Synthesis of tetrazole analogues of amino acids using Fmoc chemistry: isolation of amino free tetrazoles and their incorporation into peptides

Article abstract of DOI:10.1016/j.tetlet.2007.07.129

An efficient synthesis of tetrazole analogues of amino acids starting from N^α-Fmoc amino acid in a three-step protocol is reported. The free amino tetrazoles were obtained in good yields and with excellent purity after removal of the Fmoc group

Full text of DOI:10.1016/j.tetlet.2007.07.129

Tetrahedron Letters 48 (2007) 7038–7041
Synthesis of tetrazole analogues of amino acids using
Fmoc chemistry: isolation of amino free tetrazoles and
their incorporation into peptides
Vommina V. Sureshbabu,* Rao Venkataramanarao, Shankar A. Naik
and G. Chennakrishnareddy
Department of Studies in Chemistry, Central College Campus, Bangalore University, Dr. B. R. Ambedkar Veedhi,
Bangalore 560 001, India
Received 18 May 2007; revised 5 July 2007; accepted 20 July 2007
Available online 27 July 2007
Abstract—An efficient synthesis of tetrazole analogues of amino acids starting from Na-Fmoc amino acid in a three-step protocol is
reported. The free amino tetrazoles were obtained in good yields and with excellent purity after removal of the Fmoc group. The
synthesis of analogues of aspartic and glutamic acids in which the 5-tetrazolyl moiety is inserted at the b/c carboxyl group starting
from Fmoc-Asn and Fmoc-Gln and the incorporation of these tetrazoles into peptides are also described.
Ó 2007 Elsevier Ltd. All rights reserved.
Tetrazoles, being an interesting class of heterocycles,
find a wide range of applications as drugs in pharmaceu-
ticals,1 cis-peptide bond mimics2 and bioisosteres3 for
carboxylic acids. They are important precursors in
medicinal chemistry owing to their increased resistance
towards metabolic degradation pathways and have
found increasing applications as catalysts4 in asymmet-
ric synthesis. Tetrazoles have also been utilized in orga-
nometallic chemistry as effective stabilizers of
metallopeptide structures and as peptide chelating
agents.5
Indeed, isosteric displacement of the C-terminus carbox-
ylic acid with a tetrazole often preserves or improves the
biological activity of the parent peptides and their
mimetics.
Conventionally, 5-substituted tetrazoles are synthesized6
by [2+3] cycloaddition of an azide and a nitrile. How-
ever, disadvantages of these synthetic routes involve
the use of expensive and toxic metals, strong Lewis acids
and the in situ generation of hydrazoic acid which is
highly toxic and explosive in nature. An alternative
route to overcome some of these shortcomings was nec-
essary. Sharpless et al.,7 reported an efficient protocol
for the synthesis of tetrazoles using sodium azide and
zinc bromide in water/2-propanol mixture at reflux
employing Z-protected amino acids as key synthons.
Alternatively, tetrazole analogues of amino acids have
been reported by Grzonka et al.,8 from Z-protected ami-
no nitriles using sodium azide and aluminium chloride
in tetrahydrofuran at reflux. In continuation of our
interest in incorporating a tetrazole heterocyclic moiety
into amino acids and thereby peptides, we describe here
our efforts to isolate amino free tetrazoles of a-amino
acids employing an Fmoc-protection strategy and their
incorporation into peptides. Ley9 recently reported the
utility of free amine proline tetrazole as a catalyst in
asymmetric synthesis. This catalyst was prepared after
deprotection of the Z-group from Z-proline tetrazole
The selective modification of amino acid chains in pep-
tides and proteins is important in many areas of chemis-
try such as organic synthesis, the irreversible inhibition
of enzymes and in the improvement of pharmacokinetic
properties of peptide based drugs. Since, the acidity of
the tetrazole group corresponds closely with that of a
carboxylic acid, replacement of the C-terminal amino
acid residue in biologically active peptides with a tetra-
zole analogue would create an interesting class of com-
pounds for structure function relationship studies.
Keywords: Fmoc amino acids; Tetrazoles; Unnatural amino acids;
[2+3] Cycloaddition.
*
Corresponding author. Tel.: +91 80 22961339; e-mail: hariccb@
rediffmail.com
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.07.129

V. V. Sureshbabu et al. / Tetrahedron Letters 48 (2007) 7038–7041
7039
using a specifically designed H-cube continuous flow
hydrogenator.10 Although this process has led to com-
plete removal of the Z-group in 4 h, the expensive nature
of palladium, the special apparatus and the difficulties
encountered in isolating the product are some of the lim-
itations in scaling-up this protocol. In addition, our
efforts to obtain amino free tetrazoles from Boc-Phe
tetrazole using TFA were unsuccessful. In this context,
the use of a Fmoc protection strategy was of particular
interest since the Fmoc group can be easily removed un-
der mild basic conditions in solution and also permits
aimed at keeping the a-carboxyl group free in the case
of side chain-derived tetrazole analogues. A characteris-
tic peak around 2240 cmꢀ1 in the IR spectrum con-
firmed the presence of a nitrile group. All the Fmoc-a-
amino nitriles prepared were obtained as crystalline sol-
ids. The methodology introduced by Sharpless et al.,7
for the conversion of a-amino nitriles to 5-substituted
tetrazoles14 was found to be applicable in the case of
the Fmoc-amino nitriles described herein. The reaction
proceeds by addition of sodium azide and a catalytic
amount of zinc bromide to a-amino nitriles in water/2-
propanol (2:1) mixture at reflux for 16 h. A simple
work-up involving acidification and extraction yielded
the corresponding tetrazoles as pure solids in yields
around 80–90%.
t
the use of Trt and butyl groups for side chain protec-
tion, which can also be cleaved under mild conditions.11
We have envisaged a simple, four-step protocol for the
synthesis of amino free tetrazole analogues of amino
acids employing Fmoc chemistry in solution phase.
The Fmoc-group was deprotected using diethylamine in
CH2Cl2 in 30 min to yield a-amino tetrazoles.15 After
evaporation of the solvent and trituration with ether,
the compounds were isolated as pure solids (Table 1).
The reaction sequence was found to be free from racemi-
zation16 as determined by chiral HPLC of the methyl-
ated derivatives of the corresponding tetrazoles.
Pozdnev et al.12 reported the synthesis of Boc/Z-amino
acid amides including Fmoc-Phe and Leu using the di-
tert-butyl pyrocarbonate [(Boc)2O]-pyridine system. A
similar protocol with required modifications was
employed for our synthesis of Fmoc-protected amino
acid carboxamides (Scheme 1). Subsequent dehydration
of the amides to the corresponding nitriles was achieved
in excellent yields employing trifluoroacetic anhydride.13
This reagent was best suited for our protocol since DCC
mediated dehydration requires the use of pyridine as a
solvent, a strategy that does not fully support Fmoc-
chemistry. Another dehydrating reagent, cyanuric chlo-
ride was not used because the synthetic protocol was
Further, the utility of Fmoc chemistry for inserting the
tetrazole unit into the b/c carboxylic position of aspartic
and glutamic acids was demonstrated (Scheme 2). It was
envisaged that the carboxamido group present in the
side chains of asparagine and glutamine could be readily
exploited for the incorporation of a tetrazole moiety in
b
a
FmocNH
COOH
FmocNH
CONH2
FmocNH
CN
R
R
R
c
N
N
N
N
d
NH
NH
H2N
FmocNH
N
N
R
R
Scheme 1. Reagents and conditions: (a) (Boc)2O, pyridine, NH4HCO3, rt, 5 h, 70–80%; (b) trifluoroacetic anhydride–pyridine (1:1), THF, 0 °C, 3 h,
85–95%; (c) NaN3, ZnBr2, water/2-propanol (2:1), 80 °C, 16 h, 80–90%; (d) diethylamine, CH2Cl2, 30 min, 80–94%.
Table 1. Na-protected tetrazole analogues of amino acids and amine free tetrazoles
Entry Fmoc-amino tetrazolea Yield (%) Mp (°C) Massb (obs/calc)
Free amino Yield (%) Mp (°C) Massb (obs/calc)
tetrazolea
1
2
3
4
5
6
7
8
9
10
11
12
13
Fmoc-GlyT
Fmoc-AlaT
Fmoc-ValT
Fmoc-LeuT
Fmoc-IleT
Fmoc-PheT
Fmoc-Ser (OBz)T
Fmoc-D-PhgT
Fmoc-L-PhgT
Fmoc-MetT
89
91
85
86
88
90
82
84
82
89
81
79
81
181
189
207
202
219
204
223
211
186
193
180
227
232
321.3312 (321.3333) GlyT
335.3579 (335.3598) AlaT
363.4147 (363.4130) ValT
377.4379 (377.4396) LeuT
377.4380 (377.4396) IleT
411.4526 (411.4558) PheT
441.4832 (441.4818) Ser(OBz)T
397.4275 (397.4292) D-PhgT
397.4282 (397.4292) L-PhgT
395.4764 (395.4780) MetT
361.3949 (361.3971) ProT
93
90
88
89
90
94
85
87
85
87
83
274
257
273
262
269
278
243
181
202
233
249
227
178
99.0932 (99.0946)
113.1237 (113.1211)
141.1720 (141.1743)
155.2023 (155.2009)
155.2001 (155.2009)
189.2143 (189.2171)
219.2405 (219.2431)
175.1933 (175.1905)
175.1919 (175.1905)
173.2364 (173.2393)
139.1559 (139.1584)
247.2511 (247.2532)
261.2768 (261.2798)
Fmoc-ProT
Fmoc-Asp (OBz)T
Fmoc-Glu (OBz)T
469.4902 (469.4919) Asp(OBz)T 81
483.5164 (483.5185) Glu(OBz)T 84
a T corresponds to Tetrazole.
b High resolution mass spectrometry (HRMS).

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V. V. Sureshbabu et al. / Tetrahedron Letters 48 (2007) 7038–7041
N
N
N
N
n
O
NH
NH
N
N
CN
n
COOH
NH2
H
n
n
c
a
b
N
COOMe
FmocHN
COOH
FmocHN
FmocHN
COOH
FmocHN
O
R
n = 1, 2; R = CH2CH(CH3)2, CH3, H, CH2C6H5
Scheme 2. Reagents and conditions: (a) Trifluoroacetic anhydride–pyridine (1:1), THF, 0 °C, 6 h, 70–80%; (b) NaN3, ZnBr2, water/2-propanol (2:1),
80 °C, 16 h, 65–75%; (c) HBTU, DIEA, MeOH, DCM, 0 °C ! rt, 5 h, 80–90%.
Table 2. Synthesis of side chain derived tetrazole analogues of aspartic and glutamic acids and peptides
Entry
Compound
Yield (%)
Mp (°C)
Massa (obs/calc)
1
2
3
4
5
6
7
8
9
Fmoc-Asp(T)-OH
Fmoc-Glu(T)-OH
76
79
66
71
74
69
71
73
76
202
190
163
213
178
185
157
192
183
379.3719 (379.3734)
393.3991 (393.4008)
506.5563 (506.5581)
464.4765 (464.4789)
450.4301 (450.4513)
540.5726 (540.5758)
520.5812 (520.5855)
478.5023 (478.5058)
464.4741 (464.4785)
Fmoc-Asp(T)-Leu-OCH3
Fmoc-Asp(T)-Ala-OCH3
Fmoc-Asp(T)-Gly-OCH3
Fmoc-Asp(T)-Phe-OCH3
Fmoc-Glu(T)-Leu-OCH3
Fmoc-Glu(T)-Ala-OCH3
Fmoc-Glu(T)-Gly-OCH3
a High resolution mass spectrometry (HRMS).
the side chains. Although, initially we had started our
synthesis using Fmoc-Asn-OMe and Fmoc-Gln-OMe,
it was found that the entire protocol could be carried
out starting from Fmoc-Asn/Gln.
References and notes
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A two-step protocol including conversion of a carbox-
amido group to a nitrile followed by cycloaddition
resulted in Fmoc-Asp(T)-OH and Fmoc-Glu(T)-OH in
76% and 79% yields, respectively, which were fully char-
acterized by 1H NMR, 13C NMR and mass spectral
studies (Table 2). The tetrazolyl acids were employed
as building blocks to prepare peptides possessing a tetra-
zole unit in the side chain. Fmoc-Asp(T)-OH or Fmoc-
Glu(T)-OH was coupled with amino acid methyl ester
hydrochlorides using the standard HBTU and DIEA
methods. The coupling was carried out at 0 °C in
DCM and the reaction mixture was allowed to warm
to room temperature over 5 h. After work-up, the prod-
ucts were isolated as solids in yields around 80–90%.
In conclusion, tetrazole analogues of amino acids/pep-
tides have been synthesized through an Fmoc-amino
protection strategy. The use of Fmoc for amine protec-
tion facilitated the easy isolation of intermediate nitriles
and final tetrazoles. Amino free tetrazoles were obtained
by deprotection of the Fmoc group under mild reaction
conditions. The tetrazole-containing unnatural amino
acids isosteric to Asp/Glu have been similarly synthe-
sized from Fmoc-Asn/Gln and utilized in the synthesis
of peptides. In all cases, the final tetrazole products were
isolated in high yields and purities.
7. (a) Demko, Z. P.; Sharpless, K. B. J. Org. Chem. 2001, 66,
7945–7950; (b) Demko, Z. P.; Sharpless, K. B. Org. Lett.
2002, 4, 2525–2527.
Acknowledgements
8. (a) Grzonka, Z.; Liberek, B. Roczniki. Chem. 1971, 45,
967–980; (b) Grzonka, Z.; Rekowska, E.; Liberek, B.
Tetrahedron 1971, 27, 2317–2322.
9. Cobb, A. J. A.; Shaw, D. M.; Longbottom, D. A.; Gold, J.
B.; Ley, S. V. Org. Biomol. Chem. 2005, 3, 84–96.
We are grateful to the Department of Science and Tech-
nology (DST), Government of India, for financial assis-
tance. R.V.R. acknowledges the CSIR, New Delhi, for a
SRF.

V. V. Sureshbabu et al. / Tetrahedron Letters 48 (2007) 7038–7041
7041
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11. Atherton, E.; Sheppard, R. C. In Solid-Phase Peptide
Synthesis; Rickwood, D., Hames, B. D., Eds.; IRL Press:
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J = 6.8 Hz, 6H), 1.67 (m, 1H), 1.97 (t, J = 8.4 Hz, 2H),
4.18 (t, J = 5.9 Hz, 1H), 4.52 (d, J = 5.9 Hz, 1H), 5.13 (d,
J = 6 Hz, 2H), 5.40 (br d, J = 5.9 Hz, 1H), 7.22–7.79 (m,
8H); 13C NMR (CDCl3, 100 MHz): d 21.9, 24.1, 36.5,
39.5, 40.6, 46.6, 65.6, 120.0, 125.1, 126.9, 127.5, 140.7,
143.6, 143.8, 155.8.
15. General procedure for deprotection of the Fmoc group: To a
solution of Na-Fmoc-protected amino tetrazole (1 mmol)
in dry CH2Cl2 was added diethylamine (2 mL) and the
mixture was stirred at room temperature for 30 min. After
completion of the reaction (as monitored by TLC), the
solvent was removed in vacuo. The crude product was
recrystallized from EtOAc/hexane (1:4) to obtain the free
a-amino tetrazole as a solid.
14. General procedure for the synthesis of tetrazole analogues of
Na-Fmoc-amino acids: Na-Fmoc-amino nitrile (1 mmol),
sodium azide (2 mmol) and zinc bromide (0.5 mmol) were
dissolved in a mixture of 2-propanol (15 mL) and water
(30 mL), and stirred at reflux for 16 h. After completion of
the reaction [as monitored by TLC, using chloro-
form:methanol (9:1) as eluant], 5 mL of 3 N HCl and
30 mL of ethyl acetate were added and stirring was
continued until no solid remained. The aqueous layer was
extracted twice with ethyl acetate. The combined organic
layer was washed with water and dried over anhydrous
Na2SO4. The solvent was removed in vacuo and the
residue was recrystallized from EtOAc/hexane (1:4).
Compound characterization data for Fmoc-ValT (Table 1,
Compound characterization data for: Alanine tetrazole
(Table 1, entry 2): IR (KBr) 1535, 1442, 1266 cmꢀ1 1H
;
NMR (CDCl3, 400 MHz): d 1.49 (d, J = 6.1 Hz, 3H), 4.15
(m, 1H), 4.93 (br d, J = 6.6 Hz, 2H); 13C NMR (CDCl3,
100 MHz): d 21.5, 46.6, 151.3.
Leucine tetrazole (Table 1, entry 4): IR (KBr) 1532, 1444,
1
1263 cmꢀ1; H NMR (CDCl3, 400 MHz): d 0.86–0.88 (d,
J = 4.8 Hz, 6H), 1.65 (m, 1H), 1.97 (t, J = 5.8 Hz, 2H),
2.81 (br d, J = 6.6 Hz, 2H), 3.72 (q, J = 6.2 Hz, 1H); 13C
NMR (CDCl3, 100 MHz): d 23.8, 24.2, 42.5, 46.1, 150.3.
16. Racemization studies: The enantiomeric purities of the
synthesized tetrazoles were preserved appreciably follow-
ing the present method. The reaction sequence yields
tetrazoles with ee’s exceeding 80% as determined by chiral
HPLC studies of the corresponding methylated
derivatives.
entry 3): IR (KBr) 1692, 1533, 1263 cmꢀ1 1H NMR
;
(CDCl3, 400 MHz): d 0.91 (t, J = 6.8 Hz, 6H), 1.06 (m,
1H), 4.20 (t, J = 6.6 Hz, 1H), 4.54 (d, J = 6.6 Hz, 2H),
5.07 (br d, J = 5.8 Hz, 2H), 7.24–7.78 (m, 8H); 13C NMR
(CDCl3, 100 MHz): d 19.1, 26.3, 37.2, 39.1, 45.7, 48.5,
66.2, 120.4, 124.6, 126.4, 127.1, 140.9, 148.6, 154.5.
Fmoc-LeuT (Table 1, entry 4): IR (KBr) 1693, 1538,
1
1264 cmꢀ1; H NMR (CDCl3, 400 MHz): d 0.94–0.96 (t,