The synthesis of a geminally perfluoro-tert-butylated β-amino acid and its protected forms as a potential pharmacokinetic modulator and reporter for peptide-based pharmaceuticals

Article abstract of DOI:10.1021/jo0616308

(Chemical Equation Presented) To modulate and report the pharmacokinetics of peptide-based pharmaceuticals, a novel geminally perfluoro-tert-butylated β-amino acid (βFa) and its Fmoc- and Boc-protected forms were designed and synthesized. βFa was incorpor

Full text of DOI:10.1021/jo0616308

The Synthesis of a Geminally
Perfluoro-tert-butylated â-Amino Acid and its
Protected Forms as a Potential Pharmacokinetic
Modulator and Reporter for Peptide-Based
Pharmaceuticals
FIGURE 1. Structures of target molecules.
Zhong-Xing Jiang and Y. Bruce Yu*
Department of Pharmaceutics and Pharmaceutical Chemistry,
College of Pharmacy, UniVersity of Utah, Salt Lake City, Utah
84112
FIGURE 2. Structures of two model tripeptides, formyl-Gly-âFa-Gly-
amide (4) and formyl-Gly-L-Trp-Gly-amide (5).
bruce.yu@utah.edu
ReceiVed August 4, 2006
shown in Figure 1. We also need âFa in its Fmoc- and Boc-
protected forms (2 and 3, respectively) for solid-phase peptide
synthesis. The fluorine atoms are introduced into the amino acid
through two symmetrically positioned perfluoro-tert-butyl groups.
Such a spherically symmetric arrangement of the 18 fluorine
atoms in âFa ensures that they have an identical chemical
environment and avoids 19F-19F or 19F-1H coupling. As a
result, we anticipate this amino acid to give a single 19F NMR
signal. âFa is achiral and needs no side-chain protection,
significantly simplifying the synthesis of both the free amino
acid and its protected forms. Considering that the electron-
withdrawing capacity of perfluoro-tert-butyl groups can poten-
tially weaken the basicity of the amino group and hence its
reactivity during solid-phase peptide synthesis, a â-amino acid
is adopted to ensure sufficient separation between the amino
group and the two perfluoro-tert-butyl groups. This also allows
better steric accommodation of the bulky perfluoro-tert-butyl
groups.
To demonstrate the feasibility of âFa to be incorporated into
peptides via solid-phase synthesis, we designed the following
tripeptide, formyl-Gly-âFa-Gly-amide 4. This peptide serves
dual purposes. First, it demonstrates that protected âFa can
indeed be incorporated into peptides via solid-phase peptide
synthesis; second, it demonstrates that âFa can indeed increase
the hydrophobicity of a peptide. âFa is positioned in the middle
of the tripeptide to demonstrate that âFa can be placed in any
position of a peptide, not just the N- and C-termini. The N-
and C-termini of the tripeptide are formylated and amidated,
respectively. These modifications abolish terminal charges of
the tripeptide so that they will not interfere with hydrophobicity
and 1-octanol/water partition measurements. Note that the
disentanglement of hydrophobic interactions from electrostatic
interactions in peptides/proteins is far from trivial.4 Two
glycines, which have no side chains, flank the central âFa. The
purpose is to abolish nearest-neighbor interactions that can
interfere with hydrophobicity measurements.5 Hence, this trip-
eptide provides a “clean” model system for hydrophobicity and
To modulate and report the pharmacokinetics of peptide-
based pharmaceuticals, a novel geminally perfluoro-tert-
butylated â-amino acid (âFa) and its Fmoc- and Boc-
protected forms were designed and synthesized. âFa was
incorporated into a model tripeptide via standard solid-phase
chemistry. Both the amino acid (free and protected) and the
tripeptide show a sharp singlet 19F NMR signal. Reversed-
phase chromatography and 1-octanol/water partition mea-
surements demonstrate that âFa is extremely hydrophobic.
We are interested in the design and synthesis of fluorinated
amino acids as modulators and reporters of peptide pharmaco-
kinetics. The potential benefits brought by fluorinated amino
acids include prolonged in vivo half-life,1 enhanced membrane
permeability,2 and noninvasive detection via 19F magnetic
resonance spectroscopy.3
A generic, highly fluorinated amino acid that can dramatically
increase the lipophilicity of a peptide and give a sharp singlet
19F NMR signal is highly desirable for pharmacokinetic
modulation and reporting purposes. To this end, we designed a
geminally perfluoro-tert-butylated â-amino acid (âFa, 1), as1
(1) Hsieh, K.-H.; Needleman, P.; Marshall, G. R. J. Med. Chem. 1987,
30, 1097-1100.
(2) (a) Ortial, S.; Durand, G.; Poeggeler, B.; Polidori, A.; Pappolla, M.
A.; Bo¨ker, J.; Hardeland, R.; Pucci, B. J. Med. Chem. 2006, 49, 2812-
2820. (b) Perino, S.; Contino-Pe´pin, C.; Jasseron, S.; Rapp, M.; Maurizis,
J.-C.; Pucci, B. Bioorg. Med. Chem. Lett. 2006, 16, 1111-1114. (c) Park,
B. K.; Kitteringham, N. R.; O’Neill, P. M. Ann. ReV. Pharmacol. Toxicol.
2001, 41, 443-470. (d) Gerebtzoff, G.; Li-Blatter, X.; Fischer, H.; Frentzel,
A.; Seelig, A. ChemBioChem 2004, 5, 676-684.
(4) (a) Yu, Y.; Monera, O. D.; Hodges, R. S.; Privalov, P. L. J. Mol.
Biol. 1996, 255, 367-372. (b) Yu, Y.; Monera, O. D.; Hodges, R. S.;
Privalov, P. L. Biophys. Chem. 1996, 59, 299-314.
(5) Kovacs, J. M.; Mant, C. T.; Hodges, R. S. Biopolymers 2006, 84,
283-297.
(3) Wolf, W.; Presant, C. A.; Waluch, V. AdV. Drug DeliVery ReV. 2000,
41, 55-74.
10.1021/jo0616308 CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/23/2007
1464
J. Org. Chem. 2007, 72, 1464-1467

SCHEME 1. Synthesis of 1
SCHEME 2. Synthesis of 2
SCHEME 3. Synthesis of 3
1-octanol/water partition measurements. A reference tripeptide,
5, contains tryptophan (Trp) in place of âFa. Trp is the most
hydrophobic among the 20 natural amino acids5 and serves as
an excellent reference point for hydrophobicity measurements.
Structures of the two tripeptides (4 and 5) are shown in
Figure 2.
excellent yield. This completes the synthesis of the free amino
acid, as depicted in Scheme 1.
To obtain the Fmoc-protected form of the amino acid, the
amino group of compound 1 reacted with 9-fluorenylmethoxy-
carbonyl chloride (FmocCl) to give compound 2 with a 96%
yield on a 13.3-gram scale, as depicted in Scheme 2.
The commercially available pentaerythritol (6) provides an
ideal starting material for the synthesis of compound 1. Our
synthesis commenced with the selective protection of pen-
taerythritol (Scheme 1). Protecting two of the four hydroxyl
groups in pentaerythritol 6 with p-methoxylbenzaldehyde gave
the diol 7 with a good yield.6 As the acidity of the hydroxyl
group in perfluoro-tert-butanol is enhanced by the three electron-
withdrawing -CF3 groups, perfluoro-tert-butanol is a good
substrate for the Mitsunobu reaction to form perfluoro-tert-butyl
ethers.7 Thus, the Mitsunobu reaction was employed to introduce
two perfluoro-tert-butyl groups into compound 7 in just one
step to give fluorinated ether 8 with a 98% yield after the
reaction mixture was stirred at 45 °C for 30 h. Such a high
yield was achieved by carrying out the reaction in a sealed vessel
and in the presence of 4 Å molecular sieves. Neither FC-72
(C6F14) nor HFE-7100 (a mixture of n-C4F9OCH3 and i-C4F9-
OCH3) could extract compound 8 from the acetonitrile/water
(95%/5%) solution of the reaction mixture. Instead, standard
flash chromatography was used to purify ether 8 with a 98%
yield. The p-methoxybenzylidene acetal protecting group was
cleaved off of compound 8 by powdered aluminum chloride in
the presence of anisole to give 1,3-diol 9 with quantitative yield.
Treatment of 1,3-diol 9 with thionyl chloride gave a cyclic sulfite
intermediate which was then oxidized by in situ-generated
ruthenium tetraoxide to give the cyclic sulfate 10 with an 84%
yield in two steps. Ring opening of the cyclic sulfate 10 with
sodium azide, followed by hydrolysis of the resulting sulfonic
acid, gave the azido compound 11 with excellent yield which
was then subjected to Jones oxidation to give the carboxylic
acid 12 with an 85% yield. Hydrogenation of the azido group
in compound 12 yielded the fluorinated amino acid 1 with
To obtain the Boc-protected form of the amino acid, the azido
group of compound 12 was reduced to the amino group, which
then reacted with di-tert-butyl dicarbonate (Boc2O) to give
compound 3 with a 96% yield on a 1.3-gram scale, as depicted
in Scheme 3.
The two tripeptides (4 and 5) were synthesized using standard
Fmoc chemistry on a solid support.8 Both the carboxyl group
and the amino group of âFa showed good reactivity during solid-
phase reaction. Hence, âFa can be incorporated into any position
of a target peptide through its carboxyl group, amino group, or
both. Tripeptide 4 was purified by normal-phase HPLC, while
tripeptide 5 was purified by reversed-phase HPLC due to their
different solubilities in water and methanol. The molecular mass
and the purity of each tripeptide were verified by mass
spectrometry and analytical HPLC, respectively.
The 19F NMR spectra of the free amino acid 1 and the
tripeptide 4 are shown in Figure 3. As designed, all 18 fluorine
atoms give a sharp singlet 19F signal (line width ∼0.01 ppm)
in both the free amino acid 1 (left) and the tripeptide 4 (right).
This is also the case for the two protected forms (compounds 2
and 3) of this amino acid (see Supporting Information).
The hydrophobicity of âFa was evaluated in the context of
tripeptide 4, with tripeptide 5 serving as a reference point. To
this end, we used the reversed-phase chromatography method
developed by Hodges and co-workers, who determined the rel-
ative hydrophobicity order of 23 amino acids.5 Figure 4 shows
the chromatogram of the co-injection of the two tripeptides. Clear-
ly, tripeptide 4 is much more retentive than tripeptide 5 in
reversed-phase chromatography, proving that âFa is much more
hydrophobic than Trp, the most hydrophobic natural amino acid.
The 1-octanol/water partition coefficients (Poct) of the two
tripeptides were evaluated. Poct is a standard physicochemical
parameter in assessing membrane permeability of peptides and
(6) Aguilera, B.; Romero-Ramirez, L.; Abad-Rodriguez, J.; Corrales, G.;
Nieto-Sampedro, M.; Fernandez-Mayoralas, A. J. Med. Chem. 1998, 41,
4599-4606.
(7) Sebesta, D. P.; O’Rourke, S. S.; Pieken, W. A. J. Org. Chem. 1996,
61, 361-362.
(8) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A
Practical Approach; Oxford University Press: New York, 2000; pp 1-75.
J. Org. Chem, Vol. 72, No. 4, 2007 1465

FIGURE 3. Chemical shift (ppm) of 19F NMR in free amino acid 1 (left) and tripeptide 4 (right) (376 MHz, CD3OD, C6F6 as internal standard).
yield of ca. 65% at multigram synthesis scale. âFa can be incor-
porated into peptides using standard solid-phase synthesis. As
designed, all 18 fluorine atoms give a sharp singlet 19F NMR sig-
nal in both the free amino acid and the tripeptide context, aug-
uring well for 19F-MRS monitoring. âFa is much more hydro-
phobic than Trp, the most hydrophobic natural amino acid, and
results in a much stronger preference of organic over aqueous
phases.
Experimental Section
2-(4-Methoxyphenyl)-5,5-bis(2,2,2-trifluoro-1,1-bis-trifluo-
romethylethoxymethyl)-[1,3]-dioxane 8. To a stirred mixture of
compound 7 (24.4 g, 96.1 mmol), triphenylphosphine (75.5 g, 288.5
mmol), and 4 Å molecular sieves (20.0 g) in tetrahydrofuran (500
mL) at 0 °C was added dropwise diethylazodicarboxylate (50.2 g,
288.2 mmol). After the addition, the reaction mixture was allowed
to warm to room temperature and was stirred for an additional 20
FIGURE 4. Retention behavior of tripeptides 4 and 5 in reversed-
phase HPLC.
min. Then, perfluoro-tert-butanol (68.0 g, 288.0 mmol) was added
in one portion, and the resulting mixture was stirred at 45 °C for
30 h in a sealed vessel. The mixture was evaporated to dryness,
and the residue was dissolved in ethyl ether (600 mL). After filtered
other drugs.9 For 5 (formyl-Gly-L-Trp-Gly-amide), Poct ) 1/9.5,
as determined by analytical HPLC at 280 nm (Trp signal) of
the 1-octanol and aqueous phases. Therefore, in spite of the
hydrophobicity of Trp, 5 still prefers water to 1-octanol. As for
4 (formyl-Gly-âFa-Gly-amide), after equilibration, its existence
in water can be detected by neither analytical HPLC nor 19F
NMR, while its existence in 1-octanol can be readily detected
by both analytical HPLC and 19F NMR (see Supporting
Information). Based on this result, we conclude that, for 4, Poct
. 10. Hence, Poct(4) is over 100 times larger than Poct(5).
Results of the hydrophobicity measurement and the 1-octanol/
water partition coeffecient measurement are entirely consistent
with each other, both showing that, in the context of a peptide,
âFa is very hydrophobic and strongly prefers the organic over
the aqueous phase. All of these bode well for its potential use
as a membrane permeability enhancer for peptide-based phar-
maceuticals.
through a pad of Celite, the filtrate was washed with brine (300
mL), dried over sodium sulfate, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
(n-hexane/ethyl acetate ) 20/1) to give 8 as a solid (65.1 g, 98%):
1
mp 89-90 °C; H NMR (400 MHz, CDCl3) δ 3.78 (s, 1H), 3.80
(s, 4H), 3.89 (s, 2H), 4.13 (s, 1H), 4.16 (s, 1H), 4.49 (s, 2H), 5.39
(s, 1H), 6.88-6.92 (m, 2H), 7.33-7.37 (m, 2H); 19F NMR (376
MHz, CDCl3) δ -73.42 (s); 13C NMR (100.7 MHz, CDCl3) δ 39.2,
55.3, 66.2, 67.7, 68.7, 102.4, 113.8, 120.1 (q, J ) 292.6 Hz), 120.3
(q, J ) 292.6 Hz), 127.3, 129.9, 160.3; MS (CI) m/z 691 (M+
+
1, 100), 690 (M+, 17), 583 (22); HRMS (CI) calcd for C21H17F18O5,
691.0787; found, 691.0792.
2-Azidomethyl-3-(2,2,2-trifluoro-1,1-bis-trifluoromethylethoxy)-
2-(2,2,2-trifluoro-1,1-bis-trifluoromethylethoxymethyl)propan-
1-ol 11. Sodium azide (4.4 g, 66.9 mmol) was added to a stirred
solution of compound 10 (21.2 g, 33.4 mmol) in dimethylformal-
dehyde (120 mL). The reaction mixture was stirred at 60 °C for 4
h. The solvent was removed under vacuo, and the residue was
dissolved in tetrahydrofuran (120 mL). Sulfuric acid (0.87 mL) and
water (0.32 mL) were added to the stirred tetrahydrofuran solution,
and the resulting mixture was stirred at room temperature for an
additional 1 h. After removing the solvent, the residue was
redissolved in dichloromethane (200 mL) and extracted with
perfluorohexane (100 mL × 4). The combined extraction was
washed with dichloromethane (10 mL) and concentrated under
vacuo to give the pure azide 11 as a clear oil (19.3 g, 97%): 1H
NMR (400 MHz, CDCl3) δ 3.47 (s, 2H), 3.63 (s, 2H), 4.02 (s,
4H); 19F NMR (376 MHz, CDCl3) δ -73.21 (s); 13C NMR (100.7
In conclusion, a novel, highly fluorinated â-amino acid (âFa)
has been designed and synthesized. The Fmoc- and Boc-protec-
ted forms of this amino acid have also been synthesized with
high yield. The syntheses were highly efficient with an overall
(9) (a) Abbruscato, T.; Williams, S.; Misicka, A.; Lipkowski, A. W.;
Hryby, V. J.; Davis, T. P. J. Pharmacol. Exp. Ther. 1996, 276, 1049-
1057. (b) Gentry, C. L.; Egleton, R. D.; Gillespie, T.; Abbruscato, T. J.;
Bechowski, H. B.; Hruby, V. J.; Davis, T. P. Peptides 1999, 20, 1229-
1238.
1466 J. Org. Chem., Vol. 72, No. 4, 2007

MHz, CDCl3) δ 45.8, 49.8, 60.2, 66.8, 79.6 (m), 120.2 (q, J )
293.3 Hz); MS (CI) m/z 598 (M+ + 1, 72), 570 (100); HRMS (CI)
calcd for C13H10F18N3O3, 598.0435; found, 598.0418.
80.9 (m), 120.9, 121.7 (q, J ) 292.6 Hz), 126.2, 128.1, 128.8, 142.6,
145.2, 158.8, 174.4; MS (CI) m/z 808 (M+ + 1, 100); HRMS (CI)
calcd for C28H20F18NO6, 808.1003; found, 808.1010.
2-Aminomethyl-3-(2,2,2-trifluoro-1,1-bis-trifluoromethyl-
ethoxy)-2-(2,2,2-trifluoro-1,1-bis-trifluoromethylethoxymethyl)-
propionic Acid 1. A mixture of palladium on carbon (2.5 g) in
methanol (200 mL) was degassed for 2 min and stirred under a
hydrogen atmosphere for 30 min. A solution of acid 12 (10.7 g,
17.5 mmol) in methanol (10 mL) was then added, and the resulting
mixture was stirred at room temperature under a hydrogen
atmosphere for an additional 30 h. After solvent removal, the
resulting residue was purified by flash column chromatography on
silica gel (methanol/dichloromethane ) 1/10) to give the amino
3-tert-Butoxycarbonylamino-2,2-bis(2,2,2-trifluoro-1,1-bis-tri-
fluoromethylethoxymethyl)propionic Acid 3. A mixture of pal-
ladium on carbon (200 mg) in methanol (20 mL) was degassed for
2 min and stirred under a hydrogen atmosphere for 30 min. A
solution of acid 12 (1.2 g, 2.0 mmol) and di-tert-butyl dicarbonate
(872 mg, 4.0 mmol) in methanol (5 mL) was then added, and the
resulting mixture was stirred at room temperature under an
atmosphere of hydrogen gas for 30 h. After solvent removal, the
resulting residue was purified by flash column chromatography on
silica gel (n-hexane/ethyl acetate ) 5/1) to give the amino acid 3
1
1
acid 1 as a solid (10.1 g, 98%): mp 182-184 °C; H NMR (400
as a solid (1.32 g, 96%): mp 128-130 °C; H NMR (400 MHz,
MHz, CD3OD) δ 2.99 (s, 2H), 4.22 (d, J ) 9.2 Hz, 2H), 4.49 (d,
CD3OD) δ 1.36 (s, 9H), 3.31 (s, 2H), 4.17 (d, J ) 8.0 Hz, 2H),
4.39 (d, J ) 8.0 Hz, 2H); 19F NMR (376 MHz, CD3OD) δ -71.01
(s); 13C NMR (100.7 MHz, CD3OD) δ 28.8, 42.8, 54.1, 69.6, 80.7,
81.1 (m), 121.9 (q, J ) 293.3 Hz), 158.1, 177.2; MS (CI) m/z 686
(M+ + 1, 10), 644 (100); HRMS (CI) calcd for C18H18F18NO6,
686.0847; found, 686.0815.
J ) 8.4 Hz, 2H); 19F NMR (376 MHz, CD3OD) δ -71.00 (s); 13
C
NMR (100.7 MHz, CD3OD) δ 41.7, 52.1, 69.9, 80.9 (m), 121.7
(q, J ) 292.6 Hz), 175.0; MS (CI) m/z 586 (M+ + 1, 100); HRMS
(CI) calcd for C13H10F18NO4, 586.0322; found, 586.0285.
2-[(9H-Fluoren-9-ylmethoxycarbonylamino)methyl]-3-(2,2,2-
trifluoro-1,1-bis-trifluoromethylethoxy)-2-(2,2,2-trifluoro-1,1-
bis-trifluoromethylethoxymethyl)propionic Acid 2. To a stirred
solution of amino acid 1 (10.1 g, 17.2 mmol) in tetrahydrofuran
(100 mL) and water (100 mL) was added sodium carbonate (4.6 g,
42.9 mmol). After all of the sodium carbonate was dissolved, the
resulting mixture was cooled to 0 °C, and 9-fluorenylmethyl
chloroformate (6.7 g, 25.9 mol) was added in three portions. The
resulting reaction mixture was stirred at room temperature overnight.
The solvent was removed under vacuo, and the residue was purified
by flash column chromatography on silica gel (n-hexane/ethyl
acetate ) 5/1) to give the acid 2 as a white solid (13.3 g, 96%):
Acknowledgment. This work was supported by grants from
the NIH (EB002880 and EB004416) and the Sidney Kimmel
Foundation for Cancer Research. Y.B.Y. is a Kimmel scholar.
Supporting Information Available: Experimental procedures
and product characterization for compounds 7, 9, 10, and 12,
1
synthesis and partition procedures for 4 and 5, copies of H, 19F,
and 13C NMR spectra for compounds 8, 9, 10, 11, 12, 1, 2, and 3,
1
copies of HRMS spectra for compounds 1, 2, and 3, copies of H
NMR and HPLC spectra for compounds 4 and 5, copy of 19F NMR
spectra for compound 4, and copies of HPLC spectra of partition
test for compounds 4 and 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
mp 104-105 °C; H NMR (400 MHz, CD3OD) δ 3.45 (s, 2H),
4.16 (m), 4.27 (m, 4H), 4.46 (d, J ) 8.8 Hz, 2H), 7.26 (t, J ) 7.2
Hz, 2H), 7.35 (t, J ) 7.2 Hz, 2H), 7.60 (d, J ) 7.6 Hz, 2H), 7.74
(d, J ) 7.2 Hz, 2H); 19F NMR (376 MHz, CD3OD) δ -71.00 (s);
13C NMR (100.7 MHz, CD3OD) δ 36.9, 42.8, 53.7, 68.2, 69.0,
JO0616308
J. Org. Chem, Vol. 72, No. 4, 2007 1467