The Functionalization of a Nylon via Integration of a Diels-Alder Active 3,6 Diamide

1,2,4,5 TetrazineTyler Casselman, Jerry Britto, John SnyderA 1,2,4,5 tetrazine is a reactive, six membered ring consisting of four nitrogen atoms and two carbon atoms that has been shown to readily react with other compounds containing a double or triple bond between two atoms.. This reaction, known as the Diels-Alder reaction, is a very reliable chemical pathway that typically requires high temperatures and pressures to perform. However, the 1,2,4,5 tetrazine is highly electron deficient, allowing it to react through the inverse electron demand pathway at potentially biorthogonal conditions. Such conditions are desired because it would allow the Diels-Alder reaction to preferentially take place invivo, which can be used as a securing mechanism to fix small molecules to the target site. The carbonyl substituted tetrazine has been shown to make the most reactive tetrazine rings due to their electron withdrawing capability. The successful integration of a tetrazine within a nylon can therefore enablWe attachment various molecules, such as drugs, to the nylon through the Diels-Alder reaction, thus functionalizing the nylon. Background

• Synthesis of the 3,6 dicarboxylate tetrazine proposed by Boger et.al.3

NN N

N

OX1

O X2

Xn=OMe, NR1R2, Me

NH

NN

OX1

O X2

HN

Indenopyridazine

DCM

0oC to 40oC

N

NN

N

MeO2C

CO2Me

EDGR'

NHN N

NH

O

O

OMe

OMe

H2NNHBoc

DCM -78 to r.t.

Al(Me)3 NHN N

NH

O

O

HN

NH

4 eq

NHBoc

NHBoc

1:1 TFA:DCMN

HN NNH

O

O

HN

NH

NH2

NH2

90% 80%

Synthesis Scheme

NHN N

NH

O NR1R2

O NR1R2

Cl

O O

Cl

NHN N

NH

OR1R2N

O NR1R2

H2N NH2n

NR1R2

O

R1R2N

O

Nylon

Nylon

NHN N

NH

O

O

OMe

OMe

AlMe

NHR1R2

NHN N

NH

O

O

OMe

OMe

AlMe

NHR1R2

NHN N

NH

O OMe

NHR1R2O

NHN N

NH

O

O

OMe

OMe

AlMe

NHR1R2

NHN N

NH

O

O

OMe

OMe

Al

NHN N

NH

O OMe

O

VS

O

OtBuO

O

OtBu H2N NH2n NH2NH

n

O

tBuO

Boc2O Primary Diamine Amino-carbamate

includes secondary amines

NN N

N

O NR

O NR

NR

NR

O

RN

O

O

O

RN

DCM

40oC

MeN N

N

O NR

O NR

NR

NR

O

RN

O

O

O

RN

NMe

OO

NH

HOOH

OH

HN O

NN

O NH

HN

O

HOOH

OHXDrug

• The release of nitrogen via a [2.2.2] bicyclo transitions state allows for low temperature Diels Alder reaction to occur.2

• Bioorthogonal reactions with tetrazine have been discovered using less reactive tetrazines.

• Previous work from Snyder group has shown the use of indole to synthesize the indenopyridazine via Diels-Alder.1

Amidation vs Ketonization

Synthesis of Tetrazine Enriched Nylon

References1. Boger, D., Panek, J.; Org Snyth. 1992. 70. 79.2. Giradot, M., Nomak, R., Snyder, J.; J Org Chem.

1998. 63. 10063-10068.3. Haoxing W., Cisneros, B. J. Am. Chem. 2014. 136.

179424. Modic, M.J., Pottick, L.A.; Polymer Engineering and

Science. 1993 33(13). 819-826.

Diels-Alder Within the Nylon

Potential Work: Polysaccharide

• Reaction conditions are similar for both pathways

• The amine is more nucleophilic than trimethyl aluminum

• Trimethyl aluminum is both an alkylating agent and a lewis acid

• The similarity in structure between hexane diamine and the tetrazine allow for a small molecule embedded nylon to be synthesized

• So far, color changes have been used to postulate the success of the nylon reactions. In the future, IR, Raman, UV/vis and possibly fluorescence will be used

• Can be synthesized by peptide coupling the tetrazine to acid groups on sugar polymers

4

5

6

7

8

9

Original Synthesis: The Org. Syn. Prep

N NOEt

OOEt

O

H2N

NaNO2

H2SO4

NaOH/H2O HNN NH

N

O ONa

O ONa

HCl HNN NH

N

O OH

O OH

HNN NH

N

O OMe

O OMe

SOCl2MeOH

[NO]xNN N

N

O OMe

O OMe

Oveall Yield~15%

Boger, D. et.al Org. Synth Prep.

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Diversification of 3,6 Dicarboxylate Dihydrotetrazines

HNN NH

N

O OMe

O OMe

HNN NH

N

O

O OMe

HNN NH

N

HO

O OMe

HNN NH

N

O

O

HNN NH

N

OH

OH

HNN NH

N

O NR2

O NR2

HNN NH

N

O NR2

O OMe

AlMe3 NaBH4

Zr(OtBu)4

AlMe3HNN NH

N

O OMe

O OMe

HNN NH

N

O OMe

O OMe

NaBH4

+NHR2

• This is the only known way to make asymmetricly substituted tetrazines

• Another potential way to make asymmetric tetrazines

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Synthesis of Asymmetric Dihydrotetrazines

• So far, only the ketonization of the ester using trimethyl aluminum has been shown to be able to react with only one of the methyl esters.

HNN NH

N

O OMe

O OMe

HNN NH

N

O

O OMe

HNN NH

N

HO

O OMe

AlMe3 NaBH4

HNN NH

N

HO

O Y

AlMe3NHR1R2

HNN NH

N

HO

O NR1R2

HNN NH

N

HO

O OMe

RX

Base

HNN NH

N

RO

O Y

Y=OMe, NR1R2 Return

Diels-Alder Reactivity Series

• The donating ability from the OMe and NR1R2 raises the LUMO of the tetrazine, making it less reactive through the inverse electron demand Diels Alder pathway

• Electron withdrawing groups increase the reactivity• Aldehyde substitution would create a more reactive species, while directly

bound nucleophiles would create a relatively inert species

NN N

N

O

O

NN N

N

O

OMeO

NN N

N

OMeO

OMeO

NN N

N

OR2R1N

NR1R2O

NN N

N

HO

OH

>>>>

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Mechanism of Inverse Electron Demand Diels Alder with Tetrazine

• Heterocycle lowers HOMO and LUMO (less aromatic stabilization)

• Electron rich dienophile

NN N

NEDG

EDG

R'R

Or

R

R

NNN

N

R

R

NNN

N

R

R

NN

R

R

NN

R

R

-N2

NN

R

R

For alkynesFor alkenes

R'EDG EDG

R' EDGR'

EDG

R'

EDG

R'

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Inverse Electron Demand Diels Alder• Indole has primarily been used as the

diene in previous cycloadditions with tetrazine• Indole provides little LUMO information of

the various tetrazines

NN N

N

OX

XO

NN

OX

XO

+

NN N

N

OX

XO

NN

OX

XO

+

excess tetrazine, reoxidation

X=NR2, OR, R

•More electron deficient dienophiles, lower HOMO

•The increased HOMO-LUMO gap allows for kinetic data to be extracted

•Calculation by James Mcneely predicted 3-hexene to be the more reactive species

•The orthogonal pi system of alkyne could provide electron density to the system, lowering the HOMO

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Preliminary data: Orthogonal π cloud slows down reaction rate

• Alkene is the more reactive species

• Under same conditions, the tetrazine reaches the endpoint faster with hex-3-ene

• The reaction has the potential to use Beer’s law to determine more accurate kinetics

NN N

N

OMeO

OMeO

NN

OMeO

OMeO

+

NN N

N

OMeO

OMeO

NN

OMeO

OMeO

+

excess tetrazine, reoxidation

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Approach to Amide Substituted Tetrazine

NHN N

NH

O

O

N2

OEtO

TEA, DCM r.t.

NR1R2

NR1R2

NHN N

NH

O

O

OMe

OMe

NHR1R2

NHR1R2N

HN NNH

O

O

NR1R2

NR1R2

NHN N

NH

O

O

NR1R2

OMe

DCM r.t.

NHN N

NH

O OMe

O OMe

NHN N

NH

O NR1R2

O OR

NHN N

NH

O NR1R2

O NR1R2

[Zr(OtBu)4]NHR1R2

ROH+ +

+

+ +Al(Me)3

• The zirconium catalyst is capable of performing transesterification with the alcohol it is dissolved in (n-butanol)

• The diazo group is capable of performing an Sn2 like reaction before dimerization. Amide is made due to visualization of EtOH in NMR

• Al(Me)3 is pyrophoric, dangerous scale up but versatile

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Weinreb Amidation Vs. Trimethylaluminum alkylation

• The amidation is quicker than the alkylation

• When dihydrotetrazine and amine are mixed before addition of Al(Me)3, only amide product is formed, not ketone (shown by NMR)

NHN N

NH

O

O

OMe

OMe

AlMe

NHR1R2

NHN N

NH

O

O

OMe

OMe

AlMe

NHR1R2

NHN N

NH

O OMe

NHR1R2O

NHN N

NH

O

O

OMe

OMe

AlMe

NHR1R2

NHN N

NH

O

O

OMe

OMe

Al

NHN N

NH

O OMe

O

VS

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Solution to the Diamine polymerization

• The diamine has the potential to link two tetrazines together because of the high nucleophilicity of the free amine

• The literature has precedents for single boc protection of many diamine systems

• Four diamine systems will be to show how size, as well as primary vs secondary amines, affect the polymer.

H2N

NHBoc

NH

BocN

NH

NHBoc

H2N NHBoc

NHN N

NH

O

O

OMe

OMe

NHN N

NH

OHN

O

DCM r.t.+

Al(Me)3

H2N

NH2

NH

NH

HN

O

NNH

NHN

O

NH

HN

n

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Solution to the Diamine Polymerization cont.

• The amidation of monoboced diamines followed by the deprotection of the diamine is a successful pathway to make the bisamide tetrazines with a tethered nucleophilic amine

• The TFA was a strong enough acid to produce the ditrifalate salt of the tetrazine, it was left that way to prevent oxidation of the free amine

NHN N

NH

O

O

OMe

OMe

H2NNHBoc

DCM -78 to r.t.

Al(Me)3 NHN N

NH

O

O

HN

NH

4 eq

NHBoc

NHBoc

1:1 TFA:DCMN

HN NNH

O

O

HN

NH

NH2

NH2

90% 80%

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Utilizing the Tetrazine to Functionalize Nylon

NHN N

NH

O NR1R2

O NR1R2

Cl

O O

Cl

NHN N

NH

O NR1R2

O NR1R2

NR1R2

NR1R2

O

O

H2N NH2n

•A small molecule with a dienophile can potentially be attached to the tetrazine core within the polymer, utilizing the polymer as a transporter

•Potential drugs•Metal chelating agents•Highly specific receptors

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The Use of [6,6] nylon as a model

NHN N

NH

O NR

O NR

NR

NR

O

RN

O

O

RN

[NO]x

[Fe(III)]

NN N

N

O NR

O NR

NR

NR

O

RN

O

O

O

RN

•It is a relatively bulky polymer and requires new skills and techniques to analyze the chemistry

•Is optimal for determining the characteristic peaks of tetrazines within a polymer that can be applied to more useful polymers

• It is cheap and easy to synthesize

•Successful aromatization and Diels-Alder procedures involving the nylon can be applied to more useful polymers

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The functionalization of nylon

• The cycloaddition was performed at 40°C, which is potentially biorgthogonal conditions.

• Tetrazines have been used for biorthogonal Inverse Electron Demand Diels-Alder

NN N

N

O NR

O NR

NR

NR

O

RN

O

O

O

RN

DCM

40oC

MeN N

N

O NR

O NR

NR

NR

O

RN

O

O

O

RN

NMe

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Potential Application: Bisamide tetrazine sugar polymers

• The disubstituted diamine tetrazine will be able to attach two sugar polymers

• The tetrazine core has been shown to perform cycloadditions in nylon polymer, should be able to in other polymers such as sugars or algenates

OO

OH

HOOH

OH

NHN N

NH

OHN

O NH

NH2

NH2

EDCI, HOBt

DCM

OO

NH

HOOH

OH

HN O

NHN N

NH

O NH

HN

OO

HOOH

OH

[NO]x

OO

NH

HOOH

OH

HN O

NN N

N

O NH

HN

OO

HOOH

OH

XDrugO

O

NH

HOOH

OH

HN O

NN

O NH

HN

O

HOOH

OHXDrug

• The x-drug bond is a hydrolytic bond to release the drug from the sugar

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3,6 amino alcohol tetrazine derivatives

Two different active nucleophiles!

NHN N

NH

OHN

NHBoc

OH

NHN N

NH

O

OH

OMe

NHN N

NH

O

O

OMe

NHN N

NH

O

MeO O

OMe

Al(Me)3 NaBH4

H2N

NHBocAl(Me)3

DCM DCM DCM

NHN N

NH

OHN

NHBoc

OH

RX NHN N

NH

OHN

NHBoc

OR

Base TFA:DCM 1:1N

HN NNH

OHN

NH2

OR

• Has potential for amide bond coupling or Sn2 for diversification or cross linking of polymers

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Cross Linker vs Tethered

• Tethered- attached to one polymer by one of the nucleophilic ends

NHN N

NH

OH

HO

NHN N

NH

OHN

NH2

O NH

NH2

RCOOH, EDCI/HOBt

RX

NHN N

NH

OHN

NH

O NH

HN

O

R

O

R

NHN N

NH

OHN

NH

O NH

HN

R

R

NHN N

NH

OHN

NH2

O NH

NH2

Base

RX

Base NHN N

NH

OR

RO

NHN N

NH

OHN

NHBoc

OH

RXN

HN NNH

OHN

NHBoc

OR

Base

Cross linked- embedding the tetrazine within a polymer by both nucleophilic ends Return

Alternate route for bisamide tetrazine synthesis

• Unlike esters, the amides will not hydrolize during the protonation step, suggesting that the bisamide tetrazine can be made directly from the amide diazo compound

• The coupling reaction works with a wide range of amines

N NNHR1R2

O

NHR1R2

O

H2N

NaNO2

H2SO4OH

O

BocHN

EDCI, HOBtDCMNHR1R2

NaOH

NHN N

NH

O NHR1R2

O NHR1R2

[NO]xNN N

N

O NHR1R2

O NHR1R2

H2NOH

NBoc

HN

H2N

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