Farnesyl diphosphate analogues with ω-bioorthogonal azide and alkyne functional groups for protein farnesyl transferase-catalyzed ligation reactions

Article abstract of DOI:10.1021/jo7017747

(Chemical Equation Presented) Eleven farnesyl diphosphate analogues, which contained ω-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated

Full text of DOI:10.1021/jo7017747

Farnesyl Diphosphate Analogues with ω-Bioorthogonal Azide and
Alkyne Functional Groups for Protein Farnesyl
Transferase-Catalyzed Ligation Reactions
Guillermo R. Labadie,† Rajesh Viswanathan, and C. Dale Poulter*
315 South 1400 East Room 2020, Department of Chemistry, UniVersity of Utah,
Salt Lake City, Utah 84112
poulter@chemistry.utah.edu
ReceiVed August 14, 2007
Eleven farnesyl diphosphate analogues, which contained ω-azide or alkyne substituents suitable for
bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The
analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein farnesyl
transferase. Five of the diphosphates were good alternative substrates for farnesyl diphosphate (FPP).
Steady-state kinetic constants were measured for the active compounds, and the products were characterized
by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very
similar to those of the natural substrate.
Introduction
bound protein contributes to the development of cancer.2,3 Ras
proteins are farnesylated on the cysteine sulfur of their C-
terminal CAAX box, where A is often an aliphatic residue and
X is typically either serine or methionine (alanine, glutamine,
threonine, and, in certain cases, leucine can also serve as the X
residue). Farnesyl transferase inhibitors have been advanced to
clinical trials as anticancer agents.4,5
Recent reports suggest that the nuclear blebbing seen in cells
from patients with Hutchinson-Gilford progeria syndrome
(HGPS) results from a mutation that prevents maturation of
lamin A by blocking proteolytic cleavage of a farnesylated
C-terminal 15 amino acid peptide.6,7 Protein prenylation has also
been identified as a target for antiparasitic agents.8,9 Protein
Post-translational modification of proteins to append iso-
prenoid chains to enhance their association with membranes is
required in a variety of important biological processes, including
signal transduction pathways controlling cell growth and dif-
ferentiation, cytoskeletal rearrangement, membrane rearrange-
ment during cellular division, vision, and vesicular transport.
Approximately 1% of mammalian proteins is modified at a
C-terminal cysteine residue by C15 farnesyl or C20 geranylgera-
nyl groups.1 Among the prenylated proteins that have been
identified are nuclear lamins, the γ-subunit of heterotrimeric
small G proteins such as transducin, the Ras super family of
small G proteins, and enzymes such as protein tyrosine
phosphatases, inositol polyphosphatases, and phospholipase A.
(2) Shields, J. M.; Pruitt, K.; McFall, A.; Shaub, A.; Der, C. J. Trends
Cell. Biol. 2000, 10, 147-154.
(3) Sebti, S. M.; Hamilton, A. D. Oncogene 2000, 19, 6584-6593.
(4) Bell, I. M. J. Med. Chem. 2004, 47, 1869-1878.
(5) Appels, N. M.; Beijnen, J. H.; Schellens, J. H. Oncologist 2005, 10,
565-578.
(6) Young, S. G.; Meta, M.; Yang, S. H.; Fong, L. G. J. Biol. Chem.
2006, 281, 39741-39745.
(7) Meta, M.; Yang, S. H.; Bergo, M. O.; Fong, L. G.; Young, S. G.
Trends Mol. Med. 2006, 12, 480-487.
Several prenylated proteins are implicated in human diseases.
Ras proteins function as an on/off switch to regulate a variety
of cellular functions, including proliferation. In approximately
30% of human cancers, mutations in Ras compromise its ability
to hydrolyze GTP to GDP, and the persistently active GTP-
(8) Buckner, F. S.; Eastman, R. T.; Yokoyama, K.; Gelb, M. H.; Van
Voorhis, W. C. Curr. Opin. InVest. Drugs 2005, 6, 791-797.
(9) Gelb, M. H.; Van Voorhis, W. C.; Buckner, F. S.; Yokoyama, K.;
Eastman, R.; Carpenter, E. P.; Panethymitaki, C.; Brown, K. A.; Smith, D.
F. Mol. Biochem. Parasitol. 2003, 126, 155-163.
* Corresponding author. Tel.: (801) 581-6685; fax: (801) 581-4391.
† Present address: IQUIOS-CONICET, Universidad Nacional de Rosario,
Suipacha 531, 2000, Rosario, Santa Fe, Argentina.
(1) Ashar, H. R.; James, L.; Gray, K.; Carr, D.; Black, S.; Armstrong,
L.; Bishop, W. R.; Kirschmeier, P. J. Biol. Chem. 2000, 275, 30451-30457.
10.1021/jo7017747 CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/03/2007
J. Org. Chem. 2007, 72, 9291-9297
9291

Labadie et al.
farnesyl transferase (PFTase) inhibitors have shown a high
potency against the parasites responsible for malaria (Plasmo-
dium falciparum)10,11 and the Chagas’ disease parasite Trypa-
nosoma cruzi.12,13
The modification reactions are catalyzed by three different
protein prenyl transferases: PFTase (EC 2.5.1.58), protein
geranylgeranyltransferase-I (PGGTase-I, EC 2.5.1.59), and Rab
(a Ras-like protein) geranylgeranyltransferase (RabGGTase or
PGGTase-II, EC 2.5.1.60). The closely related PFTase and
PGGTase-I transfer prenyl groups from prenyl diphosphates to
proteins containing a C-terminal CAAX motif (also known as
a CAAX box), where C is cysteine, A is usually a small aliphatic
amino acid, and X can be a variety of amino acids (Figure 1).
moiety were developed by the Distefano and Spielmann groups,
respectively.19-21 FPP analogues appended with fluorescent
anthranilate esters were developed by Waldmann and co-
workers.22,23 Wiemer and co-workers reported the synthesis of
N-alkylated derivatives of GPP as fluorescent labels that are
resistant to esterases.24 Those compounds were subsequently
effectively transferred to peptides and proteins. Distefano and
co-workers recently reported that alkynyl ether derivatives of
GPP are alternative substrates that can be tethered to other
biomolecules after farnesylation.25 Prestwich and Liu synthe-
sized a conjugated geranylgeranyl diphosphate (GGPP) deriva-
tive (∆∆GG) with a conjugated olefinic fluorophore.26
Incorporation of a bioorthogonal functional group27,28 into
the FPP structure provides a technique for modifying proteins
for subsequent tethering and analysis. We focused our attention
on the Staudinger ligation and the Cu(I)-catalyzed Huisgen
cycloaddition (click reaction), both of which have been used in
vivo. A version of the Staudinger ligation, introduced by
Bertozzi and Saxon,29 involves intramolecular trapping of a
phosphine/azide adduct eventually to give a stable amide
linkage. The click ligation, introduced by Sharpless and cowork-
ers,30,31 is a Cu(I)-catalyzed [2 + 3] cycloaddition reaction
between azide and terminal alkyne to produce a 1,2,3-triazole.
We recently reported that proteins derivatized with suitably
functionalized analogues of FPP could be readily immobilized
on glass slides.32 Related approaches were recently reported from
the laboratories of Distefano and coworkers33 and Zhao and
coworkers34 We now report the synthesis of a family of azido-
and alkyne-substituted FPP analogues and their ability to
function as alternative substrates for yeast PFTase.
Results and Discussion
FIGURE 1. Reactions catalyzed by PFTase and PGGTase-I.
Synthesis. We designed a series of azido and alkyne
analogues as reagents with which to modify proteins for Huisgen
and Staudinger ligations (Figure 2). All of the analogues have
The X residue determines whether a farnesyl (X ) A, S, C, M,
Q) or a geranylgeranyl (X ) L, F) is added.14
The rational design of farnesyl diphosphate (FPP) analogues
for PFTase with specific functions has been facilitated by the
X-ray crystal structures for rat15 and human16 PFTases. Spiel-
mann and co-workers reported that incorporation of an aniline
moiety at the location of the ω-isoprene unit of FPP resulted in
a transferable analogue.17 Recently, successful incorporation of
this aniline-geranyl diphosphate (GPP) (AGPP) analogue was
monitored in HEK-293 cells by the use of antibodies raised
against the FPP analogue.18 Photoaffinity analogues of FPP
incorporating a benzophenone moiety or a functionalized aniline
(18) Troutman, J. M.; Roberts, M. J.; Andres, D. A.; Spielmann, H. P.
Bioconjugate Chem. 2005, 16, 1209-1217.
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istry 2003, 42, 3716-3724.
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Jefferies, M.; Xie, W.; Shintani, D. K.; McMahan, C. M.; Cornish, K.;
Distefano, M. D. J. Org. Chem. 2007, 72, 4587-4595.
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K. E.; Fierke, C. A.; Andres, D. A.; Spielmann, H. P. J. Am. Chem. Soc.
2002, 124, 8206-8219.
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R. S.; Waldmann, H. Angew. Chem., Int. Ed. 1999, 38, 509-512.
(23) Kuhn, K.; Owen, D. J.; Bader, B.; Wittinghofer, A.; Kuhlmann, J.;
Waldmann, H. J. Am. Chem. Soc. 2001, 123, 1023-1035.
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J.; Wiemer, D. F. J. Org. Chem. 2004, 69, 8186-8193.
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ChemBioChem 2007, 8, 98-105.
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Overkleeft, H. S. Org. Biomol. Chem. 2005, 3, 20-27.
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(29) Saxon, E.; Bertozzi, C. R. Science (Washington, DC, U.S.) 2000,
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(30) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
(31) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193.
(32) Gauchet, C.; Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006,
128, 9274-9275.
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Bioconjugate Chem. 2006, 17, 967-974.
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J.; Jaunbergs, J.; Weinbaum, C.; Tamanoi, F.; Falck, J.; Zhao, Y. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 12479-12484.
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Pusateri, E. E.; Fletcher, S.; Cummings, C. G.; Buckner, F. S.; Pendyala,
P. R.; Chakrabarti, D.; Sebti, S. M.; Gelb, M. H.; Van Voorhis, W. C.;
Hamilton, A. D. J. Med. Chem. 2006, 49, 5710-5727.
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Horney, C. P.; Pendyala, P. R.; Floyd, D.; Lombardo, L. J.; Williams, D.
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Chakrabarti, D.; Gelb, M. H.; Van Voorhis, W. C. J. Med. Chem. 2005,
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Klebe, G.; Schlitzer, M. J. Med. Chem. 2005, 48, 7186-7191.
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Farnesyl Diphosphate Analogues with Functional Groups
FIGURE 2. Structures of azido and alkyne FPP analogues.
SCHEME 1. Synthesis of Aldehydes 13-OAc and 13-OTBS
SCHEME 2. Synthesis of Alcohols 15-OAc and 16-OAc and
Aldehyde 17-OAc
a trisubstituted allylic diphosphate moiety in the first isoprene
unit, a requirement for all prenyl transferase reactions. Azido-
substituted analogues 1-OPP and 4-OPP contain a farnesyl unit,
while 3-OPP, 5-OPP, and 6-OPP contain a geranyl unit.
Alkyne-substituted analogues include 7-OPP and 9-OPP with
a propargyl ether moiety and 8-OPP and 10-OPP with propargyl
amine. 11-OPP contains an amide-linked alkyne unit. Analogues
1-OPP, 2-OPP, and 4-OPP are derivatives of FPP, while the
remaining compounds are derivatives of GPP substituted at the
E-ω-methyl group. In general, the analogues were prepared from
geranyl or farnesyl acetate by oxidation of the isoprenoid chain
followed by subsequent modification of the oxidized carbon.
Aldehyde 13-OAc was obtained from geranyl acetate (12-
OAc) by oxidation with SeO2/EtOH followed by hydrolysis as
shown in Scheme 1.26 Protection of the hydroxyl group with
TBSCl gave 13-OTBS. A similar oxidation of farnesyl acetate
(14-OAc) by tBuOOH/SeO2 gave 8-hydroxy- and 12-hydroxy-
farnesyl acetate (15-OAc and 16-OAc, respectively) in an
overall yield of 40% along with 33% of unreacted farnesyl
acetate (Scheme 2).35,36 Alcohol 15-OAc was oxidized to
aldehyde 17-OAc by treatment with MnO2 under basic condi-
tions that minimized isomerization of the ω-double bond.37
Treatment of 14-OAc with NBS followed by K2CO3/MeOH
gave the ω-epoxide but hydrolyzed the acetate group, which
(35) Li, J.; Lan, J.; Liu, Z.; Li, Y. J. Nat. Prod. 1998, 61, 92-95.
(36) Li, Y.; Li, W.; Li, Y. J. Chem. Soc., Perkin Trans. 1 1993, 2953-
2956.
(37) Xiao, X. Y.; Prestwich, G. D. Synth. Commun. 1990, 20, 3125-
3130.
J. Org. Chem, Vol. 72, No. 24, 2007 9293

Labadie et al.
SCHEME 4. Synthesis of Azide Analogues 1-OPP and
SCHEME 3. Synthesis of Aldehydes 19-OAc and 19-OTBS
2-OPP
was subsequently reintroduced under standard conditions.38
Epoxy acetate 18-OAc was treated with periodic acid39 to give
aldehyde 19-OAc. This aldehyde was converted to silyl-
protected 19-OTBS by hydrolysis and treatment with TBS
chloride (Scheme 3).
Syntheses of azido analogues 1-OPP and 2-OPP are outlined
in Scheme 4. Allylic alcohols 15-OAc and 16-OAc were
converted to the corresponding azides 20-OAc and 21-OAc,
respectively, by treatment with diphenylphosphorylazidate
(DPPA) according to the procedure of Thompson and co-
workers.40 The acetate group was then hydrolyzed, and the
resulting alcohols were phosphorylated as described by Davisson
et al.41,42 to give diphosphates 1-OPP and 2-OPP. 1-OPP and
2-OPP were obtained as 1:1 and 2:1 mixtures of their allylic
isomers, respectively. Allylic azides undergo a 1,3 rearrangement
at room temperature,43 and isomerization is seen when azides
are synthesized from allylic alcohols.44,45
SCHEME 5. Synthesis of Azide Analogue 3-OPP
The synthesis of azido analogue 3-OPP is shown in Scheme
5. Aldehyde 19-OAc was reduced with sodium borohydride to
give alcohol 22-OAc. When 22-OAc was treated with DPPA,
the phosphate intermediate was not displaced by azide. We then
resorted to Hu’s method,46 which uses a more reactive 2,4-
dichlorophenylphosphate leaving group. When alcohol 22-OAc
was treated with bis-(2,4-dichlorophenyl)phosphoryl chloride,
DMAP, and sodium azide, compound 3-OAc was obtained in
high yield. The acetate was hydrolyzed with K2CO3, and alcohol
3-OH was phosphorylated as described for 1-OPP.
Benzyl azido analogues 4-OPP, 5-OPP, and 6-OPP were
prepared by the four-step sequence shown in Scheme 6.
Reductive amination of aldehydes 19-OAc, 17-OAc, and 13-
OAc by treatment with 3-aminobenzyl alcohol and sodium
triacetoxyborohydride21,24,47,48 gave good yields of amino al-
cohols 23-OAc, 24-OAc, and 25-OAc, respectively. The
benzylic hydroxyl groups were displaced by azide using the
Thompson procedure,40 followed by acetate hydrolysis and
phosphorylation to provide diphosphates 4-OPP, 5-OPP, and
6-OPP. Diphosphates 5-OPP and 6-OPP were purified by
chromatography on cellulose and characterized. Diphosphate
4-OPP was unstable under these conditions. While we were
able to obtain an exact mass for the compound by HRMS, NMR
spectra were not suitable for characterization.
(38) Hanzlik, R. P. Org. Synth. 1977, 56, 112-117.
(39) Zoretic, P. A.; Fang, H.; Ribeiro, A. A. J. Org. Chem. 1998, 63,
7213-7217.
(40) Thompson, A. S.; Humphrey, G. R.; DeMarco, A. M.; Mathre, D.
J.; Grabowski, E. J. J. J. Org. Chem. 1993, 58, 5886-5888.
(41) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Muehlbacher, M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-4779.
(42) Davisson, V. J.; Woodside, A. B.; Poulter, C. D. Methods Enzymol.
1985, 110, 130-144.
(43) Gagneux, A.; Winstein, S.; Young, W. G. J. Am. Chem. Soc. 1960,
82, 5956-5957.
(44) Kale, T. A.; Distefano, M. D. Org. Lett. 2003, 5, 609-612.
(45) Feldman, A. K.; Colasson, B.; Sharpless, K. B.; Fokin, V. V. J.
Am. Chem. Soc. 2005, 127, 13444-13445.
(47) Abdel-Magid, A. F.; Maryanoff, C. A. Synlett 1990, 537-539.
(48) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. J. Org. Chem. 1996, 61, 3849-3862.
(46) Yu, C.; Liu, B.; Hu, L. Org. Lett. 2000, 2, 1959-1961.
9294 J. Org. Chem., Vol. 72, No. 24, 2007

Farnesyl Diphosphate Analogues with Functional Groups
SCHEME 6. Synthesis of Azide Analogues 4-OPP, 5-OPP,
and 6-OPP
SCHEME 8. Synthesis of Alkynyl Analogues 8-OPP and
10-OPP
SCHEME 9. Synthesis of Alkynyl Analogue 11-OPP
SCHEME 7. Synthesis of Alkynyl Analogues 7-OPP and
9-OPP
alcohols, gave modest to good yields of diphosphates 8-OPP
and 10-OPP.
Amide analogue 11-OPP was synthesized from alcohol 26-
OTBS as outlined in Scheme 9. The hydroxyl group was
replaced with a phthalimide moiety using Mitsunobu conditions
to give 28-OTBS,49,50 which was then treated with hydrazine
hydrate followed by propiolic acid in the presence of 1,2-
dihydro-2-ethoxy-1-quinolinecarboxylic acid (EEDQ)51 to give
11-OTBS. The TBS group was removed with acetic acid in
THF/H2O, and the resulting alcohol was phosphorylated to give
11-OPP in moderate yield.
Kinetic Studies with Yeast PFTase. Recombinant yeast
PFTase from an Escherichia coli clone was purified by affinity
chromatography on a Ni-NTA column as previously reported.52
Details of the enzymatic assays are provided in the Supporting
Propargyl ethers 7-OPP and 9-OPP were synthesized from
aldehydes 13-OTBS and 19-OTBS as outlined in Scheme 7.
The aldehydes were reduced with sodium borohydride to give
alcohols 26-OTBS and 27-OTBS, respectively. Propargyl ether
7-OTBS was prepared by treatment with sodium hydride
followed by the addition of propargyl bromide. The same
procedure gave low yields of 9-OTBS from 27-OTBS. After
several attempts, the best yield of the propargyl ether was
obtained when potassium hydride was used to deprotonate the
alcohol. Half of the starting alcohol was recovered despite using
rigorously anhydrous conditions and longer reaction times. The
TBS groups were then removed with tetrabutylammonium
fluoride (TBAF), and the resulting alcohols were phosphorylated
as described for the azido analogues to give ethers 7-OPP and
9-OPP.
(49) Tago, K.; Minami, E.; Masuda, K.; Akiyama, T.; Kogen, H. Bioorg.
Med. Chem. 2001, 9, 1781-1791.
Propargyl amines 8-OPP and 10-OPP were prepared by
reductive amination of aldehydes 13-OAc and 19-OAc with
propargyl amine using conditions described previously for 23-
OAc, 24-OAc, and 25-OAc (Scheme 8). Hydrolysis of the
acetate groups, followed by phosphorylation of the resulting
(50) Turek, T. C.; Gaon, I.; Gamache, D.; Distefano, M. D. Bioorg. Med.
Chem. Lett. 1997, 7, 2125-2130.
(51) Okonya, J. F.; Kolasa, T.; Miller, M. J. J. Org. Chem. 1995, 60,
1932-1935.
(52) Harris, C. M.; Derdowski, A. M.; Poulter, C. D. Biochemistry 2002,
41, 10554-10562.
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Labadie et al.
TABLE 1. Steady-State Kinetic Parameters of Active Substrates
a
compound
kcat (s-1)
Km (µM)
1.71 ( 0.04
2.12 ( 0.15
0.67 ( 0.10
1.05 ( 0.22
4.23 ( 0.47
kcat/Km (s-1 µM-1)
(kcat/Km)rel
FPP
1.31 ( 0.04
0.45 ( 0.01b
1.10 ( 0.05
0.35 ( 0.01
0.48 ( 0.01
0.77
0.21
1.6
0.33
0.11
0.55
1
0.28
2.1
0.43
0.15
0.72
1-OPP
3-OPP
5-OPP
6-OPP
9-OPP
1.06 ( 0.03
1.90 ( 0.22
a Vrel refers to kcat/Km with respect to FPP. b Rate corresponds to the incorporation of both isomers as was shown by HPLC.
SCHEME 10. 1-OPP Azide Equilibrium
Information. Preliminary studies indicated that analogues 1-OPP,
3-OPP, 5-OPP, 6-OPP, and 9-OPP were good to excellent
alternative substrates for the enzyme. The other analogues were
less active and were not characterized further.
Steady-state kinetic parameters for the most active analogues
with dansyl-GCVIA (Dn-GCVIA) as a cosubstrate are sum-
marized in Table 1. Two other groups34,53 reported that 1-OPP
was a substrate for PFTase, although a full set of kinetic
parameters was not presented. We found that the Vmax value
for the 1:1 mixture of 1-OPP and its allylic isomer (1i-OPP,
Scheme 10) was lower than for FPP, although their Km values
are similar. The rapid interconversion of the ω-allylic azides
(see Scheme 10) prevents us from obtaining individual kinetic
constants for the two isomers. The terminal alkyl azide, 3-OPP,
is an excellent substrate with a kcat value almost equal to that
of FPP and a Km value that is substantially lower, resulting in
a 2.1-fold enhancement in catalytic efficiency (kcat/Km) over the
natural substrate. Clearly, the linear carbon chain in 3-OPP
terminated by an azide moiety is a good surrogate for the
ω-prenyl unit in FPP. A similar observation was recently
reported by Nguyen et. al for an azidopropyl ether analogue of
FPP.54
SCHEME 11. Farnesylation of DansylGCVIA with FPP
Analogues
Our results for 5-OPP are similar to those reported by the
Spielmann group for a related p-substituted nitroaniline deriva-
tive with rat PFTase, where (kcat/Km)rel was 0.42.21 The catalytic
efficiency of analogue 6-OPP, with a substantially larger volume
in the region of the ω-isoprene unit than 3-OPP, was 14-fold
lower as the result of both a smaller kcat and a larger Km.
Although phenyl groups are used as surrogates for the prenyl
moiety,55 we found that the analogues with linear side chains
were better alternative substrates. Analogue 9-OPP was the only
propargyl derivative with a good kinetic profile, with a catalytic
efficiency of 72% of FPP.
Analogues 2-OPP, 4-OPP, 7-OPP, 8-OPP, 10-OPP, and 11-
OPP were substantially less reactive or inactive with yeast
PFTase in our assay. Most likely, these compounds are less
compatible with the active site of the enzyme because of
branching along the chain or bulk in the chain at the location
of the ω-isoprenoid unit in FPP.15,16,56 We were somewhat
surprised that propargyl ether 7-OPP derived from a geranyl
scaffold was a poor substrate in view of a previous report that
related that benzyl ethers were good analogues for human
PFTase.55 Recently, Distefano and coworkers used 7-OPP as a
substrate to tag proteins, although the rate of farnesylation by
the analogue in their assay was poor (∼0.08 of the rate with
FPP under the same conditions).25 The lack of activity of
propargylamino analogues 8-OPP and 10-OPP is understand-
able given the poor behavior of 7-OPP and the presence of a
charged amino group in the isoprenoid chain at the pH of the
assay. Both compounds share the same structure except for the
heteroatom. Propiolamide analogue 11-OPP is also not active.
In addition to being constructed on a geranyl scaffold, the
rigidity imposed by the conjugation of the propiolamide may
be incompatible with the active site of PFTase. The same
behavior has been seen observed for a pentaene analogue of
FPP that is not a good substrate for PFTase.57 On the other
hand, the R-azido acetamide analogue reported by Waldmann
and coworkers,54 which does not have restricted rotation, is
transferred 37 times more slower that the natural substrate,
suggesting that the alkylamide moiety is also not optimal.
Characterization of Prenylated Peptides by HPLC and
LC-MS. The products from prenylation of Dn-GCVIA by each
of our five active analogues were established using procedures
similar to those previously reported (see Supporting Informa-
tion).58 The modified peptides (see Scheme 11) were character-
ized by HPLC and LC-MS. In each case, the HPLC retention
time and the mass of the prenylated peptide (Table 2) were
(53) Rose, M. W.; Rose, N. D.; Boggs, J.; Lenevich, S.; Xu, J.; Barany,
G.; Distefano, M. D. J. Peptide Res. 2005, 65, 529-537.
(54) Nguyen, U. T. T.; Cramer, J.; Gomis, J.; Reents, R.; Gutierrez-
Rodriguez, M.; Goody, R. G.; Alexandrov, K.; Waldmann, H. ChemBio-
Chem 2007, 8, 408-423.
(55) Micali, E.; Chehade, K. A.; Isaacs, R. J.; Andres, D. A.; Spielmann,
H. P. Biochemistry 2001, 40, 12254-12265.
(56) Dunten, P.; Kammlott, U.; Crowther, R.; Weber, D.; Palermo, R.;
Birktoft, J. Biochemistry 1998, 37, 7907-7912.
(57) Liu, X.-H.; Prestwich, G. D. Bioorg. Med. Chem. Lett. 2004, 14,
2137-2140.
(58) Dolence, J. M.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 5008-5011.
9296 J. Org. Chem., Vol. 72, No. 24, 2007

Farnesyl Diphosphate Analogues with Functional Groups
1738, 1235. HRMS (CI, M + H+) Calcd for C14H25O3 241.1804;
found 241.1800.
TABLE 2. HPLC Retention Times for Dn-GCVIA and
Dn-GC(Far)VIA Peptides and LC-MS Molecular Ions for the
Products
Acetoxydecadienyl Azide 3-OAc. Sodium azide (974 mg, 14.98
equiv), DMAP (687 mg, 5.62 equiv), and bis-(2,4-dichlorophenyl)-
chlorophosphonate (1.98 g, 4.87 equiv) were added to a stirred
solution of alcohol 22-OAc (900 mg, 3.75 equiv) in DMF (20 mL)
at room temperature (rt). The reaction mixture was allowed to stir
overnight at rt before 50 mL of ethyl ether and 50 mL of brine
were added. The layers were allowed to separate. The aqueous layer
was diluted with 100 mL of brine and extracted with ethyl ether (2
× 50 mL). The combined ether extracts were washed with water
and dried over MgSO4. The solution was filtered and concentrated,
and the residue was chromatographed to give 933 mg (94%) of
compound
retention time (min)
LC-MS (M+ + H)
Dn-GCVIA
4.9
19.3
17.5
14.4
14.8
16.46
695.3
940.5
900.3
977.4
1005.5
913.6
29
30
31
32
33
consistent with alkylation of the cysteine residue in Dn-GCVIA
by the appropriate analogue. Interestingly, the LC-MS trace for
29 had two closely spaced peaks with the same mass. This
suggests that both regioisomers of 1-OPP were incorporated
into the peptide or that a single isomer, which subsequently
rearranged to its allylic isomer, was incorporated.53
1
3-OAc as a colorless oil; H NMR (CDCl3, δ): 5.34 (t, J ) 7.2
Hz, 1H), 5.13 (t, J ) 6.3 Hz, 1H), 4.58 (d, J ) 7.2 Hz, 2H), 3.23
(t, J ) 6.6 Hz, 2H), 2.16-2.00 (m, 6H), 2.06 (s, 3H), 1.70 (s, 3H),
1.68 (m, 2H), 1.60 (s, 3H). 13C NMR (CDCl3, δ): 171.3, 142.1,
134.0, 125.1, 118.6, 61.5, 51.0, 39.5, 36.6, 27.1, 26.2, 21.2, 16.6,
16.0; IR (neat): 2940, 2098, 1741, 1232. HRMS (CI, M + H+
N2) Calcd for C14H24NO2 238.1807; found 238.1812.
-
Conclusion
Decadienol Azide 3-OH. Following the general procedure,
acetate 3-OAc (1.04 g, 3.92 mmol) was treated with K2CO3 (1.63
g, 11.76 mmol) in 40 mL of MeOH. Workup followed by
purification via column chromatography gave 876 mg (100%) of
A series of 11 FPP analogues containing biologically
orthogonal functional groups as substrates for the prenylation
of cysteine residues in C-terminal CAAX recognition sequences
by yeast PFTase was synthesized. Five of these were alternative
substrates. Four of the alternate substrates were azides (1-OPP,
3-OPP, 5-OPP, and 6-OPP), and one was an alkyne (9-OPP).
Two of the compounds, azide 3-OPP and alkyne 9-OPP, were
excellent alternative substrates with respective catalytic efficien-
cies (V/K) that were 210 and 72% of V/K for FPP. We used
3-OPP and 9-OPP to regioselectively modify recombinant
versions of green fluorescent protein and glutathione S-trans-
ferase that contained a genetically engineered C-terminal CAAX
recognition motif.32 The modified proteins were subsequently
covalently attached to glass slides derivatized with complemen-
tary functional groups using azide/alkyne cycloaddition and the
Staudinger ligation. This technology offers a promise for the
attachment of any soluble protein with a C-terminal CAAX
motif to a wide variety of substrates.
1
3-OH as a light yellow oil; H NMR (CDCl3, δ): 5.41 (dt, J1 )
6.9 Hz, J2 ) 0.9 Hz, 1H); 5.14 (t, J ) 6.6 Hz, 1H); 4.16 (d, J )
6.6 Hz, 1H); 3.23 (t, J ) 6.9 Hz, 1H); 2.20-2.00 (m, 6H); 1.7 (m,
2H); 1.68 (s, 3H); 1.60 (s, 3H); 13C NMR (CDCl3, δ): 139.4, 133.8,
125.2, 123.6, 59.3, 50.9, 39.5, 36.5, 26.9, 26.2, 16.3, 15.9; IR
(neat): 3339, 2938, 2877, 2100, 1448, 1291, 1258. HRMS (CI, M
+ H+ - N2) Calcd for C12H22NO 196.1701; found 196.1683.
Decadienyl Azide Diphosphate 3-OPP. Using the standard
phosphorylation protocol, alcohol 3-OH (124 mg, 0.56 mmol) was
converted to the corresponding chloride using NCS (83 mg, 0.62
mmol) and dimethylsulfide (49 µL, 0.67 mmol). The chloride was
then treated with (NBu4)3HP2O7·3H2O (1.65 g, 1.68 mmol), and
the product was purified as described to give 127 mg (53%) of a
white solid; 1H NMR (D2O, δ): 5.40 (t, J ) 7.2 Hz, 1H); 5.19 (t,
J ) 6.6 Hz, 1H); 4.40 (t, J ) 6.6 Hz, 2H); 3.22 (t, J ) 6.9 Hz,
2H); 2.20-1.90 (m, 6H); 1.66 (s, 3H); 1.62 (m, 2H); 1.56 (s, 3H);
13C NMR (D2O, δ): δ 142.4, 134.7, 125.6, 120.7 (J ) 9.0 Hz),
62.8 (J ) 5.0 Hz), 51.1, 39.7, 36.8, 27.1, 26.6, 16.3, 15.8; 31P NMR
(D2O, δ): 6.59 (d, J ) 22.6 Hz, 1P), -10.15 (d, J ) 22.6 Hz, 1P);
HRMS (CI) Calcd for C12H22N3O7P2 382.0938, found 382.0937.
Experimental Section
Acetoxydecadienyl Alcohol 22-OAc. NaBH4 (120 mg, 3.17
mmol) was added in small portions to a solution of aldehyde 19-
OAc (630 mg, 2.65 mmol) in MeOH (30 mL) at -10 °C. The
mixture was allowed to stir at -10 °C for 2 h before ice-cold water
(50 mL) was added. The solvent was removed at reduced pressure.
The aqueous residue was saturated with solid NaCl and extracted
with ether (4 × 15 mL). The combined ether extracts were dried
over Na2SO4, and the solvent was removed. The residue was
chromatographed to give 495 mg (78%) of a colorless oil; 1H NMR
(CDCl3, δ): 5.34 (d, J ) 7.2 Hz, 1H); 5.13 (d, J ) 6.3 Hz, 1H);
4.59 (d, J ) 6.9 Hz, 2H); 3.63 (t, J ) 6.3 Hz, 2H); 2.20-2.00 (m,
6H); 2.06 (s, 3H); 1.70 (s, 3H); 1.65 (m, 2H); 1.61 (s, 3H); 13C
NMR (CDCl3, δ): 171.4, 142.2, 135.3, 124.2, 118.6, 62.7, 61.6,
39.6, 36.0, 30.8, 26.1, 21.2, 16.5, 16.0; IR (neat): 3444, 2938, 2883,
Acknowledgment. This work was supported by NIH Grant
GM21328.
Supporting Information Available: General methods; experi-
mental protocols for the synthesis of diphosphates 2-OPP-11-OPP
including all intermediates; 1H, 13C, and 31P NMR spectra for
2-OPP, 3-OPP, 5-OPP, 6-OPP, and 9-OPP-11-OPP, including
intermediates, and intermediates for 4-OPP; protocols for enzymatic
assays and protocols; and chromatogram and spectra for HPLC and
LC-MS analyses. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO7017747
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