Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs

Article abstract of DOI:10.1016/j.bmcl.2016.06.035

Protein prenylation is a type of post-translational modification that aids certain proteins in localizing to the plasma member where they activate cell signaling. To better understand the isoprenoid requirements and differences of FTase and GGTase-I, a series of saturated geranylgeranyl diphosphate analogs were synthesized and screened against both mammalian FTase and GGTase-I. Of our library of compounds, several analogs proved to be substrates of GGTase-I, with 11d having a k_rel?=?0.95 when compared to GGPP (k_rel?=?1.0).

Full text of DOI:10.1016/j.bmcl.2016.06.035

Accepted Manuscript
Exploration of GGTase-I substrate requirements. Part 2. Synthesis and bio-
chemical analysis of novel saturated geranylgeranyl diphosphate analogs
Kayla J. Temple, Elia N. Wright, Carol A. Fierke, Richard A. Gibbs
PII:
S0960-894X(16)30646-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.035
BMCL 23988
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
9 April 2016
11 June 2016
13 June 2016
Please cite this article as: Temple, K.J., Wright, E.N., Fierke, C.A., Gibbs, R.A., Exploration of GGTase-I substrate
requirements. Part 2. Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.035
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Graphical Abstract
Exploration of GGTase-I substrate
requirements. Part 2. Synthesis and
biochemical analysis of novel saturated
geranylgeranyl diphosphate analogs
Kayla J. Temple, Elia N. Wright, Carol A. Fierke, Richard A. Gibbs

Bioorganic & Medicinal Chemistry Letters
Exploration of GGTase-I substrate requirements. Part 2. Synthesis and
biochemical analysis of novel saturated geranylgeranyl diphosphate analogs
Kayla J. Templeab, Elia N. Wrightc, Carol A. Fierkecd, and Richard A. Gibbsab
aDepartment of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
bThe Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47909, USA.
cDepartment of Biological Chemistry, and dDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Protein prenylation is a type of post-translational modification that aids certain proteins in
localizing to the plasma member where they activate cell signaling. To better understand the
isoprenoid requirements and differences of FTase and GGTase-I, a series of saturated
geranylgeranyl diphosphate analogs were synthesized and screened against both mammalian
FTase and GGTase-I. Of our library of compounds, several analogs proved to be substrates of
GGTase-I, with 11d having a krel = 0.95 when compared to GGPP (krel = 1.0).
Keywords:
Protein prenylation
Saturated GGPP analogs
Farnesylation
2016 Elsevier Ltd. All rights reserved.
Geranylgeranylation
Isoprenoid Synthesis
Classified as a type of lipidation, protein prenylation
substrate of FTase into an inhibitor.12 It is still unclear whether
the other isoprene units are required for substrate activity.
occurs on a cysteine residue located four residues from the C-
terminus. Prenylated proteins contain a C-terminal motif known
as a “CaaX box”, where ‘C’ denotes cysteine, ‘a’ is typically an
aliphatic amino acid, and ‘X’ represents a small subset of amino
acid residues.1 This “CaaX” motif allows proteins to be
recognized by prenyl transferases located in the cytosol. The
prenyl transferase enzymes (PTases) catalyze the formation of a
thioether linkage between the cysteine residue of the CaaX box
and isoprenyl lipids (namely farnesyl pyrophosphate, FPP; or
geranylgeranyl pyrophosphate, GGPP).2 Farnesyl transferase
(FTase) catalyzes the covalent attachment of a 15-carbon
FPPwhile geranylgeranyl transferase-I (GGTase-I) catalyzes the
3,4
attachment of a 20-carbon GGPP to cysteine (Figure 1).
This
covalent attachment then triggers the protein to relocate to the
endoplasmic reticulum where it is proteolytically cleaved by Rce-
1 followed by methyl esterification by Icmt.1,5,6,7 The newly
modified protein is able to anchor into membranes where it
regulates various important cellular functions such as cell
signaling (Ras & others), cell division (CENP-E & CENP-F), and
organelle structure (nuclear lamins).8
Figure 1. Protein prenylation pathway of Ras and conventional method of
isoprene unit labeling (gray box).
In the past, there have been several inhibitors of
GGTase-I that contain long saturated hydrocarbon chains with
some of these inhibitors displaying submicromolar IC50’s.13 Such
compounds led us to question whether the α, β, γ, and ω isoprene
units are essential for enzyme activity. To address the query, we
synthesized a variety of diphosphate analogs in which one or
more of the isoprene units were replaced with saturated
hydrocarbon chains. Our goal was to select analogs that ranged
In the past, several other groups have investigated
unnatural isoprenoid pyrophosphates as FPP mimics; however,
these studies did not explore the importance of each individual
isoprene unit.9-11 A previous study by our laboratory indicates
that the α-isoprene is critical for substrate activity. In fact, by
adding one additional methylene between the double bond of the
first isoprene and the pyrophosphate of FPP turns the native
———
 Corresponding author. Tel.: +1-615-322-8662; e-mail: kayla.temple@vanderbilt.edu

in chain length between FPP (12 carbons long) and GGPP (16
carbons long).
To determine if the α-isoprene is sufficient to produce
substrate activity, the first set of analogs synthesized replaced the
β, γ, and ω isoprene units with aliphatic chains (compounds 3a-
c). Next, analogs that contain only the α and β isoprene units
were synthesized (compounds 11a-d), replacing the ω isoprene of
FPP and the γ and ω isoprene units of GGPP. These analogs will
determine if the first two isoprene units are sufficient for
substrate activity. Subsequently, the ω isoprene unit was re-
installed to obtain compounds that lack the internal β and γ
isoprene units (compounds 23a-b). These compounds will
examine the importance of the central isoprene units and whether
they are required for catalysis. The final two compounds
maintain the methylene-branching of isoprenoids but lack sp2
character to generate hydro-GGPP derivatives. The dihydro-
GGPP (38) analog lacks the ω double bond but retains the
methylene unit at the 15 position while the tetrahydro-GGPP (31)
analog lacks both the γ and ω double bonds but retains the
methylene units at the 11 and 15 positions (Schemes 4 & 5).
Scheme 1. Synthesis of α-containing pyrophosphates. (a) i. Me3Al,
Cp2ZrCl2, DCM, 0°C, 18 hr; ii. (CH2O)n, 3 hr; (b) NCS, DMS, DCM, 0°C to
rt, 2.5 hr; (c) (NBu4)3 HP2O7, ACN, 2.5 hr.
The synthesis of the saturated GGPP analogs began
with the compounds containing only the α isoprene unit. The
synthesis of these three analogs was simple and straight forward
(Scheme 1). Briefly, commercially available alkynes 1 were
subjected to Negishi’s zirconium asymmetric carbo-alumination
(ZACA) reaction and quenched with paraformaldehyde to afford
Scheme 2. Synthesis of α & β-containing pyrophosphates. (a) Me3Al,
Cp2ZrCl2, DCM, 0°C, 18 hr then (CH2O)n, 3hr (83%); (b) PPTS, DHP, DCM
(79%); (c) i. Me3Al, Cp2ZrCl2, DCM, 0°C, 18 hr, then I2, 3 hr; (d) i. t-BuLi,
Et2O, -78°C, ii. β-MeO-9-BBN, THF, -78°C warming to r.t., 18 hr; (e)
K3PO4, PdCl2(dppf), DMF, 85°C, 18 hr; (f) PPTS, MeOH, 60°C (Yields
given for 2 steps); (g) NCS, DMS, DCM, 0°C to rt, 2.5 hr; (h) (NBu4)3HP2O7,
ACN, 2.5 hr.
alcohols 2a-c in 50-58% yields.14
Next, these alcohols
underwent Corey-Kim chlorination with NCS followed by
pyrophosphorylation to produce the diphosphates 3a-c in
moderate to good yields.15,16
Next, we focused on the synthesis of analogs that
contain only the α and β isoprene units (Scheme 2). To
synthesize these compounds, commercially available alkynes
underwent Negishi’s ZACA reaction followed by an iodine
quench to provide vinyl iodides 8a-d. Conversion of 4-
bromobut-1-yne to alcohol 5 was accomplished using the ZACA
reaction.14 Next, alcohol 5 was THP-protected using standard
procedures to generate compound 6 which was converted into the
organoborane and Suzuki coupled to vinyl iodides 8a-d to yield
compounds 9a-d.17 After deprotection, alcohols 10a-d underwent
standard chlorination and pyrophosphorylation procedures to
yield diphosphates 11a-d in moderate to good yields.15,16
We then turned our attention to the synthesis of analogs
where the ω isoprene unit was reinstalled to obtain compounds
that lack the internal β and γ isoprene units (Scheme 3). In
general, commercially available diol 12 was subjected to mono-
iodination. Intermediates 13 and 16 were subjected to Swern
oxidations to afford aldehydes 14 and 17. These aldehydes then
underwent Wittig reactions to install the ω isoprenes of 15 and 18
in moderate yields. Next, synthesis of vinyl-iodide 20 was
accomplished by first generating the Schwartz’s reagent in situ
following a method developed by Huang & Negishi.18 Following
the addition of TBDMS-protected but-2-yn-1-ol (19),
hydrozirconation-iodinolysis proceeds to yield vinyl iodide 20.
After conversion of halides 15 and 18 into their corresponding
organoboranes, Suzuki coupling to vinyl iodide 20 generated
intermediates 21a-b. Following TBAF deprotection of the
TBDMS group, allylic alcohols 22a-b were isolated and
subjected to standard chlorination and pyrophosphorylation
procedures to yield diphosphates 23a-d in moderated yields.
Scheme 3. Synthesis of α & ω-containing pyrophosphates. (a) PPh3,
Imidazole, I2, DCM, 0°C; (b) (COCl)2, DMSO, Et3N, DCM, -78°C; (c) i-
PrPh3I, n-BuLi, THF, -78°C; (d) i. Cp2ZrCl2, DIBAL, THF, 0°C, 0.5 hr; ii.
19, warm to rt, 1.5 hr; iii. I2, THF, -78°C, 0.5 hr; (e) i. DIEA, TBDMSCl,
DCM (95%); ii. DIBAL, Cp2ZrCl2, THF, 0°C (40%); (f) i. t-BuLi, Et2O, -
78°C, ii. β-MeO-9-BBN, THF, -78°C warming to r.t., 18 hr; iii. K3PO4,
PdCl2(dppf), DMF, 85°C, 18 hr; (g) TBAF, THF, 0°C; (h) NCS, DMS, DCM,
0°C to rt, 2.5 hr; (i) (NBu4)3 HP2O7, ACN, 2.5 hr.
displacement reaction in the presence of 25 and DIEA to
generate diethyl phosphate 26. In situ formation of Grignard
reagent 28 from the corresponding bromide (27) was followed by
the slow addition of diethyl phosphate 26 to the reaction, which
lead to a SN2 displacement of the phosphate group to afford
compound 29. Following standard deprotection and
To synthesize tetrahydro-GGPP we began with THP-
protected geraniol (24) which underwent oxidation in the
presence of SeO2 followed by a NaBH4 reduction to generate
alcohol 25.19-21 Next, diethyl chlorophosphate is subjected to a

protocol.24 The first set of compounds containing only the α-
isoprene unit (3a-c) revealed that both FTase and GGTase-I can
recognize and utilize these compounds as substrates;
unexpectedly, it appears the length of the carbon chain does not
play as crucial a role as first anticipated. For example, one of the
shortest analogs, 3a, has a krel(FTase) = 0.18 versus the krel(GGTase)
=
0.08, indicating a slight preference for the enzyme whose natural
substrate (FPP) is shorter. However, this trend does not appear to
be true for analog 11b, which is of a similar length as 3a. In fact,
11b has a krel(FTase) = 0.15 versus the krel(GGTase) = 0.85, showing a
much greater preference for GGTase-I. This is potentially due to
a reduction of the amount of flexibility/rotation in the analog
after the introduction of another double bond thereby decreasing
its ability to find a binding mode that promotes rapid catalysis
and/or product release.
Scheme 4. Synthesis of tetrahydro-GGPP. (a) SeO2, t-BuOOH, salicylic
acid, DCM; ii. NaBH4, EtOH (27% - 2 steps); (b) DIEA, (EtO)2POCl, Et2O
(76%); (c) Mg powder, Et2O; (d) 28, THF, 18 hr. (15%); (e) PPTS, EtOH,
70°C (70%); (f) NCS, DMS, DCM, 2.5 hr.; (g) (NBu4)3HP2O7, ACN, 3 hr
(52% - 2 steps).
Perhaps the most telling evidence comes from
comparing analogs of a similar length (i.e. 3c, 11d, 23b, 31, 38).
These five analogs are all 16 carbons in length but differ in the
number and position of double bonds present. By removing all
but the α-isoprene, substrate activity is greatly diminished when
compared to GGPP (krel(GGTase) = 0.16). Replacing the β-isoprene
of 3c to generate 11d resulted in an increase of substrate activity
with a 6-fold increase in GGTase-I reactivity (Table 1).
Similarly, this modification led to an 8-fold increase in FTase
reactivity. Reintroduction of the ω-isoprene of 3c to generate
23b also resulted in an increase in substrate activity of both
GGTase-I and FTase; however, these increases were less
substantial with a 3-fold increase in GGTase-I and a nearly 2-fold
increase in FTase. While both the β- and ω-isoprene units are not
required for enzyme catalysis, they are necessary for enhanced
substrate activity. Based on these data, the β-isoprene unit seems
to be more essential for catalysis than the ω-isoprene unit.
Scheme 5. Synthesis of dihydro-GGPP. (a) SeO2, t-BuOOH, salicylic acid,
DCM; ii. NaBH4, EtOH (28% - 2 steps); (b) i. DHP, PPTS, DCM; (c)
saturated K2CO3/MeOH, (74% - 2 steps); (d) Mg powder, Et2O; (e) 36,
Cu(I)Br, THF, -10°C, 48 hr (17%); (f) NCS, DMS, DCM, 2.5 hr; (g)
(NBu4)3HP2O7, ACN, 3 hr (28%).
Compounds lacking the γ and/or ω isoprene double
bonds but retaining the methylene units (31 & 38) greatly
decreased substrate activity with both FTase and GGTase-I
(Table 1). When compared to other saturated molecules of
comparable length (e.g. 11d, krel(GGTase) = 0.95; and 23b, krel(GGTase)
= 0.50), analog 31 is a poorer substrate (krel(GGTase) = 0.03).
Reinstalling the γ-isoprene unit to generate analog 38 results in a
~6-fold increase of substrate activity when compared to analog
31; however, analog 38 is an overall poor substrate activity with
a krel(GGTase) = 0.17. Even though analog 38 contains 3 of the 4
isoprene units, its activity is comparable to analog 3a which only
contains the α-isoprene. One possible explanation for the results
observed for analogs 31 and 38 is molecular geometry. With the
double bonds in place, the geometry of the molecule is planar;
however, by removing the double bonds the molecular geometry
changes from planar to tetrahedral. This change in geometry
could lead to unfavorable interactions with the active site of the
enzyme and ultimately reduce substrate binding.
pyrophosphorylation procedures, analog 31 was produced in
moderate yield.
The synthesis of the final compound of this series,
dihydro-GGPP (38), was first attempted in a similar manner as
analog 31; however, efforts to displace the diethyl phosphate
group of the corresponding farnesol derivative with Grignard
reagent 36 resulted in a mixture of SN2 and SN2’ products. Thus,
a new synthetic avenue was envisioned and the synthesis of
analog 38 was accomplished using a Cu(I)-mediated Grignard
displacement of an allylic THP-ether (Scheme 5).22,23 Briefly,
acetyl-protected farnesol (32) was converted to alcohol 33 via a
SeO2 oxidation followed by a NaBH4 reduction. Next, alcohol 33
was protected as the THP-ether (34) and then deacetylated using
standard protocols to generate compound 34. After Grignard
reagent 36 was generated from the corresponding bromide (35), it
was slowly added to a cooled solution of THP-ether 34 and
Cu(I)Br to yield alcohol 37. It is crucial to keep this reaction at -
10°C to avoid degradation of the organocuprate intermediate.
Alcohol 37 was then converted into the diphosphate following
standard procedures to afford analog 38 in 28% yield.
In general, analogs with a carbon chain length of 16 (3c,
11d, 23b, & 38) are more readily turned over by GGTase-I while
the shorter analog 3a (13 carbons) is more readily turned over by
FTase. Analogs with intermediate carbon chain lengths (3a, 3b,
11b, 11c, 23a) display varying enzyme preferences. Comparing
the FTase and GGTase-I krel values of analogs containing only
the α-isoprene unit (3a & 3b) show a 2-fold and 12-fold
preference for FTase, respectively. Analogs containing both α
and β-isoprene unit (11b & 11c) revealed a ~6-fold and 3-fold
preference for GGTase-I, respectively. The analog containing
both α and ω-isoprenes with intermediate carbon chain length
(23a) shows a ~8-fold preference for FTase-I. Although chain
length is not the only factor driving reactivity, it is nonetheless
important. Analogs with a chain length of 15-carbons (3b, 11c,
and 23a) revealed 8-fold, 1.6-fold, and 25-fold decreases in
GGTase-I activity, respectively, compared to GGPP. It can be
We hypothesized that the shorter chained analogs
would be preferred as substrates by FTase while longer chained
analogs would be preferred as substrates by GGTase-I. To test
this theory, preliminary evaluation of the eleven saturated GGPP
analogs was achieved utilizing an in vitro continuous
spectrofluorometric assay versus GGTase with co-substrate
CaaX-peptide, dansyl-GCVLL, (Figure 2) or versus FTase with
co-substrate CaaX-peptide, dansyl-GCVLS (Figures 3). Analogs
displaying increased fluorescence were further evaluated and
their kcat/KM values were determined according to a published

speculated that shortening the length of the diphosphate chain
may spatially orient the terminal methylene units of the ω-
isoprene in a position where unfavorable interactions with the
enzyme and/or peptide in the binding pocket could perturb
catalysis or product release. It is also possible that the analog
binds in a mode that reduces enzyme affinity for the peptide
substrate. These results indicate that the position of the isoprene
units as well as the overall length of the molecule are crucial for
optimal activity. This work served as a preliminary study of the
isoprene requirements and their effect on FTase and GGTase-I
activity.
A library of saturated GGPP compounds were
synthesized and evaluated as co-substrates with dansyl-GCVLL
(GGTase-I) or dansyl-GCVLS (FTase) in a fluorescence-based
assay. Upon in vitro biochemical testing, it was revealed that all
saturated analogs displayed some degree of substrate activity and
provided us with interesting insights into FTase and GGTase-I
reactivity. The saturated analogs revealed both FTase and
GGTase-I can recognize and utilize compounds that contain only
the α-isoprene unit; however, the other isoprene units are
essential for enhanced turnover.
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Acknowledgments
This work was funded by NIH Grants R01 CA78819
(R.A.G.), P30 CA21328 (Purdue University Center of Cancer
Research), and GM40602 (C.A.F.). I would like to thank my
post-doctoral advisor, Dr. Craig Lindsley, for his aid in
publishing this article after the passing of a great scientist and
even greater man, my thesis advisor, Dr. Richard A. Gibbs.
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References and notes
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5
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krel
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Chain
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3.20 × 103
8.90 × 102
6.20 × 103
nd*
0.08
0.02
0.16
nd
14
15
16
13
14
15
16
15
16
16
16
12
16
8.30 × 103
1.10 × 104
4.10 × 103
1.60 × 104
7.10 × 103
9.70 × 103
3.30 × 104
6.80 × 103
7.10 × 103
3.50 × 103
nd*
0.18
0.24
0.09
0.35
0.15
0.21
0.72
0.15
0.15
0.05
nd
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(19)
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11b
11c
11d
23a
23b
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3.20 × 104
2.20 × 104
3.60 × 104
9.00 × 102
1.90 × 104
1.20 × 103
6.30 × 103
ns
0.85
0.58
0.95
0.02
0.50
0.03
0.17
ns
64, 4821.
(23)
Shull, L. W.; Wiemer, D. F. J. Organomet. Chem.
2005, 690, 2521.
(24)
Hougland, J. L.; Hicks, K. A.; Hartman, H. L.; Kelly,
R. A.; Watt, T. J.; Fierke, C. A. J. Mol. Biol. 2010, 395, 176.
38
4.60 × 104
1
FPP
GGPP
3.78 × 104
1.0
-
-
a krel = relative rate in preliminary screen, with FPP and/or GGPP kcat/KM = 1.0
b krel = relative rate in preliminary screen, with FPP = 1.0
* Showed activity in screen but unable to attain kcat/KM value