Specificity in Protein Chemistry and Analysis: I. Analysis of Deamidation and Analytical Artifact II. Chemo-Enzymatic Site-Specific Bioconjugation of Protein and Peptides
by Shanshan Liu
B.Sc. in Pharmaceutical Science, Tianjin University
A dissertation submitted to
The faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Doctor of Philosophy
August 11, 2017
Dissertation directed by
Zhaohui Sunny Zhou Professor of Chemistry and Chemical Biology
Acknowledgements
First, I would like to thank my supervisor, Professor Sunny Zhou, for giving me the chance to pursue my PhD at Northeastern, and his enormous help during the past five years. I am very grateful for his effort of leading me to be a high standard scientist. The passion and critical thinking he teach me will inspire me for my whole career.
I particularly thank my wonderful lab mates and colleagues, Dr. Tianzu Indi Zang, Dr.
Wanlu Qu, Dr. Kalli Catcott, Dr. Chris Chumsae, Lihua Yang, Kevin Moultin and Amissi Sadiki for their suggestions and assistance in my works. In addition, thanks for being part of my
Sunnyland journey, their understanding and caring are one of the best memories.
I would like to thank Dr. Richard Duclos, Dr. Poguang Wang, Prof. Jisheng Ma, for their excellent instructions and suggestions to my training in organic, biological, and analytical techniques. I would express my thanks to the ambitious undergraduate researchers I’ve worked with Mike Pablo, Ximing Gao, Joyce Chen, Erika Park and Bryan Wang.
I am indebted to my committee members, Prof. Penny Beuning, Prof. Jeffrey Agar, Prof.
Leila Deravi, and Dr. Jared Auclair for being my key expert in the fulfillment of my degree.
Finally, I would like to address my special thanks to my excellent parents, for being my greatest support in every aspect of my life, for giving me the confidence and for being an independent individual.
I couldn’t be here without any of you.
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Abstract of Dissertation
Ubiquitously occurring in protein analysis, analytical artifacts are usually neglected and underestimated. For example, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifacts can be easily introduced during sample preparation, which not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes. A method that effectively eliminates deamidation artifacts during proteolysis has been developed by simply performing proteolytic digestion at pH 4.5 using the readily available Glu-C protease. This method is generally applicable to protein analysis as it requires minimal sample preparation.
In the meantime, with the expertise in bioorganic chemistry and protein analysis, we envision that selective bioconjugation and derivatization of peptides and proteins can be achieved via a combination of engineered enzymes of tailored sequence specificity and chemical reactions of functional selectivity, hence the hybrid modality engineering of protein pharmaceuticals.
A series of novel site-specific derivatization methods of peptides, proteins and peptidyl analogs have been devised. The site specificity can be achieved either enzymatically or chemically via reactions of latent residues. For instance, one example is utilizing transglutaminase (TGase) enzymes to modify glutamine-containing peptides/proteins to generate new functionality to the peptidyl substrate. Moreover, further derivatization can be achieved via orthogonal chemistry towards the newly introduced functional groups. Such kinds of hybrid modality engineering of existing protein pharmaceuticals enable the introduction of new modifiers and/or drug conjugates to the target drug, which can bring competitive or alternative iii reactivity, as well as novel PK and PD properties. These drug entities may serve as new drugs, probes and reagents.
In practice, the chemo-enzymatic approaches had been applied to existing protein biopharmaceuticals and several novel derivatives have been obtained. Various derivatization strategies such as fluorescent tagging, PEGylation, biotinylation and click chemistry have been accomplished.
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Table of Contents
Acknowledgements ...... ii
Abstract of Dissertation ...... iii
Table of Contents ...... v
List of Schemes ...... x
List of Figures ...... xii
List of Symbols and Abbreviations ...... xiv
Chapter 1. Introduction: From Analysis of Protein Modification to Site-Specific Bioconjugation...... 1
1.1 Deamidation of asparagine and deamidation artifact during sample preparation ...... 2
1.1.1 Deamidation and formation of isoaspartic acid ...... 2
1.1.2 Deamidation artifact, the overlooked challenge ...... 7
1.2 Artifacts in protein analysis ...... 10
1.2.1 Artifacts related to sample preparation ...... 10
1.2.2 Importance of artifacts in protein analysis ...... 13
1.2.3 Reduction and elimination of artifacts ...... 14
1.3 Method development of protein site-specific bioconjugation ...... 15
1.3.1 Methodology and site-specificity ...... 15
1.3.1.1 Chemical strategy ...... 16
1.3.1.2 Enzyme introduced specificity ...... 17
1.3.2 The platform build-up ...... 18
v
1.4 References ...... 19
Chapter 2. Mildly Acidic Conditions Eliminate Deamidation Artifact during Proteolysis: Digestion with Endoprotease Glu-C at pH 4.5 ...... 25
2.1 Abstract ...... 26
2.2 Introduction ...... 27
2.3 Experimental section ...... 31
2.3.1 Materials ...... 31
2.3.2 Proteolysis ...... 31
2.3.3 Aging of ACTH peptide...... 32
2.3.4 Mass Spectrometry...... 32
2.4 Results and discussion ...... 34
2.4.1 Analysis of deamidation ...... 34
2.4.1.1 Deamidation of Calmodulin ...... 34
2.4.1.2 Deamidation of Exenatide ...... 39
2.4.1.3 Deamidation of ACTH peptide ...... 43
2.4.2 No Isomerization at pH 4.5 ...... 45
2.4.3 Nearly identical proteolytic specificity at pH 4.5 and 8.0 ...... 49
2.5 Conclusions ...... 52
2.6 References ...... 53
Chapter 3. Hybrid Modality Engineering of Exenatide, Chemo-enzymatic Site-specific Bioconjugation Mediated by Transglutaminase ...... 59
vi
3.1 Abstract ...... 60
3.2 Introduction ...... 61
3.3 Experimental section ...... 65
3.3.1 Materials ...... 65
3.3.2 Bioconjugation ...... 66
3.3.2.1 TGase-mediated propargylation ...... 66
3.3.2.2 TGase-mediated biotinylation ...... 66
3.3.2.3 TGase-mediated one-step PEGylation ...... 66
3.3.2.4 Derivatization of propargylated-exenatide with bromo-coumarin ...... 67
3.3.2.5 Derivatization of propargylated-exenatide by poly-histidine azide ...... 67
3.3.3 Analysis...... 69
3.3.3.1 Tryptic digestion ...... 69
3.3.3.2 SDS-PAGE ...... 69
3.3.3.3 MALDI-TOF/TOF mass spectrometry ...... 70
3.3.3.4 LC-QTOF mass spectrometry ...... 70
3.4 Results and Discussion ...... 72
3.4.1 Propargylation of exenatide ...... 72
3.4.2 Biotinylation of exenatide ...... 76
3.4.3 PEGylation of exenatide ...... 78
3.4.4 Additional derivatization of propargylated-exenatide through click chemistry ..... 82
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3.4.4.1 Fluorescent tagging of exenatide via click chemistry ...... 82
3.4.4.2 Poly-histidine tagging of exenatide via click chemistry ...... 86
3.5 Future directions ...... 90
3.6 References ...... 91
Chapter 4. Site-specific Bioconjugation of Peptides and Proteins by Nucleophile Trapping of Metastable Succinimide ...... 95
4.1 Abstract ...... 96
4.2 The latent reactivity behind succinimide formation: from an unavoidable problem to a
derivatization site ...... 97
4.2.1 Step 1. Formation of succinimide and latent reactivity ...... 100
4.2.1.1 Effects of conformation ...... 102
4.2.1.2 Effects of pH ...... 102
4.2.2 Step 2. Nucleophile trapping of succinimide ...... 103
4.2.3 Step 3. Further derivatization of hydrazides ...... 106
4.3 Derivatization of Vancomycin ...... 108
4.4 Derivatization of exenatide ...... 111
4.5 Derivatization of bacitracin ...... 118
4.6 Experimental section ...... 122
4.6.1 Materials ...... 122
4.6.2 Bioconjugations ...... 122
4.6.2.1 Derivatization of vancomycin ...... 122
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4.6.2.2 Derivatization of exenatide ...... 123
4.6.2.3 Dansylation of exenatide hydrazide ...... 123
4.6.2.4 Derivatization of bacitracin ...... 123
4.6.3 Mass spectrometry ...... 124
4.7 Prospective ...... 125
4.8 References ...... 126
Chapter 5. Conclusions and Prospective ...... 129
5.1 Photocaging, from simple derivatization to on-demand manipulation of protein ...... 131
5.2 A platform for on-demand design of protein and peptide ...... 133
5.3 References ...... 135
ix
List of Schemes
Scheme 1.1. Deamidation of asparagine, isomerization of aspartic acid and the formation of isoaspartic acid...... 3
Scheme 1.2. Protein L-isoaspartate O-methyltransferase catalyzed repair of isoaspartic acid. .... 5
Scheme 1.3. Methyl donor S-adenosyl L-methionine and its conversion to S-adenosyl L- homocysteine ...... 6
Scheme 1.4. 18O incorporation of succinimide hydrolysis generate additional mass shift...... 8
Scheme 2.1. Deamidation of asparagine (Asn) and the formation of aspartic acid (Asp) and isoaspartic acid (isoAsp) via a succinimide intermediate...... 30
Scheme 3.1. General scheme of transglutaminase catalyzed transamidation of substrate glutamine...... 62
Scheme 3.2. TGase mediated propargylation of exenatide...... 72
Scheme 3.3. TGase mediated biotinylation of exenatide...... 76
Scheme 3.4. TGase mediated one-step PEGylation of exenatide...... 78
Scheme 3.5. Fluorescent tagging of exenatide...... 82
Scheme 3.6. Poly-histidine tagging of exenatide via click chemistry ...... 86
Scheme 4.1. The formation of a succinimide intermediate, the subsequent trapping by hydrazine and further derivatization of hydrazide ...... 98
Scheme 4.2. Examples of functional groups that can form succinimide...... 100
Scheme 4.3. Trapping of the succinimide intermediate by nucleophiles...... 103
Scheme 4.4. Trapping of succinimide with various nucleopholes...... 105
Scheme 4.5. Further derivatization of hydrazides via biorthogonal chemistry ...... 106
x
Scheme 4.6. Selective reaction of aldehyde with hydrazides against amines at mildly acidic conditions ...... 107
Scheme 5.1. The installation of photocleavable reagent into peptide and protein via enzyme- mediated transamidation...... 131
Scheme 5.2. The installation of photocleavable reagent into peptide and protein via nucleophilic trapping of succinimide...... 132
Scheme 5.3. The generation of photolabile protein conjugation and the release of conjugation through photolysis...... 132
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List of Figures
Figure 1.1. MALDI MS spectra of PEGylated exenatide mixture at different laser intensity. .... 12
Figure 2.1. MALDI MS spectra of calmodulin Glu-C peptide under various conditions...... 36
Figure 2.2. MALDI MS/MS spectra of calmodulin Glu-C peptide...... 37
Figure 2.3. MALDI MS spectra of calmodulin Glu-C peptide 55-67 in negative ion mode ...... 38
Figure 2.4. MALDI MS spectra of exenatide Glu-C peptide under various conditions...... 40
Figure 2.5. Extracted ion chromatogram and ESI MS spectra of doubly charged exenatide Glu-C peptide after 48 h proteolysis...... 41
Figure 2.6. Extracted ion chromatograms of exenatide Glu-C peptide and ESI MS spectra of extracted aspartyl species at pH 4.5...... 42
Figure 2.7. MALDI MS spectra of the intact and ACTH Glu-C peptides...... 44
Figure 2.8. MALDI MS spectra of 18O-labeled calmodulin Glu-C peptides at pH 4.5 ...... 46
Figure 2.9. MALDI MS/MS spectra of 18O-labeled calmodulin Glu-C peptides at pH 4.5 ...... 47
Figure 2.10. Zoomed-in MS/MS spectra of 18O-labeled calmodulin Glu-C peptides at pH 4.5 . 48
Figure 2.11. MS spectra of calmodulin Glu-C digests at both pH 4.5 and pH 8.0...... 50
Figure 2.12. Sequence coverage of calmodulin by Glu-C proteolysis...... 51
Figure 3.1. Structure analysis of exenatide...... 63
Figure 3.2. MS spectra of native and propargylated-exenatide...... 74
Figure 3.3. MS/MS spectra of native and propargylated exenatide...... 75
Figure 3.4. LC-MS of exenatide biotinylation product...... 77
Figure 3.5. MS spectra of PEGylated-exenatide...... 80
Figure 3.6. SDS-PAGE of PEGylated exenatide mixture...... 81
Figure 3.7. Derivatization of propargylated-exenatide with Br-coumarin ...... 84
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Figure 3.8. MS/MS spectra of bromo-coumarin tagged exenatide tryptic peptides...... 85
Figure 3.9. MS spectra of poly-histidine tagged exenatide fragment...... 87
Figure 3.10. MS/MS spectra of poly-histidine tagged exenatide fragment...... 88
Figure 4.1. The latent reactivity control by reaction conditions...... 101
Figure 4.2. Vancomycin structure and derivatives obtained hydrazine nucleophile trapping. ... 109
Figure 4.3. MALDI-TOF MS spectra of hydrazine treated vancomycin...... 110
Figure 4.4. Chemical structure of exenatide and specific compounds observed from exenatide derivatization...... 112
Figure 4.5. LC-MS spectra of native and hydrazine treated exenatide...... 113
Figure 4.6. MS spectra of native and hydrazine treated exenatide tryptic peptide ...... 114
Figure 4.7. MS/MS spectra of exenatide tryptic peptide...... 115
Figure 4.8. Zoomed MALDI-TOF MS/MS spectra of singly charged exenatide tryptic peptide.
...... 116
Figure 4.9. MS of exenatide standard and exenatide after hydrazine treatment and dansylation.
...... 117
Figure 4.10. Structure of bacitracin A and derivatives observed ...... 119
Figure 4.11. MS spectra of bacitracin ...... 120
Figure 4.12. MS/MS spectra of singly charged bacitracin and bacitracin hydrazide...... 121
Figure 5.1. The platform for hybrid modality engineering of protein and peptides ...... 133
xiii
List of Symbols and Abbreviations
°C degrees Celsius
15N nitrogen 15
18O oxygen 18
ACTH adrenocorticotropic hormone
ADC antibody drug conjugate
AdoHcy (SAH) S-adenosylhomocysteine
AdoMet (SAM) S-adenosylmethionine
Asn (N) asparagine
Asp (D) aspartic acid
Asu aspartyl succinimide
β beta
CAS Chemical Abstracts Service
C18 octadecyl carbon chain
CHCA alpha-cyano-4-hydroxy-cinnamic acid
Da Dalton
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
xiv
E. coli Escherichia coli
EC Enzyme Commission
ECD electron capture dissociation
ESI electrospray ionization
ETD electron transfer dissociation
FA Formic acid
Gln (Q) glutamine
GLP-1 glucagon-like peptide-1
Glu (E) glutamic acid
Gly (G) glycine
H histidine
HCl hydrochloric acid
His-Tag polyhistidine-tag
HPLC high performance liquid chromatography h hour isoAsp (isoD) isoaspartic acid
K lysine kDa kilodalton
KO knock off
xv
kV kilovolt
LC-MS liquid chromatography-mass spectrometry
μL microliter
μM micromolar
μm micron, micrometer
M molar m/z mass over charge
MALDI matrix-assisted laser desorption/ionization mg/mL milligram per milliliter min Minute mM millimolar
Mn number-average molecular weight
MS mass spectrometry
MS/MS tandem mass spectrometry
Mw weight-average molecular weight
MWCO molecular weight cut off m/z mass to charge ratio
PD pharmacodynamics
PEG polyethylene glycol
xvi
pH negative logarithm of the hydrogen ion concentration
PIMT or PCMT Protein L-isoaspartate O-methyltransferase
PK pharmacokinetic
PLP pyridoxal-5-phosphate ppm parts per million
Pro (P) Proline
PTM post-translational modification
Q-ToF quadrupole time-of-flight mass spectrometer
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
TCEP tris(2-carboxyethyl)phosphine
THPTA tris(3-hydroxypropyltriazolylmethyl) amine
TFA trifluoroacetic acid
TGase transglutaminase
TIC total ion chromatography
TOF time of flight
Tris tris(hydroxymethyl)aminomethane
UV ultraviolet
U unit
xvii
U/g unit per gram v/v volume per volume w/w weight per weight
XIC extracted ion chromatogram
xviii
Introduction:
From Analysis of Protein Modification to Site-Specific
Bioconjugation
1
1.1 Deamidation of asparagine and deamidation artifact during sample preparation
1.1.1 Deamidation and formation of isoaspartic acid
In terms of mass change, deamidation is one of the smallest post translational modifications that can happen to proteins and peptides.1 As a ubiquitous, non-enzymatic process, deamidation can occur spontaneously on asparagine (Asn or N) and glutamine (Gln or Q) residues. As described in Scheme 1.1, the backbone amide acts as a nucleophile, attacks the side chain amide group of asparagine and glutamine, then forms a cyclic amide called aspartyl succinimide (Asu) with the release of the side chain amine group; the resulting succinimide can then be hydrolyzed.2-4 Since the hydrolysis can occur at two different positions, either aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD) can be generated correspondingly in an approximate ratio of 1:3.1 It worth noting that compared to asparagine deamidation, spontaneous glutamine deamidation occurs less frequently and with a much slower rate, thus it’s generally not an issue.
2
Scheme 1.1. Deamidation of asparagine, isomerization of aspartic acid and the formation of isoaspartic acid.
The deamidation process results in +1 Dalton shift in mass and +1 shift in charge to the protein. Furthermore, the formation of isoaspartic acid, which may also come from the isomerization of aspartic acid, inserts an additional carbon into the peptide backbone, thus generating a β-peptide linkage and introducing a “kink” conformation to the protein.5 The charge and structure change cause problems such as degradation, charge variant, immunogenicity and may ultimately lead to functional change of protein in vivo and in vitro.6-8
It has been implicated that isoAsp accumulation is correlated to diseases such as Alzheimer’s disease related β-amyloid aggregation, crystalline aggregates in cataract as well as autoimmunity.9-11 Besides, the accumulation of isoAsp over time is considered to be a
“molecular clock” that may be associated with aging.2 Last but not least, deamidation may affect the long term stability, efficacy and toxicity of peptide and protein pharmaceuticals.12
3
The rate of deamidation is closely related to primary sequence, 3D structure, as well as environmental aspects such as pH, ionic strength and temperature.13-14 Sequence, especially the contiguous residue C-terminal to asparagine and glutamine, is the primary determinate of the deamidation rate; typically, a linear peptide that contains certain tandem amino acids, such as
Asp-Gly tandem, is more prone to deamidation than any other sequence. The following mnemonic is used to summarize hotspots of asparagine deamidation: Not Good for NG (highly likely to deamidate); Not Happy for NH and Not Sure for NS (likely); and No Problem for NP
(not likely). Higher-order structure can alter deamidation rate as well, by enhancing or reducing the rigidity and solvent accessibility of the residue. Additionally, higher pH and temperature also accelerate the deamidation rate. The degradation half-life of deamidation varies from 24 h to 500 days. An interesting example that is worth mentioning is cytochrome C, in which previous studies indicated that the deamidation of the first residue (Asn 103) accelerates the deamidation of second residue (Asn 54).15
4
Scheme 1.2. Protein L-isoaspartate O-methyltransferase catalyzed repair of isoaspartic acid.
Protein L-isoaspartate O-methyltransferase (PIMT or PCMT, EC 2.1.1.77) is an S-adenosyl
L-methionine (AdoMet or SAM) dependent enzyme that specifically recognizes isoaspartic acid and “repairs” the isoAsp to normal L-aspartic acid in vivo and in vitro.16-18 During the process, a methyl group is firstly transferred from AdoMet to isoAsp to form an isoaspartate methyl ester, the methyl donor is then converted to S-adenosyl L-homocysteine (AdoHcy or SAH, Scheme
1.3); the labile isoaspartate methyl ester then cyclizes to the same succinimide intermediate as deamidation process, and finally is hydrolyzed to aspartic acid or isoaspartic acid (Scheme
1.2).19-20 PIMT can be found in nearly all eukaryotes and some bacteria such as E. coli, and acts as an in vivo “repair” of isoAsp. Animal tests using PIMT-KO mice suggests that without PIMT, vast accumulation of isoAsp accumulation will result in fatal consequences in a short time.21
5
Scheme 1.3. Methyl donor S-adenosyl L-methionine and its conversion to S-adenosyl L- homocysteine
Despite the detrimental impacts caused by deamidation and isoaspartic acid, the detection of deamidation and isoaspartic acid is relatively challenging due to the subtle mass and charge difference. To differentiate isoAsp from Asp is even more difficult, since there is no mass or charge difference at all. The direct way to distinguish isoaspartic acid from aspartic acid is using electron capture/transfer dissociation (ECD or ETD) mass spectrometry, the dissociation of isoaspartic acid generates unique c, z ions after fragmentation with a 57 Da mass shift compared to aspartic acid. However, the sensitivity of detection is limited due to limited ionization efficiency.22 The introduction of Asp-N proteolysis in sample preparation together with ETD enables the detection of as little as 0.5% of isoAsp.23 On the other hand, several alternative quantification and identification methods have been developed by utilizing PIMT, such as
ISOQUANT, PIMT mediated 18O-Labeling and hydrazine trapping.24-26
6
1.1.2 Deamidation artifact, the overlooked challenge
Deamidation artifacts can be easily introduced during sample preparation, which not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes. For example, human thioltransferase (Glutaredoxin-1, Grx) purified from human red blood cells in 1991, was initially found to display a mixture of Asp and Asn at residue
51, but it was later shown that the observed Asp51 was actually a deamidation artifact during purification.27 Gradually, researchers began to pay attention to the artifact identification, while the ratio of actual deamidation has been overestimated and the effect of artifacts introduced during process/sample preparation has been underestimated. In 2006, Krokhin et. al. reported that peptides containing NG tandem show 70-80% of deamidation during trypsin proteolysis in ammonium bicarbonate at 37 oC after 12 h. The NS tandem shows ~10% of degradation during the 12 h digestion.28
7
Scheme 1.4. 18O incorporation of succinimide hydrolysis generate additional mass shift.
Starting around 2007, 18O labeling strategy was introduced in the analysis of deamidation.
Then in 2008, Peter O'Connor, Hongcheng Liu etc., and their groups published a series of papers discussing the utilization of 18O water to distinguish bona fide deamidation and deamidation artifacts in sample preparation.29-30 In brief, the incorporation of 18O water will introduce up to two 18O atoms into a newly generated peptide during proteolysis (Scheme 1.4).31 The resulting additional +2 Da to +4 Da mass shift makes it readily detectable on standard mass spectrometers.
In 2007, Stroop et. al. proposed a modified trypsin digestion protocol with lower pH (6.0), temperature (4 oC), and longer digestion time (24-120 h).32 In 2009, Ren et. al. presented several tips to minimize digestion-induced modification, including the removal of guanidine from digestion buffer to maximize trypsin activity; thus minimizing the reduction and alkylation time; using 30-min in-solution digestion, etc.33 Hao et. al discussed the buffer effect on deamidation artifacts during trypsin digestion, in which four buffers were compared and found that the ammonium acetate at pH 6 was effective at reducing artifact comparatively.34 Recently,
Procopio developed a practical protocol of reducing deamidation artifacts in bone proteomics, generally handling the whole process in weakly acidic conditions, in addition to other tricks
8
including the addition of guanidine. As deamidation can be a degradative marker that has potential utility in postmortem related studies, the authors adopted the methodology from the Sze paper mentioned earlier, to practical protocol in the area of bone proteomics.35
In summary, all the current strategies can be concluded as to minimize the overall preparation time, as well as minimize the rate of deamidation. The latter basically include using lower pH and lower temperature.
9
1.2 Artifacts in protein analysis
1.2.1 Artifacts related to sample preparation
Deamidation artifact is not the only artifact that can be neglected and underestimated. It has been noted that sample preparation can introduce modifications, however, little attention has been paid to them. Volkin et. al. wrote a couple reviews about reoccurring modifications that affect protein stability in vivo and during purification, storage and handling back in 1990’s.36-37
Burgess discussed some analytical artifacts that can be easily forgotten in protein.38
Covalent modifications of proteins are the main source of analytical artifacts, some of which are relatively well known like methionine oxidation, while some can be neglected. For example, cysteine related degradations, such as oxidation, S-thiolation and disulfide scrambling are quite commonly observed during analysis and storage.39-41 Photolysis can occur on selected amino acids, particularly tyrosine and tryptophan.42-43 Some asparagine sequences, especially asparagine-proline tandem is known to be more labile than the other, bond cleavage has been observed during sample preparation of SDS-PAGE after heat.44
Some artificial modifications can be induced by buffer: conversion from cysteine to alanine has been observed during sample preparation via β-elimination of disulfide bond by heating with
TCEP.45 The ammonium cyanate in urea reacts with the amino group of lysine and the N terminus, as well as, to a lesser extent, arginine and cysteine, results in the carbamylation of protein, and generating a 43 Da mass shift.46 Moreover, it has been reported that several commonly used additives in cell culture, sample preparation, as well as storage can cause artifacts. Glycation of protein often occurs during storage, as the additive reducing sugars form
10
covalent adducts with the protein.47 Other examples such as citric acid and ascorbic acid, caused covalent modification of proteins are also reported.48-50
Besides, instrument related misassignment is another source of analytical artifacts. For example, the detection of low-abundance species is a global challenge for their insufficient ionization; aspartic acid and glutamic acid can be generated from deamidation, which can be easily misassigned to asparagine and glutamine since they usually overlap; lysine and glutamine can be misassigned because of similar molecular weight, especially with low resolving power instruments.51
Different instrumentation set-ups could result in different consequences. A recent example seen in our lab is the MALDI-TOF analysis of the PEGylation mixture of exenatide, a peptide biopharmaceutical as shown in Figure 1.1.52 The sample from the same spots ionizes differently at different laser intensity, the PEGylated exenatide appears “pure” since one of the starting material PEG amine was not observed at lower laser intensity. The residue PEG amine and
PEGylated-exenatide were both observed at higher laser intensity. Also, it has been noticed that the calculated Mn (number-average molecular weight) and Mw (weight-average molecular weight) differ from different spectrum even under the same calculation criteria, the Mn of lower intensity PEG-exenatide is larger than the high intensity one.53 One hypothesis is that due to the overlapping between PEG and PEG-exenatide as well as the limited resolution of the spectra, the calculated Mn of two cluster interferes, especially when the two species has been both well ionized.
11
Figure 1.1. MALDI MS spectra of PEGylated exenatide mixture at different laser intensity.
MALDI-TOF MS spectra of PEGylated-exenatide reaction mixture under linear ion mode at laser intensity 2700 (top, PEGylated-exenatide, calculated Mn 14801.90 Da), laser intensity 3340 (middle, PEG amine, vendor provided Mn ~10000 Da, calculated Mn 10590.70 Da; PEGylated- exenatide, calculated Mn 14708.41 Da) and laser intensity 3540 (bottom, PEG amine, vendor provided Mn ~10000 Da, calculated Mn 10799.01 Da).
12
1.2.2 Importance of artifacts in protein analysis
The existence of artifact affects the analysis of protein in various aspects. First, there are artifacts without biological implications, which are relatively simple. The artifacts only complicate the analysis by adding false positive signals, as well as shielding the real signal of low-abundance species and thus create false negative results. These artifacts can be easily depleted when processing the data. Things become more complicated when the artifacts observed are comparable or identical to the process in biological systems, i.e. the artifacts have biological implications. To distinguish the false positive artifact and bona fide modifications would be challenging.
In the case of deamidation, we’ve realized that some of our previously reported deamidation may be a combination of “real” deamidation and artifacts during proteolysis. As previously mentioned, cysteine to alanine, glutamine to glutamic acid during sample processing has been observed as well. These modifications can all cause sequence misassignment and need to be minimized. However, it is also reported that residue substitution can occur during translation with a frequency of 10-3 to 10-4 per residue in vivo; meanwhile, the spontaneous sequence variant under stress condition has been well reported in cell culture, and the misincorporation frequency can be higher.54-57 Simply ruling out the artifact may have resulted in bona fide substitution being ignored because of following modification rules and vice versa.
Hence, how to distinguish and quantify intrinsic mutation such as residue substitution and artifacts generated during sample preparation can be troublesome. Certain methods such as isobaric labeling and 18O labeling have been applied in order to distinguish artifacts and bona fide modifications, however, the increasing complexity of the sample could still weaken the bona fide signal, thus affecting the quantification.
13
1.2.3 Reduction and elimination of artifacts
Because of the existence of water, light and oxygen, there are sample preparation related artifacts such as hydrolysis and oxidation that cannot be fully avoided. However, after the awareness of their existence and mechanism, many of the artifacts can be reduced or eliminated accordingly. In 2016, we developed a method that effectively eliminated deamidation artifacts during proteolysis by simply performing Glu-C digestion at pH 4.5 in ammonium acetate. As the development of instrumentation and information technology evolve, the high resolving power of mass spectrometers have greatly improved measurement accuracy, and more and more algorithms have been developed to help identify modifications.
14
1.3 Method development of protein site-specific bioconjugation
Nature is not perfect: although natural peptides and proteins have a myriad of functions, much more is to be desired for improved properties. Traditional protein engineering relies on recombinant technology, but is limited to amino acid-based scaffolds. Nowadays, bioconjugation that builds covalent links between biomolecules and small molecules to enable additional functionalities is becoming a rapidly growing field of research. The concept, so called hybrid modality engineering, arises and explains the conjugation of hybrid molecules for extended functionalities. Bioorthogonal reactions has been applied to protein analogs as well as
DNA/RNAs to hybrid biomolecules.
The engineered conjugates can be used for therapeutic applications, since modifications may confer improved or new activities, such as pharmacokinetic (PK) and pharmacodynamics
(PD) profiles to create “biosimilar” and “biobetter”, as well as extended drug development such as antibody drug conjugate (ADC). They can also be used for biological applications, such as the design of drug delivery systems, functional probes and biomaterials (e.g. by introducing fluorophores, affinity tags and photo-caging groups).
1.3.1 Methodology and site-specificity
In the last few decades, various bioorthogonal bioconjugation strategies have been developed and applied to the bioconjugation of proteins, which can be divided into chemical and enzymatic strategies.
15
1.3.1.1 Chemical strategy
Commonly used chemical methodologies to conjugate proteins are accomplished either by utilizing the existing reactive moieties in proteins and peptides like the N-terminal amine, side chain amine of lysine residues, as well as thiol containing residues, but the site-specificity of these methods are relatively low since isomers can be obtained when multiple reactive sites exist in sequence; or by introducing functional groups or engineering residues to protein and peptides.58-62 N-terminal and C-terminal modifications are the most common site-specific derivatization strategies; for example, conjugates with functional aldehyde displays high specifically towards N-terminal amines rather than side chain amines at low pH values; N- terminus of peptides and proteins are selectively converted into ketones by pyridoxal-5- phosphate (PLP), thus allowing further modifications through oxime or hydrozone formation.63-64
Reactive residues such as cysteines have also been engineered to the C-terminal end of proteins in order to build up conjugations.
Other than the chemical conjugation mentioned above, we recently developed a bioconjugation method that has been built upon unwanted unavoidable PTMs. During deamidation, a succinimide is first formed non-enzymatically from asparaginyl and aspartyl residue; the succinimide intermediate can then be readily trapped by hydrazine or other nucleophiles. Further derivatization can occur through orthogonal chemistry such as hydrozone formation. Thus, our method turns the “bad” deamidation site to “good” site-specific derivatization. The reaction rate of succinimide formation can be affected by experimental conditions such as pH and protein conformation, hence latent reactivities, the conjugation can be modulated accordingly.
16
1.3.1.2 Enzyme introduced specificity
Compared to chemical methodologies, enzymatic strategies display higher site-specificity, as enzymes recognize substrates with strict criteria.65 To achieve site- and chemical-selectivities, bioconjugation can be achieved via a combination of enzymatic transformation with tailored substrate specificities and chemical reactions of functional selectivity. An example of enzymatic strategy is using glycosyltransferases to conjugate polyethylene glycol to the corresponding sites on glycoproteins.66
Transglutaminase is an enzyme that has been utilized quite often for bioconjugation in
Zhou’s lab, as it recognizes specific glutamine residues as acyl donors and catalyzes the transamidation reaction between glutamine and substituted amines. Existing glutamine- containing proteins (including natural products) can be site-specifically modified using transglutaminase (TGase). Further derivatizations can be achieved via orthogonal chemistry towards the newly introduced functional groups.
17
1.3.2 The platform build-up
After a decade of work on detection and analysis of deamidation and enzymology work, we started to apply our expertise into the derivatization of protein and peptides. Several new approaches of bioconjugation of peptides and proteins have been established and reduced to practice. Utilizing existing methods such as N-terminal, C-terminal modifications, along with the new strategies that we have developed, a platform can be built aiming at the introduction of a broad range of modifiers to existing proteins and peptides site-specifically. On-demand design is expected to be achievable, and the applications of this hybrid modality engineering include but are not limited to the area of biotherapeutics, biomaterials and biological probes.
18
1.4 References
1. Geiger, T.; Clarke, S., Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. Journal of Biological Chemistry 1987, 262 (2), 785-794.
2. Robinson, N. E.; Robinson, A. B., Molecular clocks. Proceedings of the National Academy of Sciences 2001, 98 (3), 944-949.
3. Aswad, D. W.; Paranandi, M. V.; Schurter, B. T., Isoaspartate in peptides and proteins: formation, significance, and analysis. Journal of Pharmaceutical and Biomedical Analysis 2000, 21 (6), 1129-1136.
4. Murray, E. D.; Clarke, S., Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues. Journal of Biological Chemistry 1984, 259 (17), 10722-10732.
5. Noguchi, S., Structural changes induced by the deamidation and isomerization of asparagine revealed by the crystal structure of Ustilago sphaerogena ribonuclease U2B. Biopolymers 2010, 93 (11), 1003-1010.
6. Moss, C. X.; Matthews, S. P.; Lamont, D. J.; Watts, C., Asparagine deamidation perturbs antigen presentation on class II major histocompatibility complex molecules. The Journal of biological chemistry 2005, 280 (18), 18498.
7. Mamula, M. J.; Gee, R. J.; Elliott, J. I.; Sette, A.; Southwood, S.; Jones, P.-J.; Blier, P. R., Isoaspartyl Post-translational Modification Triggers Autoimmune Responses to Self-proteins. Journal of Biological Chemistry 1999, 274 (32), 22321-22327.
8. D'Souza, A. J. M.; Mar, K. D.; Huang, J.; Majumdar, S.; Ford, B. M.; Dyas, B.; Ulrich, R. G.; Sullivan, V. J., Rapid deamidation of recombinant protective antigen when adsorbed on aluminum hydroxide gel correlates with reduced potency of vaccine. Journal of pharmaceutical sciences 2013, 102 (2), 454-461.
9. Roher, A. E.; Lowenson, J. D.; Clarke, S.; Wolkow, C.; Wang, R.; Cotter, R. J.; Reardon, I. M.; Zürcher-Neely, H. A.; Heinrikson, R. L.; Ball, M. J., Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease. Journal of Biological Chemistry 1993, 268 (5), 3072-3083.
10. Takata, T.; Oxford, J. T.; Demeler, B.; Lampi, K. J., Deamidation destabilizes and triggers aggregation of a lens protein, βA3-crystallin. Protein Science : A Publication of the Protein Society 2008, 17 (9), 1565-1575.
11. Doyle, H. A.; Gee, R. J.; Mamula, M. J., Altered immunogenicity of isoaspartate containing proteins. Autoimmunity 2007, 40 (2), 131-137.
19
12. Manning, M.; Chou, D.; Murphy, B.; Payne, R.; Katayama, D., Stability of Protein Pharmaceuticals: An Update. Pharmaceutical Research 2010, 27 (4), 544-575.
13. Tyler-Cross, R.; Schirch, V., Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. Journal of Biological Chemistry 1991, 266 (33), 22549-56.
14. Pace, A. L.; Wong, R. L.; Zhang, Y. T.; Kao, Y.-H.; Wang, Y. J., Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature. Journal of Pharmaceutical Sciences 2013, 102 (6), 1712-1723.
15. Wright, H. T., Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins. Protein Engineering, Design and Selection 1991, 4 (3), 283-294.
16. Clarke, S., Aging as war between chemical and biochemical processes: Protein methylation and the recognition of age-damaged proteins for repair. Ageing Research Reviews 2003, 2 (3), 263-285.
17. Hendricks, C. L.; Ross, J. R.; Pichersky, E.; Noel, J. P.; Zhou, Z. S., An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases. Analytical biochemistry 2004, 326 (1), 100-105.
18. Johnson, B.; Murray, E.; Clarke, S.; Glass, D.; Aswad, D., Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides. Journal of Biological Chemistry 1987, 262 (12), 5622-5629.
19. Dorgan, K. M.; Wooderchak, W. L.; Wynn, D. P.; Karschner, E. L.; Alfaro, J. F.; Cui, Y.; Zhou, Z. S.; Hevel, J. M., An enzyme-coupled continuous spectrophotometric assay for S- adenosylmethionine-dependent methyltransferases. Anal Biochem 2006, 350 (2), 249-55.
20. Lee, B. W.; Sun, H. G.; Zang, T.; Kim, B. J.; Alfaro, J. F.; Zhou, Z. S., Enzyme-catalyzed transfer of a ketone group from an S-adenosylmethionine analogue: a tool for the functional analysis of methyltransferases. Journal of the American Chemical Society 2010, 132 (11), 3642- 3643.
21. Dai, S.; Ni, W.; Patananan, A. N.; Clarke, S. G.; Karger, B. L.; Zhou, Z. S., Integrated Proteomic Analysis of Major Isoaspartyl-Containing Proteins in the Urine of Wild Type and Protein l-Isoaspartate O-Methyltransferase-Deficient Mice. Analytical Chemistry 2013, 85 (4), 2423-2430.
22. O’Connor, P. B.; Cournoyer, J. J.; Pitteri, S. J.; Chrisman, P. A.; McLuckey, S. A., Differentiation of aspartic and isoaspartic acids using electron transfer dissociation. Journal of the American Society for Mass Spectrometry 2006, 17 (1), 15-19.
23. Ni, W.; Dai, S.; Karger, B. L.; Zhou, Z. S., Analysis of Isoaspartic Acid by Selective Proteolysis with Asp-N and Electron Transfer Dissociation Mass Spectrometry. Analytical Chemistry 2010, 82 (17), 7485-7491.
20
24. Liu, M.; Cheetham, J.; Cauchon, N.; Ostovic, J.; Ni, W.; Ren, D.; Zhou, Z. S., Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis. Anal Chem 2012, 84 (2), 1056-62.
25. Alfaro, J. F.; Gillies, L. A.; Sun, H. G.; Dai, S.; Zang, T.; Klaene, J. J.; Kim, B. J.; Lowenson, J. D.; Clarke, S. G.; Karger, B. L.; Zhou, Z. S., Chemo-Enzymatic Detection of Protein Isoaspartate Using Protein Isoaspartate Methyltransferase and Hydrazine Trapping. Analytical Chemistry 2008, 80 (10), 3882-3889.
26. Shultz, J.; Xu, Q.-Y.; Hurst, R.; Mezei, L.; White, D.; Betlach, M.; Stevens, J.; Klekamp, M., The ISOQUANT (TM) Protein Deamidation Detection Kit. Promega Notes Magazine 1995, 53, 22-28.
27. Papov, V. V.; Gravina, S. A.; Mieyal, J. J.; Biemann, K., The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells. Protein Science 1994, 3 (3), 428-434.
28. Krokhin, O. V.; Antonovici, M.; Ens, W.; Wilkins, J. A.; Standing, K. G., Deamidation of -Asn-Gly- Sequences during Sample Preparation for Proteomics: Consequences for MALDI and HPLC-MALDI Analysis. Analytical Chemistry 2006, 78 (18), 6645-6650.
29. Li, X.; Cournoyer, J. J.; Lin, C.; O'Connor, P. B., Use of 18O Labels to Monitor Deamidation during Protein and Peptide Sample Processing. Journal of the American Society for Mass Spectrometry 2008, 19 (6), 855-864.
30. Gaza-Bulseco, G.; Li, B.; Bulseco, A.; Liu, H., Method to Differentiate Asn Deamidation That Occurred Prior to and during Sample Preparation of a Monoclonal Antibody. Analytical Chemistry 2008, 80 (24), 9491-9498.
31. Lindquist, J. A.; McFadden, P. N., Incorporation of two18O atoms into a peptide during isoaspartyl repair reveals repeated passage through a succinimide intermediate. Journal of Protein Chemistry 1994, 13 (6), 553-560.
32. Stroop, S. D., A modified peptide mapping strategy for quantifying site-specific deamidation by electrospray time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry 2007, 21 (6), 830-836.
33. Ren, D.; Pipes, G. D.; Liu, D.; Shih, L.-Y.; Nichols, A. C.; Treuheit, M. J.; Brems, D. N.; Bondarenko, P. V., An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Analytical Biochemistry 2009, 392 (1), 12-21.
34. Hao, P.; Ren, Y.; Datta, A.; Tam, J. P.; Sze, S. K., Evaluation of the Effect of Trypsin Digestion Buffers on Artificial Deamidation. Journal of Proteome Research 2015, 14 (2), 1308- 1314.
35. Procopio, N.; Buckley, M., Minimizing Laboratory-Induced Decay in Bone Proteomics. Journal of Proteome Research 2017, 16 (2), 447-458.
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36. Volkin, D. B.; Mach, H.; Middaugh, C. R., Degradative covalent reactions important to protein stability. Molecular Biotechnology 1997, 8 (2), 105-122.
37. Volkin, D. B.; Mach, H.; Russell Middaugh, C., Degradative Covalent Reactions Important to Protein Stability. In Protein Stability and Folding: Theory and Practice, Shirley, B. A., Ed. Humana Press: Totowa, NJ, 1995; pp 35-63.
38. Burgess, R. R., Important but little known (or forgotten) artifacts in protein biochemistry. Methods in enzymology 2009, 463, 813-820.
39. Ryle, A.; Sanger, F., Disulphide interchange reactions. Biochemical Journal 1955, 60 (4), 535.
40. Volkin, D. B.; Klibanov, A. M., Thermal destruction processes in proteins involving cystine residues. Journal of Biological Chemistry 1987, 262 (7), 2945-2950.
41. Auclair, J. R.; Salisbury, J. P.; Johnson, J. L.; Petsko, G. A.; Ringe, D.; Bosco, D. A.; Agar, N. Y. R.; Santagata, S.; Durham, H. D.; Agar, J. N., Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation. Proteomics 2014, 14 (10), 1152-1157.
42. Creed, D., The photophysics and photochemistry of the near ‐uv absorbing amino acids–i. Tryptophan and its simple derivatives. Photochemistry and Photobiology 1984, 39 (4), 537-562.
43. Creed, D., The photophysics and photochemistry of the near ‐uv absorbing amino acids–II. Tyrosine and its simple derivatives. Photochemistry and Photobiology 1984, 39 (4), 563-575.
44. Deutsch, D. G., Effect of prolonged 100°C heat treatment in sodium dodecyl sulfate upon peptide bond cleavage. Analytical Biochemistry 1976, 71 (1), 300-303.
45. Wang, Z.; Rejtar, T.; Zhou, Z. S.; Karger, B. L., Desulfurization of Cysteine-Containing Peptides Resulting from Sample Preparation for Protein Characterization by MS. Rapid Communications in Mass Spectrometry 2010, 24 (3), 267-275.
46. Stark, G. R., Reactions of cyanate with functional groups of proteins. III. Reactions with amino and carboxyl groups. Biochemistry 1965, 4 (6), 1030-1036.
47. Gottschalk, A., Glycoproteins: their composition, structure and function. Elsevier Pub. Co.: 1972; Vol. 5.
48. Chumsae, C.; Zhou, L. L.; Shen, Y.; Wohlgemuth, J.; Fung, E.; Burton, R.; Radziejewski, C.; Zhou, Z. S., Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody. Analytical Chemistry 2014, 86 (18), 8932-6.
49. Chumsae, C.; Hossler, P.; Raharimampionona, H.; Zhou, Y.; McDermott, S.; Racicot, C.; Radziejewski, C.; Zhou, Z. S., When Good Intentions Go Awry: Modification of a Recombinant
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Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid. Analytical Chemistry 2015, 87 (15), 7529-7534.
50. Chumsae, C.; Gifford, K.; Lian, W.; Liu, H.; Radziejewski, C. H.; Zhou, Z. S., Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species. Anal Chem 2013, 85 (23), 11401-9.
51. Nepomuceno, A. I.; Gibson, R. J.; Randall, S. M.; Muddiman, D. C., Accurate identification of deamidated peptides in global proteomics using a quadrupole orbitrap mass spectrometer. Journal of proteome research 2013, 13 (2), 777-785.
52. Mikhail, N., Exenatide: A novel approach for treatment of type 2 diabetes. In Southern Medical Journal, 2006; Vol. 99, pp 1271-1279.
53. Hanton, S., Mass spectrometry of polymers and polymer surfaces. Chemical Reviews 2001, 101 (2), 527-570.
54. Alvarez, M.; Tremintin, G.; Wang, J.; Eng, M.; Kao, Y.-H.; Jeong, J.; Ling, V. T.; Borisov, O. V., On-line characterization of monoclonal antibody variants by liquid chromatography–mass spectrometry operating in a two-dimensional format. Analytical Biochemistry 2011, 419 (1), 17-25.
55. Yu, X. C.; Borisov, O. V.; Alvarez, M.; Michels, D. A.; Wang, Y. J.; Ling, V., Identification of Codon-Specific Serine to Asparagine Mistranslation in Recombinant Monoclonal Antibodies by High-Resolution Mass Spectrometry. Analytical Chemistry 2009, 81 (22), 9282-9290.
56. Loftfield, R. B.; Vanderjagt, D., The frequency of errors in protein biosynthesis. Biochemical Journal 1972, 128 (5), 1353-1356.
57. Bogosian, G.; Violand, B. N.; Dorward-King, E. J.; Workman, W. E.; Jung, P. E.; Kane, J. F., Biosynthesis and incorporation into protein of norleucine by Escherichia coli. Journal of Biological Chemistry 1989, 264 (1), 531-539.
58. Wang, Y.-S.; Youngster, S.; Grace, M.; Bausch, J.; Bordens, R.; Wyss, D. F., Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications. Advanced Drug Delivery Reviews 2002, 54 (4), 547-570.
59. Hershfield, M. S., Biochemistry and immunology of poly (ethylene glycol)-modified adenosine deaminase (PEG-ADA). ACS Publications: 1997.
60. Bailon, P.; Palleroni, A.; Schaffer, C. A.; Spence, C. L.; Fung, W.-J.; Porter, J. E.; Ehrlich, G. K.; Pan, W.; Xu, Z.-X.; Modi, M. W., Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C. Bioconjugate Chemistry 2001, 12 (2), 195-202.
61. Goodson, R. J.; Katre, N. V., Site-directed pegylation of recombinant interleukin-2 at its glycosylation site. Nature Biotechnology 1990, 8 (4), 343-346.
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62. Sergi, M.; Caboi, F.; Maullu, C.; Orsini, G.; Tonon, G., Enzymatic techniques for PEGylation of biopharmaceuticals. PEGylated Protein Drugs: Basic Science and Clinical Applications 2009, 75-88.
63. Gilmore, J. M.; Scheck, R. A.; Esser‐Kahn, A. P.; Joshi, N. S.; Francis, M. B., N‐ terminal protein modification through a biomimetic transamination reaction. Angewandte Chemie 2006, 118 (32), 5433-5437.
64. Kinstler, O. B.; Brems, D. N.; Lauren, S. L.; Paige, A. G.; Hamburger, J. B.; Treuheit, M. J., Characterization and stability of N-terminally PEGylated rhG-CSF. Pharmaceutical Research 1996, 13 (7), 996-1002.
65. Maullu, C.; Raimondo, D.; Caboi, F.; Giorgetti, A.; Sergi, M.; Valentini, M.; Tonon, G.; Tramontano, A., Site-directed enzymatic PEGylation of the human granulocyte colony- stimulating factor. FEBS Journal 2009, 276 (22), 6741-6750.
66. DeFrees, S.; Wang, Z.-G.; Xing, R.; Scott, A. E.; Wang, J.; Zopf, D.; Gouty, D. L.; Sjoberg, E. R.; Panneerselvam, K.; Brinkman-Van der Linden, E. C. M.; Bayer, R. J.; Tarp, M. A.; Clausen, H., GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli. Glycobiology 2006, 16 (9), 833-843.
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Mildly Acidic Conditions Eliminate Deamidation Artifact during
Proteolysis: Digestion with Endoprotease Glu-C at pH 4.5
This chapter is based on a published paper with the same title Amino Acids, 2016 Apr;48(4):1059-67 Shanshan Liu, Kevin Ryan Moulton, Jared Robert Auclair and Zhaohui Sunny Zhou
Co-authors’ works in this chapter: Kevin Ryan Moulton, data analysis and revision during paper writing; Jared Auclair, mass spectrometric analysis; Zhaohui Sunny Zhou, principal investigator
25
2.1 Abstract
Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non- enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed.
This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifacts during proteolysis were effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH’s (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.
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2.2 Introduction
Asparagine deamidation is a common protein post-translational modification (PTM) that arises spontaneously and non-enzymatically. As depicted in Scheme 1, asparagine (Asn or N) is converted into aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD) through a succinimide intermediate.1-2 As isoAsp has detrimental impacts on biological systems, several repair mechanisms are present for reducing the levels of isoAsp; one prominent pathway is through protein isoaspartate O-methyltransferase (PIMT or PCMT, EC 2.1.1.77). This enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet or SAM) to isoAsp, generating an isoaspartyl methyl ester that can rapidly hydrolyze back to Asp.3-8 The repair of isoAsp via PIMT methylation is also affected by metabolites in the one-carbon and transsulfuration pathways, such as homocysteine.5, 9-12 It should be noted that deamidation may also occur on glutamine, glycosylated asparagine or other amides, which are negligible under typical proteolysis conditions; in this chapter, deamidation refers to asparagine deamidation shown in Scheme 2.1 unless noted otherwise. Naturally occurring deamidation is ubiquitous and affects both protein structure and function; therefore, its analysis is important in its own right.2, 4,
13-17 However, deamidation artifacts during sample preparation are also common, yet often overlooked, and can be highly problematic as detailed next.
The rates of deamidation depend on multiple factors, including the primary sequences and higher-order structures of the proteins, pH, temperature, and components in the solutions.18 For example, most potential deamidation sites are stabilized by higher-order structure, though not always. In comparison, in a linear peptide without defined structure, the residue immediately C- terminal to Asn is the major determinant: Asn-Gly (NG), being most flexible and acidic
(backbone amide), is most prone to deamidation with a half-life around 24 h under physiological
27
conditions (pH 7.4, 37 oC); Asn-Ser (NS) and Asn-His (NH), augmented by general acid- catalysis, deamidate faster (half-life about 10 days) than other sequences except NG.19-20 The following mnemonic may help to remember the hotspots for deamidation: Not Good for NG
(highly likely to deamidate); Not Sure for NS and Not Happy for NH (likely): and No Problem for NP (not likely). Recent surveys have revealed that at least one NG sequence (also NS) is present in over 50% of the proteins in H. sapiens, M. musculus, E. coli, and S. cerevisiae. 21
Therefore, significant and widespread deamidation of peptides or denatured proteins is expected and indeed observed during sample preparation for protein analysis.22-23 For instance, neutral to mildly basic conditions that are typically used for common proteases (e.g., trypsin, Lys-C, Glu-C and chymotrypsin) are also favorable for deamidation. All together, these cumulative factors can easily lead to deamidation artifacts during denaturation and proteolysis. In fact, as high as 70%-
80% of deamidation artifacts have been reported in some proteins after trypsin digestion for 12 h at 37 oC in ammonium bicarbonate (pH 8.0).24
Deamidation artifact has broad, significant and detrimental effects on proteins analysis.
First and obviously, these artifacts create misleading results, thus complicating both the identification and quantification of bona fide deamidation. Moreover, deamidation imparts marked changes in the physical, chemical and biochemical properties of the involved proteins and peptides.25-28 For example, the decrease in pI caused by the newly generated carboxylic group in Asp and isoAsp likely alters chromatographic behaviors.29-31 In addition, deamidation artifact may interfere with common proteolysis (e.g., peptide mapping, amino acid sequencing), as the isoAsp and Asp products may resist enzymatic hydrolysis or introduce new cleavage sites.
Asn deamidation also converts one peptide into multiple species that affects quantification.
Furthermore, the mass increase of 1 Da imparted by deamidation results in overlay of the
28
isotopic envelope and thus affects analysis based on mass accuracy. Finally, the change in pI and backbone caused by isoaspartic acid can also negatively impact proper protein refolding and induce unexpected precipitation.32-33
Conceptually, the best approach to distinguish between bona fide deamidation and artifact is to conduct sample preparation—including proteolysis—in 18O-water, as deamidation is the hydrolysis of an amide.23, 34-36 In practice, however, analysis of 18O-labeled samples can be challenging due to complicated isotopic distributions, as proteolysis also introduces varying degrees of 18O into the newly generated C-termini.37-39 In turn, deamidation artifact in 18O-water also complicates other techniques based on 18O tracing, such as labeling of C-termini and N- linked glycosylation sites using PNGase F.40 Other ramifications, such as shortening the digestion time and optimizing components of the digestion solution, have been attempted, but with only limited success, as these conditions cannot prevent deamidation.
An obvious and straightforward approach to eliminate deamidation artifact is to process the proteins under mildly acidic conditions (e.g., pH 4.5), as described herein. Deamidation rates follow an inverse bell-shaped curve with the minimum around pH 4 to 5, at which the half-lives for the Asn-Gly peptides are approximately 280 days at 37 oC; in other words, less than 1% deamidation for 24 h.41-43 In fact, preventing deamidation is a primary reason that many protein pharmaceuticals are stored under mildly acidic conditions.44 Perhaps not widely known, the commonly used endoprotease Glu-C shows maximal proteolytic activity around both pH 4 and
8.45 As detailed below, deamidation artifact is indeed eliminated during Glu-C proteolysis at pH
4.5 in ammonium acetate using bovine calmodulin, exenatide and human adrenocorticotropic hormone (ACTH) peptide, which all contain hotspots for deamidation including the most labile
NG sequence.46-47 Additionally, since Glu-C is typically used at pH 8, we also evaluated the
29
scope and limitations of proteolysis under mildly acidic conditions and compared the results between pH 4.5 and pH 8. We confirmed that the specificity of Glu-C at both pHs is nearly identical. Altogether, a simple change of buffer and pH renders a practical and general approach for protein digestion without deamidation artifact.
Scheme 2.1. Deamidation of asparagine (Asn) and the formation of aspartic acid (Asp) and isoaspartic acid (isoAsp) via a succinimide intermediate.
30
2.3 Experimental section
2.3.1 Materials
Recombinant bovine calmodulin (lyophilized powder, C4874) was from Sigma-Aldrich (St.
Louis, MO). Exenatide (lyophilized powder, AS-24464,
1 39 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS - NH2) and adrenocorticotropic hormone (ACTH) peptide (lyophilized powder, AS-20619,
18RPVKVYPNGAEDESAEAFPLEF39) were from Anaspec (Fremont, CA). The concentrations were determined by UV absorption at 280 nm using extinction coefficients calculated based on amino acid sequence. Sequencing grade Glu-C endoprotease was from Promega (V1651;
Madison, WI). Tris(2-carboxyethyl) phosphine (TCEP) hydrochloride was from Hampton
Research (Al Jodo, CA). Dithiothreitol (DTT) was from Acros Organics (New Jersey, NJ). 18O water (normalized, 97% - 98% atom percentage) was from Icon Stable Isotopes (Summit, NJ).
All aqueous solutions were prepared with MilliQ purified water. All chemicals used were reagent grade or better. The pH of all solutions was determined by EMD Colorphast pH strips with an accuracy of 0.5 unit.
2.3.2 Proteolysis
Calmodulin (final concentration 0.8 mg/mL) was dissolved in 100 mM ammonium acetate
(pH 4.5) with 10% acetonitrile or ammonium bicarbonate (pH 8.0) with 10% acetonitrile, reduced by 1 mM DTT and 1 mM TCEP at 37 oC for 20 min; then digested by Glu-C at an enzyme: protein ratio of 1:40 (w/w) at 37 oC. Exenatide (final concentration 1 mg/mL) and
ACTH peptide (final concentration 1 mg/mL) were dissolved in 100 mM ammonium acetate (pH
4.5) or ammonium bicarbonate (pH 8.0), then digested with Glu-C using an enzyme: protein
31
ratio of 1:40 (w/w) at 37 oC. Aliquots of the reactions were quenched by adding aqueous trifluoroacetic acid (TFA, 5%) to a final concentration of 0.5%; and the mixture was diluted 10- fold into a solution of 0.1% TFA in 70: 30 water/ acetonitrile (v/v) for mass spectrometry analysis. In parallel, reactions were also carried out in 18O solutions.
2.3.3 Aging of ACTH peptide
ACTH peptide was dissolved in 100 mM ammonium acetate (pH 4.5) and 100 mM ammonium bicarbonate (pH 8.0) to give a final concentration of 1.1 mg/mL (446 μM). The peptide solutions were aged by incubating at 37 oC for 48 hours, and then stored at -80 oC. The aged solutions were diluted 20-fold into a solution of 0.1% TFA in 70: 30 water/ acetonitrile (v/v) for mass spectrometry analysis.
2.3.4 Mass Spectrometry
An Applied Biosystems 5800 MALDI-TOF/TOF analyzer was calibrated using a peptide standard from Anaspec (AS-60882; Calibration mixture 1: Des-Arg1-Bradykinin 904.47 Da,
Angiotensin I 1296.68 Da, and Neurotensin 1672.92 Da). The diluted reaction mixtures were mixed 1:1 with 10 mg/mL alpha-cyano-4-hydroxy-cinnamic acid (CHCA) in a solution of 0.1%
TFA in 50:50 water/acetonitrile. The mixture (1 uL) was loaded and crystallized on MALDI target plate, and dried at room temperature prior to analysis. MALDI-TOF mass spectra were acquired in both reflectron positive mode and reflectron negative mode, with MS/MS data collected using 2 kV collision energy. Data were processed with Applied Biosystems Data
Explorer 4.6 software.
LC-MS data of exenatide was obtained using a H-Class Acquity UPLC system coupled to a
Xevo G2-S Q-ToF mass spectrometer (Waters Corp, Milford, MA). Liquid chromatography was
32
performed on a BEH-C18, 2.1 mm x 150 mm column, with pore size of 1.7 µm (Waters Corp,
Milford, MA). Mobile phase A consisted of 0.1 % formic acid (v/v) in HPLC grade water and mobile phase B consisted of 0.1 % formic acid (v/v) in 100 % HPLC grade acetonitrile (v/v) with a flow rate at 0.2 mL/min. A gradient was applied by starting at 5% mobile phase B for 2 min, increasing to 60% mobile phase B over 40 min, then increasing to 95% mobile phase B over 2 min, holding at 95% mobile phase B for 3 min, and finally decreasing to 5% mobile phase B over 5 min. After liquid chromatography, samples were introduced via an electrospray ion source in-line with the Xevo G2-S Q-ToF. External calibration of m/z scale was performed using sodium cesium iodide. Data were processed manually using Waters UNIFI 1.7.1 software.
33
2.4 Results and discussion
As expected and detailed below, deamidation artifact was eliminated during proteolysis at pH 4.5. Additionally, similar sequence specificity was observed for Glu-C digestion at pH 4.5 and 8.0, thus retaining the enzyme’s utility under mildly acidic conditions.
2.4.1 Analysis of deamidation
2.4.1.1 Deamidation of Calmodulin
There are six asparagine residues in calmodulin including two Asn-Gly (NG: “Not Good”) sequences that are deamidation hotspots4. The MS spectra of one such peptide
88AFRVFDKDGNGYISAAE104 under different proteolytic conditions are shown in Figure 2.1.
The isotopic envelope of the peptides showed a mixture of deamidated and non-deamidated species. Approximately 50% deamidation of N97 was observed after a 4 h digestion at pH 8, and nearly 80% after 24 h. On the contrary, the same peptide from Glu-C digestion at pH 4.5 for 24 h displayed an isotopic envelope that is nearly identical to the theoretical one; moreover, even after prolonged incubation at pH 4.5 (120 h), only minor deamidation (less than 10%) was observed
(see Figure 2.1). The corresponding MS/MS spectra can be found in Figure 2.2. Altogether, these data showed deamidation was eliminated at pH 4.5 under typical digestion conditions.
Since the predominant determinant of deamidation rate in peptides is their primary sequences, our method should be effective for other cases, as demonstrated later.
The Glu-C peptide containing the other deamidation hotspot, N60 (NG), was not observed in the positive mode, as the corresponding Glu-C peptide (55VDADGNGTIDFPE67, pI 3.37) may suffer from poor ionization under positive mode. On the other hand, the detection of acidic peptides can sometimes be achieved using negative ionization mode.48 Indeed, the second NG-
34
peptide and its deamidation species were detected in negative mode (see Figure 2.3).
Additionally, as expected, the peptide was fully deamidated at pH 8.0 after 24 h digestion, while digestion at pH 4.5 eliminated deamidation. Aside from N97 and N60, no appreciable levels of deamidation were observed on other asparagine residues of calmodulin: N42 (NP), N53 (NE),
N111 (NL), and N137 (NY), which was expected from the sequence dependence on kinetics of deamidation (Figure not shown).
35
Figure 2.1. MALDI MS spectra of calmodulin Glu-C peptide under various conditions.
The NG (“Not Good”) tandem (97-98) of Glu-C peptide (88AFRVFDKDGNGYISAAE104; theoretical m/z 1859.89) is prone to deamidation under neutral to basic conditions. At pH 4.5 in ammonium acetate, no deamidation was observed after 24 h digestion, while only minor deamidation (less than 10%) was present after 120 h. At pH 8.0 in ammonium bicarbonate, significant deamidation (~50%) was observed after only 4 h, with nearly complete deamidation after 24 h. Theoretical isotopic envelopes for Asn and Asp isoforms are shown in top and bottom traces respectively.
36
Figure 2.2. MALDI MS/MS spectra of calmodulin Glu-C peptide.
MALDI-TOF/TOF MS/MS spectra of singly charged precursor ion m/z 1859.8 (theoretical m/z 1859.8 for Asn, top) and m/z 1860.8 (theoretical m/z 1859.8 for Asp/isoAsp, bottom) for calmodulin Glu-C calmodulin peptide 87AFRVFDKDGNGYISAAE104 after 24 h digestion at pH 4.5 (top) and 8.0 (bottom) in positive ion mode. The letter Z denotes either Asp or isoAsp.
37
Figure 2.3. MALDI MS spectra of calmodulin Glu-C peptide 55-67 in negative ion mode
MALDI-TOF MS spectra of calmodulin Glu-C peptide (55VDADGNGTIDFPE67; theoretical m/z 1347.57) in negative ion mode. No observable deamidation was present after 120 h at pH 4.5 in ammonium acetate, while complete deamidation was observed after 24 h at pH 8.0 in ammonium bicarbonate. Theoretical isotopic envelope for Asn and Asp/isoAsp peptides are shown in top and bottom traces respectively.
38
2.4.1.2 Deamidation of Exenatide
To further evaluate the scope and applicability of our approach, analysis of other deamidation-prone systems was conducted, particularly biotherapeutics, for which deamidation is a major quality attribute. Exenatide (theoretical mass: 4185.01 Da) and adrenocorticotropic hormone peptide (18-39, theoretical mass 2465.20 Da) each contains a labile Asn-Gly sequence
(Asn28 and as Asn25 respectively), with the deamidation of ACTH having been previously reported.49-50 The MALDI mass spectra of the exenatide Glu-C fragments are shown in Figure
2.4. Again, as expected, no deamidation of N28 was detected after overnight (16 h) digestion at pH 4.5 in both 16O and 18O water. In comparison, nearly complete (≥80%) deamidation of N28 occurred after 16 h proteolysis at pH 8.0 in ammonium bicarbonate, and the corresponding 18O mass shift (+3 Da) was observed in the peptide digested in 18O water.
Electrospray ionization (ESI) mass spectrometry is complementary to MALDI, and both are common for protein analysis. Hence, deamidation of exenatide was also investigated by ESI mass spectrometry. As shown in Figure 2.5, the deamidated and asparaginyl peptides were resolved by LC and readily distinguished by mass spectrometry, thereby offering even higher sensitivity. Similar to MALDI analysis, complete deamidation was observed after 48 h at pH 8.0
(i.e., no asparaginyl peptide observed). For pH 4.5, with increased sensitivity, about 1% of deamidation was observed after prolonged incubation (48 h). Additionally, 18O labeling confirmed deamidation occurred during sample preparation (Figure 2.6). In practice, deamidation during digestion is negligible under typical digestion conditions (8-24 h).
39
Figure 2.4. MALDI MS spectra of exenatide Glu-C peptide under various conditions.
25 39 MALDI-TOF MS spectra of exenatide Glu-C peptide ( WLKNGGPSSGAPPPS -NH2 (C- terminal amide), theoretical m/z 1450.74 Da) under various conditions. After overnight digestion (16 h), no deamidation was observed at pH 4.5 in ammonium acetate, while nearly complete deamidation occurred at pH 8.0 in ammonium bicarbonate. Theoretical isotopic envelopes for Asn and Asp isoforms are shown in top and bottom traces respectively
40
Figure 2.5. Extracted ion chromatogram and ESI MS spectra of doubly charged exenatide Glu-C peptide after 48 h proteolysis.
Extracted ion chromatograms (left column) and ESI MS spectra of doubly charged exenatide 25 39 Glu-C peptide (right column, WLKNGGPSSGAPPPS -NH2 (C-terminal amide), theoretical m/z 725.87) after 48 h proteolysis. The peaks at 12.6 and 13.4 min were for the asparaginyl and deamidated species respectively as confirmed by the mass spectra to the right. Top two traces refer to peptides after proteolysis at pH 4.5 in 16O and 18O water. While bottom two traces refer to peptides after digestion at pH 8.0 in 16O and 18O water; +1 and +3 Da mass shifts that correspond to deamidated species were observed, the small +1 peaks (m/z 726.368) in 18O sample was from the residual 16O (5%) in 18O water
41
Figure 2.6. Extracted ion chromatograms of exenatide Glu-C peptide and ESI MS spectra of extracted aspartyl species at pH 4.5.
Extracted ion chromatograms (XIC) of doubly charged exenatide Glu-C peptide (left column, 25 39 WLKNGGPSSGAPPPS -NH2, (C-terminal amide), theoretical m/z 725.87) after 48 h proteolysis at pH 4.5 in 16O (top) and 18O (bottom) water and ESI MS spectra of aspartyl species extracted from XIC (right column). Because only trace amount of deamidation species was generated, considerable peak overlapping in mass spectra was observed. Due to the dominant asparaginyl species, the aspartyl species were only partially resolved by liquid chromatography.
42
2.4.1.3 Deamidation of ACTH peptide
Glu-C proteolysis of ACTH peptide also showed similar results, with deamidation artifact being completely eliminated during digestion up to 48 h at pH 4.5. Moreover, deamidation of the intact ACTH peptide was examined as well. The intact ACTH peptides and the Glu-C fragment showed nearly identical deamidation levels under the same conditions; thus indicating that digestion does not affect deamidation and vice versa (Figure 2.7).
43
Figure 2.7. MALDI MS spectra of the intact and ACTH Glu-C peptides.
MALDI-TOF MS spectra of ACTH Glu-C peptide (left traces, 18RPVKVYPNGAE28; theoretical m/z 1229.66), and ACTH intact peptide after aging (right traces, 18RPVKVYPNGAEDESAEAFPLEF39, theoretical m/z 2465.20). At pH 4.5 in ammonium acetate, no deamidation was observed after 48 h digestion and after 48 h aging of the intact ACTH peptide. At pH 8.0 in ammonium bicarbonate, nearly complete deamidation was observed in both the Glu-C digested ACTH peptide and the intact aged ACTH peptide. Theoretical isotopic envelope for Asn and Asp/isoAsp peptides are shown in top and bottom traces respectively.
44
2.4.2 No Isomerization at pH 4.5
As the proteolysis conditions change, other potential modifications may occur that should be monitored. Though unlikely, one legitimate concern is isomerization of aspartic acid that generates isoaspartic acid via a succinimide intermediate, analogous to asparagine deamidation, as shown in Scheme 2.1.51-52 Sequence dependence is similar between isomerization and deamidation, i.e., Asp-Gly is most prone to isomerization. Again, the rates of isomerization are also pH dependent: the half-lives for Asp-Gly in peptides are approximately 50 days at pH 4 and
80 days at pH 8, significantly slower than deamidation and also less sensitive to changes in pH.53
Therefore, no appreciable isomerization was expected at pH 4.5 in ammonium acetate, but was investigated in calmodulin nonetheless. Once more, 18O-labeling is the best approach to monitor isomerization during sample preparation.54-55 Among 17 aspartic acid residues, 12 were detected by mass spectrometry, including five Asp-Gly hotspots. Because 18O was incorporated into the newly formed C-termini, the b ions for the Asp residues can reveal any potential 18O incorporation at that specific residue.56 As expected, no mass shift, i.e., isomerization, was detected for any of the Asp peptides at both pH 4.5 and 8.0 (Figure 2.8-2.10).
45
Figure 2.8. MALDI MS spectra of 18O-labeled calmodulin Glu-C peptides at pH 4.5
MALDI-TOF MS spectra of calmodulin Glu-C peptide (15AFSLFDKDGDGTITTKE31, theoretical m/z 1844.89) after 96 h digestion at pH 4.5 in 16O water (top) and 18O water (bottom); the DG tandem (22-25, underlined in sequence above) is prone to isomerization. Due to varying degrees of 18O incorporation into the C-terminal, isomerization cannot be determined by MS spectra alone. Tandem MS/MS was therefore acquired to evaluate isomerization, which can be found in Figure 2.9.
46
Figure 2.9. MALDI MS/MS spectra of 18O-labeled calmodulin Glu-C peptides at pH 4.5
MALDI-TOF/TOF MS/MS spectra of precursor ion m/z 1844.8 for calmodulin Glu-C peptide 15AFSLFDKDGDGTITTKE31 after 96 h digestion at pH 4.5 in 16O water (top) and 18O water (bottom). No mass shift was detected in the b ion series between 16O and 18O proteolysis, while up to a 4 Da mass shift was observed in the y ion series. Therefore, these results indicate that isomerization did not occur during proteolysis, and that all 18O incorporation is from the newly generated C-terminus.
47
Figure 2.10. Zoomed-in MS/MS spectra of 18O-labeled calmodulin Glu-C peptides at pH 4.5
Zoomed-in MALDI-TOF/TOF MS/MS spectra of precursor ion m/z 1844.8 for calmodulin Glu- C peptide 15AFSLFDKDGDGTITTKE31 after 96 h digestion at pH 4.5 in 16O water (top) and 18O water (bottom); the isotopic envelopes of indicative fragment ions are shown. The b10 ion from 18O proteolysis shows a nearly identical isotopic envelope compared with 16O proteolysis, demonstrating that no Asp isomerization occurred during proteolysis. Additionally, the mass shift of the y11 ion (bottom trace) reveals one to two atoms of 18O incorporation at the C- terminus.
48
2.4.3 Nearly identical proteolytic specificity at pH 4.5 and 8.0
The sequence specificity of Glu-C under mildly acidic conditions has only been reported for a handful of proteins, showing similar specificity as that under neutral conditions. However, many of these studies were prior to the modern mass spectrometry era, so the specificity of Glu-
C under both conditions reported herein was compared. It is known that the enzyme specifically cleaves at the C-terminal side of glutamic acid in buffers that include Tris-HCl, bicarbonate and acetate; however, cleavage after both glutamic acid and aspartic acid has been shown to occur in phosphate buffers.57 This was one of the reasons ammonium bicarbonate and ammonium acetate were chosen for this work.
As shown in Figure 2.11, the MALDI spectra of the Glu-C digests of calmodulin at pH 4.5 and 8.0 are highly similar (both masses and relative intensity); among 35 major peaks, only three are different (highlighted in red and blue). As summarized in Figure 2.12, similar sequence coverage was observed under both pH conditions, with 88% and 87% coverage seen at pH 4.5 and 8.0 respectively. Furthermore, only glutamic acid residues were cleaved under both conditions, with no cleavage at aspartic acid observed after proteolysis at both pH 4.5 and 8.0.
While not required, having the same specificity at different pH values is convenient for the practical application of Glu-C. These observations suggest that the proposed low pH methodology can be a viable alternative to proteolysis at pH 8.
49
Figure 2.11. MS spectra of calmodulin Glu-C digests at both pH 4.5 and pH 8.0.
MALDI-TOF MS spectra of calmodulin Glu-C digests at both pH 4.5 (top) and pH 8.0 (bottom). Specific areas of spectra are zoomed in at indicated degree to improve clarity. Glu-C proteolysis at each pH generated similar peptide fragments. Colors are used to indicate peaks only observed at pH 4.5 (blue), only observed at pH 8.0 (red), and at both pH's (black)
50
Figure 2.12. Sequence coverage of calmodulin by Glu-C proteolysis.
Color codes indicate peptides observed at digestion at pH 4.5 only (blue), at pH 8.0 only (red) and at both pH values (black). The peptides between F68 and E87 were not observed by MALDI. Sequence coverage was found to be 88% and 87% at pH 4.5 and 8.0 respectively.
51
2.5 Conclusions
Deamidation artifact occurs widely and may affect various procedures in protein analysis, but can be eliminated by proteolysis at pH 4.5 in ammonium acetate using the common protease
Glu-C, which also retains the same specificity as at pH 8. Since pH is the dominant factor in the rates of deamidation, other proteases that are active under mildly acidic conditions should be equally useful for eliminating deamidation artifact. Judicious control of pH and conditions for sample preparation may also minimize or eliminate other artifacts. For example, proteolysis at low pH has been previously used to minimize disulfide scrambling.58-61 Finally, by effectively eliminating deamidation artifact, the bona fide deamidation can be analyzed with greater ease and higher confidence for both biological systems and protein pharmaceuticals alike.
52
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56. Du, Y.; Wang, F.; May, K.; Xu, W.; Liu, H., Determination of deamidation artifacts introduced by sample preparation using 18O-labeling and tandem mass spectrometry Analysis. Analytical Chemistry 2012, 84 (15), 6355-6360.
57. Houmard, J.; Drapeau, G. R., Staphylococcal protease: a proteolytic enzyme specific for glutamoyl bonds. Proceedings of the National Academy of Sciences 1972, 69 (12), 3506-3509.
58. Pompach, P.; Man, P.; Kavan, D.; Hofbauerová, K.; Kumar, V.; Bezouška, K.; Havlíček, V.; Novák, P., Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds. Journal of Mass Spectrometry 2009, 44 (11), 1571- 1578.
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59. Chen, Z.-w.; Bergman, T.; Östenson, C.-G.; Efendic, S.; Mutt, V.; Jörnvall, H., Characterization of dopuin, a polypeptide with special residue distributions. European Journal of Biochemistry 1997, 249 (2), 518-522.
60. Salzano, A. M.; Renzone, G.; Scaloni, A.; Torreggiani, A.; Ferreri, C.; Chatgilialoglu, C., Human serum albumin modifications associated with reductive radical stress. Molecular BioSystems 2011, 7 (3), 889-898.
61. Tomlinson, A. J.; Johnson, K. L.; Lam-Holt, J.; Mays, D. C.; Lipsky, J. J.; Naylor, S., Inhibition of human mitochondrial aldehyde dehydrogenase by the disulfiram metabolite S- methyl-N,N-diethylthiocarbamoyl sulfoxide: Structural characterization of the enzyme adduct by HPLC-tandem mass spectrometry. Biochemical Pharmacology 1997, 54 (11), 1253-1260.
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Hybrid Modality Engineering of Exenatide, Chemo-enzymatic Site-
specific Bioconjugation Mediated by Transglutaminase
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3.1 Abstract
Exenatide, a glucagon-like peptide-1 receptor agonist, has been used for the treatment of type II diabetes and obesity. In order to acquire enhanced properties, constant effort has been made on the derivatization of exenatide. Herein the glutamine-containing polypeptide exenatide is site-specifically modified chemo-enzymatically with functional moieties such as biotin and polyethylene glycol (PEG). The glutamine site-specificity is achieved by using transglutaminase
(TGase) enzymes. Further chemical derivatization occurs via orthogonal chemistry toward the unique reactive functionalities introduced by transglutaminase. This hybrid modality engineering enables the introduction of new conjugates to exenatide, which may confer improved or new activities, such as pharmacokinetic (PK) and pharmacodynamics (PD) profiles.
The follow up biological activity assay is ongoing with our collaborators from Tufts.
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3.2 Introduction
Human glucagon-like peptide-1 (GLP-1) is a peptide hormone that is associated with various regulatory behaviors, including the simulation of insulin secretion, inhibition of glucagon secretion, regulation of appetite and food intake by activating GLP-1 receptors in islet β-cells.1-2
In recent years, GLP-1 and its analogs have been used as drug candidates for the treatment of type II diabetes and obesity.3 Exenatide, a peptide acting as a GLP-1 receptor agonist, is the synthetic version of natural hormone exendin-4 from Gila monster, and has been approved for the treatment of type II diabetes since 2005 (Byetta or Bydureon, AstraZeneca).4 Compared to human GLP-1, exenatide binds to the receptor with similar affinity, and is refractory to in vivo degradation, thus has a much longer half-life.4-5
In order to acquire new enhanced properties, especially longer half-life, additional engineering has been built upon natural GLP-1 and exenatide. For instance, Lixisenatide
(Lyxumia or Adlyxin, Sanofi) is a new exenatide analog drug developed by Sanofi. Exenatide was modified by a C-terminal poly-lysine amide in order to achieve long-acting and oral administration.6 Albiglutide (Eperzan or Tanzeum, GlaxoSmithKline) is a GLP-1 dimer in which two peptides with engineered residues (Ala2 to Gly2) repeat as tandem, and are genetically fused to human albumin at the C-terminal of the second peptide.7 In the example of
Liraglutide (Victoza, Novo Nordisk), a sequence analog to GLP-1 was generated so that a conjugation to a fatty acid chain was built on Lys26; however, the other lysine (Lys34) was changed to arginine to achieve site-specificity.8 Generally speaking, N-terminal and C-terminal modifications are the most popular methodologies for site-specific conjugation of proteins and peptides, with the advantage of high feasibility and site-specificity. Relatively, very few work pay attention to side chain modifications, probably partially because the normally used chemical
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modification that is targeted to specific residues, such as lysine and cysteine, cannot guarantee site-specificity when multiple reactive residues exist in sequence. It is actually a universal limitation of currently used chemical strategies for site-specific modification targeted to protein and peptides.
Scheme 3.1. General scheme of transglutaminase catalyzed transamidation of substrate glutamine.
Transglutaminase (TGase, E.C. 2.3.2.13) is a large family of enzymes that catalyze the formation of an isopeptide bond between either small molecules or lysine residues in protein and the acyl group on the side chain of glutamine (Gln or Q). The enzyme recognizes specific glutamine residues as an acyl donor and catalyzes transamidation reactions between glutamine and primary amines, as well as competing reactions such as deamidation and crosslinking
(Scheme 3.1). The microbial transglutaminase (mTGase) isoforms have been widely used, and display broad amine substrate specificity, while displaying unique glutamine substrate specificity requirements.9-10 A previous study of TGase substrate glutamine selectivity shows that only one out of seventeen glutamine residues was modified after TGase mediated transamination, only glutamines that meet specific criteria can be TGase substrates, therefore the recognition by
TGase of glutamine substrate provides high specificity. This evidence provides an excellent
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platform for site-specific derivatization of glutamine containing protein and peptides, and inspired us to investigate the possibility to site-specifically modify exenatide utilizing TGase.
Figure 3.1. Structure analysis of exenatide. a) Exenatide crystal structure (PDB ID: 1JRJ), the side chain of Gln13 is shown in yellow; b) exenatide-receptor interaction (crystal structure, PDB ID: 3C59). The glutamine residue resides within the rigid α-helix region and is away from receptor binding site.
The nature of TGase substrate glutamine selectivity has not been fully understood yet; nonetheless, there are several contributing factors, such as the primary structures, region flexibility, exposure to solvent and ability to interact with the TGase active site.10 As for exenatide, an α-helix is formed from residue 8 to 28, and a C-terminal autonomously folded "Trp cage" is formed from the last nine amino acid residues, which leads to enhanced stability of the peptide.11-12 There is only one glutamine (Q13 or Gln13) in exenatide located within the α-helix region and exposed to solvent (Figure 3.1-a). A major concern was whether this Gln13 could be
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accessed by the transglutaminase enzyme, since the Gln13 residue is solvent exposed, it also resides within the rigid α-helix region, which is less favorable for TGase recognition, and again, the substrate specificity of TGase is not well understood and cannot be predicted. Moreover, the two lysine residues (K12 and K27) that exist in exenatide could be potential amine competitors and form intramolecular or intermolecular crosslinking.13 Altogether, the derivatization of exenatide on Q13 can be challenging but potentially effective. The other concern is whether the glutamine derivatization will change the original function of exenatide. The GLP-1 ligand- receptor interaction study shows that Q13 of exenatide is exposed to the solvent, and does not interact with the receptor (Figure 3.1-b), thereby making it an ideal site for bioconjugation as the derivatives are likely to maintain similar ligand-receptor interaction.14
Herein, site-specific bioconjugation of exenatide is attempted and achieved by combining
TGase-mediated transamidation and click chemistry. As a result, several novel derivatives of exenatide have been obtained. This hybrid modality engineering enables the introduction of new drug conjugates to exenatide, including biotinylated and PEGylated exenatide, which may confer improved or new activities, such as pharmacokinetic (PK) and pharmacodynamics (PD) profiles, or design as biological probes.
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3.3 Experimental section
3.3.1 Materials
Exenatide (lyophilized powder, AS-24464) and poly-histidine azide (azide-
YHHHHHH-OH, custom order) were from AnaSpec (Fremont, CA). Bromo coumarin azide
(3-azido-6-bromo 7-hydroxychromen-2-one, CAS: 1352503-77-5, PBMR142922) was from
Princeton Bio (Princeton, NJ). Microbial transglutaminase (Activa TI) was from Ajinomoto
(Hamburg, Germany, item purchased through Amazon, unit defined as the amount of enzyme that catalyzes the conversion of 1 micro mole of substrate per minute). Biotin cadaverine amine (N-(5-aminopentyl) biotinamide, trifluoroacetic acid salt) was from
Thermo Fisher Scientific (A1594; Waltham, MA). Polyethylene glycol (MW 10,000, PS1-
N-10K) amine was from Ponsure Biotech (Shanghai, China). Tris(3- hydroxypropyltriazolylmethyl) amine (THPTA) was from Click Chemistry Tools (1010-100;
Scottsdale, AZ). Propargyl amine was from Sigma Aldrich (St. Louis, MO). Copper sulfate, nickel sulfate, potassium phosphate monobasic, potassium phosphate dibasic, trifluoroacetic acid (TFA) and iodine solution (N/10, SI86-1) were from Fisher Scientific (Fair Lawn, NJ).
Tris(2-carboxyethyl) phosphine (TCEP) hydrochloride was from Hampton Research (Al
Jodo, CA). Ascorbic acid sodium salt and dithiothreitol (DTT) were from Acros Organics
(Morris Plains, NJ).
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3.3.2 Bioconjugation
3.3.2.1 TGase-mediated propargylation
To a solution of exenatide (1 mg/mL, 238 μM) in 100 mM potassium phosphate (pH 7.0) with 1 mM DTT, propargyl amine (1 M stock, final conc. 50 mM) was added. Microbial transglutaminase was added to the mixture at an enzyme: exenatide ratio of 50 U/g. The mixture was incubated at 37 oC for 4 h in the dark. Another batch of TGase was added (20 U/g), the reaction was then allowed to proceed at 37 oC for another 10 h in the dark. The reaction mixtures were then dialyzed against 100 mM potassium phosphate (pH 7.0, 2000-fold dilution, 2 h × 2 at room temperature in the dark) using a Mini dialysis kit (1 kDa MWCO, 28955966； GE
Healthcare) to remove excess propargyl amine.
3.3.2.2 TGase-mediated biotinylation
To a solution of exenatide (1 mg/mL, 238 μM) in 100 mM potassium phosphate (pH 7.0) with 1mM DTT, biotin cadaverine amine (100 mM stock, final conc 10 mM) was added.
Microbial transglutaminase was added to the mixture at an enzyme: exenatide ratio of 50 U/g.
The mixture was incubated at 37 oC for 4 h in the dark. Another batch of TGase was added (20
U/g), the reaction was then allowed to proceed at 37 oC for another 10 h. The reaction mixtures were then dialyzed against 100 mM potassium phosphate (pH 7.0, 2000 folds of dilution, 2 h × 2 at room temperature in the dark) to remove excess biotin cadaverine amine.
3.3.2.3 TGase-mediated one-step PEGylation
To a solution of exenatide (1 mg/mL, 238 μM) and polyethylene glycol amine (average Mn
~10,000, 5 mM) in 100 mM potassium phosphate (pH 7.0) with 1 mM DTT, microbial
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transglutaminase was added to the mixture at an enzyme: exenatide ratio of 50 U/g. The mixture was incubated at 50 oC for 4 h in the dark. Another batch of TGase was added (20 U/g), and the reaction was then allowed to proceed at 37 oC for another 10 h.
3.3.2.4 Derivatization of propargylated-exenatide with bromo-coumarin
To a solution of propargylated-exenatide (10 μL, ~200 μM) from previous dialysis, potassium phosphate (8.2 μL, 100 mM, pH 7) was added, then bromo-coumarin azide solution
(0.4 μL of 25 mM stock in DMSO, final conc. 0.5 mM) was added, followed by a premixed solution of CuSO4 (0.12 μL of 20 mM stock, final conc. 0.25 mM) and THPTA (Tris(3- hydroxypropyltriazolylmethyl) amine, 0.25 μL of 50 mM stock in water, final conc. 1.25 mM).
Sodium ascorbate (1 μL of 100 mM stock, final conc. 5 mM) was added in the end to make the final reaction volume 20 μL. Then the solution was mixed at room temperature for 2 h in the dark by attaching to a slow rotisserie (approx. 30 rotations per minute). The reaction mixtures were then dialyzed against 100 mM potassium phosphate (pH 7.0, 2000-fold dilution, 2 h × 2 at room temperature in the dark) to remove excess small molecules.
3.3.2.5 Derivatization of propargylated-exenatide by poly-histidine azide
To a solution of propargylated-exenatide (10 μL, ~200 μM) from previous dialysis, potassium phosphate (5.2 μL, 100 mM, pH 7) was added, then poly-histidine azide solution (0.4
μL of 25mM stock, final conc. 0.5 mM) was added, followed by NiSO4 (3 μL of 20 mM stock, final conc. 3 mM), a premixed solution of CuSO4 (0.12 μL of 20 mM stock, final conc 0.25 mM) and THPTA (tris(3-hydroxypropyltriazolylmethyl) amine, 0.25 μL of 50 mM stock, final conc.
1.25 mM) was then added. Sodium ascorbate (1 μL of 100 mM stock, final conc. 5 mM) was added in the end to make the final reaction volume to be 20 μL. Then the solution was mixed at
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room temperature for 2 h in the dark by attaching to a slow rotisserie (approx. 30 rotations per min). The reaction mixtures were then dialyzed against 100 mM potassium phosphate (pH 7.0,
2000 folds of dilution, 2 h × 2 at room temperature in the dark) to remove excess small molecules.
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3.3.3 Analysis
3.3.3.1 Tryptic digestion
To achieve better resolution of the peptide during mass spectrometric analysis, the poly-histidine tagged and the coumarin-labeled exenatide in potassium phosphate (100 mM, pH 7) were digested by trypsin at an enzyme: protein ratio of 1:40 (w/w) at 37 °C for 2 h. Reactions were quenched by adding aqueous trifluoroacetic acid (TFA, 5%) to a final concentration of 0.5%.
The reaction mixtures were then desalted by C18 tip prior to MALDI-TOF/TOF analysis.
3.3.3.2 SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a
Bio-rad Mini-PROTEIN system. Protein samples were first incubated with 4X reducing SDS-
PAGE loading buffer (BP-110R, Boston Bio Lab) at 56 oC for 10 min. In order to resolve low molecular weight exenatide, samples were loaded onto a 16.5% tris-tricine precast gel (456-3064,
Bio-Rad) in Tris/Tricine running buffer which contains 100 mM Tris, 100 mM Tricine, 0.1%
SDS (161-0744, Bio-Rad). A Dual Xtra standards protein ladder (161-0377, Bio-rad) was used for mass calibration. Electrophoresis was performed at 110 V for 20 min, then 100 V for 10 min followed by 105 V for 1 h to diminish the diffusion of peptide bands with smaller molecular weight proteins. Coomassie staining using a gel-code blue (PI24594) safe staining solution. The
PEG was stained using a barium iodine protocol by placing gel in 5% barium chloride solution for 10 min, then in 0.1 N iodine solution for an additional 10 min.15-16
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3.3.3.3 MALDI-TOF/TOF mass spectrometry
The mixtures were desalted using standard C18 tip purification (ZipTip, ZTC18S096; EMD
Millipore) prior to mass spec analysis. MALDI TOF/TOF was conducted on an AB SCIEX
5800 MALDI TOF/TOF analyzer (SCIEX, Framingham, MA). Each desalted sample was reconstituted in 70: 30 water/acetonitrile (v/v) solution with 0.1% TFA, and then co-crystallized
1:1 with 10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA, Protea Biosciences. Morgantown,
WV) in 50:50 water/acetonitrile (v/v) solution with 0.1% TFA on a MALDI sample plate. The
MS spectra were obtained in reflectron and linear positive mode, while the MS/MS spectra were obtained using 2 kV collision energy. The instrument was calibrated using a peptide standard from Anaspec (AS-60882; Calibration mixture II: ACTH (clip 1-17) 2094.46 Da, ACTH (clip
18-39) 2466.72 Da, and ACTH (clip 7-38) 3660.19 Da). MALDI data were processed using
Applied Biosystems Data Explorer 4.6 software.
3.3.3.4 LC-QTOF mass spectrometry
LC-MS data were obtained on a Waters H-Class Acquity UPLC system coupled to a Xevo G2-S
Q-ToF mass spectrometer (Waters Corp, Milford, MA). Liquid chromatography was performed on an ACQUITY UPLC Peptide BEH C18 Column (300 Å, 1.7 μm, 2.1 mm × 100 mm, Waters,
Corp, Milford, MA). A gradient using 0.1% formic acid in HPLC grade water (mobile phase A) and 0.1% formic acid in HPLC grade acetonitrile (mobile phase B) was applied with a flow rate of 0.2 μL/min. Gradient was performed by starting at 5% mobile phase B for 2 min, increasing to 60% mobile phase B over 20 min, and then to 95% mobile phase B over 2 min, holding at 95% mobile phase B for 3 min, and finally decreasing to 5% mobile phase B over 3 min. After LC, samples were introduced to mass spectrometer via an electrospray ion source in-line with the
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Xevo G2-S Q-ToF. External calibration of m/z was performed using sodium cesium iodide.
Data were processed using the UNIFI 1.7.1 software from Waters.
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3.4 Results and Discussion
3.4.1 Propargylation of exenatide
Scheme 3.2. TGase mediated propargylation of exenatide.
Propargyl amine is a known substrate of microbial TGase with excellent reactivity, which makes it a great proof-of-principle substrate for exenatide derivatization to start with (Scheme
3.2).9, 17 Propargylated-exenatide was successfully acquired initially using standard reaction conditions at 50 U/g TGase substrate ratio, 20 mM of propargyl amine at 37 oC, pH 7. However, the yield was only around 30 to 40% after overnight incubation (16 h maximum) according to the
MALDI mass spectra (Figure 3.2). The corresponding MS/MS spectra of native and propargylated-exenatide are shown in Figure 3.3. Several optimizations have been applied to the method but with little improvement. However, nearly 100% yield was observed when the mixture was firstly incubated at an increased temperature of 50 oC for 4 h, and then incubation was continued at 37 oC for an additional 10 h. The experience of increasing incubation temperature to achieve higher yield was then applied to other TGase mediated reactions and works in many of the cases not limited to exenatide.
The resulting propargylated-exenatide is a novel compound that has not been previously obtained, which itself may be worth for additional investigation about the potential of alternative therapeutic activities. Moreover, the alkyne group introduced to exenatide is highly reactive and
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additional conjugation through bioorthogonal chemistry such as click chemistry can be performed.18-19
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Figure 3.2. MS spectra of native and propargylated-exenatide.
MALDI-TOF MS spectra of native exenatide (A, theoretical m/z 4185.04, observed m/z 4185.27, difference m/z 0.23, 54 ppm) and propargylated exenatide (B, theoretical m/z 4223.05, observed m/z 4223.23, difference m/z 0.18, 42 ppm) The mass shift of +38 Da corresponding to propargylation is observed.
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Figure 3.3. MS/MS spectra of native and propargylated exenatide.
MALDI-TOF MS/MS spectra of native exenatide (top, precursor ion m/z 4185. theoretical m/z 4185.04) and propargylated-exenatide (bottom, precursor ion m/z 4223, theoretical 4223.05). The b* and y* ions with +38 Da mass shift corresponding to glutamine propargylation were observed on the bottom trace.
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3.4.2 Biotinylation of exenatide
Scheme 3.3. TGase mediated biotinylation of exenatide.
Biotinylation of exenatide is also of great interest (Scheme 3.3), for biotinylated protein and peptide enable specific purification such as immobilization and affinity purification based on the specific interaction between biotin and avidin, and often without perturbing the original functions of protein and peptide.20-21 The LC-MS chromatogram and spectra are shown in
Figure 3.4. According to chromatogram at 214 nm, no obvious side products were observed except the oxidation of methionine, and the yield of direct biotinylation using biotin cadaverine was nearly 100% including all the species of exenatide from the starting material after overnight incubation.
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Figure 3.4. LC-MS of exenatide biotinylation product.
Extracted ion chromatogram (XIC) for the 5 H+ charge state of a) native exenatide and b) 5 H+ charge state of biotinylated-exenatide. c) ESI MS spectrum of native exenatide (theoretical m/z 837.81, observed m/z 837.80, difference m/z 0.01, 11ppm) and d) biotinylated-exenatide (theoretical m/z 900.03, observed m/z 900.02, difference m/z 0.01, 11ppm)
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3.4.3 PEGylation of exenatide
Polyethylene glycol (PEG) is a widely used water soluble polymer in the field of medicine.
PEGylated biomolecules have been reported to have numerous advantages such as extended circulation time; enhanced tissue mobility, and avoided or reduced immunogenicity.22-24 Since the first approval of a PEGylated drug (PEGylated adenosine deaminase, Adagen) in 1990, a total number of 12 PEGylated biotherapeutics have been approved in the United States and
Europe till 2016, and around 5 other candidates have gone as far as Phase III clinical trials.25-26
One of which is Pegvaliase, the PEGylated enzyme phenylalanine hydroxylase for the treatment of phenylketonuria developed by BioMarin.27 The enzyme was originally from Anabaena variabilis, and the conjugation to PEG is believed to contribute to the reduction of protease sensitivity and immunogenicity.28-29 After simple mutation and PEGylation, the drug named
Pegvaliase (rAvPAL-PEG) has gone through phase III clinical trial, and been submitted to
Biologics License Application according to the recent reports.
Scheme 3.4. TGase mediated one-step PEGylation of exenatide.
In practice, exenatide has been successfully PEGylated by a 10 kDa PEG amine via the mediation of TGase (Scheme 3.4). Talking about PEGylation, there are several concepts and definition in polymer chemistry that are worth mentioning, among which Mn stands for the
78
number-average molecular weight, Mw stands for the weight-average molecular weight, and the polymer dispersion index refers to the distribution of molecular mass that calculated as
Mw/Mn.30 Figure 3.5 shows the MALDI MS spectra of exenatide PEGylation reaction mixture after C18 tip purification. The residue PEG amine and PEGylated-exenatide were both observed.
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Figure 3.5. MS spectra of PEGylated-exenatide.
MALDI-TOF MS spectra of PEGylated-exenatide reaction mixture under linear ion mode at laser intensity 3540. PEG amine (vendor provided Mn ~10000 Da, calculated Mn 10590.70 Da) and PEGylated-exenatide, calculated Mn 14708.41 Da) were observed
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Figure 3.6 shows the SDS-PAGE of TGase mediated PEGylation mixture, the gel was stained with both iodine and Coomassie blue to resolve PEG and peptide. It has been noticed that the band of PEGylated protein on SDS-PAGE is often broadened and smeared, probably due to the interaction of PEG and SDS.31 Plus, the migration patterns of PEG and protein are usually different, thus the mass observed according to protein ladder may not be accurate.32
Figure 3.6. SDS-PAGE of PEGylated exenatide mixture.
SDS-PAGE of PEGylated exenatide mixture after iodine staining (left) and Coomassie staining (right). Exenatide, TGase, and PEGylated-exenatide have been observed correspondingly.
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3.4.4 Additional derivatization of propargylated-exenatide through click chemistry
3.4.4.1 Fluorescent tagging of exenatide via click chemistry
Propargylated-exenatide generated in 3.3.2.1 has enhanced reactivity due to the functionalized alkyne. One of the popular bioorthogonal methods that can be further employed is click chemistry.
Scheme 3.5. Fluorescent tagging of exenatide.
The Zhou laboratory recently used a unique isotopic probe (i.e. brominated coumarin azide) to conjugate alkynes via copper-catalyzed azide-alkyne click chemistry (manuscript under review, confidential). The resulting product is fluorogenic and has a distinct isotopic envelope in mass spectrum generated by bromine, which can possess high sensitivity and efficiency in site- identification. In this section, the new brominated coumarin azide was selected to tag the exenatide using the standard protocol of copper-catalyzed azide-alkyne click chemistry.19 The fluorescence can be easily observed and monitored (Figure 3.7-d) during reaction, and the mass spectra of tryptic fragment of exenatide indicated that the native exenatide fragment shows normal isotopic distribution as a peptide (Figure 3.7-a) and the resulting bromo-coumarin tagged exenatide fragment shows distinct isotope pattern in which the most abundant peak are the first and third peak (Figure 3.7-b). The corresponding MS/MS spectra are also shown in Figure 3.8.
Besides, bromo-coumarin triazole has a maximum absorbance at 350 nm, while exenatide
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contains one tryptophan and has a maximum absorption at 280 nm.33 Figure 3.7-c shows the UV spectra of exenatide, bromo-coumarin triazole as well as the Br-coumarin tagged exenatide, and the resulted coumarin tagged exenatide (green) has two maximum absorption at both 280 nm and
350 nm as expected.
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Figure 3.7. Derivatization of propargylated-exenatide with Br-coumarin a) MS spectra of exenatide Gln13 containing tryptic fragment (QMEEEAVR, theoretical m/z 991.45, observed m/z 991.44, difference m/z 0.01, 10 ppm), isotopic envelope was resolved that the monoisotopic peak to be the highest peak. b) MS spectra of Br-coumarin tagged exenatide Gln13 containing tryptic fragment (Br-coumarin-QMEEEAVR, theoretical m/z 1310.40, observed m/z 1310.41, difference m/z 0.01, 8 ppm), isotopic envelope was resolved that unique distribution indicates the existence of bromine. c) normalized UV spectra of native exenatide (purple), Br-coumarin triazole (red) and Br-coumarin tagged exenatide (green). d) reaction mixture (left) and control mixture (right, no propargylated-exenatide added) of the Br-coumarin derivatization of propargylated-exenatide under 254 nm UV light.
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Figure 3.8. MS/MS spectra of bromo-coumarin tagged exenatide tryptic peptides.
MALDI-TOF MS/MS spectra of bromo-coumarin tagged exenatide tryptic peptides (Br- coumarin-QMEEEAVR, precursor ion m/z 1310.41, theoretical m/z 1310.40), the reporter ion after the fragmentation of the amide bond between glutamine side chain and coumarin triazole (y0) was observed.
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3.4.4.2 Poly-histidine tagging of exenatide via click chemistry
Scheme 3.6. Poly-histidine tagging of exenatide via click chemistry
Poly-histidine tag is a widely used separation technique in protein isolation and purification, the his-tagged protein and peptides are suitable for affinity purifications.34 The conjugation of propargylated-exenatide with poly-histidine azide was conducted (Scheme 3.6), and the poly- histidine tagged exenatide were digested by trypsin in order to achieve better resolution of the peptide during mass spectrometric analysis. Figure 3.9 and Figure 3.10 show the MS and
MS/MS spectra of exenatide tryptic fragment conjugated to poly-histidine tag at Gln13.
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Figure 3.9. MS spectra of poly-histidine tagged exenatide fragment.
MALDI-TOFMS spectra of Gln13 containing tryptic fragment of exenatide derivatized with poly-histidine tag (His-tag-QMEEEAVR, theoretical m/z 2116.12, observed m/z 2116.10, difference 0.02 m/z, 9 ppm).
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Figure 3.10. MS/MS spectra of poly-histidine tagged exenatide fragment.
MALDI-TOF MS/MS spectra of tryptic fragment of exenatide derivatized with poly-histidine tag (precursor ion m/z 2116.10, theoretical m/z 2116.12).
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Besides, there are two lysine residues (K12 and K27) existed in exenatide sequence, which could be potential intramolecular and intermolecular crosslinking sites as amine donor.35 Indeed, minor peak corresponding to intramolecular crosslinking observed on MALDI spectra, further separation and additional MS/MS may be needed to identify the crosslinking site. Another issue is that oxidation of methionine has been observed frequently during the reaction and analysis. It has been reported that the oxidation of exenatide may affect its affinity to GLP-1 receptor binding.36 However, tricks such as the addition of free L-methionine, or addition of reducing reagent (TCEP or DTT) can be used, and extensively minimize the oxidation of methionine.
In summary, multiple novel derivatives are obtained via TGase mediated conjugation, the enzyme successfully identifies Gln13 of exenatide as a substrate and build up glutamine site- specific conjugations to a variety of modifiers with primary amines, which also justifies the broad acyl acceptor specificity of TGase. Upon these derivatizations, new applications can be further utilized, such as affinity enrichment, purification and separation through biotinylated and poly-histidine tagged exenatide. Furthermore, the exenatide conjugation/derivative may confer improved or new therapeutic activities, as well as being used in biological applications, which can be used for functional probes and biomaterial design.37
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3.5 Future directions
These five novel compounds are expected to have potential to show similar or alternative biological activities as the native drug that are worth further investigation. Moreover, the poly- histidine tagged and fluorophore tagged species can be used as biomarkers or biological probes.38
We are working with collaborators from Tufts medical school on biological activity assays. For example, the binding affinity assay of GLP-1 receptor or cAMP Dynamic 2 assay are designed to conduct.39 According to the binding affinity data, additional modification of the derivatives or in vivo activity assay through animal testing can be designed in the future.
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3.6 References
1. Holst, J. J., The Physiology of Glucagon-like Peptide 1. Physiological Reviews 2007, 87 (4), 1409-1439.
2. Drucker, D. J., The biology of incretin hormones. Cell Metabolism 2006, 3 (3), 153-165.
3. Ahren, B., Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes. Nature Reviews. Drug Discovery 2009, 8 (5), 369-385.
4. Mikhail, N., Exenatide: A novel approach for treatment of type 2 diabetes. In Southern Medical Journal, 2006; Vol. 99, pp 1271-1279.
5. Göke, R.; Fehmann, H. C.; Linn, T.; Schmidt, H.; Krause, M.; Eng, J.; Göke, B., Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells. The Journal of biological chemistry 1993, 268 (26), 19650.
6. Christensen, M.; Miossec, P.; Larsen, B. D.; Werner, U.; Knop, F. K., The design and discovery of lixisenatide for the treatment of type 2 diabetes mellitus. Expert Opinion on Drug Discovery 2014, 9 (10), 1223-1251.
7. Matthews, J. E.; Stewart, M. W.; De Boever, E. H.; Dobbins, R. L.; Hodge, R. J.; Walker, S. E.; Holland, M. C.; Bush, M. A., Pharmacodynamics, pharmacokinetics, safety, and tolerability of albiglutide, a long-acting glucagon-like peptide-1 mimetic, in patients with type 2 diabetes. The Journal of Clinical Endocrinology and Metabolism 2008, 93 (12), 4810-4817.
8. Madsen, K.; Knudsen, L. B.; Agersoe, H.; Nielsen, P. F.; Thøgersen, H.; Wilken, M.; Johansen, N. L., Structure−Activity and Protraction Relationship of Long-Acting Glucagon-like Peptide-1 Derivatives: Importance of Fatty Acid Length, Polarity, and Bulkiness. Journal of Medicinal Chemistry 2007, 50 (24), 6126-6132.
9. Gundersen, M. T.; Keillor, J. W.; Pelletier, J. N., Microbial transglutaminase displays broad acyl-acceptor substrate specificity. Applied Microbiology and Biotechnology 2014, 98 (1), 219-230.
10. Maullu, C.; Raimondo, D.; Caboi, F.; Giorgetti, A.; Sergi, M.; Valentini, M.; Tonon, G.; Tramontano, A., Site‐directed enzymatic PEGylation of the human granulocyte colony‐ stimulating factor. FEBS journal 2009, 276 (22), 6741-6750.
11. Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H., Designing a 20-residue protein. Nat Struct Mol Biol 2002, 9 (6), 425-430.
12. Neidigh, J. W.; Fesinmeyer, R. M.; Prickett, K. S.; Andersen, N. H., Exendin-4 and Glucagon-like-peptide-1: NMR Structural Comparisons in the Solution and Micelle-Associated States. Biochemistry 2001, 40 (44), 13188-13200.
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13. Liu, M.; Zhang, Z.; Zang, T.; Spahr, C.; Cheetham, J.; Ren, D.; Zhou, Z. S., Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing 18O- Labeling and Mass Spectrometry. Analytical Chemistry 2013, 85 (12), 5900-5908.
14. Runge, S.; Thøgersen, H.; Madsen, K.; Lau, J.; Rudolph, R., Crystal Structure of the Ligand-bound Glucagon-like Peptide-1 Receptor Extracellular Domain. Journal of Biological Chemistry 2008, 283 (17), 11340-11347.
15. Bailon, P.; Palleroni, A.; Schaffer, C. A.; Spence, C. L.; Fung, W.-J.; Porter, J. E.; Ehrlich, G. K.; Pan, W.; Xu, Z.-X.; Modi, M. W.; Farid, A.; Berthold, W.; Graves, M., Rational Design of a Potent, Long-Lasting Form of Interferon: A 40 kDa Branched Polyethylene Glycol- Conjugated Interferon α-2a for the Treatment of Hepatitis C. Bioconjugate Chemistry 2001, 12 (2), 195-202.
16. Kurfürst, M. M., Detection and molecular weight determination of polyethylene glycol- modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analytical Biochemistry 1992, 200 (2), 244-248.
17. Gnaccarini, C.; Ben-Tahar, W.; Mulani, A.; Roy, I.; Lubell, W. D.; Pelletier, J. N.; Keillor, J. W., Site-specific protein propargylation using tissue transglutaminase. Organic & Biomolecular Chemistry 2012, 10 (27), 5258-5265.
18. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Weinheim, 2001; Vol. 40, pp 2004-2021.
19. Presolski, S. I.; Hong, V. P.; Finn, M., Copper ‐ Catalyzed Azide – Alkyne Click Chemistry for Bioconjugation. Current protocols in chemical biology 2011, 153-162.
20. Salzig, D.; Schmiermund, A.; Gebauer, E.; Fuchsbauer, H.-L.; Czermak, P., Influence of porcine intervertebral disc matrix on stem cell differentiation. Journal of functional biomaterials 2011, 2 (3), 155.
21. Chaiet, L.; Wolf, F. J., The properties of streptavidin, a biotin-binding protein produced by Streptomycetes. Archives of Biochemistry and Biophysics 1964, 106, 1-5.
22. Yang, Z.; Wang, J.; Lu, Q.; Xu, J.; Kobayashi, Y.; Takakura, T.; Takimoto, A.; Yoshioka, T.; Lian, C.; Chen, C., PEGylation confers greatly extended half-life and attenuated immunogenicity to recombinant methioninase in primates. Cancer Research 2004, 64 (18), 6673-6678.
23. Chapman, A. P., PEGylated antibodies and antibody fragments for improved therapy: a review. Advanced Drug Delivery Reviews 2002, 54 (4), 531-545.
24. Roseng, L.; Tolleshaug, H.; Berg, T., Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes. Journal of Biological Chemistry 1992, 267 (32), 22987-22993.
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25. Hershfield, M. S., Biochemistry and immunology of poly (ethylene glycol)-modified adenosine deaminase (PEG-ADA). ACS Publications: 1997.
26. Turecek, P. L.; Bossard, M. J.; Schoetens, F.; Ivens, I. A., PEGylation of biopharmaceuticals: a review of chemistry and nonclinical safety information of approved drugs. Journal of Pharmaceutical Sciences 2016, 105 (2), 460-475.
27. Bell, S. M.; Wendt, D. J.; Zhang, Y.; Taylor, T. W.; Long, S.; Tsuruda, L.; Zhao, B.; Laipis, P.; Fitzpatrick, P. A., Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.(Research Article)(Author abstract). PLoS ONE 2017, 12 (3), e0173269.
28. Alejandra, G.; Lin, W.; Mary, S.; Marianne, G. P.; Raymond, C. S., Toward PKU Enzyme Replacement Therapy: PEGylation with Activity Retention for Three Forms of Recombinant Phenylalanine Hydroxylase. Molecular Therapy 2004, 9 (1), 124.
29. Gámez, A.; Wang, L.; Sarkissian, C. N.; Wendt, D.; Fitzpatrick, P.; Lemontt, J. F.; Scriver, C. R.; Stevens, R. C., Structure-based epitope and PEGylation sites mapping of phenylalanine ammonia-lyase for enzyme substitution treatment of phenylketonuria. Molecular Genetics and Metabolism 2007, 91 (4), 325-334.
30. Hanton, S., Mass spectrometry of polymers and polymer surfaces. Chemical Reviews 2001, 101 (2), 527-570.
31. Odom, O. W.; Kudlicki, W.; Kramer, G.; Hardesty, B., An effect of polyethylene glycol 8000 on protein mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and a method for eliminating this effect. Analytical biochemistry 1997, 245 (2), 249-252.
32. Zheng, C. Y.; Ma, G.; Su, Z., Native PAGE eliminates the problem of PEG–SDS interaction in SDS‐PAGE and provides an alternative to HPLC in characterization of protein PEGylation. Electrophoresis 2007, 28 (16), 2801-2807.
33. Zang, T.; Lee, B. W.; Cannon, L. M.; Ritter, K. A.; Dai, S.; Ren, D.; Wood, T. K.; Zhou, Z. S., A naturally occurring brominated furanone covalently modifies and inactivates LuxS. Bioorg Med Chem Lett 2009, 19 (21), 6200-4.
34. Hochuli, E.; Bannwarth, W.; Döbeli, H.; Gentz, R.; Stüber, D., Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nature biotechnology 1988, 6 (11), 1321-1325.
35. Liu, M.; Zhang, Z.; Cheetham, J.; Ren, D.; Zhou, Z. S., Discovery and characterization of a photo-oxidative histidine-histidine cross-link in IgG1 antibody utilizing 18O-labeling and mass spectrometry. Analytical chemistry 2014, 86 (10), 4940-4948.
36. Janota, B.; Karczmarczyk, U.; Laszuk, E.; Garnuszek, P.; Mikołajczak, R., Oxidation of methionine - is it limiting the diagnostic properties of 99mTc-labeled Exendin-4, a Glucagon- Like Peptide-1 receptor agonist? Nuclear Medicine Review 2016, 19 (2), 104-110.
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37. Zang, T.; Pottenplackel, L. P.; Handy, D. E.; Loscalzo, J.; Dai, S.; Deth, R. C.; Zhou, Z. S.; Ma, J., Comparison of protein n-homocysteinylation in rat plasma under elevated homocysteine using a specific chemical labeling method. Molecules 2016, 21 (9), 1195.
38. Clardy, S. M.; Keliher, E. J.; Mohan, J. F.; Sebas, M.; Benoist, C.; Mathis, D.; Weissleder, R., Fluorescent exendin-4 derivatives for pancreatic β-cell analysis. Bioconjugate chemistry 2013, 25 (1), 171-177.
39. Levy, O. E.; Jodka, C. M.; Ren, S. S.; Mamedova, L.; Sharma, A.; Samant, M.; D’Souza, L. J.; Soares, C. J.; Yuskin, D. R.; Jin, L. J., Novel exenatide analogs with peptidic albumin binding domains: potent anti-diabetic agents with extended duration of action. PloS ONE 2014, 9 (2), e87704.
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Site-specific Bioconjugation of Peptides and Proteins by Nucleophile
Trapping of Metastable Succinimide
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4.1 Abstract
Site-specific bioconjugation of proteins installs enhanced functionalities to existing proteins and has become a fast-growing area of research. Current chemical conjugation strategies that have been used either focus on N-terminal and C-terminal derivatizations or utilize existing functional groups such as amino and thiol residues. However, the site-selectivity is limited since multiple reactive sites often exist, and isomers often form after derivatization.
Herein, a novel site-specific conjugation method of peptides, proteins and peptidyl analogs is devised, while nucleophile trapping is applied to a succinimide generated from latent reactivities of asparagine (Asn or N), aspartic acid (Asp or D) and other functional groups. The enhanced site-specificity can be achieved since only special tandem amino acids eligible for succinimide formation, and the reactivity can be modulated by various factors. Several novel derivatives of existing peptidyl drug have been obtained using these method, including exenatide, vancomycin and bacitracin.
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4.2 The latent reactivity behind succinimide formation: from an unavoidable problem to
a derivatization site
As mentioned in previous chapters, unwanted post-translational modifications can be troublesome. After decades of work on detection and analysis of deamidation and isoaspartic acid formation, we have started to bring up alternative thinking and potential applications upon unwanted unavoidable PTMs. It is already well known that during deamidation, a succinimide intermediate is first formed non-enzymatically from asparaginyl and aspartyl residues; the succinimide is unstable under normal conditions and can be easily hydrolyzed. Formation of the succinimide does not require additional reagents, and the reaction rates can be affected by experimental conditions such as pH and protein conformation, hence latent reactivities.1 Inspired by some of our previous work, the succinimide intermediate can be readily trapped by hydrazine or other nucleophiles.2-4
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Scheme 4.1. The formation of a succinimide intermediate, the subsequent trapping by hydrazine and further derivatization of hydrazide
A novel site-specific derivatization method is devised that peptides, proteins and related analogs can be modified via nucleophile trapping of a succinimide generated from latent reactivities of asparagine (Asn or N), aspartic acid (Asp or D) and other functional groups. The nucleophiles and their corresponding products include hydrazines (hydrazides), hydroxylamines (hydroxamic acids), amines (amides), and water (acids). Furthermore, further derivatization can be achieved via orthogonal chemistry towards the newly introduced functional groups, the general scheme of
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this whole process in
Scheme 4.1.5-7 The whole process will be discussed stepwise in the following sections.
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4.2.1 Step 1. Formation of succinimide and latent reactivity
Scheme 4.2. Examples of functional groups that can form succinimide.
Succinimide can come from multiple process, among which deamidation of asparagine or isomerization of aspartic acids are the most common source. Similar functional groups result in a succinimide as depicted in Scheme 4.2. The R1, R2 and X groups in this step can be typical
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peptide backbone residue, as well as other structures such as atypical side chain linkage in bacitracin. The blue color highlights the succinimide moiety and its precursors. The formation of succinimide is a spontaneous process, its rate can be affected by various factors, and generally considered to be “bad” in conventional views.8-10 We propose that the instability can also be considered as “latent reactivity” that can be utilized to build up site-specific derivatization.11
Moreover, this latent reactivity of peptidyl species can be modulated by various reagents and conditions.
Figure 4.1. The latent reactivity control by reaction conditions.
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4.2.1.1 Effects of conformation
Formation of the succinimide (both deamidation and isomerization) is sensitive to the conformation of the functional groups involved as illustrated in Figure 4.1. Typically, more ordered structures slow down succinimide formation. For example, one of the model peptides, exenatide, contains a Asn-Gly tandem, and is believed to be prone to deamidation. However, the formation of succinimide and hydrazide is non-detectable after 48 h incubation, as the asparagine residue is located in a rigid “Trp-Cage” region, which stabilized the asparagine under native conditions.12 The rate of succinimide formation (and the subsequent trapping by hydrazine) is significantly higher after peptide denaturation (e.g. with 4.5 M guanidine). On the other hand, more ordered structures may accelerate succinimide formation. For example, deamidation in a
PENNY (Phe-Glu-Asn-Asn-Tyr) motif in monoclonal antibodies is much faster in the native state than the denatured state.13 Disulfide linkage is another factor that helps control the latent reactivity. Different degrees of succinimide formation and deamidation have been observed with or without disulfide bonds reduction in protein, such as glycinin.14
4.2.1.2 Effects of pH
Other than conformation, the reaction itself can be modulated as well. For asparagine, deamidation and succinimide formation are in favored in neutral to basic condition (higher pH); while succinimide formation and the isomerization of aspartic acid are favored under mildly acidic conditions (lower pH). For example, conversion from Asp or isoAsp to succinimide is faster under mildly acidic conditions (e.g., pH 4 to 5) in lysozyme.15-16
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4.2.2 Step 2. Nucleophile trapping of succinimide
Scheme 4.3. Trapping of the succinimide intermediate by nucleophiles.
The labile succinimide intermediate can be trapped with various nucleophiles. Two isomers can be formed during the trapping as denoted by the green (on the peptide side chain) and pink (on the peptide backbone) dots. One of the isomers (bottom structure) confers a beta- peptide linkage (isoaspartyl or isoasparaginyl) in the backbone; in comparison, most derivatization methods only modify the side chain residues not the peptide backbone or maintain the alpha linkage of the backbone. The resulting derivatives (e.g., peptidyl hydrazides) are stable: for example, can be isolated and stored, and can be detected by standard mass spectrometry.
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These chemical transformations on asparaginyl and aspartyl residue have been previously reported by others and our laboratory. 2-3, 5, 17
Moreover, nucleophiles that can trap the succinimide are not limited to hydrazine and that previously reported, but also include water (acids as the products) substituted hydrazines
(substituted hydrazides as the products), hydroxylamines (hydroxamic acids as the products), amines (amides as the products) and others as illustrated in Scheme 4.4.
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Scheme 4.4. Trapping of succinimide with various nucleopholes.
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4.2.3 Step 3. Further derivatization of hydrazides
Scheme 4.5. Further derivatization of hydrazides via biorthogonal chemistry
The resulting peptidyl analog generates unique reactivity that allows additional modification via orthogonal chemistry (Step 3). Taking hydrazinolysis as an example, the resulting hydrazide can be further modified via known chemistry. Selected examples of chemical derivatization on hydrazinolysis products can be generated are shown in Scheme 4.5.18-21
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Compared to an amine, hydrazine can react to aldehydes and sulfonyl chloride derivatives with a high degree of selectivity.7 As shown in Scheme 4.6, the selectivity in binding hydrazide against the free amine is achieved under mildly acidic conditions (e.g., pH 5), where the majority of hydrazides (pKa ～ 3) remain neutral, and the majority of amines (pKa ～ 9) are protonated. A note is that the resulting hydrazone is usually stable enough, and further reduction is usually not needed. The reactions have been applied in the affinity enrichment or installation of multiple tags.3, 5
Scheme 4.6. Selective reaction of aldehyde with hydrazides against amines at mildly acidic conditions
The proposed hydrazine trapping methodology has been reduced to practice, several novel derivatives of existing peptidyl drug have been obtained using this method, including exenatide, vancomycin and bacitracin with considerably yield after optimization. All the derivatives obtained are novel, and with the potential to be new antibody candidates or having alternative biological activities.
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4.3 Derivatization of Vancomycin
Vancomycin is a glycopeptide antibiotic that contains two glycol groups and a pentapeptide moiety, and has been used in the treatment of severe bacterial infections for over 50 years.22
Interestingly, the structure of vancomycin was not confirmed until 20 years after it was approved.23 Due to its supreme functionality, it was once used as a last-line antibiotic for decades, however, the emergence of vancomycin-resistant bacteria and related cases made it less competitive, and that new, stronger antibiotics were highly demanded.
Efforts has been made on developing new structural and functional analogs of vancomycin in the last two decades.24 Vancomycin has recently gotten our attention because the asparagine residue in the peptide moiety was shown prone to succinimide formation and deamidation, which makes it an ideal candidate for nucleophile trapping derivatization.25-26 The attempt of derivatizing vancomycin on asparagine site was successful, and 14N, 15N isotopic labeled hydrazine was used to help characterization. Figure 4.2 shows the structure of vancomycin and the derivatives we obtained using the hydrazine trapping method, and the mass spec characterization are shown in Figure 4.3.
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Figure 4.2. Vancomycin structure and derivatives obtained hydrazine nucleophile trapping.
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Figure 4.3. MALDI-TOF MS spectra of hydrazine treated vancomycin.
MALDI-TOF MS spectra of native (top), 14N hydrazine treated (middle) and 15N hydrazine treated vancomycin. Peak A, B, and C are corresponding to native vancomycin (theoretical m/z 1470.42, observed m/z 1470.41, difference m/z 0.01, 7 ppm), 14N vancomycin hydrazide (+15 Da, theoretical m/z 1485.43, observed m/z 1485.44, difference m/z 0.01, 7 ppm) and 15N labeled vancomycin hydrazide (+17 Da, theoretical m/z 1487.43, observed m/z 1487.44, difference m/z 0.01, 7 ppm).
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4.4 Derivatization of exenatide
Exenatide is one of our favorite model biopharmaceuticals that has been depicted in the previous two chapters for the demonstration of deamidation and TGase mediated bioconjugation
(Figure 4.4).27 The NG tandem containing peptide has been investigated for the possibility of obtaining novel derivatives via hydrazinolysis. Both 14N and 15N labeled hydrazine is used during the reaction.
The challenging part of utilizing exenatide is that although the 39-amino acid peptide contains the NG tandem and located at the C terminal random coil, the flexibility is much lower than expected due to the rigid, autonomously folded “Trp-cage” at the C-terminal end, thus the deamidation rate of the asparagine is limited. 28 Hence, guanidine (4.5 M) is used to help denature the peptide and accelerate deamidation. The novel exenatide hydrazide derivative is successfully observed with the yield of 40-50%, detailed characterizations are shown in Figure
4.5 to Figure 4.8.
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Figure 4.4. Chemical structure of exenatide and specific compounds observed from exenatide derivatization.
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Figure 4.5. LC-MS spectra of native and hydrazine treated exenatide.
LC-QTOF MS spectra of native (top) and hydrazine treated (bottom) exenatide intact peptide.
(A, HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2, theoretical m/z 837.71, observed m/z 837.72, difference 0.01 m/z, 12 ppm) the m/z 840.72 peak (B) with +15 Da mass shift corresponding to exenatide hydrazide species is observed. The observed minor peak (m/z 843.92) corresponds to oxidation of methionine.
.
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Additional peptide mapping was performed in order to further confirm the hydrazide formation.
Figure 4.6. MS spectra of native and hydrazine treated exenatide tryptic peptide
MALDI-TOF MS spectra of native (top) and hydrazine treated (bottom) exenatide tryptic peptide
(LFIEWLKNGGPSSGAPPPS-NH2, theoretical m/z 1954.02, observed m/z 1954.02); the m/z 1969.00 peak with +15 Da mass shift corresponding to hydrazide species is observed. X in sequence stands for aspartyl hydrazide residue. MS/MS spectra of A and B are shown in Figure 4.7 and Figure 4.8.
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Figure 4.7. MS/MS spectra of exenatide tryptic peptide.
MALDI-TOF MS/MS spectra of singly charged exenatide tryptic peptide (precursor ion m/z 1954, theoretical m/z 1954.01 for Asn, top) and exenatide hydrazide tryptic peptide (precursor ion m/z 1969, theoretical m/z 1969.1 for Asp/isoAsp hydrazide, bottom) after 24 h incubation.
(21LFIEWLKXGGPSSGAPPPS - NH2, X denotes either asparagine or corresponding hydrazide.
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Figure 4.8. Zoomed MALDI-TOF MS/MS spectra of singly charged exenatide tryptic peptide.
Precursor ion m/z 1954 Da (theoretical m/z 1954.01 for Asn, top) and m/z 1969 (theoretical m/z 1969.1 for Asp/isoAsp hydrazide, bottom) for exenatide tryptic peptide
(21LFIEWLKXGGPSSGAPPPS-NH2, after 24 h incubation. X denotes either asparagine or corresponding hydrazide. These signature fragmentation ions indicate the modification (hydrazide formation) occurs at Asn28.
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Figure 4.9. MS of exenatide standard and exenatide after hydrazine treatment and dansylation.
Additional conjugation with fluorescent tagging is also achieved via hydrozone formation reaction (dansylation) by exenatide hydrazide and dansyl chloride.
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4.5 Derivatization of bacitracin
The formation of succinimide is not limited to between typical residue side chains and backbone, atypical side chain linkage such as bacitracin is also reported to be prone to deamidation, thus can also have the potential to be modified by the nucleus-trapping methodology.29
Bacitracin is a series of cyclic peptide antibiotics analogs with similar structure. Atypical amide bond is formed between the C-terminal end of asparagine residue and the side chain amine of lysine residue, among which bacitracin A is the major component of bacitracin (Figure
4.10).30
The drug disrupts both gram-positive and gram-negative bacteria by interfering with the cell wall and peptidoglycan synthesis. It is primarily used as a topical preparation, since it can cause kidney damage when used internally. The derivatization of bacitracin we conduct here may help with discovering alternative administration route. The initial hydrazinolysis of bacitracin failed under typical condition, however, bacitracin hydrazide was obtained after increasing the pH of the reaction mixture to pH 12.
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Figure 4.10. Structure of bacitracin A and derivatives observed
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Figure 4.11. MS spectra of bacitracin
MALDI-TOF MS spectra of native (top), phosphate buffer treated (pH 12, middle) and hydrazine treated bacitracin. Peak A, B, and C are corresponding to native bacitracin (theoretical m/z 1422.75, observed m/z 1422.76, difference m/z 0.01, 7 ppm), deamidated bacitracin (+1 Da, theoretical m/z 1423.75, observed m/z 1423.75) and bacitracin hydrazide (+15 Da, theoretical m/z 1437.75, observed m/z 1437.76, difference m/z 0.01, 7 ppm).
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Figure 4.12. MS/MS spectra of singly charged bacitracin and bacitracin hydrazide.
MALDI-TOF MS/MS spectra of singly charged bacitracin (precursor ion m/z 1422 Da theoretical m/z 1422.7 Da for Asn, top) and bacitracin hydrazide (precursor ion m/z 1437 Da, theoretical m/z 1437.7 for Asp/isoAsp hydrazide, bottom) after 24 h incubation. The cyclic moiety of bacitracin was successfully fragmented and the corresponding fragment of asparaginyl hydrazide was observed.
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4.6 Experimental section
4.6.1 Materials
Bacitracin zinc (1048007) was from U.S. Pharmacopeial Convention (Rockville, MD)
Vancomycin hydrochloride (V0045000) was from Sigma-Aldrich (St. Louis, MO). Exenatide
(AS-24464) was from Anaspec (Fremont, CA). 14N and 15N labeled hydrazine were from Sigma-
Aldrich (St. Louis, MO). Dansyl chloride, tris-HCl, dithiothretol (DTT), and tris(2- caboxyethyl)phosphine (TCEP) hydrochloride were from Fisher Scientific (Fair Lawn, NJ). C18 desalting column (ZipTip, ZTC18S096) was from EMD Millipore. All chemicals used were reagent grade or better, all aqueous solutions were prepared using water purified by a Milli-Q system (EMD Millipore, Bedford, Massachusetts).
4.6.2 Bioconjugations
4.6.2.1 Derivatization of vancomycin
To aqueous solutions of vancomycin hydrochloride (680 μM) in 2M hydrazine (14N or 15N, pH 9, pH was adjusted by sodium hydroxide) and 100 mM sodium bicarbonate/sodium carbonate buffer (pH 9.2), Dithiothretol (DTT)/ Tris(2-caboxyethyl)phosphine (TCEP) hydrochloride (1 mM each, final conc) was added. The solutions were incubated at 37 °C and aliquots were taken at 4 h and 24 h. The solutions were then dialyzed against 100 mM Tris-HCl at pH 7.5 (1:1000 v/v, 2 h × 2) with the existence of 1 mM DTT and 1 mM TECP. The mixtures were desalted using C18 ZipTip desalting column prior to analysis.
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4.6.2.2 Derivatization of exenatide
To an aqueous solution of exenatide (0.2 mM) in 1.5 M hydrazine and 4.5 M guanidine (pH
7.5-8, pH was adjusted by sodium hydroxide), Dithiothretol (DTT)/ Tris(2- caboxyethyl)phosphine (TCEP) hydrochloride (1 mM each, final conc) was added. The mixture was incubated at 37 oC for 24 h, and then dialyzed against 50 mM Tris-HCl at pH 7.5 (1:1000 v/v,
2 h × 2) to remove excess hydrazine. The mixtures were desalted using C18 desalting column prior to analysis. The resulting exenatide mixtures were digested by trypsin at an enzyme: protein ratio of 1:40 (w/w) at 37 oC for 3 h, and then quenched by 5% Trifluoroacetic acid (TFA).
4.6.2.3 Dansylation of exenatide hydrazide
To the reaction mixture resulting from hydrazinolysis of exenatide after ZipTip purification
(5 μL of ~0.2 mM exenatide/exenatide hydrazide in 50% acetonitrile 50% water with 0.1% TFA), dansyl chloride was add (5 μL of 5 mM stock in acetonitrile, fincal conc 2.5 mM), the mixture was incubated at room temperature for 12 h.
4.6.2.4 Derivatization of bacitracin
To an aqueous solution of bacitracin (1 mg/mL, 0.66 μM) in 2 M hydrazine (pH 12, pH was adjusted by sodium hydroxide), Dithiothretol (DTT)/ Tris(2-caboxyethyl)phosphine (TCEP) hydrochloride (1 mM each, final conc) was added. The solutions were aged by incubating at room temperature (24 oC) and aliquots were taken after 1 h, 2 h and 24 h. The solutions were dialyzed against 50 mM Tris-HCl at pH 7.5 (1:1000 v/v, 2 h × 2) to remove excess hydrazine.
The mixtures were desalted using C18 ZipTip desalting column prior to analysis.
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4.6.3 Mass spectrometry
Mass spectrometry was performed on a 5800 MALDI-TOF/TOF analyzer (Applied
Biosystems, Waltham, MA) and a H-Class Acquity UPLC system coupled to a Xevo G2-S Q-
ToF mass spectrometer (Waters，Milford, MA). The mixtures were mixed 1:1 with 10 mg/mL alpha-Cyano-4-hydroxycinnamic acid (CHCA) in a solution of 0.1 % Trifluoroacetic acid (TFA) in 50:50 water/acetonitrile， then loaded and crystallized on a MALDI target plate, and dried at room temperature for MALDI analysis.
LC-MS data of exenatide was acquired on a Waters H-Class Acquity UPLC system coupled to a Xevo G2-S Q-ToF mass spectrometer (Waters Corp, Milford, MA). Liquid chromatography was performed on an ACQUITY UPLC Peptide CSH C18 Column (300 Å, 1.7
μm, 2.1 mm × 100 mm, Waters, Corp, Milford, MA) with a gradient starting at 95% mobile phase A (0.1% formic acid in HPLC grade water), 5% mobile phase B (0.1% formic acid in
HPLC grade acetonitrile) for 2 min, increasing to 60% mobile phase B over 20 min, and then to
95% mobile phase B over 2 min, holding at 95% mobile phase B for 3 min, and finally decreasing to 5% mobile phase B over 3 min at a flow rate of 0.2 μL/min. After LC, samples were introduced to mass spectrometer via an electrospray ion source in-line with the Xevo G2-S
Q-ToF. Data were processed using the UNIFI 1.7.1 software from Waters.
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4.7 Prospective
As the consequence of widespread use of antibiotics, the occurrence of antibiotic resistance is becoming more and more challenging, however, the resources for antibacterial drug discovery are still limited. Under this circumstance, the peptidyl antibiotic derivatives of vancomycin and bacitracin we obtained are worth well for additional pharmacology assay, in order to develop new antibiotic candidates that have enhanced efficacy and reduced toxicity to overcome the antibiotic resistance. We are seeking for collaborations to continue working on the development of vancomycin and bacitracin analogs.31-32
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4.8 References
1. Robinson, N. E.; Robinson, A. B., Prediction of protein deamidation rates from primary and three-dimensional structure. Proceedings of the National Academy of Sciences 2001, 98 (8), 4367-4372.
2. Zhu, J. X.; Aswad, D. W., Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming. Analytical Biochemistry 2007, 364 (1), 1-7.
3. Klaene, J. J.; Ni, W.; Alfaro, J. F.; Zhou, Z. S., Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization. Journal of Pharmaceutical Sciences 2014, 103 (10), 3033-3042.
4. Mosley, S. L.; Bakke, B. A.; Sadler, J. M.; Sunkara, N. K.; Dorgan, K. M.; Zhou, Z. S.; Seley-Radtke, K. L., Carbocyclic pyrimidine nucleosides as inhibitors of S- adenosylhomocysteine hydrolase. Bioorganic & Medicinal Chemistry 2006, 14 (23), 7967-7971.
5. Alfaro, J. F.; Gillies, L. A.; Sun, H. G.; Dai, S.; Zang, T.; Klaene, J. J.; Kim, B. J.; Lowenson, J. D.; Clarke, S. G.; Karger, B. L.; Zhou, Z. S., Chemo-Enzymatic Detection of Protein Isoaspartate Using Protein Isoaspartate Methyltransferase and Hydrazine Trapping. Analytical Chemistry 2008, 80 (10), 3882-3889.
6. Chumsae, C.; Zhou, L. L.; Shen, Y.; Wohlgemuth, J.; Fung, E.; Burton, R.; Radziejewski, C.; Zhou, Z. S., Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody. Analytical Chemistry 2014, 86 (18), 8932-6.
7. Zang, T.; Dai, S.; Chen, D.; Lee, B. W. K.; Liu, S.; Karger, B. L.; Zhou, Z. S., Chemical Methods for the Detection of Protein N-Homocysteinylation via Selective Reactions with Aldehydes. Analytical Chemistry 2009, 81 (21), 9065-9071.
8. Böhme, L.; Bär, J. W.; Hoffmann, T.; Manhart, S.; Ludwig, H.-H.; Rosche, F.; Demuth, H.-U., Isoaspartate residues dramatically influence substrate recognition and turnover by proteases. Biological Chemistry 2008, 389 (8), 1043-1053.
9. Aswad, D. W.; Paranandi, M. V.; Schurter, B. T., Isoaspartate in peptides and proteins: formation, significance, and analysis. Journal of Pharmaceutical and Biomedical Analysis 2000, 21 (6), 1129-1136.
10. Noguchi, S., Structural changes induced by the deamidation and isomerization of asparagine revealed by the crystal structure of Ustilago sphaerogena ribonuclease U2B. Biopolymers 2010, 93 (11), 1003-1010.
11. Reissner, K. J.; Aswad, D. W., Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals? Cellular and Molecular Life Sciences 2003, 60 (7), 1281-95.
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12. Runge, S.; Thøgersen, H.; Madsen, K.; Lau, J.; Rudolph, R., Crystal Structure of the Ligand-bound Glucagon-like Peptide-1 Receptor Extracellular Domain. Journal of Biological Chemistry 2008, 283 (17), 11340-11347.
13. Pace, A. L.; Wong, R. L.; Zhang, Y. T.; Kao, Y.-H.; Wang, Y. J., Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature. Journal of Pharmaceutical Sciences 2013, 102 (6), 1712-1723.
14. Wagner, J. R.; Guéguen, J., Surface Functional Properties of Native, Acid-Treated, and Reduced Soy Glycinin. 2. Emulsifying Properties. Journal of Agricultural and Food Chemistry 1999, 47 (6), 2181-2187.
15. Robinson, N. E.; Robinson, A. B., Molecular clocks. Proceedings of the National Academy of Sciences 2001, 98 (3), 944-949.
16. Xie, M.; Vander Velde, D.; Morton, M.; Borchardt, R. T.; Schowen, R. L., pH-Induced Change in the Rate-Determining Step for the Hydrolysis of the Asp/Asn-Derived Cyclic-Imide Intermediate in Protein Degradation. Journal of the American Chemical Society 1996, 118 (37), 8955-8956.
17. Liu, M.; Cheetham, J.; Cauchon, N.; Ostovic, J.; Ni, W.; Ren, D.; Zhou, Z. S., Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis. Anal Chem 2012, 84 (2), 1056-62.
18. Chaiyaveij, D.; Cleary, L.; Batsanov, A. S.; Marder, T. B.; Shea, K. J.; Whiting, A., Copper(II)-catalyzed room temperature aerobic oxidation of hydroxamic acids and hydrazides to acyl-nitroso and azo intermediates, and their diels–alder trapping. Organic Letters 2011, 13 (13), 3442-3445.
19. Zang, T.; Dai, S.; Chen, D.; Lee, B. W.; Liu, S.; Karger, B. L.; Zhou, Z. S., Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes. Analytical Chemistry 2009, 81 (21), 9065-9071.
20. Zheng, J.-S.; Tang, S.; Qi, Y.-K.; Wang, Z.-P.; Liu, L., Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protocols 2013, 8 (12), 2483-2495.
21. Hoggarth, E., 250. Compounds related to thiosemicarbazide. Part I. 3-Phenyl-1 : 2 : 4- triazole derivatives. Journal of the Chemical Society (Resumed) 1949, (0), 1160-1163.
22. Fekety, R., Vancomycin. Medical Clinics of North America 1982, 66 (1), 175-181.
23. Williamson, M. P.; Williams, D. H., Structure revision of the antibiotic vancomycin. Use of nuclear Overhauser effect difference spectroscopy. Journal of the American Chemical Society 1981, 103 (22), 6580-6585.
24. Okano, A.; Isley, N. A.; Boger, D. L., Total Syntheses of Vancomycin-Related Glycopeptide Antibiotics and Key Analogues. Chemical Reviews 2017.
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25. Antipas, A. S.; Vander Velde, D. G.; Jois, S. D.; Siahaan, T.; Stella, V. J., Effect of conformation on the rate of deamidation of vancomycin in aqueous solutions. Journal of Pharmaceutical Sciences 2000, 89 (6), 742-750.
26. Antipas, A. S.; Vander Velde, D.; Stella, V. J., Factors affecting the deamidation of vancomycin in aqueous solutions. International journal of pharmaceutics 1994, 109 (3), 261-269.
27. Liu, S.; Moulton, K. R.; Auclair, J. R.; Zhou, Z. S., Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5. Amino Acids 2016, 48 (4), 1059-1067.
28. Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H., Designing a 20-residue protein. Nat Struct Mol Biol 2002, 9 (6), 425-430.
29. Pavli, V.; Kmetec, V., Pathways of Chemical Degradation of Polypeptide Antibiotic Bacitracin. Biological and Pharmaceutical Bulletin 2006, 29 (11), 2160-2167.
30. Ikai, Y.; Oka, H.; Hayakawa, J.; Matsumoto, M.; Saito, M.; Harada, K.-I.; Mayum, T.; Suzuki, M., Total Structures and Antimicrobial Activity of Bacitracin Minor Components. The Journal of Antibiotics 1995, 48, 233-242.
31. Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L., A redesigned vancomycin engineered for dual D-Ala-D-Ala and D-Ala-D-Lac binding exhibits potent antimicrobial activity against vancomycin-resistant bacteria. Journal of the American Chemical Society 2011, 133 (35), 13946-13949.
32. Zhao, G.; Wan, W.; Mansouri, S.; Alfaro, J. F.; Bassler, B. L.; Cornell, K. A.; Zhou, Z. S., Chemical synthesis of S-ribosyl-l-homocysteine and activity assay as a LuxS substrate. Bioorganic & Medicinal Chemistry Letters 2003, 13 (22), 3897-3900.
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Conclusions and Prospective
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In this thesis, artifacts in protein analysis have been overviewed and a new methodology of preventing analytical deamidation artifacts of proteins has been developed. As the continuous understanding of protein degradation and development of instrumentation, we can anticipate that the analysis of deamidation and analytical artifact will gain more accuracy and more practical methods will be developed in the field of biological analysis.
On the other hand, site-specific chemo-enzymatic bioconjugation of protein and peptide are described and has been reduced to practice in chapters 3 and 4. Nonetheless, many of the projects have yet to be described, and it is our hope that some of these may be explored in the future.
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5.1 Photocaging, from simple derivatization to on-demand manipulation of protein
Photocaging refers to a process in which a molecule is covalently modified with a photolabile reagent to form a photolabile derivative that inhibits the function of the molecule.1
The original molecule can be regenerated upon photolysis, and the lost function can be recovered.
Through photocaging, the activity of a biological compound can be controlled both temporally and spatially.2-3 Thus, photocaging has broad applications in the field of biochemistry and related fields. Photocaging of free amino acids and small peptides has been previous reported via chemical strategy.4-5 However, current methodologies have limited site-specificity in larger peptides or proteins, or only narrow scope to small peptides. There is a need for site-specific methods for photocaging of peptides and proteins.
Scheme 5.1. The installation of photocleavable reagent into peptide and protein via enzyme- mediated transamidation.
We envision that photolabile derivatives of the peptide or protein can be obtained through the methodologies described in previous chapters with high site-specificity, either enzymatically through transamidation by a transglutaminase (TGase) as illustrated in Scheme 5.1, or through nucleophilic trapping of labile succinimide as depicted in Scheme 5.2.
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Scheme 5.2. The installation of photocleavable reagent into peptide and protein via nucleophilic trapping of succinimide.
Further chemical modification of the photolabile group can also be performed to generate additional photo-releasable caged peptide or protein derivatives as shown in Scheme 5.3. A subsequent photolysis step releases the photolabile group and regenerates the original peptide or protein at a desired time and location.1 This chemo-enzymatic site-specific photocaging of peptides and proteins can have various applications, such as on-demand dosing or on-demand drug release, in vivo drug delivery system or biological probes.
Scheme 5.3. The generation of photolabile protein conjugation and the release of conjugation through photolysis.
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5.2 A platform for on-demand design of protein and peptide
Figure 5.1. The platform for hybrid modality engineering of protein and peptides
Hybrid modality engineering of proteins is not simply adding modifiers, it requires sophisticated design depending on the application. For example, some derivatizations are aimed at helping the active drug penerate specific barriers, such as the blood brain barrier or condensed cartilage, which requires the maintaince of the activity of target protein while adding alternative properties to help with the peneration.6-7 Otherwise, some derivatizations intent to targeted drug delivery usually requires the activity of drug to be temporarily blocked or the controlled release of the drug.8-9
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The ultimate goal of protein conjugation is to build up a generally applicable platform that employs mutiple derivatizaiton strategies, not limited to chemical or enzymatic site-specific methodologies, to accomplish the on-demand design of protein and peptides. i.e., based on the purpose, the structure, activity site, surface interference of target protein are taken into consideration to choose the best strategy to derivitaze a protein at the proper site and harness the desired functionality.
The application is then not limited to previously mentioned new drug candidates/conjugate development and biological probes, but also analytical aspects such as protein post translational modification analysis, biomaterial development, metastable drug delivery system design and photocleavable carrier, etc. as illustrated in Figure 5.1.
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5.3 References
1. Klán, P.; Š olomek, T. s.; Bochet, C. G.; Blanc, A. l.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J., Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chemical Reviews 2012, 113 (1), 119-191.
2. Tatsu, Y.; Nishigaki, T.; Darszon, A.; Yumoto, N., A caged sperm‐activating peptide that has a photocleavable protecting group on the backbone amide. FEBS Letters 2002, 525 (1-3), 20-24.
3. Lee, H.-M.; Larson, D. R.; Lawrence, D. S., Illuminating the chemistry of life: design, synthesis, and applications of “caged” and related photoresponsive compounds. ACS chemical biology 2009, 4 (6), 409-427.
4. Ramesh, D.; Wieboldt, R.; Billington, A. P.; Carpenter, B. K.; Hess, G. P., Photolabile precursors of biological amides: synthesis and characterization of caged o-nitrobenzyl derivatives of glutamine, asparagine, glycinamide, and. gamma.-aminobutyramide. the Journal of Organic Chemistry 1993, 58 (17), 4599-4605.
5. Hiraoka, T.; Hamachi, I., Caged RNase: photoactivation of the enzyme from perfect off- state by site-specific incorporation of 2-nitrobenzyl moiety. Bioorganic & Medicinal Chemistry Letters 2003, 13 (1), 13-15.
6. Moorman-Li, R.; Motycka, C. A.; Inge, L. D.; Congdon, J. M.; Hobson, S.; Pokropski, B., A review of abuse-deterrent opioids for chronic nonmalignant pain. Pharmacy and Therapeutics 2012, 37 (7), 412.
7. Chey, W. D.; Webster, L.; Sostek, M.; Lappalainen, J.; Barker, P. N.; Tack, J., Naloxegol for opioid-induced constipation in patients with noncancer pain. New England Journal of Medicine 2014, 370 (25), 2387-2396.
8. Li, J.; Yu, J.; Zhao, J.; Wang, J.; Zheng, S.; Lin, S.; Chen, L.; Yang, M.; Jia, S.; Zhang, X., Palladium-triggered deprotection chemistry for protein activation in living cells. Nature chemistry 2014, 6 (4), 352-361.
9. Spring, B. Q.; Abu-Yousif, A. O.; Palanisami, A.; Rizvi, I.; Zheng, X.; Mai, Z.; Anbil, S.; Sears, R. B.; Mensah, L. B.; Goldschmidt, R., Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates. Proceedings of the National Academy of Sciences 2014, 111 (10), E933-E942.
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