Easy preparation of bioactive peptides from the novel Nα-trifluoroethyl amino acids

Article abstract of DOI:10.1246/cl.2000.1052

N^α-Trifluoroethyl amino acids have been prepared for the first time and unexpectedly found to behave as conventionally N-protected amino acids. Novel unnatural peptides are easily prepared in high yields without racemization. The observed chemistry results from steric factors as well as from the acidity of the CF₃CH₂NH group, according to comparative experiments. Of ten dipeptides that were tested for antitumor activity, CF₃CH₂-L-Tyr-L-Ile-OtBu was the most active.

Full text of DOI:10.1246/cl.2000.1052

1052
Chemistry Letters 2000
Easy Preparation of Bioactive Peptides from the Novel Nα-Trifluoroethyl Amino Acids
Darryl D. DesMarteau* and Vittorio Montanari
Department of Chemistry, Box 340973, Clemson University, Clemson, SC 29634-0973, U. S. A.
(Received January 27, 2000; CL-000092)
Nα-Trifluoroethyl amino acids have been prepared for the
first time and unexpectedly found to behave as conventionally
N-protected amino acids. Novel unnatural peptides are easily
prepared in high yields without racemization. The observed
chemistry results from steric factors as well as from the acidity
of the CF3CH2NH group, according to comparative experi-
ments. Of ten dipeptides that were tested for antitumor activity,
CF3CH2–L-Tyr–L-Ile–OtBu was the most active.
Table 1) in excellent yields.6 The commercially available
amino acid t-butyl esters 2a-c are the most convenient starting
materials for preparing 4. Because t-butyl esters are especially
stable to bases, but readily hydrolyzed by dilute acids, the direct
procedure of Scheme 1 becomes possible.5 The analytically
pure amino acid products 4 are obtained by simple partitioning
between organic and aqueous phase. Chromatographic isola-
tion of the intermediate esters 3a-c is possible but unnecessary.
We wish to report a direct synthesis of unprecedented fluo-
roalkyl-substituted molecules from the common amino acids.
We reported recently the first rapid alkylations of the side
chains of cysteine, glutathione, and Nα-protected lysine in aque-
ous media at ambient temperature,1 consisting of the transfer of
a 2,2,2-trifluoroethyl group from the iodonium salt
(CF3SO2)2NI(Ph)CH2CF3 (1). Because large amounts of com-
pound 1 are prepared easily,2 we have further studied the poten-
tial of 1 as a discovery tool for novel bioactive substances.1,3
We now report that amino acid esters are alkylated at the
α-nitrogen by 1 under convenient two-phase conditions
(CH2Cl2/water/NaHCO3). Scheme 1 shows the simple prepara-
tion of the representative products Nα-2,2,2-trifluoroethyl-
phenylalanine (4a), -tyrosine (4b) and -valine (4c).
Possible racemization of the amino acid that undergoes the
coupling is a major issue of peptide chemistry.4,7 The method
of N-protection can be one of the factors in racemization.
Therefore we investigated the eight combinations (four prepara-
tions each of 5b and 5e) of 3a with L-AlaOMe and L-ValOMe
as the incoming amino acids, CH2Cl2 or DMF as the solvent,
and 1 or 2 eq of DIEA. The methyl esters of L-Ala and L-Val
and the corresponding D- and D,L- forms are commercially
available, so we could use 5d and 5f as NMR references.
Undesired diastereomers were not detected in any run by 500
MHz 1H and 470 MHz 19F NMR. The best conditions from this
screening experiment were used to prepare dipeptides from Nα-
Initially, investigating CF3CH2–L-PheOtBu 3a as a model,
we hoped to form a peptide bond to the α-nitrogen but we had
no success. We supposed that the α-nitrogen had become
essentially non-nucleophilic. Such a degree of deactivation is
the purpose of protecting groups in peptide synthesis.4 On this
basis we reasoned that standard peptide synthesis might be
applicable with the free acids 4. This proved to be true.
Standard coupling conditions gave dipeptides 5a–l (Scheme 2,
Copyright © 2000 The Chemical Society of Japan

Chemistry Letters 2000
1053
trifluoroethyltyrosine (4b) or -valine (4c) and a second amino
acid having the practical4 t-butyl or allyl ester O-protection.
All the dipeptides so prepared were essentially pure as obtained
from workup.
The financial support of this research by the National
Science Foundation is gratefully acknowledged.
References and Notes
1
D. D. DesMarteau and V. Montanari, Chem. Commun., 1998,
2241.
To determine if the electron-withdrawing character of the
CF3CH2 group could explain our findings, firstly we measured
pK2 of the water-soluble CF3CH2–Gly–OH·HCl by titration.
We found that pK2 of trifluoroethyl glycine is only slightly
lower than pKa of trifluoroethylamine, as shown in Figure 1.
However, trifluoroethylamine and Z-PheOH gave under the
same coupling conditions of Scheme 2 a 75% yield of the crys-
talline amide Z-Phe–NHCH2CF3. The importance of steric fac-
tors was confirmed when, still under the same conditions, no
product was formed from Z-PheOH and secondary N-2,2,2-tri-
fluoroethyl amines such as CF3CH2NHCH(CH3)Ph and
CF3CH2NH(CH2)2Ph.
2
Following Ref. 1 on a larger scale, HN(SO2CF3)2 (64 mmol),
CF3CH2I(OCOCF3)2 (61 mmol) and benzene (70 mmol)
reacted in CFC 113 (50 mL) during 22 h. Evaporating the
volatiles, stirring the residue with ice, filtering and freeze-
drying yielded 1 as a powder (30.7 g, 54 mmol, 89%).
Crystallization (CH2Cl2, 4 mL/g, –20 °C) gave 27.3 g (79%
overall) of 1 as transparent prisms, mp 77–79 °C, dec.
105–120 °C (TGA, 5 °C/min).
3
Fluoroalkylation reactions by CF3CH2I(Ph)OSO2CF3 (6)
were first reported in detail by Umemoto and Gotoh: T.
Umemoto and Y. Gotoh, Bull Chem Soc. Jpn., 60, 3307
(1987); T. Umemoto, Chem. Rev., 96, 1757 (1996). The
preparation of 1 is similar to that of 6, with HN(SO2CF3)2 in
place of triflic acid, but 1 requires more strictly anhydrous
conditions until workup. Compound 1 extends the reactions
of 6 to aqueous systems. The reason of the much greater sta-
bility of 1 to water relative to 6 is not yet clear; a crystallo-
graphic investigation is in progress: D. D. DesMarteau, W. T.
Pennington, and V. Montanari, J. Mol. Struct., in press.
“Chemistry and Biochemistry of the Amino Acids,” ed. by
G. C. Barrett, Chapman and Hall, London (1985); “The
Peptides: Analysis, Synthesis, Biology,”ed. by E. Gross and
J. Meienhofer, Academic Press, London (1979), Vol. 1.
Typical procedure for 4: Phenylalanine t-butyl ester
hydrochloride (2a) (9.85 mmol) was suspended in 75 mL
CH2Cl2. Water (75 mL) and Na2CO3 (7 g) were added and
the mixture stirred for 30 min. The clear organic layer was
separated. NaHCO3 (11.9 mmol), water (70 mL) and 1
(10.62 mmol) were added with stirring at 20 °C. After 45
min the CH2Cl2 phase was separated and washed with 3 ×
100 mL water. It was then stirred at 20 °C with 2 × 150 mL
6M HCl for 3 h. The combined acid solutions were dried to
constant weight, yielding 2.30 g (77%) of crystalline 4a
hydrochloride monohydrate, mp 159–160 °C. Similarly were
prepared 4b hydrochloride (78%), mp 203–204 °C, and 4c
(95%), mp 166–167 °C. The composition of 4a–c was estab-
lished by the correct elemental analyses of the bulk products.
Typical procedure for 5: CF3CH2–L-PheOH·HCl·H2O (4a)
(0.50 mmol), L-AlaOMe·HCl (0.50 mmol), HOBt·H2O (0.55
mmol) and EDC (0.55 mmol) were suspended in CH2Cl2 (5
mL) at 5 °C. DIEA (175 µL, 1.0 mmol) was added rapidly
by syringe. The reaction was run for 1 h at 5 °C, then for 3 h
at 22 °C. The reaction mixture was diluted to 50 mL with
CH2Cl2, and washed with 0.1 M NaHCO3 (50 mL), 0.5 M
HCl (50 mL), and water (2 × 50 mL). Drying on Na2SO4,
evaporating, and pumping at 0.05 mmHg gave 5a (158 mg,
95%) as a white powder, mp 73–76 °C.
4
5
6
Protecting groups such as Boc, Z, arenesulfonyl and formyl
occur in synthetic bioactive peptides and are evaluated as struc-
tural units in pharmaceutics design.8 While structure–activity
relationship is a very complex subject, a simple chemical func-
tion of such “protection” is to retard or prevent metabolic deac-
tivation by oxidative dealkylation. That is precisely the known
utility of a fluoroalkyl residue.9
Assays for anticancer activity were carried out at NIH/NCI10
on the compounds in Table 1. CF3CH2–L-Phe–L-IleOtBu (5a),
CF3CH2–L-Tyr–L-LeuOtBu (5h) and CF3CH2–L-Tyr–L-IleOtBu
(5i) reduced cancer cell growth. Compound 5i killed cancer cells
at 10–5 M concentration. These results from a very small pool of
new substances warrant further synthetic work.
7
8
L. A. Carpino, J. Org. Chem., 53, 875 (1988); J. Coste, E.
Frerot, and P. Jouin, J. Org. Chem., 59, 2437 (1994); S.
Nozaki, Chem. Lett., 1997, 1.
Y. Tamura, F. Watanabe, T. Nakatani, K. Yasui, M. Fuji, T.
Komurasaki, H. Tsuzuki, R. Maekawa, T. Yoshioka, K.
Kawada, K. Sugita, and M. Ohtani, J. Med. Chem., 41, 640
(1998).
In summary, preparative amounts of the novel type of
amino acids represented by 4a–c are available easily. Most
importantly, they undergo standard peptide chemistry. This
unexpected property of 4 affords very large numbers of poten-
tially bioactive fluoroalkylated substances possessing very
lipophilic moieties whose in vivo stability is also anticipated.
9
M. Steinman, J. G. Topliss, R. Alekel, Y-S Wong, and E. E.
York, J. Med. Chem., 16, 1354 (1973); E. H. Banitt, W. R.
Bronn, W. E. Coyne, and J. R. Schmid, J. Med. Chem., 20,
821 (1977).
10 Details on the in vitro anticancer testing protocols are avail-
able at http://dtp.nci.nih.gov.