Construction of N-1H,1H-perfluoroalkylated peptide bonds

Article abstract of DOI:10.1039/b712617d

The preparation of a variety of optically pure peptides containing an N-1H,1H-perfluoroalkyl label on a selected backbone amide bond is now possible. The Royal Society of Chemistry.

Full text of DOI:10.1039/b712617d

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Construction of N-1H,1H-perfluoroalkylated peptide bonds{
Changqing Lu and Darryl D. DesMarteau*
Received (in Cambridge, UK) 16th August 2007, Accepted 25th September 2007
First published as an Advance Article on the web 18th October 2007
DOI: 10.1039/b712617d
of both the adjacent N-trifluoroethylated peptide bond and the
The preparation of a variety of optically pure peptides
containing an N-1H,1H-perfluoroalkyl label on a selected
backbone amide bond is now possible.
activated carboxyl group, the a-carbon of the central amino acid
was racemized to give a pair of diastereomers in the process of the
C-terminal elongation with a third amino acid ester.14 The
phthaloyl protecting group employed in the latter work made
deprotection for further elongation at the N-terminus difficult due
to the formation of diketopiperazines.
Peptide based drugs suffer from their poor bioavailability and
poor proteolytic stability. To overcome these disadvantages,
several modifications have been developed, e.g. the introduction
of unnatural amino acids and the use of peptidomimetics including
peptoids.1 Among these modifications, N-alkylation of amino
acids and peptide bonds, especially N-methylation, has been
pursued for decades and continues to be of great interest for many
researchers.2
To solve the above racemization problem, we reasoned that the
C-terminal elongation should be carried out before the formation
of the trifluoroethylated peptide bond; if the more reactive acid
chloride was used instead of acid fluoride, the deprotonation of the
trifluoroethylated a-amino group of the central amino acid using a
strong base could be avoided.
To obtain site-specific N-alkylation, an amino acid was
N-alkylated, followed by the subsequent coupling reaction to
form the desired peptide bond. To date, many methods,3 e.g.
oxazolidinone formation and reduction,4 reductive amination,5
temporary protection and activation followed by SN26 or
Mitsunobu reaction,6a,7 etc., have been developed for the
N-alkylation of amino acids. The more effective coupling reagents,
e.g. triphosgene, have been used in the coupling reactions of
N-alkylated amino acids.8
In this research, the trifluoroethyl group and its two
homologues, CF3(CF2)nCH2– (n = 1, 2), were first introduced to
the a-amino group of L-phenylalanine methyl ester using iodonium
fluoroalkylating agents.12 The intermediates 1 were then elongated
at the C-terminus with a second amino acid ester, e.g. L-leucine
methyl ester, to form the dipeptides 2, Scheme 1.
The optically pure model peptide 3 was obtained by coupling
dipeptide 2 (n = 0) with Na-phthaloyl glycine acid chloride,15 in the
presence of pyridine as a catalyst and base, Scheme 2.
Fluoroalkyl groups have certain unique properties. Once
attached to biologically active compounds, fluorine(s) can be used
as a marker or tracer of the substrates by 19F NMR or 18F PET
study.9 Electron withdrawing fluoroalkyl groups alter the electron
density of the adjacent heteroatoms, and hence affect the pKa
value, hydrogen bonding, and lipophilicity of the substrates.10
Therefore, the introduction of fluoroalkyl groups to the function-
alities of amino acids and peptides has been investigated by us for
a decade.11
Introduction of the electron withdrawing trifluoroethyl group
facilitates rotation around the corresponding peptide bond. The
exchange between cis and trans isomers of the optically pure model
peptide 3 was evidenced by the cross peaks in the 19F NOESY
spectrum shown in Fig. 1.
In different solvents, both the chemical shifts and the ratio of the
two isomers of 3 changed dramatically. In less polar solvent
CDCl3, the chemical shifts of the two isomers shifted upfield with
isomer B predominating, whereas in the more polar solvent
In our recent research, the 1H,1H-trifluoroethyl group,
CF3CH2–, was introduced onto the N-terminus of small peptides
to increase the lipophilicity of the substrates.12 The trifluoroethy-
lated a-amino group did not show enough nucleophilicity towards
either the activated carboxyl group in conventional linear peptide
coupling reactions or an amino acid fluoride. However, the
trifluoroethylated N-terminus of a linear dipeptide did undergo
intramolecular cyclization reactions to form diketopiperazines.13
Subsequently, the trifluoroethylated a-amino group of an amino
acid ester was deprotonated using a strong base, e.g. NaH, and the
resulting anionic intermediate exhibited enough nucleophilicity to
couple with an Na-phthaloyl protected amino acid fluoride to form
a linear peptide bond.14 However, because of the electronic effect
Scheme 1 Reagents and conditions: (a) CF3(CF2)nCH2I(C6H5)-
N(SO2CF3)2 (n = 0–2), NaHCO3, CH2Cl2–H2O, rt, 4 h; (b) 1.0 M
NaOH, rt, 16 h, 40 h, and 64 h for n = 0–2; (c) 0 uC, conc. HCl to pH 4.5
(89%, 92%, and 83% for n = 0–2, 3 steps); (d) N-(3-dimethylaminopropyl)-
N9-ethylcarbodiimide (EDC)?HCl, 1-hydroxybenzotriazole (HOBt),
N,N-diisopropylethylamine (DIEA), CH2Cl2, 0 uC to rt, overnight (91%,
87%, and 81% for n = 0–2, respectively).
Department of Chemistry, Clemson University, Clemson, SC 29634,
USA. E-mail: fluorin@clemson.edu; Fax: +1 864 656 2545;
Tel: +1 864 656 1251
{ Electronic supplementary information (ESI) available: Experimental
procedures for the syntheses of 2 (n = 0), 3, 4 (n = 0) and 5; NMR and
HRMS spectra of 3, 4, and 5. See DOI: 10.1039/b712617d
208 | Chem. Commun., 2008, 208–210
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Table 1 Chemical shifts and ratio of two isomers of 3 in different
solventsa
Solvent
CDCl3
CD3CN
Acetone-d6
DMSO-d6
dF/ppm
268.41,
270.17
0.18
266.37,
268.87
0.62
266.60,
268.88
0.77
265.28,
267.49
1.89
A/B
a
The ratio was determined by 19F NMR spectroscopy, Fig. 2.
Scheme 2 Synthesis of optically pure model peptide 3.
Scheme 3 Resonance structures and isomer exchanges of the
N-trifluoroethylated peptide bond in different solvents.
The effect of temperature on the exchange rate between the cis
and trans isomers of 3 is shown in Fig. 3. With increasing
temperature, two well separated triplets eventually merged and
became one broad singlet. In DMSO-d6, the two peaks coalesced
at 88 uC. The rate constant for the exchange and the free energy of
activation for the rotation17 around the N-trifluoroethylated
Fig. 1 19F NOESY spectrum of 3 (282.78 MHz, CD3CN, 21 uC).
peptide bond were calculated as 1.38
67.4 kJ mol21, respectively.
6
103 s21 and
DMSO-d6, the chemical shifts of the two isomers shifted downfield
with isomer A predominating, Fig. 2 and Table 1.
To enable the further elongation at the N-terminus of the model
peptides, Fmoc15c was used as the protecting group in the
syntheses of 4. However, in the presence of pyridine as a catalyst
and base, Na-Fmoc–GlyCl underwent self-polymerization,
More polar solvents favor the charged resonance structure of
the N-trifluoroethylated peptide bond, Scheme 3. The partial
formal positive charge on the nitrogen results in downfield
chemical shifts of the two isomers of 3 in DMSO-d6. The isomer
with the CF3CH2– group trans to the carbonyl oxygen is assigned
as isomer A with downfield chemical shift based on the fact that
the larger electronic interaction is for the trans isomer.16
Fig. 2 19F NMR spectra of 3 in different solvents (282.78 MHz, 21 uC,
from top to bottom: CDCl3, CD3CN, acetone-d6, and DMSO-d6).
Fig. 3 Temperature effect on the exchange between two isomers of 3 in
DMSO-d6 shown by 19F NMR spectroscopy (282.38 MHz).
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Notes and references
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Scheme 4 Syntheses of optically pure peptide building blocks 4 in the
presence of excess 2 as a base (83%, 84%, and 82% for n = 0–2,
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Scheme 5 Elongation of the peptide building block 4 (n = 0) into a
pentapeptide 5 (27%, 4 steps).
especially at elevated temperatures, which compromised the
coupling yield. To avoid using pyridine, dipeptides 2 were used
in 2.0 equivalents in the coupling reactions to obtain optically pure
peptide building blocks 4, Scheme 4. Excess 2 was easily recovered
by chromatography.
The optically pure peptide building blocks 4 all showed cis and
trans isomers with similar solution dynamics to that of 3.
In the further elongation at the N-terminus of peptide building
block 4 (n = 0), the Fmoc protecting group was removed using
4-(aminomethyl)piperidine.15c Deprotected 4 (n = 0) was converted
into an optically pure pentapeptide 5, leucine enkephalin,
containing an N-trifluoroethylated peptide bond in the selected
position, Scheme 5.
In conclusion, the optically pure peptide building blocks 4
containing an N-1H,1H-perfluoroalkylated backbone amide bond
have been synthesized by coupling the Na-Fmoc-protected amino
acid chloride with the excess of N-terminus 1H,1H-perfluoro-
alkylated peptide fragments. The coupling reaction is straightfor-
ward and no racemization is observed. Further elaboration of 4
into 5 clearly indicates the potential of this work for the generation
of a variety of strategically labeled N-1H,1H-perfluoroalkyl
peptides.
12 D. D. DesMarteau and C. Lu, J. Fluorine Chem., 2007, 128, 1326.
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Financial support of this research by the National Science
Foundation is gratefully acknowledged.
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210 | Chem. Commun., 2008, 208–210
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