Syntheses of deuterium labeled prenyldiphosphate and prenylcysteine analogues for in vivo mass spectrometric quantification

Article abstract of DOI:10.1002/jlcr.3049

A Wittig reaction employing Li(CD₃)₂CP(C ₆H₅)₃ was used to prepare d₆- farnesol and d₆-geranylgeraniol. Reductive amination of aniline-2,3,4,5,6-d₅ was used to prepare

Full text of DOI:10.1002/jlcr.3049

Research Article
Received 13 February 2013,
Accepted 15 March 2013
Published online in 9 May 2013 Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3049
Syntheses of deuterium labeled
prenyldiphosphate and prenylcysteine
analogues for in vivo mass spectrometric
quantification
Thangaiah Subramanian,a Karunai Leela Subramanian,b Manjula Sunkara,b
Fredrick O. Onono,a Andrew J. Morris,b,c and H. Peter Spielmanna,d,e,f
*
A Wittig reaction employing Li(CD3)2CP(C6H5)3 was used to prepare d6-farnesol and d6-geranylgeraniol. Reductive amination
of aniline-2,3,4,5,6-d5 was used to prepare the unnatural isoprenoid analogues d5-anilinogeraniol and d5-anilinofarnesol. All
of these deuterated isoprenols were elaborated into their diphosphate and cysteine thioether derivatives suitable for use as
stable-isotope labeled standards for quantitative mass spectrometric analysis.
Keywords: FPP; AGPP; GGPP; AFPP; FTase; protein prenylation; mass standards; stable labeled synthesis
analysis is greatly facilitated by employing stable-isotope
Introduction
labeled standards.37
The mevalonate isoprenoid biosynthesis pathway provides the
We report the synthesis of series of natural and unnatural, stable,
cell with critical metabolic intermediates that are involved in
deuterium labeled isoprenoid analogues suitable as standards for
multiple cellular processes.1,2 Inhibition of enzymes in this
pathway with statins and bisphosphonates is clinically used in
quantitative mass spectrometric analysis and for metabolic
labeling studies. Previous preparations of d6-farnesol introduced
the isotope in multiple steps.38,39 In our strategy, common
the respective treatment of hypercholesterolemia and metabolic
bone disease.3–21 Farnesyl diphosphate (FPP) and geranylgeranyl
intermediates for the desired d6-farnesyl and d6-geranylgeranyl
derivatives were prepared by modifying a previously reported
diphosphate (GGPP) are two important isoprenoid intermediates
at branch points of this pathway. Both FPP and GGPP serve
as prenyl donors for protein isoprenylation, which is essential
for many biological functions such as cell growth and cell
proliferation. Because protein isoprenylation is an important
therapeutic target in cancer research, there is substantial interest
synthesis of d6-geranyldiphosphate to simultaneously incorporate
all of the deuteriums via a Wittig reaction.40 Analogously, the
isotope was simultaneously incorporated into d5-anilinogeranyl
and d5-anilinofarnesyl derivatives by reductive amination of
aniline-2,3,4,5,6-d5.
in understanding the role that isoprenoid levels play in disease
pathology.22 Cells can take up both natural and unnatural
isoprenols from the exogenous media and appear to convert
them to the corresponding diphosphates for use in protein
aDepartment of Molecular and Cellular Biochemistry, University of Kentucky,
Lexington, KY 40536, USA
isoprenylation.23–30 In addition, natural and unnatural isoprenoid
diphosphates are converted to their corresponding isoprenols by
the action of specific phosphatases.31 The unnatural FPP and
GGPP analogues anilinogeranyl diphosphate (AGPP) and
anilinofarnesyl diphosphate (AFPP) are particularly useful for
studying isoprenoid metabolism and protein isoprenylation
because of the desirable biochemical and mass spectrometric
properties of the aniline moiety.31–35 The accurate, quantitative
and easy measurement of isoprenoid diphosphate levels and
the products of protein isoprenylation are critical to increase
our knowledge about the regulation of isoprenoid metabolism.
Liquid chromatography coupled with tandem mass spectrometry
(LC–MS/MS) methods for detecting isoprenoid diphosphates are
two orders of magnitude more sensitive than analysis by
radio-HPLC.36 Accurate and sensitive quantitative LC–MS/MS
bDivision of Cardiovascular Medicine UK COM, University of Kentucky, Lexington,
KY 40536, USA
cLexington Veterans Affairs Medical Center, University of Kentucky, Lexington, KY
40536, USA
dMarkey Cancer Center, University of Kentucky, Lexington, KY 40536, USA
eKentucky Center for Structural Biology, University of Kentucky, Lexington, KY
40536, USA
fDepartment of Chemistry, University of Kentucky, Lexington, KY 40536, USA
*Correspondence to: H. Peter Spielmann, Department of Molecular and Cellular
Biochemistry, University of Kentucky, Lexington, KY 40536, USA.
E-mail: hps@uky.edu
J. Label Compd. Radiopharm 2013, 56 370–375
Copyright © 2013 John Wiley & Sons, Ltd.

T. Subramanian et al.
to give the desired d6-farnesylcsyteine 8 in 92% yield after
RP-HPLC.
Results and discussion
The preparation of hexadeuterated farnesyl and geranylgeranyl
derivatives is outlined in Scheme 1. Aldehydes 4a and 4b
were prepared in three steps from farnesyl acetate or
geranylgeranyl acetate respectively using a slight modification
of reported procedures.41–43 Epoxides 3a–b were obtained by
hydrobromination of farnesyl acetate 1a or geranylgeranyl
acetate 1b followed by ring closure of bromohydrins 2a–b with
NaH in THF. Oxidation of epoxides 3a–b with hydroiodic acid
provided the desired aldehydes 4a–b. The key intermediates
d6-farnesol 5a and d6-geranylgeraniol 5b were prepared in
39% yield by reaction of aldehydes 4a–b with 1.0 equivalent
of the anion of (CD3)2CDP(C6H5)3I using BuLi as the base. The
phosphonium salt (CD3)2CDP(C6H5)3I was prepared by heating
2-iodopropane-d7 (98 atom % D) and triphenylphosphine in a
sealed tube at 130ꢀC for 6 h followed by recrystallization
from EtOH/EtOAc. Reaction of d6-farnesol 5a with Ph3PBr2 in
MeCN furnished d6-farnesylbromide 6, which was divided into
two portions and used without purification to prepare d6-
farnesylcysteine 8 and d6-farnesyldiphosphate 9. Trapping
bromide 6 with ((n-Bu)4N)3HP2O7 in MeCN gave d6-FPP 9,
which was converted to the NH+4 form by ion exchange
chromatography and then purified by RP-HPLC. Condensation
of bromide 6 with L-cysteine methyl ester in 7 N NH3/MeOH
provided methyl ester 7, which was saponified with LiOH/i-PrOH
The unnatural isoprenoid analogues 16a (d5-AGPP), 16b
(d5-AFPP), 15a (d5-AG-cysteine) and 15b (d5-AF-cysteine) were
prepared using modified procedures from Spielmann and co-
workers and are outlined in Scheme 2.32 Oxidation of geranyl
acetate and farnesyl acetate with SeO2 provided aldehydes
10a and 10b, respectively.32,43 Reductive amination of
aniline-2,3,4,5,6-d5 (98 atom % D) with aldehyde 10a and
10b gave acetates 11a and 11b, which were saponified with
K2CO3 in MeOH/H2O to provide alcohols 12a and 12b. The
desired diphosphates 16a–b and cysteines 15a–b were
elaborated as described for the farnesyl derivatives above.
The final diphosphates and cysteines were characterized by
1H-NMR and both low resolution and high resolution MS. In
addition, the diphosphates were characterized by 31P-NMR. In
all cases, the levels of deuterium isotope incorporation were
identical to that in the starting synthons, and there was no
evidence of positional scrambling.
Experimental procedure
All RP-HPLC was performed on an Agilent 1200 HPLC system equipped
with a microplate autosampler, diode array and fluorescence detector
OAc
n
1a n = 1
1b n = 2
NBS
THF/H2O
HO
OAc
2a n = 1
2b n = 2
n
Br
NaH/THF
OAc
O
n
3a n = 1
3b n = 2
HIO4,THF/H2O
H
O
OAc
n
4a n = 1
4b n = 2
(CD3)2CDP(C6H5)3I
BuLi, THF
CD3
D3C
OH
n
Ph3PBr2
CH3CN
5a n = 1
5b n = 2
CD3
D3C
Br
6
7N NH3/MeOH.
L-Cysteinemethylester HCl salt
((n-Bu)4N)3HP2O7
CH3CN
O
CD3
D3C
S
OMe
NH2
7
LiOH, i-PrOH/H2O
CD3
D3C
O
S
OH
NH2
8
CD3
O
O
P
P
+
3NH4
O-
D3C
O
O
O- O-
9
Scheme 1. Synthesis deuterated isoprenoids
Scheme 2. Synthesis of deuterated unnatural isoprenoids
J. Label Compd. Radiopharm 2013, 56 370–375
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

T. Subramanian et al.
(Santa Clara, USA). Reaction temperature refers to the external bath. All
solvents and reagents were purchased from VWR (Radnor, USA) (EM
Science-Omnisolv high purity) and Aldrich (St. Louis, USA), respectively,
and used as received. Synthetic products were purified by silica gel flash
Syntheses of (2E,6E)-3,7-dimethyl-10-oxodeca-2,6-dien-1-yl
acetate 4a and (2E,6E,10E)-3,7,11-trimethyl-14-
oxotetradeca-2,6,10-trien-1-yl acetate 4b
chromatography (EtOAc/hexane) unless otherwise noted. RP-HPLC To a solution of epoxide 3a (2.5 g, 8.93 mmol) in 60 mL 1:1 THF/H2O was
purification of lipid diphosphates was carried out using a Varian (Santa added 2.6 g of periodic acid (11.4 mmol), stirred at rt for 2 h, extracted
Clara, USA) Dynamax, 10 mm, 300 Å, C-18 (10 mm  250 mm) column with EtOAc (3  50 mL), dried over MgSO4 and concentrated under
and eluted with a gradient mobile phase and flow rate of 4mL/min: 0– reduced pressure. The crude aldehyde was purified by silica gel column
3 min, 90% A; 3–18 min, 0% A; 18–20 min, 0% A; 20–23 min, 90% A; and
chromatography using EtOAc/hexanes with 0.5% triethylamine as
monitored at 254 and 210 nm [A: Water with 0.1% TFA, B: CH3CN with 0.1% solvent to give 4a (1.9 g, 90%). Epoxide 3a (100 mg) gave 54 mg of
TFA for cysteine purification; A is 25 mm aqueous ammonium acetate, B is aldehyde 4b (61%).
CH3CN for diphosphates purification]. 1H NMR and 13C NMR spectra of 4a 1H NMR (CDCl3) d 1.61 (s, 3H), 1.69 (s, 3H), 2.05 (s, 3H), 2.07 2.12 (m,
alcohols were obtained in CDCl3 and 1H and 31P of diphosphates in D2O with 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.48–2.52 (m, 2H), 4.57 (d, J = 7.2 Hz, 2H),
a Varian Inova spectrometer operating at 400 MHz (1H), 100.6 MHz (13C) and 5.09–5.13 (m, 1H), 5.29–5.34 (m, 1H), 9.74 (t, J = 2.0 Hz, 1H).
161.8 MHz (31P). Chemical shifts are reported in ppm from CDCl3 internal peak 4b 1H NMR (CDCl3) d 1.58 (s, 3H), 1.60 (s, 3H), 1.70 (s, 3H), 1.95–1.98
at 7.24 ppm for 1H and 77.4 ppm for 13C (TSP, 0ppm for 1H; H3PO4 as an (m, 2H), 2.04–2.15 (m, 6H), 2.30 (t, J = 7.2 Hz, 2H), 2.47–2.52 (m, 2H),
external reference, 0 ppm for 31P). High resolution electrospray ionization 4.58 (d, J = 6.8 Hz, 2H), 5.09 (t, J = 6.8 Hz, 1H), 5.13 (t, J = 6.8 Hz, 1H),
mass spectra were recorded with an AB Sciex Triple TOF 5600 instrument at 5.33 (t, J = 6.8 Hz, 1H), 9.74 (t, J = 2.0 Hz, 1H).
a resolution greater than 26,000. C17 Lysophosphatidyl choline with a mass
of 510.3554 was used as an internal reference to calibrate the spectrum.
Syntheses of (2E,6E)-3,7-(11-d3)-trimethyldodeca-(12-d3)-
2,6,10-trien-1-ol 5a and (2E,6E,10E)-3,7,11-(15-d3)-
tetramethylhexadeca-(14-d3)-2,6,10,14-tetraen-1-ol 5b
Syntheses of (2E,6E)-10-bromo-11-hydroxy-3,7,11-trimethyl
dodeca-2,6-dien-1-yl acetate 2a and (2E,6E,10E)-14-bromo-
15-hydroxy-3,7,11,15-tetramethylhexadeca-2,6,10-trien-1-yl
acetate 2b
The required isopropyl-d7-triphenylphosphonium iodide was prepared
by heating
a mixture of isopropyliodide-d7 (2 g, 11.3 mmol) and
triphenylphosphine (3.07 g, 11.72 mmol) in a sealed tube at 130ꢀC for
6 h, followed by recrystallizing the resultant crude product from
ethanol (3.13 g, 63%). To the d7-isopropyl triphenylphosphonium iodide
(4.6 g, 10.5 mmol) dissolved in THF(25 mL) was added a 2.5-M solution
of BuLi (4.2 mL, 10.5 mmol) dropwise at À20ꢀC and stirred for 1 h
before adding aldehyde 4a (2.51 mg, 10.5 mmol) in THF (10 mL). The
reaction mixture was stirred for another 4 h at À20ꢀC and quenched
with saturated NH4Cl solution (5 mL). The resultant mixture was
extracted with CH2Cl2 (3 Â 50 mL), and then the combined organic
extracts were washed with brine, dried over MgSO4 and concentrated
at reduced pressure. The crude product was purified by silica gel
column chromatography to give pure d6-farnesol (911 mg, 38%).
4a (74 mg) gave 25 mg of 5b (35%).
The synthesis of 2a and 2b was accomplished using a modification of
published procedures.42,44
To a stirred solution of farnesyl acetate 1a (4.68 g, 17.7 mmol) in
THF/H2O (100 mL:50 mL) at 0ꢀC was added N-bromosuccinimide
(3.53 g, 31.1 mmol) in several portions over 1 h and then stirred for
4 h. EtOAc (200 mL) was added, and the reaction mixture was
allowed to warm up to rt. The organic phase was separated, and
the aqueous phase was extracted EtOAc (3 Â 200 mL), the combined
organic layers were washed with brine, dried over MgSO4 and
concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography to give compound 2a
(4.28 g, 67%). Geranylgeranyl acetate 1b (170 mg) gave 147.4 mg of
2b (67%).
5a 1H NMR (CDCl3) d 1.58 (s, 3H), 1.66 (s, 3H), 1.94–2.12 (m, 8H), 4.13
(d, J = 7.6 Hz, 2H), 5.05–5.12 (m, 2H), 5.38–5.42 (m, 1H).
2a 1H NMR (CDCl3) d 1.31 (s, 3H), 1.33 (s, 3H), 1.57 (s, 3H), 1.68 (s, 3H),
1.70–1.8 (m, 2H ), 1.90–2.0 (m, 2H), 2.03 (s, 3H), 2.04–2.15 (m, 2H),
2.24–2.32 (m, 2H ), 3.94 (dd, J = 1.6,11.2 Hz, 1H), 4.57 (d, J = 6.8 Hz, 2H),
5.15–5.18 (m, 1H), 5.29–5.34 (m, 1H).
13C NMR (CDCl3) d 15.98, 16.25, 26.23, 26.66, 45.00, 59.38, 123.32,
123.75, 124.33, 131.17, 135.37, 139.84.
HR mass m/z (M-H2O + 1) 211.2334
2b 1H NMR (CDCl3) d 1.32 (s, 3H), 1.33 (s, 3H), 1.58 (s, 6H), 1.69 (s, 3H),
1.69–1.82 (m, 2H), 1.92–2.13 (m, 11H), 2.27–2.31 (m, 2H), 3.96 (dd,
J = 1.6, 11.2 Hz, 1H), 4.57 (d, J = 7.2 Hz, 2H), 5.09 (t, J = 6.0 Hz, 1H), 5.18
(t, J = 6.8 Hz, 1H), 5.33 (t, J = 6.8 Hz, 1H).
5b 1H NMR (CDCl3) d 1.59 (s. 3H), 1.60 (s, 3H), 1.68 (s, 3H), 1.95–2.13
(m, 12H), 4.15(d, J = 6.4 Hz, 2H), 5.08–5.12 (m, 3H), 5.42 (t, J = 7.2 Hz, 1H)
13C NMR (CDCl3) d 16.02, 16.29, 26.31, 26.61, 26.74, 39.56, 39.68, 39.73,
59.43, 123.28, 123.76, 124.15, 124.40, 131.11, 134.98, 135.40, 139.90
HR mass m/z (M-H2O + 1) 279.2960
Syntheses of (2E,6E)-9-(3,3-dimethyloxiran-2-yl)-3,7-
dimethylnona-2,6-dien-1-yl acetate 3a and (2E,6E,10E)-13-
(3,3-dimethyloxiran-2-yl)-3,7,11-trimethyltrideca-2,6,10-
trien-1-yl acetate 3b
Syntheses of (R)-methyl 2-amino-3-(((2E,6E)-3,7,11-d3-
trimethyldodeca-12-d3-2,6,10-trien-1-yl)thio)propanoate 7
To a stirred solution of bromohydrin 2a (3.68 g, 10.22 mmol) in THF
To a 0ꢀC solution of d6-farnesol (150 mg, 0.66 mmol) in acetonitrile
(70 mL) was added sodium hydride (673 mg, 16.8 mmol) in one
(10 mL) was added dibromotriphenylphosphorane (285 mg, 0.67 mmol)
portion and then stirred for 2 h. The reaction was quenched with in CH3CN (10 mL). The reaction mixture was stirred for 8 h, and then a
2 mL saturated ammonium chloride and extracted with EtOAc (50 mL
3). The combined extracts were washed with brine, dried over
MgSO4 and concentrated under reduced pressure to give epoxide
3a (2.6 g, 93%). Bromohydrin 2b (742 mg) gave 579 mg of epoxide
3b (99%).
solution of L-cysteinemethylester HCl salt (2.26 g, 13.0 mmol) in 7 M
NH3/MeOH (40 mL) was added. The resultant reaction mixture was
stirred at 0ꢀC overnight, warmed to rt and then extracted with Et2O
(5 Â 50 mL). The combined ether extracts were concentrated under
reduced pressure and purified by silica gel column chromatography to
give compound 7 (96.5 mg, 43% in two steps).
3a 1H NMR (CDCl3) d 1.26 (s, 3H), 1.30 (s, 3H), 1.59–1.60 (m, 2H), 1.62
(s, 3H), 1.70 (s, 3H), 2.05 (s, 4H), 2.06–2.22 (m, 6H), 2.69 (t, 6.4 Hz, 1H),
4.58 (d, J = 7.2 Hz, 2 Hz), 5.13–5.16 (m, 1H), 5.32–5.36 (m, 1H).
3b 1H NMR (CDCl3) d 1.25 (s, 3H), 1.29 (s, 3H), 1.55 (s, 6H), 1.59 (s, 3H),
1H NMR (CDCl3) d 1.59 (s, 3H), 1.67 (s, 3H), 1.94–2.10 (m, 8H), 2.81
(dd, J = 7.6, 14.0 Hz, 1H), 2.98 (dd, J = 4.40,13.76 Hz, 1H), 3.14–3.26
(m, 2H), 3.76 (s, 3H), 3.80–3.86 (m, 2H), 5.06–5.12 (m, 2H), 5.22
1.96–2.10 (m, 15H), 2.69 (t, J = 7.6 Hz, 1H), 4.58 (d, J = 7.2 Hz, 2H), 5.09 (t, J = 8.0 Hz, 1H)
(t, J = 6.8 Hz, 1H), 5.15 (t, J = 6.8 Hz, 1H), 5.34 (t, J = 6.8 Hz, 1H). LR mass m/z (M + 1) 346.3
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 370–375

T. Subramanian et al.
concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography using 30% EtOAc/
hexanes to give compound 12a (508 mg, 81%). compound 11b
(420 mg) gave 282 mg of compound 12b (76%).
Synthesis of (R)-2-amino-3-(((2E,6E)-3,7,11-d3-trimethyldodeca
-12-d3-2,6,10-trien-1-yl)thio)propanoic acid 8.
A mixture of compound 7 (68 mg, 0.197 mmol) and LiOH (47 mg,
1.9 mmol) was stirred in 1:1 water/i-PrOH (2 mL) overnight at rt. The pH
of resultant reaction mixture was brought to 3 by adding drops of 1 N
HCl. The cloudy solution was filtered through a syringe filter (Whatman,
13 mm CD/X disposable polypropylene prefilter). The clear solution was
purified by RP-HPLC (C13 column) using CH3CN/H2O (0.1% TFA) to give
compound 8 (51.5 mg, 79%).
d5-AGOH 12a 1H NMR (CDCl3) d 1.64 (s, 3H), 1.65 (s, 3H), 2.03
(t, J = 8.0 Hz, 2H), 2.12–2.17 (m, 2H), 3.61 (s, 2H), 4.09 (d, J = 2.8 Hz, 2H),
5.32–5.40 (m, 2H)
HR mass m/z (M + H) 251.2159
d5-AFOH 12b 1H NMR (CDCl3) d 1.58 (s, 3H), 1.62 (s, 3H), 1.65 (s, 3H),
1.95–2.08 (m, 4H), 2.09–2.18 (m, 4H), 3.60 (s, 2H), 4.18 (d, J = 4.2 Hz, 2H),
5.08 (t, J = 6.8 Hz, 1H), 5.38–5.41 (m, 2H)
1H NMR (CD3OD) d 1.60 (s, 3H), 1.73 (s, 3H), 1.94–2.14 (m, 8H), 2.87
(dd, J = 8.8, 14.8 Hz, 1H), 3.13 (dd, J = 3.8, 14.8 Hz, 1H), 3.19–3.26 (m, 1H),
4.03 (dd, J = 3.6, 8.8 Hz, 1H), 5.06–5.12 (m, 2H), 5.25 (t, J = 8.4 Hz, 1H)
HR mass m/z (M + 1) 332.2528
HR mass m/z (M + H) 319.2792
Syntheses of (R)-methyl 2-amino-3-(((2E,6E)-3,7-dimethyl-8-
(2,3,4,5,6-d5-phenylamino)octa-2,6-dien-1-yl)thio)propa
noate 14a and (R)-methyl 2-amino-3-(((2E,6E,10E)-3,7,11-trime
thyl-12-(2,3,4,5,6-d5-phenylamino)dodeca-2,6,10-trien-1-yl)
thio)propanoate 14b
Synthesis of (2E,6E)-3,7,11-d3-trimethyldodeca-12-d3-2,6,10-
trien-1-yldiphosphate 9.
To the in situ made bromide 6 from alcohol 5 (25 mg, 0.11 mmol) in
acetonitrile (5 mL) at 0ꢀC was added ((n-Bu)4N)3HP2O7 (430 mg, 0.44 mmol)
in CH3CN (10 mL). The resultant reaction mixture was stirred for 3 h at rt and
then concentrated. The resultant residue was washed with Et2O (5mL), the
organic wash discarded, the residue suspended in 25 mm NH4HCO3 solution
containing 2% i-PrOH (4 mL) and purified as for compound 7 (16 mg, 32%).
d6-FPP 1H NMR (D2O) d 1.48 (s, 3H), 1.58 (s, 3H), 1.86–1.90 (m, 2H),
1.95–2.03 (m, 2H), 4.33 (t, J = 6.8 Hz, 2H), 5.03–5.09 (m, 2H), 5.33
(t, J = 6.8 Hz, 1H)
To a stirred solution of compound 12a (200 mg, 0.8 mmol) and N,N-
diisopropylethylamine (0.24 mL, 2.9 equiv. 2.3 mmol) in dry CH3CN
(5 mL) was added Ph3PCl2 (450 mg, 1.35 mmol, 1.7 equiv.) evenly over a
7-min period. After the final addition of Ph3PCl2, the reaction mixture
was allowed to stir at 0ꢀC for an additional 40 min and then cooled to
À20ꢀC. Then, a solution of L-cysteine methyl ester hydrochloride salt
(542 mg, 3.2 mmol) in 7 M NH3/MeOH (10 mL) was added and stirred for
5 h before warming to rt. The resultant reaction mixture was partitioned
between Et2O and water and extracted with Et2O (50 mL Â 4). The
combined Et2O extracts were then dried over MgSO4, concentrated
under reduced pressure and purified by silica gel chromatography using
1:2:8 ratio of i-PrOH, EtOAc and hexanes to obtain compound
14a (38 mg, 13% in two steps). 12b (100 mg) gave 15.9 mg of 14b
(12% in two steps).
31P NMR (D2O) d À6.95 (d, J = 24.2 Hz, 1P), À10.54 (d, J = 24.2 Hz, 1P)
HR mass m/z 387.1596 (M À H)
Syntheses of (2E,6E)-3,7-dimethyl-8-oxoocta-2,6-dien-1-yl acetate 10a
and (2E,6E,10E)-3,7,11-trimethyl-12-oxododeca-2,6,10-trien-1-yl acetate
10b were accomplished by following reported procedures.32
14a (CDCl3) d 1.62 (s, 3H), 1.63 (s, 3H), 2.01–2.07 (m, 2H), 2.10–2.16
(m, 2H), 2.68 (dd, J = 7.6, 13.6 Hz, 1H), 2.87 (dd, J = 4.4,13.6 Hz, 1H),
3.09–3.19 (m, 2H), 3.60 (s, 2H), 3.64–3.67 (m, 1H), 3.71 (s, 3H), 5.16–5.23
(m, 1H), 5.33–5.37 (m, 1H)
Syntheses of (2E,6E)-3,7-dimethyl-8-(2,3,4,5,6-d5-phenyl
amino)octa-2,6-dien-1-yl acetate 11a and (2E,6E,10E)-3,7,11-
trimethyl-12-(d5-phenylamino)dodeca-2,6,10-trien-1-yl
acetate 11b
LR mass m/z (M + 1) 368.3
14b (CDCl3) d 1.53 (s, 3H), 1.60 (s, 6H), 1.91–2.07 (m, 8H), 2.61
(dd, J = 8.0, 13.6Hz, 1H), 2.81 (dd, J = 4.4,13.6Hz, 1H), 3.06–3.18 (m, 1H),
3.56 (d, J= 5.6Hz, 2H), 3.67 (s, 3H), 5.02 (t, J= 6.8Hz, 1H), 5.16
(t, J= 8.0Hz, 1H), 5.33 (t, J = 7.6Hz, 1H)
A mixture containing aldehyde 10a (1 g, 4.8 mmol), d5-aniline (550 mg,
5.6 mmol) and AcOH (0.3 mL, 315 mg, 5.3 mmol) in DCE (15 mL) was
stirred for 20 min at rt. Then, freshly made NaBH(OAC)3 (2.4 mg,
11.4 mmol) was added in THF (25 mL) and stirred overnight before
quenching with 5% NaHCO3. The reaction mixture was extracted with
CH2Cl2 (100 mL Â 2), and the combined organic extracts were washed
with brine, dried over MgSO4, filtered and concentrated under reduced
pressure. The crude product was purified by silica gel column
chromatography to give compound 11a (980 mg, 70%). Aldehyde 10b
(450 mg ) gave 422 mg of compound 11b (73%).
LR mass m/z (M + H) 436.2
Syntheses of (R)-2-amino-3-(((2E,6E)-3,7-dimethyl-8-(2,3,4,5,6-
d5-phenylamino)octa-2,6-dien-1-yl)thio)propanoic acid 15a
and (R)-2-amino-3-(((2E,6E,10E)-3,7,11-trimethyl-12-(2,3,
4,5,6-d5-phenylamino)dodeca-2,6,10-trien-1-yl)thio)
propanoic acid 15b
d5-AGOAc 11a 1H NMR (CDCl3) d 1.64 (s, 3H), 1.67 (s, 3H), 2.01–2.06
(m, 5H), 2.12–2.17 (m, 2H), 3.61 (s, 2H), 4.55 (d, J = 7.2 Hz, 2H), 5.32–5.32
(m, 1H), 5.34–5.38 (m, 1H)
A mixture of compound 14a (15 mg, 0.041 mmol), LiOH (10 mg,
0.42 mmol) in 1:1 ratio of isopropanol/water (1 mL) was stirred
overnight. The pH of reaction mixture was adjusted to 5 using drops
of 1 N HCl, filtered through a syringe Whatman filter (13 mm CD/X
disposable polypropylene prefilter). This crude product solution was
purified by RP-HPLC (C13 column) using CH3CN/H2O (0.1% TFA) to
give compound 15a (8 mg, 56%). 14b (7.0 mg) gave 3.5 mg of
compound 15b (52%).
15a (CD3OD) d 1.65 (s, 3H), 1.67 (s, 3H), 2.03 (t, J = 7.2 Hz, 2H), 2.12–2.17
(m, 2H), 2.85 (dd, J = 8.8, 14.8 Hz, 1H), 3.26–3.29 (m, 3H), 3.70 (s, 2H),
4.01 (dd, J = 4.4, 8.8 Hz, 1H), 5.18 (t, J = 6.8 Hz, 1H), 5.42 (t, J = 6.8 Hz, 1H)
HR mass m/z (M + H) 354.2267
d5-AFOAc 11b 1H NMR (CDCl3) d 1.56 (s, 3H), 1.63 (s, 3H), 1.67 (s, 3H),
1.94–1.98 (m, 2H), 2.01 (s, 3H), 2.05–2.10 (m, 2H), 3.60 (s, 2H), 4.55
(d, J = 7.2 Hz, 2H), 5.05 (t, J = 6.8 Hz, 1H), 5.31 (t, J = 6.8 Hz, 1H), 5.37
(t, J = 6.8 Hz, 1H)
Syntheses of (2E,6E)-3,7-dimethyl-8-(2,3,4,5,6-d5-phenyl
amino)octa-2,6-dien-1-ol 12a and (2E,6E,10E)-3,7,11-
trimethyl-12-(2,3,4,5,6-d5-phenylamino)dodeca-2,6,10-trien-
1-ol 12b.
A mixture of compound 11a (737 mg, 2.52 mmol), MeOH (8 mL), 15b (CD3OD) d 1.49 (s, 3H), 1.61 (s, 6H), 1.87–2.07 (m, 8H), 2.80 (dd, J = 8.8,
K2CO3 (600 mg) and water (3 mL) was stirred overnight at rt. The
resultant mixture was concentrated under reduced pressure to
remove MeOH and extracted with CH2Cl2 (50 mL Â 2). The combined
organic phases were washed with brine, dried over MgSO4 and
14.8 Hz, 1H), 3.04 (dd, J = 4.4, 15.2 Hz, 1H), 3.10–3.15 (m, 1H), 3.68 (s, 2H),
3.98 (dd, J = 4.4, 8.8 Hz, 1H), 4.99 (t, J = 6.8 Hz, 1H), 5.15 (t, J = 7.6 Hz, 1H),
5.36 (t, J = 7.6 Hz, 1H)
HR mass m/z (M + H) 422.2889
J. Label Compd. Radiopharm 2013, 56 370–375
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

T. Subramanian et al.
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Syntheses of (2E,6E)-3,7-dimethyl-8-(2,3,4,5,6-d5-
phenylamino)octa-2,6-dien-1-yl-diphosphate 16a and (2E,6E,
10E)-3,7,11-trimethyl-12-(d5-phenylamino)dodeca-2,6,10-
trien-1-yl-diphosphate 16b
To a stirred solution of compound 11a (192 mg, 0.77 mmol) in dry CH3CN
(10 mL) at 0ꢀC was added Ph3PBr2 (324 mg, 0.77 mmol). After stirring for
4 h, ((n-Bu)4N)3HP2O7 (2.96 g, 3.02 mmol) was added. Then, the resultant
reaction mixture was stirred for 1 h and concentrated. The residue was
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as for compound 9 to give diphosphate 16a as a white powder
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compound 16b (10% in two steps).
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d5-AGPP 16a 1H NMR (D2O) d 1.36 (s, 3H), 1.42 (s, 3H), 1.78–1.82
(m, 2H), 1.87–1.91 (m, 2H), 3.38 (s, 2H), 4.20 (t, J = 6.0 Hz, 2H), 5.15–5.20
(m, 2H)
31P NMR (D2O) d À6.10(d, J = 22.0 Hz, 1P), À9.39 (d, J = 22.0 Hz, 1P)
HR mass m/z (M À H) 409.6127
d5-AFPP 16b 1H NMR (D2O) d 1.39 (s, 3H), 1.41 (s, 3H), 1.53 (s, 3H), 1.80–
1.96 (m, 8H), 3.46 (s, 2H), 4.28 (t, J = 6.8 Hz, 2H), 4.94 (t, J = 6.4 Hz, 1H), 5.20
(t, J = 6.4 Hz, 1H), 5.27 (t, J = 6.4 Hz, 1H)
31P NMR (D2O) d À9.53 (d, J = 22.6 Hz, 1P), À6.80 (d, J = 22.6 Hz, 1P)
LR mass m/z (M À H) 477.3
SUPPORTING INFORMATION
1
1
The H NMR, 13C NMR, HR mass spectra of 5a-b, H, 13P and HR
mass spectra of 9a, H NMR, HR mass spectra of 13a–b, H, 13P
spectra of 16a–b, and HR and LR mass spectrum of 16a and
16b, respectively, are available.
1
1
Acknowledgements
This work was supported by NIH grant R01 GM66152 to H. P. S.,
R01 GM50388, P20 GM103527 and with resources provided by
the Lexington Veterans Affairs Medical Center to A. J. M.
Conflict of Interest
The authors did not report any conflict of interest.
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proteomics of farnesylated proteins, Proc. Natl. Acad. Sci. U. S. A.
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