Synthesis of tetrafluorinated aromatic amino acids with distinct signatures in 19F NMR

Article abstract of DOI:10.1021/ol203140n

Fluorinated amino acids serve as powerful tools in protein chemistry. We synthesized a series of para-substituted tetrafluorophenylalanines via the regioselective S_NAr chemistry of the commercially available pentafluorophenylalanine Boc-Z. Thes

Full text of DOI:10.1021/ol203140n

ORGANIC
LETTERS
2012
Vol. 14, No. 2
528–531
Synthesis of Tetrafluorinated Aromatic
Amino Acids with Distinct Signatures
in 19F NMR
Luoheng Qin, Christopher Sheridan, and Jianmin Gao*
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill,
Massachusetts 02467-3801, United States
jianmin.gao@bc.edu
Received November 23, 2011
ABSTRACT
Fluorinated amino acids serve as powerful tools in protein chemistry. We synthesized a series of para-substituted tetrafluorophenylalanines via
the regioselective SNAr chemistry of the commercially available pentafluorophenylalanine Boc-Z. These novel unnatural amino acids display
distinct 19F NMR signatures, making them powerful tools for analyzing proteinꢀmembrane interactions with NMR spectroscopy.
Fluorination has become an increasingly popular strat-
egy in medicinal chemistry and protein engineering due
to the unique properties of fluorinated compounds.1
For example, incorporating fluorinated amino acids into
synthetic proteins often leads to improved stability owing
to the added hydrophobicity by fluorination.2 In addi-
tion, fluorinated amino acids have shown promise as
supramolecular synthons to program protein assembly
in both aqueous and membrane environments.3 Another
attractive feature of fluorinated compounds lies in their
tractability by 19F NMR, which is essentially blind to
endogenous material in biological systems. Furthermore,
the chemical shift of 19F resonances is highly sensitive to
the local environment; therefore, fluorinated amino acids
serve as powerful tools for interrogating protein folding
and proteinꢀmembrane interactions.4 Ideally, multisite
incorporation of fluorinated residues should allow one to
monitor the behavior of a target protein with atomic
resolution across the protein sequence. However, the as-
signment of individual 19F resonances is often challenging5
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r
10.1021/ol203140n
Published on Web 12/23/2011
2011 American Chemical Society

and would greatly benefit from amino acids with charac-
teristic 19F NMR signatures.
used for removing the PMB group from peptides in
solution.9
There are a limited number of fluorinated amino acids
available from commercial sources as their synthesis is
nontrivial. We recently reported a facile synthesis6 of
tetrafluorotyrosine via the regioselective SNAr chemistry
of pentafluorophenylalanine (named as Z for brevity). By
using the starting material with the desired stereochem-
istry, the D- or L-isomer of tetrafluorotyrosine can be
readily synthesized in gram quantities. Herein we show
the SNAr strategy can be extended to a series of para-
X-tetrafluorophenylalanines (Scheme 1), where X repre-
sents a range of functionalities, including thiol, amine,
and halogens. For ease of discussion, we name such a
fluorinated amino acid as ZpX with X specifying the para
substituent (e.g., ZpOH stands for tetrafluorotyrosine).
Although structurally similar, these unnatural amino
acids display easily distinguishable 19F resonances, poised
for multisite labeling and analysis of proteins. All the
amino acids described in this report are in the L-config-
uration; however, the synthetic protocols should enable
the synthesis of the D-isomers as well.
Scheme 2. Synthesis of ZpSH
The SNAr chemistry of Boc-Z also applies to nitrogen
nucleophiles. The para-amino analogue ZpNH2 was
synthesized via reduction of the azide precursor ZpN3,
for which a chemoenzymatic synthesis was previously
reported by Ghadiri and co-workers.10 In their work, a
racemic mixture of ZpN3 was made through the SNAr
Scheme 1. Scope of the SNAr Reaction of Boc-Z
Scheme 3. Synthesis of ZpN3/ZpNH2
Similar to ZpOH, use of a sulfur nucleophile in the SNAr
reaction afforded the para-mercapto analogue ZpSH.
Initially we treated Boc-Z with NaSH, which predomi-
nantly gave a thioether with two Boc-Z molecules cross-
linked as a result of sequential SNAr reactions. To prevent
dimerization, we chose 4-methoxybenzenemethanethiol
(PMBSH, Scheme 2) as an alternative, which reacted
readily with Boc-Z to give Boc-ZpSPMB (2) in good yield.
Protecting group conversion yielded Fmoc-ZpSPMB (3),
which is compatible with solid phase peptide synthesis
(SPPS) via Fmoc/tBu chemistry.7 No epimerization was
observed by Marfey’s test (ref 8 and also see Supporting
Information (SI)). The PMB protecting group was effi-
ciently removed to give Fmoc-ZpSH (4) by using Hg(OAc)2
under mild conditions. A similar protocol was previously
chemistry and the L-isomer was obtained through kinetic
resolution with Aspergillus melleus acylase-I. Our synthe-
sisstarted withthe pureL-enantiomerof Boc-Z(Scheme 3).
We first protected the carboxyl group as a tBu ester
and then treated the product with Bu4NN3/NaN3 in
DMF. The SNAr reaction proceeded smoothly without
causing epimerization of the amino acid (see SI for
details). Deprotection of the carboxyl group and conver-
sion of Boc to Fmoc were accomplished through a one-
pot process. The three-step synthesis gave Fmoc-ZpN3 (7)
with an overall yield of 65%. Reduction of Fmoc-ZpN3
was best accomplished by using propylene dithiol11 under
mild conditions to afford Fmoc-ZpNH2 (8) in excellent
(9) (a) Nishimura, O.; Kitada, C.; Fujino, M. Chem. Pharm. Bull.
1978, 26, 1576. (b) Macmillan, D.; Anderson, D. W. Org. Lett. 2004, 6,
4659–4662.
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(11) Bayley, H.; Standring, D. N.; Knowles, J. R. Tetrahedron Lett.
1978, 3633–3634.
(6) Wang, F.; Qin, L.; Wong, P.; Gao, J. Org. Lett. 2011, 13, 236–239.
(7) Coin, I.; Beyermann, M.; Bienert, M. Nat. Protoc. 2007, 2
3247–3256.
(8) Bhushan, R.; Bruckner, H. Amino Acids 2004, 27, 231–247.
Org. Lett., Vol. 14, No. 2, 2012
529

yield. Propylene dithiol treatment effectively converted
ZpN3 in a peptide into ZpNH2 as well (see SI for details);
in other words, ZpN3 conveniently serves as a precursor
for the synthesis of peptides containing ZpNH2.
Table 1. Optimization of Diazonium Iodination Conditions
with the Model Compound 10
We hypothesized that ZpNH2 could be converted into a
variety of fluorinated phenylalanine analogues through
the diazonium chemistry.12 Here we describe the synthetic
protocols for the para-halogenated (Cl, Br, I) tetrafluor-
ophenylalanines (Scheme 4). Although widely used to
access halogenated aromatics,13 the diazonium reaction
is less straightforward for heavily fluorinated anilines,
as these electron-deficient anilines are less nucleophilic
and more difficult to generate the diazonium species.
Indeed, treating the model compound 10 (para-carboxyl-
tetrafluoroaniline) with the canonical conditions of
diazonium iodination (TsOH H2O/NaNO2/KI) only
3
afforded less than 60% conversion of the starting
material, indicating inefficient formation of the diazo-
nium species. In addition to the desired product 20, the
reaction yielded a major byproduct, the para-hydro-
genated compound 30. This byproduct is generated
possibly because the diazonium intermediate and the
subsequent phenyl radical are highly reactive and ex-
tract a hydrogen atom from the solvent molecules. This
hypothesis is supported by the solvent dependence of
byproduct formation: THF gives exclusively the hydro-
genated byproduct, while CH3CN favors the desired
iodo product (Table 1), affording a mixture of 20 and 30
in a ratio of 3:1. Interestingly compound 40 (with a para-
benzyl substituent) is obtained as the sole product when
toluene is used as a cosolvent. This is consistent with the
radical mechanism of the diazonium chemistry, which
leads to cross-coupling between the fluorinated phenyl
radical and toluene. This observation also led us to sus-
pect that the benzylic position of TsOH might partici-
pate in the radical chemistry and therefore should be
avoided in the diazonium chemistry. The alternative
conditions (tBuONO/I2)14 reduced the byproduct for-
mation to ∼5%. Fortuitously, these conditions also im-
proved the diazonium formation to give complete con-
version of the starting material.
a Optimized conditions: 0.05 M in CH3CN, 2 equiv of I2, and 1.5
equiv of tBuONO. b Neither 20 nor 30 was generated.
Scheme 4. Synthesis of ZpX (X = Cl, Br, I)
Optimized conditions for the model reaction were
investigated for the synthesis of the halogenated tetra-
fluorophenylanines. We initially subjected the Fmoc
protected ZpNH2 (8) to the iodination conditions, which
generated a messy mixture. NMR analysis suggested
possible degradation of the Fmoc group, presumably
because the benzylic substructure of Fmoc was attacked
by the radical species. Alternatively, we used the trifluor-
oacetyl protected ZpNH2 (Compound 10, Scheme 4) in
the diazonium chemistry, which afforded ZpI (11c) in
good yield. Similar to the model reaction, the byproduct
ZpH was found to be ∼5%.
yields. In comparison to iodination, the byproduct (ZpH)
percentage was found to be similar for bromination and
slightly higher for chlorination (∼12%), consistent with the
relatively low reactivity of chlorine in radical chemistry. The
protecting group conversion was accomplished through a
one-pot process to give Fmoc-ZpXs (12aꢀc), theform ready
for peptide synthesis (see SI). The enantiopurity of these
amino acids was confirmed by Marfey’s test.
In order to utilize these novel amino acids as protein
probes, we characterized their signatures in 19F NMR.
Despite their structural similarity, the tetrafluorinated
phenylalanine analogues display a wide distribution of
their 19F resonances, ranging from ꢀ120 to ꢀ165 ppm
(Table S1, SI). The electronegative substituents (ꢀF,
ꢀOH, ꢀNH2) on the para position elicit significant
upfield shift of the meta fluorine resonances, which
appear around ꢀ163 ppm. In contrast, the bromo and
Use of CuX2/CuX (X: Br or Cl) in the diazonium
chemistry gave ZpBr and ZpCl respectively in good
(12) Hodgson, H. H. Chem. Rev. 1947, 40, 251–277.
(13) Cowdrey, W. A.; Davies, D. S. Quart. Rev. 1952, 6, 358–379.
(14) Ek, F.; Wistrand, L. G.; Frejd, T. J. Org. Chem. 2003, 68
1911–1918.
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Org. Lett., Vol. 14, No. 2, 2012

We further examined the environmental sensitivity of 19F
chemical shifts by using chloroform and methanol as
solvents respectively. Consistent with previous reports,5
the 19F resonances are highly sensitive to environment: a
polar solvent like methanol gives resonances upfield
shifted by as much as 3 ppm in comparison to chloroform.
On the other hand, the ionizable residues (ZpOH and
ZpSH) show dramatic chemical shift changes in response
to the environment pH (Table S1), presumably as a result
of protonation/deprotonation of their side chains.
In summary, we have synthesized a series of novel
fluoroaromatic amino acids that can be readily utilized
in chemical synthesis of peptides and proteins. Our
synthetic protocol builds on the regioselective SNAr reac-
tion of the pentafluorophenylalanine and the diazonium
chemistry of ZpNH2. The new amino acids exhibit dis-
tinct 19F NMR signatures, which allow unambiguous
assignment, and therefore enable multisite labeling of
proteins for NMR characterization. Furthermore, these
fluoroaromatic residues effectively sense the local envi-
ronment by a significant shift of their 19F resonances.
Given the high abundance of aromatic residues at the
proteinꢀmembrane interface,17 we submit that the fluori-
nated aromatic residues described here are particularly
useful for the study of proteinꢀmembrane interactions.
Work along this direction is currently underway.
19
Figure 1. 19Fꢀ F COSY spectrum of a magainin mutant
incorporating three ZpX (ZpBr, ZpCl, ZpN3) residues. The
highly dispersed 19F resonances enabled facile assignment of all
peaks in 19F NMR.
iodo substituents on the para position cause a downfield
shift of the meta fluorines, with ZpBr and ZpI displaying a
resonance around ꢀ133 and ꢀ120 ppm respectively.
These characteristic chemical shifts should allow facile
assignment of 19F resonances for peptides that incorpo-
rate multiple fluorinated residues. As a proof-of-concept
experiment, we have incorporated three ZpX residues
(ZpCl, ZpBr, and ZpN3) into magainin 2, a well-known
membrane-lytic peptide.15 Due to their distinct chem-
ical shifts, the meta-fluorine resonances of ZpBr and
ZpN3 were readily identified, which in conjugation with
Acknowledgment. This work was supported partly by
Boston College, the Smith Family Foundation, and the
National Science Foundation (CHE-1112188).
Supporting Information Available. Experimental pro-
cedures and characterization of all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
the 19Fꢀ F COSY16 data enabled unambiguous assign-
19
ment of all 19F peaks (Figure 1).
(15) Matsuzaki, K. Biochim. Biophys. Acta 1998, 1376, 391–400.
(16) Yonemori, S.; Sasakura, H. J. Fluorine Chem. 1995, 75, 151–156.
(17) Killian, J. A.; von Heijne, G. Trends Biochem. Sci. 2000, 25,
429–434.
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