Protection of 3,4-dihydroxyphenylalanine (DOPA) for Fmoc solid-phase peptide synthesis

Article abstract of DOI:10.1016/S0040-4039(00)00957-6

Cyclic ethyl orthoformate (Ceof) was utilized as a protecting group to protect the catechol hydroxyl groups of 3,4-dihydroxyphenylalanine (DOPA). This protecting group is stable to strong bases and nucleophiles, and can be removed efficiently by 1 M trimethylsilyl bromide in trifluoroacetic acid in the presence of scavengers at 0°C for 60 min. Fmoc-DOPA(Ceof)-OH was synthesized in high yield and applied along with other Fmoc-amino acids to the solid-phase peptide synthesis of a DOPA-containing decapeptide from a mussel adhesive protein. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00957-6

Tetrahedron Letters 41 (2000) 5795±5798
Protection of 3,4-dihydroxyphenylalanine (DOPA) for Fmoc
solid-phase peptide synthesis
Bi-Huang Hu and Phillip B. Messersmith*
Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA
Received 18 April 2000; revised 30 May 2000; accepted 8 June 2000
Abstract
Cyclic ethyl orthoformate (Ceof) was utilized as a protecting group to protect the catechol hydroxyl
groups of 3,4-dihydroxyphenylalanine (DOPA). This protecting group is stable to strong bases and
nucleophiles, and can be removed eciently by 1 M trimethylsilyl bromide in tri¯uoroacetic acid in the
presence of scavengers at 0ꢀC for 60 min. Fmoc-DOPA(Ceof)-OH was synthesized in high yield and
applied along with other Fmoc-amino acids to the solid-phase peptide synthesis of a DOPA-containing
decapeptide from a mussel adhesive protein. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: biologically active compounds; amino acids and derivatives; solid-phase synthesis; peptides and polypeptides.
3,4-Dihydroxyphenylalanine (DOPA) 1 is a naturally occurring, unusual amino acid derived
from post-translational modi®cation of tyrosine.1 It has been one of the principal agents
administered to patients with Parkinson's disease since 1967.2 Although rarely found in proteins,
DOPA was detected in high yield (ca. 10%) in marine mussel adhesive proteins,3 as well as egg-
shell precursor proteins of Fasciola hepatica4 and Schistosoma mansoni.5 It has been postulated
that the presence of DOPA residues in these proteins could contribute to bioadhesion in sea water
and sclerotization during eggshell formation, respectively.
Our research interest is to study the role of DOPA residues in bioadhesion, and synthesize
DOPA-containing peptides and conjugates for drug delivery and new biomaterials. To
accomplish this, we investigated the synthesis of DOPA derivatives for solid-phase peptide
synthesis6 by the 9-¯uorenylmethoxycarbonyl (Fmoc) strategy. Solid-phase synthesis of peptides
by Fmoc strategy is a mild approach wherein the base-labile Fmoc protecting group protects the
a-amino group of amino acids during coupling reactions.7 The cyclic ethyl orthoformate (Ceof)
protecting group of catechol is known to be stable to strong bases8 yet acid-labile,9 and is
therefore compatible with Fmoc strategy.10 Therefore, we chose Ceof to protect the catechol
hydroxyl groups of DOPA.
The starting material l-DOPA 1 was suspended in methanol and reacted with methyl
tri¯uoroacetate in the presence of triethylamine under argon at room temperature to give
* Corresponding author. Tel: 312-503-1789; fax: 312-503-2440; e-mail: philm@northwestern.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00957-6

5796
Na-tri¯uoroacetyl-l-DOPA 2 with 95% yield (Scheme 1).11 Reaction of 2 with iodomethane in
DMF under argon in the presence of potassium bicarbonate a€orded the methyl ester 3 in 95%
yield.12
Scheme 1. Reagents and conditions: (a) CF3CO2Me, NEt3, MeOH, rt, 6 h, 95%; (b) CH3I, KHCO3, DMF, rt, 4 h,
95%; (c) HC(OEt)3, TsOH, molecular sieves 4A, benzene, re¯ux, 3 d, 80%; (d) NaOH, THF/H2O, rt, 16 h; (e) Fmoc-
OSu, THF/H2O, rt, 8 h, 80% (d and e)
Compound 3 was re¯uxed with triethyl orthoformate in anhydrous benzene with p-toluene-
sulfonic acid as catalyst in the presence of molecular sieves (4A) to give the Ceof-protected
DOPA methyl ester 4 in 80% yield.13 Alkaline hydrolysis of 4 by 0.2 M sodium hydroxide
solution in THF/water provided Ceof-protected DOPA 5, which was detected by silica gel TLC
and con®rmed by LC-ESI/MS to show a peak with molecular weight m/z 254.1 (M+1). Without
puri®cation, 5 was treated with Fmoc-Osu in a THF/water solution in the presence of sodium
carbonate14 to a€ord the ®nal product Fmoc-l-DOPA(Ceof)-OH 6,15 which was isolated as the
dicyclohexylamine (DCHA) salt (yield 80%).
To determine the stability of the protecting group Ce of toward nucleophiles used in Fmoc strategy,
.
Fmoc-l-DOPA(Ceof) DCHA was treated with 25% piperidine in DMF at room temperature for 60 h
(Scheme 2). LC-ESI/MS analysis of the reaction mixture revealed no evidence of 1; furthermore, a
new peak with molecular weight m/z 254.1 (M+1) was found, corresponding to 5. This result
indicates that the side-chain protecting group (Ceof) of DOPA is stable to the 25% piperidine
reagent that is commonly used to remove Fmoc during stepwise peptide synthesis.10
To test the suitability of the Ceof protection group to synthetic strategies involving ®nal TFA
.
deprotection of assembled peptides, Fmoc-l-DOPA(Ceof) DCHA was treated with 1 M
trimethylsilyl bromide (TMSBr) in tri¯uꢀoroacetic acid (TFA) in the presence of thioanisole,
m-cresol and 1,2-ethanedithiol (EDT) at 0 C for 60 min (Scheme 2).16 ESI/MS monitoring of the
cleavage reaction showed that the side-chain protecting group was completely removed to form
Fmoc-l-DOPA 7 within 60 min (Scheme 2).

5797
Scheme 2. Reagents and conditions: (a) 25% piperidine/DMF, rt, 60 h; (b) 1 M TMSBr/TFA, thioanisole, m-cresol,
EDT, 0ꢀC, 60 min
To demonstrate the ecacy of this new protected DOPA in an actual peptide synthesis, Fmoc-
DOPA(Ceof)-OH was applied with other Fmoc-amino acids to the solid-phase peptide synthesis
of a model decapeptide, H-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys-NH2, derived from
tandem repeats of a mussel adhesive protein (Hyp is 4-hydroxyproline).17 The model peptide was
synthesized manually on a Rink amide resin (0.8 mmol/g with Fmoc, Advanced ChemTech, KY,
USA) by Fmoc strategy with the following side-chain protecting groups: t-butyl (Ser, Tyr, Hyp
and Thr), t-Boc (Lys) and Ceof (DOPA). Fmoc deprotection was performed in 25% piperidine in
N-methyl-2-pyrrolidinone (NMP) for 20 min. Coupling reactions were performed using 2
equivalents of the mixture Fmoc-amino acid:BOP:HOBt:DIEA (1:1:1:1) in NMP, with a 10 min
pre-activation step before coupling.18 In the coupling step of Fmoc-DOPA(Ceof)-OH 6, 1.5
equivalents of 6:BOP:HOBt:DIEA (1:1:1:1) were used. The coupling time was 20 min and mon-
itored by a ninhydrin test.19
The synthesized peptide was cleaved from the resin by treatment with 1 M TMSBr in TFA in
the presence of thioanisole, m-cresol and EDT at 0ꢀC for 60 min. The crude decapeptide was
Figure 1. RP-HPLC of the crude decapeptide. HPLC was performed on a Vydac C18 reversed-phase column (250Â4.6
mm, 10 mm) with a linear gradient 5±35% CH3CN in 0.1% TFA (v/v) over 30 min, ¯ow rate 1.0 ml/min, and UV
detection at 215 nm; 2 ml (2.5 mg/ml) of sample was injected

5798
analyzed (Fig. 1) and puri®ed by RP-HPLC. ESI/MS of the puri®ed decapeptide revealed a
molecular weight m/z 1198.6 (M+1) (calculated monoisotopic molecular weight 1198.57).
.
In summary, we synthesized Fmoc-l-DOPA(Ceof) DCHA and showed that this compound is
compatible with solid-phase peptide synthesis by Fmoc strategy. Our results indicate that Fmoc-
.
l-DOPA(Ceof) DCHA is satisfactory for the solid-phase synthesis of DOPA-containing peptides.
Acknowledgements
The authors gratefully acknowledge support from the National Institute of Health (R01
DE13030 and P60 DE 13076). The authors also thank Dr. C. Miaw of the American Dental
Association for access to LC±MS facilities.
References
1. Longo, R.; Castellani, P.; Tibolla, M. Phytochemistry 1974, 13, 167±171.
2. Tolosa, E.; Marti, M. J.; Valldeoriola, F.; Molinuevo, J. L. Neurology 1998, 50 (Supplement 6), S2±S10.
3. Laursen, R. A. In Structure, Cellular Synthesis and Assembly of Biopolymers; Case, S. T., Ed. Re¯ections on the
structure of mussel adhesive proteins. Spring-Verlag: Berlin, 1992; pp. 55±74.
4. Rice-Ficht, A. C. In Structure, Cellular Synthesis and Assembly of Biopolymers; Case, S. T., Ed. Composition and
design of Fasciola hepatica eggshells. Spring-Verlag: Berlin, 1992; pp. 75±95.
5. Wells, K. E.; Cordingley J. S. In Structure, Cellular Synthesis and Assembly of Biopolymers; Case, S. T., Ed. The
cell and molecular biology of eggshell formation in Schistosoma mansoni. Spring-Verlag: Berlin, 1992; pp. 97±114.
6. Merri®eld, R. B. J. Am. Chem. Soc. 1963, 85, 2149±2154.
7. Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748±5749.
8. Dietl, F.; Gierer, G.; Merz, A. Synthesis 1985, 6/7, 626±631.
9. Merz, A.; Rauschel, M. Synthesis 1993, 797±802.
10. Fields, G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990, 35, 161±214.
11. Curphey, T. J. J. Org. Chem. 1979, 44, 2805±2807.
12. Hamada, Y.; Shibata, M.; Sugiura, T.; Kato, S.; Shioiri, T. J. Org. Chem. 1987, 52, 1252±1255.
13. Danishefsky, S.; Lee, J. Y. J. Am. Chem. Soc. 1989, 111, 4829±4837.
14. Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis 1983, 671±673.
1
15. Fmoc-l-DOPA(Ceof)-OH: H NMR (500 MHz, DMSO-d6) ꢀ ppm: 7.89±7.31 (m, 8H), 6.80 (s, 1H), 6.77 (d, 1H,
J=8.0 Hz), 6.65±6.63 (m, 2H), 4.27 (d, br, 1H, J=7.0 Hz), 4.19±4.11 (m, 3H), 3.62 (q, 2H, J=7.0 Hz), 3.08±2.82
(m, 2H), 1.12 (t, 3H, J=7.0 Hz). ESI-MS (LC/MS, single quad, m/z 150±2000) (m/z): 476.9 [M+1]+, calcd mass:
476.5. TLC: Rf=0.52 (silica gel, CHCl3:MeOH:HOAc, 100:6:1). RP-HPLC (Vydac, C18, 250Â4.6 mm, 10 mm, 41±
76% CH3CN in 0.1% TFA over 35 min) 25.82 min.
16. Yajima, H.; Fuji, N.; Funakoshi, S.; Watanabe, T.; Murayama, E.; Otaka, A. Tetrahedron 1988, 44, 805±819.
17. Waite, J. B. Chemistry & Industry 1991, 2 September, 607±611.
18. Hudson, D. J. Org. Chem. 1988, 53, 617±624.
19. Sarin, V. K.; Kent, S. B. H.; Merri®eld, R. B. Anal. Biochem. 1981, 117, 147±157.