Rational Design, Development, and Stability Assessment of a Macrocyclic Four-Hydroxamate-Bearing Bifunctional Chelating Agent for 89Zr

Accepted Article

Title: Rational Design, Chemical Development and Stability

Assessment of a New Macrocyclic, Four-Hydroxamates-Bearing

Bifunctional Chelating Agent for 89Zr

Authors: Uwe Seibold, Björn Wängler, and Carmen Wängler

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To be cited as: ChemMedChem 10.1002/cmdc.201700377

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Rational Design, Chemical Development and Stability

Assessment of a New Macrocyclic, Four-Hydroxamates-Bearing

Bifunctional Chelating Agent for ⁸⁹Zr

Uwe Seibold^[a], Björn Wängler^[b], Carmen Wängler*^[a]

Abstract: ⁸⁹Zr is a positron-emitting radionuclide being of high

interest for medical imaging applications with Positron Emission

Tomography (PET). For the introduction of this radiometal into

biologically active targeting vectors, the chelating agent

desferrioxamine B (DFO) is commonly applied which is however

known to form ⁸⁹Zr-complexes of limited in vivo stability. We herein

describe the rational design and chemical development a new,

macrocyclic, four hydroxamates-bearing chelating agent (CTH36) for

the stable complexation of Zr⁴⁺. For this purpose, we first performed

computational studies to determine the optimal chelator geometry

before we developed different synthesis pathways towards the target

structures. The best results were obtained using an efficient solution

phase-based synthesis strategy towards the target chelating agent.

To enable an efficient and chemoselective conjugation to

biomolecules, a tetrazine-modified variant of CTH36 was also

developed. The excellent conjugation characteristics of the so-

functionalized chelator were demonstrated on the example of the

model peptide TCO-c(RGDfK). In the following, we determined the

optimal ⁸⁹Zr-radiolabeling parameters for CTH36 as well as its

bioconjugate and found that the ⁸⁹Zr-radiolabeling proceeds

efficiently under very mild reaction conditions. Finally, we performed

comparative complex stability tests for ⁸⁹Zr-CTH36-c(RGDfK) and

⁸⁹Zr-DFO-c(RGDfK), showing an improved complex stability for the

newly developed chelator CTH36.

Due to these favorable characteristics, ⁸⁹Zr has a high potential

for clinical applications, being reflected in several human

studies.^[2]

For the radiolabeling of biomolecules with ⁸⁹Zr⁴⁺, the chelating

agent desferrioxamine B (DFO) is commonly applied. However,

it has been shown in many studies that the ⁸⁹Zr-DFO-complex

exhibits an only limited stability under in vivo conditions.^[3]This is

a problem as the liberated ⁸⁹Zr⁴⁺-ion accumulates in mineral

bone over time due to its affinity to hydroxylapatite^[4]and

deposits significant doses there.^[4-5]

A reason for the instability of the ⁸⁹Zr-DFO-complex lies in the

incomplete saturation of the coordination sphere of the

radiometal: The oxophilic Zr⁴⁺ion prefers an octadentate ligand

sphere but is stabilized in its DFO-complex by the three

hydroxamates (and thus 6 oxygen atoms only) and two

additional anions or water molecules, leaving a considerable

6]

cleft in the ligand sphere of the complex.^[3c,Such clefts in

ligand spheres were however shown before to have a strongly

adverse effect on the resulting complex stabilities.^[7]

Consequently, it was proposed that a chelator comprising four

hydroxamate units should able to form ⁸⁹Zr-complexes of

significantly higher stability than DFO.^[8]

Besides the incomplete saturation of the coordination sphere of

the radiometal by DFO, this chelator exhibits a linear structure

which cannot take advantage of the stabilizing macrocycle effect

upon metal complexation.

Introduction

An ideal chelator for ⁸⁹Zr⁴⁺, being able to form highly stable

complexes with the radiometal, would be able to completely

saturate the octadentate coordination sphere of ⁸⁹Zr⁴⁺and

exhibit a cavity size matching the size of the central ion. Thus,

no cleft would be present in the ligand sphere, enabling a high in

vivo stability of the complex due to the complete envelopment of

the radiometal ion. Furthermore, as macrocyclic ligands in

⁸⁹Zr is a radiometal with high relevance for PET imaging

applications due to its long half-life of 3.27 days, matching the

biological half-life of slowly-accumulating biomolecules such as

antibodies. Thus, ⁸⁹Zr allows for imaging of biological processes

at late time-points after tracer application. Furthermore, the

relatively low mean energy of the emitted positrons of 0.389

MeV enables well-resolved PET images using this nuclide.^[1]

general give more stable complexes than linear ones,^[9]

a

macrocyclic structure of the chelator could also contribute to a

high complex stability due to the macrocycle effect upon

complexation.

[a]

[b]

Dr. U. Seibold, Priv.-Doz. Dr. Carmen Wängler

Biomedical Chemistry, Department of Clinical Radiology and

Nuclear Medicine

Although several promising new chelators for ⁸⁹Zr⁴⁺have been

developed in parallel to our attempts presented here^[10]– with

some of them showing a very high potential for successful in

vivo application due to a high stability of the complexes even at

late time-points after injection as shown by their antibody

conjugates which circulate over days before getting metabolized

and excreted^{[10b, 11]}– we followed in this work another approach

towards a new chelating agent for ⁸⁹Zr⁴⁺: We intended to design

and synthesize a macrocyclic tetra-hydroxamate with optimal

Medical Faculty Mannheim of Heidelberg University

Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany

E-mail: Carmen.Waengler@medma.uni-heidelberg.de

Prof. Dr. Björn Wängler

Molecular Imaging and Radiochemistry, Department of Clinical

Radiology and Nuclear Medicine

Medical Faculty Mannheim of Heidelberg University

Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/chem.2017xxxxx.

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ring size to completely saturate the oxophilic octadentate

coordination sphere of ⁸⁹Zr⁴⁺and additionally take advantage of

the macrocycle effect upon radiometal complexation.

and the parameter set PM6^[12]and based on the structures

depicted in figure 1, giving the result that the ring has to consist

of at least 36 atoms to minimize ring strain (for details, see SI), a

result which was also recently confirmed by another group.^[13]

Thus, 36 ring atoms should form the optimal cavity size for the

Zr⁴⁺ion as this size results on the one hand in a minimal ring

strain and on the other hand in less entropy effects than larger

ring sizes. This result was likewise found for both Zr-complex

templates, Zr-II and Zr-III (figure 1). Due to the assumption that

structure III should be more favorable than II regarding

biocompatibility and solubility of the chelator as well as the

complex in aqueous media, III should represent the most

promising molecular design for the target chelator.

Furthermore, the chelator should be applicable for

a

chemoselective and highly efficient conjugation to biomolecules.

The determination of the ideal (matching) ring size aims at the

complete envelopment of the central radiometal ion without

resulting in ring strain or entropic effects limiting the resulting

complex stability. In the following, we developed an efficient

chemical synthesis pathway towards the target chelating agent,

conjugated it to a model biomolecule, and performed ⁸⁹Zr-

radiolabeling as well as complex stability tests.

Results and Discussion

As already mentioned, a four hydroxamate-bearing macrocycle

should offer ideal properties for the stable complexation of Zr⁴⁺

under in vivo conditions as it should on the one hand be able to

completely saturate the coordination sphere of the Zr⁴⁺ion and

on the other hand enable the formation of stable complexes due

to the macrocycle effect.

However, these positive effects on stability can only be taken

advantage of if a chelator cavity size is found which fits the ion

radius of the central metal ion. Otherwise, the complex geometry

gets distorted by ring strain (if the cavity is too small) or entropic

effects can impede high complex stabilities (if the cavity is too

large and the chelator thus folds too flexibly around the

radiometal, leaving room for the interaction of the central ion),

resulting in a susceptibility of the complex towards complex

challenge and ion release. This can be attributed to an

increased probability of interaction of the central ion with other

ions or challenging molecules present in vivo at the site of

incomplete ion enclosure.

Figure 1. Schematic depiction of the complexation of Zr⁴⁺by four isolated

hydroxamate units (I) and potential macrocyclic, four hydroxamates-

comprising chelating agents (II and III) derived from these isolated

hydroxamates. These three structures were used as basis for the semi-empiric

MOPAC calculations intended to determine that ring size of II and III resulting

in minimal Zr⁴⁺-complex energies and thus maximum complex stabilities. The

ideal complex geometry was found for n=4.

In addition, we also performed density functional theory

calculations using the 3-21G basis set for the Zr⁴⁺-complex of III

in direct comparison to Zr⁴⁺-DFO (for details, see SI) in order to

determine if – in contrast to Zr⁴⁺-DFO – a complete envelopment

of the central metal ion can be achieved using the 36-membered

ring. The geometry-optimized structure results of these

calculations are depicted in figure 2 and show a completely

enveloped Zr⁴⁺-ion for the Zr⁴⁺-III-complex without cleft in the

coordination sphere as it is obvious for the Zr⁴⁺-DFO-complex.

Thus, we intended to first determine the ideal cavity size of a

potential four hydroxamate-bearing macrocycle for Zr⁴⁺before

commencing the development of a chemical synthesis pathway

towards the target chelator.

Rational design of the four hydroxamate-bearing

macrocycle by computation studies

This was achieved by computational studies which are able to

predict e.g. the lowest energy and thus highest stability of

different comparable complexes. The rational design was based

on the following assumptions: i) the chelator should comprise

four hydroxamates as these structure elements are believed to

result in exceptionally high complex stabilities as Zr⁴⁺is highly

oxophilic and four hydroxamates should be able to completely

saturate the postulated eight coordination sites, ii) it should be

macrocyclic to take advantage of the macrocyclic effect, iii) it

should be rotationally symmetric to enable the formation of a

symmetrical complex, limiting the probability of complex

challenge as the coordination sphere is uniformly closed around

the central ion, iv) it should be as hydrophilic as possible to

render it highly biocompatible and soluble in aqueous media.

The computational studies were then performed by semi-empiric

geometry optimization calculations using the software MOPAC

A

B

Figure 2. Geometry-optimized structure results of the DFT calculations of the

Zr⁴⁺-III-complex (A) and Zr⁴⁺-DFO(H₂O)₂(B). Hydrogen atoms were omitted for

clarity.

As consequence of our calculation results and due to the

assumption that structure III should be more favorable than II

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hydroxamate-building block 4 (for m=2 and n=1 (4_2,1): overall yield of 6–25%

over four steps; for m=1 and n=2 (4_1,2): overall yield of 4–46% over four steps).

B shows the tetramerization of 4, the synthesis of the benzyl-protected

hydroxamate-tetramer 6 (for m=2 and n=1 (6_2,1): percentage of product in raw

mixture: 20%, product not isolated; for m=1 and n=2 (6_1,2): overall yield of 37%

over 9 steps), its cyclization and deprotection to 7_2,3(overall yield of 37% over

two steps). DIPEA: N,N-diisopropylethylamine, DMF: N,N-dimethylformamide,

DPPA: diphenyl phosphoryl azide, HBTU: N,N,N’,N’-tetramethyl-O-(1H-

benzotriazol-1-yl)uronium hexafluorophosphate, HFIP: 1,1,1,3,3,3-hexafluoro-

2-propanol.

(figure 1) regarding biocompatibility and solubility in aqueous

media, we developed in the following a synthesis pathway for

the macrocyclic chelator based on structure III with a ring size of

36 ring atoms, termed in the following CTH36 (Cyclic Tetra-

Hydroxamate with a ring size of 36 atoms).

Synthesis of CTH36 via solid phase-assisted pathways

Initially, we developed a solid phase-assisted synthesis towards

CTH36, as by this approach, large excesses of reagent can be

applied resulting in principle in high reaction yields and pure

products. Furthermore, repeated purifications can be omitted,

simplifying and expediting the overall chemical synthesis.

In the initial attempt, we used the standard α-amino acid glycine

for the synthesis of 4_2,1(with the intention to later just replace

one glycine unit by a branched amino acid to obtain a backbone-

functionalized derivative of CTH36), resulting in a monomeric

hydroxamate building block exhibiting a short distance (one

methylene unit) between the hydroxamate and the amino

functionality (scheme 1A, m=2, n=1). This building block could

be synthesized using a 2-chloro-trity resin in four steps in

varying yields of 6–25%. From 4_2,1, the protected hydroxamate

tetramer 6_2,1could be assembled on solid support in moderate

yields of about 20% as determined by analytical HPLC of the

crude product mixture. However, 6_2,1showed to be highly

For this solid phase synthesis approach, we intended to develop

a procedure for the preparation of a benzyl protected tetra-

hydroxamate as a key intermediate for the macrocycle synthesis.

This intermediate should in the following be cyclized in solution

to the target chelator CTH36. In a first attempt, we tested if it is

possible to build the entire hydroxamate tetramer chain by

successive coupling of the components 5-bromopentanoic acid,

O-benzyl-hydroxylamine and Fmoc-glycine on resin. However,

this procedure showed to be unsuccessful – despite optimization

of several reaction steps – as during every reaction step, side

products were formed which resulted in an exponentiating

number of side products with increasing chain length.

susceptible to hydrolysis which prevented

a

successful

purification of the product. This observation is in accordance

with previously described findings for the synthesis of DFO-

analogs, reporting that the use of α-amino acids can result in

sequence deletions or the cleavage of the hydroxamate.^[14]

Therefore, we developed an alternative approach for the solid

phase-assisted hydroxamate-tetramer synthesis which consisted

of two steps: i) the preparation of monomeric protected

hydroxamate building blocks on solid support (4, scheme 1A),

and ii) their repeated conjugation on solid support to form the

linear tetra-hydroxamates which were intended – after cleavage

from the resin in hydroxamate-protected form (6) – to be

cyclized in solution and deprotected to the target macrocycle

CTH36 (scheme 1B).

As a result, we developed an analogous synthesis approach

using β-alanine and 4-bromo butanoic acid in contrast to the

previously applied α-amino acid glycine and 5-bromo pentanoic

acid to result in an unaltered ring size of 36 atoms while

“shifting” the position of the acid amides within the ring (scheme

1B). Following this strategy, again, the respective hydroxamate

monomer building block 4_1,2was synthesized first and obtained

in varying yields of up to 46%. For the subsequent assembly of

the protected hydroxamate-tetramer 6_1,2on solid support using

the new hydroxamate monomer 4_1,2, the synthesis parameters

were optimized to maximize product yields and purities. One

parameter which showed to influence product specifications was

the solid support used and the best results were obtained using

a

rink acid instead of

a

2-chloro-trityl resin. Further

improvements were made using PyBOP ((benzotriazol-1-

yloxy)tripyrrolidinophosphonium hexafluorophosphate) instead of

HBTU as activation agent during coupling. By these

improvements, the overall synthesis yields for 6_1,2(over four

steps) were increased to 37%. This new tetra-hydroxamate 6_1,2

also showed to be much more resistant against hydrolysis than

its tautomer 6_2,1and could be purified by HPLC without any

observed fragmentation.

Before the cyclization of 6_1,2to the protected macrocycle

intermediate could be performed in solution, the formate

counterion – introduced during the HPLC purification of 6_1,2

–

was removed by C18 cartridge purification to prevent the

formylation of the linear tetra-hydroxamate during the cyclization

reaction. The following cyclization was then performed in

solution under similar dilution conditions as described for the

Scheme 1. Schematic depiction of the solid phase-assisted synthesis towards

CTH36 (7_2,3). A shows the on-resin-synthesis and cleavage of the protected

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synthesis of the macrocyclic three hydroxamate moieties-

containing desferrioxamine-E.^[15]We however observed that

much longer reaction times of up to 20 days were obligatory to

achieve satisfying reaction rates. The following de-benzylation to

the target macrocycle CTH36 (7_2,3) (scheme 1B, n=2, p=3) was

performed by catalytic hydrogenation under standard conditions.

It is noteworthy that the reaction progress had to be monitored

carefully as otherwise, – if conducted for long reaction times – a

reduction of the hydroxamates took place. This observation is in

accordance with former reports.^[16]

of the non-functionalized macrocycle 7_2,3to give the fully

protected, backbone-modified macrocycle 15.

By this approach, a small amount of the desired macrocyclic

target molecule CTH36 (7_2,3) could be obtained. However, the

whole multistep preparation of 7_2,3unfortunately yielded

unsatisfactory low amounts of the target substance due to the

very low overall synthesis yield of only 0.3 % (referred to the

loading of the starting rink acid solid support). This can first be

attributed to the low yields for the solid phase-assisted synthesis

of 4_1,2and more important to the required excesses of at least

11 equivalents of 4_1,2which had to be applied during each step

of the tetramerization to 6_1,2to achieve complete conversions.

Incomplete conversions resulted in product mixtures containing

besides the target substance also shorter hydroxamate chains

which were nearly inseparable from the target compound.

Scheme 2. Schematic depiction of the solution phase synthesis pathway

towards the branched, hydroxamate-bearing β-amino acid building block 12

(A) and the solid phase-based synthesis towards the protected and backbone-

amino-functionalized

tetra-hydroxamate

macrocycle

15.

DIC:

diisopropylcarbodiimide, HOBt: 1-hydroxy-benzotriazole, TFA: trifluoro acetic

acid.

In parallel, we intended to establish a corresponding synthetic

approach towards

a backbone-functionalized derivative of

CTH36 (fCTH36) (scheme 2B), being applicable for further

modification and biomolecule derivatization reactions. For this

purpose, an appropriate, branched, hydroxamate-bearing β-

amino acid derivative with a suitable side chain functionality for

further derivatization and potential conjugation (12) had to be

synthesized first. This was initially performed analogously to the

synthesis of 4_1,2on solid support which however showed to be

rather inefficient, rendering it difficult to obtain the product in

gram scale for the following syntheses. Thus, an alternative

solution phase synthesis approach towards the required building

block was developed (scheme 2A).

Although not only the non-functionalized chelator CTH36 (7_2,3),

but also the protected and backbone-functionalized macrocycle

15 could be synthesized using the described solid phase-based

synthesis pathway, an alternative, solution phase-based

synthesis approach towards CTH36 and fCTH36 had to be

developed in order to be able to obtain larger amounts of the

target compounds. Of 15, e.g., only a few milligrams could be

obtained although starting with gram amounts of hydroxamate

monomers; the obtained product quantities were thus too small

for further deprotection, modification and biomolecule

derivatization being however the prerequisite for the application

of this new chelating agent in molecular imaging.

The synthesis of this monomeric hydroxamate building block

started from O-benzyl-hydroxylamine which was protected to its

N-(tert-butoxycarbonyl) derivative 8 and N-alkylated with 4-

bromo-butyric acid to 9, as described before.^[17]After Boc-

deprotection using dilute TFA, the resulting amine 10 was

conjugated to Fmoc-D-Dbu(Boc)-OH similar to a published

procedure^[18]using HOBt and DIC activation in order to receive

the fully protected building block 11 in good yields of 62%. 12

could be obtained from the protected intermediate 11 by

treatment with 1M aqueous NaOH solution which removed not

only the ethyl ester but also the Fmoc-protecting group which

thus had to be re-introduced by reacting the intermediate with

Fmoc-Cl in aqueous solution to give 12 in good yields over two

steps of 60%.

The solid phase-based approach – although being in principle

successful – did thus not measure up with our expectations as

we initially intended to develop a solid phase-assisted synthesis

strategy to achieve high conversion rates and thus high yields

for our target compounds, an aim which was not achieved due to

a large number of side reactions or required building block

excesses.

Solution phase synthesis of CTH36 and its protected,

backbone-functionalized analog fCTH36

Thus, a new synthesis route towards CTH36 and its backbone-

functionalized analog had to be developed. For this reason, we

in the following focussed on the development of a conventional

organic liquid phase synthesis – enabling a comparatively

convenient upscaling – to significantly improve the synthesis

This building block 12 was in the following applied during the

synthesis of the linear, backbone-functionalized tetra-

hydroxamate 14 on solid support (scheme 2B) and also during

the later on developed solution phase approach (vide infra). 14

could be cyclized in solution as established during the synthesis

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efficiency and to be able to obtain sufficient amounts of

substance for further processing.

obtained after treatment of the Boc-protected educts with dilute

TFA, to remove remains of TFA completely before the next

conjugation step can be performed. For this purpose, the crude

deprotected amines were thoroughly washed with aqueous

carbonate solution. Otherwise, the TFA remains can significantly

decrease the yields of the target compounds due to the

formation of trifluoro-amides whose removal from the product

mixture is furthermore highly intricate.

A challenge during the development of this new synthesis

strategy was the susceptibility of the hydroxamates to various

reaction conditions, easily resulting in product fragmentation.

Furthermore, a suitable and complementary protecting group

chemistry had to be found to enable a chain elongation to the

target tetra-hydroxamates without side reactions in order to

obtain the desired products with high synthesis efficiency.

The linear, terminally protected tetra-hydroxamate 21 was in the

following treated first with acid and then base to remove the Boc

protecting group as well as the ethyl ester to give the terminally

deprotected intermediate 22. This intermediate interestingly

showed to be only moderately stable in aqueous acid and base

and attempts to purify larger amounts than a few milligrams

were not successful. However, 22 can also be used without

further purification for the following cyclization reaction being

performed in diluted solution with DPPA over 18 days, giving the

protected macrocycle 23 in 20% overall yield over three steps,

starting from 21.

The new synthesis strategy comprised – just as the solid phase-

based approach – first the synthesis of suitable, protected,

hydroxamate monomer building blocks (17 and 18, scheme 3)

which subsequently can be assembled to the respective

hydroxamate di-, tri- and tetramers. Using four non-branched

hydroxamate monomer units, the protected, non-backbone-

functionalized hydroxamate tetramer 21 (scheme 4) could be

obtained. Applying three non-branched hydroxamate monomers

and 12, the protected, backbone-functionalized hydroxamate

tetramer 25 (scheme 5) could be synthesized. These protected,

linear tetra-hydroxamates were then terminally deprotected and

cyclized to the respective protected macrocycles 23 and 26.

23 was in the following further reacted to the non-functionalized

target chelator 24 (CTH36).

In the final reaction step, the hydroxamate units were de-

benzylated by hydrogenation, giving the deprotected, non-

backbone-functionalized macrocyclic tetra-hydroxamate CTH36

(24) – which demonstrated to exhibit a poor solubility in most

organic solvents and in water – in good yields of 79%.

Scheme 3. Schematic depiction of the synthesis of the monomeric

hydroxamate building blocks 17 and 18.

The synthesis of the monomeric hydroxamate building blocks 17

and 18 started from the amine 10 (vide supra) which was

reacted with Boc-β-alanine using HOBt and DIC activation to

receive 16 in high yields of 95%. The complementarily protected

monomeric hydroxamate building blocks 17 and 18 were

obtained from 16 by treatment with dilute TFA (to obtain the

carboxyl-reactive monomer 17) or NaOH (to obtain the amino-

reactive monomer 18) in almost quantitative or quantitative

yields. These monomers could in the following be reacted with

each other or 12 to achieve the required chain elongation to the

desired linear hydroxamate di-, tri- and tetramer intermediates

19, 20, 21 and 25 (schemes 4 and 5).

Scheme 4. Schematic depiction of the solution phase synthesis of the

deprotected, non-backbone-functionalized macrocyclic tetra-hydroxamate

target chelator CTH36 (24).

In detail, the non-backbone-functionalized, protected tetra-

hydroxamate 21 was obtained by threefold subsequent reaction

of 17 with 18 under standard HOBt / DIC conjugation conditions,

giving the linear protected di-, tri- and tetra-hydroxamates 19, 20

and 21 in high yields of 86%, 97% and 90%, respectively.

It should be noted here – although being self-evident – that it is

essential for the amino-deprotected intermediates, which were

The

corresponding

protected,

backbone-functionalized

macrocyclic tetra-hydroxamate pfCTH36 (26), whose backbone

amino functionality was intended for further derivatization and

finally biomolecule-derivatization, was synthesized in a similar

way than 23 (scheme 5) and was the highly stable key

intermediate for the development of a suitable conjugation

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chemistry of the backbone-functionalized tetra-hydroxamate

macrocycle. To obtain 26, first, the protected, branched tetra-

hydroxamate 25 was synthesized from 20 by according

synthesis protocols as applied for the synthesis of 21 and

obtained in good yields of 59% referring to 20. 25 was then

further reacted under basic conditions to simultaneously remove

the ethyl ester as well as the Fmoc protecting group and the

obtained intermediate was cyclized without previous purification

(due to its instability in solution as observed before for 22) to

give the target intermediate pfCTH36 (26) in good yields of 53%

over two steps, starting from 25.

purpose, the terminal amino functionality of DFO is first modified

with succinic anhydride to generate a carboxylic acid which can

be converted (after temporary blocking of the hydroxamates by

Fe³⁺-complexation) into the respective active ester. After the

conjugation of the chelator to the biomolecule, the iron ion can

be removed from the complex via an excess of DTPA. This

approach – although working quite well for DFO – can however

not be adapted to fCTH36 since Fe³⁺is able to prevent only

three of the four hydroxamate units from participating in the

activation of the carboxylic acid, resulting in the formation of a

significant number of side-products during the formation of an

active ester of CTH36.

Our attempts thus focussed on developing another approach to

obtain a reactive species of fCTH36 which can be used for the

biomolecule conjugation of the chelator. In detail, we tested the

following approaches depicted in scheme 6.

Scheme 5. Schematic depiction of the solution-phase synthesis of the

protected, backbone-functionalized macrocyclic tetra-hydroxamate pfCTH36

(26).

Scheme 6. Schematic depiction of the different attempts made to generate a

reactive species of fCTH36 applicable for conjugation of the macrocycle to

biomolecules.

Thus, and although comprising multi-step syntheses and being

challenging due to the susceptibility of the hydroxamates, the

solution phase-approach towards the target chelators CTH36

and its backbone-functionalized analog pfCTH36 showed to be

successful. The synthesis was based on a complementary

protecting group chemistry and optimized so that the target

chelators could be obtained in satisfactory overall yields.

Of the studied synthesis pathways, approach i) (to activate and

conjugate unprotected fCTH36 via its free amino functionality

being converted into an active ester in situ) was – as expected –

not successful due to the formation of a large number of side

products; approach ii), aiming at the protection of the

hydroxamates with highly acid-labile trimethyl-silyl or tert-butyl-

dimethyl-silyl protecting groups during the conversion of the

amino function of the chelator into first an acid and afterwards

into an NHS active ester was also not successful as the

formation of the target silyl-protected intermediates could neither

be proven nor could the products been isolated; approach iii), to

react the free amino functionality of the chelator with the bis-

NHS ester di(N-succinimidyl) glutarate (with the aim to obtain an

NHS active ester derivative of the chelator) demonstrated to be

feasible for DFO but not for fCTH36 due to the low solubility of

the latter which resulted in such long reaction times of fCTH36

with the bis-NHS ester that the formation of a significant number

of hydroxamate hydrolysis and other side products was

observed.

Development of a suitable conjugation chemistry for the

introduction of the backbone-functionalized macrocycle

into biomolecules

Next step in the development of

a macrocyclic tetra-

hydroxamate chelator applicable for the stable ⁸⁹Zr-labeling of

biomolecules was the development of a suitable conjugation

chemistry allowing the introduction of

a deprotected and

activated version of the backbone-modified chelator 26 into

biomolecules. This biomolecule conjugation should ideally be

able to proceed under mild reaction conditions to principally

enable the conjugation not only to relatively robust peptides but

also to susceptible biomolecules such as antibodies.

Furthermore, the formed conjugates should be stable under in

vivo imaging conditions. These requirements are fulfilled by

different conjugation chemistries and we tested some of them

regarding their applicability towards fCTH36 conjugation.

In contrast to the before mentioned attempts to generate a non-

chemoselectively reacting activated species of fCTH36,

approach iv) finally demonstrated to be successful: the

derivatization of fCTH36 with a tetrazine moiety (scheme 7).

This activated CTH36 analog is able to react via the inverse

The most established route for DFO introduction into

biomolecules is its conjugation via active esters. For this

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electron demand Diels-Alder (iEDDA) reaction with TCO-

modified bioactive molecules (TCO: trans-cyclooctene). This

iEDDA reaction type has gained increasing popularity over the

last years as it proceeds chemoselectively under mild conditions

via very fast reactions kinetics and has thus also found

widespread application in radiochemistry.^[19]Furthermore,

another advantage of this tetrazine-derivatized fCTH36 analog is

that it does not require blocking or de-blocking steps for the

hydroxamate units during and after macrocycle conjugation.

To evaluate the chosen tetrazine/trans-cyclooctene conjugation

method regarding its applicability for the introduction of the

newly developed chelator into bioactive molecules, a TCO-

modified model peptide was synthesized (TCO-c(RGDfK), 33).

To obtain 33, first, c(RGDfK) (31) was synthesized using

standard peptide synthesis methods and the amino functionality

of the peptide was in the following reacted with the commercially

available TCO-p-nitrophenyl active ester 32 (scheme 8). TCO-

c(RGDfK) was subsequently used as model reaction partner for

tCTH36 (30) to show its applicability in biomolecule-

derivatization via the chemoselective iEDDA reaction.

Scheme 8. Schematic depiction of the synthesis of the TCO-modified

c(RGDfK) analog 33 used as model biomolecule for the following chelator-

conjugation reaction.

Scheme 7. Schematic depiction of the reaction pathway towards the tetrazine-

modified tetra-hydroxamate macrocycle tCTH36 (30). EDC: (1-ethyl-3-(3-

dimethylamino-propyl)carbodiimide.

Besides evaluation of the chosen approach regarding its

applicability to biomolecule modification with tCTH36, we also

intended to synthesize a biomolecule conjugate of the chelator

to be able to evaluate the ⁸⁹Zr-radiolabeling properties of the

newly developed chelator under radiotracer conditions, where

the chelator is usually conjugated to a more complex carrier

molecule. Furthermore, a biomolecule conjugate of the chelator

further allows to study the ⁸⁹Zr-complex stability under more

realistic radiotracer conditions as if the non-conjugated, “naked”

complex were used.

As we further intended to directly compare the ⁸⁹Zr-complex

stability of our new chelator CTH36 to that of DFO, we also

synthesized a tetrazine-modified DFO analog tDFO (34) which

served as the according reaction partner to tCTH36 in the

reaction with TCO-c(RGDfK). In detail, the tetrazine-modified

DFO analog tDFO (34) could be prepared according to

published procedures: After reaction of DFO-mesylate with

succinic anhydride,^[21]the resulting N-succinyl-desferrioxamine B

was conjugated to the commercially available amino-tetrazine

In detail, the synthesis of 30 was performed starting from the

fully protected backbone-functionalized tetra-hydroxamate

intermediate 26 of which the Boc-protecting group was removed

by treatment with dilute TFA, yielding 27. The liberated amino

functionality of 27 was in the following reacted with the tetrazine-

building block 28, obtained by the reaction of (4-(1,2,4,5-tetrazin-

3-yl)benzylamine with succinic anhydride according to

a

published procedure.^[20]During the conjugation reaction of 28 to

27, the formation of side products could be observed which likely

were formed by rearrangements of 27 under the basic conditions

applied. The formed side products could however be separated

by semipreparative HPLC from the desired product 29. In the

final reaction step, the benzyl protecting groups were removed

from the hydroxamates by hydrogenation, giving 30 in

quantitative yields. Interestingly, the tetrazine moiety became

also reduced under these conditions (observable by a color shift

from pink to colorless during hydrogenation) but readily re-

oxidized in air (observable by the reverse color shift from

colorless to pink within minutes).

containing

building

block

4-(1,2,4,5-tetrazine-3-yl)-

benzenemethanamine.^[22]

Thus, we were successful in developing an appropriate

approach to obtain a highly reactive backbone-functionalized

analog of CTH36. With this tetrazine-modified macrocycle

tCTH36 at hand, the introduction of the chelator into bioactive

molecules became feasible and was performed in the following

on the example of a TCO-modified peptide.

Both chelator-tetrazine derivatives tCTH36 (30) as well as tDFO

(34) showed very fast reaction kinetics with TCO-c(RGDfK) (33):

Both reactions were finished within seconds (comprehensible by

means of the immediate decolorization of the solution) and gave

the peptide-chelator-conjugates CTH36-c(RGDfK) (35) and

DFO-c(RGDfK) (36) in good yields of 65% and 42%,

respectively (scheme 9). As determined by analytical HPLC, the

formed products were obtained as a mixture of different isomers

and tautomers, an effect having been described for this reaction

Conjugation of tCTH36 to bioactive molecules on the

example of TCO-modified c(RGDfK)

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type before,^[23]and being not adverse especially in case of the in

future intended modification of antibodies using this approach.

In the following, we intended to determine the optimal pH range

enabling a highly efficient ⁸⁹Zr-incorporation into CTH36. For this

purpose, 24 was incubated with ⁸⁹Zr-oxalate in HEPES-buffered

(HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid)

solution at different pH values and the progress of the

radiolabeling reactions was monitored by analytical radio-HPLC.

The results of these experiments are depicted in figure 3 and

show the most efficient radiometal incorporation rates in a pH

range of 7 to 9, enabling a complexation of ≥90% of the activity

within 5 minutes reaction time at ambient temperature.

H

O

N

O

HN

N

OH

O

HO

N

HN

O

H

N

33

DMSO

65%

O

N

O

N

30

OH

O

NH

OH

NH

H

OH

N

O

N

NH

HN

N

NH

O

H

O

35

H

N

O

H₂

N

H

O

NH

H

N

H₂N

N

HN

O

OH

N

O

33

DMSO

42%

H

N

O

N

34

HN

NH

N

OH

N

OH

N

H

O

N

H

O

HN

H

N

36

OH

O

Scheme 9. Schematic depiction of the conjugation of both chelating agents,

tCTH36 and tDFO to TCO-modified c(RGDfK), giving the peptide-chelator-

conjugates CTH36-c(RGDfK) (35) and DFO-c(RGDfK) (36).

Thus, it could be demonstrated that tCTH36 is able to react

chemoselectively and with extremely high efficiency with

appropriately modified biomolecules.

Using 24 and the CTH36- and DFO-chelator-peptide-conjugates

35 and 36, we in the following performed the ⁸⁹Zr-radiolabeling

and comparative complex stability experiments.

Determination of optimal ⁸⁹Zr-radiolabeling conditions, pH

dependence of the labeling reaction and comparative

complex stability

Figure 3. Depiction of the results of the ⁸⁹Zr-radiolabeling experiments

investigating the pH dependence of the CTH36-⁸⁹Zr-labeling reaction. The

values are given SD and each experiment was performed thrice.

At first, the optimal ⁸⁹Zr-radiolabeling conditions were

determined for the non-functionalized chelator CTH36 (24). In

these initial experiments, we observed a rapid and almost

quantitative complexation of the radiometal within 30 to 60

minutes under mild reaction conditions such as ambient

temperature and neutral pH as it was already reported for

DFO.^[24]

Next step was the determination of the optimal ⁸⁹Zr-radiolabeling

conditions for the peptide conjugates CTH36-c(RGDfK) (35) and

DFO-c(RGDfK) (36). Both peptide conjugates could be labeled

under similar conditions as the non-conjugated chelators at

ambient temperature and neutral pH and showed ⁸⁹Zr-

incorporation rates of ≥96% and non-optimized molar activities

of 8.4–9.2 GBq/µmol for [⁸⁹Zr]35 and 4.0–4.6 GBq/µmol for

[⁸⁹Zr]36 after a reaction time of one hour, being comparable to

the results being recently reported for other ⁸⁹Zr-

bioconjugates.^{[10, 11b]}

Thus, the cyclization of the tetra-hydroxamate to a macrocycle

did not result in slowed complex formation kinetics resulting in

prolonged reaction times or the necessity to perform the

radiolabeling reaction at elevated temperatures to achieve a

complete radiometal incorporation as it is often observed for

macrocyclic chelating agents compared to their linear

counterparts.

With these ⁸⁹Zr-labeled peptide conjugates, the ⁸⁹Zr-complex

stability of the new chelator CTH36 could be directly compared

to that of the commonly used ⁸⁹Zr-DFO. For this purpose,

transchelation challenge experiments were performed: [⁸⁹Zr]35

and [⁸⁹Zr]36 were incubated with large excesses of EDTA (100-,

1000- and 9000-fold) as competing chelator at neutral pH and

ambient temperature. The transchelation rate from [⁸⁹Zr]35 and

[⁸⁹Zr]36 to [⁸⁹Zr]EDTA was monitored by radio-HPLC and the

results of these experiments are depicted in figure 4.

This is an important finding as the long-term objective for the

application of tCTH36 is the derivatization and subsequent ⁸⁹Zr-

radiolabeling of antibodies. ⁸⁹Zr is ideally suited for radiolabeling

and molecular imaging purposes using this biomolecule class as

the half-life of the radiometal matches the biological half-life of

the antibody molecule well. However, antibodies are susceptible

to harsh radiolabeling conditions such as elevated reaction

temperatures and highly acidic or basic reaction parameters.

Thus, the observed mild and efficient ⁸⁹Zr-labeling of tCTH36-

conjugates is well-compatible with antibody-⁸⁹Zr-labeling.

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efficiently under very mild reaction conditions. Complex

challenge stability evaluations of ⁸⁹Zr-CTH36-c(RGDfK) in direct

comparison to ⁸⁹Zr-DFO-c(RGDfK) revealed an improved

complex stability for the newly developed chelator ⁸⁹Zr-CTH36

compared to ⁸⁹Zr-DFO, indicating its high potential for immuno-

PET imaging applications.

Experimental Section

General

All commercially available reagents and solvents were of analytical grade

or better and were used without further purification. The resins for

peptide synthesis, coupling reagents, and Fmoc-protected amino acids

were obtained from NovaBiochem (Darmstadt, Germany). (S)-3-(Fmoc-

amino)-4-(Boc-amino)butyric acid (Fmoc-D-Dbu(Boc)-OH) was obtained

from Iris-Biotech (Marktredwitz, Germany), Desferrioxamine B (DFO)

mesylate and Palladium on carbon were obtained from Sigma-Aldrich

(Schnelldorf, Germany) and (E)-Cyclooct-4-ene p-nitrophenyl active ester

(32) and (4-(1,2,4,5-tetrazine-3-yl)phenyl)methanamine formate were

purchased from Sirius Fine Chemicals GmbH (Bremen, Germany). For

column chromatography, silica gel 60 for chromatography, 0.040–0.063

mm, 230–400 mesh (Merck, Germany) was used. ⁸⁹Zr-oxalate solution

for radiolabeling experiments was obtained from Cyclotron VU

(Amsterdam, Netherlands). The synthons 8^{[17a, 17b]}, 9^[17c], 28^[20]and 34^[3d,

Figure 4. Depiction of the results of the complex challenge experiments of the

⁸⁹Zr-labeled chelator-peptide conjugates [⁸⁹Zr]35 and [⁸⁹Zr]36 with 100-, 1000-

and 9000-fold excess of EDTA as challenging chelating agent. The graph

shows the transchelation rates from [⁸⁹Zr]35 and [⁸⁹Zr]36 to [⁸⁹Zr]EDTA as

monitored by radio-HPLC. The values are given SD and each experiment

was performed thrice. Different time points were used for each challenge

experiment so that the curves for 100-fold and 1000-fold excess of EDTA were

cut off for a clearer overall presentation of the data.

25]

were prepared according to published procedures. For analytical and

semi-preparative chromatography, a Dionex UltiMate 3000 system was

used together with a Chromolith Performance (RP-18e, 100–4.6 mm,

Merck, Germany) and a Chromolith (RP-18e, 100–10 mm, Merck,

Germany) column, respectively, operated at a flow rate of 4 mLmin^-1and

H₂O and MeCN +0.1% formic acid as eluents. For radio-HPLC

chromatography, a Dionex UltiMate 3000 system was used together with

a Phenomenex Gemini (5µm, C18, 250–4.6 mm) column, operated at a

flow rate of 2 mLmin^-1(5 min at 15% MeCN in H₂O with 0.1% TFA,

followed by 15 to 50% MeCN in H₂O with 0.1% TFA within 15 minutes as

the gradient). Unless otherwise stated, product purities were ≥95%.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric

data were collected using a Bruker Daltonics Microflex spectrometer

(Bremen, Germany). NMR spectra were recorded on a Varian 500 MHz

NMR system.

The ⁸⁹Zr-CTH36-complex in all experiments showed less and

slower transchelation than the respective ⁸⁹Zr-DFO-complex.

The difference in transchelation is thereby – as expected –

depending on the excess of competing chelator and most

pronounced using an excess of 9000 equivalents of EDTA.

These results thus indicate a higher kinetic complex stability for

⁸⁹Zr-CTH36 compared to ⁸⁹Zr-DFO. Comparative in vivo stability

determinations with ⁸⁹Zr-labeled antibody conjugates of tCTH36

and tDFO and PET imaging studies using these ligands are

underway.

General description of the deprotection and coupling procedure

applied during the synthesis of substances 10, 11, 16, 17, 19, 20, 21,

25 and 27. Deprotection: To a solution of Boc-amino ester (9, 16, 19, 20,

21 and 26; 1.0 equiv.) in a small amount of CH₂Cl₂(just enough to solve

the educt), TFA (5–8 equiv.) was added and the mixture was stirred for 5

h at room temperature before being evaporated to dryness. The residue

was redissolved in a small amount of CH₂Cl₂and washed three times

with 10% Na₂CO₃-solution to remove the TFA completely. After

evaporation of the organic solvent, the resulting deprotected amino ester

was used for further coupling without purification. Coupling: Amino ester

(10, 17; 1.0 equiv.), protected amino acid (Boc-β-Alanine, 12, 18; 1.02

equiv.) and 1-hydroxybenzotriazole (0.1 equiv.) were dissolved in ice-

cooled, dry acetonitrile. N,N’-diisopropylcarbodiimide (1.1 equiv.) was

added and the reaction mixture was kept at 0–3°C for 4 h and in the

following at ambient temperature overnight. The formed precipitate was

removed by filtration and the solution was concentrated in vacuo before

the product was purified by column chromatography on silica gel. Details

for every substance are given in the following.

Conclusions

We developed a new chelating agent for the complete and

symmetrical encapsulation and thus stable complexation of

⁸⁹Zr⁴⁺. For this purpose, we performed a rational design and

determined the optimal target chelator geometry by

computational studies. The preparation of the target structures –

comprising multi-step syntheses and a sophisticated protecting

group chemistry – was successful, showed to be most feasible

by solution phase procedures and gave the non- as well as the

tetrazine-functionalized chelators CTH36 and tCTH36 in good

overall yields. The highly efficient and chemoselective

conjugation of tCTH36 to biomolecules could be demonstrated

on the example of the bioactive model peptide TCO-c(RGDfK)

and the ⁸⁹Zr-radiolabeling of the new chelator proceeds

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Ethyl-(S)-4-(N-(benzyloxy)-4-(Boc-amino)-3-(Fmoc-amino)-

¹H NMR (300 MHz, [D₃]CDCl₃,

25°C): δ=8.37 (s_broad, 1H, H-

13), 7.75 (d, ³J(H,H)=7.4 Hz,

2H, H-42 + H-43), 7.59 (d,

³J(H,H)=7.4 Hz, 2H, H-39 + H-

46), 7.38 (td, ^3,4J(H,H)=7.6 +

1.3 Hz, 2H, H-41 + H-44),

7.35 (s_broad, 5H, H-1 – H-3 +

butanamido)-butanoate (11).

11 was obtained as described before in the general description by

deprotection of Boc amino ester (2.64 g, 11.13 mmol) and

9

condensation with (S)-3-(Fmoc-amino)-4-(Boc-amino)butyric acid (5.00 g,

11.35 mmol). The product was purified by column chromatography using

cyclohexane / ethyl acetate 1:1 as eluent and obtained as colorless oil in

62% yield (4.53 g, 6.90 mmol).

H-5

+

H-6), 7.29 (td,

³J(H,H)=7.4 + 1.3 Hz, 2H, H-40 + H-45), 6.05 (s_broad, 1H, H-20), 5.05

(s_broad, 1H, H-25), 4.79 (s, 2H, H-7), 4.33 (d, ³J(H,H)=7.2, 2H, H-33), 4.18

¹H NMR (300 MHz,

[D₃]CDCl₃, 25°C): δ=7.76

(d, ³J(H,H)=7.4 Hz, 2H, H-

(t, ³J(H,H)=7.0 Hz, 1H, H-34), 4.04 (s_broad, 1H, H-19), 3.71 (t_broad

³J(H,H)=5.4 Hz, 2H, H-9), 3.31 (s_broad

2H, H-24), 2.79 (d_broad

,

²J(H,H)=15.4 Hz, 1H, H-17b), 2.54 (dd, ^2,3J(H,H)=16.2 + 6.1 Hz, 1H, H-

17a), 2.37 (t, ³J(H,H)=7.0 Hz, 2H, H-11), 1.95 (p, ³J(H,H)=6.8 Hz, 2H, H-

10), 1.42 (s, 9H, H-30 + H-31 + H-32). ¹³C NMR (75 MHz, [D₃]CDCl₃,

25°C): δ=176.99 (C-12), 172.64 (C-16), 156.45 + 157.06 (C-21 + C-26),

144.04 (C-35 + C-38), 141.39 (C-36 + C-37), 134.14 (C-4), 129.45 (C-2 +

C-6), 129.25 (C-1), 128.90 (C-3 + C-5), 127.78 (C-41 + C-44), 127.17 (C-

42 + C-43), 125.32 (C-40 + C-45), 120.05 (C-39 + C-46), 79.86 (C-29),

76.47 (C-7), 67.00 (C-33), 49.29 (C-19), 47.31 (C-34), 44.41 (C-24),

43.85 (C-9), 34.19 (C-17), 31.30 (C-11), 28.47 (C-30 + C-31 + C-32),

22.06 (C-10). MALDI-MS (calculated): m/z=632.34 [M+H]⁺(632.30),

654.41 [M+Na]⁺(654.28), 670.31 [M+K]⁺(670.25).

18

³J(H,H)=7.4 Hz, 2H, H-21

H-22), 7.39 (td,

^3,4J(H,H)=6.8 + 1.2 Hz, 2H,

H-19 + H-24), 7.37 (s_broad

+

H-25), 7.60 (d,

+

,

5H, H-31 – H-35), 7.30 (td,

^3,4J(H,H)=7.4 + 1.2 Hz, 2H, H-20 + H-23), 5.95 (s_broad, 1H, H-7), 4.99

(s_broad, 1H, H-38), 4.80 (s, 2H, H-29), 4.35 (dt, ^3,4J(H,H)=7.6 + 3.2 Hz, 2H,

H-11), 4.20 (t, ³J(H,H)=7.0 Hz, 1H, H-12), 4.10 (q, ³J(H,H)=7.1 Hz, 2H, H-

47), 4.04 (m, 1H, H-6), 3.70 (m, 2H, H-8), 3.33 (m, 2H, H-46), 2.82 (d_broad

,

²J(H,H)=15.4 Hz, 1H, H-5b), 2.55 (dd, ^2,3J(H,H) = 16.0 + 6.0 Hz, 1H, H-

5a), 2.33 (t, ³J(H,H)=7.2 Hz, 2H, H-3), 1.96 (p, ³J(H,H)=7.0 Hz, 2H, H-4),

1.43 (s, 9H, H-43 – H-45), 1.21 (t, ³J(H,H)=7.1 Hz, 3H, H-48). ¹³C NMR

(75 MHz, [D₃]CDCl₃, 25°C): δ=173.00 (C-2 + C-36), 156.24 + 156.74 (C-

9 + C-39), 144.12 (C-14 + C-17), 141.40 (C-15 + C-16), 134.17 (C-30),

129.46 (C-32 + C-34), 129.23 (C-33), 128.89 (C-31 + C-35), 127.77 (C-

19 + C-24), 127.16 (C-18 + C-25), 125.32 (C-20 + C-23), 120.05 (C-21 +

C-22), 79.74 (C-42), 76.47 (C-29), 66.89 (C-11), 60.61 (C-47), 49.29 (C-

6), 47.36 (C-12), 44.57 (C-8), 43.82 (C-46), 34.09 (C-5), 31.52 (C-3),

28.49 (C-43 – H-45), 22.25 (C-4), 14.31 (C-48). MALDI-MS (calculated):

m/z=660.27 [M+H]⁺(660.32), 682.32 [M+Na]⁺(660.31), 698.40 [M+K]⁺

(698.28).

Ethyl 4-(N-(benzyloxy)-3-((tert-butoxycarbonyl)-amino)-

propanamido)-butanoate (16).

16 was obtained as described before in the general description by

deprotection of Boc amino ester 9 (6.93 g, 29.19 mmol) to 10 and

condensation with Boc-β-alanine (5.53 g, 29.21 mmol). The product was

purified by column chromatography using cyclohexane / ethyl acetate 2:1

as eluent and obtained as colorless oil in 95% yield (11.38 g, 27.73

mmol).

¹H NMR (300 MHz, [D₃]CDCl₃,

(S)-4-(N-(benzyloxy)-4-(Boc-amino)-3-(Fmoc-amino)-butanamido)-

25°C): δ=7.37 (s_broad, 5H, H-1 –

H-3 + H-5 + H-6), 5.16 (s_broad,

butanoic acid (12).

1H, H-22), 4.78 (s, 2H, H-7),

4.10 (q, ³J(H,H)=7.1 Hz, 2H, H-

11 (4.53 g, 6.87 mmol) was treated with aqueous sodium hydroxide

solution (1M, 2.75 equiv., 18.9 mL, 18.9 mmol) and afterwards, the

mixture was diluted with methanol (4 mL) and stirred overnight at

ambient temperature to remove the ethyl ester. Under these conditions,

the Fmoc-protecting group is removed as well. The resulting suspension

was concentrated in vacuo and stored at 3 °C for several hours. The

precipitate was removed by filtration and rinsed with H₂O (thrice ∼10 mL).

The resulting, cloudy solution was filtrated over Celite, the Celite was

washed with H₂O (twice ∼50 mL) and the solution was concentrated to a

volume of 40 mL. NaHCO₃(4 g, 47.6 mmol) was added and the mixture

was cooled in an ice bath. To this mixture was dropwise added a solution

of Fmoc-Cl (2.32 g, 9.00 mmol) in dioxane. After complete addition, the

reaction mixture was stirred another two hours at 0–4 °C and overnight at

ambient temperature before it was lyophilized to a colorless solid, which

was extracted two times with diethylether (100 mL). This solution was

further diluted with ethyl acetate (100 mL) and acidified to pH 2 with

aqueous sodium hydrogen sulfate solution (20%). The aqueous layer

was separated and extracted with ethyl acetate (50 mL). The combined

organic extracts were dried over MgSO₄and evaporated to dryness. The

product was purified by column chromatography with ethyl acetate /

cyclohexane 1:1 containing 1 % acetic acid as eluent and obtained as

colorless, non-crystalline solid in 60% yield (2.62 g, 4.12 mmol).

15), 3.69 (t, ³J(H,H)=6.7 Hz, 2H,

H-9), 3.37 (t, ³J(H,H)=5.8 Hz,

2H, H-21), 2.60 (t, ³J(H,H)=5.7 Hz, 2H, H-19), 2.31 (t, ³J(H,H)=7.3 Hz, 2H,

H-11), 1.94 (p, ³J(H,H)=7.2 Hz, 2H, H-10), 1.41 (s, 9H, H-27 – H-29),

1.22 (t, J(H,H)=7.1 Hz, 3H, H-16). ¹³C NMR (75 MHz, [D₃]CDCl₃, 25°C):

3

δ=173.7 (weak, C-18), 172.91 (C-12), 156.02 (C-23), 134.21 (C-4),

129.37 (C-2 + C-6), 129.17 (C-1), 128.84 (C-3 + C-5), 79.21 (C-26),

77.16 (CDCl₃), 76.51 (C-7), 60.54 (C-15), 44.59 (C-9), 36.12 (C-21),

32.81 (C-19), 31.40 (C-11), 28.51 (C-27 – C-29), 22.33 (C-10), 14.31 (C-

16). MALDI-MS (calculated): m/z=408.98 [M+H]⁺(409.23), 430.95

[M+Na]⁺(431.22), 447.04 [M+K]⁺(447.19).

4-(N-(benzyloxy)-3-(Boc-amino)propanamido)butanoic acid (18).

To an ice-cooled solution of 16 (7.50 g, 18.4 mmol) in methanol (100 mL)

was slowly added an aqueous sodium hydroxide solution (1M, 20 mL, 20

mmol). After warming to ambient temperature and stirring overnight, the

solvent was removed in vacuo. The residue was dissolved in ethyl

acetate (50 mL) and washed two times with 20% aqueous sodium

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hydrogen sulfate solution. The aqueous phase was extracted with ethyl

acetate (20 mL) and the combined organic solutions were dried over

MgSO₄, evaporated to dryness and afforded the product acid in

quantitative yield (7.03 g, 18.4 mmol).

20 was obtained as described before in the general description by

deprotection of 19 (1.33 g, 2.18 mmol) and condensation with

4-(N-(benzyloxy)-3-(Boc-amino)propanamido)butanoic acid (18) (0.87 g,

2.29 mmol). The product was purified by column chromatography using

first cyclohexane / ethyl acetate 1:3 and in the following ethyl acetate /

ethanol 9:1 as eluents to obtain the product as colorless oil in 97% yield

(1.96 g, 2.11 mmol).

¹H NMR (300 MHz, [D₃]CDCl₃,

25°C): δ=8.88 (s, 1H, H-13), 7.37

(s_broad, 5H, H-1 + H-2 + H-3 + H-5 +

H-6), 5.30 (s, 1H, H-20), 4.79 (s,

2H, H-7), 3.71 (t, ³J(H,H)=6.5 Hz,

2H, H-9), 3.37 (t_broad,

³J(H,H)=4.8

Hz, 2H, H-19), 2.61 (t, ³J(H,H)=5.6

Hz, 2H, H-17), 2.36 (t, ³J(H,H)=7.2 Hz, 2H-11), 1.95 (p, ³J(H,H)=7.0 Hz,

2H, H-10), 1.41 (s, 9H, H-25 + H-26 + H-27). ¹³C NMR (75 MHz,

[D₃]CDCl₃, 25°C): δ=177.30 (C-12), 173.93 (C-16), 156.18 (C-21), 134.16

(C-4), 129.40 (C2 + C-6), 129.22 (C-1), 128.87 (C-3 + C-5), 79.36 (C-24),

76.59 (C-7), 44.56 (C-9), 36.05 (C-19), 32.77 (C-17), 31.15 (C-11), 28.51

(C-25 + C-26 + C-27), 22.16 (C-10). MALDI-MS (calculated): m/z=402.43

[M+Na]⁺(403.18), 418.43 [M+K]⁺(419.16).

¹H NMR (300 MHz, [D₃]CDCl₃, 25°C): δ=7.36 (s_broad, 15H, H-1 – H-6 + H-

27 – H-29 + H-31 + H-32 + H-48 – H-50 + H-52 + H-53), 6.93 (s, 2H, H-

60 + H-67), 5.33 (s_broad, 1H, H-19), 4.78 + 4.79 (2s, 6H, H-7 + H-33 + H-

3

54), 4.09 (q, J(H,H)=7.1 Hz, 2H, H-41), 3.69 (t_broad, 6H, H-9 + H-35 + H-

56), 3.49 (s_broad, 4H, H-47 + H-66), 3.35 (t, ³J(H,H)=5.7 Hz, 2H, H-18),

2.58 – 2.65 (3t, ³J(H,H)=5.6 Hz, 6H, H-16 + H-45 + H-64), 2.30 (t,

³J(H,H)=7.2 Hz, 2H, H-37), 2.18 (q, ³J(H,H)=7.3 Hz, 4H, H-11 + H-58),

1.86 – 2.02 (m, 6H, H-10 + H-36 + H-57), 1.41 (s, 9H, H-24 – H-26), 1.22

(t, ³J(H,H)=7.1 Hz, 3H, H-42).¹³C NMR (75 MHz, [D₃]CDCl₃, 25°C):

δ=173.68 + 173.76 (C-15 + C-44 + C-63), 172.87 (C-38), 172.47 (C-12 +

C-59), 156.06 (C-20), 134.16 (C-4 + C-30 + C-51), 129.43 (C-2 + C-6 +

C-28 + C-32 + C-49 + C-53), 129.18 (C-1 + C-27 + C-48), 128.86 (C-3 +

C-5 + C-29 + C-31 + C-50 + C52), 79.29 (C-23), 76.41 + 76.49 (C-7 + C-

33 + C-54), 60.60 (C-41), 44.52 (C-9 + C-35 + C-56), 36.22 (C-18), 34.89

(C-47 + C-66), 33.08 + 33.21 (C-11 + C-58), 32.31 + 32.51 + 32.68 (C-37

+ C45 + C-64), 31.44 (C-16), 28.54 (C-24 – C-26), 23.10 (C-10 + C-57),

22.28 (C-36), 14.33 (C-42). MALDI-MS (calculated): m/z=933.63 [M+H]⁺

(933.50), 955.74 [M+Na]⁺(955.50), 971.86 [M+K]⁺(971.45).

Ethyl 12-(benzyloxy)-3-(3-(Boc-amino)propanoyl)-7,11-dioxo-1-

phenyl-2-oxa-3,8,12-triazahexadecan-16-oate, Boc protected amino

ester dimer (19).

19 was obtained as described before in the general description by

deprotection of 16 (0.95 g, 3.09 mmol) to 17 and condensation with

4-(N-(benzyloxy)-3-(Boc-amino)propanamido)butanoic acid (18) (1.23 g,

3.24 mmol). The product was purified by column chromatography using

first cyclohexane / ethyl acetate 1:3 and in the following 100% ethyl

acetate as eluents to obtain the product as colorless oil in 86% yield

(1.78 g, 2.66 mmol).

¹H NMR (300

MHz, [D₃]CDCl₃,

25°C): δ=7.36

(s_broad, 10H, H-1

– H-5 + H-6 +

H-22 – H-24 +

Ethyl-12,21,30-tris(benzyloxy)-3-(3-(Boc-amino)propanoyl)-

7,11,16,20,25,29-hexaoxo-1-phenyl-2-oxa-3,8,12,17,21,26,30-

heptaaza-tetratriacontan-34-oate, Boc protected amino tetramer

ethyl ester (21).

H-26

6.76 (s_broad, 1H,

H-34), 5.31

+ H-27),

3

21 was obtained as described before in the general description by

deprotection of 20 (570 mg, 684 µmol) and condensation with

4-(N-(benzyloxy)-3-(Boc-amino)propanamido)butanoic acid (18) (276 mg,

725 µmol). The product was purified by column chromatography using

acetone / CH₂Cl₂3:2 containing 1% H₂O as eluent and obtained as

colorless oil in 90% yield (737 mg, 616 µmol).

(s_broad, 1H, H-41), 4.78 + 4.79 (2s, 4H, H-7 + H-28), 4.10 (q, J(H,H)=7.1

Hz, 3H, H-15), 3.69 (q, ³J(H,H)=5.7 Hz, 4H, H-9 + H-30), 3.44 – 3.55 (m,

2H, H-21), 3.35 (t, ³J(H,H)=5.6 Hz, 2H, H-40), 2.62 (q, ³J(H,H)=6.2 Hz,

4H, H-19 + H-38), 2.30 (t, ³J(H,H)=7.2 Hz, 2H, H-11), 2.16 (t, ³J(H,H)=7.1

Hz, 2H, H-32), 1.88 – 2.01 (m, 4H, H-10 + H-31), 1.41 (s, 9H, H-46 – H-

48), 1.22 (t, ³J(H,H)=7.3 Hz, 2H, H-16). ¹³C NMR (75 MHz, [D₃]CDCl₃,

25°C): δ=173.75 (C-18 + C-37), 172.39 + 172.90 (C-12 + C-33), 156.06

(C-42), 134.18 (C-4 + C-25), 129.42 + 129.43 (C-2 + C-6 + C-23 + C-27),

129.19 (C-1 + C-22), 128.86 (C-3 + C-5 + C-24 + C-26), 79.30 (C-45),

76.46 + 76.52 (C-7 + C-28), 60.60 (C-15), 44.53 (C-9 + C-30), 36.19 (C-

21), 34.87 (C-40), 32.30 + 32.69 + 33.08 (C-19 + C-32 + C-38), 31.45 (C-

11), 28.53 (C-46 – C-48), 23.03 (C-10), 22.29 (C-31), 14.33 (C-16).

MALDI-MS (calculated): m/z=671.41 [M+H]⁺(671.80), 693.66 [M+Na]⁺

(693.35), 709.58 [M+K]⁺(709.32).

¹H NMR (300 MHz, [D₃]CDCl₃, 25°C): δ=7.35 + 7.36 (4 s_broad, 20H, H-1 –

H-3 + H-5 + H-6 + H-19 – H-21 + H-23 + H-24 + H-40 – H-42 + H-44 + H-

45 + H-60 – H-62 + H-64 + H-65), 6.38 (s_broad, 3H, H-52 + H-59 + H-72),

5.36 (s_broad, 1H, H-79), 4.74 – 4.82 (4s, 8H, H-7 + H-25 + H-46 + H-66),

4.09 (q, ³J(H,H)=7.1 Hz, 2H, H-33), 3.68 (t_broad, 8H, H-9 + H-27 + H-48 +

Ethyl-12,21-bis(benzyloxy)-3-(3-(Boc-amino)propanoyl)-7,11,16,20-

tetraoxo-1-phenyl-2-oxa-3,8,12,17,21-pentaazapentacosan-25-oate,

Boc protected amino ester trimer (20).

3

H-68), 3.50 (t_broad, 6H, H-18 + H-39 + H-58), 3.34 (t, J(H,H)=5.6 Hz, 2H,

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H-78), 2.58 – 2.66 (m, 8H, H-16 + H-37 + H-56 + H-76), 2.16 – 2.38 (m,

8H, H-11 + H-29 + H-50 + H-70), 1.83 – 2.04 (m, 8H, H-10 + H-28 + H-49

+ H-69), 1.40 (s, 9H, H-84 – H-86), 1.21 (t, ³J(H,H)=7.2 Hz, 3H, H-34).

¹³C NMR (75 MHz, [D₃]CDCl₃, 25°C): δ=173.40 + 173.51 + 173.62 +

173.71 + 173.75 (C-15 + C-30 + C-36 + C-55 + C-75), 172.86 + 173.07 +

173.13 (C-12 + C-51 + C-71), 156.08 (C-80), 134.12 (C-4 + C-22 + C-43

+ C-63), 129.42 (C-2 + C-6 + C-20 + C-24 + C-41 + C-45 + C-61 + C-65),

129.19 (C-1 + C-19 + C-40 + C-60), 128.85 (C-3 + C-5 + C-21 + C-23 +

C-42 + C-44 + C-62 + C-64), 79.33 (C-83), 76.40 + 76.43 + 76.49 +

76.51 (C-7 + C-25 + C-46 + C-66), 60.59 (C-33), 44.46 + 44.50 + 44.53 +

44.58 (C-9 + C-27 + C-48 + C-68), 36.27 (C-78), 35.07 + 35.12 + 35.16

(C-18 + C-39 + C-58), 32.19 + 32.34 + 32.49 + 32.66 + 32.90 + 33.02 (C-

11 + C-16 + C-37 + C-50 + C-56 + C-70 + C-76), 31.43 (C-29), 28.46 +

28.58 (C-84 – C-86), 23.16 (C-10 + C-49 + C-69), 22.27 (C-28), 14.36

(C-34). MALDI-MS (calculated): m/z=1195.32 [M+H]⁺(1195.63), 1217.15

[M+Na]⁺(1217.61), 1233.11 [M+K]⁺(1233.58).

2.14 (t, ³J(H,H)=6.6 Hz, 8H, H-4 + H-10 + H-17 + H-24), 1.95 (p,

³J(H,H)=6.0 Hz, 8H, H-3 + H-9 + H-16 + H-23). ¹³C NMR (75 MHz,

[D₃]CDCl₃, 25°C): δ=174.05 (C-5 + C-11 + C-18 + C-25), 172.43 (C-34 +

C-36 + C-37 + C-39), 134.14 (C-50 + C-57 + C-64 + C-66), 129.42 (C-52

+ C-54 + C-59 + C-61 + C-68 + C-70 + C-73 + C-75), 129.23 (C-53 + C-

60 + C-69 + C-74), 128.89 (C-51 + C-55 + C-58 + C-62 + C-67 + C-71 +

C-72 + C-76), 76.41 (C-49 + C-56 + C-63 + C-65), 44.24 (C-2 + C-8 + C-

15 + C-22), 34.80 (C-13 + C-20 + C-27 + C-28), 33.25 (C-4 + C-10 + C-

17 + C-24), 32.51 (C-1 + C-7 + C-14 + C-21), 23.16 (C-3 + C-9 + C-16 +

C-23). MALDI-MS (calculated): m/z=1049.43 [M+H]⁺(1049.53), 1071.40

[M+Na]⁺(1071.52), 1087.23 [M+K]⁺(1087.30).

1,10,19,28-tetrahydroxy-1,5,10,14,19,23,28,32-octaaza-

cyclohexatriacontan-2,6,11,15,20, 24,29,33-octaone, CTH36 (24).

To a solution of the benzyl protected macrocycle 23 (27 mg, 25.7 µmol)

in a mixture of methanol (5 mL), H₂O (1 mL) and formic acid (60 µL), was

added 10% Pd/C (2 mg) and the mixture was stirred under a hydrogen

atmosphere for 3.5 h. The suspension was centrifuged and the remaining

solid was extracted with MeCN / H₂O 2:1 (5 mL). The methanol fraction

was dried in vacuo, the residue was taken up with the MeCN / H₂O

fraction and the resulting mixture was lyophilized to afford the product as

white powder in 96% yield (17 mg, 24.7 µmol) and a purity of 78%. This

material was further purified by semipreparative HPLC (10 minutes

gradient from 0 to 20% MeCN in H₂O with 0.1% formic acid as additive,

R_t=7.28 min) and subsequent lyophilization, giving the pure product

(determined by analytical HPLC) in 79% yield (14.1 mg, 20.5 µmol).

1,10,19,28-tetrakis(benzyloxy)-1,5,10,14,19,23,28,32-octaaza-

cyclohexatriacontan-2,6,11, 15,20,24,29,33-octaone, benzyl

protected CTH36 (23).

21 (138 mg, 115 µmol) was deprotected as described before in the

general description of deprotection and condensation. The resulting, Boc-

deprotected intermediate was dissolved in methanol (2.5 mL) and treated

with aqueous sodium hydroxide solution (0.5M, 690 µL, 345 µmol, 3

equiv.) and reacted overnight at ambient temperature. In the following,

the solvent was removed by evaporation, the residue was taken up with

H₂O and lyophilized to give

a colorless powder. The deprotected

intermediate was purified by semipreparative HPLC (10 minutes gradient

from 20 to 70% MeCN in H₂O with 0.1% formic acid as additive, R_t=5.52

min) and subsequently lyophilized. Afterwards, formic acid was removed

by cartridge purification. For this purpose, the intermediate product was

dissolved in a mixture of MeCN (500 µL) and phosphate buffer (0.05M,

pH 7.2, 50 mL) and divided into three portions which were independently

purified using Waters Sep-Pak tC18 Plus Short cartridges by first

trapping the product on the cartridge, washing the cartridge with

phosphate buffer (0.05M, pH 7.2, 3 mL) and elution of the product with

MeCN (15 mL). The combined product solution was concentrated, diluted

with H₂O and lyophilized, giving tetramer 22 as colorless solid in 45%

yield (55 mg, 51.8 µmol). To an ice-cooled solution of this intermediate

22 in dry DMF (40 mL) were added N,N-diisopropylethylamine (DIPEA,

9.5 µL, 55.8 µmol, 1.1 equiv.) and DPPA (diphenyl phosphoryl azide,

13.1 µL, 60.8 µmol, 2.75 equiv.) and the mixture was kept at 3 °C for 18

days. The solvent was removed in vacuo and the residue was taken up in

MeCN / H₂O 1:1 (1 mL) and lyophilized. The product was precipitated in

MeCN (300 µL) at 3 °C for three days. The precipitate was washed four

times with cold MeCN (50 µL each) and dried before the product was

purified by column chromatography with acetone / CH₂Cl₂3:1 containing

5% H₂O as eluent, giving the desired macrocycle 23 as colorless solid in

20% overall yield (23.5 mg, 23 µmol) over three steps, starting from 21.

The product could not be characterized by NMR due to a very limited

solubility of the substance in organic solvents as well as aqueous media,

preventing the achievement of the required substance concentration in

the NMR sample. MALDI-MS (calculated): m/z=689.17 [M+H]⁺(689.35),

711.18 [M+Na]⁺(711.33), 727.3 [M+K]⁺(727.15).

Ethyl-(S)-12,21,30-tris(benzyloxy)-3-(3-(Fmoc-amino)-4-(Boc-

amino)butanoyl)-7,11, 16,20,25,29-hexaoxo-1-phenyl-2-oxa-

3,8,12,17,21,26,30-heptaazatetratriacontan-34-oate, Boc/Fmoc

bisprotected tetramer ethyl ester (25).

25 was obtained as described before in the general description by

deprotection of 20 (238 mg, 285 µmol) and condensation with (S)-4-(N-

(benzyloxy)-4-(Boc-amino)-3-(Fmoc-amino)-butanamido)-butanoic acid

12 (180 mg, 285 µmol). The product was purified by column

chromatography using acetone / CH₂Cl₂1:1 containing 1% H₂O as eluent

and obtained as colorless, non-crystalline solid in 59% yield (245 mg,

168 µmol).

¹H NMR (300 MHz, [D₃]CDCl₃,

25°C): δ=7.36 (s_broad, 20H, H-51 –

H-55 + H-58 – H-60 + H-61 + H-62

+ H-67 – H-76), 7.06 (s_broad, 4H, H-6

+ H-12 + H-19 + H-26), 4.78 (s, 8H,

H-49 + H-56 + H-63 + H-65), 3.69 (t,

³J(H,H)=6.1 Hz, 8H, H-2 + H-8 + H-

15 + H-22), 3.49 (dt, ^3,3J(H,H) = 5.8

+ 5.1 Hz, 8H, H-13 + H-20 + H-27 +

H-28), 2.64 (dd, ^3,3J(H,H)=5.8 + 4.9

Hz, 8H, H-1 + H-7 + H-14 + H-21),

3

¹H NMR (300 MHz, [D₃]CDCl₃, 25°C): δ=7.73 (d, J(H,H)=7.5 Hz, 2H, H-

100 + H-101), 7.57 (d, ³J(H,H)=7.4 Hz, 2H, H-97 + H-104), 7.31 – 7.40

(m, 22H, H-1 – H-3 + H-5 + H-6 + H-19 – H-21 + H-23 + H-24 + H-40 –

H-42 + H-44 + H-45 + H-60 – H-62 + H-64 + H-65 + H-99 + H-102), 7.28

(t, ³J(H,H)=7.5 Hz, 2H, H-98 + H-103), 6.08 (s_broad, 1H, H-78), 5.24 (s_broad

,

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1H, H-83), 4.97 (s_broad, 3H, H-52 + H-59 + H-72), 4.70 – 4.84 (2s, 8H, H-7

+ H-25 + H-46 + H-66), 4.32 (d, ³J(H,H)=7.5 Hz, 2H, H-91), 4.16 (t,

³J(H,H)=7.0 Hz, 1H, H-92), 4.08 (q, ³J(H,H)=7.1 Hz, 2H, H-33), 3.95 –

4.06 (m, 1H, H-77), 3.67 (4t_broad, 8H, H-9 + H-27 + H-48 + H-68), 3.49

(3t_broad, 6H, H-18 + H-39 + H-58), 3.28 (t_broad, 2H, H-82), 2.72 – 2.84 (m,

1H, H-76b), 2.47 – 2.70 (m, 7H, H-16 + H-37 + H-56 + H-76a), 2.18 –

2.34 (m, 8H, H-11 + H-29 + H-50 + H-70), 1.85 – 2.02 (m, 8H, H-10 + H-

28 + H-49 + H-69), 1.40 (s, 9H, H-88 – H-90), 1.21 (t, ³J(H,H)=7.1 Hz, 3H,

H-34). ¹³C NMR (75 MHz, [D₃]CDCl₃, 25°C): δ=173.11 + 173.17 + 173.28

+ 173.46 + 173.57 (C-15 + C-30 + C-36 + C-55 + C-75), 171.80 + 172.42

+ 172.88 (C-12 + C51 + C-71), 156.88 (C-79), 156.26 (C-84), 144.01 +

144.04 (C-93 + C-96), 141.34 (C-94 + C-95), 134.10 (C-4 + C-22 + C-43

+ C-63), 129.40 + 129.47 (C-2 + C-6 + C-20 + C-24 + C-41 + C-45 + C-

61 + C-65), 129.18 (C-1 + C-19 + C-40 + C-60), 128.84 (C-3 + C-5 + C-

21 + C-23 + C-42 + C-44 + C-62 + C-64), 127.15 + 127.76 (C-99 – C-

102), 125.26 (C-98 + C-103), 120.02 (C-97 + C-104), 79.70 (C-87), 76.35

+ 76.41 + 76.43 + 76.48 (C-7 + C-25 + C-46 + C-66), 66.83 (C-91), 60.59

(C-33), 49.31 (C-77), 47.30 (C-92), 43.78 + 44.14 + 44.57 (C-9 + C-27 +

C-48 + C-68 + C-82), 35.07 + 35.22 (C-18 + C-39 + C-58), 34.15 (C-76),

32.19 + 32.31 + 32.50 + 32.78 + 32.89 + 33.04 (C-11 + C-16 + C37 +

C-50 + C-56 + C-70), 31.42 (C-29), 28.44 + 28.53 (C-88 – C-90), 23.08 +

23.19 + 23.27 (C-10 + C-49 + C-69), 14.27 (C-34), 22.27 (C-28). MALDI-

MS (calculated): m/z=1447.39 [M+H]⁺(1446.72), 1469.87 [M+Na]⁺

(1468.71), 1485.57 [M+K]⁺(1484.68).

2.81 (m, 8H, H-1 + H-7 + H-13 + H-20), 2.11 – 2.33 (m, 8H, H-4 + H-10 +

H-16 + H-23), 1.94 (p_broad, ³J(H,H)=6.4 Hz, 8H, H-3 + H-9 + H-15 + H-22),

1.40 (s, 9H, H83 + H-84 + H-85). ¹³C NMR (75 MHz, [D₃]CDCl₃, 25°C):

δ=172.51 + 173.13 – 173.77 (C-5 + C-11 + C-17 + C-24 + C-33 + C-35 +

C-36 + C-38), 156.71 (C-78), 134.16 (C-49 + C-56 + C-63 + C-65),

129.44 (C-51 + C-53 + C-58 + C-60 + C67 + C-69 + C-72 + C-74),

129.20 (C-52 + C-59 + C-68 + C-73), 128.87 + 128.91 (C-50 + C-54 +

C-57 + C-61 + C-66 + C-70 + C-71 + C-75), 79.50 (C-82), 76.37 + 76.43

(C-48 + C-55 + C-62 + C-64), 47.44 (C-26), 43.78 + 44.04 + 44.09 +

44.41 (C-2 + C-8 + C-14 + C-21 + C-80), 35.03 + 35.17 (C-19 + C-27 +

C-76), 34.52 (C-13), 32.30 + 32.43 + 32.81 (C-1 + C-4 + C-7 + C-10 + C-

16 + C-20 + C-23), 28.54 (C-83 – C-85), 23.07 + 23.35 (C-3 + C-9 + C-15

+

C-22). MALDI-MS (calculated): m/z=1178.48 [M+H]⁺(1178.61),

1200.21 [M+Na]⁺(1200.60).

N¹-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-N⁴-((5,14,23,32-tetrakis-

(benzyloxy)-4,9,13,18,22,27, 31,36-octaoxo-1,5,10,14,19,23,28,32-

octaaza-cyclohexatriacontan-2-yl)methyl)succinamide, benzyl

protected tetrazine-modified CTH36 (29).

26 (12.0 mg, 13.9 µmol) was deprotected as described before in the

general description of deprotection and condensation, giving the

deprotected aminomethyl-CTH36 derivate 27 (11.6 mg, 10.8 µmol) which

was dissolved together with 4-((4-(1,2,4,5-tetrazin-3-yl)benzyl)amino)-4-

oxobutanoic acid (28, 3.1 mg, 10 µmol)^[20]in MeCN (800 µL). To this

mixture was added EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,

2.1 mg, 11 µmol, 1.1 equiv.) and the mixture was stirred at ambient

temperature overnight. The solvent was evaporated, the residue was

dissolved in CH₂Cl₂(1 mL) and washed with aqueous sodium hydrogen

sulfate solution (10%, 100 µL). After evaporation of the solvent, the

purification of the product was performed by semipreparative HPLC (15

minutes gradient from 35 to 40% MeCN in H₂O with 0.1% formic acid as

additive, R_t=11.08 min). After lyophilization, the pure product (determined

by analytical HPLC) was obtained as pink-colored powder in 48% overall

yield (7 mg, 4.8 µmol) over two steps, starting from 26.

1,10,19,28-Tetrakis(benzyloxy)-4-((Boc-amino)methyl)-

1,5,10,14,19,23,28,32-octaaza-cyclohexatriacontan-

2,6,11,15,20,24,29,33-octaone, Boc/benzyl protected functionalized

CTH36 (26).

To an ice-cooled solution of bis-protected tetramer 25 (227 mg, 157µmol)

in methanol (3.4 mL) was slowly added an aqueous sodium hydroxide

solution (0.5M, 940 µL, 470 µmol, 3 equiv.). After warming up to ambient

temperature and stirring overnight, the solvent was removed in vacuo.

The obtained residue was taken up in H₂O (1 mL) the precipitate was

removed by filtration and washed twice with H₂O (0.5 mL each portion).

The aqueous solution was lyophilized and the residue (201 mg) was

used without further purification for the next reaction step. To an ice-

cooled solution of this intermediate in dry DMF (50 mL) was added DPPA

(42 µL, 195 µmol, 1.24 equiv.) and the mixture was stirred at 3°C for two

hours. The solvent was removed in vacuo, the residue was taken up in

MeCN (5 mL) and the precipitate was removed by filtration. Further

purification was performed by column chromatography with acetone /

CH₂Cl₂/ H₂O 20:10:1 as eluent. After isolation of the product by

evaporation, it was redissolved in MeCN / H₂O 1:1 and lyophilized to

afford the desired macrocycle 26 as colorless solid in 53% overall yield

(97.4 mg, 83 µmol) over two steps, starting from 25.

The product could not be characterized by NMR due to a very limited

solubility of the substance in organic solvents as well as aqueous media,

preventing the achievement of the required substance concentration in

the NMR sample. MALDI-MS (calculated): m/z=1347.63 [M+H]⁺

(1347.65), 1369.76 [M+Na]⁺(1369.63), 1385.65 [M+K]⁺(1385.61).

N¹-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-N⁴-((5,14,23,32-tetrahydroxy-

4,9,13,18,22,27,31,36-octaoxo-1,5,10,14,19,23,28,32-

octaazacyclohexatriacontan-2-yl)methyl)succinamide, tetrazine-

modified CTH36 (tCTH36, 30).

¹H NMR (300 MHz,

[D₃]CDCl₃,

25°C):

To a solution of 29 (6.5 mg, 5.0 µmol) in a mixture of methanol (3 mL),

H₂O (30 µL) and formic acid (1 µL) was added Pd/C (10%, 2mg) and the

mixture was stirred under a hydrogen atmosphere for 3.5 h. The catalyst

was removed by filtration and the solution was evaporated to dryness.

The residue was dissolved in MeCN / H₂O 1:1 (1 mL) and lyophilized

before the product was purified by semipreparative HPLC (10 minutes

gradient from 10 to 20% MeCN in H₂O with 0.1% formic acid as additive,

R_t=8.40 min) and subsequent lyophilization. The pure product

(determined by analytical HPLC) was obtained in quantitative yield (4.9

mg, 5.0 µmol).

δ=7.73 (s_broad, 4H, H-6 +

H-12 + H-18 + H-25),

7.35 + 7.36 (2s_broad, 20H,

H-50 – H-54, H-57 – H-

61, H-66 – H-75), 5.78 (s,

1H, H-77), 4.76 – 4.81

(4s, 8H, H-48 + H-55 +

H-62 + H-64), 4.35 (s,

1H, H-26), 3.56 – 3.92

(m, 8H, H-2 + H-8 + H14

+ H-21), 3.36 – 3.56 (m,

,

³J(H,H)=5.7 Hz, 2H, H-80), 2.50 –

6H, H-19 + H-27 + H-76), 3.28 (d_broad

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The product could not be characterized by NMR due to a very limited

solubility of the substance in organic solvents as well as aqueous media,

preventing the achievement of the required substance concentration in

the NMR sample. MALDI-MS (calculated): m/z=987.28 [M+H]⁺(987.46).

33 (3.25 mg, 4.31 µmol) in DMSO (135 µL). The pink color of 30

disappeared during the reaction. The mixture was evaporated to dryness

and the product was purified by semipreparative HPLC (10 minutes

gradient from 0 to 40% MeCN in H₂O with 0.1% formic acid as additive,

retention times of isomers: R_t1=8.14 min, R_t2=9.39 min and R_t3=9.58 min).

The product was obtained after lyophilization as a mixture of isomers and

colorless solid in 65% yield (4.77 mg, 2.80 µmol).

c(RGDfK) Peptide (31).

MALDI-MS (calculated): m/z=1714.96 [M+H]⁺(1714.86), 1737.08

[M+Na]⁺(1736.84).

The cyclic pentapeptide c(RGDfK) was synthesized in 0.2 mmol scale by

solid phase peptide synthesis on solid support using the standard Fmoc-

strategy on commercially available H-Asp(tBu)-2-Chlortrityl-resin

(loading: 0.80 mmol/g). For amino acid conjugation, 3.9 equiv. HBTU

(N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluoro-

DFO-c(RGDfK) conjugate (36).

phosphate), 4.0 equiv. of Fmoc protected amino acids and 4.0 equiv. of

DIPEA were used in DMF as solvent. Coupling times were 30 minutes for

each amino acid. After coupling of the last amino acid and Fmoc-removal,

the linear, protected peptide was cleaved from the resin using 1% TFA in

CH₂Cl₂. The crude intermediate was isolated by evaporation of the

volatile components of the mixture and was in the following dissolved in

dry DMF (170 mL). To this solution, DIPEA (119 µL, 700 µmol, 3.5

equiv.) was added and the solution was cooled to 0-4°C before DPPA

(54 µL, 250 µmol, 1.25 equiv.) were added. The mixture was allowed to

warm to ambient temperature and reacted for three days until the

cyclisation was complete. The volatile components of the mixture were

removed in vacuo and the residue was treated for 2.5h with a mixture of

TFA / TIS (triisopropylsilane) 97.5:2.5 (5 mL) to completely deprotect the

peptide. After concentration in vacuo and precipitation of the product in

cooled diethylether (25 mL), the crude product was obtained by

centrifugation, washing with diethylether (5 mL) twice and drying of the

obtained solid. Purification of the product was performed by

semipreparative HPLC (6 minutes gradient from 0 to 15% MeCN in H₂O

with 0.1% formic acid as additive, R_t=3.65 min). The product was

obtained after lyophilization as white solid in 69% overall yield (90 mg,

138 µmol).

To a solution of tDFO 34 (5.92 mg, 7.13 µmol) in DMSO (200 µL) was

added in portions a solution of the TCO-modified c(RGDfK) peptide 33

(3.78 mg, 5.00 µmol) in DMSO (140 µL). The pink color of 34

disappeared during the reaction. The mixture was evaporated to dryness

and the product was purified by semipreparative HPLC (10 minutes

gradient from 0 to 50% MeCN in H₂O with 0.1% formic acid as additive,

retention times of isomers: R_t1=8.56 min, R_t2=9.38 min and R_t3=9.58 min).

The product was obtained after lyophilization as a mixture of isomers and

colorless solid in 42% yield (3.30 mg, 2.1 µmol).

MALDI-MS (calculated): m/z=1558.24 [M+H]⁺(1557.84).

Radiolabeling of CTH36 (24) with ⁸⁹Zr⁴⁺and determination of the

optimal pH for radiometal incorporation.

First, a stock solution of the radiolabeling precursor 24 (220 µg, 320

nmol) in H₂O (320 µL) was prepared. For the radiolabeling experiments,

⁸⁹Zr-oxalate solution (0.1M, 8–12 µL, 13.5–18.5 MBq) was added to

HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffers of

different pH (0.25M, 150 µL, pH 4, 5, 6, 7, 8, 9, 10, 11 and 12) and the

pH was controlled and if necessary adjusted to the initial pH by addition

of NaOH (0.1M). Aliquots of the stock solution of 14 (40 µL, 40 nmol)

were added to each ⁸⁹Zr solution and the radiolabeling reaction mixtures

were analyzed by radio-HPLC after 5, 25 and 45 minutes. Each

experiment was performed thrice.

MALDI-MS (calculated): m/z=604.11 [M+H]⁺(604.32), 626.29 [M+Na]⁺

(626.30), 642.17 [M+K]⁺(642.28).

TCO-modified c(RGDfK) derivative (33).

To a solution of 31 (15.3 mg, 20 µmol) and DIPEA (6.8 µL, 40 µmol) in

DMF (400 µL) was added a solution of trans-cyclooct-4-en-1-yl p-

nitrophenyl carbonate (32) (5.8 mg, 20 µmol) in DMF (50 µL). After 6h

reaction at ambient temperature, the solvent was removed in vacuo and

the obtained yellow-colored solid was washed twice with acetone (200

µL) and dried. The product was purified by semipreparative HPLC (10

minutes gradient from 0 to 50% MeCN in H₂O with 0.1% formic acid as

additive, R_t=8.46 min) and obtained after lyophilization as white solid in

52% yield (7.8 mg, 10.4 µmol).

Radiolabeling of tCTH36- and tDFO-peptide conjugates 35 and 36

with ⁸⁹Zr⁴⁺

.

First, stock solutions of the radiolabeling precursors 35 and 36 in H₂O (1

nmol/µL) were prepared. For the radiolabeling experiments, ⁸⁹Zr-oxalate

solution (0.1M, 16–25 µL, 20–23 MBq) was added to HEPES buffer

(0.25M, 150 µL, pH 9) and the pH of the solution was controlled and if

necessary adjusted to 7.0 by addition of NaOH (0.1M, ∼20 µL). To this

mixture was added 35 (2.5 µL of the before prepared stock solution, 2.5

nmol) or 36 (5 µL of the before prepared stock solution, 5 nmol) and

reacted for 60 minutes at ambient temperature. The radiolabeling

reaction mixtures were analyzed by radio-HPLC and showed ⁸⁹Zr-

incorporation rates of ≥96%. [⁸⁹Zr]35 and [⁸⁹Zr]36 were thus obtained in

radiochemical yields of ≥96% and non-optimized molar activities of 8.4–

9.2 GBq/µmol for [⁸⁹Zr]35 and 4.0–4.6 GBq/µmol for [⁸⁹Zr]36.

MALDI-MS (calculated): m/z=755.95 [M+H]⁺(756.40), 778.03 [M+Na]⁺

(778.39).

CTH36-c(RGDfK) conjugate (35).

To a solution of the tCTH36 30 (6.30 mg, 6.38 µmol) in DMSO (100 µL)

was added in portions a solution of the TCO-modified c(RGDfK) peptide

10.1002/cmdc.201700377

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Challenge experiments of ⁸⁹Zr-labeled peptide conjugates [⁸⁹Zr]35

and [⁸⁹Zr]36 with EDTA.

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Entry for the Table of Contents

FULL PAPER

Uwe Seibold, Björn Wängler, Carmen

Wängler*

Page No. – Page No.

Rational Design, Chemical

Development and Stability

Assessment of a New Macrocyclic,

Four-Hydroxamates-Bearing

Bifunctional Chelating Agent for ⁸⁹Zr

Text for Table of Contents

Complex four-leaf clover: Symmetry meets high complex stability.

Here we present the development of a macrocyclic tetra-hydroxamate ⁸⁹Zr-chelator, applicable in bioconjugation and symmetrically

encapsulating the radiometal without cleft in the ligand sphere for high ⁸⁹Zr-complex stability.

Article Doi

Rational Design, Development, and Stability Assessment of a Macrocyclic Four-Hydroxamate-Bearing Bifunctional Chelating Agent for 89Zr

DOI: 10.1002/cmdc.201700377

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