Microelectrode Arrays, Dihydroxylation, and the Development of an Orthogonal Safety-Catch Linker

Orthogonal Safety-Catch Linker

Letter

Microelectrode Arrays, Dihydroxylation, and the Development of an

Nai-Hua Yeh, Ruby Krueger, and Kevin D. Moeller*

Cite This: Org. Lett. 2021, 23, 5440 −5444

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sı Supporting Information

ABSTRACT: Construction of larger molecular libraries on an

addressable microelectrode array requires a method for recovering

and characterizing molecules from the surface of any electrode in

the array. This method must be orthogonal to the synthetic

strategies needed to build the array. We report here a method for

achieving this goal that employs the site-selective dihydroxylation

reaction of a simple oleﬁn.

ecause they can serve as tools for evaluating biological

interactions in real time, we have been developing the

Scheme 1. Requirements for a Safety-Catch Linker to Be

Used for Building Molecular Libraries

synthetic chemistry needed to place the members of a

molecular library by any electrode or combination of

electrodes in a microelectrode array. For each method

developed, the transformation uses the selected electrode or

electrodes to generate the required catalyst or reagent. That

catalyst or reagent is then conﬁned to the surface of the

selected electrode with the use of a solution-phase “conﬁning

agent” that destroys it before it can migrate to any other site on

the array. The strategy allows for the site-selective function-

alization of any electrode in the array, even at electrode

densities up to 12,544/cm .

While the chemistry works well, it is currently limited in

terms of the size of the library that can be generated. We can

place molecules by any electrode in an array, but to assemble a

larger addressable library requires building the molecular

library elsewhere and then transferring the individual members

of the library to the electrodes on the array one at a time.

Obviously, a better method would be to synthesize the

molecular library directly on the arrays, a challenge that

amounts to the total synthesis of a complex, two-dimensional

addressable array.

cyclization reaction to form a lactone cleaves the molecule

from the surface. For the arrays, the safety-catch linker used

has taken advantage of a t-Boc protected alcohol (XP = Ot-

Boc) that could be cleaved by generating acid at a selected

electrode.

While the use of the t-Boc group worked ﬁne for assessing

the success of individual reactions run on an array, it is a poor

choice for eﬀorts to build a molecular library directly on an

array. Consider the proposal shown in Scheme 1. In this

approach, a core scaﬀold would contain a series of protected

amines and/or alcohols that could each be independently

deprotected and utilized as a site for diversiﬁcation. The

diversiﬁcation step would use the alcohol or amine as a

nucleophile in a coupling step that is frequently catalyzed with

base. To do the coupling step site-selectively on an array, the

Of course, the development of synthetic strategies for

building molecules directly on the arrays also requires a

method for characterizing those molecules once they are made.

To do so, the molecules on the surface of any electrode in a

high-density microelectrode array must be selectively removed.

To ensure the ﬁdelity of the process, the same electrode used

for the synthesis and monitoring of the molecules in question

must also be used for their recovery. In addition, the method

must be orthogonal to the synthetic chemistry used to make

the molecules and stable to the analytical methods employed.

To date, characterization methods of this nature have taken

Received: May 28, 2021

Published: June 29, 2021

advantage of a Kenner-type safety-catch linker strategy. In this

approach, a protected alcohol or amine (XP in Scheme 1) is

unmasked after the synthesis of a molecule. A subsequent

2021 American Chemical Society

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Org. Lett. 2021, 23, 5440−5444

Organic Letters

Letter

array needs to be submerged in an acidic solution (the

reaction desired for the safety-catch strategy to aﬀord a 95%

isolated yield of the alcohol product 3.

conﬁnement strategy for the generation of a base) and then

the selected electrodes used as a cathode to generate the base-

catalyst needed. Under such conditions, a safety-catch linker

with an acid cleavable t-Boc group would not be stable.

As an alternative, it is tempting to suggest a reductively or

oxidatively cleavable protecting group for the alcohol or amine

in the safety-catch linker. However, the use of such a group in

the linker precludes its use for the diversity oriented synthesis

portion of the strategy, a scenario that is again less than ideal

given the short supply of such protecting groups and the need

to avoid the use of acid- and base-cleavable protecting groups

for the coupling steps in the sequence. What is needed is a

safety-catch linker that avoids the use of an alcohol or amine

protecting group altogether but can still be cleaved with the

use of any electrode or set of electrodes in the array. We report

here that the dihydroxylation of a simple monosubstituted

A similar reaction with the related six-membered ring

precursor 1b was conducted. In this case, the oxidation led to

the dihydroxylated product 2b in a 91% isolated yield, but

generation of the diol did not spontaneously lead to the

cleavage step. The diﬀerence between the two substrates

reﬂected the rate of the cyclization reaction with the ﬁve-

membered ring cyclization being much faster. The ﬁve-

membered ring strategy was selected for further development,

since cleavage from an array in a single step was highly

desirable. It was for this same reason that an ester linkage was

chosen for attachment of the substrate to the array rather than

an amide linkage. While amide linkages are certainly

compatible with Kenner-type safety-catch linker strategies,

we wanted to use a leaving group for the required addition−

elimination strategy that would depart faster under neutral

conditions.

oleﬁn provides an ideal solution to this challenge (Scheme 2).

To test the deprotection strategy on an array, substrate 4

was synthesized and placed by every electrode in an array

Scheme 2. Use of a Dihydroxylation Strategy

(

Scheme 3B). For this experiment, the Alloc group used in

substrate 1 was replaced with a 4-pentenoic acid derivative in

order to avoid the use of a protecting group that could be

employed to mask a site of diversiﬁcation in future synthetic

eﬀorts. The placement of the substrate by the electrodes in the

array was achieved by coating the array with a diblock

copolymer containing a 4-bromo-polystyrene block and a

cinnamate functionalized methacrylate block and then photo-

cross-linking the cinnamate groups in order to add stability to

The oleﬁn is stable, the oxidation can be conducted selectively

at any electrode in an array, and the subsequent cyclization

and cleavage of the molecule from the electrode surface occurs

spontaneously.

Development of this approach began with a solution-phase

test of the overall concept (Scheme 3A). For the initial “proof

the surface. Each electrode in the array was then used as a

cathode to drive a Cu(I)-catalyzed cross-coupling reaction

between the aryl bromide on the polymer surface and substrate

. In this experiment, the potential listed is the potential drop

across the cell and therefore controls the current used or the

rate at which the catalyst is generated. Typically, the electrode

is cycled on and oﬀ to help with conﬁnement of the catalyst to

the electrode used for its generation. Since this was a ﬁrst time

trial with the substrate being placed by every electrode in the

array, the conditions used for previous placement reactions

Scheme 3. Initial Studies

were selected.

The dihydroxylation reaction was performed on the

functionalized array by using blocks of 12 microelectrodes

each (a 4 × 3 pattern) as anodes in order to recycle the Fe(III)

co-oxidant needed to generate catalytic amounts of the active

Os(VIII) reagent. 4-Phenyl-1-butene was added to the

solution above the array as a conﬁning agent so that any

Os(VIII) that escaped from the surface of the selected

electrodes would be consumed before it could reach a remote

site on the array. This experiment was accomplished by

treating the array with a 1:1 tBuOH/H O solution containing

.07 g of AD-mix-β, 28 μL of 68 mM K OsO ·2H O, K CO

2 4 2 2 3

as base, and 1.0 equiv of the 4-phenyl-1-butene conﬁning

agent. This mixture was stirred overnight in order to make sure

that all of the Os(VIII) reagent was consumed prior to the start

of the array reaction. For the reaction shown in Scheme 3B (a

reaction run on a 12K array), the selected electrodes were set

of principle” experiment, an Alloc protecting group was used,

since it could be synthesized with available reagents (Scheme

at a potential of +2.0 V relative to the counter electrode. This

was done for a period of 30 s followed by a period of 10 s

where the electrodes were turned oﬀ. This was repeated 60

times. The cycling of the electrodes was done in order to

balance the rate of Os(VIII) generation with the rate that it

was consumed by the surface substrate, and in so doing

A). Following its synthesis, substrate 1a was treated with AD-

mix-β using the standard tBuOH/water reaction conditions to

generate the cis-hydroxylated product. The product could not

be isolated but instead immediately underwent the cleavage

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Org. Lett. 2021, 23, 5440−5444

Organic Letters

Letter

optimize conﬁnement of the reaction to the selected

electrodes.

methanesulfonamide, potassium carbonate, and 4-phenyl-1-

butene as a conﬁning agent. The selected electrodes in the

array were set to a potential of +2.0 V relative to the counter

electrode for a period of 0.5 s and then turned oﬀ again for 0.1

s. This pattern was repeated for 1800 cycles.

After the dihydroxylation reaction and cleavage of the linker,

all of the electrodes in the array were used to reduce vitamin

B and generate a base for catalyzing an esteriﬁcation reaction

between any alcohol on the surface of the array and a pyrene-

labeled activated ester. The array was then examined using

ﬂuorescence microscopy. From the image provided in Scheme

The success of the reaction can be seen by comparing the

ﬂuorescence images of the array taken before and after the

dihydroxylation. In this case, the substrate for the deprotection

reaction contained a pyrene group that was cleaved from the

surface of the array, leading to decreased ﬂuorescence at the

selected electrodes. A small amount of ﬂuorescence did remain,

indicating the potential for an incomplete reaction. This is not

a problem as long as enough of the cleavage occurs for the

product to be characterized, which turned out to be the case.

The material cleaved from the array was collected, examined

by HRMS, and shown to be lactone 6. The cleavage reaction

on the array clearly worked as designed and aﬀorded the same

product as the solution-phase reaction.

B, it was clear that the esteriﬁcation reaction only occurred on

the electrodes used for the dihydroxylation reaction. This

indicated that the dihydroxylation was selective, but it did not

prove that the cleavage reaction had occurred, since the

esteriﬁcation reaction used to ﬂuorescently label the product

would also occur with the dihydroxylated intermediate.

The issue was addressed by taking advantage of the

chemistry the linker was designed for. To this end, the lactone

product from the cleavage reactions was independently

synthesized (Scheme 4A) and fully characterized. In this

In order to verify that the cleavable linker was compatible

with array-based coupling reactions, the experiment shown in

Scheme 5 was conducted. In this case, the t-Boc-protected

Scheme 4. Schematic Showing the Array-Based Reaction

Does Form the Same Product as the Solution-Phase Study

Scheme 5. Testing Linker Compatibility with Peptide

Synthesis

case, the instability of the lactone made it diﬃcult to isolate in

high yield relative to the corresponding alcohol product.

Nevertheless, the solution-phase reaction did provide enough

of the lactone product 6 for characterization. Due to the

diﬀerences between solution-phase reactions and array-based

reactions, further optimization of the solution-phase reaction

was not pursued.

allyl-glycine substrate 8 was placed by every electrode in a 1K

array and then the t-Boc group removed by using the

electrodes as anodes to oxidize diphenylhydrazine and generate

an acid. The electrodes were then used as cathodes to induce

The safety-catch linker substrate 7 was then synthesized and

added to the surface of every electrode in an array having 1024

a base-catalyzed coupling reaction between the deprotected

amine and the NHS-ester of 4-pyrene butanoic acid to form

microelectrodes/cm , as shown in Scheme 4B. In this case, a

the pyrene-labeled allylglycine.

longer reaction time (more cycles) and a higher voltage

With the substrate for the dihydroxylation reaction

assembled, the electrodes were once again used as anodes to

conduct the dihydroxylation reaction and cleave the molecule

from the surface of the array. As in the previous experiment,

the transformation led to both a decrease in ﬂuorescence on

the arrays and the isolation and characterization by HRMS of

lactone 6 from the solution above the array. The safety-catch

linker strategy was clearly compatible with the acid- and base-

catalyzed deprotection and coupling steps used for the

synthesis of peptides on a microelectrode array.

(

higher current and faster catalyst generation) were used

relative to the experiment shown in Scheme 3B in order to

maximize the amount of substrate placed on the array. Note

that the image provided in Scheme 3B was not as intense as

one might like and showed incomplete coverage of the

electrodes. Since our goal for this experiment was to recover

material from the electrode surface for characterization, we

maximized the amount of material placed by each electrode.

Next, the dihydroxylation was performed using a checker-

board pattern of electrodes. This was done by submerging the

array along with a remote Pt-wire counter electrode in a 1:1

solution of tBuOH and water containing AD-mix β,

The synthetic sequence shown in Scheme 5 was intriguing in

that it illustrated the versatility of the arrays for synthesis. For

the chemistry, the electrodes were used alternatively as

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Org. Lett. 2021, 23, 5440−5444

Organic Letters

(1) For selected examples, see: (a) Chandra, S.; Siraj, S.; Wong, D.

Letter

cathodes (the Cu(I) coupling reaction and the amide

coupling) and anodes (the t-Boc deprotection and dihydrox-

ylation steps) with no change required to the electrolysis setup.

Each reaction was a simple constant current electrolysis where

the polarity of the electrode was set either negative or positive

and then the working potential of the electrode allowed to

adjust to whatever catalyst was being used. In addition, it

should be noted that each of the reactions conducted is not

actually an “electrochemical reaction”. The electrochemistry is

used to locate a standard chemical reagent or catalyst at a site

on the array. The chemistry takes place on the surface of the

polymer coating away from the electrode. Therefore, the

selectivity of the reactions should be the same as that for any

other solution-phase or solid-phase synthesis. The cis-

hydroxylation reaction should show the same selectivity

between oleﬁn substrates it normally does.

In conclusion, we have demonstrated that a simple oleﬁn can

form the basis of a synthetically orthogonal safety-catch linker.

For cleavage of the linker on an addressable array, the

electrodes in the array were used to generate an Os(VIII)

species that in turn triggered a cis-hydroxylation of the oleﬁn.

One of the resulting alcohols then underwent a spontaneous

cyclization to generate a lactone and remove the molecule

from the surface of the electrode. The success of the reaction

was indicated by both ﬂuorescence microscopy and HRMS

characterization of the lactone produced. In this fashion, a

molecule at any electrode in the array can be cleaved from the

array for characterization. This development provides the

foundation for constructing molecular libraries directly on the

surface of a microelectrode array and then verifying the

structure of the synthesized molecules. Since the safety-catch

linker is simply allylglycine, allylglycine is used frequently as a

building block in peptide synthesis, and no naturally occurring

amino acid contains an oleﬁn, the strategy appears particularly

well suited for the synthesis, analysis, and characterization of

peptides on a microelectrode array.

ACKNOWLEDGMENTS

■

We thank the National Institutes of Health (1R01 GM122747)

for their generous support of our work.

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ASSOCIATED CONTENT

sı Supporting Information

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AUTHOR INFORMATION

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Saint Louis, Missouri 63130, United States; orcid.org/

Corresponding Author

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000-0002-3893-5923 ; Email: moeller@wustl.edu

Authors

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Ruby Krueger − Washington University in Saint Louis, Saint

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