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1
Supplementary Information
Facile one-pot synthesis of tetrahydroisoquinolines from amino acids via hypochlorite-mediated
decarboxylation and Pictet-Spengler condensation
Justin J. Maresh, Sean O. Crowe, Arthur A. Ralko, Mark D. Aparece, Casey M. Murphy, Mark Krzeszowiec,
Michael W. Mullowney
Contents
Supporting Figures and Tables ........................................................................................................ 5
Table S1. ...................................................................................................................................... 5
Figure S1...................................................................................................................................... 5
Figure S2...................................................................................................................................... 6
Figure S3...................................................................................................................................... 6
Figure S4...................................................................................................................................... 7
Figure S5...................................................................................................................................... 7
Scheme S1. .................................................................................................................................. 8
Table S2 ....................................................................................................................................... 9
Supporting Methods ..................................................................................................................... 10
General information. ................................................................................................................ 10
Determination of sodium hypochlorite concentration ............................................................ 11
Standardization of sodium thiosulfate ................................................................................. 11
Titration of 10-15% NaOCl .................................................................................................... 11
Sodium hypochlorite oxidations ............................................................................................... 12
General Procedure for sodium hypochlorite oxidation ........................................................ 12
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4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 12
3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 12
3-bromo-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 13
3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 13
2-phenylacetaldehyde (2e) ................................................................................................... 13
2-(1H-indol-3-yl)acetaldehyde (2f) ....................................................................................... 14
Measurement and analysis of kinetics for oxidative decarboxylation of tyrosine. .............. 14
Pictet-Spengler reactions .......................................................................................................... 15
General procedure for synthesis of racemic tetrahydroisoquinolines from α-amino acids 15
1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e) ......................................................... 17
1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f). .................................. 18
Expression of recombinant norcoclaurine synthase enzyme. .................................................. 18
General procedure for synthesis of (S)-enantiomer tetrahydroisoquinolines from α-amino
acids with norcoclaurine synthase............................................................................................ 19
Norcoclaurine synthase kinetic assays for aldehyde substrates 4a – 4e. ................................ 20
Halogenation of tyrosol ............................................................................................................ 20
2-chloro-4-(2-hydroxyethyl)phenol (6b) ............................................................................... 20
2-bromo-4-(2-hydroxyethyl)phenol (6c)............................................................................... 21
2-iodo-4-(2-hydroxyethyl)phenol (6d) .................................................................................. 21
Parikh-Doering oxidation reactions .......................................................................................... 22
4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 22
3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 22
3-chloro-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 23
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3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 23
3-Halogenation of tyrosine ....................................................................................................... 24
3-chloro-tyrosine hydrochloride (1b) ................................................................................... 24
3-bromo-tyrosine hydrochloride (1c) ................................................................................... 24
3-iodo-tyrosine (1d) .............................................................................................................. 25
Characterization Data ................................................................................................................... 26
1H
NMR spectra and GC-MS data for 3-halogenated tyrosine compounds 5b-d ..................... 26
2-chloro-4-(2-hydroxyethyl)phenol (5b) ............................................................................... 26
2-bromo-4-(2-hydroxyethyl)phenol (5c)............................................................................... 27
2-iodo-4-(2-hydroxyethyl)phenol (5d) .................................................................................. 28
1H
NMR spectra for 3-halogenated tyrosine analogs in D2O. ................................................... 30
3-chloro-tyrosine·HCl ............................................................................................................ 30
3-bromo-tyrosine·HCl ........................................................................................................... 30
3-iodo-tyrosine...................................................................................................................... 31
1H NMR spectra for extracted 4-hydroxyphenylacetaldehyde products (2a-2d) generated via
oxidative decarboxylation of halogenated tyrosine. ................................................................ 32
4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 32
3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 32
3-bromo-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 33
3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 33
GC-MS data for 3-halogenated-4-hydroxyphenylacetaldehyde products (2a-f) generated via
oxidative decarboxylation of halogenated tyrosine. ................................................................ 34
4-hydroxyphenylacetaldehyde (2a) ...................................................................................... 34
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3-chloro-4-hydroxyphenylacetaldehyde (2b) ....................................................................... 35
3-bromo-4-hydroxyphenylacetaldehyde (2c) ....................................................................... 35
3-iodo-4-hydroxyphenylacetaldehyde (2d) .......................................................................... 36
1H
NMR spectra for racemic tetrahydroisoquinoline products 4a-f after extraction. ............. 37
Norcoclaurine (4a) ................................................................................................................ 37
3’-chloro-norcoclaurine (4b) ................................................................................................. 38
3’-bromo-norcoclaurine (4c) ................................................................................................. 38
3’-iodo-norcoclaurine (4d) .................................................................................................... 39
1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e) ......................................................... 39
1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f) ............................... 40
References .................................................................................................................................... 40
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Supporting Figures and Tables
X
H
Cl
Br
I
6a
6b
6c
6d
Yield
-72 %
79 %
72 %
2a
2b
2c
2d
Yield
67 %
35 %
6%
26 %
Table S1. The yields of 3-halogenated-4-HPAA analogs via Parikh-Doering oxidation of tyrosol.
Figure S1. Reverse phase HPLC chromatogram of a typical Parikh–Doering oxidation of tyrosol.
As expected, pyridine, DMSO, and 4-hydroxyphenyl-acetaldehyde (HPAA) are present along
with several other unknown side products. The plot intensity represents the maximum
absorbance at each time point. Addition of pyridine has been suggested to eliminate side
products that result from presence of sulfuric acid in the sulfur trioxide solution.1 We observed
no significant improvement from this modification. Data obtained using Condition A (see
General Information). The maximum intensity wavelength for each time is plotted.
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Figure S2. The effect of tyrosine concentration on the efficiency of oxidative decarboxylation by
NaOCl. For each reaction, a 0.1 mM solution of NaOCl was added dropwise to the tyrosine
solution in 75 mM pH 7.0 phosphate buffer over 10 minutes via syringe pump at 37 °C. The
undiluted reaction mixture was analyzed by reverse phase HPLC after 1.5 to 2 hours. Data
obtained using Condition B (see General Information). The maximum intensity wavelength for
each time is plotted.
Figure S3. Time dependent behavior of oxidative decarboxylation by NaOCl. Solid lines
represent a two-step irreversible kinetic model (k1 = 16 M-1s-1 and k2 = 2.6×10-3 s-1).
Concentrations for intermediates are approximated by arbitrarily assigning their extinction
coefficients to that of 4-HPAA. These data correspond to the experiment in Figure 1.
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Figure S4. Regioselectivity of the reaction of 0.500 mM dopamine and 100 mM propanal with
varied concentrations of phosphate buffer at pH 6.90 and 23.0 °C. The equation of the trend
line is Ratio = 277×[Phosphate] + 6.05 (R² = 0.9994).
Figure S5. In the presence of Tris buffer, a significant oxazolidine side product accumulates due
to reaction of Tris with aldehydes. HPLC chromatogram at 280 nm of gradient 0-70%
acetonitrile in 0.1 % trifluoroacetic acid (TFA) over 10 minutes monitoring a norcoclaurine
synthase enzyme reaction in 100 mM TRIS at pH 7.5. Dopamine, HPAA, norcoclaurine, and 1naphthalene-acetic acid (NAA, an internal reference compound) are present along with a
significant undesired oxazolidine side product (see Scheme S1). Additionally, it is apparent that
Tris buffer generated numerous minor side products that are not observed in phosphate,
HEPES, BES, ADA, or maleic acid buffers.
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Scheme S1. Tris is not recommended for enzyme catalyzed tetrahydroisolquinoline synthesis as
it generates oxazolidine side products that may consume as much as 50% of the aldehyde
reactant, thus reducing yield and complicating isolation of tetrahydroisoquinoline products.
Figure S6. Chiral HPLC chromatograms at 230 nm for norcoclaurine synthase enzyme reactions
compared to reactions of the same substrates by phosphate catalyzed synthesis. Enzyme
reactions A – C were carried-out in 100 mM BES buffer at pH 7.0. Enzyme reaction D was
carried out in 50 mM maleic acid buffer. All data was acquired using Condition C.
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Buffer
pH of reaction
Phosphate
ACES
ADA
BES
Bicine
Bis-tris
HEPES
Imidazole
Maleic acid
MOPSO
Tris
7.0
7.0
7.0
7.0
7.5
7.0
7.0
7.0
6.8
7.0
7.5
pKa at 25 mM
and 23 °Ca
7.20
6.83
6.61
7.17
8.32
6.57
7.54
7.12
6.03
6.96
8.30
Rate of Pictet-Spengler reaction
(relative to phosphate)b
100%
1.2%
4.3%
0.8%
15.4%
3.6%
0.9%
0.7%
13.2%
1.0%
7.1%c
Inhibition
of NCSd
None
0.32 ± 0.05
None
None
None
None
0.27 ± 0.03
0.58 ± 0.08
None
0.28 ± 0.08
None
Table S2. The effects of 25 mM buffer on the relative rates of catalysis for the Pictet-Spengler
reaction 2.0 mM dopamine and 2.0 mM 4-HPAA reacted in 25 mM buffer at 23 °C and inhibition
of norcoclaurine synthase.
a. The listed pKa values are calculated using the simplified Debye-Hückel equation described by
Ellis2 for I = 0.025 M and 23 °C using the pKa values from that reference. The thermodynamic
pKa2 for maleic acid was taken as 6.233. These values are given as reasonable estimates of
the effective pKa. No effort was made to quantitatively control ionic strength.
b. The rate of chemically-catalyzed reaction was estimated as initial rates from the linear slope
the time dependent change in dopamine concentration as monitored by HPLC using
Condition B (see General Information, page 10) at 225 nm. For phosphate-catalyzed data,
samples were injected every 7 minutes. For other buffers, samples were injected every 14 or
21 minutes.
c. Tris reacts with 4-HPAA (Scheme S1), thus the 4-HPAA concentration was not controlled.
d. Inhibition of norcoclaurine synthase (NCS) in units of “percent inhibition per mM of buffer.”
The initial rate was observed following the procedure for Norcoclaurine Synthase Kinetic
Assays for Aldehyde Substrates (page 20). In each experiment, 20.0, 40.0, 60.0, and 80.0 mM
buffer concentrations evaluated. When present, inhibition followed a linear trend. No
sigmoidal trend was apparent. Uncertainty is given as the standard deviation from two
replicate measurements. When “None” is stated, any change in activity from 20 to 80 mM
was no greater than the detection limit (the standard deviation of the four samples).
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Supporting Methods
General information. All reagents were purchased from commercial suppliers at the highest
available purity and used without further purification. Milli-Q water refers to water purified to
resistivity of 18.2 MΩcm (25 °C) using an EMD Millipore Ultrapure Milli-Q reverse osmosis
water purification system outfitted with ion exchange and organic removal cartridge filters.
All reactions involving air or water sensitive reagents were performed using flame dried
glassware under an inert atmosphere of nitrogen.
Reactions were monitored using high pressure liquid chromatography on a Waters Acquity
Ultra Performance Liquid Chromatography instrument with a photodiode array detector. The
solvent and gradient conditions used for HPLC analysis were as follows. Condition A was used
for routine analysis. Condition B provides better resolution of dopamine, aldehyde, and product
peaks for kinetics and for Figure 2. Condition C was used for resolution of chiral products.



Condition A: Acquity UPLC BEH C18 column (1.7 µm, 2.1x50 mm); 0.4 mL/min; 0-70%
acetonitrile in 0.1 % trifluoroacetic acid (TFA) over 4.75 minutes, holding at 70% acetonitrile
for 0.25 minutes.
Condition B: Acquity UPLC BEH C18 column (1.7 µm, 2.1x50 mm); 0.4 mL/min, 0-17.5%
acetonitrile in 0.1% TFA over 2.5 minutes followed by 17.5% to 70% acetonitrile in 0.1 % TFA
over 1.5 minutes, holding at 70% for 1.0 minute.
Condition C: Astec Chirobiotic™ T2 column (5 µm, 2.1x150 mm); 0.4 mL/min; isocratic
mobile phase composition of 30% acetonitrile, 0.25% triethylamine, and 0.50% acetic acid in
methanol.
Percent conversion of reactions by HPLC was estimated from the HPLC peak areas measured at
225 nm. Area extinction coefficients for starting and product compounds were measured based
on a calibration curve prepared from pure sample in water covering a concentration range from
roughly 0.1 to 3000 μM. For reactions with multiple side products, the extinction coefficient
was estimated by assuming that it was the same as the product.
All details of UV spectra are listed from most to least intense wavelength maximum.
Gas Chromatography Mass Spectroscopy analysis of products was performed using a Hewlett
Packard HP6890 Gas Chromatography System with an Agilent Technologies 5975 inert mass
selective detector. High resolution mass data (HRMS) were obtained on a Waters SYNAPT G1
High Definition Mass Spectrometer using an ESI ionization source.
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1H-
and 13C[1H]- NMR spectroscopy were performed using a Bruker Avance 300 MHz NMR
spectrometer at 300 MHz and 75.4 MHz respectively. All 1H-NMR chemical shifts were
referenced to a tetramethylsilane (TMS) internal standard. For 13C-NMR, the residual solvent
peaks were used to reference the spectra as follows: in methanol-d4 was referenced as 49.00
ppm and D2O with formic acid was as 166.22 ppm. Addition of acid was required to solubilize
tetrahydroisoquinoline samples. The NMR data for tetrahydroisoquinoline samples were
acquired as saturated solutions.
Infrared spectroscopy analysis of reaction products were performed using an ABB FT-IR FTLA
2000 spectrometer.
Prepared compounds 1b-d, 2a-f, 4a, 4e, and 5b-d are known compounds, whereas 4b-d and 4f
are new compounds. For new compounds, 1H-NMR, 13C-NMR, UV-Vis, and high resolution mass
spectral data are provided.
Determination of sodium hypochlorite concentration
We found that fresh commercial solutions of NaOCl may be used by assuming the lower end of
the manufacturer’s stated concentration. However, measurement of concentration appears to
be essential for solutions more than six months after date of manufacture.
Standardization of sodium thiosulfate.4 A solution of was prepared from 3.2 g of sodium
thiosulfate pentahydrate with 0.025 g sodium carbonate (as a preservative) in 250.0 mL of MilliQ deionized water. This was standardized against a potassium iodate (KIO3) solution by
dissolving approximately 2 g of potassium iodide in 5.0 mL of 6 M H2SO4 and 10.0 mL of 0.03740
M KIO3 solution. The resulting solution was titrated with the sodium thiosulfate solution until it
was a pale yellow solution was observed. At this point, 1 mL of a starch indicator solution was
added. The resulting blue solution was then titrated until a colorless endpoint was observed.
This procedure was repeated four times yielding a concentration of (5.14 ±0.05) x 10 -2 M for the
sodium thiosulfate solution. Standardized sodium thiosulfate solutions are also commercially
available.
Titration of 10-15% NaOCl.4 2.4476 g of a 10-15% solution of sodium hypochlorite was diluted
into a 250.0 mL volumetric flask with Milli-Q water. For titration, 50.0 mL of the resulting
solution was then added to a flask, with 50.0 mL of Milli-Q water, 10 mL of glacial acetic acid,
and approximately 2 g of KI to yield a brown solution. This solution was titrated with
standardized sodium thiosulfate until pale yellow. At this point, 1 mL of a starch indicator was
added. The resulting blue solution was then titrated until the endpoint was reached, as
indicated by a colorless solution. This was measurement was repeated in triplicate to determine
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the percent of reactive NaOCl by mass.
Sodium hypochlorite oxidations
General Procedure for sodium hypochlorite oxidation.5, 6 To a vigorously stirring solution of
amino acid (1 mmol) in 250 mL of sodium phosphate buffer (10 mM, pH 7.0) was added a
solution of NaOCl (1 equivalent as a 0.1 M aqueous solution) slowly over 10 minutes via syringe
pump in a 37 °C warm room or water bath. The resulting solution was stirred vigorously at this
temperature until the reaction was judged to be complete by HPLC (1-2 hours). In general, the
reaction may be stopped after 2 hours if monitoring is inconvenient. The solution was then
extracted into dichloromethane (4 x 40 mL), washed with brine (2 x 50 mL), dried over MgSO4,
and the solvent removed under reduced pressure in an unheated water bath to afford the
desired aldehyde.
4-hydroxyphenylacetaldehyde (2a). DL-Tyrosine (1a, 0.200 g, 1.1 mmol), was dissolved in 250
mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield
0.107 g of 2a (71 %) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 9.72 (t, J = 2.4 Hz, 1H), 7.09 (d, J =
8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 4.82 (s, 1H), 3.62 (d, J = 2.3 Hz, 2H). LRMS (EI+)m/z 136 (M+
17.6), 137 (M+1, 1.7), 108 (9.5), 107 (100), 89 (.63), 78 (3.7), 77 (21.6). HPLC retention time 1.63
mins condition A. Absorbance spectra λmax 222 nm minor λmax 276 nm.
3-chloro-4-hydroxyphenylacetaldehyde (2b). 3-chloro-DL-Tyrosine (0.253 g, 1 mmol), 1b, was
dissolved in 250 mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 2
hours to yield 0.143 g of 2b (83 %) as a brown oil. 1H NMR (300 MHz, CDCl3) δ 9.75 (t, J = 2.1 Hz,
1H), 7.21 (s, 1H), 7.04 (s, 2H), 5.55 (s, 1H), 3.64 (d, J = 2.1 Hz, 2H). LRMS (EI+)m/z 170 (M+, 5.89),
172 (M+2, 18.8), 143 (32.2), 141 (100), 105 (5.4), 77 (25.6). HPLC retention time 2.17 mins
condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm.
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3-bromo-4-hydroxyphenylacetaldehyde (2c). 3-bromo-DL-Tyrosine (0.298 g, 1 mmol), 1c, was
dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2
hours to yield 0.177 g of 2c (82 %) as a brown crystalline semisolid. 1H NMR (300 MHz, CDCl3) δ
9.73 (t, J = 2.2 Hz, 1H), 7.33 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 8.3, 1.9 Hz, 1H), 7.01 (d, J = 8.3 Hz,
1H), 5.55 (s, 1H), 3.62 (d, J = 2.1 Hz, 2H). LRMS (EI+) m/z 214 (M+, 17.4), 215 (M+1, 2.5), 216 (M+2,
18.0), 188 (8.7), 187 (100), 186 (9.7), 185 (97), 107 (6.1), 106 (4.9), 78 (15.6), 77 (26). HPLC
retention time 2.33 mins condition A. Absorbance spectra λmax 223 nm minor λmax 281 nm.
3-iodo-4-hydroxyphenylacetaldehyde (2d). 3-iodo-DL-Tyrosine (0.302 g, 1 mmol), 1d, was
dissolved in 250 mL of 10 mM pH 7 phosphate buffer and reacted by the above procedure for 2
hours to yield 0.197 g of 2d (77 %) as a brown crystalline semisolid. 1H NMR (300 MHz, CDCl3) δ
9.74 (t, J = 2.2 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.11 (dd, J = 8.3, 2.1 Hz, 1H), 7.00 (d, J = 8.3 Hz,
1H), 5.36 (s, 1H), 3.63 (t, J = 3.2 Hz, 2H). LRMS (EI+) 262 (M+, 26.6), 263 (M+1, 2.2), 234 (8.4), 233
(100), 136 (0.19), 127 (2.41), 107 (3.5), 106 (16.5), 78 (10), 77 (9). HPLC retention time 2.63
mins condition A. Absorbance spectra λmax 284 nm.
2-phenylacetaldehyde (2e). DL-Phenylalanine (0.165 g, 1 mmol) was dissolved in 250 mL of 10
mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield 72 mg of
2e (60 %) as a yellow oil. 1H-NMR, LRMS, and HPLC data matched an authentic commercial
sample (Sigma-Aldrich).
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2-(1H-indol-3-yl)acetaldehyde (2f). L-Tryptophan (205 mg, 1 mmol) was dissolved in 250 mL of
10 mM pH 7 phosphate buffer and reacted by the above procedure for 45 minutes. A second
equivalent of NaOCl was added and the reaction continued for a total of 2 hours to yield 118
mg of crude 2f (72%) as a brown semi-solid crude oil with impurities. HPLC analysis indicated
that 35% of the tryptophan had not reacted. Additional equivalents of NaOCl resulted in the
formation of significant side products. Product was not purified further.
Measurement and analysis of kinetics for oxidative decarboxylation of tyrosine.
The kinetics of oxidative decarboxylation were measured by quantification of HPLC peaks in a
time course reaction using Condition B and observation of absorbance at 275 nm. To convert
observed area to concentration, the area-extinction coefficients for starting and product
compounds were measured based on a calibration curve of the pure compound dissolved in
water covering a concentration range from roughly 0.1 to 3000 μM. Pure solutions of 4hydroxyphenyl-acetaldehyde (4-HPAA) were prepared by two alternative methods. By the first
method, multiple solutions were prepared in volumetric glassware using the mass of pure 4HPAA oil prepared by NaOCl oxidation. By the second method, a tyrosine solutions of known
concentration were reacted with NaOCl to generate 4-HPAA solutions of known concentration,
assuming complete conversion (verified by HPLC). Both methods gave comparable areaextinction coefficients, however the second method generated calibration curves with
significantly less deviation from linearity. The area-extinction coefficient from the latter method
was used for analysis of kinetics data.
A 1.00 mM solution of commercial tyrosine was prepared in 1.00 L of pH 7.0 phosphate buffer.
The concentrations of phosphate buffer evaluated were 0, 3.96, 8.0, 16.0, 20.0, 40.0, and 120.0
mM. A 50 μL sample of the initial solution was removed for concentration analysis by HPLC. The
solution was equilibrated with vigorous stirring at 37.0 °C in a warm room. A concentrated
solution of 1.0 mmol of NaOCl was added slowly by syringe pump over 5 minutes. The change in
volume resulting from this addition was less than 0.1% of the total and was treated as negligible
for later rate estimates. Once addition was complete, three replicate 1 mL samples of the
reaction mixture were transferred to HPLC sample vials. These vials were immediately
transferred to an autosampler chamber that maintained 37.00 ± 0.01 °C for the course of the
reaction. Continuous time course measurements were injected and analyzed at 7 minute
15
intervals using HPLC Condition B. As observed in Figure 2, there was no noticeable variation
between replicate samples. Since three unstable intermediates were observed, it was not
possible to interpolate an extinction coefficient for each of these species. Thus, the
concentrations of intermediates were excluded from any analysis.
The time dependent disappearance of tyrosine and appearance of 4-HPAA were fit to various
models using the Gepasi computer program7. Time course curves from a two-step irreversible
kinetic model most closely matched the experimental data. The rate constants for tyrosine
disappearance and 4-HPAA appearance were found using a genetic algorithm (population of 10
and 1000 generations). The rates were not dependent on buffer concentration. The reported
rate constant values represent the average and standard deviation from replicate
measurements of all phosphate buffer concentrations from 3.96 – 40.0 mM. Samples prepared
with no buffer gave inconsistent kinetic results and formed of side products. These samples
were not included in the reported average. The rate of tyrosine disappearance at 120 mM
phosphate buffer was slower than the other samples (5.8 ± 0.5 M-1s-1), thus data from 120 mM
phosphate buffer was excluded.
Pictet-Spengler reactions
General procedure for synthesis of racemic tetrahydroisoquinolines from α-amino acids. Each
1 mmol of amino acid was suspended with 2.0 mL of 1.0 M phosphate buffer at pH 6.9 (this
concentrated buffer gives pH 7.0 upon dilution) in 200 mL of purified water at 37 °C. A solution
of 1 molar equivalent of sodium hypochlorite (e.g. 700 μL of 9.16 % NaOCl) was dissolved in 10
mL of purified water. The NaOCl was added dropwise over 10 minutes via syringe pump with
vigorous stirring by magnetic stir bar. This solution was allowed to stir at 37 °C for 1.5 - 2 hours
and was quenched by the addition of ascorbic acid (88 mg, 0.5 mmol). Additional phosphate
catalyst, 100 mL of 1.0 M phosphate buffer at pH 6.9, was then added to the solution followed
by dopamine hydrochloride (291 mg, 1.5 mmol). The reaction was monitored until all of the
aldehyde was consumed as monitored by HPLC. If monitoring is not convenient, reactions are
generally complete after 2 hours. The reaction was then extracted into ethyl acetate (6 X 50
mL), the combined organic layers were washed brine (2 X 50 mL), dried with MgSO4, and the
solvent was removed under reduced pressure to yield the product as a solid. When scaling-up
the reaction, we have found that the volume of extraction solvent can be minimized by
exhaustively extracting the reaction mixture more times rather than scaling-up the extraction
solvent volume.
16
Norcoclaurine (4a). DL-Tyrosine (0.189 g, 1 mmol), 3a, was reacted by the above procedure for
75 minutes to yield 0.187 g of 4a (69%) as a pale yellow solid. HPLC retention time 1.74 mins
condition A. Absorbance spectra λmax 225 nm minor λmax 283 nm. 1H NMR (300 MHz, Methanold4) δ 7.10 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 8.5 Hz, 2H), 6.66 (s, 1H), 6.53 (s, 1H), 3.18 (s, 2H), 2.84
(s, 2H), 2.69 (s, 2H), 2.02 (d, J = 2.9 Hz, 1H). HRMS (C16H18NO3+): calc. 272.1287 [M+H]+; found
272.1290.
3-chloro-norcoclaurine (4b). 3-chloro-DL-Tyrosine (0.309 g, 1.5 mmol), 3b, was dissolved in 500
mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield
0.252 g of 4b (83%) as a yellow solid. HPLC retention time 1.98 mins condition A. Absorbance
spectra λmax 284 nm. 1H NMR (300 MHz, methanol-d4) δ 7.25 (d, J = 2.1 Hz, 1H), 7.07 – 7.02 (m,
1H), 6.90 (d, J = 8.2 Hz, 1H), 6.64 (s, 1H), 6.56 (s, 1H), 3.29 – 3.16 (m, 3H), 2.98 (d, J = 6.7 Hz, 1H),
2.87 – 2.74 (m, 3H). 1H NMR (300 MHz, DMSO-d6) δ 8.66 (broad s, 2H), 7.24 (s, 1H), 7.02 (d, J =
8.1 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.59 (s, 1H), 6.43 (s, 1H), 3.94 (d, J = 6.1 Hz, 1H), 3.13 – 2.89
(m, 2H), 2.89 – 2.53 (m, 3H). 13C NMR (75 MHz, D2O with 35% formic acid) δ 151.62, 144.67,
143.45, 131.40, 129.93, 128.41, 124.22, 123.39, 121.00, 117.73, 116.32, 114.24, 56.74, 39.99,
38.75, 24.60. HRMS (C16H17NO3Cl+): calc. 306.0897 [M+H]+; found 306.0910 [M+H]+.
3-bromo-norcoclaurine (4c). 3-bromo-DL-Tyrosine (0.5235, 2 mmol), 3c, was dissolved in 500
mL of 20 mM pH 7 phosphate buffer and reacted by the above procedure for 2 hours to yield
0.575 g of 4c (82%) as a tan solid. HPLC retention time 2.05 mins condition A. Absorbance
spectra λmax 284 nm. 1H NMR (300 MHz, acetonitrile-d3 with 0.3% trifluoroacetic acid) δ 7.47 (d,
J = 2.1 Hz, 1H), 7.13 (dd, J = 8.3, 2.1 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.67 (d, J = 4.9 Hz, 2H), 4.62
17
– 4.51 (m, 1H), 3.47 – 3.32 (m, 2H), 3.27 – 3.12 (m, 1H), 3.04 – 2.78 (m, 3H). 1H NMR (300 MHz,
DMSO-d6) δ 10.20 – 9.87 (m, 1H), 8.73 (s, 1H), 8.68 – 8.50 (m, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.11
– 7.01 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.60 (s, 1H), 6.43 (s, 1H), 3.98 – 3.92 (m, 1H), 3.09 – 3.03
(m, 1H), 2.94 (s, 1H), 2.80 – 2.73 (m, 1H), 2.67 (d, J = 13.9 Hz, 1H). 13C NMR (75 MHz, methanold4 with 3% formic acid). δ 155.05, 146.86, 145.74, 135.10, 130.84, 128.92, 123.72, 123.53,
117.75, 116.23, 114.19, 111.36, 57.66, 40.88, 39.96, 25.67. HRMS (C16H17NO3Br+): calc.
350.0392 [M+H]+; found 350.0405 [M+H]+.
3-iodo-norcoclaurine (4d). 3-iodo-DL-Tyrosine (0.615, 2 mmol), 3d, was dissolved in 250 mL of
20 mM pH 7 phosphate buffer and reacted by the above procedure for 3 hours to yield 0.955 g
of 4d (75%) as a yellow solid. HPLC retention time 2.21 mins condition A. Absorbance spectra
λmax 285 nm. 1H NMR (300 MHz, methanol-d6 with 0.3% trifluoroacetic acid) δ 7.68 (d, J = 1.7
Hz, 1H), 7.13 (dd, J = 8.4, 1.8 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.62 (s, 1H), 6.60 (s, 1H), 4.58 (dd, J
= 8.4, 5.7 Hz, 1H), 3.53 – 3.33 (m, 2H), 3.29 – 3.16 (m, 1H), 3.09 – 2.80 (m, 3H). 1H NMR (300
MHz, methanol-d6) δ 7.61 (d, J = 1.8 Hz, 1H), 7.08 (dd, J = 8.2, 1.8 Hz, 1H), 6.80 (d, J = 8.2 Hz,
1H), 6.62 (s, J = 6.6 Hz, 1H), 6.53 (s, J = 9.8 Hz, 1H), 4.12 (dd, J = 9.1, 4.3 Hz, 1H), 3.25 – 3.10 (m,
2H), 2.95 – 2.61 (m, 4H). 13C NMR (75 MHz, methanol-d4 with 6% formic acid) δ 163.39, 157.71,
146.81, 145.70, 141.24, 131.75, 129.20, 123.71, 123.52, 116.20, 114.17, 85.32, 57.74, 40.95,
39.75, 25.65. HRMS (C16H17NO3I+): calc. 398.0253 [M+H]+; found 398.0271 [M+H]+.
1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e). Prepared according to the general
procedure above using L-phenylalanine (0.165 g, 1 mmol) reacted for 4 hours to yield 4e (0.149
g, 58%) as a yellow powder. 1H NMR (300 MHz, methanol-d4) δ 7.40 – 7.17 (m, 5H), 6.65 (s, 1H),
6.51 (s, 1H), 4.07 (dd, J = 9.2, 3.9 Hz, 1H), 3.29 – 3.04 (m, 2H), 2.95 – 2.75 (m, 2H), 2.74 – 2.53
(m, 2H). 1H NMR (300 MHz, DMSO-d6) δ 8.66 (broad s, 1H), 8.55 (broad s, 1H), 7.35-7.13 (m,
5H), 6.62 (s, 1H), 6.41 (s, 1H), 3.92 (d, 1H), 3.01 (d, 2H), 2.73 (t, 2H), 2.00 (s, 1H). 13C NMR (75
MHz, methanol-d4) δ 145.27, 144.71, 139.92, 130.45, 129.74, 127.67, 126.79, 116.37, 114.12,
57.95, 43.15, 41.52, 29.20. HRMS (C16H18NO2+): calc. 256.1338 [M+H]+; found 256.1324 [M+H]+;
18
additional fragments 239.1058, 161.0592.
1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f). Prepared according to
the general procedure above using L-tryptophan (0.204 g, 1 mmol) and 10 mL of NaOCl solution
(0.0925 M, 1 mmol). After 45 minutes of stirring, a second equivalent of 10 mL of NaOCl
solution (0.0925 M, 1 mmol) was added and the reaction stirred for 90 minutes before adding
ascorbic acid (88 mg, 0.5 mmol) followed by dopamine hydrochloride (291 mg, 1.5 mmol). The
resulting solution was stirred for 3 hours. A dark purple powder was removed by filtration
before extracting as describe to yield 4f (0.112 g, 38%) as a pale yellow solid. The yellow solid
may be further purified by dissolving in minimal methanol, adding 10 volumes of chloroform,
and precipitating with diethyl ether to yield a fluffy crystalline solid. The product partially
overlap either the residual water or solvent peaks from all deuterated solvents in which it is
soluble. Two 1H NMR spectra in different solvents were acquired for an unambiguous
assignment. HPLC retention time 2.84 mins in condition A. Absorbance spectra λmax 222, 279.5
nm. 1H NMR (300 MHz, Methanol-d4) δ 7.61 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.19 (s,
1H), 7.18 – 7.10 (m, 1H), 7.06 (dd, J = 10.9, 4.0 Hz, 1H), 6.76 (s, 1H), 6.62 (s, 1H), 4.68 (dd, J =
9.2, 5.0 Hz, 1H), 3.53 – 3.39 (m, 2H), 3.28 – 3.14 (m, 2H), 2.99 – 2.83 (m, 2H). 1H NMR (300 MHz,
CD3CN with 0.33% trifluoroacetic acid) δ 7.67 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.21 (s, J
= 7.4 Hz, 1H), 7.26 – 7.17 (m, 1H), 7.16 – 7.08 (m, 1H), 6.89 (s, 1H), 6.69 (s, 1H), 4.79 – 4.63 (m,
1H), 3.63 (dd, J = 15.3, 4.5 Hz, 1H), 3.50 – 3.37 (m, 1H), 3.27 – 3.11 (m, 2H), 3.01 – 2.81 (m, 2H).
13C NMR (75 MHz, methanol-d with 1.5% formic acid) δ 146.82, 145.91, 138.50, 128.23,
4
125.59, 124.14, 123.70, 123.04, 120.36, 119.12, 118.17, 116.21, 114.11, 112.72, 108.81, 56.68,
40.85, 31.42, 25.71. HRMS (C18H19N2O2+): calc. 295.1447 [M+H]+; found 295.1429 [M+H]+; major
fragment 164.0698.
Expression of recombinant norcoclaurine synthase enzyme. We followed a modification of the
procedure described by Luk et al.9 except using the plasmid construct of Ruff et al.10 This
plasmid contains a codon-optimized sequence of TfNCSΔ19 (5071-5769), Thalictrum flavum
norcoclaurine synthase with the N-terminal 19 amino acids deleted. This sequence was cloned
into the pET-28a(+) vector between the NdeI and EcoRI restriction sites to generate plasmid
pNCSa. This plasmid was transformed into BL21(DE3) chemically competent cells (Sigma).
19
The cells were incubated with shaking at 250 rpm in 50 mL of Luria-Bertani (LB) medium
containing BL21(DE3)/pNCSa and 30 μg/mL kanamycin overnight at 37 °C. The overnight
cultures were poured into 1.0 L of LB medium containing 30 μg/mL kanamycin and grown at 37
°C with shaking at 250 rpm until an OD600 of 0.6 was reached. Cells were induced for
overexpression by addition of 238 mg (1 mM) of isopropyl-β-D-galactopyranoside (IPTG), and
the cultures were allowed to continue growth at 25 °C until an OD600 of 3.0 - 4.0 was reached
(~24 h). Cells were harvested by centrifugation at 4000 rcf for 20 min and resuspended in
binding buffer (50 mM phosphate, 20 mM imidazole, 300 mM NaCl at pH 8.0). The cells were
lysed by sonication at 250 Watts for 120 secs in cycles of 10 seconds on and 10 seconds of cool
down. The cell lysate was clarified by centrifugation at 20,000 rcf for 25 min. The supernatant
was drawn through a 10 mL syringe with 22 gauge needle 80 times to shear DNA and filtered
through a 0.22 μm membrane filter. A 1 mL GE Healthcare HisTrap FF nickel affinity column was
washed with 10 column volumes (CV) of water, 10 CV of binding buffer (50 mM phosphate, 20
mM imidazole, 300 mM NaCl at pH 8.0) to prime the column. The filtered cell lysate was loaded
at a rate of 1 mL/min. Binding buffer (10 CV) was passed through the column at 1 mL/min. A
wash with wash buffer (20 mM imidazole, 500 mM NaCl at pH 7.8) was used to remove
nonspecifically bound proteins until no more flow-through protein eluted, as determined by
measurement of A280 (~50 CV). Histidine-tagged protein was eluted with 21 CV of elution buffer
(300 mM imidazole and 500 mM NaCl at pH 7.0). The enzyme was concentrated by
ultrafiltration (Amicon Ultra-4, 10 000 MWCO) to a volume of 500 μL. This solution diluted into
equal volume glycerol to yield 3 -7 mg/mL as determined by Bradford assay. The solution was
divided into 500 μL aliquots and stored at -20 °C without freezing and with no noticeable loss of
activity after several months. The enzyme was not allowed to warm before use.
General procedure for synthesis of (S)-enantiomer tetrahydroisoquinolines from α-amino
acids with norcoclaurine synthase. Each 1 mmol of amino acid was suspended in 250 mL of 25
mM pH 6.80 maleic acid buffer at 37 °C. A solution of 1 molar equivalent of sodium
hypochlorite (e.g. 700 μL of 9.16 % NaOCl) was dissolved in 10 mL of purified water. The NaOCl
was added dropwise over 10 minutes via syringe pump with vigorous stirring by magnetic stir
bar. This solution was allowed to stir at 37 °C for 1.5 – 2 hours and was quenched by the
addition of ascorbic acid (88 mg, 0.5 mmol). After 5 minutes, 1.5 molar equivalents of
dopamine hydrochloride (291 mg, 1.5 mmol) was added followed by 2.5 mg of norcoclaurine
synthase (isolated by the procedure above). After stirring for 2 hours at 37 °C, the reaction
mixture was analyzed. HR-MS and HPLC-UV properties were identical to racemic norcoclaurine
synthesized in phosphate buffer. Chiral HPLC revealed only one detectable enantiomer. All
other characterization data were identical to the equivalent enantiomeric mixture prepared in
phosphate buffer.
20
Norcoclaurine synthase kinetic assays for aldehyde substrates 4a – 4e. The initial rate of
enzyme reaction was measured for substrates 4a – 4e by preparing a 500 μL reaction
containing a final concentration of 1.00 mM dopamine HCl (from 10.0 mM stock solution in
deionized water), 1.00 mM aldehyde (from 100 mM DMSO stock solution), 100 mM HEPES (or
BES, both gave identical results) at pH 7.2, and 10 μg of norcoclaurine synthase enzyme
(prepared as described above). For the total number of aldehyde samples, all other
components of the reaction except for the aldehyde (dopamine, buffer, and enzyme) were
mixed as a single stock solution and then divided into equal aliquots. The reaction was started
by the addition of aldehyde. The reactions were quenched every 60 seconds by removing 80 µL
of the reaction and mixing it with 20 µL of 1 M HCl. The rate of the reaction was determined by
monitoring the area of the dopamine peak by HPLC (condition B) at 280 nm. The resulting areas
were then plotted as a function of time to yield straight lines, the slopes of which gave the rates
in units of µV*s /s. All rates are reported as a percent of the rate of positive control 4a within
the same experiment. All analyses were repeated three independent times under identical
conditions.
Halogenation of tyrosol
2-chloro-4-(2-hydroxyethyl)phenol (6b).11 A suspension containing 4-(2-hydroxyethyl)phenol
(6a, 2.990, 21.6 mmol) and NaCl (1.266 g, 21.6 mmol) in acetone (55 mL) was cooled to 0 oC
while a solution of Oxone (19.818 g, 32.2 mmol) in water (100 mL) was added dropwise over
three hours. The reaction was then allowed to stir overnight at room temperature, after which
the reaction was extracted with ethyl acetate (3 x 60 mL) and the combined organic layers
washed with brine (1 x 30 mL). The resulting solution was then dried with MgSO 4, filtered and
the solvent removed under reduced pressure yielding an amber colored oil. The resulting oil
was then dissolved in a minimal amount of hot dichloromethane and cooled yielding 3.735 g of
6b (72 %) as white crystals. 1H NMR (300 MHz, Acetone-d6) δ 8.64 (s, 1H), 7.39 (d, J = 2.0 Hz,
1H), 7.08 (dd, J = 8.2, 2.1 Hz, 1H), 6.94 – 6.88 (m, 1H), 3.73 – 3.70 (m, 2H), 2.74 – 2.69 (m, 2H).
LRMS (EI+)m/z 172 (M+, 25), 173 (M+1, 2,23), 174 (M+2, 8.4), 143 (33.2), 142 (9.5), 141 (100), 107
(5.4), 105 (4.5), 77 (19.3). HPLC retention time 2.18 mins condition A. Absorbance spectra λmax
222 nm minor λmax 280 nm.
21
2-bromo-4-(2-hydroxyethyl)phenol (6c).11 A suspension containing 4-(2-hydroxyethyl)phenol
(6a, 3.463, 25 mmol) and NaBr (2.589 g, 25.2 mmol) in acetone (55 mL) was cooled to 0 °C while
a solution of Oxone (23.153 g, 32.2 mmol) in water (100 mL) was added dropwise over three
hours. The reaction was then allowed to stir for three additional hours at room temperature,
after which the reaction was extracted with ethyl acetate (3 x 60 mL) and the combined organic
layers washed with brine (1 x 30 mL). The resulting solution was then dried with MgSO 4, filtered
and the solvent removed under reduced pressure yielding an amber colored oil. The resulting
oil was then dissolved in a minimal amount of hot dichloromethane and cooled yielding 4.292 g
of 6c (79 %) as a white solid 1H NMR (300 MHz, Acetone D-6) δ 8.64 (s, 1H), 7.39 (d, J = 2.0 Hz,
1H), 7.07 (dt, J = 8.1, 4.1 Hz, 1H), 6.95 – 6.89 (m, 1H), 3.72 (dd, J = 6.1, 4.4 Hz, 2H), 2.79 – 2.66
(m, 2H). LRMS (EI+)m/z 216 (M+, 23.8), 218 (M+2, 24.3), 173 (16), 188 (9.0), 187 (97.9), 186 (9.1),
185 (100), 107 (10) 106 (4.4) 78 (9.3), 77 (21.7). HPLC retention time 2.31 mins. Absorbance
spectra λmax 218 nm minor λmax 281 nm.
2-iodo-4-(2-hydroxyethyl)phenol (6d).12 A solution of I2 (5.074 g, 20 mmol) and KI (4.374 g, 26
mmol) in 100 mL of a 1:1 water and ethanol was added to a solution containing 4-(2hydroxyethyl)phenol (6a, 2.733 g, 20 mmol) dissolved in 20 mL of a 40% solution of dimethyl
ammine. The resulting solution was allowed to stir at root temp for 4 hours after which it was
acidified with a 2 M solution a HCl and extracted into ethyl acetate (3x100 mL). The combined
organic layers were then washed with a 10% (m/V) solution of sodium thiosulfate (2x50 mL)
and brine (1x40mL). The resulting organic layer was then dried with MgSO4, filtered, and the
solvent removed under reduced pressure yielding a brown oil. The resulting oil was dissolved in
a minimal amount of hot water and cooled yielding 3.7602 g of 6d (72 %) as white needle like
crystals. 1HNMR (300 MHz, CDCl3) δ 7.55 (d, J = 2.0 Hz, 2H), 7.12 (dd, J = 8.2, 2.0 Hz, 2H), 6.93 (d,
J = 8.3 Hz, 2H), 5.40 (s, 2H), 3.84 (t, J = 6.5 Hz, 5H), 2.84 – 2.63 (m, 5H), 1.60 (d, J = 33.9 Hz, 4H).
LRMS (EI+) m/z 264 (M+ 35.2), 265 (M+1 3.0), 234 (9.8), 233 (100), 128 (1.4), 107 (5.0), 106 (13.9)
78 (7.8), 77 (6.5). HPLC retention time 2.48 mins condition A. Absorbance spectra λmax 217 nm
minor λmax 284 nm.
22
Parikh-Doering oxidation reactions
4-hydroxyphenylacetaldehyde (2a).1, 8, 13-15 In a flame dried flask purged with N2, sulfur trioxide
pyridine complex (3.114 g, 20 mmol) was dissolved in 15 mL of dimethyl sulfoxide dried over 3A
molecular sieves.16 This solution was then transferred by cannula into a second flame dried
flask purged with N2 containing 4-(2-hydroxyethyl)phenol (6a, 0.951 g, 6.8 mmol) and
triethylamine (3 mL, 21 mmol), dissolved in 15 mL of sieve dried dimethyl sulfoxide, slowly over
a period of 15 mins. The resulting solution was allowed to stir at room temperature for 1.5
hours under a N2 atmosphere, after which it was poured into 100 mL of ice cold water and
acidified with 10 mL of 2 M HCl. The resulting solution was extracted into ethyl acetate (3x100
mL), and the combined organic fractions washed with brine (2x100 mL), dried with MgSO 4,
filtered and the solvent removed under reduced pressure yielding a crude amber colored oil.
The oil was purified by flash chromatography (20% ethyl acetate in hexanes) yielding 0.2272g of
2a (24%) as a clear oil. 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.4 Hz, 1H), 7.15 – 7.06 (m, 2H),
6.90 – 6.82 (m, 2H), 4.80 (s, 1H), 3.63 (dd, J = 8.4, 2.9 Hz, 2H). LRMS (EI+) m/z 136 (M+ 17.6), 137
(M+1, 1.7), 108 (9.5), 107 (100), 89 (.63), 78 (3.7), 77 (21.6). HPLC retention time 1.63 mins
condition A. Absorbance spectra λmax 222 nm minor λmax 276 nm.
3-chloro-4-hydroxyphenylacetaldehyde (2b). In a flame dried flask purged with N2, sulfur
trioxide pyridine complex (0.311 g, 1.9 mmol) was dissolved in 5 mL of dimethyl sulfoxide dried
over 3A molecular sieves.16 This solution was then transferred by cannula into a second flame
dried flask purged with N2 containing 6b (0.129 g, 0.75 mmol) and triethylamine (0.33 mL, 3.7
mmol), dissolved in 5 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15 mins. The
resulting solution was allowed to stir at room temperature for 4 hours under a N 2 atmosphere,
after which it was poured into 100 mL of ice cold water. The resulting solution was extracted
into ethyl acetate (3x50 mL), and the combined organic fractions washed with brine (1x40 mL),
dried with MgSO4, filtered and the solvent removed under reduced pressure yielding a crude
amber colored oil. Flash chromatography was performed (0-40% ethyl acetate in hexanes)
yielding 0.127 g of 2b (35.5%). 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.2 Hz, 1H), 7.20 (d, J = 6.4
23
Hz, 1H), 7.12 – 7.01 (m, 2H), 5.62 (s, 1H), 3.66 (t, J = 11.0 Hz, 2H). LRMS (EI+)m/z 170 (M+, 5.89),
172 (M+2, 18.8), 143 (32.2), 141 (100), 105 (5.4), 77 (25.6). HPLC retention time 2.17 mins
condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm.
3-chloro-4-hydroxyphenylacetaldehyde (2c). In a flame dried flask purged with N2, sulfur
trioxide pyridine complex (0.897 g, 5.6 mmol) was dissolved in 10 mL of of dimethyl sulfoxide
dried over 3A molecular sieves.16 This solution was then transferred by cannula into a second
flame dried flask purged with N2 containing 6c (0.5858 g, 2.7 mmol) and triethyl amine (0.75
mL, 5.4 mmol), dissolved in 10 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15
mins. The resulting solution was allowed to stir at room temperature for 1 hour under a N 2
atmosphere, after which it was poured into 150 mL of ice cold water. The resulting solution was
extracted into ethyl acetate (3x75 mL), and the combined organic fractions washed with brine
(1x50 mL), dried with MgSO4, filtered and the solvent removed under reduced pressure yielding
a crude orange oil. Flash chromatography was performed (25% ethyl acetate in hexanes)
yielding 0.0361 g of 2c (6.2%). 1H NMR (300 MHz, CDCl3) δ 9.72 (d, J = 16.0 Hz, 1H), 7.35 (s, 1H),
7.09 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 5.61 (s, 1H), 3.61 (d, J = 22.4 Hz, 2H). LRMS (EI+)
m/z 214 (M+, 17.4), 215 (M+1, 2.5), 216 (M+2, 18.0), 188 (8.7), 187 (100), 186 (9.7), 185 (97), 107
(6.1), 106 (4.9), 78 (15.6), 77 (26). HPLC retention time 2.33 mins condition A. Absorbance
spectra λmax 223 nm minor λmax 281 nm.
3-iodo-4-hydroxyphenylacetaldehyde (2d). In a flame dried flask purged with N2, sulfur trioxide
pyridine complex (0.869 g, 5.5 mmol) was dissolved in 10 mL of dimethyl sulfoxide dried over
3A molecular sieves.16 This solution was then transferred by cannula into a second flame dried
flask purged with N2 containing 6d (0.7060 g, 2.7 mmol) and triethylamine (0.75 mL, 5.4 mmol),
dissolved in 10 mL of sieve dried dimethyl sulfoxide, slowly over a period of 15 mins. The
resulting solution was allowed to stir at room temperature for 1 hour under a N 2 atmosphere,
after which it was poured into 100 mL of ice cold water. The resulting solution was extracted
into ethyl acetate (3x80 mL), and the combined organic fractions washed with brine (1x100
24
mL), dried with MgSO4, filtered and the solvent removed under reduced pressure yielding a
crude orange oil. Flash chromatography was performed (30% ethyl acetate in hexanes) yielding
0.1804 g of 2d (26%) as a slightly colored oil. 1H NMR (300 MHz, CDCl3) δ 9.74 (t, J = 2.2 Hz, 1H),
7.54 (d, J = 2.0 Hz, 1H), 7.12 (dd, J = 8.3, 2.1 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 5.29 (s, 1H), 3.63 (d,
J = 2.2 Hz, 2H). LRMS (EI+) 262 (M+, 26.6), 263 (M+1, 2.2), 234 (8.4), 233 (100), 136 (0.19), 127
(2.41), 107 (3.5), 106 (16.5), 78 (10), 77 (9). HPLC retention time 2.63 mins condition A.
Absorbance spectra λmax 284 nm.
3-Halogenation of tyrosine
3-chloro-tyrosine hydrochloride (1b).17 Tyrosine (1a, 7.567 g, 41.8 mmol) was suspended in 75
mL of glacial acetic acid and SO2Cl2 (3.44 mL, 44 mmol) was slowly added to the suspension
over 10 minutes. The resulting solution was allowed to stir for 5.5 hours, after which the solid
precipitate was collected by vacuum filtration and washed with glacial acetic acid (3x10 mL) and
diethyl ether (3x25mL). The resulting product was then recrystallized from boiling concentrated
hydrochloric acid and dried under vacuum to yield 6.910 g of 1b (66%) as white crystals. 1H
NMR (300 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.25 (s, 3H), 7.24 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.3,
2.1 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 4.14 (s, 1H), 3.00 (t, J = 5.7 Hz, 2H). HPLC retention time 1.56
mins condition A. Absorbance spectra λmax 222 nm minor λmax 280 nm.
3-bromo-tyrosine hydrochloride (1c).18 A solution containing tyrosine (1a, 5.541 g, 30.6 mmol)
suspend in 25 mL of glacial acetic acid and 15 mL of a 33% solution of HBr in acetic acid (61
mmol), was prepared. A solution of bromine (1.7 mL, 33.2 mmol) in 25 mL of glacial acetic acid
was then added to the previously prepared solution over a period of 3 hours, and the resulting
solution was allowed to stir for 24 hours. Upon completion of the reaction the solid precipitate
was collected by vacuum filtration, washed with glacial acetic acid (3x25 mL) and diethyl ether
25
(3x25mL) yielding an orange powder. The powder was recrystallized from boiling concentrated
hydrochloric acid and dried under vacuum to yield 6.023g of 1c (70%) as white crystals. 1HNMR
(300 MHz, D2O) δ 7.40 (d, J = 2.1 Hz, 1H), 7.06 (dd, J = 8.3, 2.1 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H),
4.12 (d, J = 5.4 Hz, 1H), 3.15 (dd, J = 14.7, 5.6 Hz, 1H), 3.02 (dd, J = 14.8, 7.6 Hz, 1H). HPLC
retention time 1.64 mins condition A. Absorbance spectra λmax 280 nm.
3-iodo-tyrosine (1d).19 A solution of 1 (4.968 g, 27 mmol) dissolved in concentrated NH4OH was
cooled in an ice water bath, and iodine (7.0325 g, 27.8 mmol)dissolved in 150 mL of ethanol
was slowly added dropwise via syringe pump. The resulting solution was allowed to stir
overnight, after which the solvent was removed under reduced pressure, yielding an off white
solid, which was suspended in 150 mL of ice cold water. The solid was collected by vacuum
filtration and re-suspended in ice cold acetone and stirred for 1.5 hours. The resulting solid was
collected by vacuum filtration yielding 2.528 g of 1d (30 %) as a white powder. 1H NMR (300
MHz, D2O) δ 7.60 (d, J = 2.1 Hz, 1H), 7.12 – 7.03 (m, 1H), 6.83 (d, J = 8.3 Hz, 1H), 3.84 – 3.78 (m,
1H), 3.10 – 3.01 (m, 1H), 2.91 (dd, J = 14.7, 7.8 Hz, 1H). HPLC retention time 1.78 mins condition
A. Absorbance spectra λmax 283 nm.
26
Characterization Data
1H
NMR spectra and GC-MS data for 3-halogenated tyrosine compounds 5b-d
2-chloro-4-(2-hydroxyethyl)phenol (5b)
27
2-bromo-4-(2-hydroxyethyl)phenol (5c)
28
2-iodo-4-(2-hydroxyethyl)phenol (5d)
29
30
1H
NMR spectra for 3-halogenated tyrosine analogs in D2O.
3-chloro-tyrosine·HCl
3-bromo-tyrosine·HCl
31
3-iodo-tyrosine
32
1H NMR spectra for extracted 4-hydroxyphenylacetaldehyde products (2a-2d) generated via
oxidative decarboxylation of halogenated tyrosine.
4-hydroxyphenylacetaldehyde (2a)
3-chloro-4-hydroxyphenylacetaldehyde (2b)
33
3-bromo-4-hydroxyphenylacetaldehyde (2c)
3-iodo-4-hydroxyphenylacetaldehyde (2d)
34
GC-MS data for 3-halogenated-4-hydroxyphenylacetaldehyde products (2a-f) generated via
oxidative decarboxylation of halogenated tyrosine.
4-hydroxyphenylacetaldehyde (2a). Expect M+ 136.15 Da.
35
3-chloro-4-hydroxyphenylacetaldehyde (2b). Expect M+ 170.01 and (M+2)+ 172.02 Da
3-bromo-4-hydroxyphenylacetaldehyde (2c). Expect M+ 213.96 and (M+2)+ 215.96 Da.
36
3-iodo-4-hydroxyphenylacetaldehyde (2d). Expect M+ 262.04 Da.
37
1H
NMR spectra for racemic tetrahydroisoquinoline products 4a-f after extraction.
Norcoclaurine (4a). Solvent peaks at 4.8 and 3.6 ppm are water and methanol-d4.
38
3’-chloro-norcoclaurine (4b).
3’-bromo-norcoclaurine (4c). Solvent peaks at 1.94 and 5.10 ppm are residual acetonitrile-d3
and TFA (added to promote solubility). Phenol protons appear in the aromatic region under
these conditions.
39
3’-iodo-norcoclaurine (4d). Solvent peaks at 4.8 and 3.6 ppm are water and methanol-d4. The
peaks at 1.22 (t), 2.03 (s), and 4.06 (q) are residual ethyl acetate.
1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4e). Solvent peaks at 4.85, 3.31, and 3.34
ppm are water, methanol-d4, and methanol.
40
1-((1H-indol-3-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol (4f). Solvent peaks at 4.85,
and 3.31 ppm are water and residual methanol-d4 respectively. Inset shows the methylene
region acquired in acetonitrile-d3 with 0.33% trifluoroacetic acid.
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