Chapter 26: Biomolecules: Amino Acids, Peptides, and...

Click here to load reader

  • date post

    06-Mar-2018
  • Category

    Documents

  • view

    221
  • download

    4

Embed Size (px)

Transcript of Chapter 26: Biomolecules: Amino Acids, Peptides, and...

  • 177

    348

    Chapter 26: Biomolecules: Amino Acids, Peptides, and Proteins

    monomer unit: -amino acids biopolymer: peptide (< 50 amino acids)

    protein (> 50 amino acids)

    R CO2H

    NH2H

    !- Amino Acid

    R = sidechain

    Peptide or protein

    NH

    O

    R3

    HN

    O

    R2

    NH

    O

    R1

    HN

    NH

    O

    R7

    HN

    O

    R6

    NH

    O

    R5

    R4 HN

    O

    Amino acids are linked together through amide bonds (peptide bonds)

    349

    26.1 Structures of Amino AcidsAmino acids exist as a zwitterion: a dipolar ion having

    both a formal positive and formal negative charge (overall charge neutral); internal salts

    CO2H

    R

    H

    H2N CO2

    R

    H

    H3N_+

    Amino acids are amphoteric: they can react as either an acid or a base. Ammonium ion acts as an acid, the carboxylate as a base

  • 178

    350

    20 common amino acids19 are 1-amines, one (proline) is a 2-amine

    19 amino acids are chiral; one (glycine) is achiral (R=H)

    The configuration natural amino acid is L19 are of the S stereochemistry, one (cysteine) is R

    R CO2H

    NH2H

    R = sidechain

    primary -amino acid

    N CO2_

    H H

    +

    prolinesecondary -amino acid

    CHO

    CH2OH

    H OH

    D-glyceraldehyde

    CHO

    CH2OH

    HO H

    L-glyceraldehyde

    CO2H

    CH3

    H2N H

    CO2H

    CH2SH

    H2N H

    L-alanine L-cysteine

    351

    Neutral amino acids

    (S)-(+)-Valine (Val, V)(S)-()-Tryptophan (Trp, W)

    (S)-()-Proline (Pro, P)(S)-()-Phenylalanine (Phe, F)

    COO

    NH3

    H

    N

    H

    COO

    NH3

    COO

    NH3

    COO

    NH3

    NH3

    COO

    COO

    (2S,3S)-(+)-Isoleucine (Ile, I)

    (S)-(+)-Alanine (Ala, A)

    (S)-()-Leucine (Leu, L)

    NH3

    COOS

    (S)-()-Methionine (Met, M)

    (S)-(+)-Glutamine (Gln, Q)

    Glycine (Gly, G)

    NH3

    COO

    H2N

    O

    NH3

    COO

    (S)-()-Tyrosine (Tyr, Y)

    (2S,3R)-()-Threonine (Thr, T)(S)-()-Serine (Ser, S)

    COO

    NH3

    COO

    NH3

    COO

    NH3 NH3

    COO

    NH3

    COO

    O

    H2NHS

    HO

    OH

    HO

    (R)-()-Cysteine (Cys, C)(S)-()-Asparagine (Asn, N)

    pKa ~ 13 pKa ~ 13

    pKa ~ 8.2

    pKa ~ 10.1

    COO

    NH3NH

  • 179

    352

    Acidic amino acids

    Basic amino acids

    (S)-()-Histidine (His, H)

    NH3

    COOCOO

    NH3 NH3

    COON

    H

    NN

    H

    H

    (S)-(+)-Lysine (Lys, K) (S)-(+)-Arginine (Arg, R)

    pKa ~ 10.5 pKa ~ 6.0 pKa ~ 12.5

    H2N

    NH H

    H3N

    -O

    O NH3

    COO

    (S)-(+)-Aspartic Acid (Asp, D)

    COO

    NH3

    O

    -O

    (S)-(+)-Glutamic Acid (Glu, E)

    pKa ~ 3.6 pKa ~ 4.2

    353

    Humans can produce (biosynthesize) ten of the twenty aminoacids; the remaining ten must be obtained by diet andare called essential amino acids: tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucineand isoleucine.

    26.2 Isoelectric points pI: pH at which the amino acid exists largely in a neutral, zwitterionic form (influenced by the nature of the sidechain)

    CO2H

    R

    H

    H2N CO2

    R

    H

    H3N_+

    CO2H

    R

    H

    H3N+H3O

    +

    pKa1

    CO2

    R

    H

    H2N

    HO_

    pKa2

    low pH

    high pH

    _

  • 180

    354

    pI =pKax + pKay

    2

    pI =pKa1 + pKa2

    2

    pI =pKa1 + pKa3

    2

    pI =pKa2 + pKa3

    2

    pI = 9.7

    pI = 2.7

    CO2

    CH3

    H

    H3NCO2H

    CH3

    H

    H3N

    low pH

    CO2

    CH3

    H

    H2N+ +

    high pH

    pKa1(2.3)

    pKa2(9.7)

    pI = 6.0

    CO2H

    (CH2)4

    H

    H3N

    NH3

    pKa2(9.0)

    CO2

    H

    H3N

    (CH2)4

    NH3

    CO2

    H

    H2N

    (CH2)4

    NH3

    pKa3(10.5)

    pKa1(2.2)

    CO2

    H

    H2N

    (CH2)4

    NH2

    low pH high pH

    CO2H

    CH2

    H

    H3N

    CO2H

    pKa3(3.6)

    CO2

    H

    H3N

    CH2

    CO2H

    CO2

    H

    H3N

    CH2

    CO2

    pKa2(9.6)

    pKa1(1.9)

    CO2

    H

    H2N

    CH2

    CO2

    low pH high pH

    355

    Henderson-Hasselbalch equation: allows the calculation of the relative amounts of the protonated, neutral, and deprotonated forms at a given pH from the pKa values of the amino acid (please read)

    Electrophoresis: separation of polar compounds based on theirmobility through a solid support. The separation is based on charge (pI) or molecular mass.

    + _

    _ _ _ _ + + + +

    + _

  • 181

    356

    26.3 Synthesis of Amino AcidsFrom Chapter 24: HVZ reaction followed by SN2 reaction

    with NH3 or ammonia equivalents

    Reductive amination

    R-CH2-CO2H

    R C CO2H

    Br

    R C CO2H

    NH2

    R C CO2H

    N3

    Br2, PBr3

    NH3NaN3

    H2, Pd/C

    R C CO2H

    N OOKOH, H2O

    N

    O

    O

    KH

    H

    H

    C CO2HR

    O NH3

    H2, Pd/C-or-

    NaB(CN)H3

    C CO2HR

    NH2

    H

    357

    RCH2 CO2EtC

    CO2EtHN EtO Na

    RCH2X

    H3O

    - CO2 RCH2 CO2HC

    HH2N

    O

    RCH2 CO2EtC

    CO2EtHN

    O

    Amidomalonate Synthesis

    26.4 Enantioselective Synthesis of Amino AcidsThe syntheses in the previous section give racemic products!!

    R-CH2-CO2H

    Br2, PBr3

    RCH Br

    O

    Br Br

    R CH

    CO2H

    Br

    R

    H O

    Br

    Br Br

    Br Br

    Br

    R

    HCO2H

    Br

    R

    HCO2H

    R

    S

    50:50mixture of

    enantiomers

  • 182

    358

    Resolution: separation of enantiomers

    RCH2 CO2HC

    NH2H

    N

    N

    H

    H

    (-)-sparteine(chiral base)

    RCH2 CO2

    C

    NH2H

    N

    N

    H

    HH

    N

    N

    H

    HH

    RCH2 CO2

    C

    HH2N

    +

    Diastereomeric salts(separate)

    H3O H3O

    RCH2 CO2

    C

    NH3H

    RCH2 CO2

    C

    HH3N

    racemic

    Resolutions are inherently inefficient

    359

    Enantioselective Synthesis of Amino Acids (please read)

    R

    H CO2H

    NHAc

    H H

    H H

    R NHAc

    H H

    H CO2H

    R NHAc

    H H

    H CO2HR

    S

    Rh(I) L*, H2

    O

    O

    CO2Me

    HO

    OH

    CO2

    NH3

    L-DOPA

    O

    O

    CO2Me

    HN

    O

    PhHN

    O

    Ph

    H3O+

    Chiral hydrogenation catalysts can differentiate the faces of the C=C double bond leading a products that is highly enriched in one enantiomer.

  • 183

    360

    26.5 Peptides and ProteinsProteins and peptides are polymers made up of amino acid units

    (residues) that are linked together through the formationof an amide bonds (peptide bonds) from the amino group of one residue and the carboxylate of a second residue

    By convention, peptide sequences are written left to right from the N-terminus to the C-terminus

    H2N CO2H

    Alanine

    H2N CO2H

    HO

    Serine

    + - H2OH2N

    HN

    O

    CO2H

    OH

    Ala - Ser(A - S)- H2O

    H2N

    HN

    O

    CO2H

    HO

    Ser - Ala(S - A)

    C-terminus

    N-terminus

    C-terminus

    N-terminus

    NH

    O

    R3

    HN

    O

    R2

    NH

    O

    R1

    HN

    NH

    O

    R7

    HN

    O

    R6

    NH

    O

    R5

    R4 HN

    O

    backbone

    361

    26.6 Covalent Bonding in PeptidesI. The amide bond

    II. Disulfide bonds: the thiol groups of cysteine can be oxidized to form disulfides (Cys-S-S-Cys)

    C=N double bond characterdue to this resonance structure

    restricts rotationsresistant to hydrolysis

    R1

    HN

    N

    O R2 HN

    O

    amide bond

    R1

    HN

    N

    O R2 HN

    O

    _

    +

    H H

    SHHO2C

    NH22

    1/2 O2 H2O

    SHO2C

    NH2

    SCO2H

    NH2

    NH

    O

    R2

    HN

    O

    R1

    NH

    HN

    O

    R5

    NH

    O

    R4

    HN

    O

    NH

    OHN

    O

    R6

    NH

    HN

    O

    R10

    NH

    O

    R9

    R8 HN

    O

    SH

    NH

    O

    R2

    HN

    O

    R1

    NH

    HN

    O

    R5

    NH

    O

    R4

    HN

    O

    NH

    OHN

    O

    R9

    NH

    HN

    O

    R13

    NH

    O

    R12

    R11 HN

    O

    S

    SHS 1/2 O2

  • 184

    362

    Epidermal Growth Factor (EGF):53 amino acid, 3 disulfide linkages

    Cys14-Cys31Cys6-Cys20

    Cys33-Cys42

    1986 Nobel Prize in Medicine:Stanley Cohen Rita Levi-Montalcini

    363

    26.7 Structure Determination of Peptides: Amino Acid Analysis

    Primary (1) structure of a peptide or protein is the amino acid sequence

    Amino acid analyzer- automated instrument to determine the amino acid content of a peptide or protein

    peptide-or-

    protein

    [H] reduce anydisulfidebonds

    Enzymaticdigestion

    R CO2

    NH3 individualamino acids-or-

    H3O+,

    1972 Nobel Prize in ChemistryWilliam Stein Stanford Moore

    liquidchromatography

    derivatize w/ninhydrin

    Detected w/UV-vis

    Different amino acids have different chromatographicmobilities (retention times)

  • 185

    364

    Reaction of primary amines with ninhydrin

    Intense purple color

    R CO2

    NH3

    O

    O

    O+-H2O

    O

    O

    N

    R

    O

    O H

    O

    O

    N

    R

    H2O

    - RCHO

    O

    O

    NH2

    - CO2

    O

    O

    OO

    O

    N

    O

    O

    Amino Acid Analysis Chromatogram

    365

    26.8 Peptide Sequencing: The Edman Degradationchemical method for the sequential cleavage andidentification of the amino acids of a peptide, one at a time starting from the N-terminus.

    Reagent: Ph-N=C=S, phenylisothiocyanate (PITC)

    S

    C

    NPh

    +H2N

    O

    R1 HN CO2

    pH 9.0

    NH

    O

    R1 HN CO2

    NH

    S

    Ph

    H+

    H+

    HN

    S

    OH

    HN CO2

    N

    R1

    PhH+

    HN

    S OH2N CO2

    N

    R1

    Ph

    H+

    HN

    N OS

    R1

    Ph

    +

    N-phenylthiohydantoin:separated by liquid chromatography (based of the R group) and detected by UV-vis

    -1 peptide with a new N-terminal amino acid (repeat degradation cycle)

  • 186

    366

    Peptide sequencing by Edman degradation: cycle the pH to control the cleavage of the N-terminal

    amino acid by PITC. Monitor the appearance of the new N-phenylthiohydantoin

    for each cycle. Good for peptides up to ~ 25 amino acids long. Longer peptides and proteins must be cut into smaller

    fragments before Edman sequencing.

    Enzymatic cleavage of peptides and proteins at defined sites

    trypsin: cleaves at the C-terminal side of basic residues, Arg, Lys but not His

    NH

    O

    HN

    O

    NH

    R3

    O

    HN

    R1O

    H3NCO2

    NH3

    NH

    O

    HN

    O

    H3N

    R3

    O

    HN

    R1O

    H3NCO2

    NH3

    O +trypsin

    H2O

    367

    chymotrypsin: cleaves at the C-terminal side of aromatic residuesPhe, Tyr, Trp

    NH

    O

    HN

    O

    NH

    R3

    O

    HN

    R1O

    H3NCO2 N

    HO

    HN

    O

    H3N

    R3

    O

    HN

    R1O

    H3NCO2

    O +chymotrypsin

    H2O

    trypsin

    H2NValPheLeuMetTyrProGlyTrpCysGluAspIleLysSerArgHis-CO2H

    H2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-CO2HH2N-His-CO2HH2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-CO2HH2N-Ser-Arg-CO2H

    H2N-Val-Phe-CO2HH2N-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2HH2N-Val-Phe-Leu-Met-Tyr-CO2HH2N-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2HH2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-CO2HH2N-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2H

    chymotrypsin

  • 187

    368

    Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-ArgVal-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys Ser-Arg

    His

    Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-HisVal-Phe-Leu-Met-Tyr-Pro-Gly-Trp Cys-Glu-Asp-Ile-Lys-Ser-Arg-HisVal-Phe-Leu-Met-TyrVal-Phe

    Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His

    If given the sequence of the enzyme digest fragements, allignthe sequences of the fragments to get the sequence ofthe full peptide

    Trypsin:

    Chymotrypsin:

    369

    EPIDERMAL GROWTH FACTOR (EGF): 53 amino acids

    H2N-ASN1SER2TYR3PRO4GLY5CYS6PRO7SER8SER9TYR10ASP11GLY12TYR13CYS14LEU15ASN16GLY17GLY18VAL19CYS20MET21HIS22ILE23GLU24SER25LEU26ASP27SER28TYR29THR30CYS31ASN32CYS33VAL34ILE35GLY36TYR37SER38GLY39ASP40ARG41CYS42GLN43THR44ARG45ASP46LEU47ARG48TRP49TRP50GLU51LEU52ARG53-CO2H

    Trypsin Chymotrypsin Cyanogen Bromide (BrCN)

    Cys14-Cys31Cys6-Cys20

    Cys33-Cys42

    26.9 Peptide Sequencing: C-Terminal ResidueDetermination (please read)

  • 188

    370

    Peptide sequencing by mass spectrometry (Lagniappe)Peptides can be sequenced rapidly by tandem mass spectrometry.

    Peptide and proteins are product of gene expression

    The Central Dogma (F. Crick):

    DNA mRNA Protein (genome) (proteome)

    Can we understand . . .. . . the biological functions of proteins . . . . . . the relationship between protein function / expression

    and disease . . . . . . the relationship between protein modification, either

    biochemically or by toxicants and alter protein function / expression and disease . . .

    . . . the biological target of drugs or toxicants . . .

    . . . by profiling the proteins in a cell or tissue?

    371

    Mass spectrometry is a gas phase technique. Peptides (and proteins) are charged, polar, high molecular weight molecules (ions). How can peptides and proteins becoaxed into the gas phase?

    Electrospray ionization (ESI): analyte is introduced into the massspectrometer as an aerosol.

    +++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    +

    - +

    +

    to the mass analyzer

    liquid chromatographyor capillary electrophoresis(separate the analytes)

    +++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    + +++++

    - +

    + ++++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    + + ++++

    - +

    +++++

    ++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    + +++++

    - +

    + ++++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    + +++ ++

    - +

    + ++++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    + +++ ++

    +

    +

    ++

    +

    - +

    + ++

    Coulombicfission

    +++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    +

    +

    +

    ++

    +

    - +

    ++

    +++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    +

    - +

    +

    +

    +

    +

    +

    ++

    +++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    +

    - +

    +

    +

    +

    +

    +

    +

    +++++

    +++

    ++++

    +++++

    ++++

    +++

    ++++

    +++++

    +

    +

    - +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    ++++ ++

    +

    +

    ++

    +

    ++

    Coulombic

    fission

    +++++

    ++ ++++

    +++++++

  • 189

    372

    MALDI ionization (matrix-assisted laser desorption): analyte isco-crystallized with an organic molecule that has anintense UV absorption. A laser that is tuned to the absorption of the matrix, is pulsed at the MALDImatrix and energy is indirectly transferred to the analyte.

    ++

    +

    +

    +

    +

    to the mass analyzer

    Laser pulse

    ++

    +

    +

    +

    +

    +

    +

    2002 Nobel Prize in ChemistryJohn Fenn (ESI)Koichi Tanaka (MALDI)

    373

    Peptide sequencing by tandem mass spectrometry

    to thedetector

    Q1ElectrosprayIon Source Q3Collision Cell (Q2)NanosprayCapillary

    Select peptide to be analyzed

    fragment thepeptide

    Analyze thepeptide fragments

    1000 1500 2000 2500 3000 m/z

    1116

    .67

    1287

    .73

    1375

    .76

    1424

    .85 1505

    .77

    1665

    .89

    1811

    .85

    2005

    .07

    2476

    .21

    2550

    .52

    2719

    .48

    1849

    .12

    1574

    .20

    1247

    .70

    Peptides fragment ina predictable manner

    H2N CH C

    R1

    OHN CH C

    R2

    OHN CH C

    R3

    OHN CH C

    R4

    OH

    O

    b1

    y1

    b2

    y2 y3 charge to C-terminus

    charge to N-terminus

    b3

    Select m/z 1228.7 for Q2

  • 190

    374

    average exact - HN-CHR-COGlycine G 75.07 75.03 57Alanine A 89.10 89.05 71Serine S 105.09 105.04 87Proline P 115.13 115.05 97Valine V 117.15 117.08 99Threonine T 119.12 119.06 101Cysteine C 121.16 121.02 103Isoleucine I 131.18 131.09 113Leucine L 131.18 131.09 113Asparagine N 132.12 132.05 114Aspartic Acid D 133.11 133.04 115Glutamine Q 146.15 146.07 128Lysine K 146.19 146.11 128Glutamic Acid E 147.13 147.13 129Methionine M 149.21 149.05 131Histidine H 155.16 155.02 137Phenylalanine F 165.19 165.19 147Arginine R 174.20 174.11 156Tyrosine Y 181.19 181.07 163Tryptophan W 204.23 204.09 186

    Amino Acids Sorted by Mass

    H2N CH C

    R1

    OHN CH C

    R2

    OHN CH C

    R3

    OHN CH C

    R4

    OH

    O

    b1

    y1

    b2

    y2 y3

    b3

    375

    112.2Leu/Ile

    173.1Arg

    96.9Pro

    101.1Thr

    113.1Leu/Ile

    115.3Asp

    57.1Gly

    146.8Phe

    146.9Phe

    71.2Ala

    57.1Gly

    71.1Ala

    128.0Lys/Gln128.2

    Lys/Gln

    57.2Gly

    57.0Gly

    70.9Ala147.1

    Phe

    147.0Phe115.1

    Asp

    57.0Gly

    57.1Gly

    57.1Gly

    71.0Ala

    57.0Gly

    H2N-Ile-

    -Arg-CO2H

    -Pro-

    -Phe-

    -Ile-

    -Gly-

    -Gly-

    -Gly-

    -Phe--Ala-

    -Gln--Ala-

    -Gly--Ala-

    -Gly-

    -Gln-

    -Ala-

    -Gly-

    -Phe-

    -Gly-

    -Gly-

    -Phe--Asp-

    -Asp--Thr-

  • 191

    376

    Analysis of the proteome: separate proteins of a cell by two-dimensional electrophoresis

    _ _ _ _ + + + +

    separateby pI

    separateby mass(PAGE)

    377

    5.1 5.95.7 6.3 9.6

    16.9

    50

    107

    42

    128

    pI

    MW

  • 192

    378

    26.10: Peptide Synthesis

    H2N CO2H

    Ala

    H2N CO2H

    Val

    +- H2O

    H2N

    HN

    O

    CO2H

    Ala - Val(A - V)

    H2N

    HN

    O

    CO2H

    Val - Ala(V - A)

    - H2O

    The need for protecting groups

    Orthogonal protecting group strategy: the carboxylateprotecting group must be stable to the reaction conditions for the removal of the -aminoprotecting group (and vice versa)

    NH

    Ala

    H2N

    Val

    +

    - H2O

    NH

    HN

    O

    Ala - Val(A - V)

    Pn OH

    O

    OPc

    O

    PnOPc

    Oselectivelyremove Pn

    H2N

    HN

    O

    Ala - Val(A - V)

    OPc

    O

    peptidecoupling

    peptidecoupling(-H2O)

    NH

    Pn OH

    O

    Ph

    Phe (F)

    NH

    HN

    O

    OPc

    OOHN

    Ph

    Pn

    Phe - Ala - Val(F - A - V)

    Repeat peptide synthesis

    379

    C-terminal protecting group:benzyl ester: removes by catalytic hydrogenation

    (H2, Pd/ C)

    -amino protecting group: tert-butyloxycarbamoyl (BOC): removed with strong

    protic acid (F3CCO2H)

    Peptide coupling reagent: dicyclohexylcarbodiimide (DCC)

    C6H11 N C N C6H11

    R OH

    O

    C6H11 N C NH

    C6H11

    R O

    O

    +

    R O

    O

    C

    N

    C6H11

    C6H11

    NH

    R'-NH2

    R O

    O

    C

    HNC6H11

    C6H11

    NH

    NHR' +

    R NH

    O

    R'NH

    NH

    O

    C6H11 C6H11+

    (DCC)

    Amide DCU

    "activated acid"

    R O

    O

    C

    N

    C6H11

    C6H11

    NH

    NR'

    ++H

    H

    Mechanism: Figure 26.5, page 1006

  • 193

    380

    In order to practically synthesize peptides and proteins, time consuming purifications steps must be avoided until the very end of the synthesis.

    Large excesses of reagents are used to drive reactions forward and accelerate the rate of reactions.

    How are the excess reagents and by-products from the reaction,which will interfere with subsequent coupling steps, removed without a purification step?

    NH

    Ala

    H2N

    Val

    + NH

    HN

    OBOC OH

    O

    OBn

    O

    BOCOBn

    O

    selectivelyremove N-protecting

    group

    H2N

    HN

    O

    OBn

    O

    NH

    BOC OH

    O

    Ph

    Phe (F)

    NH

    HN

    O

    OBn

    OOHN

    Ph

    BOC

    DCC

    peptidecoupling

    CF3CO2HDCC

    CF3CO2H

    NH

    HN

    O

    OBn

    OO

    H2N

    Ph

    H2, Pd/CNH

    HN

    O

    OH

    OO

    H2N

    Ph

    Phe - Ala - Val(F - A - V)

    381

    26.11 Automated peptide synthesis: The Merrifield Solid-Phase Techniques: Peptides and proteins up to ~ 100 residues long are synthesized on a solid, insoluble, polymer support. Purification is conveniently accomplishedafter each step by a simple wash and filtration.

    The solid support (Merrifield resin): polystyrene polymer

    ~ 1 - 10% of the available phenyl groups are functionalized

    O

    R

    OHN_

    O

    O

    R

    NH

    CF3CO2H

    O

    O

    R

    NH2

    BOC

    BOC

    +

    polymerization

    styrene

    divinylbenzene(crosslinker, ~1 %)

    initiator

    Ph

    Ph Ph Ph Ph

    PhPhPh Ph

    Ph

    Ph

    Ph

    Ph

    Ph

    Ph

    Ph

    H3COCH2ClZnCl2

    CH2Cl

    commericallyavailable

  • 194

    382

    H2N

    Val

    O

    Opeptidecoupling

    NH

    BOC OH

    ODCC

    O

    O

    HN

    BOC NH

    Opurify:

    wash & filter

    remove N-protecting

    group

    CF3CO2H

    O

    O

    H2NNH

    O

    NH

    BOC OH

    O

    Ph

    Phe (F)

    DCC

    O

    O

    NH

    O

    purify:wash & filter

    NH

    BOCHN

    O

    Ph

    purify:wash & filter HF

    remove N-protecting

    group and cleavefrom solid-support

    OH

    O

    NH

    O

    H2N

    HN

    O

    Phpurify by liquidchromatograrphy

    or electrophoresis

    Solid-phase peptide synthesis

    383

    LYS GLU THR ALA ALA ALA LYS PHE GLU ARG GLN HIS MET ASP SER SER THR SER ALA ALA SER SER SER ASN TYR CYS ASN GLN MET MET LYS SER ARG ASN LEU THR LYS ASP ARG CYS LYS PRO VAL ASN THR PHE VAL HIS GLU SER LEU ALA ASP VAL GLN ALA VAL CYS SER GLN LYS ASN VAL ALA CYS LYS ASN GLY GLN THR ASN CYS TYR GLN SER TYR SER THR MET SER ILE THR ASP CYS ARG GLU THR GLY SER SER LYS TYR PRO ASN CYS ALA TYR LYS THR THR GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE ASP ALA SER VAL

    His-12 AHis-119 A

    His-12 BHis-119 B

    pdb code: 1AFL

    Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNASolid-phase synthesis of RNase A:B. Gutte & R. B. Merrifield, J. Am. Chem. Soc. 1969, 91, 501-2.

    Synthetic RNase A: 78 % activity 0.4 mg was synthesized 2.9 % overall yield average yield ~ 97% per coupling step

    R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry: for his development of methodology for chemical synthesis on a solid matrix.

  • 195

    384

    26.12 Protein Classification (please read)

    26.13 Protein Structureprimary (1) structure: the amino acid sequence.secondary (2) structure: recurring sub-structural motifs of

    proteins. These include -helices, -sheets, turns, disulfide bonds and others. These sub-structures arelargely held together by H-bonds and other non-covalentinteractions.

    tertiary (3) structure: The overall three-dimensional structure(conformation) of a singe polypeptide chain.

    quaternary (4) structure: overall organization of non-covalentlylinked subunits of a functional protein

    385

    Common secondary (2) sub-structural motifs; -helix: collagen 3.6 amino acids per coil, 5.4

    3.6 AA,5.4

  • 196

    386

    Common secondary (2) sub-structural motifs; -helix:

    387

    Hydrophobic (on the inside) and hydrophilic (on the outside)residues of myoglobin

    Pro Ile Lys Tyr Leu Glu Phe Ile Ser Asp Ala Ile Ile His Val His Ser Lys

  • 197

    388

    26.14 Enzymes: a catalyst of a biological reaction.accelerates the rate of a reaction by lowering the

    activation energy (G)

    Proteases: catalyzes the hydrolysis of peptide bonds

    NH

    HN

    NH

    O

    RO

    HN

    N

    O R

    RO

    R

    H

    O protease

    H2OH3N

    CO2 NH

    HN

    H3N

    O

    RO

    HN

    N

    O R

    RO

    R

    H

    O

    H3NCO2O

    +

    chymotrypsin: cleaves at the C-terminal side of aromatic residues Phe, Tyr, Trptrypsin: cleaves at the C-terminal side of basic residues Arg, Lys but not His

    389

    Many enzymes catalyze reactions by using the function groupson the amino acid sidechains.

    Ser-195

    His-57

    Asp-102

    Catalytic triad of -chymotrypsin

    pdb code: 5CHA

  • 198

    390

    Some reactions require additional organic molecules or metalions. These are referred to as cofactors (coenzymes)

    N N

    SO

    P-O O

    O PO-

    O-

    O

    NH2

    N

    +

    Thiamin diphosphate (Vitamin B1)

    N

    2-O3POOH

    O H

    Pyridoxal Phosphate (Vitamin B6)

    N

    N

    NH

    N O

    O

    O

    OH

    HO

    OH

    O-

    P

    O

    P O

    O

    O-

    O

    HO

    N

    OH

    O

    N

    N

    N

    NH2

    Flavin Adenine Diphosphate(Vitamin B2)

    NN

    N N

    H 2N

    O NH2

    O

    NH2

    NH2O

    O

    O

    Co

    O

    H

    H2N

    O

    NH

    O

    C

    PO

    O

    -O

    O

    N

    N

    HO H

    H

    NH2

    H

    H

    OH

    N

    Cyanocolbalmin(Vitamin B12)

    N

    NN

    N

    O

    HOO

    OH

    Fe(III)

    Heme

    NHHN

    S

    O

    CH4CO2H

    Biotin

    N

    HNN

    N

    O

    H2N

    HN

    O

    HN CO2H

    CO2H

    Folic acid

    391

    The protein part in such an enzyme is called an apoenzyme, and the combination of apoenzyme plus cofactor is called a holoenzyme

    26.15 How Do Enzymes Work? Citrate SynthaseBy bringing reactants together and holding them in the optimal

    orientation for the reaction to occur

    26.16 Protein DenaturationThe unfolding of the three-dimensional structure of a protein to a random coil state. This results in the lossof its secondary, tertiary and quaternary structures andany biological activity. Proteisn can be denatured byheat, pH or chemicals (urea)