Efficient asymmetric synthesis of Tryptophan Analogues having useful Photophysical properties

Article abstract of DOI:10.1021/ol403429e

Two new fluorescent probes of protein structure and dynamics have been prepared by concise asymmetric syntheses using the Sch?llkopf chiral auxiliary. The site-specific incorporation of one probe into dihydrofolate reductase is reported. The utility of these tryptophan derivatives lies in their absorption and emission maxima which differ from those of tryptophan, as well as in their large Stokes shifts and high molar absorptivities.

Full text of DOI:10.1021/ol403429e

Letter
pubs.acs.org/OrgLett
Efficient Asymmetric Synthesis of Tryptophan Analogues Having
Useful Photophysical Properties
Poulami Talukder, Shengxi Chen, Pablo M. Arce, and Sidney M. Hecht*
Center for BioEnergetics, The Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe,
Arizona 85827, United States
S
* Supporting Information
ABSTRACT: Two new fluorescent probes of protein structure and
dynamics have been prepared by concise asymmetric syntheses using the
Schollkopf chiral auxiliary. The site-specific incorporation of one probe into
̈
dihydrofolate reductase is reported. The utility of these tryptophan
derivatives lies in their absorption and emission maxima which differ from
those of tryptophan, as well as in their large Stokes shifts and high molar
absorptivities.
he aromatic amino acid tryptophan (Trp) is often
Timportant to protein function and structural integrity
and is also the main source of intrinsic protein fluorescence.
Therefore, it has long been used as a probe for protein structure
and function studies.1 However, due to its complicated
photophysics and multiple occurrence in many proteins of
interest, Trp cannot always be used as a suitable optical probe.2
Therefore, it seemed of interest to design novel tryptophan
analogues which have more useful spectroscopic properties and
do not perturb the local environment of the target protein.
Recently, the incorporation of azatryptophans as fluorescence
probes in structurally modified proteins has been described.2
Among the azaindole chromophores, 4-azaindole has been
identified as a promising candidate. It has strongly red-shifted
fluorescence with a large Stokes shift (∼130 nm).2 As its 4-
azatryptophan derivative, it has been incorporated into target
proteins.1 However, the fluorescence intensity and quantum
yield of 4-azatryptophan are much lower than those of
tryptophan itself.1 In addition, azatryptophans are hydrophilic,
such that their presence in the hydrophobic core of proteins
would likely modify protein structure and function.1 To
overcome these limitations we have prepared more hydro-
phobic tricyclic tryptophan derivatives which have larger Stokes
shifts (∼150−160 nm) and exihibit significantly higher molar
absorptivities than that of tryptophan.
Figure 1. Structure of 4-azaindole and the chromophoric moieties of
the new tryptophan analogues.
same strategy, we describe the incorporation of Trp-1 into
position 17 of dihydrofolate reductase (DHFR).
For the asymmetric synthesis of Trp-1, the required indole 4
was prepared in four steps from commercially available 4-
bromoisoquinoline according to the method of Briere et al.10
with some modifications. The synthesis began with amination
of 4-bromoisoquinoline (Scheme 1). The reaction was carried
out using the Cu/CuCl couple, which induced the trans-
formation at a much lower temperature11 than that for the
reported procedure (autoclave, aq NH4OH, CuSO4, 150−200
°C).12 Iodination with I2 and Ag2SO4 provided 4-amino-3-
iodoisoquinoline (2) in satisfactory yield.13 Sonogashira
coupling of trimethylsilylacetylene (TMSA) and 4-amino-3-
Scheme 1. Synthesis of Pyrroloisoquinoline 4
The 1H-pyrrolo[3,2-c]isoquinoline molecule consists of a
benzene ring fused to a bicyclic 4-azaindole ring (Figure 1)
whereas 1H-pyrrolo[2,3-f ]quinoline can be considered to be 4-
azaindole modified with a benzo ring spacer that separates the
pyridine ring and pyrrole ring.
To realize the asymmetric synthesis of the Trp analogues
Trp-1 and Trp-2, a stereoselective strategy utilizing the
Schollkopf chiral reagent3 has been adopted. This strategy
̈
has enabled the successful synthesis of the unnatural S-amino
acids. In recent years, the use of misacylated tRNAs has
facilitated the introduction of unnatural amino acids into
predetermined positions in peptides and proteins.4−9 Using the
Received: November 26, 2013
© XXXX American Chemical Society
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Letter
Scheme 2. Synthesis of Cyanomethyl Ester 13
Scheme 3. Synthesis of Cyanomethyl Ester 22
iodoisoquinoline (2) proceeded very smoothly at room
temperature. The desired ethynyl derivative 3 was obtained in
91% yield, whereas the reported procedure involved coupling
with 4-amino-3-bromoisoquinoline at a higher temperature in
lower yield.10 4-Amino-3-ethynyl derivative 3 was then heated
to reflux with NaNH2 in DMF to effect cyclization into key
intermediate 4.10
Pyrroloisoquinoline 4 was next converted to its respective
amino acid derivative. Key intermediate 13 was obtained in
nine steps from 4 (Scheme 2). Pyrroloisoquinoline 4 was first
formylated under Duff reaction conditions14 to yield aldehyde
5, the latter of which was subjected to N-tosylation with p-
toluenesulfonyl chloride (p-TsCl) and sodium hydride to afford
6 in 46% overall yield.15 Reduction of aldehyde 6 to alcohol 7
using NaBH4 in EtOH proceeded almost quantitatively.14
Chlorination of alcohol 7 with thionyl chloride afforded 8 in
77% yield.16 Asymmetric synthesis of the amino acid precursor
was carried out using the Schollkopf reagent (R)-2,5-dihydro-
̈
3,6-dimethoxy-2-isopropylpyrazine.3 Regioselective lithiation
(n-BuLi, THF, −78 °C) of the chiral auxiliary produced the
lithium enolate which afforded the adduct 9 from 8 with high
diastereoselectivity (only one diastereomer was detectable in
1
the H and 13C NMR spectra). Mild hydrolysis (2 N HCl)
afforded the α-substituted amino acid methyl ester 10.3 Amine
10 was protected as the NVOC carbamate to yield 11 in 90%
yield.17 The NVOC group was chosen, as it can be removed by
UV irradiation.18,19 N-Detosylation of 11 was performed using
cesium carbonate in 2:1 THF−MeOH to yield 12 in 73%
yield.20 Methyl ester 12 was then hydrolyzed to afford the free
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Table 1. Spectroscopic Properties of N-Acetylated Methyl Esters of Trp Analogues in MeOH
Trp analogue
λabs, max (nm)
ε (M−1 cm−1
)
λex, max (nm)
λem, max (nm)
ΦF
Trp
280
260
272
6900
18 100
15 500
280
260
272
346
420
421
0.18
0.10
0.03
Ac-Trp-1-OMe
Ac-Trp-2-OMe
Scheme 4. Preparation of Suppressor tRNACCCG Activated with Trp-1
acid which was subsequently treated with chloroacetonitrile to
afford the requisite cyanomethyl ester 13 in 54% yield.17
The synthesis of N-NVOC cyanomethyl ester 22 of the 1H-
pyrrolo[2,3-f ]quinoline followed a similar procedure (Scheme
3). Initially, the commercially available pyrroloquinoline was
formylated to yield 1414 which was N-tosylated with p-TsCl
and NaH, affording 15 in 55% overall yield.15 Reduction of
aldehyde 15 with NaBH4 in EtOH afforded alcohol 16 in 82%
yield.14 Chlorination of 16 with thionyl chloride then provided
17.16 Regioselective lithiation (n-butyllithium, THF, −78 °C)
acids) make Trp-1 and Trp-2 potentially attractive probes for
protein conformational studies.
In order to evaluate the properties of Trp-1 as a constituent
of the protein DHFR, this amino acid was used to activate a
suppressor tRNA (Scheme 4). Treatment of cyanomethyl ester
13 with the tris(tetrabutylammonium) salt of pdCpA22 in
anhydrous DMF afforded pdCpA ester 23 in 52% yield.17 The
aminoacylated dinucleotide was ligated to an abbreviated
tRNACCCG-COH transcript in the presence of T4 RNA ligase
and ATP17 to afford NVOC-aminoacyl-tRNA 24. Activated
tRNA 24 was deprotected by UV irradiation to afford free
aminoacyl-tRNA 25.17
The activated suppressor tRNA was employed in an in vitro
protein synthesizing system programmed with DHFR mRNA
having a CGGG codon at position 17 of the elaborated protein
to introduce Trp-1 into position 17 of DHFR (Figure S1,
Supporting Information). To ensure that the modified protein
was properly folded, the ability of this modified enzyme to
catalyze the reduction of dihydrofolate to tetrahydrofolate was
studied.17 Substitution of position 17 with Trp-1 was
reasonably well tolerated (Figure S2), affording a construct
which consumed NADPH 65% as efficiently as wild-type
DHFR under turnover conditions at pH 7.0 and 37 °C. The
modified DHFR was subjected to irradiation at 260 nm and
exhibited an emission spectrum centered at 409 nm for the
incorporated Trp-1 (Figure 2, red curve). Thus, it was possible
to selectively excite and monitor Trp-1 in the modified DHFR,
which also contains five Trp’s.
of the Schollkopf reagent produced the lithium enolate which
̈
afforded adduct 18 with high diastereoselectivity (only one
diastereomer was detectable in the 1H and 13C NMR spectra).3
Mild hydrolysis (2 N HCl) provided α-substituted amino acid
methyl ester 193 which was protected as the NVOC carbamate
to yield 20 in 92% yield.17 N-Detosylation of 20 using cesium
carbonate in 2:1 THF−MeOH afforded ester 21.20 Hydrolysis
of 21 afforded the free acid, which was activated as cyanomethyl
ester 22 in 40% overall yield.17 The syntheses of 13 and 22
were quite robust, providing workable routes to these
compounds preparatively.
Next, we evaluated the photophysical properties of the
tryptophan analogues as potentially useful fluorescence probes.
The study was carried out using the respective N-acetylated
methyl esters to mimic their anticipated behavior when present
in proteins. The synthesis of the N-acetylated methyl esters of
the Trp analogues (Trp-1 and Trp-2) is illustrated in Scheme
S1 in the Supporting Information. Both of the methyl esters
had larger Stokes shifts and greater molar extinction coefficients
than those of tryptophan (Table 1).
Tryptophan is commonly used as a fluorophore in
biophysical studies, and the emission maxima for both Ac-
Trp-1-OMe and Ac-Trp-2-OMe are substantially red-shifted
from that of tryptophan.21 This means that the emission signals
from these Trp analogues can be easily isolated from those of
the native tryptophans in the protein. Both the increased molar
absorptivities and the shifted emission spectra (distinct from
normal tryptophans and the other normal aromatic amino
In summary, we report the synthesis of two new tricyclic
tryptophan derivatives (Trp-1 and Trp-2) and demonstrate that
Trp-1 can be introduced into DHFR with minimal disruption
of function and could be selectively monitored in the presence
of five Trp residues in that DHFR, underscoring its potential as
a fluorescence probe for studying protein conformation and
dynamics.
C
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(15) Yao, C. H.; Song, J. S.; Chen, C. T.; Yeh, T. K.; Hsieh, T. C.;
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(18) (a) Patchornik, A.; Amit, B.; Woodward, R. B. J. Am. Chem. Soc.
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1974, 39, 192.
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1991, 113, 2722.
(20) Bajwa, J. S.; Chen, G.; Prasad, K.; Repic, O.; Blacklock, T. J.
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Figure 2. Fluorescence emission spectra of a modified DHFR (Trp-1
at position 17) (red) and wild-type DHFR (blue). λex = 260 nm.
(21) Tucker, M. J.; Ovola, R.; Gai, F. J. Phys. Chem. B 2005, 109,
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ASSOCIATED CONTENT
* Supporting Information
■
C.; Schultz, P. G. Nucleic Acids Res. 1989, 17, 9649.
S
Experimental procedures, characterization data, and spectra are
included. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: sidney.hecht@asu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Stephen Benkovic and C. Tony Liu,
Pennsylvania State University, for helpful discussions regarding
the photophysical properties of the Trp derivatives, and for a
critical reading of the manuscript. This work was supported by
NIH research Grant GM 092946.
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