The effect of counterion and tertiary amine on the efficiency of N-triazinylammonium sulfonates in solution and solid-phase peptide synthesis

Article abstract of DOI:10.1002/ejoc.201402862

A collection of N-triazinylammonium sulfonates, designed according to the concept of "superactive esters", was obtained by treatment of ammonium sulfonates with 2-chloro-4,6-dimethoxy-1,3,5-triazine. The structure of the tertiary amine as well as sulfonate anion influenced their reactivity and stability in N,N-dimethylformamide (DMF) solution. The reagents were successfully used in solution- and solid-phase synthesis of Z-, Boc-, and Fmoc-protected peptides containing natural and unnatural sterically hindered amino acids as well as in [2+1] fragment condensation approaches, yielding the final products in 80-100% yield and high optical purity. In manual SPPS of the [Aib]²[Aib]⁴-enkephalin analogue and the ACP(65-74) peptide fragment VQAAIDYINEG, several sulfonates yielded peptides significantly faster than TBTU or HATU. Comparative analyses demonstrated that 4-(4,6-di-methoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 4-toluenesulfonate was the most versatile reagent in a wide range of coupling procedures.

Full text of DOI:10.1002/ejoc.201402862

FULL PAPER
DOI: 10.1002/ejoc.201402862
The Effect of Counterion and Tertiary Amine on the Efficiency of N-
Triazinylammonium Sulfonates in Solution and Solid-Phase Peptide Synthesis
[
a]
[a]
[a]
[a]
Beata Kolesinska,* Kamil K. Rozniakowski, Justyna Fraczyk, Inga Relich,
Anna Maria Papini,[ and Zbigniew J. Kaminski
b,c]
[a]
Keywords: Synthetic methods / Coupling reagents / Peptides / Amino acids / Esters
A collection of N-triazinylammonium sulfonates, designed
well as in [2+1] fragment condensation approaches, yielding
according to the concept of “superactive esters”, was ob- the final products in 80–100% yield and high optical purity.
2
4
tained by treatment of ammonium sulfonates with 2-chloro-
,6-dimethoxy-1,3,5-triazine. The structure of the tertiary
In manual SPPS of the [Aib] [Aib] -enkephalin analogue and
the ACP(65–74) peptide fragment VQAAIDYINEG, several
sulfonates yielded peptides significantly faster than TBTU or
HATU. Comparative analyses demonstrated that 4-(4,6-di-
4
amine as well as sulfonate anion influenced their reactivity
and stability in N,N-dimethylformamide (DMF) solution. The
reagents were successfully used in solution- and solid-phase
synthesis of Z-, Boc-, and Fmoc-protected peptides contain-
ing natural and unnatural sterically hindered amino acids as
methoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
enesulfonate was the most versatile reagent in a wide range
of coupling procedures.
4-tolu-
ters”)[3] as acylating intermediates and confirmed the en-
hanced propensity of the triazinyl moiety as a leaving group
in the process of amine acylation. This characteristic was
attributed to the energetically favoured isomerisation of 2-
hydroxy-1,3,5-triazine 3 to the appropriate triazinone 4
Introduction
The increasing pool of naturally occurring peptides bear-
ing noncoded amino acid residues, peptides decorated with
additional structural elements, as well as benefits attended
by incorporation of unnatural building blocks into native
peptide chains, provide strong motivation for the systematic
search for new coupling reagents and more efficient pro-
[
4]
(Figure 1).
In this way, the triazinyl “superactive ester” 1 activation
“push” was amplified by the “pull” caused by additional
[
1]
cedures. To overcome problems caused by the instability
and poor reactivity of some building blocks and to improve
the process of peptide synthesis, we developed an innovative
coupling strategy, avoiding over-activation. This novel strat-
egy was achieved by introducing an additional, synchro-
nous, and thermodynamically highly favoured process into
the synthetic pathway leading to peptide (i.e., amide) bond
formation. Thus, this approach prevents (or at least re-
duces) side reactions and racemisation at the stereogenic
centre brought about by strong activation of the carboxylic
function. The previously[ developed triazine-based cou-
pling reagents (TBCRs) display the structural features we
postulated for this novel concept.[ Previous studies proved
the efficacy of 2-acyloxy-1,3,5-triazines 1 (“superactive es-
stabilisation of the side-product 3 by its transformation into
thermodynamically more stable 4. This was expected to give
better synthetic results than those obtained solely due to
more powerful activation. The TBCRs that were designed
according to this concept were demonstrated to be useful
[
5,6,7]
[8]
in the synthesis of amides,
carboxylic acids, in peptide and glycopeptide synthesis by
conventional and microwave-assisted strategies,
esters, and anhydrides of
[
9]
[
6,10]
in the
synthesis of seleno- and thiono-phosphoranes analogues of
[
11]
nucleic acid,
and in many other synthetic procedures.
2]
The modular structure of TBCRs allows extensive modifi-
cation of their structure to fine-tuning their properties in
user-friendly forms, at a reasonably low cost and low haz-
ard levels. There are three ways to modulate the properties
of triazine-based condensing reagents encompassing: incor-
poration of substituents on the triazine ring, structurally
diversification of the ammonium fragments, and modifica-
tion of the counterion structures.
3]
[
[
[
a] Institute of Organic Chemistry,Technical University of Lodz,
9
0-924 Lodz, Poland
E-mail: beata.kolesinska@p.lodz.pl
www.chorg.p.lodz.pl
b] French-Italian Laboratory of Peptide and Protein Chemistry
and Biology, Dipartimento di Chimica “Ugo Schiff”,
Universita di Firenze,
Thus, introduction of substituents on the triazine ring
such as highly fluorinated hydrocarbon side chains led to
5
0019 Sesto Fiorentino, Firenze, Italy
c] Laboratoire SOSCO-PeptLab@UCP EA4505, University of fluorinated coupling reagents designed for peptide synthesis
Cergy-Pontoise,
5
[12]
in fluorophorous media. Moreover, hydrophilic triazines
Mail Gay Lussac, 95031 Cergy-Pontoise Cedex, France
were successfully used for peptide bond formation in aque-
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402862.
[
13]
ous solution, whereas the introduction of a benzyl group
Eur. J. Org. Chem. 2015, 401–408
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
401

B. Kolesinska et al.
FULL PAPER
Figure 1. Peptide bond formation by using triazine-based coupling reagents is accompanied by energetically favoured isomerisation of 2-
hydroxy-1,3,5-triazine 3 into the more stable triazinone 4.
into the triazine ring enhanced reactivity in the case of but nonpolarisable 10-camphorsulfonic acid (2b), the hy-
SPPS on polystyrene resins.[ The advantageous properties drophilic nonpolarisable methanesulfonic acid (2c), and the
of triazines have been confirmed by designing and syn- extremely acidic, non-nucleophilic trifluoromethanesulfonic
thesising N-methyl-N-triazinylmorpholinium tetrafluoro- acid (2d) (see Scheme 1, Figure 2).
borate, which outperformed N-hydroxybenzotriazole-based
14]
reagents in terms of reactivity and stability in a broad range
of possible applications.[
15]
Further structural improve-
ments based on replacing methoxyl groups of the triazine
ring with 2,2,2-trifluoroethoxyl substituents substantially
enhanced the efficiency of new N-triazinylmorpholinium
tetrafluoroborate analogues in the synthesis of extremely
difficult sequences, such as MeVal-MeVal and MeLeu-Me-
Leu, in the enkephalin analogues TyrMeValMeValPheLeu
and TyrMeLeuMeLeuPheLeu, making them much more
efficient than the classic benzotriazole-based coupling rea-
[
16]
gents
and comparable only with Oxyma-based rea-
Scheme 1. Synthesis of N-(4,6-dimethoxy-1,3,5-triazin-2-yl)-N,N,N-
trisubstituted ammonium sulfonates 5a–l.
[
17]
gents.
With these considerations in mind, we focused our atten-
tion on increasing the versatility of N-triazinylammonium
reagents by replacing the small, nonpolarisable tetrafluoro-
borate anion or hexafluorophosphate with the more lipo-
philic alkyl(aryl)sulfonate counterion. Most sulfonate
anions are stable, inexpensive, environmentally friendly, and
The new TBCRs 5a–l were obtained in 55–99% yield as
white solids isolated after crystallisation in acetonitrile/di-
ethyl ether or as oils, and used in coupling reactions without
additional purification (see Table 1).
As expected, in all cases the activation of 4-methoxy-
benzoic acid (6) with 5a–l gave the triazine “superactive es-
ter” 7, which was isolated in 64–98.5% yield (see Scheme 2
and Table 2). Its structure was confirmed by comparison
with an authentic sample obtained by using the classical
procedure and by the presence of the characteristic IR band
readily available in a broad range of structural forms. In
our preliminary report,[18] it was confirmed that a wide col-
lection of N-triazinylammonium sulfonates were easily af-
fordable. Herein, we present a systematic study in order to
establish how modifications of the tertiary amine compo-
nent and counterion could influence the stability, versatility
and coupling efficiency of this family of coupling reagents.
This proposition was additionally supported by the pre-
–1
at 1750–1780 cm .
To determine the compatibility of the new TBCRs 5a–l
in solution and solid-phase peptide synthesis, both by man-
ual and automatic strategies, we evaluated their solubility
viously reported influence of the counterion on the stability
of coupling reagents[
19a]
as well as disruption of the peptide
1
chain conformation in the presence of the ions.[
19b]
and stability by HPLC and H NMR techniques.
As expected, it was found that all the sulfonates 5a–l,
both solid and oil compounds, were stable enough to be
stored at low temperature for at least six months, and,
Results and Discussion
–
moreover, in the case of DMT/NMM/TsO (5a) NMR stud-
The reagents 5a–l were obtained by treatment of 2- ies after one year of storage at 4 °C confirmed their sta-
chloro-4,6-dimethoxy-1,3,5-triazine (1) with sulfonates of bility. However, their stability in N,N-dimethylformamide
the tertiary amines 4a–l in the presence of sodium hydrogen (DMF) solution varied at room temperature (see Tables 3
[
20]
carbonate.
and 4).
Taking advantage of the modular structure of triazine
The most stable compounds were the most lipophilic N-
reagents, 5a–l were prepared starting from the weakly basic triazinylammonium 4-toluenesulfonates 5a,e,i and 10-cam-
N-methylmorpholine (3a), as well as the more basic N- phorosulfonates 5b,f,j, and their stability was comparable to
methylpyrrolidine (3b) and N-methylpiperidine (3c). As that of conventional coupling agents such as TBTU, HBTU
counterions, we selected the moderately lipophilic and and COMU. On the other hand, N-triazinylammonium
highly polarisable 4-toluenesulfonic acid (2a), the lipophilic methanesulfonates 5c,g,k were less stable, which restricts
402
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 401–408

N-Triazinylammonium Sulfonates in Peptide Synthesis
Figure 2. The new collection of TBCRs, sulfonates of the N-triazinylammonium salts 5a–l.
Table 1. Characteristics of sulfonates 5a–l.
Table 2. Activation time and yield in the synthesis of 4,6-dimeth-
oxy-1,3,5-triazin-2-yl 4-methoxybenzoate (7) using the new TBCRs
Entry Sulfonates of
Yield
[%]
M.p.
[°C]
– [15]
5a–l and DMT/NMM/BF
4
.
N-triazinylammonium salts 5
Entry Coupling reagent
Activation time [min]
Yield [%]
1
2
3
4
5
6
7
8
9
1
1
1
DMT/NMM/TsO–
5a
5b
5c
5d
5e
5f
5g
5h
5i
72
96
55
86
99
86
87
98
99
84
84
94
57–61
127–129
96–98
112–114
oil
110–115
oil
85–90
81–85
oil
DMT/NMM/CsO–
1
2
3
4
5
6
7
8
DMT/NMM/TsO–
DMT/NMM/CsO–
5a
5b
5c
5d
30
30
30
30
30
30
45
30
30
45
30
30
90
98.5
76.1
97.0
90.0
98.0
79.2
80.7
78.6
94.0
87.2
90.0
64.0
86
–
DMT/NMM/MsO
DMT/NMM/TfO–
DMT/NMPip/TsO–
DMT/NMPip/CsO–
DMT/NMPip/MsO
DMT/NMPip/TfO–
DMT/NMPyr/TsO–
DMT/NMPyr/CsO–
–
DMT/NMM/MsO
DMT/NMM/TfO–
DMT/NMPip/TsO– 5e
DMT/NMPip/CsO– 5f
–
–
DMT/NMPip/MsO 5g
DMT/NMPip/TfO– 5h
–
0
1
2
5j
5k
5l
9
10
11
DMT/NMPyr/TsO
5i
–
DMT/NMPyr/CsO– 5j
DMT/NMPyr/MsO
oil
85–90
DMT/NMPyr/TfO–
DMT/NMPyr/MsO 5k
–
–
1
2
DMT/NMPyr/TfO
DMT/NMM/BF
4
5l
–
13
derived from N-hydroxybenzotriazole in the presence of a
stoichiometric amount of DIPEA, the stability decreased
with time, but not for COMU for which the degradation
products were not observed under the same conditions.
These observations have a practical implication both for so-
lid-phase and solution strategies involving extremely poorly
reactive carboxylic components, and for cyclisation steps
proceeding slowly under high dilution conditions.
Scheme 2. Synthesis of triazine “superactive ester” 7 from 4-meth-
oxybenzoic acid (6) and TBCRs 5a–l.
their possible use in automatic SPPS. As expected, stability
The effects of limited stability were, however, barely vis-
of 5a–l in DMF solution further decreased in the presence ible in the case of coupling of sterically hindered Z-Aib-
of a stoichiometric amount of DIPEA. In solution, in the OH, with H-Aib-OMe proceeding in dichloromethane
presence of a base (Table 4), the most stable compounds (Table 5). Under such conditions, a 65–89% yield was ob-
were again N-triazinylammonium 4-toluenesulfonates 5a,e,i tained even with the less stable 5b,c and 5g. The most pure
and trifluoromethanesulfonates 5d,h,l but not 10-camphor- products were obtained with reagents derived from the
sulfonates 5b,f,j or N-triazinylammonium methanesulfon- weakly basic N-methylmorpholine 5a–d and 5l, although
ates 5c,g,k. In the case of the classical coupling reagents prolonged coupling time was required in the latter case.
Eur. J. Org. Chem. 2015, 401–408
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
403

B. Kolesinska et al.
FULL PAPER
Table 3. Stability of the sulfonates 5a–l, DMT/NMM/BF
TBTU, HBTU and COMU in DMF solution.
4
–,[15]
Table 5. The synthesis of Z-Aib-Aib-OMe (8a–l) by using TBCRs
–
[15]
5a–l, DMT/NMM/BF
4
,
TBTU and COMU.
Coupling
reagent
Stability [%][a]
1 h 6 h
Entry Dipeptide Coupling reagent
Activation
time [h]
Yield
[%]
Purity
[%]
12 h 1 d
2 d
4 d
7 d
8
5
a
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
1
2
3
4
5
6
7
8
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
1
1
1
1
1
1
1
3
1
1
0.5
3
1
1
83
69
65
71
77
77
89
79
72
51
72
97
75.5
95.8
82
98.8
90.1
93.3
85.9
79.3
81.4
82.2
80.8
77.1
73.7
82.5
86.5
96.8
81.2
94.2
5
b
5
c
Ͼ 99 12
–
–
–
–
–
5
d
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
5
e
Ͼ 99 Ͼ 99 99
Ͼ 99 Ͼ 99 Ͼ 99 98
52 21 2.5
Ͼ 99 Ͼ 99 Ͼ 99 99
Ͼ 99 99 99 99
98
98
98
–
98
98
98
97
–
97
98
97
97
–
96
98
5
f
5
g
–
5
h
5
i
9
5
j
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
10
11
12
13
14
15
5k
Ͼ 99 Ͼ 99 86
Ͼ 99 Ͼ 99 Ͼ 99 99
85
74
99
61
99
60
99
5
l
TBTU
HBTU
COMU
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99 Ͼ 99
TBTU
COMU
DMT/NMM/BF
8
8
–
4
2.5
–
DMT/NMM/BF
4
100
Ͼ99 Ͼ99 Ͼ99 Ͼ98
–
–
[
a] Stability studies were performed by HPLC (Aqua C-18) column
analysis of aliquots from stock solutions (0.25 m) of the various ates 5a–l to promote chirality retention. The activation of
coupling reagents in DMF at indicated times, yields were calculated
the epimerisation-prone C-terminal Phe provided a suitable
according to the integration of the peak area at 220 and 254 nm.
scenario for evaluating the performance of the new cou-
pling reagents described herein, in peptide fragment con-
densation strategy (Table 6).
Table 4. Stability of sulfonates 5a–l, DMT/NMM/BF –,[15] TBTU,
4
HBTU, and COMU in DMF in the presence of a stoichiometric
amount of DIPEA.
Table 6. Activation time, yield, purity and epimerisation in the syn-
thesis of Z-Ala-Phe-Leu-OBz (9a–l) ([2+1] fragment condensation
strategy) using TBCRs sulfonates 5a–l.
Coupling
reagent
Stability [%][a]
5 min
30 min 1 h
2 h
3 h
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
97
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ99
Ͼ 99
66
Ͼ 99
Ͼ 99
Ͼ 99
Ͼ 99
88
Ͼ 99
99
99
98
99
Ͼ 95
Ͼ 99
Ͼ 99
Ͼ99
Ͼ 99
–
15
Ͼ 99
–
11
98
Ͼ 99
99
35
99
99
99
93
99
Ͼ 90
Ͼ80
Ͼ 99
Ͼ99
Ͼ 99
–
–
98
Ͼ 99
99
35
99
99
99
93
99
Ͼ 80
Ͼ 70
Ͼ 99
Ͼ99
Tri-
Coupling Activation Yield Purity Extent of epimerisation
peptide reagent
9
time
[h]
[%]
[%]
2 1 2 3
H N–X –X –X –COOH
99
[%L]/[%D]
Ͼ 99
Ͼ 99
37
99
99
99
98
99
Ͼ 90
Ͼ 90
Ͼ 99
Ͼ99
X
1
X
2
X
3
9
a
5a
5b
5c
5d
5e
5f
5g
5h
5i
1
2.5
1
88
63
99.5
97.3
97.4
95.5
99.0
98.7
89.4
95.2
99.1
99.3
97.8
90.1
100:0 100:0 99:1
100:0 100:0 100:0
100:0 100:0 100:0
100:0 100:0 100:0
9
b
9
c
74.4
72.5
78.6
53.4
81.5
80.2
81.6
75.3
71.2
77.3
9
d
1
j
k
l
9
e
1.5
1.5
1.5
3.5
1
1
0.5
3.5
98:2
100:0 100:0 95:5
99:1 100:0 99:1
100:0 97:3 99:1
99:1 100:0 100:0
98:2
100:0
9
f
9
g
TBTU
9
h
HBTU
COMU
DMT/NMM/BF
9
i
9
j
5j
5k
5l
100:0 100:0 100:0
100:0 100:0 100:0
–
4
9
k
[a] See footnote in Table 3.
9l
100:0 95:5
98:2
Comparative studies on efficiency of other representative
It could be expected that in this type of coupling, the
condensing reagents have shown that the application of nature of the base and the rate of coupling would play a
COMU allows the final peptide to be obtained with com- crucial role in preserving the chirality of the activated di-
parable yield, but with moderate purity. On the other hand, peptide starting material and the amino component. Im-
in the case of TBTU, Z-Aib-Aib-OMe was obtained with portantly, the extent of racemisation of the Phe residue de-
moderate yield, but high purity.
termined by GC on a ChirasilVal capillary column after
The loss of chiral integrity is more severe during the acti- hydrolytic degradation of the tripeptide 9 into amino acids,
vation of peptide fragments (even at the dipeptide stage) was maintained in all cases below 0.1% using all the
due to the open pathway leading to the formation of the TBCRs 5a–d, derived from the weakly basic N-methyl-
epimerisation-prone oxazolone intermediate. Therefore, the morpholine.
ability of reagents 5a–l to preserve the enantiomeric homo-
On the other hand, prolonged coupling time was ac-
geneity in fragment condensation was carefully examined. companied by partial epimerisation on all three stereogenic
The [2+1] coupling strategy in the synthesis of Z-Ala-Phe- centres in the case of all N-methylpiperidine TBCRs 5e–h.
Leu-OBz (9) was selected as a suitable peptide model to High levels of inversion of Phe and Leu configurations were
highlight the relative ability of the different TBCRs sulfon- also observed in the case of the TBCR 5l, which was pre-
404
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 401–408

N-Triazinylammonium Sulfonates in Peptide Synthesis
Table 7. Manual SPPS of the (65–74) ACP fragment 10 on 2-chlorotrityl resin using TBCRs 5a–l and DMT/NMM/BF
4
–:[15] single
coupling and a threefold excess of acylating reagents were used at all stages of the synthesis.
Coupling
reagent
Condensation time [min]
Yield
(purity)
[%]
1
2
3
4
5
6
7
8
9
10
2
H N–V –Q –A -A –I –D -Y –I –N -G –2-chlorotrityl resin
1
2
3
4
5
6
Y7
I8
N9
V
Q
A
A
I
D
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
15
45
30
30
30
60
15
30
15
60
15
30
15
15
45
30
30
30
60
15
30
15
60
15
30
15
15
45
30
30
30
60
15
30
15
60
15
30
15
15
45
30
30
30
60
15
30
15
60
15
30
15
15
45
30
30
30
60
15
30
15
60
15
30
15
15
45
30
30
30
60
15
20
15
60
15
30
15
15
45
30
30
30
60
15
30
15
60
15
30
15
15
45
30
30
30
60
15
30
15
60
30
45
15
30
120
30
45
45
90
30
45
30
90
30
60
15
97
66
82
94
92
67
82
72
83
65
89
84
84
j
k
l
DMT/NMM/BF
4
pared from the relatively basic N-methylpyrrolidine and the from methanesulfonic acid and the relatively basic N-meth-
strongly acidic trifluoromethanesulfonic acid 2d. Interest- ylpyrrolidine and N-methylpiperidine, respectively. The
ingly, TBCRs 5h and 5l, prepared from 2d, were substan- highest yields (94–99%) were afforded by the coupling rea-
tially less reactive also in the tests involving coupling of gents 5a–c, prepared from the less basic N-methylmorph-
sterically hindered substrates (Table 5). As expected, epi- oline (see Table 8). The latter results do not correlate with
merisation in Leu, used as amino component, was observed the stability tests (see Table 3 and Table 4) in the case of
only when a prolonged coupling time was required.
5b,c. However, these results are in compliance with the
The next assay used to verify the usefulness of TBCRs highest purity obtained in the case of the solution-phase
5a–l in peptide bond formation was based on SPPS of the synthesis of Z-AibAib-OMe. To explain this surprisingly
(65–74) ACP fragment (10) starting from Fmoc-Gly-2- poor correlation, it has to be taken into consideration that
chlorotrityl resin. after a short preactivation time, the unstable TBCRs 5b,c
To emphasise the differences in terms of reactivity, we were transformed in a reaction with Fmoc-Aib-OH into the
used a threefold excess of the acylating mixture in a single substantially more stable 4,6-dimethoxy-1,3,5-triazin-2-yl
coupling procedure. The progress of all coupling reactions esters of appropriate N-protected amino acids. Thus, by
[21]
were monitored by Kaiser tests. In particular, diverse re- using a freshly prepared DMF solution of N-triazinylam-
activity of TBCRs 5a–l in terms of time needed to complete monium sulfonates, acceptable synthetic results could be
each coupling step was observed. In fact, in the case of obtained even in the case of the less stable TBCRs.
TBCR 5a, the coupling reactions of each amino component
Table 8. Synthesis of the enkephalin analogue H
Phe-Leu-COOH (11a–l) with TBCR sulfonates 5a–l.
2
N-Tyr-Aib-Aib-
proceeded considerably faster (15 min) than those reported
in experiments performed with HATU (30 min) or TBTU
(
45 min) as commonly used coupling reagents under the Peptide Coupling Condensation time [h]
Yield
[
15]
11
reagent H N-Tyr-Aib-Aib-Phe-Leu-2-chlorotrityl resin (purity)
2
same conditions. Considering the relatively fast coupling
process and satisfactory yield, methanesulfonates 5g, 5i and
Tyr
Aib
Aib
Phe
[%]
1
1
1
1
1a
1b
1c
1d
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
0.5
1
0.75
1
1
0.75
1
1.5
1
0.75
1
2
0.5
2
1
2
1
1
0.75
2
0.5
1.5
1
0.75
1.5
0.5
2
0.5
2
1
99
97
94
85
87
92
83
98
91
94
79
86
5k also appear to be extremely promising (see Table 7), al-
though their very limited stability poses a major problem.
The reagents prepared with 10-camphorsulfonic acid 5b,f,j
were less efficient due to the prolonged coupling time re- 11e
quired and because of the relatively low yield of the final 11f
peptide.
Another useful test for rating the versatility of the new
TBCRs involved the solid-phase synthesis of peptides con-
taining difficult fragments because of the presence of steri- 11k
1
1
1
1
1g
1h
1i
1j
5j
5k
5l
2
0.5
0.75
0.75
11l
cally hindered α,α-disubstituted amino acid residues. There-
4
3
fore, we prepared manually the [Aib] [Aib] -enkephalin an-
–
[15]
alogue 11 on 2-chlorotrityl resin in a single coupling mode.
In comparative studies using DMT/NMM/BF , cou-
4
The rate of coupling in each synthetic step was monitored pling proceeded less readily. The assembly of the first
with the Kaiser test. The purity of isolated products was Fmoc-Aib-OH to the growing peptide chain on the resin
estimated by standard chromatographic procedures. The was completed within 2 h, but incorporation of the second
obtained results showed the lowest yields (in the range of Fmoc-Aib-OH required extension of condensation time up
79–83%) for the less stable TBCRs 5k and 5g, prepared to 4 h. The observed decrease of coupling rate in incorpora-
Eur. J. Org. Chem. 2015, 401–408
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
405

B. Kolesinska et al.
FULL PAPER
tion of lipophilic and sterically hindered Fmoc-Aib-OH 7000 HPLC manager) using a Supelco Discovery BIO Wide Pore
C18 column (25 cmϫ4.6 mm, 5 mm, Sigma). MS spectra were re-
into the peptide chain could be due to the nature of the
corded with an IonSpec Ultima 4.7-T-FT ion cyclotron resonance
counterion because in both cases the structure of the acti-
spectrometer (ICR. HR-MALDI, in 2,5-dihydroxybenzoic acid
vated intermediate was identical.
matrix). LC/MS spectra were recorded with a Dionex UltiMate
3
000. IR spectra were recorded as KBr pellets or film with a Bruker
1
13
ALPHA spectrometer or a PerkinElmer Spectrum 100. H and
C
Conclusions
NMR spectra were recorded with a Bruker Avance DPX 250
(
(
250 MHz) spectrometer or a Varian (300 MHz). Chemical shifts
ppm) are relative to TMS used as an internal standard. Multi-
There are several reports presenting the general opinion
that the nature of the counteranion has no practical influ-
ence on the outcome of coupling reactions mediated by
uronium/ammonium tetrafluoroborates or hexafluorophos-
plicities are marked as s = singlet, d = doublet, t = triplet, q =
quartet, quint = quintet, m = multiplet. Melting points were deter-
mined with a Büchi apparatus, model 510.
[
22a]
phates.
efficiency of TBCR tetrafluoroborates
The preliminary results of our research on the
[15]
Gas chromatography (GC) was conducted with a Shimadzu GC-
14A, FID (H
(
and hexafluoro-
were also in agreement with this suggestion.
[
22b]
2
/air), split 1:50. Column: Chirasil-Val capillary
25 mϫ0.32 mm), film thickness 0.2 μm, carrier gas: helium, pres-
phosphates
However, the present study reporting the diversified sta-
bility and reactivity of the novel TBCR sulfonates 5a–l sur-
prisingly contradicts that. As a consequence, there is a need
to re-evaluate this conclusion. The current comparative
study using TBCRs 5a–l derived from the three tertiary
amines 3a–c and four sulfonic acids 2a–d with very diverse
basic/acid, lipophilic/hydrophilic, and polarisation charac-
teristics, yielding exactly the same structure of the reactive
intermediate 7 after the activation of the carboxylic func-
sure 0.45 atm. Temperature program: 4 min at 90 °C, next 4 °C/min
to 190 °C, and 3 min at 90 °C.
Hydrolysis of Coupling Products to Amino Acid (GP 1): Peptide
(
5 mg) was treated with redistilled, constant-boiling HCl in a sealed
tube at 100 °C for 20 h. The solution was concentrated to dryness,
and the residue was dissolved with redistilled water and concen-
trated again. The remaining salt was dried overnight in a vacuum
dessicator under P
2 5
O and then treated for 12 h at room tempera-
ture with anhydrous methanol saturated with dry HCl (2 mL).
tion, documented significant differences in stability, ten- Methanol was evaporated, the residue was dried overnight in a vac-
dency to epimerisation, coupling rate, yield, and purity of uum dessicator under P O , then suspended in dichloromethane
2
5
the final products. The differences in synthetic versatility and treated with trifluoroacetic acid anhydride (50 μL) for 12 h at
room temperature. The solution was analysed on a Chirasil-Val
capillary column (25 mϫ0.25 mm) with a split of 1:50; helium was
used as carrier gas.
was noted both in the syntheses proceeding in solution as
–
well as in manual SPPS. The TBCR (DMT/NMM/TsO ,
a) prepared with the highly polarisable p-toluenesulfonate
5
anion and the less basic N-methylmorpholine was found to Manual Solid-Phase Peptide Synthesis (SPPS): Protected amino
be the most universal, and the coupling results surpassed acids were purchased from Novabiochem or Bachem. All peptides
were synthesised in an appropriate glass reactor or syringe by Fmoc
methodology.
those of its tetrafluoroborate analogue (DMT/NMM/
–
BF ). An additional, very important advantage of DMT/
4
–
NMM/TosO (5a) is its crystalline state, high stability Loading of the 2-Chlorotrityl Chloride Resin (GP 2): The amino
(
which allows long-term storage and shipping), as well as acid (3 equiv. rel. to the resin) and DIPEA (6 equiv.) were dissolved
stability in solution under the conditions of SPPS. If the in CH Cl (10 mL per 1 g of the resin), containing, if necessary, a
small amount of DMF to facilitate dissolution of the amino acid.
The 2-chlorotrityl chloride resin was preswollen in CH Cl for 1 h,
2
2
advantageous effects of polarisability of salt components
are also observed in the case of other salt-type coupling
reagents, a new pathway to improve the effectivness of sev-
eral classical coupling reagents will be opened. This is par-
ticularly important for the fascinating area dedicated to
syntheses of difficult peptides containing highly function-
alised or noncoded amino acids.
2
2
and then the solution containing the protected amino acid was
added and the resin was shaken for 30–120 min. The resin was
washed with CH
2 2
Cl /MeOH/DIPEA (17:2:1, 3ϫ), then DMF (2ϫ)
and CH Cl (3ϫ).
2
2
Standard Coupling Procedure (GP 3): Protected amino acid
3 equiv.), 5 (3 equiv.) and DIPEA (6 equiv.) were mixed and added
to the resin. The resin was shaken for 1–2 h. The progress of the
(
reaction was monitored by Kaiser[ or TNBS tests.
21]
[23]
Experimental Section
Deprotection (GP 4): The Fmoc protecting group was removed by
treatment with a solution of 20% piperidine in DMF (2ϫ 5 min).
General Information: Thin-layer chromatography experiments
(TLC) were carried out on silica gel (Merck; 60 Å F254), and spots
were located with UV light (254 and 366 nm) and with 1% ethan-
olic 4-(4-nitrobenzyl)pyridine (NBP).
Cleavage from the Resin (GP 5): The peptides were cleaved from
the resin by using TFA/Et
resin). Cleavage was performed during 4 h, then the resin was fil-
tered off, and the filtrate was evaporated. To the oily residue, Et
was added to precipitate the peptide. The resulting solid was fil-
tered off, washed with Et O, dried and lyophilised.
3 2
SiH/H O, 9.5:2.5:2.5 (ca. 2 mL/0.1 g of
Analytical RP-HPLC was performed with a Waters 600S HPLC
system (Waters 2489 UV/Vis detector, Waters 616 pump, Waters
2
O
7
17 plus autosampler, HPLC manager software from Chromax)
using a Vydac C18 column (25 cmϫ4.6 mm, 5 mm; Sigma). HPLC
was performed with a gradient of 0.1% TFA in H O (A) and 0.08%
TFA in MeCN (B), at a flow rate of 1 mL/min with UV detection
at 220 nm, t in min; or on a Merck/Hitachi HPLC system (La-
Chrome; L-6200 pump, L-4000 UV detector, D-6000 interface, D-
2
2
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
Tolu-
ene-4-sulfonate (DMT/NMM/TosO-5a); Typical Procedure: To a
vigorously stirred solution of 4-methylmorpholinium toluene-4-
sulfonate (5.46 g, 20 mmol) and sodium hydrogen carbonate
R
406
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 401–408

N-Triazinylammonium Sulfonates in Peptide Synthesis
–1
(
4
5.04 g, 60 mmol) in acetonitrile (60 mL), cooled to 5 °C, 2-chloro-
,6-dimethoxy-1,3,5-triazine (CDMT) (3.50 g, 20 mmol) was
1299, 1251, 1226, 1193, 1170, 1152, 1087, 1066, 1021 cm . 1H
NMR (250 MHz, CDCl ): δ = 1.52 (s, 12 H, CH ), 3.72 (s, 3 H,
CH -O-), 5.10 (s, 2 H, Ph-CH -O), 5.22–5.30 (m, 1 H, -CO-NH),
6.89–6.91 (m, 1 H, CO-NH-), 7.29–7.40 (m, 5 H, ArH) ppm.
): δ = 24.4, 25.2, 52.5, 56.9, 65.4, 67.0,
128.0, 128.1, 128.5, 156.2, 171.6, 174.0, 176.5 ppm. LC/MS: m/z
3
3
added. The mixture was stirred at 5 °C for 20 h and the progress
of the reaction was monitored by TLC (staining with 0.5% solution
of NBP in EtOH). Upon complete consumption of CDMT, the NMR (75 MHz, CDCl
precipitate was filtered off, and the filtrate was evaporated to dry-
ness at a temperature not exceeding 20 °C. The solid residue was
washed with THF and recrystallised from acetonitrile/diethyl ether
to afford 5a (5.93 g, 72%); m.p. 57–61 °C. IR (film/NaCl): ν˜ =
3
2
1
3
C
3
+
+
calcd. for C17
HPLC (3–97%B in 30 min): t
Z-Ala-Phe-Leu-OBz (9); Typical Procedure: Z-Ala-Phe-OH
0.370 g, 1 mmol) and DIPEA (0.088 mL, 0.5 mmol) were added
to a vigorously stirred solution of 5 (1 mmol) in CH Cl (5 mL),
H
24
N
2
O
5
336.39; found 337.3 [M + H] . Anal. RP-
R
= 9.55 min (purity 98.8%).
3
1
1
C
)
7
)
6
418, 3041, 2957, 2111, 1630, 1540, 1483, 1457, 1408, 1386, 1338,
319, 1308, 1285, 1271, 1227, 1198, 1132, 1122, 1060, 1037,
(
2
2
–1 1
014 cm . H NMR (300 MHz, CD
-), 3.42 (s, 3 H, CH -N), 3.73–4.03 (m, 6 H, -N-CH
, 4.12 (s, 6 H, 2ϫCH -O), 4.20–4.48 (m, 2 H, -N-CH -CH
.22 (d, J = 7.5 Hz, 2 H, -C -), 7.62 (d, J = 7.5 Hz, 2 H, -C
ppm. 13C NMR (75 MHz, CD
CN): δ = 22.7, 55.6, 58.2, 59.2,
2.4, 128.0, 131.1, 142.5, 145.3, 172.6, 176.3 ppm. HRMS: m/z
3
CN): δ = 2.35 (s, 3 H, CH
-CH -O-
-O-),
3
-
cooled to 0 °C. Stirring was continued until the disappearance of a
condensing reagent 5 was observed (TLC, staining with 0.5% solu-
tion of NBP), after which time TsOH·Leu-OBz (0.393 g, 1 mmol)
and DIPEA (0.176 mL, 1 mmol) were added, and stirring was con-
tinued for an additional 2 h at 0 °C and overnight at room tempera-
6
H
4
3
2
2
3
2
2
6
H
4
6 4
H -
3
ture. The mixture was diluted with CH
tion was washed successively with water, 0.5 m aqueous NaHSO
water, 0.5 m aqueous NaHCO , and water again. The organic layer
was dried with MgSO , filtered, and concentrated to dryness. The
2 2
Cl (10 mL) and the solu-
+
+
calcd. for C17
H
25
N
4
O
6
S
413.48; found: 413.1490 [M ], 414.1525
4
,
+
[
M + H] . C17
H
24
N
4
O
6
S (413.47): calcd. C 49.50, H 5.87, N 13.58,
3
O 23.27, S 7.77; found C 49.51, H 5.86, N 13.58, S 7.77.
,6-Dimethoxy-1,3,5-triazin-2-yl 4-Methoxybenzoate (7); Typical
Procedure: 4-Methoxybenzoic acid (1 mmol) and DIPEA (88 μL,
4
residue was dried under vacuum with P
weight to afford Z-Ala-Phe-Leu-OBz.
2 5
O and KOH to constant
4
0
.5 mmol) were added at 0 °C to a vigorously stirred solution of 5 Z-Ala-Phe-Leu-OBz (9a): Obtained from Z-Ala-Phe-OH (0.
(
1 mmol) in CH
pearance of condensing reagent 5 (TLC analysis, staining with
.5% solution of NBP), after which time the mixture was diluted
with CH Cl (10 mL), and the solution was washed successively
with water, 0.5 m aqueous KHSO , water, 0.5 m aqueous NaHCO
and water again. The organic layer was dried with MgSO , filtered,
and concentrated to dryness. The residue was dried under vacuum
with P and KOH to constant weight.
2
Cl
2
(3 mL). Stirring was continued until the disap-
0.370 g, 1 mmol), TsOH·Leu-OBz (0.393 g, 1 mmol), DMT/NMM/
TsO (5a) (0.413 g, 1 mmol), and DIPEA (0.264 mL, 1.5 mmol).
Activation time: 1 h, yield 0.505 g (88%). IR (film/NaCl): ν˜ = 3276,
3068, 3033, 2956, 2871, 2035, 1950, 1738, 1687, 1644, 1533, 1484,
1453, 1386, 1368, 1326, 1255, 1234, 1192, 1146, 1113, 1075,
–
0
2
2
4
3
,
–
1 1
1028 cm . H NMR (300 MHz, CDCl
(CH CH, 6 H], 1.47–1.49 [m, 1 H, (CH
7.2 Hz, 3 H, CH -CH-), 1.82–1.89 (m, 2 H, -CH-CH
dd, J = 8.3, J = 4.1 Hz, 2 H, -CH-CH -), 4.41 (t, J = 5.2 Hz, 1
H, -CH -CH-), 4.68 (q, J = 7.2 Hz, 1 H, CH -CH-), 4.80–4.88, (m,
H, -CH-CH -), 5.09 (s, 2 H, -CH -Ph), 5.34 (s, 2 H, -CH -Ph),
.25–7.40 (m, 15 H, Ph) ppm. C NMR (75 MHz, CDCl ): δ =
3
): δ = 0.91 [d, J = 6.5 Hz,
CH-], 1.48 (d, J =
-), 3.19, 3.44
4
3
)
2
3 2
)
2
O
5
3
2
(
1
2
2
4
,6-Dimethoxy-1,3,5-triazin-2-yl 4-Methoxybenzoate (7a): Obtained
2
3
from 4-methoxy-benzoic acid (0.152 g, 1 mmol), DIPEA (88 μL,
1
7
1
1
1
2
5
2
2
2
0
3
3
1
1
.5 mmol), and DMT/NMM/TsO– (5a) (0.413 g. 1 mmol), in
0 min, yield 0.287 g (98.5%). IR (film/NaCl): ν˜ = 3461, 3078,
023, 2953, 2844, 2604, 2337, 2172, 2072, 1945, 1759, 1744, 1590,
570, 1544, 1512, 1470, 1444, 1427, 1406, 1355, 1318, 1242, 1233,
1
3
3
8.5, 21.8, 22.6, 24.6, 38.1, 40.8, 41.9, 50.4, 53.9, 54.1, 66.8, 126.7,
27.8, 128.0, 128.1, 128.3, 128.4, 135.3, 136.1, 136.3, 155.8, 170.5,
72.1, 172.3 ppm. Anal. RP-HPLC (3–97%B in 30 min): t
3.82 min (purity 99.5%). LC/MS: m/z calcd. for C33H N O
73.70; found 574.4 [M + H] . GC (hydrolyzate was derivatised
R
=
–1
1
202, 1189, 1172, 1164, 1108, 1085, 1042, 1021 cm . H NMR
+
39 3 6
(300 MHz, CDCl
3
): δ = 3.76 (s, 3 H, CH
3
O-C
-), 7.98 (d, J = 7.5 Hz,
): δ = 55.6, 55.9,
13.8, 119.9, 132.6, 163.8, 170.1, 173.6 ppm. LC/MS: m/z calcd. for
6 4
H -), 3.97 (s, 6 H,
+
CH
2
1
C
3
O-CN), 6.84 (d, J = 7.5 Hz, 2 H, C
6
H
4
according to GP 1; chromatography conditions: temp. 90 °, 4 min,
0–200 °, 4 °/min, 200 °, 3 min) t = 3.07 (d-Ala), 3.13 (l-Ala) min
L/D = 100:0; t = 17.27 (l-Phe), 17.38 (d-Phe) min L/D = 100:0;
= 6.56 (d-Leu), t 6.91 (l-Leu) min L/D = 99:1.
Synthesis of (65–74) ACP: H N-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-
H, C
6
H
4
-) ppm. 13C NMR (75 MHz, CDCl
3
9
R
+
+
R
13 13 3 5
H N O 291.27; found 292.1 [M + H] .
t
R
R
Z-Aib-Aib-OMe (8); Typical Procedure: Z-Aib-OH (0.237 g,
mmol) and DIPEA (0.088 mL, 0.5 mmol) were added to a vigor-
ously stirred solution of 5 (1 mmol) in CH Cl (5 mL), cooled to
°C. Stirring was continued until the disappearance of condensing
2
1
Asn-Gly-OH (10): 2-Chlorotrityl chloride resin (600 mg, 1.1 mmol/
g, 0.66 mmol) was esterified with Fmoc-Gly-OH (0.589 g,
2
2
0
1.98 mmol) in the presence of DIPEA (0.713 mL, 3.96 mmol) ac-
reagent 5 was observed (TLC analysis, staining with 0.5% solution
of NBP), after which time HCl·Aib-OMe (0.154 g, 1 mmol) and
DIPEA (0.176 mL, 1 mmol) were added, and the mixture was
stirred for an additional 2 h at 0 °C and overnight at room tempera-
cording to GP 2, followed by Fmoc deprotection (GP 4). Modified
resin was divided into 12 portions (50 mg, 0.055 mmol) and used
for further reaction steps in separate reactors. Subsequently, the
peptide chains were elongated (GP 3), respectively, with Fmoc-
Asn(Trt)-OH (98 mg, 0.165 mmol), Fmoc-Ile-OH (58 mg,
ture. The mixture was diluted with CH
tion was washed successively with water, 0.5 m aqueous NaHSO
water, 0.5 m aqueous NaHCO , and water again. The organic layer
was dried with MgSO , filtered, and concentrated to dryness. The
2 2
Cl (10 mL), then the solu-
4
,
0
.165 mmol), Fmoc-Tyr(tBu)-OH (76 mg, 0.165 mmol), Fmoc-
Asp(tBu)-OH (68 mg, 0.165 mmol), Fmoc-Ile-OH (58 mg,
.165 mmol), Fmoc-Ala-OH (51 mg, 0.165 mmol), Fmoc-Ala-OH
51 mg, 0.165 mmol), Fmoc-Gln(Trt)-OH (101 mg, 0.165 mmol)
3
4
0
(
residue was dried under vacuum with P
weight, to afford the neutral peptide.
2 5
O and KOH to constant
and Fmoc-Val-OH (56 mg, 0.165 mmol) in the presence of appro-
priate reagent 5a–l (0.165 mmol) and DIPEA (60 μL, 0.33 mmol).
After the last deprotection (GP 4), the peptides were cleaved from
Z-Aib-Aib-OMe (8a): Obtained from Z-Aib-OH (0.237 g, 1 mmol),
HCl·Aib-OMe (0.154 g, 1 mmol), DMT/NMM/TsO (5a) (0.413 g,
–
the resin (GP 5).
1
mmol), and DIPEA (0.264 mL, 1.5 mmol). Activation time: 1 h,
–
yield 0.279 g (83%). IR (film/NaCl): ν˜ = 3380, 3362, 3315, 3276, Compound 10a: Coupling reagent: DMT/NMM/TsO (5a) (68 mg,
3
033, 2985, 2939, 1726, 1713, 1655, 1517, 1453, 1409, 1385, 1363,
0.165 mmol) at each step of an elongation peptide chain. Product:
407
Eur. J. Org. Chem. 2015, 401–408
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org

B. Kolesinska et al.
FULL PAPER
1
9
1
0a: Anal. RP-HPLC (3–97%B in 30 min): t
R
= 22.91 min (purity [2] Z. J. Kami n´ ski, Biopolymers 2000, 55, 140–165.
+
[3] Z. J. Kami n´ ski, Int. J. Pept. Protein Res. 1994, 43, 312–319.
[4] Z. J. Kami n´ ski, J. Prakt. Chem. 1990, 332, 579–581.
7.9%). LC/MS: m/z calcd. for C47
064.5 [M + H] .
H
74
N
12
O
16 1063.18; found
+
[
5] Z. J. Kami n´ ski, P. Paneth, M. O’Leary, J. Org. Chem. 1991, 56,
H
2
N-Tyr-Aib-Aib-Phe-Leu-OH (11): 2-Chlorotrityl chloride resin
600 mg, 1.1 mmol/g, 0.66 mmol) was esterified with Fmoc-Leu-
OH (0.700 g, 1.98 mmol) in the presence of DIPEA (0.713 mL,
.96 mmol) according to GP 2, followed by Fmoc deprotection
GP 4). Modified resin was divided into 12 portions (50 mg,
.055 mmol) and used for the further reaction steps in separate
5716–5719.
[
[
[
6] Z. J. Kami n´ ski, Tetrahedron Lett. 1985, 26, 2901–2904.
(
7] Z. J. Kami n´ ski, Synthesis 1987, 917–920.
8] J. E. Kami n´ ska, Z. J. Kami n´ ski, J. Góra, Synthesis 1999, 593–
3
(
0
596.
[
[
9] Z. J. Kami n´ ski, B. Kolesi n´ ska, M. Marcinkowska, Synth. Com-
mun. 2004, 34, 3349–3358.
10] I. Paolini, F. Nuti, M. F. Pozo-Carrero, F. Barbetti, B. Kolesin-
ska, Z. J. Kaminski, M. Chelli, A. M. Papini, Tetrahedron Lett.
2007, 48, 2901–2904.
reactors. Subsequently, the peptide chains were elongated (GP 3),
respectively, with Fmoc-Phe-OH (64 mg, 0.165 mmol), Fmoc-Aib-
OH (54 mg, 0.165 mmol), Fmoc-Aib-OH (54 mg, 0.165 mmol) and
Fmoc-Tyr(tBu)-OH (76 mg, 0.165 mmol) in the presence of 5a–l [11] a) L. A. Wozniak, M. Gora, Z. J. Kami n´ ski, W. J. Stec, Phos-
phorus Sulfur Silicon Relat. Elem. 2008, 183, 1076–1081; b)
L. A. Wozniak, M. Gora, W. J. Stec, J. Org. Chem. 2007, 72,
(0.165 mmol) and DIPEA (60 μL, 0.33 mmol). After the last depro-
tection (GP 4), the peptides were cleaved from the resin (GP 5).
8584–8587.
–
Compound 11a: Coupling reagent: DMT/NMM/TsO (5a) (68 mg, [12] M. W. Markowicz, R. Dembinski, Synthesis 2004, 80–86.
0
1
9
.165 mmol) at each step of an elongation peptide chain. Product:
[13] a) M. Kunishima, C. Kawachi, F. Iwasaki, K. Terao, S. Tani,
Tetrahedron Lett. 1999, 40, 5327–5330; b) M. Kunishima, C.
Kawachi, J. Morita, F. Iwasaki, K. Terao, S. Tani, Tetrahedron
1999, 55, 13159–13170; c) M. Kunishima, C. Kawachi, K.
Hioki, K. Terao, S. Tani, Tetrahedron 2001, 57, 1551–1558; d)
K. Zaj a˛ c, Z. J. Kami n´ ski, Acta Pol. Pharm. (Engl. Transl.)
1a: Anal. RP-HPLC (3–97%B in 30 min): t
R
+
= 16.81 min (purity
9.5%). LC/MS: m/z calcd. for C32H N O 611.74; found 612.4
45 5 7
+
[M + H] .
Supporting Information (see footnote on the first page of this arti-
cle): Synthetic procedures for 5b–l, chromatographic characterisa-
tion of products as well as H NMR, C NMR, MS and IR spec-
tra.
2010, 67, 725–728.
1
13
[14] K. G. Jastrz a˛ bek, B. Kolesi n´ ska, G. Sabatino, F. Rizzolo, A. M.
Papini, Z. J. Kami n´ ski, Int. J. Pept. Reas. Therap. 2007, 13,
229–236.
[
[
15] Z. J. Kami n´ ski, B. Kolesi n´ ska, J. Kolesi n´ ska, G. Sabatino, M.
Chelli, P. Rovero, M. Błaszczyk, M. L. Główka, A. M. Papini,
J. Am. Chem. Soc. 2005, 127, 16912–16920.
16] K. G. Jastrzabek, R. Subiros-Funosas, F. Albericio, B. Kolesin-
ska, Z. J. Kaminski, J. Org. Chem. 2011, 76, 4506–4513.
Acknowledgments
This study was supported by the Ministry of Science and High
Education, Poland under the Research Project N N405 669540.
[17] a) R. Subirós-Funosas, S. N. Khattab, L. Nieto-Rodríguez, A.
El-Faham, F. Albericio, Aldrichim. Acta 2013, 46, 21–40; b) A.
El-Faham, R. S. Funosas, R. Prohens, F. Albericio, Chem. Eur.
J. 2009, 15, 9404–9416.
[1] For classical coupling reagents, see: a) A. El-Faham, F. Alber-
icio, Chem. Rev. 2011, 111, 6557–6602; b) E. Valeur, M. Brad- [18] B. Kolesi n´ ska, J. Fr a˛ czyk, A. M. Papini, Z. J. Kami n´ ski, Chim-
ley, Chem. Soc. Rev. 2009, 38, 606–631; c) C. A. G. N. Montal-
betti, V. Falque, Tetrahedron 2005, 61, 10827–10852; d) F. Al-
bericio, R. Chinchilla, D. J. Dodsworth, C. Nájera, Org. Prep.
Proced. Int. 2001, 33, 203–303; e) B. L. Bray, Nat. Rev. Drug
ica Oggi 2007, 25, 26–29.
[19] a) O. Marde, F. Albericio, Chimica Oggi 2003, 21, 6–11; b) K.
Ito, K. Nagase, N. Morohashi, Y. Ohba, Chem. Pharm. Bull.
2005, 53, 90–94.
Discovery 2003, 2, 587–593; f) S. Kent, J. Pept. Sci. 2003, 9, [20] Z. J. Kami n´ ski, B. Kolesi n´ ska, J. Kolesi n´ ska, K. Jastrz a˛ bek,
5
2
74–593; g) A. Basso, F. Wrubl, Spec. Chem. Mag. 2003, 23,
8–30; h) C. U. Pittman, Polym. News 2003, 28, 183–184; i) P.
Eur. Pat. EP1586566 A1 from 19.10.2005, Chem. Abstr. 2006,
143, 387385.
Li, J. C. Xu, J. Pept. Res. 2001, 58, 129–139; j) D. T. Elmore, [21] E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal.
Amino Acids Pept. Proteins 2002, 33, 83–134; k) L. A. Carpino,
M. Beyermann, H. Wenschuh, M. Bienert, Acc. Chem. Res.
Biochem. 1970, 34, 595–598.
[22] a) S.-Y. Han, Y.-A. Kim, Tetrahedron 2004, 60, 2447–2467; b)
Z. J. Kami n´ ski, B. Kolesi n´ ska, unpublished results.
[23] W. S. Hancock, J. E. Battersby, Anal. Biochem. 1976, 71, 260–
264.
1996, 29, 268–274; l) J. M. Humphrey, A. R. Chamberlin,
Chem. Rev. 1997, 97, 2243–2266; m) P. D. Bailey, An Introduc-
tion to Peptide Chemistry, Wiley, Chichester, UK, 1990; n) M.
Bodanszky, Principles of Peptide Synthesis Springer, Berlin,
Received: July 3, 2014
1984, and references cited therein.
Published Online: November 21, 2014
408
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 401–408