
--
0.18
a 2 equiv of NMM was used instead of the regular 6 equiv. b Aib-Aib coupling.
Table 4. Synthesis of Tyr-MeLeu-MeLeu-Phe-Leu Enkephaline Analogue (9) with Coupling Time of 5 Min Each
entry
coupling reagent
base
preactivation time [min]
solvent
pentapeptide [%]
des-MeLeu
des-Tyr
des-MeLeu,Tyr
1
2
3
4
TBTU
NMM
NMM
NMM
NMM
1
0
1
0
DMF
10.75
1.37
53.25
24.91
6.05
13.81
5.93
20.59
66.95
1.14
“
“
“
“
3b
3b
78.74
66.14
10.32
11.66
15.65
2.78
Table 5. Synthesis of Tyr-MeVal-MeVal-Phe-Leu Enkephaline Analogue (10), Preactivation 1 min
entry coupling reagent
base
coupling time [min]
solvent
pentapeptide [%] des-MeVal des-Tyr des-2 MeVal des-MeVal,Tyr
1
2
3
4
TBTU
TBTU
3b
NMM
NMM
NMM
NMM
5 min each
DMF
0.15
0.22
8.87
4.36
16.05
37.18
32.31
5.11
7.34
0.07
0.51
0.26
0.72
90.31
75.89
22.49
7.76
2 ꢁ 120 min each
5 min each
DMF/DCM (1:8)
DMF
31.21
12.31
3b
2 ꢁ 120 min each
DMF:DCM (1:8)
46.9
10 (Table 5). TBTU gave unsatisfactory results even if the total
coupling time was prolonged to 240 min. Much better results
were obtained using DFET/NMM/BF4 (3b) (Table 5, entries 3,
4 vs 1, 2). Even for short 5 min couplings in DMF as solvent,
identified previously as unfavorable, superior performance was
observed. Besides the pentapeptide, obtained in 9% yield,
tetrapeptides with the single amino acid deletion dominated in
the mixture of products.
A substantial increase of yield to 47% was obtained by
prolongation of coupling time and reduction of solvating power
of the solvent used in the coupling procedure, by increasing the
percentage of DCM.
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’ CONCLUSIONS
TLC: Rf (chloroform) = 0.65. 1H NMR (250 MHz; CDCl3) δ =
4.89 (q, J3HꢀF = 7.9 Hz, 4H, CH2) [ppm]. MS: 312.03 [M þ 1]þ.
13C NMR (175 MHz; CDCl3) δ = 64.46; 122.29; 171.32; 173.67
[ppm]. Anal. Calcd for C7H4ClF6N3O2: C, 26.99%; H, 1.29%; Cl,
11.38%; F, 36.59%; N, 13.49%; O, 10.27%. Found: C, 27.31%;
H, 1.36%.
4-(4,6-Di[2,2,2-Trifluoroethoxy]-1,3,5-triazin-2-yl)-4-me-
thylomorpholinium Tetrafluoroborate (3b). The suspension of
NaHCO3 (16.8 g, 0.2 mol) and 2-chloro-4,6-di[2,2,2-trifluoroethoxy]-
1,3,5-triazine (31.16 g, 0.1 mol) in acetonitrile (50 mL) was cooled to 0 °C
in the iceꢀsalt bath, and then a solution of N-methylmorpholinium
tetrafluoroborate (18.90 g, 0.1 mol) in 20 mL of acetonitrile was added.
Stirring was continued until 2-chloro-4,6-di[2,2,2-trifluoroethoxy]-1,3,5-
triazine was not detected by TLC (about 4 h). The mixture was filtered,
and precipitate was washed with acetonitrile (4 ꢁ 20 mL). The combined
filtrates were concentrated to dryness. Crude oil was diluted with 5 mL of
acetonitrile and participated 100 mL of THF. After recrystallization of the
solid residue from the mixture acetonitrile/THF, 4-(4,6-di[2,2,2-
trifluoroethoxy]-1,3,5-triazyn-2-yl)-4-methylomorpholinium tetrafluoro-
borate was obtained (19.95 g; 43%) as a white crystalline solid which
decomposed when heated above 250 °C.
The increased steric hindrance of substrates always disturbed
coupling of amino acids. In the search for the universal remedy,
the contraction of the leaving group gave some effects and simple
acyl chlorides, and more reactive acyl fluorides supervene more
sophisticated approaches. Careful analysis of the couplings
involving triazine reagent strongly suggest a presence of the
additional factor controlling efficiency of coupling sterically
hindered substrates, besides the acidity of the leaving group.
These are the structure of the leaving group permitting formation
of a cyclic intermediate or a cyclic transition state and the absence
of solvents strongly solvating hydrogen of the acylated nucleo-
phile. It has to be considered as highly probable that, in the
case of activation of the carboxylic function by a triazine-based
coupling reagent triazine “superactive esters” which are able
to acylate amino components (and especially N-alkylated amio
components) via cyclic intermediates or cyclic transition state, in
the weakly solvating dichloromethane solution, this mechanism
of acylation is dominating. The consequence of arrangement of
both components into the cyclic intermediate or cyclic transition
state is their stabilization by accumulation of geminal (vicinal)
substituents, known as the ThorpeꢀIngold effect.26 This would
compensate (at least partially) retardation of the coupling
process caused by steric hindrance, and the excellent results with
Aib-Aib coupling evaluate the compensation as extensive to
consider it as the important factor in designing the structure of
coupling reagents.
1H NMR (250 MHz; CD3CN) δ = 3.43 (s, 3H); 3.74ꢀ3.83 (m, 4H);
4.00ꢀ4.05 (m, 2H); 4.46ꢀ4.51 (m, 2H); 5.07 (q, J3HꢀF = 8.4 Hz, 4H)
[ppm]. 13C NMR (175 MHz; CD3CN) δ = 53.6; 60.4; 61.5; 65.0; 122.6;
171.4; 172.6 [ppm]. MS: 377.12 [M ꢀ BF4]þ. Anal. Calcd for
C12H15BF10N4O3: C, 31.06%; H, 3.26%; B, 2.33%; F, 40.94%; N,
12.07%; O, 10.34%. Found: C, 31.84%; H, 3.68%.
2-Acyloxy-4,6-di[2,2,2-trifluoroethoxy]-1,3,5-triazine (5a,
b): Typical Procedure. The appropriate carboxylic acid (1 mmol)
and NMM (22 μL, 0.2 mmol) were added to a vigorously stirred
solution of 3b (0.464 g, 1 mmol) in THF (5 mL) and CH3CN (1 mL),
cooled to 0 °C. Stirring was continued until 4-(4,6-di[2,2,2-
trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoro-
borate was not detected by TLC (about 15 min). The mixture was
concentrated to dryness and the residue suspended in THF (5 mL) and
filtered. Filtrate was concentrated to dryness again. The residue was dried
under vacuum with P2O5 and KOH to constant weight affording neutral
products 5a or 5b.
4-Methoxybenzoic Acid 4,6-Di[2,2,2-trifluoroethoxy]-[1,
3,5]triazin-2-yl Ester (5a). The solution of 4-(4,6-di[2,2,2-trifl-
uoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate
(0.464 g, 1 mmol) and 4-methoxybenzoic acid (0.152 g, 1 mmol) in 5 mL
of THF and 1 mL of CH3CN was cooled to 0 °C in the iceꢀsalt bath, and
then NMM (22 μL, 0.2 mmol) was added. Stirring was continued until
4-(4,6-di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpho-
linium tetrafluoroborate was not detected by TLC (about 15 min). The
mixture was concentrated to dryness and the residue suspended in THF
(5 mL) and filtered. Filtrate was concentrated to dryness again. 4-Methox-
ybenzoic acid 4,6-di[2,2,2-trifluoroethoxy]-[1,3,5]triazin-2-yl ester was
obtained (yield 0.346 g; 81%) as oil.
’ GENERAL METHODS
NMR spectra were recorded on 250 or 700 MHz spectrometers using
the solvent peak as an internal reference. Multiplicities are indicated, s
(singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sept
(septet), m (multiplet); coupling constants (J) are in Hertz (Hz).
IR spectra were recorded as KBr pellets or film.
Analysis of experiments regarding dipeptide formation in solution
were carried out using a Vydac C-18 column, detection at 220 nm,
gradient from 10% to 50% B in 15 min, flow 1 mL/min; A: 0.1% TFA in
water, B: 0.1% TFA in mixture 90% acetonitrile and 10% water.
Experiments of efficiency during assembly of enkephalin analogues in
the solid phase were performed using a Waters SunFire C18 (3.5 μm, 4.6 ꢁ
100 mm) column, with detection at 220 nm. Solvents used were A: H2O/
0.045% TFA and B: CH3CN/0.036% TFA,with a flow of 1 mL/min.
Gas chromatography (GC)ꢀFID (H2/air), Split 1:50, column capil-
lary Chirasil-Val (25 m ꢁ 0.32 mm, thickness film 0.2 μm, carier gas
hel ꢀpressure 0.45 atm. Temperature program: 4 min in 90 °C, next
4 °C/min to 190 °C and 3 min in 190 °C.
2-Chloro-4,6-di[2,2,2-trifluoroethoxy]-1,3,5-triazine (2b).
Cyanuric chloride (92.2 g; 0.5 mol) was gradually added to the
vigorously stirred suspension of sodium bicarbonate (126 g; 1.5 mol)
in 2,2,2-trifluoroethanol (86.3 mL; 1.2 mol) diluted with chloroform
(40 mL) in such a rate to maintain the temperature of the reacting
mixture in the range 25ꢀ30 °C. Stirring was continued at 25ꢀ30 °C
until no more cyanuric chloride was detected (TLC; Rf = 0.85;
chloroform), then heated to gentle reflux until all intermediate product
was consumed (TLC; Rf = 0.72; chloroform). Then, the reacting mixture
was cooled to room temperature, further diluted with chloroform
(50 mL), and thoroughly washed with 1% aqueous NaHCO3 solution
(4 ꢁ 500 mL). The organic layer was dried with MgSO4, filtered, and
concentrated under reduced pressure. Crude oil was distilled under
reduced pressure using a column with a rotating band. Fraction
152ꢀ155 °C/27 mmHg. was collected yielding 2-chloro-4,6-di[2,2,2-
trifluoroethoxy]-1,3,5-triazine (2b) (81 g; 52% yield).
IR (film) = 1757 [cmꢀ1]. 1H NMR (700 MHz; CDCl3) δ = 3.90 (s,
3H, CH3ꢀOꢀPh); 4.87 (q, J3HꢀF = 7.9 Hz, 4H, CF3ꢀCH2ꢀO); 6.96
(d, J3HꢀH = 7 Hz, 2H, CHꢀCꢀOꢀCH3); 8.09 (d, J3HꢀH = 7 Hz, 2H,
CHꢀCꢀCO) [ppm]. 13C NMR (175 MHz; CDCl3) δ = 55.5; 64.2;
114.2; 119.4; 122.4; 133.0; 161.3; 165.0; 171.3; 172.7 [ppm].
2,2-Dimethylpropionic Acid 4,6-Di[2,2,2-Trifluoroethox-
y]-[1,3,5]triazin-2-yl Ester (5b). The solution of 4-(4,6-di[2,2,2-
trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoro-
borate (0.464 g, 1 mmol) and 2,2-dimethylpropionic acid (0.102 g,
1 mmol) in 5 mL of THF and 1 mL of CH3CN was cooled to 0 °C in
the iceꢀsalt bath, and then NMM (22 μL, 0.2 mmol) was added. Stirring
was continued until 4-(4,6-di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-
4-methylomorpholinium tetrafluoroborate was not detected by TLC
(about 15 min). The mixture was concentrated to dryness and the
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The Journal of Organic Chemistry
ARTICLE
residue suspended in THF (5 mL) and filtered. Filtrate was concentrated
to dryness again. 2,2-Dimethylpropionic acid 4,6-di[2,2,2-trifluoro-
ethoxy]-[1,3,5]triazin-2-yl ester was obtained (yield 0.275 g; 73%) as oil.
IR (film) = 1786 [cmꢀ1]. 1H NMR (250 MHz; CD3CN) δ = 1.36
(s, 9H); 4.86 (q, J3HꢀF = 7.9 Hz, 4H) [ppm]. 13C NMR (175 MHz;
CDCl3) δ = 26.4; 39.6; 62.2; 122.4; 171.5; 172.8; 173.5 [ppm].
Formation of the Peptide Bond in Solution, Synthesis
of Z-Aib-Aib-OMe. Typical Procedure. The solution of 4-(4,
6-di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tet-
rafluoroborate (0.464 g, 1 mmol) and Z-Aib-OH (0.237 g, 1 mmol) in
5 mL of THF was cooled to 0 °C in the iceꢀsalt bath, and then NMM
(30 μL, 0.27 mmol) was added. Stirring was continued until 4-(4,6-
di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tet-
rafluoroborate was not detected by TLC (about 20 min). After this time,
HPLC: tR = 11.42 min, purity = 82.4%. GC: tR = 7.37 min (D-Leu); tR =
7.97 (L-Leu) L/D = 98,24/1,76; tR = 4,57 (D-Val); tR = 7,76 (L-Val) L/D =
99.75/0.25. 1H NMR (250 MHz; CDCl3) δ = 0.89ꢀ0.96 (m, 12H); 1.44
(s, 9H); 1.49ꢀ1.53 (m, 1H); 1.61ꢀ1.64 (m, 3H); 3.74 (s, 3H); 4.03ꢀ4.16
(m, 1H); 4.51ꢀ4.56 (m, CH); 4.85ꢀ4.88 (m, 1H); 6.52ꢀ6.58 (m, 1H)
[ppm].
Boc-Pro-Phe-OMe. The solution of 4-(4,6-di[2,2,2-trifluoroethoxy]-
1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (0.464 g, 1
mmol) and Boc-Pro-OH (0.215 g, 1 mmol) in 5 mL of THF was cooled
to 0 °Cintheiceꢀsalt bath, and then NMM (30 μL, 0.27 mmol) was added.
Stirring was continued until 4-(4,6-di[2,2,2-trifluoroethoxy]-1,3,5-triazin-2-
yl)-4-methylomorpholinium tetrafluoroborate was not detected by TLC
(about 20 min). After this time H-Phe-OMe HCl (0.216 g, 1 mmol) and
3
NMM (110 μL, 1 mmol) were added. The reaction was left overnight at
room temperature. The mixture was concentrated to dryness and dissolved
in 30 mL of ethyl acetate and washed with water (2 ꢁ 15 mL), 1 NNaHSO4
(2 ꢁ 15 mL), water (2 ꢁ 15 mL), 1 N NaHCO3 (3 ꢁ 15 mL), water (3 ꢁ
15 mL), and brine (1 ꢁ 20 mL). The organic layer was dried with MgSO4,
filtered, and concentrated under reduced pressure. Boc-Pro-Phe-OMe was
obtained (354 mg; 94%) as oil. Lit.40 mp. 65ꢀ66 °C.
H-Aib-OMe HCl (0.154 g, 1 mmol) and NMM (110 μL, 1 mmol) were
3
added. The reaction was left overnight at room temperature. The mixture
was concentrated to dryness, and the residue was dissolved in 30 mL of
ethyl acetate and washed with water (2 ꢁ 15 mL), 1 N NaHSO4 (2 ꢁ
15 mL), water (2 ꢁ 15 mL), 1 N NaHCO3 (3 ꢁ 15 mL), water (3 ꢁ
15 mL), and brine (1 ꢁ 20 mL). The organic layer was dried with MgSO4,
filtered, and concentrated under reduced pressure. Z-Aib-Aib-OMe was
obtained (0.299 g; 89%) as a white crystalline solid, mp 98ꢀ100 °C, lit.37
mp 99ꢀ100 °C.
HPLC: tR = 10.09 min, purity = 96.4%. GC: tR = 10,91 min (L-Pro) L/D =
100; tR = 18.52 (D-Phe); tR = 19.36 (L-Phe) L/D = 99.79/0.21. 1H NMR
(250 MHz; CDCl3) δ = 1.43 (s, 9H); 1.50ꢀ1.80 (m, 2H); 1.79ꢀ2.10 (m,
2H); 2.97ꢀ3.23 (m, 2H); 3.29ꢀ3.32 (m, 2H); 3.72 (s, 3H); 4.13ꢀ4.34
(m, 1H); 4.72ꢀ4.93 (m, 1H); 6.40ꢀ6.52 (m, 1H); 7.08ꢀ7.31 (m, 5H)
[ppm].
HPLC: tR = 13.28 min, purity = 94.2%. 1H NMR (250 MHz; CDCl3)
δ = 1.49 (s, 12H); 3.76 (s, 3H); 5.08 (s, 2H); 5.22ꢀ5.27 (m, 1H);
6.86ꢀ6.91 (m, 1H); 7.28ꢀ7.36 (m, 5H) [ppm].
Z-Aib-Leu-OMe. The solution of 4-(4,6-di[2,2,2-trifluoroethoxy]-
1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (0.464 g,
1 mmol) and Z-Aib-OH (0.237 g, 1 mmol) in 5 mL of THF was cooled
to 0 °C in the iceꢀsalt bath, and then NMM (30 μL, 0.27 mmol) was
added. Stirring was continued until 4-(4,6-di[2,2,2-trifluoroethoxy]-
1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate was not
Fmoc-Phe-Ala-OMe. The solution of 4-(4,6-di[2,2,2-trifluoroeth-
oxy]-1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (0.464 g,
1 mmol) and Fmoc-Phe-OH (0.387 g, 1 mmol) in 5 mL of THF was
cooled to 0 °C in the iceꢀsalt bath, and then NMM (30 μL, 0.27 mmol)
was added. Stirring was continued until 4-(4,6-di[2,2,2-trifluoroethoxy]-
1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate was not de-
detected by TLC (about 20 min). After this time, H-Leu-OMe HCl
3
tected by TLC (about 15 min). After this time H-Ala-OMe HCl (0.140 g,
3
(0.181 g, 1 mmol) and NMM (110 μL, 1 mmol) were added. The
reaction was left overnight at room temperature. The mixture was
concentrated to dryness, and the residue was dissolved in 30 mL of ethyl
acetate and washed with water (2 ꢁ 15 mL), 1 N NaHSO4 (2 ꢁ 15 mL),
water (2 ꢁ 15 mL), 1 N NaHCO3 (3 ꢁ 15 mL), water (3 ꢁ 15 mL), and
brine (1 ꢁ 20 mL). The organic layer was dried with MgSO4, filtered,
and concentrated under reduced pressure. Z-Aib-Leu-OMe was ob-
tained (0.332 g; 91%) as a white crystalline solid which melted in the
range 81ꢀ84 °C, lit.20 mp 78ꢀ80 °C.
1 mmol) and NMM (110 μL, 1 mmol) were added. The reaction was left
overnight at room temperature. The mixture was concentrated to dryness
and dissolved in ethyl acetate (30 mL) and washed with water (2 ꢁ
15 mL), 1 N NaHSO4 (2 ꢁ 15 mL), water (2 ꢁ 15 mL), 1 N NaHCO3
(3 ꢁ 15 mL), water (3 ꢁ 15 mL), and brine (1 ꢁ 20 mL). The organic
layer was dried with MgSO4, filtered, and concentrated under reduced
pressure. Fmoc-Phe-Ala-OMe was obtained (0.463 g; 98%) as a white
crystalline solid, mp 172ꢀ175 °C, lit.38 mp 177ꢀ179 °C.
HPLC: tR = 14.13 min, purity = 97.1%. GC: tR = 3.36 min (D-Ala);
tR = 3.87 (L-Ala) L/D = 98,87/1,13; tR = 18.40 (D-Phe); tR = 18.95
(L-Phe) L/D = 99.47/0.53. 1H NMR (250 MHz, CDCl3) δ = 1.26 (d, J =
7.1, 3H), 2.98 (d, J = 7.0 Hz, 2H), 3.63 (s, 3H), 4.12 (t, J = 6.8 Hz, 1H),
4.25ꢀ4.45 (m, 4H, 2ꢁCH), 5.31 (s, 1H), 6.26 (s, 1H), 7.10ꢀ7.70
(m, 13H) [ppm].
HPLC: tR = 13.56 min, purity = 96.2%. GC: tR = 3.22 min (Aib); tR =
7.46 (D-Leu); tR = 8.11 (L-Leu) L/D = 99.23/0.77. 1H NMR (250 MHz;
CDCl3) δ = 0.91 (d, 6H, J3HꢀH = 6.5 Hz); 1.53ꢀ1.55 (s, 6H);
1.57ꢀ1.68 (m, 3H); 3.71 (s, 3H); 4.52ꢀ4.64 (t, 1H, J = 6 Hz);
5.09 (s, 2H); 5.32ꢀ5.35 (m, 1H); 6.69ꢀ6.77 (m, 1H); 7.33ꢀ7.36
(m, 5H) [ppm].
Boc-Leu-Val-OMe. The solution of 4-(4,6-di[2,2,2-trifluoroethoxy]-
1,3,5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate (0.464 g,
1 mmol) and Boc-Leu-OH (0.231 g, 1 mmol) in 5 mL of THF was cooled
to 0 °C in the iceꢀsalt bath, and then NMM (30 μL, 0.27 mmol) was
added. Stirring was continued until 4-(4,6-di[2,2,2-trifluoroethoxy]-1,3,
5-triazin-2-yl)-4-methylomorpholinium tetrafluoroborate was not detected
Synthesis of H-Phe-Leu-RinkAmide-AM-PS. Dipeptide H-Phe-
Leu-RinkAmide-AM-PS was manually assembled on Fmoc-Rink
Amide-aminomethyl-PS-resin (5 g, 0.59 mmolgꢀ1), after Fmoc removal
with piperidine in DMF (20%, 2 ꢁ 10 min). The resin was washed
with DMF (ꢁ 10), DCM (ꢁ 10), and DMF (ꢁ 10). Residues were
introduced after 30 min coupling, with preactivation of Fmoc-amino acids
(3 equiv) with HBTU (3 equiv) and DIPEA (6 equiv) in 30 mL
of DMF for 1 min. The resin was then washed with DMF (ꢁ 10),
DCM (ꢁ 10), and DMF (ꢁ 10) prior to the next cycle of deprotection/
coupling. Quantitative incorporation wascheckedat each stepbyuse of the
Kaiser test for primary amines. Sample cleavage (10 mg) with TFA/H2O
(95:5) confirmed the dipeptide in >99.5% purity, as analyzed by reversed-
phase HPLC and ESI-MS ([M þ H]þ = 278.21). The purity was checked
onreversed-phase HPLC, using a 0% to 100% linear gradient of A in B over
8 min, with detection at 220 nm. The tR of the dipeptide H-Phe-Leu-NH2
was 3.55 min.
by TLC (about 20 min). After this time H-Val-OMe HCl (0.168 g,
3
1 mmol) and NMM (110 μL, 1 mmol) were added. The reaction was left
overnight at room temperature. The mixture was concentrated to dryness,
and the residue was dissolved in 30 mL of ethyl acetate and washed with
water (2 ꢁ 15 mL), 1 N NaHSO4 (2 ꢁ 15 mL), water (2 ꢁ 15 mL), 1 N
NaHCO3 (3 ꢁ 15 mL), water (3 ꢁ 15 mL), and brine (1 ꢁ 20 mL). The
organic layer was dried with MgSO4, filtered, and concentrated under
reduced pressure. Boc-Leu-Val-OMe was obtained (0.320 g; 93%) as
a white crystalline solid which melted in the range 95ꢀ98 °C, lit.39 mp
125ꢀ126 °C.
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The Journal of Organic Chemistry
ARTICLE
General Procedure of the Synthesis of H-Tyr-Sar-Sar-Phe-
Leu-NH2 on the Solid Phase (7). H-Tyr-Sar-Sar-Phe-Leu-NH2 was
synthesized on the solid phase from H-Phe-Leu-RinkAmide-AM-PS
(0.1 g, 0.59 mmol gꢀ1). The resin was swelled with DMF (ꢁ 10) and
DCM (ꢁ 10), and the next step was started by washing with solvent
(ꢁ 5). Residues were introduced after using different coupling times
(see Table 3), with 1 min preactivation of Fmoc-amino acids (3 equiv)
with the corresponding coupling reagent (3 equiv) and base (6 equiv) in
0.6 mL of solvent. After coupling, resin was washed with DMF (ꢁ 10),
DCM (ꢁ 10), and DMF (ꢁ 5). The Fmoc residue was removed by
using 20% piperidine in DMF (2 ꢁ 10 min) and washed with DMF
(ꢁ 10) and DCM (ꢁ 10). The peptide chain was cleaved from the resin
with TFA/H2O (95:5) over 2 h at room temperature. The solution was
filtered, and the resin was washed with DCM (5 ꢁ 1 mL), which was
removed together with TFA under nitrogen flow. The crude peptide
was purified with cold Et2O (3 ꢁ 2 mL), and after lyophilization, purity
was checked on reversed-phase HPLC, using a 17% to 18% linear
gradient of A in B over 15 min.
General Procedure of Synthesis of H-Tyr-MeVal-MeVal-
Phe-Leu-NH2 on the Solid Phase (10). H-Tyr-MeVal-MeVal-Phe-
Leu-NH2 was assembled on the solid phase from H-Phe-Leu-RinkA-
mide-AM-PS (0.1 g, 0.59 mmol gꢀ1). The resin was swelled with DMF
(ꢁ 10) and DCM (ꢁ 10). The step was started from washing with
solvent (ꢁ 5). Residues were introduced after various coupling times
(see Table 5), with 1 min preactivation of Fmoc-amino acids (3 equiv)
with coupling reagent (3 equiv) and base (6 equiv) in 0.6 mL of solvent.
After coupling, resin was washed with DMF (ꢁ 10), DCM (ꢁ 10), and
DMF (ꢁ 5). Fmoc residue was removed by using 20% piperidine in
DMF (2 ꢁ 10 min) and washed with DMF (ꢁ 10) and DCM (ꢁ 10).
The peptide chain was cleaved from the resin with TFA/H2O (95:5)
over 2 h at room temperature. The solution was filtered, and the resin
was washed with DCM (5 ꢁ 1 mL), which was removed together with
TFA under nitrogen flow. The crude peptide was purified with cold Et2O
(3 ꢁ 2 mL), and after lyophilization, purity was checked on reversed-
phase HPLC, using a 15ꢀ35% linear gradient of A in B over 8 min.
The purity of H-Tyr-MeVal-MeVal-Phe-Leu-NH2 was determined
based on signal tR = 8.04 min. [M þ H]þ = 667.48. Des-MeVal tR = 6.41
min. [M þ H]þ = 554.38. Des-Tyr, tR = 7.51 min. [M þ H]þ = 504.36.
Des-2 MeVal tR = 4.90 min. [M þ H]þ = 441.31. Des-TyrMeVal tR =
4.11 min. [M þ H]þ = 391.30.
The purity of H-Tyr-Sar-Sar-Phe-Leu-NH2 was determined based on signal
tR =9.35min,[Mþ H]þ = 583.36. Des-Sar tR =8.72min,[Mþ H] = 512.90.
General Procedure of the Synthesis of H-Tyr-Aib-Aib-Phe-
Leu-NH2 on the Solid Phase (8). H-Tyr-Aib-Aib-Phe-Leu-NH2 was
assembled on the solid phase from H-Phe-Leu-RinkAmide-AM-PS (0.1 g,
0.59 mmolꢀ1g). The resin was swelled with DMF (ꢁ 10) and DCM (ꢁ
10). The step was started from washing with solvent (ꢁ 5). Residues were
introduced after different coupling times (see Table 4), with 1 min
preactivation of Fmoc-amino acids (3 equiv) with coupling reagent
(3 equiv) and base (6 equiv) in 0.6 mL of solvent. After coupling, resin
was washed with DMF (ꢁ 10), DCM (ꢁ 10), and DMF (ꢁ 5).
The Fmoc residue was removed by using 20% piperidine in DMF (2 ꢁ
10 min) and washed with DMF (ꢁ 10) and DCM (ꢁ 10). The peptide
chain was cleaved from the resin with TFA/H2O (95:5) over 2 h at room
temperature. The solution was filtered, and the resin was washed with
DCM (5 ꢁ 1 mL), which was removed together with TFA under nitrogen
flow. The crude peptide was purified with cold Et2O (3 ꢁ 2 mL), and after
lyophilization, purity was checked on reversed-phase HPLC, using a
15ꢀ35% linear gradient of A in B over 8 min.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures, char-
b
acterization data, and copies of 1Hand13C NMR and MS spectra for
new compounds, and copies of HPLC and GC chromatograms.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ 48-42 631 31 51. Fax: þ 48 42 636 55 30. E-mail:
zbigniew.kaminski@p.lodz.pl (Z. J. K.) Phone: þ 34 93 403 70
88. Fax: þ 34 93 403 71 26. E-mail: fernando.albericio@
irbbarcelona.org.
The purity of H-Tyr-Aib-Aib-Phe-Leu-NH2 was determined based
on signal tR = 7.30 min, [M þ H]þ = 611.40. Des-Aib tR = 7.39 min,
[M þ H]þ = 526.31. Des-Tyr tR = 5.74 min, [M þ H]þ = 448.29. Des-
2Aib tR = 4.75 min, [M þ H]þ = 441.32. Des-TyrAib tR = 4.12 min,
[M þ H]þ = 363.30.
’ ACKNOWLEDGMENT
General Procedure of the Synthesis of H-Tyr-MeLeu-Me-
Leu-Phe-Leu-NH2 on the Solid Phase (9). H-Tyr-MeLeu-MeLeu-
Phe-Leu-NH2 was elongated on the solid phase from H-Phe-Leu-
RinkAmide-AM-PS (0.1 g, 0.59 mmol gꢀ1). The resin was swelled with
DMF (ꢁ 10) and DCM (ꢁ 10). The step was started from wash with
solvent (ꢁ 5). Residues were introduced by means of 5 min couplings
and various preactivation times (see Table 5) of Fmoc-amino acids
(3 equiv) with coupling reagent (3 equiv) and base (6 equiv) in 0.6 mL
of DMF. After coupling, resin was washed with DMF (ꢁ 10), DCM
(ꢁ 10), and DMF (ꢁ 5). Fmoc residue was removed by using 20%
piperidine in DMF (2 ꢁ 10 min) and washed with DMF (ꢁ 10) and
DCM (ꢁ 10). The peptide chain was cleaved from the resin with TFA/
H2O (95:5) over 2 h at room temperature. The solution was filtered, and
the resin was washed with DCM (5 ꢁ 1 mL), which was removed
together with TFA under nitrogen flow. The crude peptide was purified
with cold Et2O (3 ꢁ 2 mL), and after lyophilization, purity was checked
on reversed-phase HPLC, using a 15ꢀ50% linear gradient of A in B
over 8 min.
Financial support of these studies from the Technical Uni-
versity of Lodz is gratefully acknowledged. The work in Barce-
lona was partially supported by CICYT (CTQ2009-07758), The
Generalitat de Catalunya (2009SGR 1024), the Institute for
Research in Biomedicine, and the Barcelona Science Park.
’ REFERENCES
(1) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243.
(2) Albericio, F.; Chinchilla, R.; Dodsworth, D. J.; Najera, C. Org.
Prep. Proced. Int. 2001, 33, 203.
(3) Han, S.-Y.; Kim, Y.-A. Tetrahedron 2004, 60, 2447.
(4) Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61, 10827.
(5) Valeur, E.; Bradley, M. Chem. Soc. Rev. 2009, 38, 606.
(6) Bacsa, B.; Horvati, K.; Bosze, S.; Andreae, F.; Kappe, C. O. J. Org.
Chem. 2008, 73, 7532.
(7) Sewald, N. Angew. Chem., Int. Ed. 2002, 41, 4661.
(8) Nishiuchi, Y.; Nishio, H.; Ishimaru, M.; Kimura, T. Int. J. Pept.
Res. Ther. 2007, 13, 119.
Purity of H-Tyr-MeLeu-MeLeu-Phe-Leu-NH2 was determined based
on signal tR = 7.93 min, [M þ H]þ = 695.46. Des-MeLeu tR = 6.19 min,
[M þ H]þ = 568.36. Des-Tyr tR = 6.83 min, [M þ H]þ = 532.40. Des-
TyrMeLeu tR = 4.56 min, [M þ H]þ = 405.29.
(9) Sabatino, G.; Mulinacci, B.; Alcaro, M. C.; Chelli, M.; Rovero, P.;
Papini, A. M. Lett. Pept. Sci. 2002, 9, 119.
(10) Santagada, V.; Fiorino, F.; Perissutti, E.; Severino, B.; De Filippis,
V.; Vivenzio, B.; Caliendo, G. Tetrahedron Lett. 2001, 42, 5171.
4512
dx.doi.org/10.1021/jo2002038 |J. Org. Chem. 2011, 76, 4506–4513

The Journal of Organic Chemistry
ARTICLE
(11) Wehrstedt, K. D.; Wandrey, P. A.; Heitkamp, D. J. Hazard.
Mater. 2005, 126, 1.
(12) El-Faham, A.; Funosas, R. S.; Prohens, R.; Albericio, F. Chem.—
Eur. J. 2009, 15, 9404.
(13) Subiros-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.;
Albericio, F. Chem.—Eur. J. 2009, 15, 9394.
(14) El-Faham, A.; Albericio, F. Org. Lett. 2007, 9, 4475.
(15) El-Faham, A.; Albericio, F. J. Org. Chem. 2008, 73, 2731.
(16) Dutta, A. S.; Morley, J. S. J. Chem. Soc. (C) 1971, 2896.
(17) Albericio, F.; Bailen, M. A.; Chinchilla, R.; Dodsworth, D. J.;
Najera, C. Tetrahedron 2001, 57, 9607.
(18) Garner, P.; Sesenoglu, O.; Kaniskan, H. U. Tetrahedron Lett.
2006, 47, 483.
(19) Kamiꢀnski, Z. J. Biopolymers 2000, 55, 140.
(20) Kamiꢀnski, Z. J.; Kolesiꢀnska, B.; Kolesiꢀnska, J.; Sabatino, G.;
Chelli, M.; Rovero, P.; Bzaszczyk, M.; Gzꢀowka, M. L.; Papini, A. M. J. Am.
Chem. Soc. 2005, 127, 16912.
(21) Kolesiꢀnska, B.; Kamiꢀnski, Z. J. Org. Lett. 2009, 11, 765.
(22) Kolesiꢀnska, B.; Kasperowicz, K.; Sochacki, M.; Mazur, A.; Jankowski,
S.; Kamiꢀnski, Z. J. Tetrahedron Lett. 2010, 51, 20.
(23) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(24) Gzowka, M. L.; Iwanicka, I.; Kaminski, Z. J. Acta Crystallogr.
1990, C46, 2211.
(25) Gzꢀowka, M. L.; Kamiꢀnski, Z. J.; Sobaꢀnska, A. W. Pol. J. Chem.
1994, 68, 1731.
(26) Jung, M. E.; Piizzi, G. Chem. Rev. 2005, 105, 1735.
(27) Bruice, T. C.; Pandit, U. K. J. Am. Chem. Soc. 1960, 82, 5858.
(28) Bruice, T. C.; Pandit, U. K. Proc. Natl. Acad. Sci. U.S.A. 1960,
46, 402.
(29) Jung, M. E.; Nichols, C. J. Tetrahedron Lett. 1996, 37, 7667.
(30) Jung, M. E. Synlett 1999, 843.
(31) Page, M. J.; Jencks, W. P. Proc. Natl. Acad. Sci. U.S.A. 1971, 68,
1678.
(32) Kamiꢀnski, Z. J.; Paneth, P.; O’Leary, M. J. Org. Chem. 1991, 56,
5716.
(33) Kaminski, Z. J.; Kolesiꢀnska, B.; Kolesiꢀnska, J.; Jastrza-bek, K.;
Papini, A.M.l Bianchini, R.; Sabatino, G. Patent WO/2007/051496 from
2007. 05. 10.
(34) Raw, S. A. Tetrahedron Lett. 2009, 50, 946.
(35) Carpino, L. A.; El-Faham, A. Tetrahedron 1999, 55, 6813.
(36) Subiros-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.;
Albericio, F. Chem.—Eur. J. 2009, 15, 9394.
(37) Li, P.; Xu, J. C. Tetrahedron Lett. 1999, 40, 8301–8304.
(38) Kolesinska, B.; Kaminski, Z. J. Org. Lett. 2009, 11, 765–768.
(39) Luebke, K.; Schroeder, E. Ann. Chem. 1963, 665, 205–212.
(40) Mohapatra, D. K.; Datta, A. J. Org. Chem. 1999, 64, 6879–6880.
4513
dx.doi.org/10.1021/jo2002038 |J. Org. Chem. 2011, 76, 4506–4513