Chemical Synthesis of Peptides and Proteins: Solid Support

Transcription

1 Chemical ynthesis of Peptides and Proteins: olid upport PG Protected Amino Acid (PG = Fmoc, Boc, Cbz, etc.) Activation olid PG LG 2 upport Linker Basic Conditions PG Linker olid upport Polystyrene-based resins: ynthesize solid support, add linker, add first amino acid 1-2 % DBP or AIB particle size depends on agitation speed cross-linked polystyrene functionalize FG olid upport 1-2 % FG = C 2 X (X = Cl,, 2 ) FG DBP or AIB cross-linked polystyrene FG = -C 2 Cl, -C 2 FG PEG-FG = (C 2 C 2 ) n -C 2 -X These resins swell to 5 times their volume in non-polar solvents (TF, dioxan, DCM), but not in polar solvents (Me, Et). The swelling properties of the resin in polar solvents can be improved by adding polyethylene glycol (PEG-FG) chains, or by using polyacrylamide resins.

2 Chemical ynthesis of Peptides and Proteins: Linkers Linkers: Linkers are frequently used to control: 1) the functional group at the C-terminus (e.g. acid or amide), and 2) the conditions required for release of the peptide chain, which in turn determines whether side-chain protecting groups are removed or not. FG olid upport olid upport FG = -C 2 2, -C 2 Cl, -C 2 Linker The following resin linkers are used to produce C-terminal carboxylic acids: Cl Merrifield resin Me Cl Cl Wang resin Me 2-Chlorotritylchloride resin -PG Loaded resin: -PG -PG Me Me Cl -PG Cleavage conditions: F (for Boc) % TFA (for Fmoc) 5 % TFA (Fmoc) 0.5 % TFA (for Fmoc)

3 Chemical ynthesis of Peptides and Proteins: Peptide Cleavage C-terminal amides can be released from : -Peptide ame for: -Peptide -Peptide Me Me C 3 Anhydrous F for BC (very harsh) 95 % TFA for Fmoc (much more gentle) C 3 -Peptide Me Me Me -Peptide C 3 -Peptide Me 2 -Peptide 2 Me C 3 Me

4 Chemical Peptide ynthesis: Products of Peptide Cleavage -Peptide Peptide Me 95 % TFA Me Me Me C 2 Peptide -Peptide Me Me 95 % TFA 2 i Diethyl ether (to precipitate peptide) Peptide x TFA x=#basicgroups

5 Chemical ynthesis of Peptides and Proteins: First esidue ormal coupling conditions: 2 C, PyBP, DIPEA, DMF PG olid upport Facile 2 reaction: More difficult reaction: Cl -C, Cs 2 C 3,DMF (C) 2, DIPEA, DMF PG olid upport ynthesis of (C) 2 : PG 1 Protected Amino Acid C = cylclohexyl (DCC) = isopropyl (EDC) PG 1 C Determination of esin Loading : Calculated as moles per gram, method of determination depends upon identity of PG on the first amino acid. PG 1 Protected Amino Acid PG 1 1 ymmetric Anhydride "(C) 2 " GP C Urea

6 Chemical Peptide ynthesis: Example 1 From Prof. obinson s Lab: The aim was to prepare a fragment of the apical membrane antigen-1 (AMA-1) from the malaria parasite Plasmodium falciparum, as a potential malaria vaccine candidate to elicit an immune response against the parasite: 2 Me Fmoc-Gly- BTU/Bt, DIEA, MP Fmoc Gly Me Differences for BC chemistry 1. BC-Xaa- Me a) 20% piperidine, DMF b) Fmoc-potected amino acid BTU/Bt (4eq.) DIEA, DMF c) Ac 2, Bt, DIEA, DMF Me 2. esin must withstand TFA 3. TFA, not piperidine deprotection side chain protection: Boc (K), tbu (,D,E,Y), Pbf (), Trt (,Q,C) 4. ide chain protecting groups must withstand TFA Fmoc GGCYKDEIKKEIEEKIKLDDDEGKKIIAPIFIDDKDLKC G Me 5. Cleavage form resin in anhydrous F and p-cresol 1) TFA, ipr 3 i, 2 EDT (94:1:2.5:2.5) 2) I 2 in 80% Ac/ 2 ) Me Yield = 0.3 % (good yield ~30%) GGCYKDEIKKEIEEKIKLDDDEGKKIIAPIFIDDKDLKCG- 2 Can try BC chemistry: different resin, etc. r, can incorporate pseudoproline at problem spots

7 Chemical ynthesis of Peptides and Proteins: Monitoring Coupling eaction PG 2 Activation olid PG LG 2 upport Linker Protected Amino Acid (PG = Fmoc, Boc, Cbz, etc.) 1 Basic Conditions PG 1 2 Linker olid upport The identities of PG, LG, 1 and 2, and even the solid support can have a dramatic effect on the reaction rate and yield. Methods for monitoring the coupling reaction: 1. Chemical stains for free amines: 2 Linker olid upport PG Linker olid upport tain tain inhydrin Fluorescamine Colored / fluorecent resin or solution o color change 2. Measuring conductivity or UV-absorbance of subsequent deprotection solution: PG Linker olid upport Deprotect aromatic PG alt 2 Linker olid upport

8 Chemical Peptide ynthesis: Example 1 Fmoc Gly 2 Me Fmoc-Gly- BTU/Bt, DIEA, MP Me Me Me a) 20% piperidine, DMF b) Fmoc-potected amino acid BTU/Bt (4eq.) DIEA, DMF c) Ac 2, Bt, DIEA, DMF 120 elative Abs (313 nm) Amino acid position

9 Chemical ynthesis of Peptides : Problem equences 3 C 3 C C 3 C 3 3 C 3 C Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- 2 3 C C C 3 C C 3 C Aggregation, secondary structure, folding, collapse, etc 3 C 3 C C 3 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- Xaa- 3 C Xaa- C 3 Xaa- 3 C Xaa- 2 -Xaa- Xaa- Xaa- Xaa- By incorporating pseudoproline dipeptide units at the appropriate place in a "difficult" sequence, problems associated with peptide chain aggregation can sometimes be eliminated. Fmoc- 1 Peptide ynthesis Peptide- 1 Peptide 95 % TFA Peptide- 1 Peptide = any 2 =, C 3 Limited to Xaa-er and Xaa-Thr

10 Chemical Peptide ynthesis: A Brief istory 1901: Fischer's synthesis of glycyl glycine is the first reported synthesis of a dipeptide and the first use of the term "peptide" to refer to a polymer of amino acids. (Ber. Deutsch. Chem. Ges. 1901, 34, 2868). 1907: Fischer's synthesis of an octadecapeptide consisting of 15 glycines and 3 leucines, but without control of the amino acid sequence. (J. Chem. oc. Chem. Comm. 1907, 91, ). Cl xytocin:. Kunz, Angew. Chem. Int. Ed. 2002, 41, : Introduction of the benzyloxycarbonyl (Z) group for α-amino protection by Max Bergmann. (Ber. Deutsch. Chem. Ges. 1932, 65, ). 1953: olution phase synthesis of the octapeptide hormone oxytocin by du Vigneaud and coworkers. (J. Am. Chem. oc. 1953, 75, ). 1963: The first stepwise solid-phase synthesis of a peptide by Bruce Merrifield (J. Am. Chem. oc. 1963, 85, ; cience, 1986, 232, 341).

11 Chemical Peptide ynthesis: A Brief istory 1966: The first automated machine for peptide synthesis by Merrifield. (Anal. Chem. 1966, 38, ). 1969: The first synthesis of an enzyme: ribonuclease A by Merrifield and Gutte, and ase by alf irschmann and co-workers. (J. Am. Chem. oc. 1969, 91, 501-2; J. Am. Chem. oc. 1969, 91, 507) 1978: Introduction of the -(a)-fluorenylmethoxycarbonyl- (Fmoc-) protecting group for solid-phase peptide synthesis. (J. Chem. oc. Chem. Comm. 1978, 537-9; Int. J. Pept. Prot. es. 1978, 11, 246-9). 1989: The chemical synthesis and crystal structure of the IV-1 protease. (A. Wlodawer et. al., cience 1989, 245, 616). 1994: Introduction of native chemical ligation for coupling unprotected peptide fragments and so allowing routine access to synthetic proteins. (cience, 1994, 266, ) : The first peptide-based drug manufactured on a multi-ton scale - the anti-iv drug T20, or Fuzeon, or Enfuvirtide. (M. C. Kang et al., (2000) U Patent 6,015,881; see also B. L. May, ature evs. Drug Disc. 2003, 2, ).

12 Chemical ynthesis of Peptides: Modified esidues osphorylation is one of the most common post-translational modifications: % of all proteins phosphorylated P osphorylation site(s) on protein surface erine (Thr, or Tyr) Kinase, ATP, Mg 2 osphatase osphoserine Each kinase has loose consensus sequence, ex. PKA phosphorylates (/K)-(/K)-X- Kinases account for ~2% of the human proteome. ynthetic monomers for the synthesis of phosphopeptides and proteins: For sulfopeptides: P P P P - (nbu) 4 Fmoc- Fmoc- Fmoc- Fmoc- Fmoc- Fmoc-osphoserine(Bzl) Fmoc-osphothreonine(Bzl) Fmoc-osphotyrosine(Bzl) Fmoc-osphotyrosine Fmoc-ulfotyrosine

13 Chemical ynthesis of Peptides: Modified esidues ome post translational modifications are highly complex and not fully amenable to standard solid-phase synthesis: Glycopeptides and Proteins Approx. 50% of all proteins are glycosylated. Essential for proper folding of certain proteins, as well as cell-cell communication. (er or Thr) Variable linkages and monosaccharide units allow many glycoforms of the same protein. This often complicates biological production and isolation. Asn-Xaa-(er/Thr/Cys)

14 Chemical ynthesis of Peptides: Modified esidues Although the -glycosidic and interglycosidic linkages are acid labile, the glycosidic linkages of fully acylated carbohydrates can withstand a short treatment with TFA. ence: Ac Ac Ac Ac Fmoc- =, C 3 PP TFA a Ac C 3 Ac-er-Tyr-Pro-Thr-er-Pro-er-Tyr-er- 2 In eukaryotes, one of the most prevalent types of -linked glycosylation is where -acetylgalactosamine (GalAc) or - acetylglucosamine (GluAc) is linked in the α-anomeric configuration to the ß-hydroxy group of er or Thr. Formation of the - glycosyl amino acid requires a suitably protected monosaccharide, with a leaving group at the anomeric position: X = alogen (Cl) X = - X= CCl 3 (trichloroacetimidate) X Activator '- ' ' ote that α- and ß-glycosides may be produced, depending upon the starting materials and conditions. But when a neighbouring group is present that can participate in stabilizing the intermediate carbocation, then often the 1,2-trans-glycosides (ß-gluco) anomer is preferred: X Me Me Me Ac ' Ac preferred ' Minor product Main product

15 Chemical ynthesis of Peptides: Modified esidues For the synthesis of more complex glycopeptides, it is an advantage to use a relatively simple glycosyl-amino acid, and then to elaborate the glycopeptide enzymatically, e.g.: UDP-Galactose P P CMP-ialic Acid 2 Ac P C 2 GDP-Fucose P P 2

16 Chemical ynthesis of Peptides: Modified esidues For the synthesis of more complex glycopeptides, it is an advantage to use a relatively simple glycosyl-amino acid, and then to elaborate the glycopeptide enzymatically, e.g.: The glycopeptide can then be made into a glycoprotein using native chemical ligation: But how is the C-terminal thioester made? For proteins: see inteins..

17 Chemical ynthesis of ite-pecifically Modified Proteins We can use native chemical ligation (review previous slide and homework) to fuse peptide fragments into proteins, this requires an -terminal Cys and a C-terminal thioester which can be prepared according to the choice of linker: From previous examples, one might think the following is a good route: This has motivated the development of a safty catch resin: Peptide 2 95 % TFA Peptide Br-C 2 - Peptide C 2 PyBP/DIPEA Peptide synthesis Me Me owever, thioesters are not very stable in the presence of primary amines: -PG Alkylation: (ex. IC 2 C) -Peptide 2 Me Me ucleophile: (ex. 2 -) -Peptide Me Me -PG 2 -Peptide

18 Chemical ynthesis of ite-pecifically Modified Proteins This has motivated the development of a safty catch resin: By modifying this approach, the same resin can be used to make cyclic peptides: 2 C 2 PyBP/DIPEA Peptide synthesis "Backbone" Cyclic Peptide -Peptide Alkylation: (ex. IC 2 C) Peptide- 2 Deprotect -Peptide

19 What To Do With Modified Peptides and Proteins? Biophysical Analysis (in vitro): tructure (M, X-ray crystallography, CD) tability Dynamics Biological Analysis (in vitro or in cell lysates): Enzymatic activity Identification of binding partners Inhibition of binding interactions ignal transduction Biological Analysis (in living cells): Enzymatic activity? Identification of binding partners Inhibition of binding interactions ignal transduction Drug-like properties Biological Analysis (in animals): Drug-like properties Immunological response ow do each of these properties change upon site-specific modification (glycosylation, phosphorylation, etc.)? equires cellular uptake of modified peptides and proteins. Alternatively, these studies can be enabled by site-specific modification of proteins produced by the cell.

20 Labeling of Proteins with Fluorescent Probes In Vitro ere are some very common methods involving residue-selective electrophiles: C 2 C 2 I iodoacetamide succinimidyl ester (p = 7-8) 2 (p = 8-10) _ C 2 _ C 2 Angew Chem Int Ed Engl. 2009, 48,

21 Methods for Labeling Proteins in Vivo Method 1 Genetic engineering to introduce new sequence into a fusion protein which can be detected directly: Genetic fusion (like GFP) Any protein Method 2 Introduction of modified metabolite that is incorporated into protein: * Any protein * Method 3 Combination of Methods 1 and 2 where fusion proteins are subsequently modified: Any protein * Any protein * Method 4 Use of modified enzyme substrates for covalent enzyme modification followed by ligation with reporter molecule.

22 Methods for Labeling Proteins in Living Cells: Method 1 4-ydroxybenzylidene-imidazolidone Variable Fluorescence Properties In vivo imaging Disadvantages Q = ε = 32, ,000 cm -1 M -1 τ = ~3 ns equires transfection of DA Large size (238 amino acids) low fluorophore maturation Multimerization ature Biotechnology 1999, 17, 969.

23 Methods for Labeling Proteins in Living Cells: Method 2 Introduction of modified metabolite that is incorporated into protein: * Any protein * In addition to chultz s approach (review Expanded Genetic Code ), a number of other examples have been reported: 3 2 Media depleted of Met 3 All proteins 3 3 Azidohomoalanine Bacteria C C / Cu(I) Azido groups (- 3 ) and terminal alkynes are examples of bioorthogonal functional groups. All proteins

24 Methods for Labeling Proteins in Living Cells: Method 2 Introduction of modified metabolite that is incorporated site-specifically into a protein using the expanded genetic code approach, followed by a bioorthogonal chemical reaction : * expanded genetic code Any protein * bioorthogonal reaction Any protein * Metal-Catalyzed eactions Metal-Free eactions Biomolecule Biomolecule Biomolecule C C I - 3 Biomolecule Cu(I) Biomolecule Cu(I) B Pd(0) Biomolecule Biomolecule Biomolecule P Biomolecule P Biomolecule Biomolecule I Pd(0) Biomolecule Biomolecule Biomolecule - 2

25 ther Examples of Bio-orthogonal Functional Groups ketone hydrazide expanded genetic code ketone alkoxyamine expanded genetic code

26 Methods for Labeling Proteins in Living Cells: Method 2 An example using modified saccharides is from Bertozzi s group: Ac Ac Ac Ac Media lacking ManAc 3 uman cells Capture with.a. agarose beads BITI P 2 BITI C 2 - P 2 CELL UFACE 3 C 2 - The azide-modified ManAc was converted into azide-modified sialic acid units expressed on cell surface proteins

27 Methods for Labeling Proteins in Living Cells: Method 3 Combination of Methods 1 and 2 where fusion proteins are subsequently modified: * Any protein * The first example utilizes native chemical ligation: Met Cys Protease site Any protein Express Protease Any protein The second example utilizes thiol-arsenic exchange chemistry: - As As - C 2 -CCPGCC- Cys 4 protein FlAs B. A. Griffin,.. Adams,. Y. Tsien, cience. 1998, 28,

28 Methods for Labeling Proteins in Living Cells: Method 3 The second example utilizes thiol-arsenic exchange chemistry: As As - eas FLCCPGCCMEP ptimized Cys 4 motif ela cells expressing tetracysteine-fused connexin were treated with FlAs (green), incubated in medium for 4 hours, then treated with eas (red) and imaged. This two-color pulse-chase labeling experiment demonstrated that newly synthesized connexin is incorporated at the outer edges of existing gap junctions (indicated by white arrows).

29 Methods for Labeling Proteins in Living Cells: Method 4 The last example utilizes biotin, a 15 residue biotinylation sequence and E. Coli biotin ligase (BirA): 2 Any protein GLDIFEAQKIEW Biotin BirA ATP BirA - P 2 Any protein GLDIFEAQKIEW K eq = ~10 14 Labeled streptavidin Any protein GLDIFEAQKIEW

30 E. Coli biotin ligase (BirA): Methods for Labeling Proteins in Living Cells: Method 4 ther examples: 2 Any protein GLDIFEAQKIEW Any protein GLDIFEAQKIEW Angew Chem Int Ed Engl. 2009, 48,