To perform a photo-affinity pulldown experiment, the photo-affinity analog of the parent inhibitor must be designed with significant preexisting SAR knowledge. The point of attachment for the photocrosslinker and the affinity handle must conserve the
selectivity of the parent molecule for the target. To achieve this, we designed a
photoaffinity probe by the addition of a photoaffinty label and a biotin handle to our most potent inhibitors. The SAR of dimeric chloroquine (DC) and dimeric quinacrine (DQ) inhibitors indicated that inhibitors containing a 6/6 triamine linker are the most potent inhibitors of autophagy and the most potent anti-cancer agents. SAR studies of both classes molecules identified the importance of the basic nitrogen atom within the linker of the dimeric compound. The linker size and the presence of a tertiary amine was to be maintained in the photoaffinity analog. Studies of DQ inhibitors established the central nitrogen atom alkylation governs whether a molecule induces or inhibits autophagy. We identified that alkylation of the central nitrogen of our triamine linker could serve as an attachment point for the required PAL functional groups. We used these criteria to design a series of photoaffinity probes to identify the target of tertiary amine containing DQ and DC inhibitors.
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A linear synthetic strategy was chosen (Fig 3.5, A), where a photocrosslinker could be attached to the DC660 68 (Figure 3.5, B) inhibitor through a flexible linker via
alkylation of the central linker nitrogen. The attached photoaffinity moiety would contain an alkyne handle where click chemistry could be used either synthetically to attach an affinity handle or in situ utilizing biorthogonal click chemistry. Benzophenone was selected as the first choice for a photoreactive functional group because of the synthetic ease of its incorporation and its specificity in photolabeling reactions (Figure 3.5, C).
Biotin is a natural product ubiquitous in biological systems and is commonly used as a handle in affinity chromatography. Multiple homologs of biotin-binding proteins exist which form extremely tight non-covalent binding interactions. The interaction
Figure 3.5: (A) A linear scheme for the construction of a photoaffinity probe. (B) a DC inhibitor warhead. (C) Benzophenone, the photocrosslinking functional group chosen for our photoaffinity probe. (D) Two affinity handles which can be used to pulldown proteins from the biological milieu.
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between biotin and streptavidin, a common biotin-binding protein, is non-covalent. The tight binding affinity of this interaction allows for solid-supported streptavidin to rapidly bind biotinylated molecules in solution. The non-covalent interaction between biotin and a biotin binding protein is strong enough that the supernatant can be removed from a mixture of lysate and solid support without breaking the interaction. Further, washing the solid support with detergent will not break the biotin-enzyme interaction. Desthiobiotin is an analog of biotin which maintains a significant binding interaction with biotin-binding enzymes. However, additional biotin can be utilized to displace desthiobiotin from biotin binding proteins (Figure 3.5, D). The advantages of using a biotin analog become
apparent when separating biotin labeled targets from native biotinylated molecules.
Polyethylene glycol (PEG) was selected as a linker between each component of the photo-affinity probe. With binding conformation of the parent inhibitor unknown, a flexible linker was chosen to maximize the likelihood of crosslinking. PEG specifically was chosen as the linker because of its lower cLogP value when compared to an aliphatic linker of similar length (Fig 3.6). The addition of benzophenone adds a significant
amount of hydrophobic surface area to the probe; therefore, the addition of the
hydrophilic PEG chain was designed to maintain the aqueous solubility of the molecule. Tetraethylene glycol was selected as the PEG linker for multiple reasons: first, PEG linkers between two and four monomer units are commodity chemicals; second, tetraethylene glycol afforded a significant amount of hydrophilic surface area to help solubilize the large probe molecule; third, the increased flexibility of the extended linker would maximize the chances of photocrosslinking success, even if the inhibitor warhead
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was buried deep within the protein of interest; fourth, the longer linker was selected to space the benzophenone far enough away from the parent warhead so as to not influence the binding mode of the probe molecule.
With advances in the sensitivity of mass spectrometry, it is becoming increasingly possible to generate large profiles of data for multiple photocrosslinking probes. If the number and cost of biological experiments were not a factor, each photoreactive
functional group would be combined with each length of spacer. A protein pulled down most often among all analogs would be the most attractive putative target. However, in a case where three photoreactive chemistries are available and three linkers are considered, nine biological experiments would be required prior to the reproduction of the results. Each biological experiment consists of one set of experimental conditions and two controls, which would result in 27 proteomic profiles for analysis of each inhibitor warhead. Such efforts were beyond the scope of our capabilities, and therefore we proceeded with inhibitors utilizing one PEG length and one photoaffinity probe per inhibitor warhead.
Figure 3.6: The structures of PEG chains and the corresponding aliphatic diols of equal length.
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With the design of a photoaffinity probe in hand, the synthetic effort focused on the synthesis of three separate regions. The dimeric inhibitor was synthesized as the secondary amine-linked analog, which would allow for the attachment of a PEG chain via reductive amination. The benzophenone could be attached to a PEG chain, which could be used to alkylate the inhibitor. The other side of benzophenone could contain a propargyl group which could facilitate the attachment of biotin via click chemistry.
Figure 3.7: A. General structure of a DC photoaffinity pulldown analog. Key synthetic disconnections are noted by dotted lines. B. Retrosynthesis of a photoaffinity analog of DC661.
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Tetraethylene glycol was mono-tosylated using p-toluene sulfonyl chloride as the limiting reagent in the presence of pyridine to yield compound 143.104 4,4-
dihydroxybenzophenone was monoalkylated with propargyl bromide in ethanol heated to reflux using potassium carbonate as a base to yield 142.105 4-propargyloxy-4-
hydroxybenzophenone 142 was deprotonated by potassium carbonate in ethanol, and the
resulting phenolate was then alkylated with tosylate 143 to give 141. Compound 141 was
oxidized by Swern conditions yielding an aldehyde, 144. Aldehyde 144 was used in
slight excess, without purification, in the subsequent reductive alkylation of the secondary amine containing DC660 68 with sodium triacetoxyborohydride in CH2Cl2
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The affinity handle for the probe was synthesized starting from the bis-mesylation of tetraethylene glycol in methylene chloride, using triethylamine as a base. The crude mesylate was then reacted with sodium azide in situ using a mixture of water and ethanol heated to reflux.105 Monoreduction of the bisazide was facilitated by Staudinger reduction using triphenylphosphine in a biphasic solvent system of diethyl ether and 0.9 M
phosphoric acid (1:1, 50% v/v) to give compound 147.105 The resulting primary amine
147 was then coupled to desthiobiotin 131 via a peptide coupling reaction using TBTU
and Hünig’s base in DMF to give 140.105 (Figure 3.9)
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The alkyne-containing photoprobes 139, 145, and 146 were then linked to the
desthiobiotin affinity handle 140 by click chemistry using copper (II) sulfate and sodium
ascorbate in a methanol and water solvent system. Click chemistry was selected as the final synthetic step due to its broad functional group compatibility. The click reaction yielded photoaffinity compounds 139, 148, and 149 in serviceable yield. (Figure 3.10).
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CQ 1 and HCQ 4 have structural differences in the linker region when compared
to DC inhibitors, specifically a methyl group and a diethylamine (Figure 3.11). Both CQ
1 and HCQ 4 are similarly effective in inhibiting autophagy, and thus we hypothesized
the hydroxyl of HCQ 4 was not necessary and could be utilized as a handle to attach
photoaffinity functional groups to commercial HCQ 4. We envisioned the construction of
a photoaffinity analog for CQ/HCQ 1/4 would contain a PEG chain linked to the tertiary
amine found in the linker of CQ/HCQ 1/4 (Figure 3.12). Multiple attempts were made to
utilize the hydroxyl of HCQ 4 as a free nucleophile in the SN2 alkylation of a tosylated
PEG derivative. Each of these attempts proved unsuccessful, where no alkylated product
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was observed. We attempted to reverse the polarity of the SN2 reaction by exchanging the hydroxyl of HCQ 4 with a leaving group via synthesis of the mesylate and the bromide.
TLC analysis of both reactions indicated a consumption of starting material; however, a baseline spot was observed which was found to be immobile on silica. We hypothesized that either the molecule is undergoing a decomposition reaction with the unprotected aminoquinoline acting as a nucleophile, or that the tertiary amine could be contributing to the premature ejection of the new leaving group via aziridinium formation.
Unfortunately, any attempts to trap the electrophile with triethylene glycol proved to be unsuccessful. This reaction should be studied in future derivatizations of chloroquine. However, in the interest of synthetic ease we pivoted our design strategy to synthesize a CQ 1 derivative de novo.
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In 1946 Professor Marvin Carmack and coworkers synthesized monoalkylated versions of CQ.106 In their paper they described a compound which was referred to as desethyl chloroquine, a chloroquine analog containing a secondary amine rather than the standard tertiary amine. This led us to hypothesize that by accessing this chloroquine derivative, we could apply the same chemistry we had utilized to functionalize secondary amine containing DC inhibitors. The route published by Carmack and coworkers
contained no spectroscopic data for the compounds and required high pressure reductions
Figure 3.11: A. A structural comparison of CQ and HCQ. B. Retrosynthesis of a chloroquine photoaffinity analog. The synthesis proceeds through a secondary amine containing CQ analog which is synthesized de novo.
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and extremely low-pressure distillations. Therefore, we proposed to improve the route through the utilization of modern organic chemical methods.
We envisioned synthesizing desethyl chloroquine by first synthesizing an amino alcohol and arylating the nitrogen via Buchwald-Hartwig conditions. We elected to arylate prior to synthesizing the diamine because we feared the length of the carbon linker (four) would make any attempts to synthesize the diamine prone to cyclization. We hypothesized that arylation of the primary amine of the amino alcohol 153 with the
electron withdrawing chloroquine ring would help prevent the cyclization from occurring.
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We began by synthesizing 4-nitropentanoate, 154, from nitroethane and ethyl
acrylate, according to literature precedent.107 Ester 154 was then reduced with lithium
aluminum hydride to afford the 4-amino-1-pentanol 153 in good yield. Buchwald-
Hartwig arylation of amino alcohol 153 produced the arylated product 152 in serviceable
yield. Alcohol 152 was then oxidized to aldehyde 155 using Swern conditions. Initial
experiments which used CH2Cl2 as a solvent were unsuccessful. Changing the solvent to THF proved critical to the success of the Swern oxidation. Aldehyde 155 was found to
decompose on purification. Therefore, the crude aldehyde was reacted without
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purification in a reductive amination reaction with ethylamine and sodium triacetoxyborohydride to yield desethyl chloroquine, 151. Compound 151 was
reductively alkylated with aldehyde 144 using sodium triacetoxyborohydride in CH2Cl2.
Alkyne 150 analog was then ligated to desthiobiotin azide 140 via click chemistry,
yielding the final CQ-P 156.
For a pulldown probe to be successfully utilized in a competitive
photocrosslinking experiment, it must mimic the in vivo activity of the inhibitor analog from which it was derived. We assayed A375P melanoma cells treated with each of the photoaffinity pulldown molecules to determine whether autophagy was inhibited
similarly to A375P melanoma cells treated with the parent inhibitors. DC661-P, (Figure
3.10, 138) caused the accumulation of LC3-B at doses as low as 0.3 nM. DC221-P, 148,
and CQ-P, 156, both lost significant potency in comparison to their parent inhibitors
(Figure 3.13). However, inhibition of autophagy at high micromolar doses demonstrated that the molecules were still functional, and that the molecules still caused autophagy
Figure 3.13: Western blots of LC3B for DC inhibitors. DC661-P (pulldown) was observed to significantly inhibit autophagy. Lys05 and CQ-P also inhibited autophagy, although, significantly less potently than their parent inhibitors.
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inhibition. This indicated to us that they would be acceptable molecules for a competitive PAL experiment.