The Development of VIP Antagonist (ANT) Peptide Derivatives and Synthetic Approaches towards the Ryptide Macrocycle Open Access
Lester, Christina (Spring 2024)
Abstract
In part 1 of this dissertation, we will discuss the development of VIP antagonist peptide (ANTs)
derivatives. ANTs improve T cell-dependent anti-tumor response in acute myeloid leukemia (AML)
murine models. Despite this, peptide therapeutics tend to suffer from poor metabolic stability and
subsequently short half-life circulation in vivo. We propose three modified ANT derivatives: Ac-
ANT308, ANT308C13C17 stp, and ANT308-PEG with the purpose of improving their drug
properties. In vitro studies found that Ac-ANT308 exhibited diminished T cell activation compared to
parental ANT308, indicating N-terminus conservation was critical for antagonist activity.
Furthermore, incorporation of cysteines at residues 13 & 17 to accommodate a staple resulted in
diminished overall survival and increased tumor burden when dosed in leukemic mice. However, the
incorporation of the staple at this position increased survival and reduced tumor burden relative to its
unstapled counterpart. Notably, ANT308-PEG had a significant positive effect, and required
significantly fewer doses to achieve comparable overall survival and tumor burden in leukemic mice
dosed with parental ANT308. In part 2 of this dissertation, we will discuss our investigation of
synthetic strategies to access the ribosomally post-translationally modified peptide (RiPP) Ryptide. To
date, there is no known synthetic strategy to access the C-C cross-linked RRY macrocycle of the
Ryptide family. We explored three synthetic routes to access this macrocycle: metallophotoredox
cross-coupling, ortho-hydroxylation, and allylic amination. While the first two strategies were
unsuccessful, we were able to access the key allylic guanidine tyrosine motif via the allylic amination
strategy. Despite accessing the linear protected VGly-Arg-Tyr trimer, we were unable to effectuate
ring closing metathesis (RCM) to complete the last major transformation prior to global deprotection
to generate the Ryptide macrocycle.
Table of Contents
Table of Contents
Chapter 1: Background of VIP and the inception of the VIP antagonist peptides 1
1.1 Introduction to VIP 1
1.1.1 VIP and its receptors: implications in physiological properties 1
1.1.2 Implication of VIP and its receptors in disease states 3
1.2 Introduction to ANT peptide series 6
1.2.1 Historical perspective of VIP agonists 6
1.2.2 Historical perspective of VIP antagonists & design rationale for 1st
generation VIP antagonist peptide 9
1.3 Concluding remarks 11
1.4 References
Chapter 2: Introduction to peptide therapeutics & peptide modifications 23
2.1 Historical perspective and applications of peptide therapeutics 23
2.1.1 Protein targets in drug discovery 23
2.1.2 Emergence of peptide therapeutics and their associated challenges 26
2.2 Introduction to peptidomimetics and peptide modifications 27
2.2.1 Historical perspective of peptide modifications 27
2.2.2 D-amino acid substitutions 28
2.2.3 Macrocyclization of peptides 31
2.2.4 Peptoid and other backbone modifications 36
2.3 Concluding remarks 40
2.4 References 40
Chapter 3: Development of ANT308 derivatives & their performance in T cell
studies of AML murine models 54
3.1 Derivatization of ANT peptides 54
3.1.1 Introduction to ANT derivatives 54
3.2 Peptide stapling of ANT308 55
3.2.1 Incorporation of a covalent staple in ANT308 55
3.2.2 Peptide stapling effects on secondary structure of ANT308 56
3.3 PEGylation of ANT308 57
3.4 in vitro T cell proliferation studies 59
3.5 in vivo AML murine studies 62
3.5.1 AML: an unmet need for clinical therapeutic development 62
3.5.2 Survival challenge studies of ANT308 derivatives 63
3.6 Plasma stability assay of ANT308 derivatives 67
3.7 Concluding remarks 68
3.8 References 69
3.9 Supporting Information 72
3.9.1 Materials 72
3.9.2 Linear peptide synthesis 72
3.9.3 Cleavage and purification of crude peptides 73
3.9.4 Peptide stapling and purification 74
3.9.5 Synthesis and purification of peptide-PEG conjugates 74
3.9.6 Peptide analysis and characterization 75
3.9.7 Circular dichroism spectroscopy 75
3.9.8 α-helical content 76
3.9.9 Plasma stability assay 76
3.9.10 in vitro T cell studies 76
3.9.11 in vivo AML studies 77
3.9.12 HPLC and MS data 78
3.9.13 Peptide-PEG Analysis and Characterization 84
Chapter 4: Introduction to RiPP macrocyclic peptides 85
4.1 Introduction to macrocyclic peptides in drug discovery 85
4.1.1 Historical perspective of natural products in drug discovery – Introduction
of Ribosomally synthesized and post-translationally modified peptides (RiPPs) 85
4.1.2 Genome sequencing and biosynthesis of RiPPs 87
4.2 Synthetic approaches toward RiPP peptides 88
4.2.1 Synthetic approaches towards RiPPs with C-C crosslinks to Tyrosine 88
4.2.2 Synthetic approaches towards RiPPs with C-C crosslinks of Arg
(Xenorceptide) 97
4.2.3 Introduction to Ryptide & retrosynthetic analysis 98
4.3 Concluding remarks 99
4.4 References 100
Chapter 5: Progress towards the synthesis of Ryptide 107
5.1 Synthetic strategies leveraging metallophotoredox cross-coupling to access the
Tyr fragment 107
5.1.1 Historical perspective of metallophotoredox chemistry of amino acids 107
5.1.2 Metallophotoredox chemistry towards ryptide core 111
5.2 Synthetic strategy leveraging o-hydroxylation of Tyr precursor 114
5.2.1 Background on o-hydroxylation of aryl halides 114
5.2.2 ortho-hydroxylation chemistry towards Ryptide macrocycle 117
5.3 Synthetic strategy leveraging allylic amination towards a Tyr precursor 119
5.3.1 Background on stereoselective allylic amination 119
5.3.2 Allylic amination chemistry towards Ryptide macrocycle 122
5.4 Synthesis towards dipeptide fragment for ring closing metathesis 125
5.4.1 Synthesis of vinylglycine 125
5.4.2 Background on RCM to generate macrocyclic peptides 130
5.4.3 Exploration of RCM and CM of dipeptide and tyrosine fragments 134
5.5 Concluding remarks 138
5.6 References 138
5.7 Supporting information 150
5.7.1 General information 150
5.7.2 General procedures 151
5.7.3 References 193
List of Figures
Chapter 1
Figure 1.1 VIP docking and binding to its VPAC receptor 2
Figure 1.2 VIP plays an important role in immunomodulation 4
Figure 1.3 Sequence deviation of VIP agonists in literature 6
Figure 1.4 Sequence deviation of VIP antagonists in literature 9
Chapter 2
Figure 2.1 Comparison of small molecule vs. peptide properties when binding to
shallow vs. defined binding sites 24
Figure 2.2 Implications of D-amino acids on peptide secondary structure 29
Figure 2.3 Different types of peptide macrocycles 31
Figure 2.4 Different classes of peptidomimetics possessing modified peptide
backbones 36
Figure 2.5 Different classes of biomolecules employed in therapeutic peptide
conjugates 38
Chapter 3
Figure 3.1 m-xylene bisalkylation to generate stapled ANT308 55
Figure 3.2 Circular dichroism spectra for ANT308 and ANT308 derivatives 56
Figure 3.3 Generation of ANT308-PEG via strain-promoted copper-free click
chemistry 57
Figure 3.4 in vitro T-cell activation study of acetylated and stapled ANT308 59
Figure 3.5 ANT308 & ANT308-PEG activate human T cells 60
Figure 3.6 In myeloid sarcoma murine models, ten doses of ANT308C13C17 stp
has similar prolonged survival of tumor burden mice as ten doses of ANT308,
compared to control mice 62
Figure 3.7 In myeloid sarcoma models, ten doses of ANT308 control and
ANT308C13C17 stp prolonged tumor burden suppression relative to
ANT308C13C17 unstp and control mice 63
Figure 3.8 Four doses of ANT308-PEG significantly prolonged survival tumor
burden mice as fourteen doses of ANT308, compared to control mice 64
Figure 3.9 Four doses of ANT308-PEG significantly prolonged tumor burden
suppression relative to fourteen doses of ANT308 and control mice 65
Figure 3.10 Control study monitoring propantheline bromide degradation 66
Chapter 4
Figure 4.1 The emergence of the RiPP subclass 86
Figure 4.2 General biosynthetic pathway towards generation of RiPPs 87
Figure 4.3 Different RiPP subclasses containing C-C crosslinks to tyrosine 89
Figure 4.4 Structure of Xenorceptide 98
Figure 4.5 Structure of Ryptide 99
Chapter 5
Figure 5.1 Simplified schematic depicting approaches towards allylic C-H amination 119
Figure 5.2 Predominant olefin metathesis catalysts used in CM and RCM 130
Figure 5.3 Pentenyl-alanine (top) and allylglycine (bottom) are commonly employed
in RCM of peptides, unlike vinylglycine 132
List of Schemes
Chapter 2
Scheme 2.1 Lactamization and disulfide bridging are common methods that leverage
the native functionalities of amino acids 32
Scheme 2.2 Hydrocarbon stapling and Click chemistry utilize non-canonical
residues to effectuate chemoselective cyclizations 33
Scheme 2.3 Utility of copper & copper-free click chemistry 35
Scheme 2.4 Solid phase synthesis of N-glycine peptoids 37
Chapter 4
Scheme 4.1 Weinreb synthesis of PQQ 90
Scheme 4.2 Corey synthesis of PQQ 91
Scheme 4.3 Hendrickson synthesis of PQQ 92
Scheme 4.4 Büchi synthesis of PQQ 93
Scheme 4.5 Boger synthesis of PQQ 94
Scheme 4.6 Zhu synthesis of the Cittilin western macrocycle 95
Scheme 4.7 Zhu total synthesis of the Cittilin B atropisomer 96
Scheme 4.8 Boger synthesis of the eastern macrocycle of Cittilin B 97
Scheme 4.9 Retrosynthetic analysis of Ryptide 100
Chapter 5
Scheme 5.1 General strategy leveraged by MacMillan and co-workers to forge
stereoselective C-C crosslinks via decarboxylative α-amino radical formation 107
Scheme 5.2 Forward synthesis leveraging metallophotoredox cross-coupling 110
Scheme 5.3 A&B Asymmetric decarboxylative metallophotoredox cross-couplings 111
Scheme 5.4 A&B Racemic ligand model systems 112
Scheme 5.5 A&B Racemic ligand cross-coupling of Ser/phenol and Ser/Tyr 113
Scheme 5.6 ortho-hydroxylation of Cbz-benzylic amines developed by Zhao & coworkers
114
Scheme 5.7 Re-worked total synthesis of the ryptide core, starting with orthohydroxylation
of a protected glycinol derivative 115
Scheme 5.8 Potential entries (XEC or Negishi) to access the tyrosine fragment to
furnish the Ryptide RRY macrocycle. 116
Scheme 5.9 Initial investigations of ortho-hydroxylation on benzylamine model
systems 117
Scheme 5.10 Strategy to access asymmetric allylic aminations of 2. racemic allyic
alcohols developed by Carreira and co-workers 120
Scheme 5.11 Revised total synthesis of ryptide macrocyclic core leveraging
enantioselective allylic amination 121
Scheme 5.12 Macmillan and co-workers’ metallophotoredox XEC strategy to access
aryl amino acids 123
Scheme 5.13 Jackson and co-workers’ utility of both inter- and intramolecular
Negishi cross-coupling to forge cyclic peptides 124
Scheme 5.14 Our utility of Negishi cross-coupling to furnish protected allylic
guanidine derived tyrosine 125
Scheme 5.15 A-C Different synthetic strategies developed towards L-vinylglycine via
chrions 126
Scheme 5.16 Different synthetic strategies developed towards L-vinylglycine via
chrions 127
Scheme 5.17 Acidolysis of differentially protected benzyl ester vinylglycine
derivatives 128
Scheme 5.18 Our synthetic attempts to access the dipeptide vinylglycine-arginine
fragment 129
Scheme 5.19 Total synthesis of Syringolin A utilizes a vinylglycine to furnish the
macrocycle via RCM 133
Scheme 5.20 Our model systems for RCM 134
Scheme 5.21 Our investigation of RCM towards the protected and unsaturated
Ryptide macrocycle 135
Scheme 5.22 Our investigation of CM to enable lactamization to furnish the
protected, unsaturated Ryptide macrocycle 136
Scheme 5.23 Schrock catalysts have been employed to effectuate CM of protected
vinylglycine 137
List of Tables
Chapter 2
Table 2.1 Peptides possess advantageous drug profiles of both small molecules and
biologics 26
Chapter 3
Table 3.1 Calculated helicity values of ANT308 and ANT308 derivatives 56
Table 3.2 Area under the curve replicate averaged to quantitate remaining intact
control 67
Table 3.3 HPLC gradient for ANT peptide purification 74
Table 3.4 List of antibodies used for flow cytometry 77
Table 3.5 Comparison of calculated & experimental m/z 78
Table 3.6 SDS-PAGE analysis of purified ANT308-PEG 84
Chapter 5
Table 5.1 PIFA screen of the ortho-hydroxylation model reaction 118
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