Synthesis of (L)-4-fluorotryptophan

Article abstract of DOI:10.1080/00397911.2010.523154

A new synthesis of stereochemically pure (L)-4-fluorotryptophan [i.e., (2S)-4-fluorotryptophan] in seven steps from 4-fluoroindole is described. A key reaction in the synthetic strategy is a diastereoselective alkylation of the Schollkopf chiral auxiliary

Full text of DOI:10.1080/00397911.2010.523154

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Synthesis of (L)-4-Fluorotryptophan
David W. Konas a , Drilona Seci a & Safa Tamimi a
a Department of Chemistry and Biochemistry, Montclair State
University, Montclair, New Jersey, USA
Accepted author version posted online: 22 Jul 2011.Version of
record first published: 14 Sep 2011.
To cite this article: David W. Konas , Drilona Seci & Safa Tamimi (2012): Synthesis of (L)-4-
Fluorotryptophan, Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 42:1, 144-152
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Synthetic Communications1, 42: 144–152, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.523154
SYNTHESIS OF (L)-4-FLUOROTRYPTOPHAN
David W. Konas, Drilona Seci, and Safa Tamimi
Department of Chemistry and Biochemistry, Montclair State University,
Montclair, New Jersey, USA
GRAPHICAL ABSTRACT
Abstract A new synthesis of stereochemically pure (L)-4-fluorotryptophan [i.e., (2S)-4-
fluorotryptophan] in seven steps from 4-fluoroindole is described. A key reaction in the
synthetic strategy is a diastereoselective alkylation of the Schollkopf chiral auxiliary with
a fluorinated electrophile. All reaction steps are efficient and proceed in at least 80% yield.
Keywords Asymmetric synthesis; fluoroamino acids; 4-fluorotryptophan; organofluorine
chemistry
INTRODUCTION
Fluorinated amino acids are important tools for biological and medicinal che-
mists. Substitution of fluorine for hydrogen within the natural amino acid structure
often results in a product with significantly altered physical and chemical properties,
which is still recognized and accepted in biological systems. As a result, fluoroamino
acids and larger compounds containing them have been widely used as biochemical
probes, alternate enzyme substrates, and enzyme inhibitors.[1,2] The result of this
popularity is a significant body of literature describing the synthesis of these com-
pounds.[3–6] Despite such progress, a demand for improved synthetic methods and
strategies for preparing these compounds, particularly in stereochemically pure
forms, remains.
4-Fluorotryptophan (4-FTrp, 9) is a nonfluorescent analog of tryptophan.[7,8]
Thus, selective or complete replacement of tryptophan with 4-FTrp within a pro-
tein sequence suppresses the native tryptophan fluorescence at those positions[7]
and has enabled a variety of experiments aimed at gaining information on protein
structure and dynamics in the years since it was first characterized (see Hogue
Received June 14, 2010.
Address correspondence to David W. Konas, Department of Chemistry and Biochemistry,
Montclair State University, 1 Normal Ave., Montclair, NJ 07043, USA. E-mail: konasd@mail.montclair.edu
144

SYNTHESIS OF (L)-4-FLUOROTRYPTOPHAN
145
et al.[9] and Acchione et al.[10] for examples). Some 19F NMR structural studies on
4-FTrp labeled proteins have also been reported (see Lian et al.,[11] Pearson
et al.,[12] and Seifert et al.[13] for examples), along with others investigating the
biological activity of the free amino acid, such as a recent study revealing its
cytotoxic effects against a human cancer cell line.[14]
The most readily available and commonly used commercial source of 4-FTrp is
a mixture of both (D) and (L) stereoisomers. Such material can also be prepared in
the laboratory through condensation of 4-fluoroindole with a formamidomalo-
nate.[15] Biosynthetic incorporation of such samples into expressed proteins using
tryptophan auxotropic bacteria or other techniques provides a natural means of
resolving the enantiomers because these organisms will utilize only the natural (L)
isomer (S configuration at the asymmetric carbon) required for a bioactive structure.
Unfortunately, in such cases half of the material is wasted. Incorporation of only
(L)-4-FTrp into a smaller synthetic peptide starting with the racemic material
requires a resolution prior to use or separation of diastereomers after peptide coup-
ling, and still has the drawback of wasted material. The selective biosynthetic prep-
aration of (L)-4-FTrp using tryptophan synthase and (L)-serine was recently
described,[14] but we are unaware of any other reported preparations that selectively
yield single enantiomers of this compound. Based on our ongoing interest in the syn-
thesis of optically active fluoramino acids and their application in biological systems,
we now report a new efficient and enantioselective synthesis of (L)-4-FTrp.
RESULTS AND DISCUSSION
Our synthesis of (L)-4-FTrp, or (S)-4-fluorotryptophan (9), is shown
in Scheme 1. The overall strategy relies on regioselective functionalization of
Scheme 1. Synthesis of (L)-4-fluorotryptophan.

146
D. W. KONAS, D. SECI, AND S. TAMIMI
4-fluoroindole (1) followed by diastereoselective coupling to the enantiopure
Schollkopf chiral auxiliary (6) derived from glycine and (D)-valine[16] to establish
the desired product stereochemistry. Deprotection of the resulting intermediate 7
yields the final fluorinated amino acid 9.
4-Fluoroindole (1) was regioselectively formylated at the 3-position under
Vilsmeier–Haack conditions in dimethylformamide (DMF). The resulting aldehyde
2 was recovered by extraction of the final aqueous hydrolysis mixture but can also
be recovered by filtration from this mixture, particularly on larger scales. The
compound can be recrystallized in petroleum ether, but the reaction was quite
clean and the product was typically used as obtained without any further purifi-
cation being necessary. The indole nitrogen of 2 was deprotonated with NaH
and protected with a tosyl group by reaction with TsCl to yield the protected
indole 3. The aldehyde proton peaks in the 1H NMR spectra of compounds 2
and 3 appear as a closely spaced doublets. This indicates the possibility that
two distinct conformations of their formyl groups relative to the indole rings exist
in solution. After the nitrogen protection step, the aldehyde carbonyl of 3 was
cleanly reduced to the corresponding alcohol (4) with NaBH4 in tetrahydrofuran
(THF)=EtOH.
Protected 3-bromomethyl indoles are valuable compounds for use in conjunc-
tion with chiral glycine anion equivalents such as the Schollkopf chiral bis-lactim
ether 6 in the synthesis of unnatural tryptophans and their derivatives.[17–26] These
compounds can be prepared through radical bromination of 3-methyl indoles or
via an alcohol such as 4. One drawback of the brominated products is their reactivity
and instability, which affects their handling and usually precludes purification by
silica-gel flash chromatography. Thus, they must be obtained through an efficient
bromination reaction followed by minimal workup, and they are often used quickly
in the alkylation reaction. Our initial work involved preparation of the alcohol cor-
responding to 4 with an N-Boc protecting group. While a detailed bromination of
such an unfluorinated alcohol has been reported,[27] we were unsuccessful in bromi-
nating ours cleanly using several methods (PBr3; P(Ph)3=Br2; P(Ph)3=NBS). These
problems were caused by the loss of the Boc protecting group due to in situ gener-
ated HBr along with numerous other side reactions. Instead, the Boc group
was replaced with N-tosyl, and we observed a much cleaner and more reliable
bromination reaction using P(Ph)3=NBS. Furthermore, we were pleased to find that
this reaction could be set up and left at room temperature for 12–24 h with no
problems.
The bromide 5 was dried and immediately added in solution to the lithiated
Schollkopf compound 6 at ꢀ78 ꢁC. The 19F NMR spectrum of the crude reaction
mixture obtained after quenching and workup typically indicated one major peak
corresponding to the desired product 7 plus a small number of minor peaks (includ-
ing unreacted 5), which combined were less than 10% of the total spectrum inte-
gration. Compound 7 was easily purified to a single diastereomer by flash
chromatography, and the pyrazine ring was hydrolyzed under acidic conditions to
give the amino ester 8. A second hydrolysis under basic conditions removed the tosyl
group along with the ester and yielded the final amino acid 9. Characterization of the
product as described yielded data consistent with expected and previously published
results for the desired product (L)-4-FTrp.[13–15,28]

SYNTHESIS OF (L)-4-FLUOROTRYPTOPHAN
147
EXPERIMENTAL
4-Fluoroindole (1) was purchased from Aldrich ($184 USD=g). The Schollk-
opf chiral bis-lactim ether (6) was either purchased from Aldrich ($123 USD=g)
or prepared from glycine and (D)-valine.[29,30] All other reagents and solvents were
obtained from Sigma-Aldrich or Fisher-Acros and used without further purifi-
cation. Moisture-sensitive reactions were run in oven-dried (T > 100 ꢁC overnight)
glassware using anhydrous solvents in an atmosphere of dry argon. Column chro-
matography was performed with 100 to 200 m particle size silica gel obtained from
Selecto Scientific (Georgia, USA), and thin-layer chromatography (TLC) was per-
1
formed using Merck silica-gel 60 F-254 plates. H, 13C, and 19F NMR spectra were
obtained with a Bruker Avance DRX300 spectrometer using the X-WinNMR soft-
ware. Chemical shifts are reported in parts per million (ppm) upfield or downfield
from either tetramethylsilane (1H, 13C) or trifluoroacetic acid (19F). Specific rota-
tions were obtained using a Polyscience SR-6 polarimeter. Melting points were
determined using an SRS DigiMelt MPA160 device. Liquid chromatographic–mass
spectrometric (LC-MS) analysis was carried out with an Agilent 1100 Series instru-
ment using atmospheric pressure ionization–electrospray (API-ES) and positive ion
mode. Liquid chromatography prior to mass analysis was carried out using a C18
column and an isocratic mobile phase consisting of 35% MeOH and 64% water
plus 0.1% ammonium acetate in both solvents.
4-Fluoro-3-formylindole (2)
DMF (2.0 mL) was stirred and cooled in an ice-water bath, and POCl3 (0.9 mL,
9.6 mmol) was added. After 15 min, a solution of 4-fluoroindole (650 mg, 4.8 mmol)
in DMF was slowly added. After 15 min, the ice bath was removed, and the reaction
flask was warmed to 40 ꢁC. The bright yellow reaction mixture remained stirring at
this temperature until it became an opaque paste (approximately 1 h). The heat
source was removed, and ice chips were added to the mixture until it was once again
homogeneous. The solution was gradually made basic with the slow addition of 5%
aqueous NaOH until a white precipitate was present and the pH was greater than 10,
at which point the suspension was rapidly heated to reflux for 5 min. The heat was
removed, and the mixture was cooled to room temperature and extracted with
EtOAc. The combined organic extracts were dried (Na2SO4) and concentrated to
give the desired product as an off-white solid, which was used without further puri-
fication (640 mg, 3.9 mmol, 82% yield). Mp 182–187 ꢁC; 1H NMR (300 MHz,
DMSO) d 7.0 (ddd, 1H, J ¼ 10.9, 7.7, 0.5 Hz), 7.20–7.27 (m, 1H), 7.34–7.37 (m,
1H), 8.30 (s, 1H), 10.0 (d, 1H, CHO), 12.5 (br, 1H, NH); 19F NMR (282 MHz,
DMSO) d ꢀ34.5 (br).
4-Fluoro-3-formyl-1-tosylindole (3)
Compound 2 (640 mg, 3.9 mmol) was dissolved in THF and cooled in an
ice-water bath. NaH (140 mg, 5.8 mmol) was added, followed by tosyl chloride
(818 mg, 4.3 mmol). The ice-water bath was allowed to warm to rt, and the cloudy
mixture stirred overnight. The reaction was recooled in an ice-water bath and

148
D. W. KONAS, D. SECI, AND S. TAMIMI
quenched with saturated aqueous NH4Cl. The solvents were removed in vacuo, and
the resulting residue was partitioned between EtOAc and water. After separating
layers, the aqueous layer was extracted further with EtOAc, and the combined
organic layers were dried (Na2SO4) and concentrated to give a light yellow solid.
This crude material was purified by silica-gel column chromatography (hexanes=
EtOAc, 2:1) to give the desired product as a white solid (1.0 g, 3.3 mmol, 85% yield).
Rf ¼ 0.55 (hexanes=EtOAc, 2:1); mp 131–133 ꢁC; 1H NMR (300 MHz, CDCl3) d 2.40
(s, 1H), 7.08 (dd, 1H, J ¼ 10.0, 8.0 Hz), 7.31–7.40 (m, 3H), 7.80–7.88 (m, 3H),
8.31 (s, 1H), 10.28 (d, 1H, CHO); 13C NMR (75.5 MHz, CDCl3) d 21.9, 110.0
(d, JC-F ¼ 4.0 Hz), 110.7 (d, JC-F ¼ 19.3 Hz), 116.1 (d, JC-F ¼ 22.3 Hz), 120.8 (d, JC-F
¼
5.0 Hz), 126.2 (d, JC-F ¼ 7.6 Hz), 127.5, 130.6, 132.0, 134.2, 137.1 (d, JC-F ¼ 10.5 Hz),
146.7, 156.7 (d, JC-F ¼ 251 Hz), 185.5 (d, JC-F ¼ 3.5 Hz); 19F NMR (282 MHz,
CDCl3) d ꢀ37.8 (ddd, J ¼ 10.0, 5.1, 2.1 Hz).
4-Fluoro-3-hydroxymethyl-1-tosylindole (4)
Compound 3 (1.0 g, 3.3 mmol) was dissolved in 2:1 THF=EtOH and cooled in
an ice-water bath. NaBH4 (185 mg, 4.9 mmol) was added, and then the mixture was
slowly warmed to rt and allowed to stir overnight. The reaction was recooled in an
ice-water bath and slowly quenched with saturated aqueous NH4Cl. The solvents
were removed in vacuo, and the resulting residue was partitioned between CH2Cl2
and brine. After separating layers, the aqueous layer was extracted further with
CH2Cl2, and the combined organic layers were dried (Na2SO4) and concentrated
to give a white solid. This crude material was purified by silica-gel column chroma-
tography (hexanes=EtOAc, 2:1) to give the desired product as a white solid (954 mg,
1
3.0 mmol, 91% yield). Rf ¼ 0.35 (hexanes=EtOAc, 2:1); mp 98–100 ꢁC; H NMR
(300 MHz, CDCl3) d 2.12 (br, 1H), 2.36 (s, 3H), 4.85 (br, 2H), 6.92 (dd, 1H,
J ¼ 10.0, 8.0 Hz), 7.23–7.30 (m, 3H), 7.53 (s, 1H), 7.76–7.80 (m, 3H); 13C NMR
(75.5 MHz, CDCl3) d 21.8, 57.8 (d, JC-F ¼ 2.1 Hz), 109.1 (d, JC-F ¼ 19.2 Hz), 110.1
(d, JC-F ¼ 3.9 Hz), 118.1 (d, JC-F ¼ 21.1 Hz), 120.9 (d, JC-F ¼ 3.9 Hz), 124.0, 126.0
(d, JC-F ¼ 7.8 Hz), 127.1, 130.2, 135.1, 137.7 (d, JC-F ¼ 10.1 Hz), 145.5, 156.5 (d,
JC-F ¼ 247 Hz); 19F NMR (282 MHz, CDCl3) d ꢀ46.1 (dd, J ¼ 10.0, 5.3 Hz).
3-Bromomethyl-4-fluoro-1-tosylindole (5)
Compound 4 (500 mg, 1.6 mmol) was dissolved in CH2Cl2 and cooled in an
ice-water bath. Triphenylphosphine (446 mg, 1.7 mmol) was added, and then after
10 min NBS (303 mg, 1.7 mmol) was added. The reaction was maintained near
0 ꢁC for 1 h before the ice-water bath was allowed to warm to rt. The reaction was
then stirred overnight. The solvents were removed in vacuo with minimal heating,
and the resulting residue was dissolved in EtOAc and applied to a short plug
silica-gel column. The column was rapidly eluted with 4:1 hexanes=EtOAc, and
the resulting collected solution was concentrated with minimal heating to give the
crude desired product as an off-white solid, which was kept under high vacuum
and used immediately for the next reaction without further purification (585 mg).
1H NMR (300 MHz, CDCl3) d 2.38 (s, 3H), 4.70 (s, 2H), 6.95 (dd, 1H, J ¼ 10.0,

SYNTHESIS OF (L)-4-FLUOROTRYPTOPHAN
149
8.0 Hz), 7.24–7.32 (m, 3H), 7.62 (s, 1H), 7.74–7.80 (m, 3H); 19F NMR (282 MHz,
CDCl3) d ꢀ46.7 (dd, J ¼ 10.0, 5.3 Hz).
(3S, 6R)-3-((4-Fluoro-1-tosyl-3-indoyl)methyl)-3,6-dihydro-6-
isopropyl-2,5-dimethoxypyrazine (7)
A solution of compound 6 (310 mg, 1.7 mmol) in THF was cooled to ꢀ78 ꢁC in
a dry ice–iPrOH bath, and a solution of nBu-Li (2.0 mmol, 1.26 mL of 1.6 M reagent
in hexanes) was added dropwise. The bright yellow solution was stirred for 40 min,
and then a solution of the crude bromide 5 (585 mg, 1.5 mmol) in THF was added
dropwise. The reaction was maintained at ꢀ78 ꢁC for 1.5 h before it was quenched
with saturated aqueous NH4Cl and allowed to warm to rt. The solvents were
removed in vacuo, and the resulting residue was partitioned between CH2Cl2 and
brine. After separating layers, the aqueous layer was extracted further with CH2Cl2,
and the combined organic layers were dried (Na2SO4) and concentrated to give a col-
orless residue. This crude material was purified by silica-gel column chromatography
(CHCl3=EtOAc, 18:1) to give the desired product as a thick colorless oil (631 mg,
1.3 mmol, 80% yield in two steps from 4). Rf ¼ 0.64 (CHCl3=EtOAc, 18:1); 1H
NMR (300 MHz, CDCl3) d 0.64 (d, 3H, J ¼ 6.8 Hz), 0.94 (d, 3H, J ¼ 6.8 Hz),
2.09–2.20 (m, 1H), 2.36 (s, 3H), 3.17 (dd, 1H, J ¼ 14.4, 6.6 Hz), 3.36–3.45 (m, 2H),
3.61 (s, 3H), 3.68 (s, 3H), 4.29–4.35 (m, 1H), 6.85 (dd, 1H, J ¼ 10.7, 8.0 Hz),
7.14–7.28 (m, 4H), 7.70–7.75 (m, 3H); 13C NMR (75.5 MHz, CDCl3) d 16.7, 19.2,
21.8, 30.7 (d, JC-F ¼ 2.5 Hz), 31.7, 52.4, 52.5, 56.0, 60.7, 109.0 (d, JC-F ¼ 19.6 Hz),
109.7 (d, JC-F ¼ 3.9 Hz), 117.0 (d, JC-F ¼ 3.4 Hz), 120.2 (d, JC-F ¼ 18.6 Hz), 124.8,
125.3 (d, JC-F ¼ 7.8 Hz), 127.0, 130.1, 135.3, 137.1 (d, JC-F ¼ 9.8 Hz), 145.2, 156.9
(d, JC-F ¼ 249 Hz), 162.9, 164.1; 19F NMR (282 MHz, CDCl3) d ꢀ46.0 (dd,
J ¼ 10.8, 5.1 Hz).
(S)-1-Tosyl-4-fluorotryptophan Methyl Ester (8)
Compound 7 (500 mg, 1.0 mmol) was dissolved in THF (16 mL) and cooled
in an ice-water bath. Next, 2 N HCl (8 mL) was added, and after 10 min the sol-
ution was allowed to warm to rt. The reaction continued stirring for a total of 1 h.
The THF was removed in vacuo, and the remaining aqueous solution was diluted
with water (8 mL). The product hydrochloride was neutralized by slowly adding
aqueous NH4OH until the pH of the solution reached 9–10. The resulting mixture
was extracted with EtOAc, and the combined organic layers were dried (Na2SO4)
and concentrated to give a cloudy oil. This crude product was purified by
silica-gel column chromatography (EtOAc with 5% MeOH and 0.5% NH4OH(aq)
)
to give the desired product as a thick colorless oil (338 mg, 0.87 mmol, 87% yield).
Rf ¼ 0.70 (EtOAc with 5% MeOH and 0.5% NH4OH(aq)); 1H NMR (300 MHz,
CDCl3) d 1.61 (br, 2H, NH2), 2.36 (s, 3H), 2.97 (dd, 1H, J ¼ 14.4, 8.0 Hz), 3.27
(dd, 1H, J ¼ 14.4, 5.4 Hz), 3.69 (s, 3H), 3.79–3.85 (m, 1H), 6.90 (dd, 1H,
J ¼ 10.7, 8.1 Hz), 7.20–7.28 (m, 3H), 7.40 (s, 1H), 7.74–7.79 (m, 3H); 13C NMR
(75.5 MHz, CDCl3) d 21.8, 32.1, 52.2, 54.8, 109.2 (d, JC-F ¼ 19.4 Hz), 110.1
(d, JC-F ¼ 3.9 Hz), 116.7 (d, JC-F ¼ 3.2 Hz), 119.3 (d, JC-F ¼ 19.4 Hz), 124.8 (d, JC-F
¼
1.0 Hz), 125.8 (d, JC-F ¼ 7.8 Hz), 127.0, 130.1, 135.1, 137.6 (d, JC-F ¼ 9.7 Hz),

150
D. W. KONAS, D. SECI, AND S. TAMIMI
145.43, 156.7 (d, JC-F ¼ 248 Hz), 175.4; 19F NMR (282 MHz, CDCl3) d ꢀ46.7 (dd,
J ¼ 11.0, 5.0 Hz).
(S)-4-Fluorotryptophan (9)
Compound 8 (300 mg, 0.77 mmol) was dissolved in EtOH (9.0 mL), and 2 N
NaOH (5.0 mL) was added. The reaction mixture was heated at reflux for 0.5 h.
The solution was cooled to rt, and the pH was adjusted to 2.0 with 6 N HCl. The
resulting aqueous solution was extracted with CH2Cl2. The pH of the aqueous phase
was raised to 6.0, and the water was removed in vacuo to leave a residue containing
the crude product, which was purified by crystallization in EtOH=H2O to obtain the
final product as a white solid (140 mg, 0.63 mmol, 82% yield). Mp 230–233 ꢁC (lit. mp
25
25
for racemic 4-FTrp ¼ 241 ꢁC)[15]; ½aꢂD ¼ ꢀ28.5 (c ¼ 0.9, H2O) (lit. ½aꢂD for
(L)-Trp ¼ ꢀ31.5 (c ¼ 1, H2O)[28]
;
1H NMR (300 MHz, D2O) d 3.25 (dd, 1H,
J ¼ 15.1, 9.2 Hz), 3.61 (dd, 1H, J ¼ 15.0, 4.4 Hz), 4.09 (dd, 1H, J ¼ 9.2, 4.7 Hz),
6.88 (dd, 1H, J ¼ 11.6, 7.8 Hz), 7.17–7.25 (m, 2H), 7.29 (s, 1H); 13C NMR
(75.5 MHz, D2O) d 25.5, 56.5, 109.2 (d, JC-F ¼ 19.8 Hz), 110.1 (d, JC-F ¼ 4.1 Hz),
116.7 (d, JC-F ¼ 2.9 Hz), 119.3 (d, JC-F ¼ 19.0 Hz), 124.8 (d, JC-F ¼ 1.0 Hz), 125.8
(d, JC-F ¼ 7.1 Hz), 137.6 (d, JC-F ¼ 9.5 Hz), 156.7 (d, JC-F ¼ 249 Hz), 173.5; 19F
NMR (282 MHz, D2O) d ꢀ48.8 (dd, J ¼ 11.7, 5.2 Hz). LC-MS analysis of the sample
yielded a single peak with kmax ¼ 265 nm and [M þ 1] molecular ion mass corre-
sponding to 4-FTrp.
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