Introduction

The

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University of Aberdeen, The School of Natural and Computing Sciences

Assembly-assisted peptide ligation in native conditions.Protein Synthesis

Author: Mikolaj ZabrockiSupervisor: Dr. Laurent TrembleauAssessor: Dr. John Plater

25 April 2014

following study focuses on exploring opportunities in peptide and protein synthesis, with a

particular attention to peptide bond formation in native conditions. Proteins and peptides are

built up of amino acids – primary metabolites with neighbouring amino and carboxylic acid

groups, being an essential building material for living organisms. Their polymers are called

peptides (up to 100 amino acids) and proteins above this length. The characteristic feature

deciding of their activity is their secondary and higher order structure – folding of the linear

chains according to the intramolecular interactions between various amino acids involved.

Peptides are products of multiple peptide bond formations – process that has a certain

complexity to it. Yielded predominantly by dehydration between an amine and a carboxylic

acid groups [fig.1], peptide bond involves a chiral center and, in case of multiple group

availability – which must be accounted for in the case of at least two-fold chemical

functionality of each amino acid – may give more than one possible combination product.

These issues are some of the concerns relevant to the synthetic peptide bond formation (called

“ligation” afterwards).

Several methodological approaches to ligation have emerged since the breakthrough of

Merrifield who introduced solid-phase synthesis to construct complex organic molecules from

simple building blocks (e.g. amino acids). This method uses polymeric beads (“resin”) as a

support binding the first amino acid usually at its C-terminus, and following amino acids of

the peptide (called “residues” exchangeably) are attached sequentially at the following N-

termini. For that to happen the terminal carboxylic group at attaching amino acids must be

activated which is achieved by special a reactant called coupling reagent. To assure selectivity

of activation and attachment, protecting groups removable under defined conditions are bound

to these sites which are to remain unreacted. This method quickly replaced solution synthesis

due to, the much simplified and more efficient purification and retrieval, as well as attachment

of following amino acids (called “coupling” afterwards) steps all due to better control in

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handling of the product anchored on the large resin particles. It did not, however, have an

influence on the achievable peptide length which remained around 50 residues.

This threshold was surpassed by convergent synthesis of peptides using Dawson's Chemical

Ligation. The selectivity of this method relies on a selective activation of terminal carboxylic

acid into a thioester. Then such thioester terminus is capable of reacting with sulphide on

cysteine yielding thioester connection between peptides. [fig.2] Even though the bond is easy

to selectively form and the product presents properties majorly comparable to the peptide

bonded (native) variant, it requires presence of a sulphide instead of an amine group, which

limits the method to applications involving cysteine. Later observation that the said thioester

is capable of exchanging with neighbouring amino group led to development of Native

Chemical Ligation, yielding peptides and proteins without any odd bonds. Furthermore, a

catalytic desulphurization reaction have been designed to remove sulphur, tranforming the

cysteine into an alanine expanding the pool of achievable peptides. This reaction is, on the

other hand, non-specific, giving a product ridden of any cysteine residues. Finally the reaction

of carboxylate into a thioester is a process requiring a specific chemistry that may be

incompatible with some peptides.[1][2]

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fig.1 On the left: a generic structure of naturally significant L or S enantiomer amino acid.

On the right: a general reaction of peptide bond formation. Typically occurs with additional

carboxylic acid activation.

The available methodology gives access to synthesis of a wide spectrum of natural peptides

and proteins longer than 200 amino acids. [1] Additionally, it also allows their modification

by introduction of non-proteogenic and hindered amino acids or isotopic labels. It is possible

due to discoveries of new coupling reagents. Another advancement in the field of coupling

reagents is identification of the racemization suppressants. Yet, contemporary protein

synthesis still faces many challenges. Some of them are overcoming the necessity of cysteine

or alanine presence in the final product, or simplified synthesis of even longer chains. [2][3]

Native conditions are conditions allowing the intramolecular interactions within proteins like

they occur in physiological environment. Particularly important factors are warm temperature

(ideally approx. 37oC) and mild pH (often above 7) in aqueous solution. Conversely, a key

element of the contemporary total protein synthesis are denaturing conditions. Some organic

solvents like dimethylformamide (DMF) and altered pH may prevent folding of the chains up

to a certain length assuring control over the peptide secondary structure, and the reaction site

availability. However, there are several discoveries pointing towards a potential of the native

conditions in ligation of assembled peptides. The phenomenon of protein fragment

complementation is described as capability of the protein fragments to assemble - with or

without assistance of peripheral groups - into protein-resembling clusters by means of non-

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fig.2 General mechanisms of chemical ligation (left) and native chemical ligation (right)

Picture from: [2]

covalent interactions. In the cases of smaller, simpler proteins, the assemblies of their

fragments may even show functionality corresponding to whole protein. [4] The phenomenon

occurs in condition allowing the natural intramolecular interactions – native conditions. So far

it found analytical and biochemical applications but it is also interesting from the synthetic

point of view.

Understanding the principle of shape attainment without a necessity of chain continuity

implies that in native conditions the termini of assembled peptides may be spontaneously

brought into a close proximity. Provided they occur on the protein surface in an accessible site

an attempt of their ligation would be possible. Farther studies confirmed that, indeed

proximity of the termini enhances the ligation rate and, moreover, possibly occurs without the

need of cysteine as N-terminus stepping beyond the scope of native chemical ligation.[8]

Furthermore there are some reasons to hypothesize, that the thioester formation step may not

5

fig.3 Two halves of modified zif268 structure. a) termini at wich ligation was designed to

occur. b) site of zinc ion complexing. C) the synthesised half with following amino acid

sequence: RHVDQHRSLASKG

a)

b)

c)

be necessary. While the coupling reagents are non-selective in carboxylic acid activation, they

are also prone to hydrolysis in aqueous environment – the reason why they are usually applied

in an organic solvent. However, this means that in native conditions the non-selective

activation is going to be temporary due to the ongoing hydrolysis, for period perhaps

sufficient to allow a reaction with approximate amine, especially when an excess of coupling

reagent is provided. The required proximity is achievable in native conditions if the peptides

to be ligated are complementing fragments. However subtle an idea, this process is a good

explanation of what was observed in some of the studies on cleaved proteins.

Phenylmethylsulfonymfluoride (PMSF) is a protease inhibitor, that in a biological experiment

was also observed, to exhibit an activity of coupling reagent on some assembled peptides

causing religation of proteins locally cleaved by a protease enzyme. [5] It is one coupling

reagent that this project aimed to study. In an another experiment a triose phosphate isomerase

(TIM) protein was similarly cleaved by the same protease - subtilisin Carlsberg, and in this

case efficiently religated by the protease itself given particular conditions. [6] This points

towards the possible utility in coupling of assembled peptides, of enzymes like ligases or

perhaps the PatGmac protease shown recently to ligate neighbouring termini of a peptide

resulting in cyclization[7]. There is need for further studies in the area to identify the degree

of fragmentation allowed, and strength of interactions required for a successful assembling,

coupling reagents and the exact conditions optimal for this kind of reactions.

To put the proposed assembly-assisted ligation in native conditions into test a convergent

synthesis involving such ligation step of small proteins like crambin [9] or 7HVP HIV

protease were considered due to their reasonable size and strong intramolecular interactions.

Finally it was decided upon a modified structure of Zif268 (fig.4) a Cys2His2 zinc finger

peptide instead, due to simplicity and material availability. It was predicted that the assembly

of two halves: would be assisted by presence of zinc cations and provide the proximity of

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termini. (fig.3) To test possibility of full convergent synthesis two experimental steps where

designed: firstly traditional solid-phase synthesis of the two halves of the zinc finger complex,

secondly attempt of assembly-assisted ligation in native conditions with PMSF as the

coupling reagent.

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a)

b)

c)

Another similar function of the coupling reagents is cyclisation of peptides. If peptide folding,

possibly combined with its short length, provides a similar termini proximity to that observed

in a protein fragment complementation, the coupling reagents of similar qualities are required

for their successful ligation. For sake of that resemblance, and much simplified experimental

design some intramolecular ligations were examined as well. These were cyclization of

ornithine, a non-proteogenic amino acid and assisted lasso-cyclisation of Microcin J25

(McJ25) precursor. (fig.4)

The first reaction in concern is a cyclisation of L-ornithine, a non-proteogenic aminoacid

capable of forming a six-membered lactam ring on dehydration. It was used to test HBTU

which is an uronium coupling reagent active in denaturating conditions[3] and 1-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC) a water-soluble coupling reagent[3]. In this study

their activity was examined in an aqueous solution (with an addition of methanol for HBTU to

facilitate its dissolution). Important matter of interest other than understanding their activity in

native conditions is observing whether the coupling reaction would occur at rate sufficiently

higher than the hydrolysis of activated amine and the coupling reagent itself to observe

product formed in a decent amount. Another issue is verification if the selectivity of the

cyclisation over oligomerisation can be observed.Microcin J25 is a peptide consisting of 21

residues produced by some of the Escherichia coli bacteriae, exhibiting some antibacterial-

properties. It consists of 21 amino acids (fig.5) and is not cyclic as initially predicted, but

exists in a somewhat complex “lassoed tail” structure. [10] The center of the peptide attains a

half-fold hairpin conformation and it is the predominant structure of the McJ25 precursor

which deprived of the 'lassoed tail'. It is worth pointing out that the middle of the peptide, or

“U-turn” of the hairpin, involves a particularly hydrophiobic set of amino acids. It is hence

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fig.4 Summary of reactions carried out in the project. a) ligation of modified zif268

structure (dotted line) b) cyclization of ornithine c) cyclization of McJ25 precursor, either

lasso-cyclization (smaller dotted curve) or head-to-tail cyclization (large dotted curve)

hypothesized that the precursor exhibits surfactant properties. A solubility test was conducted

to confirm that. It is hypothesized that in the presence of another surfactant above the critical

micelle concentration (CMC) the hairpin conformation would be enhanced if the hydrophobic

region enters said micelles. The reaction site on their surface would then exposed to the

solvent with EDC improving reaction selectivity. Hence experiments in an aqueous solution

and with surfactant presence were designed aiming to observe surfactant-assisted lasso-

cyclisation.

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fig.5 Structure of examined McJ precursor peptide. The aminoacid sequence as follows:

GGAGHVPEYFVGIGTPISPYG a) Site of head-to-tail cyclization yeialding natural

peptide. b) hydrophobic region with two isoleucines.

a)

b)

Results and discussion

Zinc finger peptide

The histidine half of Zif268, whilst successfully synthesized and precipitated as a fine white

powder that turned into yellow, sticky granules upon drying, was not of a satisfactory quality,

to continue with the assembly and ligation steps. All couplings were deemed successful as

confirmed by the Kaiser tests, with a peculiarity observed in some of the last couplings when

the test after positive result on heating was becoming dark blue after hours of storage in room

temperature, but that is considered insignificant. In the product, the peptide was identified as a

doubly charged ion molecule with 745.9001m/z and the characteristic isotopic pattern in the

LCMS data [app.A] at retention time between 6 and 8 minutes. Unfortunately, the low yield

of 34% combined with low peptide content – present amongst minor peaks – implied a

significant contamination and small total quantity of the peptide. There is however a result

promising for confirmation of the hypothesized spontaneous assembly of the peptide from

two halves in the future. That is identification of a peak corresponding to the triply charged

complex of the peptide with a zinc cation in the same LCMS spectrum, eluting at a similar

time and matching exactly the predicted mass to charge ratio at 518.2467m/z, but with

distorted isotopic pattern [app.A]. Conclusion that even one half of the modified zif268

structure, even when present in a low concentrations, is capable of complexing the zinc ions

in solution – where they were not introduced on purpose in a significant concentration – at a

noticeable ratio, makes the perspective of full assembly more viable.

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Ornithine

The predicted cyclization mediated by HBTU occurred, however not very efficiently as it

appears in LCMS results. The compounds eluted early, sharply at 0.84 minute [app.B] and

admittedly little of the desired lactam can be observed at 115.0867m/z and far more of the

unreacted ornithine with 133.08971m/z. [app.C] The low efficiency, may be due to apremature

hydrolysis of HBTU. There seems to be a trace of it eluting after 6 minutes in a peak with

234.1423m/z of a barely detectible intensity. [app.A] On the other hand the ornithine dimer*

was not detected neither singly charged with 247.18m/z nor doubly charged with 124.09m/z,

which gives an evidence of reaction selectivity.

A similar, comparison experiment was carried out as part of another project on PyBOP, an

organophosphonium reagent, but no reaction was observed in this case.[3]

Less conclusive results gave the test of EDC tracked by H1 NMR. The most noticeable

differences between the reactnats [app.H] and the products [app.J] are residues of EDC

hydrolysis at 0.95ppm, 2.75ppm and in the higher order spin system around 3ppm, since they

are already emerging before the coupling reagent activation by lowered pH [app.G]. The

hydrogens of ornithine that would be most affected in the reaction, would be these next to the

amine group taking part in the lactam formation: the peak around 2.90ppm. If a cyclic product

is formed a deshielded equivalent of that triplet would appear after a time for the reaction is

given [app.J] and it could be speculated to be some of the trace peaks around 3.45 or 3.85, but

there is not enough evidence to support that suggestion.

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Microcin J25

In this experiment a linear precursor of McJ25 prepared in course of some previous laboratory

projects is used. In the test of solubility in water McJ25 precursor formed a suspension not

prone to sedimentation – a colloid. This confirms its surfactant properties: a strong

hydrophilic nature counterbalanced by some hydrophobic interactions preventing from

complete dissolution. With addition of some Soduim dodecyl sulphate (SDS) - another

surfactant, the mixture becomes clear. In spite of this promising interaction between

surfactants observed, the results of coupling could not completely confirm predictions. Firstly

it is worth noticing that the LCMS results of the precursor in water [app.D] before the reaction

already suggest trace presence of cyclized peptide. Even though the peak proportions seem to

be distracted, possibly by the presence of some heavy water in the solution. The larger set can

be matched with doubly charged precursor of 1063.53m/z and the accompanying group of

peaks corresponds to 1054.52m/z predicted for the cyclized peptide, both with isotopic

intervals of 0.5m/z which is appropriate for doubly charged molecules. Corresponding peaks

can be identified in LCMS results for the sample including reaction with EDC in water,

[app.E] with the difference in their relative intensity. That could be caused by the precursor

decreasing in concentration while reacting into oligomers, however the shape of peaks

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fig.6 Ornithine dimers.

Their precence amongst

products of ornithine

ligation would imply that

the reaction is not

selective for cyclisation,

and the coupling reagent

in unsuitable for proposed

assembly-assisted peptide

ligation in native

conditions.

corresponding to the cyclized product suggests otherwise. In reactant spectrum they are very

flat but become sharp after the reaction giving an evidence of cyclization occurrence.

Unfortunately, the LCMS data obtained from the sample of the reaction carried out in

presence of SDS [app.F] does not include the peak set recognized in the former two. Absence

of the peaks could be caused by the strong interactions between the peptide and surfactant

influencing the results, and making the analytic method unreliable. However, the H1 NMR

results do bear some evidence of the same reaction occurring. For the reaction in absence of

SDS the region undisturbed by the oversaturated EDC peaks between 5.5ppm and 9ppm

shows certain change of peak pattern from unreacted [app.K] to reacted [app.L] precursor

sample. A strikingly similar pattern shift can be observed for the reaction in presence of SDS

correspondingly [app.M] and [app.N]. This confirms that the same reaction would occur in

presence of SDS despite the lack of evidence from LCMS results. Although the reaction is

confirmed to be successful its full characterization cannot be concluded. Firstly, the

selectivity cannot be confirmed since the presence of oligomers cannot be ruled out, because

their mass to charge ratio falls beyond the measurement range. Similarly, there is no evidence

for or against the hypothesized facilitation of the reaction in presence of another surfactant.

Finally the nature of reaction cannot be deduced from obtained data since the analysis carried

out does not distinguish between the lasso and the head-to-tail isomers. The crystallization

carried out to obtain MCJ25 precursor crystals for a structural analysis was successful. A

sample was submitted and the results are due in to be obtained.

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Experimental

Zinc finger peptide

The first experiment consisted of two parts first one being a solid-phase synthesis of the 13

amino acid dihistidine half of modified Zif268. Second part was intended to involve a test of

PMSF as a coupling reagent for the hypothesized assembly-assisted ligation in native

conditions of the zinc finger structure however, due to an unsatisfactory quality of the product

from first stage and the time constrain this step of the experiment was not conducted. The

sequence (fig.3) was synthesized starting from arganine on the surface of 0.4g of NOVASyn®

TGA 90 µm resin bonding by an ester linkage. All the steps were carried out in a syringe with

a filter which simplified changing of reaction environment, and rinsing and retrieval of the

product, which remained in the syringe at all times until the last step: cleavage. Attachment of

the first amino acid was carried out according to a standard procedure from the manual [11] of

the resin manufacturer, while the choice of resin and the details of following coupling steps

were decided upon basing on optimum results achieved in previous in-house experiments. All

the protected amino acids and the resin were manufactured by Novabiochem®. The solvent

used as an environment for the reaction and for rinsing between steps was DMF. Deprotection

of the Fmoc-protected N-termini was achieved in basic conditions (addition of 20%

piperidine), and the following residue attachments in proportion of 5 equivalents per mol of

total resin loading, were carried out mediated by 5 equivalents of the HBTU coupling reagent

operating in basic conditions provided by 10 equivalents of N,N-Diisopropylethylamine

(DIEA). All the reactions and quadruple rinsing steps with DMF in between were carried out

with 4 ml of the solvent. Success of each coupling was confirmed with Kaiser test.[11] Finally

the peptide unattachment and full deprotection were carried out according to a standard

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procedure from Applied Biosystems Technical Bulletin[13] and relied upon acidic conditions

obtained by addition of some tetrafluoroacetic acid (TFA) spiked with water phenol and TIPS.

The solvents were evaporated and the product was precipitated in diethyl ether and dried over

vacuum. A sample dissolved in methanol was analyzed by Liquid Chromatography – Mass

Spectrometry (LCMS).

Ornithine

Approximately 100mg of L-ornithine was dissolved in 5ml of water and approximately 2

equivalents of HBTU were added. pH of the solution was adjusted to 6.8 with NaOH, and

approximately 3ml of methanol added to facilitate the HBT dissolution, which was not

completely achieved. The solution was agitated for two hours and then stored overnight

before a sample was submitted for the LCMS analysis.

The experiment with EDC was carried out in NMR tubes what allowed to track its progress

with H1 NMR starting from pure reactant: around 10mg of ornithine in 0.6ml of deuterated

water. Then the reaction solution was analysed with added 1.5 equivalents of EDC, but before

pH adjustment from 7 to below 6, then afterwards and finally after a night allowed for the

reaction to occur.

Microcin J25

The hypothesized surfactant properties are verified testing how does the precursor behave

when mixed with water, and how does the mixture change with addition of some SDS – a

known surfactant. The reaction itself is carried out in 0.6ml of deuterated water.

Approximately 7 equivalents of EDC were used in two separate, parallel reactions to compare

ligation of 5 mg of the precursor with and without presence of approximately 10 mg of SDS,

which is 16,7g/L – equal to its CMC. It is expected, that the formation of the natural “lassoed

15

tail” structure would be facilitated in the presence of SDS micelles. The NMR measurements

were carried out on the peptide in water and after the addition of SDS as blanks, and then in

an hour after addition of EDC to each. A sample of the peptide and each reaction was also

diluted in water and submitted for LCMS analysis. Meanwhile some of the McJ25 precursor

was dissolved in methanol and left for crystallization with dichloromethane.

Summary

The project did not strictly accomplish many of its posed aims, but it gave some support to

proposed hypothezes, and presented some perspectives for future research. The zinc finger

peptide was synthesized in poor quality, which might be due to the numerous basic residues it

includes, that are capable of interacting strongly to fold the peptide despite the denaturing

conditions if an inappropriate resin or reagents are used. Repetition of this scheme in search

of better method is recommended given the observed strength of the peptide affinity for zinc

that is a promising result for a successful zinc finger structure assembly in the second step.

The cyclization of ornithine with HBTU confirmed expected outcomes, whlist it was not very

efficient. A better efficiency could possibly be obtained by using larger excess of the coupling

reagent. Although this could negatively influence the selectivity of reaction with approximate

amine, especially in ornithine being a small molecule with accessible reaction site, it may be

less of a concern for possible applications in ligation of large and hindered peptide assembles.

Much less can be said about the same reaction mediated by EDC. Given previous succes with

a similar reaction on gamma aminobutyric acid forming five-membered lactam ring, much

seems to suggest that acidified pH is crucial for the EDC to act in aqueous solution.

Finally there is some evidence for succesful McJ25 precursor cyclization, which could serve

to support the effectiveness of the proposed mechanism. However, to make a convicted

statement about the selective EDC activity in water or the influence of added surfactant on the

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reaction path either a different experimental method or analytic technique would be necessary

to verify possibility of oligomerisation and receive a relaible measurement of the reaction in

presence of added surfactant.

References

1. “Expressed protein ligation: Method and applications”, R. David, M. P.O. Richter, A.

G. Beck-Sickinger, Eur. J. Biochem., 2004, vol. 271, pg 663–677

2. “Total chemical synthesis of proteins”, S. B. H. Kent, Chem. Soc. Rev., 2009, vol. 38,

pg 338–351

3. “Recent development of peptide coupling reagents in organic synthesis”, S. Han, Y.

Kim, Tetrahedron 60, 2004, pg 2447-2467

4. “Exploring bioanalytical applications of assisted protein reassembly”, S. K. Deo, Anal.

Bioanal. Chem., 2004, vol. 379, pg 383–390

5. “Conformationally Assisted Protein Ligation Using C-Terminal Thioester Peptides”,

Gangamani S. Beligere and Philip E. Dawson, J. Am. Chem. Soc., 1999, vol. 121, pg

6332-6333

6. “Serine protease inhibitor mediated peptide bond re-synthesis in diverse protein

molecules”, A. Sharma, K. V. R. Kishan, FEBS Letters 585, 2011, pg 3465–3470

7. “Rapid and Efficient Resynthesis of Proteolyzed Triose Phosphate Isomerase”, K.

Vogel and J. Chmielewski, J. Am. Chem. SOC., 1994, vol. 116, pg 11163-11164

8. “The mechanism of patellamide macrocyclization revealed by the characterization of

the PatG macrocyclase domain”, J. Koehnke, A. Bent, W. E. Houssen, D. Zollman, F.

Morawitz, S. Shirran, J. Vendome, A. F. Nneoyiegbe, L. Trembleau, C. H. Botting, M.

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C. M. Smith, M. Jaspars, J. H. Naismith, Nat. Struct. Mol. Biol., 2012, vol. 19, pg 767-

772

9. “Total Chemical Synthesis of Crambin”, D. Bang, N. Chopra, S. B. H. Kent, J. Am.

Chem. Soc., 2004, vol. 126, pg 1377-1383

10. “Structure of Microcin J25, a Peptide Inhibitor of Bacterial RNA Polymerase, is a

Lassoed Tail”, K. Wilson, M. Kalkum, J. Ottesen, J. Yuzenkova, B. T. Chait, R.

Landick, T. Muir, K. Severinov, S. A. Darst, J. Am. Chem. Soc., 2003, vol. 125, pg

12475-12483

11. Novabiochem® Catalog 2006/2007, Merck Biosciences

12. “Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase

Synthesis of Peptides”, E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Short

Communications, 1969, pg 595-598

13. Technical Bulletin, “Cleavage, Deprotection, and Isolation of Peptides after Fmoc

Synthesis”, Applied Biosystems

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Appendix A

20

Appendix B

21

Appendix C

22

Appendix D

23

Appendix E

24

Appendix F

25

Appendix G

26

Appendix H

27

Appendix I

28

Appendix J

29

Appendix K

30

Appendix L

31

Appendix M

32

Appendix N