Problem Set

Bioconjugation methods

1 jantonio@[Link]
 Exercises

Indicate which reagent was used to obtain the following bioconjugate.

Indicate possible side reactions of this reagent in aqueous conditions and with other common functionalities in proteins.
Draw the secondary products.

S H
N
Bioconjugation methods

NH 4

Answer

Classical protocol of isothiocyanate labelling:

5–10 equivalents of ITC at basic pH in the range of 9.0–9.5

2
 Exercises

Write two different reactants and respective reaction conditions (pH, solvent, temperature) to obtain the following conjugate.
Explain the differences between the chosen reagents.
What are the main advantages of this type of amide connection?

O H
N
4
Bioconjugation methods

Answer

H 2N
O O 4

O N O O H
S Buffer pH 7.4, 20 ºC N
O O O- Na+ 4
Buffer pH 7.4, 20 ºC
Amine-reactive
Sulfo-NHS Ester
(dry-stable)

May require a co-solvent due to the low

Better water solubility
solubility of the NHS ester

Advantages of bonds with activated esters:

- Formed amide is very stable under physiological conditions
- NHS esters are easy to synthesize from corresponding acids
- Selectivity for lysines
3 - Work well at room temperature and physiological pH
 Exercises

State two methods to achieve the following transformation.

Draw the regents and intermediate products.
Mention the advantages and disadvantages of each of them.

H
H 2N N
4 4
Bioconjugation methods

N
O NaBH3(CN)
NH2 HN
pH 6.5–8.5 pH 6.5–8.5
4

Advantages: Cheap and non-toxic

Disadvantages: Poor stability in water and must be used excessively; May reduce other functionalities of the protein

O
H
N N H 2N
4 4 4

Advantages: More selective reagent, does not reduce disulfide bridges

Disadvantages: Iridium Toxicity

4
 Exercises

Based on the reaction intermediates, explain why the N-terminal amine is not modified.

NH2
4 NH
4
O

H H
O
CuI, 1,10-Phenanthroline O
R
N kPi, 25ºC R
H N
NH2 H
Bioconjugation methods

NH2

Answer
latent electrophile

NH2
4
N Ph
4
4 O Ph NH
4
NH3 4
H H NH2
O 4
CuI, 1,10-Phenanthroline NH3
R O kPi, 25ºC
N 4
H O
NH2 NH3 N 4
R NH3 N 4
4 N R NH3
H 3N H
4 N
H 3N H
H 3N 4

Protected N-terminal

N-terminal deprotection

Ph
NH
4

4
NH3

5 R N
H
4
NH3
NH2
H 3N 4
 Exercises

Indicate which reagent was used to obtain the following bioconjugate.

Indicate possible side reactions of this reagent in aqueous conditions and with other common functionalities in proteins.
Draw the secondary products.

O H
Bioconjugation methods

S N
O 4

Answer

O
S S
O

Cys

H 2N
O H 2O 4
O H
O
S OH S N
S Cl
O O
O 4

Ser

O
6 S O
O
 Exercises

If you use 2 equivalents of maleimide for the bioconjugation reaction, match the products (letters) with the corresponding
reactants (numbers).
Bioconjugation methods

S S S S O O
O O
S S S S
N N
O O O O N N
N N
O O O O

A B C D

O N O N O N O
N O O
O O

4
Br PhS SPh OPh

1 2 3
Answer

A-3 B - 4 (2 may also form) C-2 D-1

7
 Exercises

Which of the following bioconjugates best resists exchange with other thiols? Justify
Bioconjugation methods

O O S OH
S S NH
N N O
O O

Answer

Open thiosuccinimides are more stable to retro-Michael/thiol exchange

Ring hydrolysis makes thiosuccinimide protons less acidic and does not eliminate
O

S OH
NH
O

8
 Exercises

Write two linkers that favor the formation of the following conjugate.
Explain the mechanism involved in each of the processes.
Bioconjugation methods

SH S OH
NH
O
Linker

Answer

Resonance-promoted thiosuccinimide hydrolysis

Intramolecular Acid catalysis
EWG EWG

mc-vc
Payload mc-vc
O O Payload mc-vc
Payload
Payload
SH O O S O O
N Enhanced HN O NH S
O O HO NH H 2O NH
Hydrolysis N N HN
O S Intramolecular O
S O NH3 Rapid
O H 3N base catalysis HO H 3N
Stable bioconjugate

9
 Exercises

Complete the following scheme:

Bioconjugation methods

Answer

Glutathione
Br
O SH
O O O NH2 O
H H
N N HO N OH
HO N OH N
H H
O NH2 O O O O
S
O

N
O O
S
N SH
SH O

10
 Exercises

Draw the expected end products for each of the following reactions in aqueous environment.
If present, include reaction intermediates.
What are the advantages and disadvantages of each method.

SH O

A
S O
O NH N
N
O H

O N O
Bioconjugation methods

O
SH S NH
OH

B
NH
NH
O
O N O

SH O
S

C
N
O N O
O

A- Advantage: Greater stability of the reagent; Disadvantage: Retro-michael and thiol exchange, Formation of diastereoisomers
B- Advantage: Ring opening and greater stability of thiosuccinimide; Disadvantage: Hydrolysis of the maleimide reagent
11 C- Advantage: Does not form isomers and does not exchange with other thiols; Disadvantage: Slower kinetics
 Exercises

Which maleimide would you choose to obtain the following bioconjugation? Explain in detail.

O N O

Br
Bioconjugation methods

S S

O N O O N O

OPh

Somatostatin Bioconjugate
O O
Answer PhO N
k2>k1
N

k1 k2 O
O
1.5 eq.
Br
1.5 eq. Bromo-maleimides generate di-
H Ala Gly Cys Lys Asn Phe
Phe
H Ala Gly Cys Lys Asn Phe
modification and rebridging mixtures
Phe
O O
S Trp S Trp
N N
O
SH Lys
O
SH Lys Aryloxymaleimides can rebridge without
HO Cys Ser Thr Phe Thr HO Cys Ser Thr Phe Thr
57 57
generating di-modified conjugates
k3 k3
>k <k
k3 1 k3 2
H Ala Gly Cys Lys Asn Phe
H Ala Gly Cys Lys Asn Phe H Ala Gly Cys Lys Asn Phe Phe
Phe Phe O
O
O O S Trp
S Trp S Trp + N
N
N N S
S S O Lys
Lys Lys O
O O
HO Cys Ser Thr Phe Thr
HO Cys Ser Thr Phe Thr HO Cys Ser Thr Phe Thr
12 58 58 Mixture
59

Single product
 Problem Set

Indicate which of the following reagents allow:

a) Rebuild the bridge between two cysteines in a protein

b) A light-promoted addition
c) Addition of a second thiol
d) Selectively modify the N-terminal cysteine
e) Form a disulfide bond

O
Bioconjugation methods

RO2SS S ArO2S
N O C N
N

A B C D

F
F F
H
OH • N N
O O
F F
OH O
F
OPh

E F G H
Answer

a) D, G, H b) E c) D, G d) C e) B
13
 Problem Set

Considering the two molecules, propose a binding strategy between them that allows the bridge between the two cysteines
to be recreated. Draw the missing reagents.

S
O
Bioconjugation methods

S
HN O
R
Answer

O O R
O
N3 N
N O H
H HN O
PhO2S R
S TCEP O O
SH S S
S
SH S HN S HN
N3 N
N N

This is just an example. Other reagents that allow rebridging can be used

14
 Problem Set

Considering the following molecules, propose two binding strategies between them by choosing two of the reagents
shown. Draw reaction products and intermediates.

SH
HS
Bioconjugation methods

Answer

O O H 2N O

HS
H 2N Br Br NH2 +
S
SH S

H 2N O

R HS
R R
SH S S
S
+ hυ + hυ

15
 Problem Set

The following scheme shows the structure of an IgG1 monoclonal antibody and the fluorescent probe AlexaFluor azide.
Considering the two molecules, propose a strategy to connect them by drawing the missing reactants. Discuss the
proposed strategy in terms of functionalization location
Bioconjugation methods

1) TCEP
2) O

N
PhO
O
3) Cu (I), AlexaFluor Azide

This strategy allows the rebridging of cysteines and retains the structure of the antibody, which is important for their
structure

16
 Problem set

Which amino acids have a known conjugation reaction with diazonium salts?

Cl

N N

Answer
Protein Modification

N N
N
NH

N N
N N
pH 8.0 OH
NH
NH

N N

N
pH > 8.0 NH OH
OH
N
N

OH

N
N

17
 Problem set

Bromoacetamides are known to alkylate the lysine side chain. Why does this not happen in the following case?
Can this method be applied generally to large proteins with activity? Justify

NH2 NH2
Protein Modification

O
Br
N
H O
0.2 M formic acid N
20 ºC H
S S

Br

Answer

In the presence of a strongly acidic medium, lysines are protonated and unable to react with bromide, making it possible to
obtain some selectivity for methionine.
However, strongly acidic media cause denaturation and consequent loss of structure and activity of complex proteins.

18
 Problem set

Match the following reagents and reaction conditions (numbers) with the corresponding amino acid residues.
Each amino acid can have more than one correspondence.
Write the products.
N
O
O N
O OAc
H O
N O
N N
O H Pd(OAc)2, TPPTS O O

pH 7.4, 25 ºC NaOH, pH 11-12, 80 ºC

pH 8-9 (3 equiv)
NaNO2 80% TFA PBS, pH = 7.3
H2O/AcOH (200/1)
rt, air, 30 min

1 2 3 4 5 6
Protein Modification

OH

N O
S
O
NH NH

A B C D E
Answer

1- E 2- A 3- B 4- B 5- D 6- C

19
 Problem set

Write the products formed from the reaction of tryptophan with the following reagents:

O
H

O
1
2 3 4
80% TFA N
NH
Protein Modification

Rh2(OAc)2

O
EWG Ph
O
[Ir] N N2
O
υ = 450 nm
h
N
O (3 equiv)
NaNO2

NH

NH N

20
 Problem Set

The following figure shows the structure of Mylotarg®. This ADC was the first approved by the FDA and was used in the
treatment of acute myeloid leukemia.

Taking into account the structure of Mylotarg, propose a strategy for building it starting from the individual components.
Bioconjugation methods

O
O
N O
O O Drug
O N S
N Br HS
H O
NH2 O
HN O
N S
N Br
H

O
O
HN O Drug
N S
21 N S
H