Synthesis of Non-natural, Frame-Shifted Isoprenoid Diphosphate Analogues

Article abstract of DOI:10.1021/acs.orglett.6b02977

A set of synthetic approaches was developed and applied to the synthesis of eight frame-shifted isoprenoid diphosphate analogues. These analogues were designed to increase or decrease the methylene units between the double bonds and/or the pyrophosphate m

Full text of DOI:10.1021/acs.orglett.6b02977

Letter
pubs.acs.org/OrgLett
Synthesis of Non-natural, Frame-Shifted Isoprenoid Diphosphate
Analogues
Kayla J. Temple,*,†,§ Elia N. Wright,‡ Carol A. Fierke,‡ and Richard A. Gibbs†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, Indiana
47907, United States
‡Departments of Biological Chemistry and Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: A set of synthetic approaches was developed and applied to the synthesis of eight frame-shifted isoprenoid
diphosphate analogues. These analogues were designed to increase or decrease the methylene units between the double bonds
and/or the pyrophosphate moieties of the isoprenoid structure. Evaluation of mammalian GGTase-I and FTase revealed that
small structural changes can result in substantial changes in substrate activity.
soprenoids are found in nearly all life forms and are the largest
and most structurally diverse class of natural products.1 As
flexibility in relation to FTase activity. Preliminary evaluation
revealed four analogues are substrates of FTase (2,2,1,1-OPP;
1,2,1-OPP; 1,3,1-OPP, 3,1,1-OPP) and one analogue, homo-
farnesyldiphosphate(2,2,2-OPP), isaninhibitorofFTasewithan
IC50 below 1 μM. The naming scheme refers to the number of
carbon spacers between the double bonds and between the first
isoprene double bond and the pyrophosphate group (Figure 1).
With the preliminary data in hand, our goal was to expand upon
this theme and develop non-natural, frame-shifted isoprenoid
diphosphate analogues in an effort to explore the enzyme
specificity and requirements of GGTase-I versus FTase. The
target compounds were 3,3,1-OPP, 2,3,1-OPP, 1,2,2,1-OPP,
2,2,2,2-OPP, 3,2,1-OPP, 4,2,1-OPP, 5,2,1-OPP, and 6,2,1-OPP.
Unlike the previously synthesized frame-shifted analogues, these
analogues are much more flexible and vary greatly in length
between FPP and GGPP. Additionally, 6,2,1-OPP is essentially
the same as GGPP with the exception that the third (γ) isoprene
unit has been removed. We believe that increased flexibility may
aid in binding ability.
The synthesis began with the preparation of 3,3,1-OPP
(Scheme 1). By taking advantage of commercially available
alkynyl alcohols represented by compound 1, we could easily
incorporate thedesiredcarbonchainlengthaswellasinstallthe α-
isoprene unit using Negishi’s zirconium-catalyzed asymmetric
carbo-alumination (ZACA) reaction. Quenching with parafor-
maldehyde afforded iodo alcohol 2.8 Similarly, 5-hexyn-1-ol (4)
underwent a ZACA reaction and quenching with iodine yielded
the vinyl iodide 5. To install the last isoprene unit, 5 was subjected
to a Swern oxidation to yield aldehyde 6 followed by a Wittig
I
such, they are responsible for a multitude of biochemical
functions, including their use as hormones (e.g., steroids,
gibberellins, and abscisic acid) and roles in cell membrane
structure (e.g., cholesterol), electron transfer (e.g., quinones),
andphotosynthesis(e.g., carotenoids).2 Asprecursors toamyriad
of lipid moieties, isoprenoids are important biosynthetic
intermediates that lead to the production of sterols, triterpenes
(e.g., squalene), carotenoids, and hopanoids.2 Isoprenoids can
also serve as lipid anchors for proteins and carbohydrates.3
Perhaps the most interesting and complex group of isoprenoid
biosynthetic products is the vast set of cyclic terpene natural
products such as monoterpenes, sesquiterpenes, and diterpe-
nes.1b,4 Because of the extensive diversity of isoprenoid natural
products, it is not surprising that many promising and effective
pharmaceuticals such as Taxol (cancer), artemisinin (malaria),
vinblastine(cancer), andprostratin(HIV)havebeendiscovered.2
Cyclic isoprenoids are not only of importance to the
pharmaceutical industry but also of great interest in the materials,
chemical, and fuel industries.5 Therefore, there is significant
interest in generating novel isoprenoid diphosphate analogues to
use as chemical tools to further explore these multifarious
processes.
Our laboratory has a long-standing interest in the design and
synthesis of non-natural FPP and GGPP analogues as chemical
tools to explore the enzyme specificity and requirements of FTase
and, more recently, GGTase-I.6 Prior to these studies, our
laboratory developed a method for the preparation of a small
library of frame-shifted FPP analogues.7 The design of these FPP
analogues increases and/or decreases the carbon spacers of the
FPP backbone to examine the relevance of chain length and
Received: October 3, 2016
© XXXX American Chemical Society
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Scheme 2. Synthesis of 2,3,1-OPP (16)
coupled with vinyl iodide 7 under Suzuki cross-coupling
conditions. The THP ether was cleaved under mild acidic
conditions to afford 3,3,1-OH (8). Chlorination and pyrophos-
phorylation were accomplished in a similar manner as described
by Davisson et al. to produce pyrophosphate 9 (3,3,1-OPP).9
Attempts to synthesize compounds 16 and 21 in a similar
manner as compound 9 proved unsuccessful. Shortening the
carbon chain prior to aldehyde formation resulted in
intermediates that quickly decomposed. Therefore, this route
was abandoned in order to explore alternate methods that would
allow the installation of shorter chain ω-isoprene units. To
synthesize 2,3,1-OH (Scheme 2), we first synthesized alcohol, 12,
which was obtained in one step from commercially available (E)-
geranyl bromide by employing a method first described by
Kuwajima and Doi and later used in our laboratory with great
success.10 Briefly, LDA was added to an equimolar amount of
ethyl acetate in the presence of Cu(I)I at −110 °C. The solution
was then allowed to slowly warm to −30 °C, at which point
geranyl bromide (10) was added to the reaction to give ester 11 in
34% yield. Subjecting the ester to DIBAL reduction followed by
iodination afforded bishomogeranyl iodide 13, which was
converted into the corresponding organoborane and coupled to
vinyl iodide 14 via Suzuki cross-coupling. Following deprotection
of the TBS group with TBAF, alcohol 15 (2,3,1-OH) was
produced in 56% yield. Subsequent chlorination and pyrophos-
phorylation resulted in compound 16 in moderate yield.
Figure 1. Evaluation of frame-shifted isoprenoid diphospate analogue
substrate ability versus GGTase-I and FTase. GGTase-I assays utilized
the cosubstrate dansyl-GCVLL peptide, and rates were compared to the
native substrate, GGPP; kGrel = relative rate derived from the preliminary
GGTase-I screen by setting kcat/KM of GGPP = 1.0. FTase assays utilized
the cosubstrate dansyl-GCVLS peptide, and rates were compared to the
native substrate, FPP; kFrel = relative rate derived from the preliminary
FTase screen by setting kcat/KM of FPP = 1.0 (NS = nonsubstrate; nd =
not determined; TC = total carbons; L = length of carbon chain).
Scheme 1. Synthesis of 3,3,1-OPP (9)
The strategy for the synthesis of 1,2,2,1-OH was based on the
displacement of allylic diethyl phosphates with Grignard reagents
as utilized by Snyder and colleagues (Scheme 3).11 First, alcohol
17 was generated following published protocols from commer-
cially available (2E,6E)-farnesol.12 Next, diethyl chlorophosphate
underwent a displacement reaction in the presence of alcohol 17
and DIEA to generate diethyl phosphate 18. Previously, we found
that using the diethyl phosphate derivative rather than the
Scheme 3. Synthesis of 1,2,2,1-OPP (21)
reaction to generate vinyl iodide 7. Previous synthetic efforts in
our laboratory relied on both Stille and Negishi couplings;
however, we wished to eliminate the use of toxic tin-based
reagents as well as employ more aqueous-friendly cross-coupling
reactions.7 Thus, following THP protection of alcohol 2, alkyl
iodide 3 could be converted into the organoborane and then
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Scheme 4. Synthesis of 3,2,1-OPP (28)
Scheme 6. Synthesis of 4,2,1-OPP, 5,2,1-OPP, and 6,2,1-OPP
(39a−c)
corresponding halide afforded several advantages. For example,
the diethyl phosphate derivatives could be stored for longer
periods. Additionally, the corresponding halides generally
underwent Grignard displacement reactions to give mixtures of
the SN2 and SN2′ products that could not easily be separated via
traditional chromatography methods.6a Displacement of the
phosphate group with (2-methylprop-1-en-1-yl)magnesium bro-
mide followed by THP deprotection produced allylic alcohol 20.
Subsequent chlorination and pyrophosphorylation resulted in
compound 21.
In the synthesis of 3,2,1-OH (Scheme 4), we decided to take
advantage of the “2,1-OH” motif inherent to commercially
available (2E,6E)-farnesol. Iodide 26 was obtained by first
converting the TBDPS ether of farnesol (22) to epoxide 23 via a
bromohydrin intermediate. Cleavage of the epoxide with periodic
acid followed by sodium borhydride reduction and iodination
afforded intermediate 25, and subsequent deprotection with
TBAF afforded iodo alcohol 26. Substitution of alkyl iodide 26
was performed in the presence of Cu(I)I and (2-methylprop-1-
en-1-yl) magnesium bromide to produce 3,2,1-OH (27).7,13
Subsequent chlorination and pyrophosphorylation resulted in
compound 28.
We hypothesized that homogeranylgeranyl pyrophosphate
would behave in a similar manner as the corresponding farnesyl
derivative.7 When choosing the route to synthesize compound 32
(Scheme 5), we wanted to explore the possibility of quenching
Negishi’s ZACA reaction with oxirane to generate homoallylic
isoprenoid alcohols. To synthesize compound 32, commercial
(2E,6E)-farnesyl chloride (29) was treated with TMS-propynyl
anion followed by TMS deprotection with TBAF to afford
intermediate 30. This alkyne underwent a ZACA reaction and
quenching with oxirane afforded homogeranylgeraniol (31).14
The alcohol was first converted into the mesylate and
subsequently pyrophosphorylated to yield compound 32.9
Finally, we turned to synthesizing the remaining three target
analogues (Scheme 6). Conversion of commercially available
bromoethyl esters (33) into the corresponding iodoalkenes
(34a−c) was accomplished utilizing a series of Finkelstein,
DIBAL reduction, and Wittig reactions. In order to complete the
unique transformation of 34 to 36, our laboratory has successfully
employed a strategy first developed by Wenkert et al. and later
used by Kocienski et al.7,15 This method relies upon the nickel-
catalyzedringopeningofdihydrofuranswithGrignardreagentsto
producestereodefinedtrisubstitutedalkenes. First, iodides34a−c
underwent alkylation with 5-lithio-2,3-dihydrofuran using the
modified procedure of Placzek et al.7 Next, Suzuki cross-coupling
with vinyl iodide 14 and then TBS deprotection with TBAF
afforded alcohols 38a−c. Subsequent chlorination and pyrophos-
phorylationproducedcompounds39a−c.Priortotheconversion
to diphosphates, analysis of our frame-shifted isoprenoid alcohol
precursors by 13C NMR confirmed all new compounds exhibited
(E)-olefin stereochemistry (see Supporting Information for brief
discussion).
A preliminary evaluation of these frame-shifted analogues
utilized an in vitro continuous spectrofluorometric assay with
either FTase or GGTase-I.16 With this data, it was evident that
seven of the eight frame-shifted analogues were substrates of
FTase and six were substrates of GGTase-I to varying degrees
(Figure 1). The preliminary evaluation of these compounds
revealed several interesting trends. The first observation was that
increasing or decreasing the number of carbons by one methylene
unit between the α- and β-isoprenes resulted in a significant
decreaseinbothFTaseandGGTase-Isubstrateactivity(16vs28;
9 vs 39a). One possible reason for this observation could be that
extending the β-isoprene into the binding pocket of GGTase-I
could result in unfavorable interactions between the analogue and
the enzyme and/or peptide substrate. Alternatively, changes in
flexibility of the frame-shifted analogues could potentially hinder
Scheme 5. Synthesis of 2,2,2,2-OPP (32)
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ORCID
product release from the enzyme, which is dependent on the
movement of the isoprene chain into an exit groove.
Kayla J. Temple: 0000-0001-5290-574X
The second observation is that the length of the carbon chain
appears to be more important than flexibility in regard to
GGTase-I substrate activity. Comparing compounds of 15
carbons in length, 39b (kGrel = 0.23; kFrel = 0.11) lacks the γ-
isoprene unit of 21 (kGrel = 0.24; kFrel = 0.11); however, both
analogues have very similar reactivity. Thus, the lack of flexibility
in the ω-isoprene of 21 seems not to be an important factor. The
similarity between these two compounds also indicates the γ-
isoprene is not required to produce substrate activity.
Moreover, when an analogue has two carbons between the α-
and β-isoprene units (i.e. y = 2), increasing the carbon chain
length from 13 to 15 carbons has a more pronounced effect on
GGTase-IactivityversusFTase(28vs39b). Increasingthelength
to16carbons(39c)hasa∼3-foldincreaseinkcat/KM forGGTase-
I substrate activity; however, this trend does not carry over to
FTase. Analogue 39c also reveals the γ-isoprene unit of GGPP is
not necessary for enzyme recognition but is desirable for higher
enzyme turnover. Adding a methylene unit between the
diphosphate moiety and the α-isoprene unit led to the
homoallylic analogue 32. Preliminary data suggests analogue 32
is an inhibitor of GGTase-I.
Thisstudyaddressestheeffectsofdecreasingorincreasingboth
flexibility and length of non-natural, frame-shifted isoprenoid
diphosphate analoges. Our results indicated that a key factor in
frame-shifted isoprenoid diphosphate substrate reactivity was the
chain length and the position of the β-isoprene unit (y = 2 vs y =
3). Further analysis would reveal if these analogues could be
selective substrates, meaning that although these analogues are
poor co-substrates with dansyl-GCVLL in our preliminary
GGTase-I assay they could potentially be great co-substrates
with other CaaX sequences.
Present Address
§(K.J.T.) Vanderbilt Center for Neuroscience Drug Discovery,
Vanderbilt University Medical Center, Nashville, TN 37232.
Notes
The authors declare no competing financial interest.
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 postdoctoral
research advisor, Dr. Craig Lindsley (Vanderbilt University), 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
(Purdue University).
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ASSOCIATED CONTENT
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* Supporting Information
TheSupportingInformationisavailablefreeofchargeontheACS
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Spectral data (1H NMR, 13C NMR, 31P NMR, LRMS,
HRMS) for all newly synthesized compounds; detailed
experimental procedures for the synthesis of 9, 16, 21, 28,
32, 39a−39c. Detailed protocols for determination of
analog reactivity parameters with FTase and/or GGTase-I
(PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: kayla.temple@vanderbilt.edu.
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DOI: 10.1021/acs.orglett.6b02977
Org. Lett. XXXX, XXX, XXX−XXX