Copy of Topic 3.2 - Amino Acids, Peptides, and Proteins Part 2 PDF

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ORGO TOPIC 3.2: AMINO ACIDS, PEPTIDES, AND PROTEINS PART II LAB SYNTHESIS OF PEPTIDES Synthesizing Peptides - Why synthesize peptides? o Can be used as drugs o Allows us to modify proteins/enzymes and study how they work - Making a peptide is synthetically challenging; they all have the same terminal functional groups, so how do we get them in the correct order? o We also need to consider that side-chains can react (but we won’t focus on this for the sake of the course) - To prevent undesirable reactions from taking place, we use protecting groups o Convert the functional groups to something less reactive (temporary) o Squared groups on the right are the ones we need to protect METHOD 1: Solution Phase Synthesis - Choice of protecting group depends on: o Type of functional group we want to protect o Conditions under which the groups are stable o Conditions used to remove the protecting group  cannot be too strong or else it will destroy the product a) Protection and Deprotection of COOH - We can protect the COOH group by converting it to an ester o Most common choice is the benzyl ester  Synthesized using Fischer Esterification (carboxylic acid + alcohol)  Removed using strong acid  use acid that is NOT strong enough to hydrolyze amide bonds (mechanism shown)  Catalysts: HBr and acetic acid (HOAc) o Esters are more acid-labile (unstable) then amides Fischer Esterification Review 1. Nucleophilic addition o Carbonyl is protonated by acid. Alcohol binds to carbon and is deprotonated by CB 2. Elimination o Acid protonates on of the OH to make good LG. Water is removed, remaining OH is deprotonated and carbonyl group is restored b) Protection and Deprotection of NH2 - Protected as t-butyloxycarbamate o Nucleophilic acyl substitution of tert-butyloxycarbonyl to amino acid o Resulting protecting group is called BOC group - BOC group is easily removed by TFA (CF3COOH) o Too weak to remove benzyl esters and hydrolyze amides Deprotection by TFA - E1 elimination is the first step  TFA protonates leaving group - Amine is protonated in the final step Note: The peptide in the example can have any length

c) Formation of Amide Bonds - We CANNOT make a peptide bond directly by mixing amino acids together o Need to us coupling agents - Coupling agents make the COOH more reactive by converting it to a reactive acid derivative o i.e. We could convert the COOH to acid chloride, but this leads to enolization (tautomerism) which destroys the stereochemistry of the amino acid (we only want Lamino acids) o We want to avoid using coupling agents which will alter stereochemistry - Best Reagent: DCC o DCU (urea) is formed as a by-product and acts as good leaving group Mechanism 1) DCC nitrogen deprotonates carboxylic acid functional group 2) Nucleophilic oxygen attacks central carbon in DCC, forming a new covalent bond 3) Amino group from second amino acid attacks carbonyl carbon, forming new covalent bond 4) Amide group is deprotonated 5) Carbonyl double bond reforms, ejecting DCU and leaving newly synthesized peptide 6) DCU is protonated Steps after formation of Amide bonds - If we want to add another amino acid, we need to deprotect the N-terminal end so that a third BOC protected residue can be added 1) TFA 2) Et3N: base that ensures that the amino group is deprotonated (NH2) and not protonated - Following deprotection, we use DCC again to add the next amino acid, and the cycle repeats - We synthesize the amino acid from the C-terminus to the N-terminus (contrary to how it is done in the body) o Therefore, the benzyl protecting group on the first amino acid is a permanent protecting group because it is not removed until the end of the synthesis o BOC groups are temporary protecting groups - At the END, we need to remove the last BOC groups as well as the benzyl group o Treatment with HBr/HOAc can remove both protecting groups (HOAc is a stronger acid than TFA which is needed to remove BOC) - End product is fully deprotected synthesized amino acid SERIOUS DRAWBACK: The process is tedious, and we need to purify at each step (remove all unwanted byproduct) METHOD 2: Solid Phase Synthesis - The same protecting groups are used with one exception o The COOH is protected as an ester connected to an insoluble polymer o The polypeptide will be linked to the polymer - Polymer: chloromethylpolystyrene o SN2 reaction with carboxylate salt (COO-) of a BOC-protected amino acid - Keep in mind that a lot of reactions are taking place on the polymer (not just one) Steps: 1. Attach first amino acid to polymer 2. React with TFA to remove the BOC group deprotection 3. React with Et3N to neutralize NH3+ to NH2 4. React with next amino acid with the help of DCC  new amide bond formed

5. Rinse and repeat until done 6. When done; react with HBr/HOAc o Cleaves the peptide from the solid support and removes BOC group from last amino acid, as well as any side chain protecting groups Advantages to Solid Phase Synthesis - Can use excess reagent  forces reaction to go to completion - We can wash and filter to remove reagents/by-products  polypeptide will remain stuck to polymer - Automated (simple liquid handler is all you need) Yield for Polypeptide Synthesis - For polypeptide synthesis, each step needs to proceed with HIGH efficiency o Small changes in the yield of each step can lead to tremendous changes in overall yield (example on the right for a 100 amino acid polypeptide) - It is crucial that the coupling step (formation of amide bond) has high efficiency o At the end of the synthesis, we’ll have similar products, but some will be missing one or more amino acids because it is not 100% efficient o For solid phase synthesis, we can easily synthesize peptides up to 30 amino acids in length; but it becomes difficult once we surpass 30 ENZYMES AND CATALYSIS Enzymes - Biological catalysts - Increase rates of chemical reactions up to a sextillion fold (1021) Interactions in Substrate-Protein Binding - For an enzyme to function: o It needs to recognize and bind a substrate through chemical interactions o The substrate must be able to physically fit in the enzyme’s binding site - Some drugs mimic substrates and competitively bind to the active site, inhibiting the enzyme 1) Hydrogen bonding - Interaction between δ+ Hydrogen covalently bonded to an electronegative atom, and the lone pair of another electronegative atom (δ–—δ+H - - - δ–) o For us δ– = O or N - There is no change in energy when substrates H-bond to enzymes o Any group that can H-Bond is already H-bonded to H2O o The binding of the substrate to protein replaces this H-bond (ΔG = 0) o If the substrate is incorrect or incorrectly orientated, the interaction is unfavorable (ΔG = +5) 2) Hydrophobic forces - Entropically driven grouping of hydrophobic regions o Expulsion of water from non-polar regions is favored by entropy (ΔG = negative) o There is NO “attraction” between hydrophobic regions they are just grouped together 3) Ionic interactions - Attraction of oppositely charged molecules - In water, this attraction is broken because water is highly polar and stabilizes ions (i.e. NaCl dissociates into Na+ and Cl-)

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In active site: polarity is much lower (ions are not stabilized), ion pairs can form between the site and the substrate  the larger the charge the greater the attraction (ΔG = negative)

Chiral Environment of Enzymes - Enzymes are chiral molecules o Enzymes will recognize one stereoisomer of a substrate over another o Molecules that are mirror images of each other are known as enantiomers - Enzymes can also bind achiral substrates and turn them in to chiral products o The substrate is considered prochiral: one step away from being chiral  Glycine is a prochiral amino acid (we need to swap ONE of the hydrogens to make it chiral)  Prochiral molecules have two identical groups around the chiral center o Conversion to chiral molecules is stereospecific  We change a specific atom of the two identical ones because the substrate can only fit in the enzyme in one orientation (can’t be either or) Proximity and Orientation Effects - Enzymes increase rates by bringing substrates in to proximity and holding their functional groups together in proper orientation How does Proximity work? (Fischer Esterification example) The following are listed in order of reactivity (refer to image) 1) Intermolecular reaction  two separate entities come together 2) Intramolecular reaction  groups are tethered to the same molecule, proximity is greater as a result, increasing reaction rates (4 bonds that can rotate) 3) Constrained orientation  ring structures are constrained (minimal rotation), the groups are constrained to the same side of the molecule, increasing proximity and orientation effects (3 bonds that can rotate) 4) Perfect alignment  the groups are constrained and perfectly aligned in order to achieve maximum reaction rates (2 bonds that can rotate) Notes: - When it comes to orientation, think about how many bonds can rotate relative to each other, the more rotation, the less likely that the two groups will be in close proximity - The COOH and OH groups CANNOT be on opposite sides of the constrained molecule, nor can one be facing up and the other down  both proximity and orientation are important Enzyme Kinetics - Enzymes accelerate rate of reactions by lowering the activation barrier (Ea) between starting materials and products o They DO NOT change the equilibrium constant between reactants and products To lower Ea enzymes: can - Orient the reacting functional groups - Provide a solvent environment that favors the chemical reaction (often non-polar) - Perform a complex reaction in a series of smaller steps (uses a different mechanism but results in the same products as the uncatalyzed version) - Binds and stabilizes the transition state very well - Act as acid or base catalyst (able to protonate and deprotonate; often carried out by histidine) - Form covalent bonds with the substrate after initial binding EXAMPLE OF ENZYME MECHANISM: Chymotrypsin

Chymotrypsin - Digestive enzyme found in the small intestine o Belongs to the family of serine proteases o Related to trypsin (hydrolyzes after basic residues), and elastase - Hydrolyzes peptides with sequence specificity o Hydrolyzes amide bonds on the C-terminal side of hydrophobic amino acids (after)  Trp, Tyr, and Phe are the best (aromatics are favored)  Tyr has hydrophobic aromatic ring despite being polar o Hydrolysis creates a new C-terminal on the N-terminal fragment and a new N-terminal on the other C-terminal fragment Why does amide hydrolysis need to be enzyme-catalyzed? - Amide bonds are very stable and won’t break readily in physiological pH How Chymotrypsin helps - Biological pH is ~7  OH- is not present in significant amounts for basic hydrolysis o Serine residue on Chymotrypsin acts as the base/nucleophile instead - Recall that for the basic mechanism for hydrolysis has a tetrahedral intermediate (oxyanion) o The oxyanion is not stable at physiological pH  stabilized by enzyme through hydrogen bonding - The newly created N-terminus is not a good leaving group  needs to be protonated by enzyme as it leaves Overview of Chymotrypsin Mechanism - The enzyme divides the hydrolysis mechanism in to two smaller reactions o Each has an Ea lower than that of the uncatalyzed reaction Reaction 1: OH group on serine residue acts as a nucleophile and attacks the amide, removing Cterminal fragment of the substrate  remaining N-terminal fragment is covalently bonded to the enzyme

Reaction 2: Water acts as nucleophile which hydrolyzes the enzyme-bound N-terminal fragment and regenerate the original state of the enzyme.

NOTE: BOTH of these reactions are nucleophilic acyl substitutions Mechanism of Chymotrypsin Action What’s involved: - The area of the enzyme that stabilizes the oxyanion is known as the oxyanion hole - Make a note of how the substrate is orientated in the active site of the enzyme***

Step 1: - His deprotonates Ser-OH to make a stronger nucleophile - Nucleophilic attack of the carbonyl carbon on the substrate occurs at the SAME time  prevents serine from forming an ion o Oxyanion is formed and stabilized through hydrogen bonds with the amino acid backbone Step 2: - Oxyanion collapses to reform carbonyl group, leaving group (amine group of C-terminal fragment) leaves o Amine is a poor leaving group and is protonated by HisH+ before it leaves Step 3: - Substrate is bonded to enzyme as an ester (known as acyl enzyme) - Amine leaves the active site and is replaced by water, which hydrogen bonds with His Step 4: - Water is a poor nucleophile; His acts as a base and deprotonates it - At the same time, the resulting OH- ion undergoes a nucleophilic attack (nucleophilic acyl substitution) on the carbonyl carbon of the ester o Oxyanion is formed and again stabilized by amino acid backbone through H-bonds - Same mechanistic procedure as Step 1 Step 5: - Oxyanion collapses and the carbonyl C=O is regenerated - Ser-O- is the leaving group, but it is a poor leaving group; HisH+ will protonate the residue at the same time - Same concept as Step 2 Step 6: - Enzyme is returned to original state, and N-terminal fragment has been cleaved from the enzyme Note: The function of Asp is to hold His in place, in older theories it was believed to also act as a base Cofactors and Coenzymes - Cofactors are non-protein components which are required by many enzymes o Can be inorganic ions (Mg2+) o Can be small organic molecules known as coenzymes - Coenzymes have a specific task during the enzymatic reaction and may donate/accept a portion of their structure to/from the starting material/intermediate/product o Most are derived from vitamins; nutrients that we need to consume Examples (red regions are the part that participate in reactions) 1) TPP (Vitamin B1): a-decarboxylation reactions 2) Biotin (Vitamin B7): carboxylation reactions (carries CO2 during glucose synthesis) 3) NADH/NADPH (Vitamin B3): hydride (H-) donor/acceptor (redox reactions) Acetylated Lipoic Acid 4) FAD (Vitamin B2): redox reactions 5) ATP: Energy and phosphate transfer (phosphorylation of proteins and lipids) 6) Vitamin B6: Transamination of amino acids

7) Lipoic Acid: transfer of acyl groups (usually acetyl) (important in CAC) - Often exists in disulfide form 8) CoA (Vitamin B5): transfer of acyl groups (usually acetyl) - When acetylated, acetyl group adds to the sulfur - Made up of ADP, linker region (from B5), and thiol - Key for synthesis of fatty acids OVERVIEW OF PROTEIN BIOSYNTHESIS - 100,000 proteins in the human body, the primary sequence for each is coded in the DNA RNA -

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All RNA is made of a sugar phosphate backbone, as well as the bases A, C, G and U o Purines: A and U o Pyrimidines: G and C o OH group on 2’ carbon o Phosphodiester linkage RNA polymerase uses DNA template to synthesize RNA o Three types are involved in protein biosynthesis

1. mRNA - Carries amino acid sequence of to-be synthesized proteins o Subdivided in to codons (groupings of three nucleobases) (64 total) o Each codon codes for an amino acid o Degenerate; some codons code for the same amino acid 2. tRNA - Carry amino acids and deliver them to the ribosome during translation - Each amino acid has a different tRNA with a specific anticodon that is complementary to codons on the mRNA o Complementary base pairing through H-bonds - Ribose OH at the 3’ end of the tRNA carries the amino acid tRNA Charging - Amino-acyl tRNA synthase attaches amino acid to appropriate tRNA via an ester linkage o There is a specific amino-acyl tRNA synthase for each amino acid - Reaction could proceed by Fischer Esterification, but this requires acidic conditions - Instead, we turn the carboxylic acid in to a more reactive acid derivative to make it more efficient o Endothermic reaction  requires input of energy from ATP o Usually we add a phosphate or a phosphate derivative, creating a mixed anhydride  Phosphates are good leaving groups for nucleophilic acyl substitution o Mg2+ is a common cofactor Mechanism: - Two nucleophilic acyl substitutions (basic conditions) 1) Phosphorylation of the amino acid - Mg2+ holds negative charges to make phosphorous more electrophilic - Amino acid attacks first phosphate, nucleophilic acyl substitution - Products: o AA + AMP = Mixed anhydride: anhydride with two different atoms on either side of the oxygen (C and P instead of C and C)

o Pyrophosphate (PP2)  quickly hydrolyzed to phosphate in water 2) Attachment of amino acid to tRNA - AMP moiety acts as a “handle” which is recognized by enzyme - Exothermic (anhydride  ester) - Base deprotonates 3’ ribose end of tRNA, which acts as the nucleophile for nucleophilic acyl substitution 3. rRNA (ribosomes) - Two subunit complexes made of ~65% rRNA and ~35% protein o Small subunit binds mRNA o Large subunit has three tRNA binding sites  E site: exit site for tRNA  P site: peptidyl-tRNA site  A site: aminoacyl-tRNA site - Bring together mRNA, loaded tRNA, and other accessory protein (elongation factors) for translation Protein Elongation (N terminus  C terminus) - Ribosomes use peptidyl transferase reactions to catalyze amide formation 1. One loaded tRNA is in the P site, the other is in the A site 2. The amino acid in the P site forms an amide bond and is transferred to the amino acid in the A site 3. The entire complex shifts over by one codon (3 nucleotides) o Free tRNA exits through the E site o Growing peptide is now in P site o New amino-acyl tRNA comes in at A site 4. Reaction repeats  (it is the growing peptide that is transferred each time a new amide bond is formed) Once the last amino acid is added and a stop codon is read, the protein is released from the last tRNA by hydrolysis of the ester linkage Peptidyl Transferase Mechanism - It was originally believed that the mechanism was the reverse of the chymotrypsin mechanism, with histidine functioning as a base o The growing peptide bound to the tRNA as an ester would act the same as the acylenzyme intermediate - X-ray crystallography was used to get a high-resolution structure of the ribosome o They found that the active site was made of RNA and NOT protein, disproving the idea that His was used as a base o Crystallography requires a solid ribosome, however they are naturally not solid o Reinforces theory of prebiotic RNA world (all three kingdoms of life use RNA catalysis) - The catalytic ability of the ribozyme (RNA catalysts are NOT enzymes) is attributed to an adenine residue o Adenine deprotonates the amino-acyl tRNA to make a good nucleophile  basic amino acyl substitution follows o Unlike in chymotrypsin, there is no oxyanion hole in the ribosomal active site  Protonated adenine is close enough to stabilize the oxyanion...