Enantiospecific synthesis of genetically encodable fluorescent unnatural amino acid l-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid

Article abstract of DOI:10.1021/jo2007626

Fluorescent unnatural amino acids (UAAs), when genetically incorporated into proteins, can provide unique advantages for imaging biological processes in vivo. Synthesis of optically pure l-enantiomer of fluorescent UAAs is crucial for their effective application in live cells. An efficient six-step synthesis of l-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (l-Anap), a genetically encodable and polarity-sensitive fluorescent UAA, has been developed. The synthesis takes advantage of a high-yield and enantiospecific Fukuyama-Mitsunobu reaction as the key transformation.

Full text of DOI:10.1021/jo2007626

NOTE
pubs.acs.org/joc
Enantiospecific Synthesis of Genetically Encodable Fluorescent
Unnatural Amino Acid L-3-(6-Acetylnaphthalen-2-ylamino)-2-
aminopropanoic Acid
Zheng Xiang and Lei Wang*
The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines
Road, La Jolla, California 92037, United States
S Supporting Information
b
ABSTRACT:
Fluorescent unnatural amino acids (UAAs), when genetically incorporated into proteins, can provide unique advantages for imaging
biological processes in vivo. Synthesis of optically pure L-enantiomer of fluorescent UAAs is crucial for their effective application in
live cells. An efficient six-step synthesis of L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap), a genetically
encodable and polarity-sensitive fluorescent UAA, has been developed. The synthesis takes advantage of a high-yield and
enantiospecific FukuyamaꢀMitsunobu reaction as the key transformation.
he past decade has witnessed the dramatic progress on site-
specific incorporation of unnatural amino acids (UAAs) into
T
proteins in live cells through the expansion of the genetic code.1ꢀ4
Using engineered tRNA-synthetase pairs orthogonal to endo-
genous counterparts in the host cell, more than 70 UAAs have
been incorporated into proteins in Escherichia coli, yeast, or
mammalian cells, which enable new opportunities to study biological
problems with expanding nonproteinogenic chemistries. Fluor-
escent UAAs (Figure 1), including L-dansylalanine (1),5ꢀ7
L-(7-hydroxycoumrin-4-yl)ethylglycine (2),8 and L-Anap (3),9 are of
particular interest for molecular and cellular biology, which may
complement and enchance the widely used fluorescent proteins
(FPs).10 These fluorescent UAAs can be incorporated at virtually
any site of a protein, whereas the fusion of FPs is often limited to
the N- or C-terminus or certain loop regions. In addition, fluo-
rescent UAAs are much smaller than FPs, which helps to mitigate
undesired perturbations to the structure and function of the tar-
get protein. Moreover, when developing fluorescent reporters,
sensitivity to different environment cues such as pH and polarity
can be chemically designed and integrated11 into the fluorescent
UAAs, a flexibility not readily available for FPs.
Figure 1. Genetically encoded fluorescent UAAs.
When racemic UAAs are fed to cells, the D-enantiomer competes for
cellular uptake yet cannot be incorporated into proteins by the
ribosome.2 This reduces the net intracellular concentration of the
bioactive L-enantiomer, leading to lower incorporation efficiency.
For fluorescent UAAs in particular, the intracellular D-enantio-
mer further increases background fluorescence, which is a critical
issue for cell imaging with UAAs. Synthetic routes to enantio-
merically pure 1 and 2 have been developed,5,12 but only a race-
mic synthesis is available for UAA 3.9 Anap is sensitive to polarity
with changes in intensity and emission wavelength.9 UAA 3,
when selectively incorporated into proteins, has the potential to
Despite the potential advantages, effective application of
fluorescent UAAs in live cells faces multiple challenges. Efficient
synthesis of optically pure UAAs is a foremost demand.
Received: April 12, 2011
Published: July 06, 2011
r
2011 American Chemical Society
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|

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Scheme 1. Synthetic Approach for D,L-Anap (A) and Synthetic Strategy for L-Anap (B)
fluorescently report protein conformational changes, interac-
tions, modifications, and activities in live cells. Herein, we report
a concise and efficient synthetic approach of L-Anap, which offers
significant improvements over the current synthesis.
We commenced with the synthesis of 6-acyl-2-naphthylamine
(13) via two pathways (Scheme 2). In approach A, the selective
Heck reaction20ꢀ22 and Cu(I)-catalyzed CꢀN23,24 bond forma-
tion were employed to introduce the acetyl and amino groups.
6-Bromo-2-naphthol (14) was transformed to triflate 15, which was
coupled with butyl vinyl ether through Cabri’s procedure,20ꢀ22
followed by treatment with acid, to give intermediate 16. Under
Buchwald’s condition,25 compound 16 underwent the CꢀN
bond formation to give intermediate 17 in 75% yield. After de-
protection, compound 13 was obtained in 54% yield over four
steps. Although this approach provided gram-scale synthesis of
13 with satisfactory yield, approach B, which took three steps less
than A, was explored in parallel. Approach B features the direct
conversion of the hydroxyl group of compound 4 to an amino
group. Although this transformation via Burcherer reaction26 has
been reported by Cho and his co-workers,27 the procedure in-
volves stirring the reaction mixture at 140 °C for 96 h in a steel-
bomb reactor, which is not viable in many biology laboratories.
Inspired by recent advances in one-pot alkylationꢀSmiles re-
arrangementꢀhydrolysis28 sequence, we applied Mizuno and
Yamano’s procedure to the transformation of 4 to 13. Monitoring
the reaction with TLC showed the complete conversion of the
alkylation and Smiles rearrangement steps. However, the in situ
hydrolysis of 19 under basic conditions was incomplete and the
final product 13 was difficult to separate from intermediate 19.
Therefore, the crude product of 19 was separated and hydrolyzed
under acidic condition, whichprovidedcompound13 in73%yield.
With compound 13 in hand, we set out to explore the
FukuyamaꢀMitsunobu reaction (Scheme 3). Compound 13
was treated with o-nitrobenzenesulfonyl chloride and pyri-
dine to give compound 10. In the presence of diisopropyl
L-Anap has been incorporated into proteins in Saccharomyces
cerevisiae by Schultz and co-workers.9 They developed a synthetic
approach of racemic Anap, which gives 8.1% of total yield over eight
steps (Scheme 1A). The synthesis was based on the nucle-
ophilic substitution of ethyl bromopyruvate oxime (6) developed
by Gilchrist and co-workers.13,14 Presumably due to the low nucle-
ophilicity of the Burcherer reaction product of 4 and 5, com-
pound 7 was synthesized in only 26% yield over two steps from
compound 4.15 Another drawback of this synthesis resides in the
nonstereoselective reduction of 7, which leads to the final race-
mic product.
To synthesize sufficient amounts of L-Anap, we decided to
develop an efficient and enantiospecific synthetic pathway toward
L-Anap (Scheme 1B). Our synthetic strategy involves Fukuyamaꢀ
Mitsunobu reaction16ꢀ18 as the key step, which couples com-
pound 10 with N-trityl-L-serine methyl ester 1119 to give inter-
mediate 12. This approach has two features: First, both the
nucleophile and the electrophile are channeled to facilitate the
intermolecular coupling by tuning the electronic effect of the pro-
tecting groups. o-Nitrobenzenesulfonyl group makes 10 a good
nucleophile under Mitsunobu condition and can be removed
easily under mild conditions. Trityl group of compound 11 can
conduct the reactive intermediate through the intermolecular
coupling and circumvent β-elimination as the side reaction.
Second, the Mitsunobu reactions of 11 with different nucleophiles
have been proved to be free of racemization;19 therefore, inter-
mediate 12 is expected to be synthesized enantiospecifically.
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Scheme 2. Synthesis of Compound 13
’ EXPERIMENTAL SECTION
1-(6-Bromonaphthalen-2-yl)ethanone 16. To a solution of
6-bromo-2-naphthol 14 (2.231 g, 10 mmol) in CH2Cl2 (50 mL) were
added DIPEA (1.92 mL, 11 mmol) and PhNTf2 (3.930 g, 11 mmol) at
room temperature. The reaction mixture was stirred under nitrogen for
12 h and concentrated under vacuum. The residue was filtered through a
short silica gel column and eluted with EtOAc/hexanes (1/19). The
solution was concentrated under vacuum to give the crude product of
15 as a yellow oil. 15 was dissolved in DMF (20 mL). Et3N (2.79 mL,
20 mmol), butyl vinyl ether (6.47 mL, 50 mmol), dppp (113.4 mg,
0.275 mmol), and Pd(OAc)2 (56.1 mg, 0.25 mmol) were added to the
solution sequentially. The mixture was stirred under nitrogen at 80 °C
for 2 h and then cooled to 0 °C. A 2 M HCl solution (20 mL) was added
to the reaction mixture slowly, and the mixture was stirred for 0.5 h.
Water (120 mL) and EtOAc (50 mL) were added to the mixture. The
two phases were separated, and the aqueous phase was washed twice
withEtOAc(50mL). The organicphaseswerecombinedandwashedwith
brine (30 mL), dried over anhydrous Na2SO4, concentrated, and purified
with flash chromatography (EtOAc/hexanes = 1/7) to give compound 16
1
(1.798 g, 72%) as white solid. Rf = 0.23 (EtOAc/hexances = 1/6). H
NMR (500 MHz, CDCl3): δ = 8.41 (s, 1 H), 8.05 (d, J = 1.5 Hz, 1H),
8.04 (s, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.62 (dd,
J = 9.0, 2.0 Hz, 1H), 2.71 (s, 3H); 13C NMR (125 MHz, CDCl3):
δ = 197.8, 136.6, 134.9, 131.2, 131.1, 130.4, 130.1, 127.6, 125.2, 123.0, 26.8.
HRMS (EI): calcd for C12H9BrO: 247.9831, found 247.9832.
Scheme 3. Synthesis of L-Anap
tert-Butyl 6-Acetylnaphthalen-2-ylcarbamate 17. To the
mixture of compound 16 (1.993 g, 8 mmol), tert-butyl carbamate (1.125
g, 9.6 mmol), CuI (76.2 mg, 0.40 mmol), and K2CO3 (2.211 g, 16 mmol)
was added N,N0-dimethylethylenediamine (86.1 μL, 0.80 mmol) and
toluene (16 mL). The mixture was stirred under nitrogen at 110 °C for
24 h. After being cooled to room temperature, the reaction mixture was
filtered and washed with EtOAc (20 mL). The filtrate was concentrated
and purified by flash chromatography (EtOAc/hexanes = 1/3) to give
compound 17 (1.719 g, 75%) as white solid. Rf = 0.29 (EtOAc/hexanes =
3/7). 1H NMR (500 MHz, CDCl3): δ = 8.37 (s, 1H), 8.07 (s, 1H), 7.99
(dd, J = 9.0, 1.5Hz, 1H), 7.86(d, J = 8.5Hz, 1H), 7.78(d, J = 8.5 Hz, 1H),
7.42 (dd, J = 8.5, 2.0 Hz, 1H), 6.85 (s, 1H), 2.70 (s, 3H), 1.56 (s, 9H);
13C NMR (125 MHz, CDCl3): δ = 198.1, 152.7, 138.5, 136.7, 133.3,
130.7, 130.0, 128.9, 127.9, 124.8, 119.9, 114.0, 81.2, 28.5, 26.7. HRMS
(ESI-FT): [M + H]+ calcd for C17H20NO3, 286.1438; found 286.1441.
6-Acyl-2-naphthylamine 13. To a solution of compound 17
(1.427 g, 5 mmol) in CH2Cl2 (15 mL) at 0 °C was added TFA (5 mL)
dropwise. The reaction mixture was stirred at room temperature for 3 h
and concentrated under vacuum. The residue was dissolved in dichlor-
omethane (100 mL) and washed with saturated Na2CO3 aqueous solu-
tion and brine. The organic phase was dried over Na2SO4 and concen-
trated to give compound 13 (0.919 g, 99%) as a yellow solid. 1H NMR
(500 MHz, CDCl3): δ = 8.31 (d, J = 1.5 Hz, 1H), 7.92 (dd, J = 8.5,
1.5 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 6.97 (dd,
J = 8.5, 2.5 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 4.08 (br s, 2 H), 2.66 (s,
3 H); 13C NMR (125 MHz, CDCl3): δ = 198.0, 146.9, 137.8, 131.5,
131.3, 130.6, 126.6, 126.1, 124.8, 118.9, 108.0, 26.6.
6-Acyl-2-naphthylamine 13. To a solution of compound 4
(558.6 mg, 3 mmol) in DMA (6 mL) was added NaOH (360 mg,
9 mmol). The resulting mixture was stirred at room temperature for 1 h.
2-Bromo-2-methylpropanamide (1.494 g, 9 mmol) was added to the
mixture. After the mixture was stirred at room temperature overnight,
TLC showed complete conversion of compound 4. NaOH (1.080 g,
27mmol) was added, andtheresulting mixture wasstirredat50°C for5 h.
Water (60 mL) was added to the mixture and was extracted with CH2Cl2
(25 mL) for three times. The combined organic phase was concentrated
and redissolved in ethanol (15 mL) and 6 M HCl solution (15 mL). The
mixture was refluxed until TLC showed complete conversion of
azodicarboxylate and triphenylphosphine, compounds 10 and 11
were coupled to give intermediate 12 in 88% yield over two steps.
Sequential deprotection of trityl group and o-nitrobenzensulfo-
nyl group furnished L-Anap methyl ester (20). Deprotection of
20 under acidic condition29,30 furnished L-Anap as its hydro-
chloride salt. The enantiomeric purity of 3 was examined with the
Mosher method,31 and the dr value was above 40:1, which
indicated that less than 3% racemization had occurred during
deprotection (see Supporting Information).
In conclusion, we developed an efficient synthetic route to
enantiomerically pure L-Anap, which gives 51% of total yield.
Compared to the previous method, our synthetic approach not
only afforded a bioactive L-enantiomer but also enabled a 12-fold
increase in yield with fewer steps. All the reactions of our ap-
proach proceeded under mild conditions as well as in gram-scale.
The general approach demonstrated here can also be applied to
the synthesis of other 3-N-aryl-2,3-diaminopropanoic acids,
which serve as important building blocks for medicinal small
molecules.32ꢀ34 The application of L-Anap to image protein
modifications and activities in mammalian cells is in process and
will be reported in due course.
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NOTE
compound 19. After the mixture was cooled to room temperature, most
of EtOH was removed under reduced pressure. The residue was diluted
with EtOAc and water. The aqueous solution was neutralized with 1 M
NaOH solution. The mixture was extracted with EtOAc for three times.
The combined organic layers were dried over anhydrous Na2SO4,
concentrated, and purified by flash chromatography (EtOAc/hexanes =
1/1) to give 13 (406.8 mg, 73%) as a yellow solid.
’ ASSOCIATED CONTENT
S
Supporting Information. General experimental informa-
b
tion and spectra for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(S)-Methyl 3-(N-(6-Acetylnaphthalen-2-yl)-2-nitrophenyl-
sulfonamido)-2-(tritylamino)propanoate 12. To a solution of
13 (555.7mg, 3mmol) inCH2Cl2 (30mL) wereaddedpyridine(0.27mL,
3.3 mmol) and o-NsCl (698.1 mg, 3.15 mmol) sequentially at 0 °C. The
reaction was stirred at room temperature overnight. The reaction mixture
was washed with 1 M HCl solution (20 mL), water, and brine and dried
over Na2SO4. The solution was concentrated, and the resulting red solid
was dissolved in toluene (30 mL) and stirred at 0 °C. To this mixture were
added 11 (2.169 g, 6 mmol) and PPh3 (1.574 g, 6 mmol). DIAD (1.18 mL,
6 mmol) was added dropwise to the solution. The reaction mixture was
stirred overnight at room temperature. The solution was concentrated and
purified by flash chromatography (EtOAc/hexanes = 2/3) to give compound
12 (1.892 g, 88%) as a red solid. Rf = 0.28 (EtOAc/hexanes = 1/1).
1H NMR (500 MHz, CDCl3): δ = 8.43 (s, 1H), 8.06 (dd, J = 8.5, 1.5 Hz,
1H), 7.89 (d, J = 9.0 Hz, 1H), 7.88 (s, 1H), 7.60 (dd, J = 8.0, 1.5 Hz, 1H),
7.56 (dt, J = 8.0, 1.0 Hz, 1H), 7.45 (dd, J = 8.5, 2.0 Hz, 1H), 7.42 (dd, J =
8.0, 1.0 Hz, 1H), 7.33ꢀ7.29 (m, 1H), 7.28ꢀ7.26 (m, 6H), 7.11ꢀ7.05 (m,
9H), 4.36 (dd, J = 15.0, 4.5 Hz, 1H), 4.29 (dd, J = 14.5, 7.0 Hz, 1H), 3.53
(br s, 1H), 3.16 (s, 3 H), 2.72 (s, 3H), 2.66 (br s, 1H); 13C NMR (125
MHz, CDCl3): δ = 197.8, 173.0, 145.5, 138.6, 135.7, 135.4, 133.9, 132.3,
131.8, 131.7, 131.2, 131.0, 129.7, 128.8, 128.69, 128.66, 128.0, 127.9, 127.0,
126.5, 125.0, 124., 71.4, 56.7, 56.6, 52.0, 26.9. HRMS (ESI-FT):
[M + Na]+ calcd for C41H35N3O7SNa, 736.2089; found 736.2086.
[R]23D +69.6 (c 1.00, CHCl3).
(S)-Methyl 3-(6-Acetylnaphthalen-2-ylamino)-2-amino-
propanoate 20. To a solution of 12 (1.428 g, 2 mmol) in CH2Cl2
(16 mL) were added TFA (2.0 mL) and water (2.0 mL) at 0 °C. The
reaction mixture was stirred at room temperature for 3 h and concen-
trated under vacuum. The residue was dissolved in DMF (10 mL).
Thiophenol (440.4 mg, 4.0 mmol) and K2CO3 (2.76 g, 20 mmol) were
added to the solution sequentially. The reaction mixture was stirred at
room temperature for 2 h. Water (80 mL) was added to the mixture, and
the solution was extracted with EtOAc (40 mL) three times. The
combined organic phase was washed with brine (40 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated and purified by flash
chromatography (EtOAc/hexanes = 1/1, then MeOH/MeOH = 1/20)
to give compound 20 (459.6 mg, 80%) as a yellow solid. Rf = 0.15
(MeOH/CH2Cl2 = 1/20). 1H NMR (500 MHz, CDCl3): δ = 8.29 (s,
1 H), 7.92 (dd, J = 9.0, 2.0 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.61 (d, J =
8.5 Hz, 1H), 6.95 (dd, J = 8.5, 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 4.75
(br s, 1H), 3.82 (br s, 1H), 3.78 (s, 3H), 3.65ꢀ3.63 (m, 1H), 3.35 (dd,
J = 12.0, 7.5 Hz, 1H), 2.66 (s, 3H), 1.70 (br s, 2H); 13C NMR (125 MHz,
CDCl3): δ = 197.9, 147.9, 138.0, 131.2, 131.0, 130.4, 126.3, 126.2,
124.9, 118.9, 104.4, 53.6, 52.5, 46.9, 26.5. HRMS (ESI-FT): [M + H]+
calcd for C16H19N2O3, 287.1390; found 287.1395. [R]24D +74.0 (c 1.00,
CHCl3).
Corresponding Author
*E-mail: lwang@salk.edu
’ ACKNOWLEDGMENT
We thank Dr. Michael Burkart, Mr. Christopher Vickery, and
Dr. Jing Xu for help with the HRMS and optical rotation
measurements. This work was supported by the March of Dimes
Foundation (5-FY08-110), California Institute for Regenerative
Medicine (RN1-00577-1), and National Institutes of Health
(1DP2OD004744-01).
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L-Anap 3. A solution of compound 16 (286.3 mg, 1 mmol) in 2 M
HCl solution (10 mL) was stirred at 60 °C for 8 h. The reaction mixture
was lyophilized to give a yellow solid. After being washed with diethyl ether,
the solid was dried under vacuum to give L-Anap (306.8 mg, 99%) as a
yellow solid. 1H NMR (500 MHz, D2O): δ = 7.82 (s, 1H), 7.45 (dd, J =
8.5, 2.0 Hz, 1H), 7.39 (d, J= 8.5 Hz, 1H), 7.32 (d, J= 8.5 Hz, 1H), 6.87 (dd,
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4.5 Hz, 1H), 3.23 (dd, J = 13.0, 7.0 Hz, 1H); 13C NMR (125 MHz, DMSO-
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