Synthesis of orthogonally protected azahistidine: Application to the synthesis of a GHK analogue

Article abstract of DOI:10.1007/s00726-009-0248-5

The synthesis of various orthogonally protected azahistidine derivatives are obtained via 1,3-dipolar cycloaddition reactions. The newly obtained amino-acids can be selectively deprotected either at the side chain or at the N-terminus of the amino acid and should thus allow the use of these derivatives in (solid phase) peptide synthesis.

Full text of DOI:10.1007/s00726-009-0248-5

Amino Acids (2010) 38:279–286
DOI 10.1007/s00726-009-0248-5
ORIGINAL ARTICLE
Synthesis of orthogonally protected azahistidine: application
to the synthesis of a GHK analogue
´
Stephane Roux Æ Melinda Ligeti Æ David-Alexandre Buisson Æ
Bernard Rousseau Æ Jean-Christophe Cintrat
Received: 1 July 2008 / Accepted: 15 January 2009 / Published online: 15 February 2009
ꢀ Springer-Verlag 2009
Abstract The synthesis of various orthogonally protected
azahistidine derivatives are obtained via 1,3-dipolar
cycloaddition reactions. The newly obtained amino-acids
can be selectively deprotected either at the side chain or at
the N-terminus of the amino acid and should thus allow the
use of these derivatives in (solid phase) peptide synthesis.
applications. One of the most obvious parent compounds is
azahistidine where the imidazole ring has been replaced by
a 1,2,3-triazole (Fig. 1).
Recent applications based on this analogue include the
synthesis of 99mTc radiolabelled amino-acids as well as
metal chelating compounds (Mindt et al. 2006). It has also
been demonstrated that histidine-auxotrophic bacteria
(E. coli) can be supplemented with compound 1 leading to
in vivo incorporation of this amino-acid in proteins (Ikeda
et al. 2003).
Keywords Click chemistry ꢀ Huisgen cycloaddition ꢀ
Triazole ꢀ Azahistidine ꢀ GHK
Although the preparation of such a histidine surrogate
has been already described (Losikina and Sokolov 1991),
to the best of our knowledge, the synthetic routes published
to date to obtain the fully deprotected amino-acid are
usually rather long (seven steps) (Ikeda et al. 2003).
Introduction
Unnatural a-amino-acids are key components for recent
developments in peptides or proteins research. These syn-
thetic compounds have been incorporated in biologically
active peptides mostly to enhance proteolytic stability or to
confer peculiar properties to these peptides. In addition,
conformational flexibility, selectivity, pharmacokinetics
and bioavailability can also be tuned based on well-
designed amino-acids. In the field of nanotechnology, there
is also a growing interest in synthetic amino-acids that can
present specific properties (metal binding ability, poly-
merisation precursors, electron transfer…). Among the
natural amino-acids, histidine is often strongly involved in
the chemistry of enzymes due to the ability of the imid-
azole side chain to take part in acid-base catalysis. Indeed,
histidine can act both as a donor or an acceptor of protons:
developing analogues could pave the way to new
Materials and methods
Chemicals
Amino acid derivatives were purchased from Bachem,
Fluka and Acros Organics. Reagents and solvents for
synthesis and RP-HPLC were from Sigma, Fluka or
Normapur products and used without further purification.
Aryl-azide derivatives were prepared by standard protocol.
Equipment
LC/MS analysis
S. Roux ꢀ M. Ligeti ꢀ D.-A. Buisson ꢀ B. Rousseau ꢀ
J.-C. Cintrat (&)
CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage,
91191 Gif sur Yvette Cedex, France
e-mail: jean-christophe.cintrat@cea.fr
HPLC Waters system (2525 binary gradient module,
in-line degasser, 2767 sample manager, 2996 Photodiode
Array Detector), eluents: A: 99.9% water/0.1% HCOOH;
B: 99.9% ACN/0.1% HCOOH.
123

280
S. Roux et al.
Fig. 1 Triazole analogue of
histidine
Boc-b-(1-(4-methoxybenzyl)-1,2,3-triazol-4-yl)-Alanine
3 was purified by flash chromatography using an
isocratic elution consisting in DCM/MeOH/acetic acid
90:10:1. The fractions were pooled, evaporated and
dissolved in chloroform and crystallised from hexane.
Boc-b-(1-(3,5-dimethoxybenzyl)-1,2,3-triazol-4-yl)-Ala-
nine 4 was obtained as a colourless oil without
crystallization.
Analytical RP-HPLC column: X-bridge C18 column
˚
(100 9 4.6 mm, 3.5 lm particle size, 135 A pore size),
Boc-b-(1-pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine
5 was purified by flash chromatography using an
isocratic elution consisting in DCM/MeOH/acetic acid
96:4:5%.
1 ml/min flow rate, 20 ll sample is injected.
Preparative RP-HPLC column: X-bridge C18 column
˚
(150 9 19 mm, 5 lm particle size, 135 A pore size),
17 ml/min flow rate.
Fmoc-b-((1-benzyl)-1,2,3-triazol-4-yl)-Alanine
6 was
Using a 8 or 25 min gradient of 95% A/5% B to 100% B
for analysis and purification, respectively.
dissolved in ethyl acetate and crystallised with pentane.
Fmoc-b-(1(-4-methoxybenzyl)-1,2,3-triazol-4-yl)-Alanine
7 was dissolved in chloroform and crystallised with
hexane.
MS Waters Micromass ZQ system (electrospray ioni-
zation), ZQ2000 quadrupole analyser, Mass Lynx 4.0
software, source temperature 120ꢁC, cone voltage 20 V,
continuous sample injection at 0.3 ml/min flow rate, mass
spectra were recorded in positive ion mode in the m/z 100–
1,000 range.
Fmoc-b-(1-(3,5-dimethoxybenzyl)-1,2,3-triazol-4-yl)-
Alanine 8 was dissolved in ethyl acetate and crystallised
with cyclohexane.
Fmoc-b-(1-pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine
9 was purified by flash chromatography using an
isocratic elution consisting in DCM/MeOH/acetic acid
97:3:5%.
NMR analysis
1H- and 13C-NMR spectra were recorded at room temper-
ature at a frequency of 400 and 100 MHz, respectively,
using a Bruker Avance 400 Ultrashield. Samples were
dissolved in 400 ll of deuterated solvent. Chemical shifts
are given in ppm and the coupling constants in Hz.
General method (B): synthesis of Ac-(1-benzyl)-1,2,3-
triazol-5-yl)-Ala-OEt 10
In a 50 ml two-necked round bottom flask was introduced
10 ml of anhydrous toluene. This solvent was then degas-
sed by three vacuum freeze/thaw cycles. 100 mg
(0.54 mmol) of N-acetylpropargylglycine ethyl ester were
then introduced together with 145 mg (1.09 mmol, 2 eq.)
of benzyl azide and the mixture was degassed once more.
Finally 21.7 mg (27.3 lmol, 0.05 eq.) of Cp*RuCl(PPh3)2
were introduced and the mixture was degassed once again.
The reaction mixture was then heated to 80ꢁC under
nitrogen for 15 h. The resulting mixture was then evapo-
rated to afford 205.9 mg of crude product. This product
was dissolved in acetonitrile and filtered. The liquid was
further purified by preparative HPLC using a water/aceto-
nitrile/formic acid gradient, yielding 97.12 mg of viscous
oil for a yield of 44%.
General method (A): synthesis of Boc-b-(1-benzyl)-
1,2,3-triazol-4-yl)-Alanine 2
N-Boc-propargylglycine, measuring 107 mg (0.5 mmol)
was introduced in a 100 ml round-bottom flask in 15 ml of
tBuOH together with 70 mg (0.5 mmol, 1 eq.) of benzy-
lazide. 10 mg (0.05 mmol, 0.01 eq.) of Cu(OAc)2 ꢀ xH2O
dissolved in 3 ml of water and 20 mg (0.1 mmol, 0.02 eq.)
of Na-ascorbate in 2 ml of water were further introduced.
The mixture was stirred overnight at room temperature.
tBuOH was then removed by rotary-evaporation and the pH
of the resulting aqueous mixture was adjusted to 2 with a
1 M HCl solution. The aminoacid was extracted three
times with ethylacetate (50 ml) and the organic phases,
pooled together were washed with brine and dried over
anhydrous MgSO4. After filtration, the crude product was
evaporated and dissolved in ethyl acetate. After crystalli-
sation in cyclohexane and filtration 104.3 mg of the
Selective removal of BOC protecting group, affording
H-b-((1-pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine
12 (Stahl et al. 1978)
expected Boc-b-(1-benzyl)-1,2,3-triazol-4-yl-Alanine
were obtained (60% yield).
2
HCl 35% measuring 2.2 ml was introduced on 100 mg
(0.27 mmol) of Boc-b-(1-pivaloyloxymethyl)-1,2,3-tria-
zol-4-yl)-Ala-OH 5 in 7.8 ml of ethyl acetate. Under
Purification conditions for the other derivatives were as
follows:
123

Synthesis of orthogonally protected azahistidine
281
stirring at R.T. the reacting mixture, initially biphasic,
turned rapidly to a homogenous yellowish liquid. After 3 h,
the mixture is diluted in water and washed three times with
dichloromethane. The aqueous phase was then freeze dried
to obtain 94 mg of a yellowish solid.
Pd(OH)2 on charcoal (20% pure, 50% water in weight) in
2 ml of MeOH. The tank was flushed with N2 for 5 min
and then 10 bars of H2 were admitted. The reaction was
stirred at 45ꢁC over 72 h.
The crude mixture was clarified on a syringe Millipore
separation filter and analysed by LC/MS, revealing a peak
of expected m/z accounting for more than 90% of the total
UV signal area
The crude product was purified by preparative HPLC
using a water/acetonitrile gradient with formic acid as ion
pairing agent. 32.2 mg of pure white solid were obtained
with a yield of 44%.
Boc-b-(1-benzyl)-1,2,3-triazol-4-yl)-Alanine 2 1H NMR
t
(400 MHz; CDCl3): d 1.27 (s, 9H, Bu), 3.15 (br. s, 2H,
CbHH0), 4.32 (bs, 1H, CaH), 5.32 (d, J = 15.0, 1H, CH-
Bzl), 5.38 (d, 1H, J = 15.0, CH0-Bzl), 5.97 (br. s, 1H,
HNCa), 7.13-7.19 (m, 2H, Bzl), 7.23-7.28 (m, 3H, Bzl),
7.32 (br. s, 1H, CH triazol).
Selective removal of Fmoc protectivng group, affording
H-b-((1-pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine 12
Ninety-nine milligram (0.20 mmol) of Fmoc-b-(1-piva-
MS (ESI): 347.1 (M ? H)?
loyloxymethyl)-1,2,3-triazol-4-yl)-Ala-OH
9
were
introduced in 5 mL of DMF with 13.5 ll (0.22 mmol,
1.1 eq.) of piperidine. The mixture was stirred at R.T. for
3 h 45 min and evaporated to obtain a white solid. The
crude product was then triturated with diethyl ether to
remove the dibenzofulvene-piperidine adduct. After dry-
ing, 29.3 mg (53%) of purified solid were obtained.
Boc-b-(1-(4-methoxybenzyl)-1,2,3-triazol-4-yl)-Alanine
3
1H NMR (400 MHz; CDCl3): d 1.42 (s, 9H, tBu), 3.15-
3.25 (m, 1H, CbH), 3.29-3.39 (m, 1H, CbH0), 3.81 (s, 3H, p-
methoxy), 4.49 (bs, 1H, CaH), 5.43 (s, 2H, CH2-Bzl), 5.73
(br. s, 1H, HNCa), 6.90 (d, J = 8.4, 2H, H-3,5 Bzl), 7.22
(d, J = 8.4 Hz, 2H, H-2,6 Bzl), 7.31 (br. s, 1H, CH triazol)
MS (ESI): 377.1 (M ? H)?
Boc-b-(1-(3,5-dimethoxybenzyl)-1,2,3-triazol-4-yl)-Alanine
4
Selective removal of POM protecting group, affording
Boc-b-(1, 2, 3-triazol-4-yl)-Alanine 13 (Bock et al. 2006)
1H NMR (400 MHz; CD3OD): d 1.36 (s, 9H, tBu), 3.05
(dd, J = 14.9, J = 4.5, 1H, CbH), 3.25 (dd, J = 14.9,
J = 8.9, 1H, CbH0), 3.74 (s, 6H, m,m-dimethoxy), 4.37 (dd,
J = 8.9, J = 4.5, 1H, CaH), 5.48 (s, 2H, CH2-Bzl), 6.43
(bs, 3H, Bzl), 7.73 (s, 1H, CH triazol)
150 mg (0.40 mmol) of Boc-b-(1-(pivaloyloxymethyl)-
1,2,3-triazol-4-yl)-Ala-OH 5 were added to 2.1 ml of
methanol with 2.5 ml of KOH 1 M (2.5 mmol, 6.2 eq.).
The mixture was stirred for 1 day at R.T., then acidified to
pH 3 and extracted with ethyl acetate. The organic
fractions were pooled and dried with MgSO4. After evap-
oration 92.6 mg of amorphous solid were obtained with a
yield of 61%.
MS (ESI): 407.1 (M ? H)?
Boc-b-(1-(pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine
t
5
1H NMR (400 MHz; d6-DMSO): d 1.11 (s, 9H, Bu),
t
1.45 (s, 9H, Bu), 2.94 (dd, 1H, J = 14.5, J = 9.7, CbH),
3.09 (dd, 1H, J = 14.5, J = 4.1, CbH), 4.10–4.19 (m, 1H,
CaH), 6.27 (s, 2H, CH2 POM), 7.08 (d, 1H, J = 7.9, NH),
7.94 (s, 1H, CH triazol).
Selective removal of benzyl protecting group
13C NMR {1H} (100 MHz, d6-DMSO): d 26.87, 27.44,
28.53, 38.60, 53.80, 70.27, 78.52, 124.45, 144.14, 155.76,
173.52, 176.84.
•
affording Ac-b-(1,2,3-triazol-5-yl)-Ala-OEt
30 mg (95 lmol) of Ac-b-(1-benzyl)-1,2,3-triazol-5-yl)-
Ala-OEt 10 were introduced in 1.5 ml of MeOH into a one-
neck round-bottom flask. 101.3 mg (95 lmol, 1 eq.) of
palladium 10% on charcoal were added and the mixture
was put under H2 atmosphere and stirred at R.T. during
72 h. The crude mixture was then filtered through celite
and a Millipore HVLP membrane, to yield to 22.9 mg of
product. The yield was quantitative.
MS (ESI): 371.7 (M ? H)?
IR (KBr): 3386, 3151, 2981, 2938, 2576, 1747, 1716,
1517 cm-1
Fmoc-b-((1-benzyl)-1,2,3-triazol-4-yl)-Alanine 6 1H NMR
(400 MHz; CDCl3): d 3.36 (dd, J = 14.8, J = 5.4, 1H,
CbH), 3.42 (dd, J = 14.8, J = 3.8, 1H, CbH0), 4.16
(t, J = 6.6, 1H, CH Fmoc), 4.30–4.41 (m, 2H, CH2 Fmoc),
4.57–4.66 (m, 1H, CaH), 5.37 (d, J = 14.9, 1H), 5.51 (d,
J = 14.9, 1H, CH2-Bzl), 5.93 (d, J = 5.6, 1H, HNCa),
7.12–7.20 (m, 2H, Ar), 7.23 (bs, 1H, CH triazol), 7.27–7.33
(m, 5H, Ar), 7.34–7.44 (m, 3H, Ar), 7.55 (d, J = 7.3, 2H,
Ar), 7.74 (t, J = 8.2, 2H, Ar).
•
affording Boc-b-(1,2,3-triazol-4-yl)-Alanine
In a pressure tank, 5 mg (14.4 lmol) of Boc-b-(1-ben-
zyl)-1,2,3-triazol-4-yl)-Alanine
2 were introduced to
3.84 mg (3.6 lmol, 0.25 eq.) of Pearlman catalyst i.e.
123

282
S. Roux et al.
13C NMR {1H} (400 MHz; CDCl3): d 27.51, 47.10,
3.16 (dd, J = 15.8, J = 6.2, 1H, CbH0), 4.12–4.24 (m, 2H,
CH2 ethyl), 4.73 (ddd, J = 7.1, J = 6.2, J = 5.9, 1H,
HCa), 5.46 (d, J = 16.0, 1H, CH-Bzl), 5.60 (d, J = 16.0,
1H, CH0-Bzl), 6.38 (d, J = 7.1, 1H, NH), 7.12–7.20 (m,
2H, Bzl), 7.30–7.38 (m, 3H, Bzl), 7.43 (s, 1H, CH triazol)
13C NMR {1H} (100 MHz, CDCl3): d 14.00, 22.95,
25.72, 51.28, 51.65, 51.18, 127.15, 128.37, 128.97, 132.20,
133.36, 134.70, 170.05, 170.51.
53.25, 54.35, 66.78, 119.93, 123.18, 125.10, 127.06,
127.69, 127.92, 128.80, 129.08, 134.03, 141.19, 142.33,
143.67, 155.72, 172.55.
MS (ESI): 469.1 (M ? H)?
Fmoc-b-(1(-4-methoxybenzyl)-1,2,3-triazol-4-yl)-Alanine
7
1H NMR (400 MHz; CDCl3): d 3.28 (dd, J = 14.8,
J = 6.7, 1H, CbH), 3.40 (bd, J = 14.8, 1H, CbH0), 3.74 (s,
3H, p-methoxy), 4.19 (t, J = 6.9, 1H, CH Fmoc), 4.37 (d,
J = 6.9 Hz, 2H, CH2 Fmoc), 4.59 (bs, 1H, CaH), 5.35 (d,
J = 14.8, 1H, CH-Bzl), 5.46 (d, J = 14.8, 1H, CH-Bzl),
5.95 (d, J = 5.4, 1H, HNCa), 6.84 (d, J = 8.2, 2H, Ar),
7.16 (d, J = 8.6, 2H, Ar), 7.21 (s, 1H, CH triazol), 7.28–
7.34 (m, 2H, Ar), 7.37–7.43 (m, 2H, Ar), 7.55–7.61 (m, 2H,
Ar), 7.74–7.80 (m, 2H, Ar).
MS (ESI): 317.2 (M ? H)?
Ac-b-(1-(3,5-dimethoxybenzyl)-1,2,3-triazol-5-yl)-Ala-
OEt 11 1H NMR (400 MHz; CDCl3): d 1.21 (t, J = 7.1,
3H, CH3 ethyl), 1.96 (s, 3H, acetyl), 3.08 (dd, J = 15.8,
J = 5.5, 1H, CbH), 3.15 (dd, J = 15.8 Hz, J = 6.4 Hz,
1H, CbH0), 3.71 (s, 6H, m,m-dimethoxy), 4.13 (qd, J = 9.6,
J = 7.1, 1H, CH ethyl), 4.18 (qd, J = 9.6, J = 7.1, 1H,
CH ethyl), 4.73 (ddd, J = 7.1, J = 6.4, J = 5.5, 1H, CaH),
5.38 (d, J = 15.6, 1H, CH-Bzl), 5.51 (d, J = 15.6, 1H,
CH-Bzl), 6.27 (d, J = 2.1, 2H, Bzl), 6.35 (t, J = 2.1, 1H,
Bzl), 6.59 (d, J = 7.1, 1H, NH), 7.41 (s, 1H, CH triazol).
MS (ESI): 377.1 (M ? H)?
MS (ESI): 499.1 (M ? H)?
Fmoc-b-(1-(3,5-dimethoxybenzyl)-1,2,3-triazol-4-yl)-Ala-
nine 8 1H NMR (400 MHz; CD3CN): d 3.10 (dd,
J = 14.9, J = 7.6, 1H, CbH), 3.20 (dd, J = 14.9, J = 4.7,
1H, CbH0), 3.70 (bs, 6H, m,m-dimethoxy), 4.20 (t, J = 7.1,
1H, CH Fmoc), 4.30 (d, J = 7.1, 2H, CH2 Fmoc), 4.41–
4.46 (m, 1H, CaH), 5.40 (s, 2H, CH2-Bzl), 6.38 (bs, 3H,
Bzl), 7.32 (t, J = 7.5, 2H, Fmoc), 7.42 (dd, J = 7.5,
J = 7.1, 2H, Fmoc), 7.58–7.64 (m, 3H, Fmoc ? CH tria-
zol), 7.83 (d, J = 7.5, 2H, Fmoc).
H-b-(1-(pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine
t
12 1H NMR (400 MHz; D2O): d 1.26 (s, 9H, Bu), 3.30
(dd, 1H, J = 15.8, J = 6.8, CbH), 3.36 (dd, 1H, J = 15.8,
J = 5.2, CbH), 4.06 (dd, 1H, J = 6.8, J = 5.2, CaH), 6.33
(s, 2H, CH2 POM), 8.06 (s, 1H, CH triazol).
13C NMR {1H} (100 MHz, D2O): d 25.81, 26.16, 38.41,
54.16, 71.22, 125.42, 142.14, 172.91, 179.74.
MS (ESI): 271.1 (M ? H)?
MS (ESI): 529.2 (M ? H)?
Fmoc-b-(1-pivaloyloxymethyl)-1,2,3-triazol-4-yl)-Alanine
t
9
1H NMR (400 MHz; d6-DMSO): d 1.07 (s, 9H, Bu),
IR (KBr): 3142, 3044, 2969, 2591, 2088, 1740, 1617,
1586 cm-1
3.00 (dd, 1H, J = 14.5, J = 9.6, CbH), 3.15 (dd, 1H,
J = 14.5, J = 4.6, CbH0), 3.30 (s, 1H, CH Fmoc), 4.10–
4.30 (m, 3H, CaH ? CH2 Fmoc), 6.26 (s, 2H, CH2 POM),
7.31 (bt, J = 7.6, 2H, Fmoc), 7.41 (bt, 2H, J = 7.4, Fmoc),
7.67 (bt, 2H, J = 6.2, Fmoc), 7.88 (d, 1H, J = 7.6 triazol),
7.96 (s, 1H, CH triazol).
13C NMR {1H} (100 MHz, CDCl3): d 26.72, 27.47,
38.73, 47.05, 53.18, 67.06, 69.81, 119.93, 124.57, 124.89,
125.08, 127.07, 127.68, 141.20, 141.21, 142.66, 143.64,
143.73, 155.89, 172.75, 177.65.
Boc-b-(1,2,3-triazol-4-yl)-Alanine
13 1H
NMR
t
(400 MHz; CD3OD): d 1.37 (s, 9H, Bu), 3.06 (dd, 1H,
J = 14.9, J = 8.7, CbH), 3.24 (dd, 1H, J = 14.9, J = 4.6,
CbH), 4.38 (dd, J = 8.7, J = 4.6, CaH), 7.57 (s, 1H, CH
triazol).
13C NMR {1H} (100 MHz, d6-DMSO): d 26.72, 28.15,
53.39, 78.19, 131.5, 142.8, 155.30, 173.08
MS (ESI): 257.1
IR (KBr): 3159, 3981, 2602, 2360, 1694, 1520 cm-1
MS (ESI): 493.1 (M ? H)?
IR (KBr): 3411, 3129, 2966, 1738, 1725, 1529 cm-1
Ac-b-(1,2,3-triazol-5-yl)-Ala-OEt from 10
Ac-b-(1-benzyl)-1,2,3-triazol-5-yl)-Ala-OEt 10 1H NMR
(400 MHz; CDCl3): d 1.22 (t, J = 7.2, 3H, CH3 ethyl),
1.96 (s, 3H, acetyl), 3.10 (dd, J = 15.8, J = 5.9, 1H, CbH),
AcHN
CO2Et
H
N
N
N
1H NMR (400 MHz; CDCl3): d 1.19 (t, J = 7.0, 3H,
CH3 ethyl), 1.89 (s, 3H, acetyl), 3.10–3.30 (m, 2H,
CbHH0), 4.13 (q, J = 7.0, 2H, CH2 ethyl), 4.86 (bs, 1H,
CaH), 7.18 (bs, 1H, CaMH), 7.41 (s, 1H, CH triazol),
7.75 (bs, NH triazol).
Scheme 1 Azahistidine analogues via [3 ? 2] Huisgen cycloaddition
123

Synthesis of orthogonally protected azahistidine
283
Table 1 Synthesis of azahistidine analogues
Entry
1
Propargyl glycine derivative
Azide
Conditions
A
Product
Yield (%)
60
Bn–N3
2
3
4
PMB–N3
DMB–N3
A
A
A
40
15
73
5
6
Bn–N3
A
A
70
40
PMB–N3
7
DMB–N3
A
30
8
9
A
B
57
84
Bn–N3
123

284
S. Roux et al.
Table 1 continued
Entry
10
Propargyl glycine derivative
Azide
Conditions
B
Product
Yield (%)
44
DMB–N3
t
Conditions A Cu(OAc)2/sodium ascorbate in BuOH/H2O at r.t.; Conditions B Cp*RuCl(PPh3)2 in toluene at 80ꢁC
Gly-D-azaHis-D-Lys 14
The synthesis of the tripeptide was achieved according to a
literature protocol using DIC and HOBt as coupling
reagents (Liakopoulou et al. 1997). Fmoc-Lys(Mtt)-OH
and Fmoc-His(Mtt)-OH were replaced by Fmoc-D-Lys(-
Boc)-OH and Fmoc-D-azaHis(POM)-OH, respectively.
Cleavage from the resin was performed using 10 ml of a
Scheme 3 Selective deprotection of Fmoc-protected azahistidine
derivative 9
cocktail composed of TFA/TIS/H2O (9.5/0.3/0.2) for 4
hours at room temperature. After filtration, TFA was
removed by evaporation, the resulting solid was dissolved
in water and the solution was lyophilized. About 100 mg of
the peptide TFA salt was then subjected to deprotection
(Fmoc and POM) by addition of 650 ll of a 1 M NaOH
aqueous solution (2.2 eq.) in 3.5 ml of MeOH for 5 hours
at room temperature. After addition of HCl (1 M) to reach
pH 4, the solution was evaporated and the solid was taken
up in MeOH. NaCl was filtrated and the resulting solution
was evaporated. The resulting solid was triturated in cold
diethyl ether yielding peptide 14 (yield 36%) as a white
Results and discussion
In order to efficiently use such a compound in (solid phase)
peptide synthesis, easy access to orthogonally protected
derivatives is strongly a necessity. Although there have been
some routes to protected 1 or derivatives, none of them deals
with the orthogonally protected analogues. The recent
development of the copper catalysed version of Huisgen
cycloaddition (so called click chemistry) allows the rapid
access to 1,2,3-triazoles (Kolb et al. 2001; Lipshutz and Taft
2006; Bock et al. 2006). Using this approach, azahistidine
derivatives have been obtained but no real attention has been
paid to the removal of the protecting groups. In this paper, we
describe the click chemistry based access to fully protected
azahistidine, suitable for peptide synthesis and we briefly
assay the deprotection steps (Scheme 1).
1
solid with a purity of 90% (estimated by LC/MS and H
NMR).
1H NMR (400 MHz; D2O): d 1.19–1.31 (m, 2H, CH2 d
Lys), 1.50–1.80 (m, 4H, CH2 b and c Lys), 2.88 (t, 2H,
J = 7.2 CH2 e Lys), 3.13 (dd, 1H, J = 7.7, J = 15.2, CH2
b azaHis), 3.20 (dd, 1H, J = 6.2, J = 15.2, CH2 b azaHis),
3.74 (AB system, 2H, J = 16.1, CH2 Gly), 4.13 (dd, 1H,
J = 5.4, J = 7.5, CH a Lys), 4.51–4.82 (m, 1H superim-
posed with water, CH a azaHis), 7.68 (s, 1H, CH triazol).
13C NMR {1H} (100 MHz, D2O): d 21.84, 26.14, 26.69,
30.36, 39.08, 40.24, 53.26, 53.84, 127.29, 140.06, 166.8,
171.58, 176.73.
Azahistidines were obtained using either copper-cata-
lysed click chemistry or the more recently described
ruthenium-catalysed cycloaddition (Zhang et al. 2005;
Oppilliart et al. 2007), affording the 1,5-disubstituted tri-
azole. Nevertheless, the latter requires the fully protected
propargylglycine as starting material, since the catalyst
does not allow the use of free carboxylic acids. Using
HRMS (ES-) calcd for C13H22N7O4:340.1733;
found:340.1723.
Scheme 2 Selective
deprotection of Boc-protected
azahistidine derivative 5
123

Synthesis of orthogonally protected azahistidine
285
Scheme 4 SPPS of a GHK
analogue, Gly-D-azaHis-D-Lys
either condition, rapid access to protected azahistidines was
demonstrated and the expected regioisomers were obtained
with fair to satisfactory yields (Table 1).
cycloaddition starting from tBoc or Fmoc protected propar-
gylglycine yielded the expected pivaloyloxymethyl (POM)
azahistidine cleanly (Entries 4 and 8).
Using the copper-catalyzed version of the reaction, we
t
were able to react either Fmoc- or Boc- protected prop-
At this stage, selective deprotection of the tBoc group on
compound 5 was achieved using HCl in ethyl acetate to
furnish 12. In parallel, selective removal of the pivaloyl
group was cleanly achieved using 1 M NaOH in MeOH
argylglycine with a range of azides (Entries 1–8) yielding
the expected triazoles. It was found that the purification of
the acid products was sometimes difficult resulting in low
yields. When the N-acetyl, ethyl ester of propargylglycine
was used as a starting material, in conjunction with
ruthenium catalyst, the expected 1,5-regioisomer was also
cleanly obtained (Entries 9 and 10).
t
without affecting the Boc group to afford compound 13
(Scheme 2).1
In a similar fashion, when compound 9 was treated
under classical conditions for removal of the Fmoc pro-
tecting group (piperidine), we obtained compound 12. This
last result should permit the use of compound 9 for inser-
tion of azahistidine derivatives in peptides using standard
SPPS strategies (Scheme 3).
With the above histidine analogues in hand, the next step
was to selectively remove the protective group. As expected
the hydrogenolysis of benzyl, p-methoxybenzyl and dime-
thoxybenzyl derivatives turned out to be very difficult. By
using harsh conditions we managed to remove the protecting
groups on compounds 2 and 10 but the same protocols
applied to compounds bearing a Fmoc protecting group (6–
8) afforded debenzylated compounds along with partial
hydrogenation of the benzyl ring as well as removal of Fmoc
group. Alternative, oxidising conditions using CAN were not
satisfactory. We then decided to test the recently proposed
protected azide by Sharpless id est azidomethyl pivalate
(Loren et al. 2005). Noteworthy is that this protecting group
has been recently used in a very elegant synthesis of
a-CF3-substitued azahistidine (Shchetnikov et al. 2007). The
As an example, compound 9 was used for the solid
phase synthesis of a Gly-His-Lys analogue, an endogenous
tripeptide known as a growth-modulating factor, as a strong
activator of wound healing and as a copper chelator. The
1
As suggested by one of the referees, we checked the potential
racemization of the azahistidine compounds under the deprotection
conditions. Treatment of compound 5 (5 mg) overnight with NaOD/
D2O (100 ll of a 40% solution) in CD3OD (400 ll) resulted in
removal of the POM group without incorporation of deuterium at the
alpha position (checked by 1H NMR). This finding was moreover
corroborated by the synthesis of the G-azaH-K peptide that resulted in
no epimerisation of the two D-amino acids.
123

286
S. Roux et al.
Ikeda Y, Kawahara S-I, Taki M, Kuno A, Hasegawa T, Taira K
(2003) Synthesis of a novel histidine analogue and its efficient
incorporation into a protein in vivo. Protein Eng 16:699–706
Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse
chemical function from a few good reactions. Angew Chem Int
Ed Engl 40:2004–2021
Liakopoulou-Kyriakides M, Pachatouridis C, Ekateriniadou L,
Papageorgiou VP (1997) A new synthesis of the tripeptide Gly-
His-Lys with antimicrobial activity. Amino Acids 13:155–161
Lipshutz BH, Taft BR (2006) Heterogeneous copper-in-charcoal-
catalyzed click chemistry. Angew Chem Int Ed 45:8235–8238
Loren JC, Krasinkski A, Fokin VV, Sharpless KB (2005) NH-1,2,3-
triazoles from azidomethyl pivalate and carbamates: base labile
N-protecting groups. Synlett 18:2847–2850
synthesis was performed on a chlorotrityl resin using
Fmoc-glycine, compound 9 (D enantiomer) and Fmoc-D-
lysine(Boc). After cleavage of the peptide from the resin
using TFA, the two remaining protecting groups (id est
Fmoc on the glycine and POM on the azahistidine side
chain) were removed using NaOH in MeOH. After tritur-
ation in cold ether, GHK analogue 14 was obtained as a
single isomer (Scheme 4).
In conclusion, we have described the synthesis of
numerous protected azahistidine derivatives. Derivatives
bearing a POM group on the triazole ring appeared to be
good candidates for SPPS since selective deprotection of
either the side chain or the amino-acid nitrogen group
proved to be possible. Azahistidine bearing an Fmoc group
and a POM group on the side chain is of particular interest
since the deprotection of the Fmoc group takes place with
piperidine while the POM deprotection requires more basic
conditions thus allowing the use of compound 9 in SPPS as
exemplified in the synthesis of a GHK analogue described
herein.
Losikina VI, Sokolov VI (1991) Asymetric synthesis and reactions of
the triple bond of propargylglycine a precursor of N-substituted
triazolalanine. Doklady Akademii Nauk SSSR 320:339–342
Mindt TL, Struthers H, Brans L, Anguelov T, Schweinsberg C, Maes
´
V, Tourwe D, Schibli R (2006) Click to chelate: synthesis and
installation of metal chelates into biomolecules in a single step.
J Am Chem Soc 128:15096–15097
´
Oppilliart S, Mousseau G, Zhang L, Jia G, Thuery P, Rousseau B,
Cintrat J-C (2007) 1-protected 5-amido 1,2,3-triazoles via
ruthenium-catalyzed [3?2] cycloaddition of azides and yna-
mides. Tetrahedron 63:8094–8098
Shchetnikov GT, Peregudov AS, Osipov SN (2007) Effective
pathway to the alpha-CF3-substituted azahistidine analogues.
Synlett 1:136–140
Stahl GL, Walter R, Smith CW (1978) General procedure for the
synthesis of mono-N-acetylated 1,6-diaminohexanes. J Org
Chem 43:2285–2286
References
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123