Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether as coupling reagent for peptide synthesis

Article abstract of DOI:10.1002/cbdv.201200369

Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether (4) was prepared by treatment of 2-hydroxy-4,6-dimethoxy-1,3,5-triazine with 2-chloro-4,6-dimethoxy-1,3,5- triazine in 61% yield. Ether 4, isoelectronic with pyrocarbonates, was found capable to activate carboxylic acids in the presence of 1,4-diazabicyclo[2.2.2] octane (DABCO) to yield, under mild reaction conditions, superactive triazine esters. Versatility of this new coupling reagent was confirmed by condensation of lipophilic and sterically hindered carboxylic acids with amines in 71-98% yield, and by synthesis of peptides, including those containing Aib-Aib sequence, in solution with high yield and high enantiomeric purity. Copyright

Full text of DOI:10.1002/cbdv.201200369

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) Ether as Coupling Reagent for Peptide
Synthesis
by Konrad Jastrzabek, Przemyslaw Bednarek, Beata Kolesinska, and Zbigniew J. Kaminski*
Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, PL-90-924 Lodz
(phone: þ48-6313151; fax: þ48-426365530; e-mail: zbigniew.kaminski@p.lodz.pl)
Dedicated to Professor Ignacy Z. Siemion on the occasion of his 80th birthday
Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether (4) was prepared by treatment of 2-hydroxy-4,6-
dimethoxy-1,3,5-triazine with 2-chloro-4,6-dimethoxy-1,3,5-triazine in 61% yield. Ether 4, isoelectronic
with pyrocarbonates, was found capable to activate carboxylic acids in the presence of 1,4-
diazabicyclo[2.2.2]octane (DABCO) to yield, under mild reaction conditions, superactive triazine
esters. Versatility of this new coupling reagent was confirmed by condensation of lipophilic and sterically
hindered carboxylic acids with amines in 71–98% yield, and by synthesis of peptides, including those
containing Aib-Aib sequence, in solution with high yield and high enantiomeric purity.
Introduction. – In recent years, incorporation of an unnatural residue into the
peptide chain became a standard synthetic practice. The main motivation for this
approach arose from the expected increase of the resistance of unnatural structures
toward metabolic degradation, prolonged activity, reduction of dosage, and expected
less ambiguous interpretation of effects caused by incorporation of unnatural building
blocks. There is also a substantially increased pool of naturally occurring peptides with
non-coded amino acids or with additional structural elements active as antibiotics,
toxins, and others [1]. In both cases, the available reagents and synthetic strategies
turned out to be insufficient or even to fail completely in the case of poorly reactive and
sterically hindered building blocks, the so-called difficult sequences or coupling
building blocks with opposite disposition such as hydrophilicityꢀhydrophobicity. An
example is the well-recognized group of peptides isolated from fungi, known as
peptaibols, bearing several characteristic structural features [2]. These include the
presence of multiple residues of a-aminoisobutyric acid (Aib) and amino alcohol at the
C-terminal position. Due to the accumulation of a,a-disubstituted amino acid residues
in sequences, synthesis of peptaibols has always been the synthetic challenge [3].
Synthetic problems caused by sterically hindered amino acids led to the search for the
new, more-efficient coupling procedures and reagents, including phosphinium,
iminium, and ammonium derivatives of 1-hydroxybenzotriazole [4], 8-azabenzotria-
zole [5], or oxyma (¼ethyl 2-cyano-2-(hydroxyimino)acetate) [6].
Our efforts in this area were focused on the development of a new generation of
triazine-based coupling reagents, designed according to the concept of ꢀsuperactive
estersꢁ [7]. Following the concept of ꢀsuperactive estersꢁ, increased efficiency of
coupling reagents was achieved with an innovative strategy based on the introduction
ꢂ 2013 Verlag Helvetica Chimica Acta AG, Zꢃrich

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
953
of an additional synchronous and thermodynamically highly favored process. The
accompanying process (i.e. an additional, fast, and energetically favored reaction or
rearrangement of the co-products) synchronous with a release of the leaving group
from tetrahedral intermediates should facilitate the rate-determining step [7]. By this
way, the ꢀpushꢁ from the relatively mild activation and the ꢀpullꢁ from additional
stabilization of the products should afford better synthetic results than those obtained,
when the same change of the thermodynamic potential of the reaction is solely due to
more powerful activation. The versatility of the idea of acceleration of the coupling
process in the case of ꢀsuperactive estersꢁ, based on facile departure of the leaving group
by its rearrangement in an energetically favored, consecutive process to a stable,
chemically inert, and neutral side product, has been thoroughly confirmed. The
usefulness of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoro-
borate (DMT/NMM/BF4) was compared with standard coupling reagents, and its
supremacy in peptide synthesis in solution and in solid phase was demonstrated [8].
Modification of substituents in the native structure of triazine-based coupling reagent
afforded
4-[4,6-bis(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]-4-methylmorpholinium
tetrafluoroborate, which was found highly efficient for incorporation of sterically
hindered N-methylleucine and N-methylvaline into the peptide chain [9]. Replacement
of 4-methylmorpholine (NMM) fragment by chiral tertiary amines provided highly
enantioselective coupling reagents. These reagents, named ꢀtracelessꢁ, when applied
peptide synthesis from racemic N-protected amino acids for the first time, gave
optically pure products with configuration and enantiomeric enrichment precisely
predicted by a single model experiment [10].
Recently, we noticed the increased trends in application of strongly lipophilic
building blocks used in modifications of the native peptide structures [11]. Moreover,
there is an increasing number of new natural products with identified strongly lipophilic
fragments crucial for the activity of the native molecules. Examples are peptaibols
containing several a,a-disubstituted a-amino acid residues [12], N-alkylated amino
acids, b-amino acids [13], lipidated peptides for gene delivery, fluorinated amino acids,
and many others [14]. As the lipophilic nature of amino acids is essential for activity,
there is increased demand for suitable methods of incorporation which reconciles high
efficiency with tolerance of increased lipophilicity of reagents and reacting media.
Following the motto that ꢀsimilia similibus solvunturꢁ, we attempted to improve the
synthetic versatility of triazine-based coupling reagents by modification of ionic
structure of N-triazinylammonium salts in order to obtain more lipophillic analogs.
Crystallographic analyses of structures of ꢀsuperactiveꢁ triazine esters [15], aryloxy-
1,3,5-triazines [16], and bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether (4) suggested that
the latter, due to unsymmetrical charge distribution [17], should be considered as
potentially reactive intermediate (aza analog of pyrocarbonates) (see Fig.).
According to this assumption, bis-triazinyl ethers, after treatment with carboxylic
acids, should yield ꢀsuperactiveꢁ triazine esters identical to active acylating species
obtained under classic coupling conditions involving triazine-based coupling reagents.
To verify this assumption, we prepared bis-1,3,5-triazine ether 4 and studied its
potential for activation of the carboxylic group.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Figure. Stick-and-ball representation of X-ray structure of bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether (4)
demonstrating different arrangements of substituents in both triazine rings [17] (above), and analogous
fragments of pyrocarbonates and triazine ethers in bold (below)
Results and Discussion. – Synthesis of Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) Ether
(4). The procedure has been already described in [18]. In this work, however, expecting
that ether synthesis according to Williamson-type procedure from aromatic substrates
is not a trivial process [19], ether 4 was prepared in two separate steps (see Scheme 1).
Scheme 1. Synthesis of Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) Ether (4)
Substrate 3 was obtained in good overall yield by hydrolysis of 1, presumably, via
intermediate N-triazinylammonium salt 2. The main advantage of this two-step
procedure was reduced contamination of 3 with side-products formed by excessive
cleavage of Me groups under strongly basic conditions. Then, the final product 4 was
obtained in good overall yield by treatment of 2-hydroxy-4,6-dimethoxy-1,3,5-triazine
(3) with 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT; 1) activated with 1,4-diazabi-
cyclo[2.2.2]octane (DABCO) under anhydrous conditions.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
955
Crystallographic studies of 4 confirmed asymmetry of structure with one ether CꢀO
bond length of 1.371 ꢄ and a dihedral angle of CꢀOꢀCꢀtriazine ring of 40.778, with the
other CꢀO bond length being 1.365 ꢄ and the corresponding dihedral angle 23.978 [17].
In spite of the observed alternation of ether CꢀO bonds and appropriate dihedral
angles resulting from the strong AGIBA effect (Angular Group-Induced Bond
Alternation) [20], preliminary experiments of carboxylic acid activation using 4 were
not encouraging.
Activation of Carboxylic Acids Using 4. Carboxylic acids were not prone to react
with 4. Moreover, an addition of equimolar amount of NMM or 4-(dimethylamino)-
pyridine (DMAP) in order to generate a more nucleophilic carboxylate anion did not
accelerate the process very much. However, it has been found that, in the presence of
equimolar amount of DABCO, activation of carboxylic acids proceeded relatively fast,
and that, under mild condition, 4 was completely consumed within 40 min. As the
mechanism of this unique effect of DABCO is not known, it was essential to identify
the reactive intermediate. The experiments involving 2,2-dimethylpropanoic acid (¼
pivalic acid; 5a) and 4-methoxybenzoic acid (5b) treated with 4 in the presence of
DABCO gave already known 2-(acyloxy)-4,6-dimethoxy-1,3,5-triazines (6; see
Scheme 2) with characteristic IR signal of the active ester group at 1776 cmꢀ1. These
compounds were spectroscopically and chromatographically identical with those
prepared by standard procedure [7b].
Scheme 2. Activation of Carboxylic Acids 5 Using 4 and 1,4-Diazabicyclo[2.2.2]octane (DABCO) to
Yield Appropriate 2-Acyloxy-4,6-dimethoxy-1,3,5-triazines 6 and, in the Presence of Amines, the
Corresponding Amides 7
This alternative pathway to ꢀsuperactiveꢁ triazine esters displays synthetic potential
of 4 in the application for coupling strongly lipophilic substrates, because the approach
based on 4 avoids ionic activators in contrast to all of the most advanced, typical
condensing reagents (N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethy-
lene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[(1H-ben-

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
zotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluorobo-
rate N-oxide (TBTU), DMT/NMM/BF4, etc.).
Formation of the peptide bond from amino acids is a complex, multistage process.
Therefore, although it is known that activation of carboxylic function yields well known
and highly reactive intermediates 6, the versatility of 4 has to be cautiously evaluated.
In the preliminary assessment, 4 was used as condensing reagent in the synthesis of
amides. It has been found that the one-pot procedure is efficient for a broad range of
substrates affording appropriate amides with 71–98% yield and satisfactory purity of
the crude isolated products 7a–7f. Ether 4 was found efficient in coupling amines with
sterically hindered 2,2-dimethylpropanoic acid (5a), 2-phenylbutanoic acid (5c), and
strongly hydrophobic (Z)-octadec-9-enoic acid (5d), as well as benzoic acid (5e) with
amino acid esters.
Peptide Synthesis. In further studies, 4 was used as coupling reagent in the synthesis
of protected peptides 8a–8i (Table). Both hydrophobic as well as hydrophilic peptides
protected with (tert-butoxy)carbonyl (Boc) or (benzyloxy)carbonyl (Z) group were
prepared according to non optimized, standard procedure, with good yields, also in the
case of sterically hindered AibꢀAib peptide bond useful in the synthesis of peptaibols.
Their purities determined by HPLC at l 220 nm were in the range of 61–97%, which is
satisfactory considering strong UV absorption characteristics for any triazine-derived
impurities at this wavelengths [21].
Table. Peptide Synthesis Using 4 as Coupling Reagent
Entry
Peptide
Yield [%]
Purity [%]
Racemization er [% l/% d]a)
N-terminalb)
C-terminalb)
1
2
3
4
5
6
7
8
9
ZLeu-PheOMe (8a)
ZAib-LeuOMe (8b)
ZAib-PheOMe (8c)
ZAib-AibOMe (8d)
BocVal-AibOMe (8e)
BocVal-LeuOMe (8f)
BocLeu-ValOMe (8g)
BocPro-PheOMe (8h)
Z-PheAib-LeuOMe (8i)
87
83
83
69
69
75
87
91
78
75
96
93
87
87
96
70
97
91
96.5/3.5
–
–
97.4/2.4
99.0/1.0
99.6/0.4
–
–
99.0/1.0
98.9/1.1
97.6/2.4
100.0/0.0
99.0/1/0
–
96.9/3.1
99.8/0.2
99.9/0.1
99.5/0.5
a) Enantiomeric ratio expressed as normalized ratio % l/% d determined by GC on Chirasil-ValTM
capillary column after hydrolysis of peptides to amino acids and subsequent derivatization. b) N-
Terminal and C-terminal amino acid residue.
Enantiomeric homogeneity at all stereogenic centers was confirmed by GC on a
Chirasil-ValTM capillary column after total hydrolyses of peptides. In most of the cases,
er % l/% d ratio exceeded 99 :1 [22], even in the cases of difficult couplings involving
Aib residue as a reaction components.
Conclusions. – In contradistinction to the well-known chemical inertness of all
members of the ether family [23], bis(4,6-dimethoxy-1,3,5-triazin-2-yl) ether (4) was
found surprisingly reactive. To our knowledge, these heterocyclic ethers, isoelectronic

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
957
with pyrocarbonates, have never been considered before as candidates for coupling
reagents. Taking into account the advantages of 4 and a very broad range of possible
modification of the parent heterocyclic structures, it seems that a brand new family of
coupling reagents is accessible and careful studies on their properties are continued.
Experimental Part
General. HPLC: Vydac C-18 column, detection at 220 nm, gradient from 10 to 50% B in 15 min, flow
rate, 1 ml/min; A: 0.1% TFA in H2O, B: 0.1% CF3COOH (TFA) in mixture 90% MeCN and 10% H2O.
TLC: Merck ꢀSilicagel-Alufolieꢁ; mobile phase, CH2Cl2, visualization, UV 254 nm and then spraying with
0.5% soln. of 4-(4-nitrobenzyl)pyridine (NBP) in EtOH. IR Spectra: KBr pellets or film; n in cmꢀ1
.
1H-NMR Spectra: 250-MHz spectrometers; solvent peak as an internal reference; d in ppm, J in Hz.
Elucidation of enantiomeric homogeneity of peptides was performed by GC equipped with a flame-
ionization detector on Chirasil-ValTM column (25 mꢁ0.25 mm), H2/air split, 1:50; carrier gas, He;
pressure, 135 kPa. Hydrolysis of peptides (2–4 mg) was performed by treating with redistilled, constant-
boiling aq. HCl (5 ml) in glass tube sealed under vacuum and heating to 1008 in a water-bath for 24 h.
After evaporation of the acids, the remaining residue was dried and derivatized according to the
procedure described in [24] to obtain N-(trifluoroacetyl) amino acid methyl esters. In all cases, minor
signal of d-enantiomer was identified by co-injection with appropriate racemate used as reference, and
enantiomeric purity was calculated by normalization of signals intensities of both enantiomers.
4,6-Dimethoxy-1,3,5-triazin-2-ol (3). To a vigorously stirred soln. of 1,4-diazaybicyclo[2.2.2]octane
(DABCO; 22.44 g, 200 mmol) and NaOH (8.80 g, 220 mmol) in H2O (400 ml), cooled in an ice/water
bath, 2-chloro-4,6-dimethoxy-1,3,5-triazine (1; 35.10 g, 200 mmol) was added. Stirring was continued for
2 h, then the mixture was heated to 808 until all 1 was consumed (TLC). The clear soln. was cooled to r.t.,
and then acidified with AcOH to pH 6. The precipitate was filtered, washed with H2O (2ꢁ30 ml), dried
in vacuum desiccators, and recrystallized from EtOH to yield 3 (17.5 g, 55.8%). M.p. 144–1468 ([25]:
183–1848). 1H-NMR (CDCl3): 3.86 (s, 2 Me). Anal. calc. for C5H7N3O3: C 38.22, H 4.49, N 26.74; found:
C 38.55, H 4.35, N 27.00.
Bis(4,6-dimethoxy-1,3,5-triazin-2-yl) Ether (4). A soln. of 1 (17.60 g, 100 mmol) in THF (300 ml) was
vigorously stirred and cooled to 08, then DABCO (11.22 g, 100 mmol) was added dropwise. After 30 min,
3 (15.71 g, 100 mmol) was added. Stirring was continued at r.t. for 10 h, then the solvent was evaporated
in vacuo, and the dry residue was dissolved in CH2Cl2 (100 ml). The org. phase was washed with H2O (2ꢁ
50 ml) and then with sat. aq. NaCl soln. (50 ml), dried (MgSO4), filtered, and concentrated to dryness.
Recrystallization of the solid residue from CH2Cl2/hexane gave 4 (18.1 g, 61%). Rf (CH2Cl2) 0.15. M.p.
148–1528 ([18a]: 157–1588). IR (film): 1041, 1312, 1456, 1585, 1768, 1912, 2550, 2879, 2975, 3023, 3438,
3777. 1H-NMR (CDCl3): 4.02 (s, 4 Me). Anal. calc. for: C10H12N6O5: C 40.54, H 4.08, N 28.37; found: C
40.54, H 3.98, N 28.71.
4,6-Dimethoxy-1,3,5-triazin-2-yl 2,2-Dimethylpropanoate; 6a). A vigorously stirred soln. of 2,2-
dimethylpropanoic acid (5a; 0.102 g, 1 mmol) in MeCN (5 ml) was cooled to 0–58, then 4 (0.296 g,
1 mmol) and DABCO (0.112 g, 1 mmol) were added. Stirring and cooling was continued for 40 min, the
solvent was evaporated in vacuo to avoid excessive heating, and the isolated oily residue crystallized
when stored in a vacuum desiccators. M.p. 49–508 ([7b]: 50–528). IR (film): 1096, 1199, 1366, 1585, 1696,
1776 (C¼O), 2599, 2879, 2975, 3521.
4,6-Dimethoxy-1,3,5-triazin-2-yl 4-Methoxybenzoate (6b). Synthesis proceeded as described above,
with 4-methoxybenzoic acid (5b; 0.152 g, 1 mmol), MeCN (5 ml), 4 (0.296 g, 1 mmol), and DABCO
(0.112 g, 1 mmol). After evaporation of solvent, the obtained oily residue crystallized slowly when stored
under vacuum. M.p. 96–988 ([8]: 97–998). IR (film): 1018, 1103, 1361, 1512, 1600, 1775 (C¼O), 2584,
2846, 2953, 3640.
(2E)-3-Phenyl-1-(piperidin-1-yl)prop-2-en-1-one (7a). General Procedure (GP). The vigorously
stirred soln. of cinnamic acid (5f; 0.148 g, 1 mmol) in MeCN (5 ml) was cooled to 0–58, then 4 (0.296 g,
1 mmol) and DABCO (0.112 g, 1 mmol) were added, and stirring was continued for 40 min. Piperidine
(0.085 g, 1 mmol) was added and stirring and cooling were continued for 2 h at 0–58, and overnight at r.t.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
After evaporation of the solvent, the residue was dissolved in AcOEt (20 ml) and washed successively
with H2O (15 ml), 1m KHSO4 (2ꢁ15 ml), H2O (2ꢁ15 ml), 1m NaHCO3 (2ꢁ15 ml), H2O (2ꢁ15 ml),
and sat. aq. NaCl soln. (15 ml). The org. phase was dried (MgSO4), filtered, and the filtrate was
concentrated to dryness in vacuo yielding crude 7a (0.207 g, 98%). HPLC (gradient): purity 99%. M.p.
113–1168 ([26]: 115–1168).1H-NMR (CDCl3): 1.61–1.67 (m, 3 CH2); 3.62–3.63 (m, 2 CH2); 6.91 (d, J¼
15.5, CH); 7.36 (m, 3 arom. H); 7.52 (m, 2 arom. H); 7.64 (d, J ¼ 15.5, CH).
2,2-Dimethyl-N-(4-methylphenyl)propanamide (7b). Condensation proceeded according to the GP
with 5a (0.102 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), and 4-toluidine (0.107 g,
1 mmol). After evaporation, crude 7b was obtained (0.147 g, 77%). HPLC (gradient): purity 82%. M.p.
100–1028 ([27]: 119–1218). 1H-NMR (CDCl3): 1.31 (s, 3 Me); 2.31 (s, Me); 3.99 (s, COꢀNH); 7.14 (d, J¼
8, 2 arom. H); 7.40 (d, J¼8, 2 arom. H).
N-Butyl-2-phenylbutanamide (7c). Condensation proceeded according to the GP with 2-phenyl-
butanoic acid (5c; 0.164 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), and BuNH2 (0.073 g,
1 mmol). After evaporation, crude 7c was obtained (0.156 g, 71%) as an oil [28]. HPLC (gradient):
1
purity 94%. H-NMR (CDCl3): 0.73–0.92 (m, MeCH2CH, MeCH2CH2); 1.11–1.20 (m, MeCH2CH2);
1.20–1.35 (m, MeCH2CH2); 1.69–1.73 (m, 1 H, MeCH2CH); 2.07–2.13 (m, 1 H, MeCH2CH); 3.00–3.21
(m, MeCH2CH, NHCH2); 5.33–5.43 (br., CONH); 7.11–7.27 (m, 5 arom. H).
(9E)-N-(4-Methylphenyl)octadec-9-enamide (7d). Condensation proceeded according to the GP
with (E)-octadec-9-enoic acid (5d; 0.322 g, 1.14 mmol), 4 (0.337 g, 1.14 mmol), DABCO (0.128,
1.14 mmol), and 4-toluidine (0.122 g, 1.14 mmol). After evaporation, 7d was obtained (0.347 g, 93%). Oil
([29]: m.p. 42.58). 1H-NMR (CDCl3): 0.95 (t, J¼6.5, Me); 1.12 (s, Me); 1.47–1.54 (m, 12 CH2); 1.95–2.00
(m, CH2CH¼CHCH2); 2.28–2.34 (m, CH¼CH); 5.23–5.34 (m, NH); 7.09 (d, J¼8, 2 arom. H); 7.37 (d,
J¼8, 2 arom. H).
Methyl N-Benzoyl-l-phenylalaninate (7e). Condensation proceeded according to the GP with
benzoic acid (5e; 0.122 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), l-phenylalanine
methyl ester hydrochloride (0.216 g, 1 mmol), and NMM (0.11 ml, 1 mmol). After evaporation, crude 7e
was obtained (0.257 g, 91%). HPLC (gradient): purity 85%. GC: tR 18.42 (d-Phe), 18.71 min (l-Phe); %
l/% d 97.9 :2.1. M.p. 115–1178 ([30]: 90–978). 1H-NMR(CDCl3): 3.26 (t, J¼5.5, CH2Ph); 3.77 (s, MeO);
5.046–5.16 (dd, J¼5.5, CHCH2Ph); 6.50–6.54 (br. s, NH); 7.12–7.30 (m, PhCH2); 7.38–7.54 (m, 3 H,
PhCONH); 7.70–7.74 (m, 2 H, PhCONH).
Benzyl N-Benzoyl-l-alaninate (7f). Condensation proceeded according to the GP with 5e (0.122 g,
1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), l-alanine benzyl ester 4-toluenesulfonate
(0.351 g, 1 mmol), and NMM (0.11 ml, 1 mmol). After evaporation, crude 7f was obtained (0.202 g,
71%). HPLC (gradient): purity 94%. GC: tR 3.61 (d-Ala), 3.89 min (l-Ala); % l/% d 94.9 :5.1. M.p. 55–
578 ([31]: 978). 1H-NMR (CDCl3): 1.53 (d, J¼7, Me); 4.85 (q, J¼7, MeCH); 5.21 (s, 1 H, CH2); 5.22 (s,
1 H, CH2); 6.77–6.79 (m, NH); 7.32–7.37 (m, PhCH2); 7.42–7.82 (m, PhCONH).
Peptide Synthesis with 4 as Coupling Reagent. Z-Leu-Phe-OMe (8a; Table, Entry 1). Condensation
proceeded according to the GP with Z-Leu-OH (0.265 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112,
1 mmol), HCl·PheOMe (0.216 g, 1 mmol), and NMM (0.11 ml, 1 mmol) to afford crude 8a (0.371 g,
87%). Oil ([32]: m.p. 87–888). HPLC (gradient): purity 75%. GC: tR 7.81 (d-Leu), 8.47 min (l-Leu); %
l/% d 96.5 :3.5; tR 18.33 (d-Phe), 18.76 min (l-Phe); % l/% d 97.4 :2.6. 1H-NMR (CDCl3): 0.84–0.93 (m,
Me2CH); 1.54–1.68 (m, Me2CHCH2); 1.71–1.88 (m, Me2CHCH2); 3.00–3.21 (m, CHCH2Ph); 3.70 (s,
MeO); 4.38–4.70 (m, CHCH2Ph); 4.77–4.87 (m, Me2CHCH2CH); 5.09 (s, PhCH2O); 5.25–5.38 (m,
NH); 6.39–6.58 (m, CONH); 7.04–7.46 (m, 10 arom. H).
Z-Aib-Leu-OMe (8b; Table, Entry 2). Condensation proceeded according to the GP with Z-Aib-OH
(0.237 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl·Leu-OMe (0.181 g, 1 mmol), and
NMM (0.11 ml, 1 mmol) to afford crude 8b (0.301 g, 83%). HPLC (gradient): purity 96%. GC: tR 3.22
(Aib), 7.77 (d-Leu), 8.44 min (l-Leu); % l/% d 99.0 :1.0. M.p. 80–838 ([8]: 78–808). 1H-NMR (CDCl3):
0.91 (dd, J¼6.5, Me2CHCH2); 1.53–1.55 (s, 2 Me); 1.57–1.68 (m, Me2CHCH2); 3.71 (s, MeO); 4.52–4.64
(t, J¼6, CHCH2CH); 5.09 (s, OCH2Ph); 5.32–5.35 (m, CONH); 6.69–6.77 (m, CONH); 7.33–7.36 (m,
Ph).
Z-Aib-Phe-OMe (8c; Table, Entry 3). Condensation proceeded according to the GP with Z-Aib-OH
(0.237 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl·Phe-OMe (0.216 g, 1 mmol), and

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
959
NMM (0.11 ml, 1 mmol) to give crude 8c (0.301 g, 83%). HPLC (gradient): purity 93%. GC: tR 3.23
(Aib), 18.21 (d-Leu), 18.49 min (l-Leu); % l/% d 99.6 :0.4. M.p. 74–768 ([33]: 93–948). 1H-NMR
(CDCl3): 1.48 (s, Me2C); 3.11 (d, J¼5.6, PhCH2CH); 3.71 (s, MeO); 4.84 (q, J¼5.6, PhCH2CH); 5.07 (s,
PhCH2O); 5.17–5.31 (m, CONH); 6.66–6.93 (m, CONH); 7.07–7.28 (m, PhCH2CH); 7.33–7.39 (m,
PhCH2O).
Z-Aib-Aib-OMe (8d; Table, Entry 4). Condensation proceeded according to the GP with Z-Aib-OH
(0.237 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl·Aib-OMe (0.154 g, 1 mmol), and
NMM (0.11 ml, 1 mmol) to furnish crude 8d (0.255 g, 75%). M.p. 94–978 ([34]: 107–1098). HPLC
1
(gradient): purity 87%. H-NMR (CDCl3): 1.49 (s, 4 Me); 3.76 (s, MeO); 5.08 (s, PhCH2O); 5.22–5.27
(m, CONH); 6.86–6.91 (m, CONH); 7.28–7.36 (m, 5 arom. H).
Boc-Val-Aib-OMe (8e; Table, Entry 5). Condensation proceeded according to the GP with Boc-Val-
OH (0.237 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl·Aib-OMe (0.154 g, 1 mmol),
and NMM (0.11 ml, 1 mmol) to yield crude 8e (0.181 g, 57%). HPLC (gradient): purity 85%. GC: tR 4.79
(d-Val), 4.99 min (l-Val); % l/% d 99.0 :1.0; tR 3.21 (Aib). M.p. 134–1368 ([35]: 1408). 1H-NMR
(CDCl3): 0.92 (dd, J¼6.8, Me2CHCH); 1.42 (s, tBu); 1.52 (s, Me2C); 1.56–1.68 (m, Me2CHCH); 3.70 (s,
MeO); 3.77–3.85 (m, Me2CHCH); 5.01–5.07 (m, CONH); 6.47–6.54 (m, CONH).
Boc-Val-Leu-OMe (8f; Table, Entry 6). Condensation proceeded according to the GP with Boc-Val-
OH (0.237 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl Leu-OMe (0.182 g, 1 mmol),
and NMM (0.11 ml, 1 mmol) to affording crude 8f (0.260 g, 75%). HPLC (gradient): purity 96%. GC: tR
4.81 (d-Val), 5.04 min (l-Val); % l/% d 99.0 :1.0; tR 7.80 (d-Leu), 8.48 min (l-Leu); % l/% d 97:3. M.p.
1
t
115–1178 ([36]: 121–1248). H-NMR (CDCl3): 0.92–0.99 (m, 2 Me2CH); 1.45 (s, Bu); 1.58–1.71 (m,
Me2CHCH2); 2.09–2.23 (m, Me2CHCH); 3.73 (s, MeO); 3.89 (dd, J¼9, Me2CHCH); 4.58–4.67 (m,
COCH); 4.96–5.11 (m, CONH); 6.11–6.25 (m, CONH).
Boc-Leu-Val-OMe (8g; Table, Entry 7). Condensation proceeded according to the GP with Boc-
Leu-OH (0.231 g, 1mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl Val-OMe (0.168 g,
1 mmol),·and NMM (0.11 ml, 1 mmol) to yield crude 8g (0.299 g, 87%). HPLC (gradient): purity 70%.
GC: tR 7.37 (d-Leu), 7.97 min (l-Leu); % l/% d 98.0 :2.0; tR 4.57 (d-Val), 7.76 min (l-Val); % l/% d
1
t
99.7:0.3. M.p. 112–1158 ([37]: 1168). H-NMR (CDCl3): 0.89–0.96 (m, 4 Me); 1.44 (s, Bu); 1.49–1.53
(m, Me2CHCH2); 1.61–1.64 (m, Me2CHCH2 , Me2CHCH); 3.74 (s, MeO); 4.03–4.16 (m,
Me2CHCH2CH); 4.51–4.56 (m, CH); 4.85–4.88 (m, CONH); 6.52–6.58 (m, CONH).
Boc-Pro-Leu-OMe (8h; Table, Entry 8). Condensation proceeded according to the GP with Boc-
Pro-OH (0.265 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl·Leu-OMe (0.181 g,
1 mmol), and NMM (0.11 ml, 1 mmol) to give crude 8h (0.245 g, 71%). HPLC (gradient): purity 97%.
GC: tR 10.41 min (l-Pro); % l/% d 100 :0; tR 7.81 (d-Leu), 8.46 min (l-Leu); % l/% d 98.1:1.9. M.p. 94–
1
t
968 ([38]: 69–708). H-NMR (CDCl3): 0.92 (d, J ¼ 4.4, Me2CH); 1.47 (s, Bu); 1.54–1.75 (m, (CH2)2);
1.87–1.88 (m, Me2CHCH2CH); 2.09–2.37 (m, Me2CHCH2CH); 3.40–3.51 (m, CH2); 3.72 (s, MeO);
4.20–4.33 (m, Me2CHCH2CH); 4.23–4.34 (m, CH); 4.50–4.67 (m, CONH); 6.36–6.44 (m, CONH).
Z-Phe-Aib-Leu-OMe (8i; Table, Entry 9). Condensation proceeded according to the GP with Z-
Phe-Aib-COOH (0.384 g, 1 mmol), 4 (0.296 g, 1 mmol), DABCO (0.112, 1 mmol), HCl·Leu-OMe
(0.182 g, 1 mmol), and NMM (0.11 ml, 1 mmol) to afford crude 8i (0.470 g, 78%). HPLC (gradient):
purity 91%. GC: tR 18.18 (d-Phe), 18.54 min (l-Phe); % l/% d 99.2 :0.8; tR 3.22 (Aib), 7.69 min (d-Leu),
8.37 min (l-Leu); % l/% d 98.9 :1.1. M.p. 96–988. 1H-NMR (CDCl3): 0.92 (d, J¼6, Me2CH); 1.35 (s, 3 H,
Me2C); 1.43 (s, 3 H, Me2C); 1.51–1.71 (m, Me2CHCH2); 3.03 (d, J ¼ 7.5, CHCH2Ph); 3.71 (s, MeO); 4.25
(q, J¼7, Me2CHCH2CH); 4.51–4.50 (m, PhCH2CH); 5.08 (s, CH2Ph); 5.33 (d, J¼6, CONH); 6.04–6.06
(m, CONH); 6.83–6.89 (m, CONH); 7.18–7.39 (m, 10 arom. H).
This study was supported by the Ministry of Science and High Education under the Research Project
No. N204 326737 and Research Project No. N405 669540.

960
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Received October 30, 2012