coupling was monitored by HPLC. The results are presented in
Fig. 3. The reaction without addition of catalyst was a control
reaction, affording only 3% formation of an Aib᎐Aib bond.
Addition of HOBt or HOAt gave 6 and 38% conversion to
product respectively, whereas compound 7 afforded 43%
conversion to the peptide containing the Aib᎐Aib bond.
Compound 7 is therefore superior to HOAt in the catalysis of
αα-dialkylglycine-coupling reactions.
min. 1H and 13C NMR spectra were recorded on a 200 MHz or
300 MHz Bruker instrument. J-Values are given in Hz.
Hammer tests were performed on compounds 7–10 by placing a
2–4 mg sample of a compound on a block of iron and dropping
a hammer-head on the compound from height of 50 cm.
Compounds 7 and 8 were not explosive whereas compounds 9
and 10 both exploded.
The synthesis of fluorine-substituted analogs of compound 7
was attempted by a range of different strategies with both elec-
trophilic and nucleophilic substitution reactions; however, none
were successful.
1-Hydroxy-5-(methoxymethyl)-1,2,3-triazole 4
A solution of 1-benzyloxy-5-formyl-1,2,3-triazole 114 (163 mg,
0.80 mmol) and MeOH (10 cm3) was cooled to 0 ЊC and was
then treated with NaBH4 (41 mg, 1.08 mmol). After 40 min, the
solvent was removed in vacuo. The obtained solid was dissolved
in CH2Cl2 (15 cm3) and washed with saturated aq. NaCl (3 × 10
cm3). The organic phase was dried (MgSO4) and the solvent was
removed in vacuo to obtain 160 mg (97%) of 1-benzyloxy-5-
(hydroxymethyl)-1,2,3-triazole 2, Rf (EtOAc–heptane, 1:1)
0.05; mp 71–72 ЊC; δH (CDCl3) 7.49 (1 H, s), 7.42–7.31 (5 H, m),
5.47 (2 H, s), 4.33 (2 H, d, J 4.9) and 3.06 (1 H, br t, J ~5); δC
(CDCl3) 132.5 (s), 131.6 (s), 131.2 (d), 130.1 (d), 130.0 (d), 128.9
(d), 82.6 (t) and 52,1 (t) (Calc. for C10H11N3O2: C, 58.53; H,
5.40; N, 20.48%. Found: C, 58.60; H, 5.59; N, 20.24).
In conclusion, efficiency of the formation of a peptide bond
and measurement of the racemization during peptide coupling
have been determined using 6 new hydroxy-triazole and
-tetrazole derivatives and 3 commonly used compounds as
coupling catalysts in DIPCDI mediated couplings on a solid
phase. The compounds were modified 1-hydroxytriazoles and
N-hydroxytetrazoles.11–14 The synthesis of compounds 4 and 6
was achieved and afforded between 85% and 99% yield in the
four reaction steps reported. The results showed that three of
the new triazole and tetrazole derivatives could compete with
HOAt and HOBt. One of the catalysts was even found to be
superior to HOAt with respect to coupling efficiency. These
compounds contain only one five-membered ring and thus are
less sterically crowded. In particular, compound 7 could have an
advantage over HOAt and HOBt in coupling reactions, espe-
cially between sterically hindered amino acids such as Aib
residues. Furthermore, 5-chloro-1-hydroxy-1,2,3-triazole 7 was
a superior catalyst, giving faster coupling reactions than HOAt
but with less suppression of racemization. The results showed
that 2-hydroxytetrazole was as effective as HOAt in forming
peptide bonds. 2-Hydroxytetrazole afforded optical purity
comparable to that obtained with HOBt. 1-Hydroxytetrazole
had no significant catalytic activity but suppressed racemiz-
ation quite efficiently. CAUTION: The N-hydroxytetrazoles
were both explosive in a hammer test.
1-Benzyloxy-5-(hydroxymethyl)-1,2,3-triazole
2 (160 mg,
0.78 mmol) was dissolved in dry THF (5 cm3) and MeI (166 mg,
1.17 mmol) was added. The reaction mixture was cooled to
Ϫ78 ЊC and then NaH (24 mg, 1.0 mmol) was added. The cool-
ing bath was removed and the solution was stirred at room
temperature for 1 h. Water (2 cm3) was added and the solution
was extracted with CH2Cl2 (3 × 10 cm3). The organic phase was
dried (MgSO4) and the solvent was removed in vacuo. Purifica-
tion by column chromatography (EtOAc–heptane, 1:1) gave
145 mg (85%) of 1-benzyloxy-5-(methoxymethyl)-1,2,3-triazole
3, Rf (EtOAc–heptane, 1:1) 0.43; δH (CDCl3) 7.58 (1 H, s),
7.46–7.22 (5 H, m), 5.45 (2 H, s), 4.17 (2 H, s) and 3.25 (3 H, s);
δC (CDCl3) 132.2 (s), 131.7 (s), 129.7 (d), 129.5 (d), 128.5 (d),
128.3 (d), 82.4 (t), 60.8 (q) and 57.8 (t).
1-Benzyloxy-5-(methoxymethyl)-1,2,3-triazole 3 (315 mg,
1.43 mmol) was dissolved in EtOH (20 cm3), the solution was
cooled to 0 ЊC, 10% palladium on charcoal (50 mg) was added,
and the reaction mixture was stirred under hydrogen for 30 min.
After filtration through Celite and evaporation in vacuo, 182 mg
(99%) of 1-hydroxy-5-(methoxymethyl)-1,2,3-triazole 4 was
obtained, mp 108–110 ЊC; δH (D2O) 8.04 (1 H, s), 4.53 (2 H, s)
and 3.36 (3 H, s); δC (D2O) 128.7 (s), 127.4 (d), 61.4 (q) and 58.6
(t) (Calc. for C4H7N3O2: C, 37.21; H, 5.46; N, 32.54%. Found:
C, 37.52; H, 5.25; N, 32.30).
Experimental
General
Analytical TLC was performed on Merck silica gel 60F254
alumina sheets with detection of the protected triazoles by
charring with mostain [Ce(SO4)2ؒH2O (0.4 g) and
(NH4)6Mo7O24 (20 g) dissolved in 10% H2SO4 (400 ml)]. All
organic solvents were purchased from Labscan Ltd. (Dublin,
Ireland). Concentrations were performed under reduced
pressure at temperatures <40 ЊC. Suitable protected Nα-Fmoc-
amino acids, Z-Phe-Val and the Rink-amide-linker were pur-
chased from Nova Biochem (Switzerland); TBTU, NaBH4,
DIPCDI, HOBt and Dhbt-OH from Fluka (Switzerland);
NEM from Merck (Germany); NaCl and MgSO4 from Aldrich;
and HOAt from Millipore. The optical purity of Z-Phe-Val-
Pro-NH2 was analyzed by analytical HPLC. The catalysts
1-hydroxy-1,2,3-triazole 812,14 5-chloro-1-hydroxy-1,2,3-triazole
711,14 1-hydroxytetrazole 10,12 and 2-hydroxytetrazole 912 were
prepared as previously described. The relative molecular masses
of the peptide compounds were determined, using matrix-
assisted laser desorption time-of-flight mass spectroscopy
(MALDITOF-MS), recorded on a Lasermat 2000 (Finnigan
Mat) using a matrix of α-cyano-4-hydroxycinnamic acid. Quan-
titative amino acid analyses were performed on a Pharmacia
LKB Alpha Plus amino acid analyser following hydrolysis with
6 HCl at 110 ЊC for 36 h. Analytical HPLC was performed
using a Waters RCM 8 × 10 module and with a Deltapak C-18
column (19 × 300 mm). The solvent system for the analytical
HPLC was buffer A; 0.1% TFA in water and buffer B; 0.1%
TFA in 90% acetonitrile–10% water and UV detection was at
215 and 280 nm. The gradient for analytical HPLC (1 cm3
minϪ1); a linear gradient of 0–100% buffer B over a period of 50
5-Acetyl-1-hydroxy-1,2,3-triazole 6
5-Acetyl-1-benzyloxy-1,2,3-triazole 5 (138 mg, 0.64 mmol)13
was dissolved in MeOH (10 cm3) and the solution was cooled to
0 ЊC. Then, palladium on charcoal (30 mg) was added and the
reaction mixture was stirred under hydrogen for 30 min. After
filtration through Celite and evaporation in vacuo 76 mg (93%)
of 5-acetyl-1-hydroxy-1,2,3-triazole 6 was obtained, mp 143–
145 ЊC (decomp.); δH (CD3OD) 8.40 (1 H, s) and 2.65 (3 H, s);
δC (CD3OD) 186.2 (s), 132.3 (d), 128.7 (s) and 27.22 (q) (Calc.
for C4H5N3O2: C, 37.80; H, 3.97; N, 33.06%. Found: C, 37.91;
H, 3.81; N, 32.71).
Solid-phase synthesis and analysis, general procedure
The synthesis of the peptides using the PEGA resin15 were per-
formed by the plastic syringe technique24 or a 20-column
library generator.25 The protected Nα-Fmoc--amino acid
pentafluorophenyl (OPfp) esters (3 equiv.)22 were coupled in
DMF with the addition of Dhbt-OH (1 equiv.) as an acylation
catalyst and an indicator of the end-point of the acylation reac-
tion. The dipeptide Z-Phe-Val was coupled as its free acid using
TBTU in situ activation22 to prepare the two diastereoisomers
Z-Phe-Val-Pro-NH2 and Z-Phe-val-Pro-NH2.7 The Nα-Fmoc
group was removed by 20% piperidine in DMF. The resin was
1730
J. Chem. Soc., Perkin Trans. 1, 1998