The radiolabelling group at Almac have synthesised a number of peptide APIs containing carbon-14 amino acid residues using Solid Phase Peptide Synthesis (SPPS) approach. A number of these carbon-14 labelled peptides were modified by the addition of polyethylene glycols (PEGs) to produce a new chewmical entity with different pharmacological profile. In some cases carbon-14 labelled peptides can undergo biotinylation to provide targeted drug substances. This poster gives a general overview of SPPS, PEGlyation and biotinylation towards the synthesis of carbon-14 labelled peptides

1. Carbon-14 Labelled Peptide APIs
Solid Phase Peptide Synthesis, BIOTINylation & PEGylation
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2. Objective
• This presentation will focus on a brief
introduction to carbon-14
• Leading onto synthetic strategies towards
labelling peptides with carbon-14
14C
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3. Introduction to C
14
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4. Discovery of 14C
Martin Kamen & Sam Ruben (27-FEB-1940)
T1/2 ~ 5730 Years
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5. 14
C Starting Materials
Ba(OH) 2
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6. Barium 14C carbonate staircase
OH MeO
CH3
OH H
H314CO N O
14C
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OMe
OH
14C H3C
6 [ C]Combretastatin A-1
14
OMe N
14CH
R T Brown et al. JLCR 2009, 52, 567-570
H14C HO [14C]ZT-1
[ C]Apomorphine
14
H14CHO *
MeO Cl
14CH I
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14 N S L Kitson & L Leman et al. JLCR 2011, 54 760-770
C CH3 14CH OH
H 3
HO
[14C]XEN-D0401 Cu14CN
H
N O K14CN
HO
14CO
14 2
F3C C OH
S L Kitson. JLCR 2007, 50, 290-294
S L Kitson. JLCR 2006, 49, 517-531 OH Ba14CO3
Cl S L Kitson, S Jones. JLCR 2010, 53, 140-146
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7. 14
C Drug Molecules
C Labelled drugs are used in human mass
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balance (AME) or ADME studies to evaluate:
• Mass balance and the routes of elimination
• Identify circulatory and excretory metabolites
• Determination of clearance mechanisms
• To determine the exposure of parent compound
and its metabolites
• Used to validate animal species used for
toxicological testing
• To explore whether metabolites contribute to the
pharmacological / toxicological effects of the
drug - MIST
C Prakash et al. Biopharm. Drug Dispos; 2009, 30, 185-203
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8. 14
C Labelling Strategy
When designing a 14C labelled synthesis it is important to
consider the following:
• Identify simple starting materials from the barium 14C
carbonate ‘staircase’ which are commercially available or
alternatively easily made
• Plan, develop and execute the synthetic methodology to
the final drug substance. This approach can often restrict
the position of the label in the drug and will cause a change
in the drug purity profile from the original laboratory
synthesis route
• Locate a biologically stable position for the 14C label
S L Kitson ‘Accelerated Radiochemistry’,PMPS Manufacturing 2010, 68-70
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9. 14
C Amino acids
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10. Algae to [U-14C]-Amino Acids
Ba14CO3
14
CO2
14
C O
14
14 C
C OH
NH2
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11. 14
C Labelling
• The simplest approach to 14C labelling
involves acetylation of free amino groups in
the peptide with 14C-acetic acid via activation
to provide peptides with a specific activity of
up to 120 mCi/mmol
O O O
14 14
C 14 C 14
OH C OH C OH
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12. 14
C - Glycine Family
NH2 NH2 NH2
* CO2H *
CO2H CO2H
* *
• C-Glycine can be prepared with one or both
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carbon atoms labelled with carbon-14 leading to a
maximum specific activity of 100-120 mCi/mmol
• Incorporated during peptide assembly
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13. A Synthesis of [1-14C]Glycine
O O
NaI / Acetone K 14CN
N N
Cl I
Acetone
O O
O
AcOH / HCl aq NH2
N
CO 2H
CN
* heat *
O
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14. 14
C Peptide Strategy
S L Kitson. ‘Keeping Tags on Biomolecules’, Manufacturing Chemist April 2012
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15. • Stage 1 involves the synthesis of the peptide up to the
step prior to introduction of the 14C label
• This is most typically performed by incremental growth of
the peptide chain by solid phase peptide synthesis
(SPPS) within a peptide synthesiser
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16. • Stage 2 sees the introduction of the 14C amino acid
• This is shown ideally as the final amino acid in the
sequence although in practice further unlabelled
amino acids may need to be added thereafter
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17. • Stage 3 involves cleavage of the crude labelled peptide
from the resin support and subsequent purification by
preparative HPLC
• At this stage a full batch of analytical tests can be run to
confirm identity, purity and, over time, stability
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18. • Stage 4 sees the (optional) further functionalisation of the
labelled peptide (e.g. by PEGylation, BIOTINylation or
conjugation to other high molecular weight biomolecules)
• This additional chemistry is followed by further purification
and analytical characterisation
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19. 14
C Peptide API Case Studies
14C
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20. CASE STUDY 1:
Synthesis of [1-14C]Valine 46-mer
• Manufactured by SPPS using the Fmoc
approach
• First 32 amino acids sequence were
coupled using a 433 peptide synthesiser
by the Almac Peptide Group
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21. 14
C Radiolabelling
• Step 1 involved the synthesis of Fmoc-[1-14C]-L-
valine
• The 14-amino acid sequence containing the
Fmoc-[1-14C]-L-valine residue were coupled
manually
• Cleavage of the labelled peptide from the resin
and simultaneous deprotection using TFA
• Purification by reverse phase HPLC
• Conversion to acetate salt by preparative ion
exchange HPLC
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22. H2N 32-mer Resin
1) Coupling of 14 14
CO2H CO2H
Fmoc-OSu
NHFmoc NH2
9% aq Na2CO3
2) Capping
3) Deblock V*
H2N V* 32-mer Resin [1-14C]-L-VALINE
1) Coupling of
the 13 AAs
2) Capping
3) Deblock
H2N 13-mer -V* 32-mer Resin
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23. H2 N 13-mer -V* 32-mer Resin
TFA, Water
Thioanisole
TIS, EDT
Phenol
H2 N 13-mer -V* 32-mer OH
Purification by RP-HPLC (C18)
in 0.1 % TFA Water / 0.1 % ACN
H2N 13-mer -V* 32-mer OH
TFA Salt
Ion exchange HPLC
H2N 13-mer -V* 32-mer OH
Acetate Salt
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24. Analysis
• 0.22 mCi (8.7 MBq) of labelled [14C]-peptide
acetate salt
• Radiochemical purity = 98%area
• Specific activity = 54 mCi/mmol
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25. Case Study 2:
[14C]-BIOTINylated Peptide
14C BIOTIN
Customer Requirements:
• 2 mg [14C]-BIOTINylated peptide (84-mer)
• S.A. ≥ 300 mCi/mmol
• Terminal amino acid radiolabelled with [U-14C]-L-isoleucine
• Chemical and radiochemical purity ≥95%area
• Stability Study at 2oC and –20oC for 4 weeks
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26. Peptide Group: SPPS of Fmoc-Peptide
RESIN
ivDde Automated
Peptide
Synthesis
Fmoc ivDde
RESIN
83-mer
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27. Peptide Group: SPPS of Fmoc-Peptide
Fmoc ivDde
RESIN
83-mer
Fmoc cleavage
ivDde
RESIN
83-mer
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28. Radiolabelling: [14C]-Peptide
ivDde
RESIN
83-mer
*
CH3
14C Boc * *
H3C CO2H
* *
Boc *
NHBoc
14C ivDde
RESIN
84-mer
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29. Radiolabelling: Boc-[14C]-Peptide-Biotin
Boc
14C ivDde
RESIN
84-mer
1. Cleavage of ivDde
2. Biotin
Boc
14C BIOTIN
RESIN
Biotinylated 84-mer
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30. Radiolabelling: [14C]-Peptide-BIOTIN
Boc
14C BIOTIN
RESIN
Biotinylated 84-mer
1. Boc cleavage
2. Resin cleavage
14C BIOTIN
Biotinylated 84-mer [14C]-Peptide
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31. Project Strategy: Peptide & Radiolabelling Group
Peptide Group Core Tasks:
• Fmoc protected 83-mer peptide on resin preparation
• Trials on final peptide coupling with reduced equivalents of
radiolabelled amino acid in collaboration with radiochemistry
• Trials on ivDde cleavage
• Trials on BIOTINylation
• Trials on resin cleavage (prevention of methionine oxidation)
• Identification of suitable purification conditions
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32. Project Strategy: Peptide & Radiolabelling Group
Radiolabelling Core Tasks :
• Conversion of [U-14C]-L-isoleucine to Boc-[U-14C]-L-
isoleucine
• Trials on final peptide coupling with reduced equivalents of
radiolabelled amino acid in collaboration with the Peptide
Group
• Radiolabelled [14C]-BIOTINylated peptide synthesis
• Stability Study
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33. Summary
• 4 mg of [14C]-BIOTINylated peptide delivered on schedule
• HPLC Purity 98.9%area (RCP), 99.3%area (UV)
• SA = 338 mCi/mmol
Stability Study:
• Material stable at –20oC over 4 weeks
• 1% drop in RCP at 2oC over 4 weeks
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34. Case 3: PEGylation & Bio-conjugation
• Stage 1: In corporation of [1-14C]glycine
into the peptide sequence
• Stage 2: PEGylation
• Stage 3: Bio-conjugation to protein-SH
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35. Stage 1: [14C]-Peptide
H2N AA-SEQUENCE LINKER Resin
*
14 CO2H
C Boc Coupling
NHBoc
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Boc C AA-SEQUENCE LINKER Resin
Deprotection
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Boc C AA-SEQUENCE LINKER
SA Dilution
14
Boc C AA-SEQUENCE LINKER
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36. Stage 2: PEGylation
14
Boc C AA-SEQUENCE LINKER
O O PE
N O PEG N PEG
O O
O
14
Boc C AA-SEQUENCE LINKER PEG N
O
Boc Deprotection
O
14
C AA-SEQUENCE LINKER PEG N
O
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37. Stage 3: Bio-conjugation
O
14
C AA-SEQUENCE LINKER PEG N
O
O
14
C AA-SEQUENCE LINKER PEG N
S
O
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38. Conclusion
• Biomolecules are well recognised as a
significantly growing area within the
pharmaceutical and biotechnology sectors.
Especially in the area of peptide APIs, many of
which are being developed as potential new
therapies for a range of indications
• A critical element of the development of any drug
is an assessment of its ADME profile, most
commonly performed using 14C labelled versions
of the parent drug
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39. Conclusion
• For peptide labelling there are other options such
as tritium labelling or radio-iodination
• One clear benefit of using a 14C for the ADME
programme is the fact that the label is placed
within the core of the drug, without any risk of
wash out or need to use a modified structure
• One limitation of 14C is its rather modest maximum
specific activity (62 mCi/mmol), a limitation that
becomes ever more significant as the molecular
weight of the molecule increases
• This limitation can be overcome through the use of
Accelerated Mass Spectrometry (AMS)
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