Convenient preparation of 4-(tetrazol-5-yl)-phenylalanine for use in Fmoc-based solid-phase peptide synthesis

Article abstract of DOI:10.1016/S0040-4039(00)01135-7

To create a novel amino acid incorporating both acidic and aromatic features, trimethyltin azide was used to synthesize L-4-(tetrazol-5-yl)-phenylalanine (Tpa) and the corresponding D,L racemate by [3+2] cycloadditition of azide to the nitrile group of co

Full text of DOI:10.1016/S0040-4039(00)01135-7

Tetrahedron Letters 41 (2000) 6555±6558
Convenient preparation of 4-(tetrazol-5-yl)-phenylalanine for
use in Fmoc-based solid-phase peptide synthesis
John S. McMurray,* Olga Khabashesku, James S. Birtwistle and Wei Wang
University of Texas, M. D. Anderson Cancer Center, Department of Neuro-Oncology, Box 316, 1515 Holcombe Blvd.,
Houston, TX 77030, USA
Received 8 May 2000; revised 3 July 2000; accepted 6 July 2000
Abstract
To create a novel amino acid incorporating both acidic and aromatic features, trimethyltin azide was
used to synthesize l-4-(tetrazol-5-yl)-phenylalanine (Tpa) and the corresponding d,l racemate by [3+2]
cycloadditition of azide to the nitrile group of corresponding 4-cyanophenylalanine analogs. Na-Fmoc-Tpa
derivatives, possessing no protection of the tetrazole ring, were well-behaved monomers in the synthesis of
a model peptide using standard solid-phase methods. # 2000 Elsevier Science Ltd. All rights reserved.
Unnatural amino acids are useful building blocks in drug discovery e€orts in that they can
provide interactions with receptors or enzymes that are not possible with the 20 genetically coded
(natural) residues. For example, 4-carboxyphenylalanine (4-Cpa) combines the acidic features of
aspartic acid and glutamic acid with the aromatic features of phenylalanine and tyrosine. The
preparation of Fmoc-4-Cpa(O-t-Bu)-OH and its incorporation into a peptide was reported by us
earlier.1 Among structural units that mimic pharmacophoric functional groups, the 5-yl tetrazole
group is widely used in medicinal chemistry as an isostere of the carboxyl group.2 Compounds
containing this heterocycle have pKa values close to the corresponding carboxylic acids.3 The
tetrazole ring serves as a tyrosinate or carboxylate mimic in the non-peptide angiotensin II
mimetic, losartan,4 and has been used as a replacement of a- and g-carboxyl units of the glutamic
acid moiety of methotrexate analogues5 as well as the side chain carboxyl group of the aspartic
acid residue in the C-terminal gastrin tetrapeptide, Trp-Met-Asp-Phe-NH2.6 The sulfate group in
cholecystokinin7 and the phosphate group of phospholipase A2 inhibitors8 have also been
replaced by tetrazole rings. To extend the concept of acidic and aromatic amino acid hybrids, we
report here a simple procedure for the preparation of Fmoc-4-(tetrazol-5-yl)-phenylalanine
(Fmoc-Tpa) for use in peptide synthesis.
Tilley et al.7 reported a synthesis of Nꢀ-acetyl-d,l-Tpa, in which the tetrazole unit was
introduced by alkylation of N-(diphenylmethylene)glycine benzyl ester with 4-(tetrazol-5-yl)-
benzylbromide. In this case the tetrazole ring was protected by alkylation of position 3 with a
* Corresponding author.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01135-7

6556
tert-butyl group. After deprotection of the amino and carboxy groups and N-acetylation, this
amino acid was incorporated as the N-terminal residue of a cholecystokinin analogue using solid-
phase synthesis with Boc/benzyl strategy. The tert-butyl group was removed from the tetrazole
ring during the ®nal deprotection/cleavage step with HF.
We chose to introduce the tetrazole group by transformation of the nitrile group of previously
formed 4-cyanophenylalanine derivatives. The synthesis of tetrazoles by cycloaddition of azide to
nitriles is a well-known reaction,2 and one of the classic methods is treatment with NaN3 in DMF
at elevated temperature for periods of 24 h or more. Trimethyl and tributyl tin azide9 have been
shown to be e€ective reagents for this conversion.4b,5b,c,10 We found that clean transformations
were achieved by stirring 4-cyanophenylalanine derivatives with Me3SnN3 in toluene at elevated
temperature.5b
Stereospeci®c synthesis of Fmoc-l-Tpa (2) was readily achieved by merely treating commercially
available Fmoc-l-4-cyanophenylalanine (1) with 2 equivalents of Me3SnN3 in dry toluene at
80±90ꢀC for 24 h (Scheme 1).11,12 The free carboxyl group did not interfere with the reaction and
the Fmoc group was perfectly stable to these conditions. In contrast, we found that treating
Fmoc±amino acid nitriles with NaN3 in DMF at elevated temperatures resulted in premature
cleavage of the amino protecting group.
Scheme 1.
The synthesis of Fmoc-d,l-Tpa is depicted in Scheme 2. Commercially available N-(diphenyl-
methylene)glycine ethyl ester 3 was alkylated with a-bromo-para-tolunitrile in the presence of
KOtBu13 to produce the racemic N-protected 4-cyanophenylalanine derivative, 4. Nitrile 4 was
converted to the corresponding tetrazolyl derivative, 5, by reaction with azidotrimethylstannane
in dry toluene 80±90ꢀC for 24 h.5b The imine moiety was stable to these conditions. Acidic
hydrolysis of the Schi€'s base followed by saponi®cation13 provided the amino acid, which was
N-protected by treatment with Fmoc-OSu in aqueous sodium carbonate to a€ord Fmoc-d,l-Tpa 6.
Scheme 2. (a) Me3SnN3, ~; (b) H3O+; (c) NaOH; (d) Fmoc-OSu, Na2CO3

6557
Treatment of 2 and 6 with 1% DBU in ACN followed by Marfey's reagent14 revealed no
detectable d-isomer in preparations of Fmoc-l-Tpa.
The new tetrazolyl phenylalanine derivatives were incorporated into a test peptide, [Val5]
angiotensin II, by replacing the tyrosine at position 4 to give Asp-Arg-Val-Tpa-Val-His-Pro-Phe-
NH2. Standard solid-phase Fmoc/tert-butyl protocols employing polymethylacrylamide resin,15
derivatized with the Rink handle, were carried out in a manual reactor. Side chain protection was
Asp, tert-butyl; Arg, Pbf; and His, trityl. A 3-fold excess of amino acid was used and coupling
was mediated with DIPCDI and HOBt in DMF:DCM (1:1). Fmoc removal was achieved with
20% piperidine in DMF. Coupling of both 2 and 6 to the sterically hindered Val5 was complete in
30 min, as judged by Kaiser ninhydrin tests.16 Addition of the subsequent valine also proceeded
in less that 30 min.
HPLC analysis of the crude peptides incorporating 2 and 6 show very clean products (Fig. 1).
The diastereomers resulting from incorporation of d,l-Tpa are remarkably well separated and
the d-Tpa-containing isomer elutes second. The very small peak corresponding to peptide
possessing d-Tpa in the crude product incorporating 2 indicates that very little racemization
(<5%) occurred during activation or coupling of the Fmoc-Tpa derivatives.
Figure 1. Reverse phase HPLC of crude [Tpa4,Val5] angiotensin II analogues.17 Upper chromatogram: peptide incor-
porating l-Tpa. Lower chromatogram: peptide incorporating d,l-Tpa
In conclusion, we have prepared 4-(tetrazol-5-yl)-phenylalanine derivatives by [3+2] cycloaddition
of azide to the nitrile of corresponding 4-cyanophenylalanine derivatives. The transformation of
the nitrile was readily achieved with Me3SnN3. Fmoc-Tpa analogues were well behaved in peptide
synthesis and will serve as useful tools in peptide-based structure±activity studies.
Abbreviations: DIPCDI, diisopropylcarbodiimide; ESI-MS, electrospray ionization mass
spectrometry; Fmoc, 9-¯uorenylmethoxycarbonyl; Fmoc-OSu, N-(¯uorenylmethoxycarbonyl-
oxy)succinimide, HOBt, 1-hydroxybenzotriazole; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-
5-sulfonyl.

6558
Acknowledgements
This work was supported by the National Cancer Institute (CA53617) and the Texas Higher
Educational Control Board (ARP 15-073). Mass spectometry was performed at the University of
Texas, M. D. Anderson Cancer Center, Analytical Chemistry Center.
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11. Procedure for the synthesis of 2: Fmoc-l-4-cyanophenylalanine (0.50 gm, 1.2 mmol) and Me3SnN3 (0.43 gm, 2.1
mmol) in 5 ml toluene were stirred for 24 h at 80±90ꢀC. More Me3SnN3 (0.4 mmol) was added and stirred for 4 h
at the same temperature. The solvent was evaporated o€, the solids were dissolved in EtOAc and this was washed
with aqueous HCl (pH 2). After drying (MgSO4) and evaporation the residue was puri®ed by silica
chromatography using gradients of EtOAc, MeOH and AcOH. Yield: 0.47 gm, 86%.
12. Analytical Data. Compound 2: ESI-MS (M+H) calcd: 456.42; found: 456.10. 1H NMR (300 MHz) ꢁ (CDCl3) 7.8±
7.9 (4H, m, arom.), 7.5±7.7 (m, 3H, arom, NH), 7.2±7.6 (m, 6H, arom.) 4.2 (m, 4H, Fmoc CH, Fmoc CH2, aCH),
3.1 (m, 1H, bCHa), 2.9 (m, 1H, bCHb). Compound 6: ESI-MS (M+H) calcd: 456.42; found: 456.09. 1H NMR (300
MHz) identical to 2. [Tpa4,Val5] ATII: ESI-MS [M+H] calcd: 1084.4; found: 1083.1. [d,l-Tpa4,Val5] ATII: ESI-
MS [M+H] calcd: 1084.4; found: 1083.0.
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