Organic Chemistry Option II: Chemical Biology

Dr Stuart Conway Organic Option II: Chemical Biology University of Oxford

1

Organic Chemistry Option II: Chemical Biology

Dr Stuart Conway Department of Chemistry, Chemistry Research Laboratory, University of Oxford email: [email protected] Teaching webpage (to download hand-‐outs): http://conway.chem.ox.ac.uk/Teaching.html

Recommended books: Biochemistry 4th Edition by Voet and Voet, published by Wiley, ISBN: 978-‐0-‐470-‐57095-‐1. Foundations of Chemical Biology by Dobson, Gerrard and Pratt, published by OUP (primer) ISBN: 0-‐19-‐924899-‐0


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Information flow in cells

• How does cellular information encoded in gene flow, via RNA, to form proteins?

• We must understand this process in order to harness it for exploration of biological problems.

The central dogma of molecular biology

• How does DNA in genes direct the synthesis of RNA and protein?

• How is DNA replicated?

• Francis Crick encapsulated the outlines of this process in the “central dogma of molecular biology”

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3

The central dogma of molecular biology

• DNA directs its own replication and transcription to yield RNA, which is translated to form proteins.

• Solid lines indicate the genetic information transfers that occur in all cells.

• Dotted lines indicate special transfers.

• The missing lines indicate transfers that the central dogma postulates never occur. The structure of DNA and RNA

• DNA and RNA comprise a polymeric phosphate-‐sugar backbone attached to a nucleic acid base.

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4

The structure of DNA and RNA

• Nucleotides are phosphate esters of pentose (furanose) sugars.

• Deoxynucleotides lack the hydroxyl group at the 2’ position of the sugar ring.

• A nitrogen-‐containing base is linked to the 1’-‐position of the sugar. The structure of DNA and RNA

• RNA, but NOT DNA, is susceptible to base-‐catalysed hydrolysis.

• DNA lacks the 2’-‐hydroxyl group, which makes it

resistant to base-‐catalysed hydrolysis.

• It is possible that this chemical stability is why DNA has evolved to be the store of genetic information.

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5


• The nitrogen bases are planar, aromatic and heterocyclic.

• They are usually either purine or pyrimidine derivatives.


• The major purine components of nucleic acids are adenine and guanine.

• The purines form glycosidic bonds to ribose via their N9 atoms.


• The major pyrimidine components of nucleic acids are cytosine, uracil and thymine (5-‐methyluracil).

• Uracil occurs mainly in RNA whereas thymine occurs mainly in DNA.

• The pyrimidinesform glycosidic bonds to ribose via their N1 atoms.

N

N N

NN

N

N N

NO

H

NHH

adenine guanine

H H

H

H

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6


• Some DNAs contain bases that are derivatives of the standard set.

• For example N6-‐methylation of adenine and 5-‐methylation of cytosine can occur. The structure of DNA and RNA

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Nucleotide: adenosine monophosphate (R = OH in RNA and H in DNA)

Nucleoside: adenosine (R = OH in RNA and H in DNA)

Base: adenine


• Nucleic acids are usually linear polymers

of nucleotides.

• The phosphate groups bridge the 3’-‐ and 5’-‐positions of successive sugar residues.

• The phosphate groups are deprotonated

at physiological pH, hence nucleic acids are polyanions in the cell.

• Polynucelotides have directionality:

each has a 3’ end and a 5’ end.

N

N

N

N

NH2

OO

HO R

PO

HOHO

N

N

N

N

NH2

OHO

HO R

NH

N

N

N

NH2

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• Nucleic acids were first isolated in 1869 and the presence of these molecules in cells was demonstrated a few years later.

• In the 1930s and 1940s it was widely believed that nucleic acids had a monotonously

repeating sequence of all four bases = the so called “tetranucleotide hypothesis”.

• It was generally assumed that genes, known to be carriers of genetic information, were proteins.

• See Biochemistry pages 85-‐89 to see the experiments that proved DNA is the carrier of

genetic information. The structure of DNA and RNA

• Erwin Chargaff was the first to show that DNA contains equal numbers of adenine and thymine residues (A = T) and equal numbers of cytosine and guanine residues (C = G).

• These relationships are known as

“Chargaff’s rules”.

• Although not specifically stated by Chargaff, this observation suggests some form of base pairing in the (then unknown) structure of DNA.

N

N N

NNH2

N

N N

NO

H

H2NX

X

N

N

N

N

XO

NH2

X

O

O

H CH3

adenine(A, X=H)

thymine(T, X=H)

guanine(G, X=H)

cytosine(C, X=H)

=

=

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• The Watson-‐Crick structure of B-‐DNA consists of two strands that wind about a common axis with a right-‐handed twist to form a ~20 Å diameter double helix. The two strands are anti parallel (run in opposite directions).

• The planes of the bases are nearly perpendicular to the helix axis.

• Each base is hydrogen bonded to a base on the opposite strand to form a planar base pair.

Complementary base pairing

• The most remarkable feature of the Watson and Crick structure is that it can accommodate

only two types of base pairs.

• Each adenine residue must pair with a thymine residue and vice versa.

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• Each guanine residue must pair with a cytosine residue and vice versa.

• The geometries of these A:T and G:C pairs , the so-‐called Watson-‐Crick base pairs, mean

that these base pairs are interchangeable in the double helix.

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Hydrogen bonding

• Hydrogen bonds are one of the most important non-‐covalent interactions in biological systems.

• They take place between an electron-‐rich heteroatom (acceptor) and an electron-‐deficient hydrogen atom (donor).

• The hydrogen atom is usually covalently linked to an electronegative atom, such as O or N.

• There is a significant electrostatic component to H-‐bonding.

Hydrogen bonding

• However, orbital interactions are also an important component of H-‐bonding.

• H-‐bonds can be viewed as having a σ-‐bonding component.

• Consequently, there is an optimum orientation for H-‐ bonding.

Hydrogen bonding

• The optimum angle for H-‐bonding is where the X-‐H bond points directly to the lone pair, such that the angle is 180°.

• H-‐bond strength can vary between 16 and 60 kJmol-‐1.

• H-‐bonds are typically 1.5-‐2.2 Å compared to 1.0-‐1.5 Å for covalent bonds.

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• The H-‐bond donor and acceptor patterns are such that A can only bind to T and G can only bind to C.

• As A can only bind to T and G can only bind to C, we can immediately understand

Chargaff’s rules.

• In addition, the Watson-‐Crick structure allows for any sequences of bases on one polynucleotide strand if the opposite strand has the complementary sequence.

• This structure also suggests that hereditary information is encoded in the sequence of

bases on either strand.

NN

NH

HN

NX N

NH

O CH3

XOHN

N

ON

NX N

N

N

XON

H

HH

HHdonor

acceptor

acceptor

donor

acceptor

donor

donor acceptor

donor

acceptor

adenine thymine guanine cytosine

NN

NH

HN

NX N

N

N

XOH

donor

acceptor

donor

acceptor

adenine cytosine

HH

acceptor

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DNA structure

• DNA has three major helical forms, B-‐DNA, A-‐DNA and Z-‐DNA.

• B-‐DNA is the biologically predominant form of DNA it forms a right-‐handed helix with major and minor grooves.

• When relative humidity is reduced to 75%, B-‐DNA undergoes a reversible conformational

change to A-‐DNA.

• A-‐DNA forms a wide, flatter helix than B-‐DNA.

• The base pairs of A-‐DNA are tilted 20 ° with respect to the helix axis.

• Certain DNA sequences can form a left-‐handed helix that has been called Z-‐DNA.

• It is not clear whether Z-‐DNA has any biological significance -‐ it may play a role in regulating DNA transcription.

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RNA structure

• RNA can also adopt defined conformations.

• Transfer RNA (see later) resembles an “L” shape, being made up of two short helical regions connected by a hinge.

• Each helical segment comprises two portions of

the single RNA chain running in opposite directions.

RNA structure

• Hydrogen bonding in helical RNA occurs between cytosine and guanine as in DNA.

• Cytosine is replaced by uracil, which forms complementary hydrogen bonds with adenine.

NN

NH

HN

NX N

NH

O

XOHadenine uracil

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DNA replication

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for genetic material.”

• The division of cells must be accompanied by the replication of DNA.

• In this process, mediates by DNA polymerase enzymes, each DNA strand acts as a template for the formation of its complementary strand.

• Consequently, every progeny cell contains a complete copy of the DNA from the parent cell.

• Mutations arise when, through rare copying errors, one or more wrong bases are incorporated into a daughter strand.

• DNA replication is a highly complex process.

• This complexity, when compared to the chemically similar transcription process, arises from the need for extreme accuracy in DNA replication so as to preserve the integrity of the genome from generation to generation.

Translation and transcription

• DNA directs its own replication and transcription to yield RNA, which is translated to form proteins.

• “Transcription” indicates that the “language” of the encoding information remains the same.

• “Translation” indicates that the “language” changes from that of the base sequence to that of the amino acid sequence.

• Individual portions of a DNA molecule provide the information for the construction of various RNA molecules and proteins.

• RNA corresponding to the region of interest is produced by transcription (the synthesis of an RNA strand from a DNA template). The RNA produced in this case is called messenger RNA or mRNA.

• This mRNA is then translated when molecules of transfer RNA (tRNA) align with the mRNA via complementary base pairing between segments of three consecutive nucleotides (codon).

• Each type of tRNA carries a specific amino acid, which are covalently joined by the ribosome to form a protein.

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RNA synthesis: Transcription

• The enzyme that synthesises RNA is called RNA polymerase.

• It catalyses the DNA-‐directed coupling of nucleotide triphosphates to synthesise new RNA.

• The newly synthesised RNA is complementary to the template DNA.

Transcription

• RNA synthesis proceeds in a stepwise manner in the 5’→3’ direction.

• Hence, the incoming nucleotide is added to the free 3’-‐OH of the growing RNA chain.

• RNA polymerase selects the nucleotide it incorporates into the growing RNA chain based on the requirement that it forms a Watson-‐Crick base pair with the DNA strand that is being transcribed (the template strand -‐ only one strand of DNA is transcribed at a time).

• The RNA polymerase moves along the DNA duplex that it is transcribing and separates a short (~14 base pairs) segment of the DNA helix to form a transcription bubble.

• The DNA in the transcription bubble forms a short DNA-‐RNA helix with the newly synthesised RNA.

• The DNA-‐RNA hybrid helix consists of antiparallel strands, hence the DNA’s template strand is read in its 3’→5’ direction.

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RNA polymerase

• RNA polymerase has the overall structure of a crab claw with two “pincers”.

• The outer surface of the protein is almost uniformly negatively charges, whereas the surfaces that interact with nucleic acids are positively charged.

• The DNA occupies the main channel, which directs the template strand to the active site.

• There the DNA base-‐pairs with the incoming nucleotide triphosphate (not in structure).

Translation

• Translation is the RNA-‐directed synthesis of polypeptides.

• Although the formation of a peptide bond is relatively simple, the translational process in highly complicated.

• This complexity arises from the need to link 20 different amino acids residues accurately in the order specified by a particular mRNA.

• According to the one gene-‐one polypeptide hypothesis, the genetic message dictates the amino acid sequences of proteins.

• As the base sequence of DNA is the only variable element in this otherwise monotonously repeating polymer, the base sequence and the protein sequence must be linked.

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Translation

“The problem of how a sequence of four things can determine a sequence of twenty things is known as the coding problem.” Translation

• With only 4 bases in DNA to code for 20 amino acids, a group of several bases (a codon) is necessary to specify a single amino acid.

• A triplet code (3 bases per codon) is minimally required since there are 43 = 64 different

triplets of four bases.

• A doublet code would only allow 42 = 16 codons, which is insufficient to specify 20 amino acids.

• In a triplet code as many as 44 codons might not code for amino acids.

• Alternatively, some amino acids might be specified by more than one codon -‐ a degenerate

code.

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The genetic code

• How is DNA’s continuous sequence grouped into codons?

• Is the code overlapping? E.g. ABC codes for the first amino acids and BDC codes for the second etc.

The genetic code

• Or is the code non-‐overlapping?

• E.g. ABC specifies the first amino acid and DEF the second etc.

The genetic code

• The genetic code (right) is a non-‐

overlapping, comma free, degenerate, triplet code.

• The genetic code is highly degenerate:

Three amino acids (L, R, S) are each specified by six codons.

• Only Met and Trp, two of the least

common amino acids in proteins, are specified by a single codon.

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The genetic code

• Sydney Brenner and Francis Crick formed the following hypotheses on the genetic code:

1. The code is a triplet code.

2. The code is read in a sequential manner starting from a fixed point in the gene. The insertion or deletion of a nucleotide shifts the frame (grouping) in which in which the succeeding nucleotides are read as codons. Thus the code has no internal punctuation that indicates the reading frame -‐ the code is comma free.

3. All (or nearly all) of the 64 triplet codons code for an amino acid. Therefore some amino

acids are specified by more than one codon -‐ the code is degenerate. The genetic code

• The sentence represents a gene in which the words (codons) each contain three letters (bases).

• The spaces have no physical significance; they only present to indicate the reading frame.

• The deletion of the fourth letter (B) shifts the reading frame so that all of the words after the deletion are meaningless -‐ specify the wrong amino acids.

The genetic code

• Insertion of a letter (X) passed the point of the original mutation restores the original reading frame.

• Hence on the words (codons) between the two changes (mutations) are altered.

• Therefore the sentence may still be intelligible (the gene could still specify a functional protein), particularly if the changes are close together.

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The genetic code

• The major breakthrough in deciphering

the genetic code came in 1961 when Nirenberg and Matthaei established that UUU is the codon specifying Phe.

• They added poly(U) to a cell-‐free protein synthesising system and showed that this stimulated synthesis of only poly(Phe).

• In similar experiments, poly(A) was shown to specify poly(Lys) and poly(C) was found to specify poly(Pro).

• UAG, UAA and UGA are “stop” codons, which act as a signal to the ribosome to terminate protein synthesis.

• These stop codons are also known (somewhat inappropriately) as nonsense codons as they are the only codons that do not specify amino acids.

• UAG, UAA and UGA are sometimes referred to as amber, ochre and opal codons.

• AUG and (less frequently) GUG codons form part of the chain initiation sequence.

• These codons also specify amino acids, Met and Val, respectively.

• The arrangement of the genetic code is not random.

• Most synonyms (codons that only differ in their third nucleotide) occupy the same box in the table.

• XYU and XYC always specify the same amino acids; XYA and XYG do so in all by two cases.

• Changes in the first codon position tend to specify the same or similar amino acids.

• Codons with second position pyrimidines (C AND U) tend to specify hydrophobic amino

acids.

• Codons with second position purines (A and G) encode mostly polar amino acids.

• It seems that the genetic code has evolved so as to minimise the deleterious effects of mutations.

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The genetic code

• How does the information in DNA actually translate into polypeptide sequences?

• In 1955 Francis Crick proposed the adaptor hypothesis stating that translation occurs through the mediation of adaptor molecules.

• Crick suggested that the adaptors might contain RNA as codon recognition could occur by complementary base pairing.

• Each adaptor was postulated to carry a specific amino acid and to recognise the corresponding codon.

• At a similar time it was shown that in the course of protein synthesis 14C labelled amino acids become bound to low molecular mass fractions of RNA.

• This RNA is known as transfer RNA or tRNA and is Crick’s putative adaptor molecule.

Translation

• All tRNAs contain:

• A 5’-‐terminal phosphate.

• A 7-‐base pair step that includes the 5’-‐terminal nucleotide and may include non-‐Watson-‐Crick base pairs, such as G ⋅ U. This assembly is known as the acceptor stem as the amino acid is appended to the 3’-‐OH group.

• A 3-‐ or 4-‐base stem ending in a loop that that frequently contains the modified base dihydrouridine (D), known as the D arm.

• A 5-‐base-‐pair stem ending in a loop that usually contains the sequence TΨC (Ψ = pseudouridine).

• All tRNAs terminate in the sequence CCA, with a free 3’-‐OH group.

• There are 15 invariant positions and 8 semi-‐invariant (only a purine or only a pyrimidine) positions.

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23

Modified nucleotides that occur in tRNA

The structure of yeast tRNAPhe

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24

Synthesis of tRNA

• The amino acid is activated by reaction

with ATP to form aminoacyl-‐adenylate.

• This mixed anhydride then reacts with tRNA to form aminoacyl-‐tRNA and AMP.

Ribosome

• For translation to occur, mRNA and tRNA must bind to each other, and the amino acids carried by the tRNA must react to form the polypetide chain.

• This process is mediated by the ribosome.

• The ribosome is a ribozyme, comprising mainly ribosomal RNA (rRNA).

• Elucidating the molecular structure of the ribosome has been extremely challenging.

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25

Ribosome

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26

Translation

Translation

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27

Translation

• The ribosomal peptidyl transfer reaction occurs ~107-‐fold faster than the uncatalysed reaction.

• The rate enhancement does NOT occur via general acid or general base catalysis. Translation

• The ribosome enhances the rate of peptide bond formation by properly positioning and

orienting its substrates.

• The ribosome may also play a role in excluding water from the preorganised electrostatic environment of the active site.

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28

Translation

• Ribosomes act very fast -‐ a rate of 6-‐9 amino acids per second in eukaryotic cells and 17-‐21 amino acids per second in prokaryotic cells.

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29

Naturally occurring amino acids

Protein structure –α-‐helices

• Only one helical polypeptide conformation has simultaneously allowed conformational

angles and a favourable hydrogen-‐binding pattern.

• This striking element of secondary structure is known as the α-‐helix.

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30

Protein structure – anti-‐parallel β-‐sheets

• Anti-‐parallel β-‐sheets are an important type of protein secondary structure.

• This arrangement results in a strong hydrogen bond with a near optimal N-‐O distance. Protein structure – parallel β-‐sheets

• β-‐sheets can also have a parallel arrangement.

• This results in a staggered pattern of hydrogen-‐bonding. Protein structure – β-‐turns

• There are two types of β-‐turns, Type I and Type II.

• Each comprises four key amino acids.

ONH

O

Phe

NHLys

ON

Val

H

ON

Phe

H

ONH

Gln

H ON N

ONH

Trp

Ala H

N

O

HThr

N

O

HIle

Gln

NH

ON

Leu

H

ON

Phe

H

ONH

Ile ONH

Asp

NH

ON

Ile

H

ON

Trp

H

ONH

Ile ONH

AlaN

O

AspHO

Leu

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31

Enzymes

• Almost all chemical reactions that comprise life are catalysed by enzymes.

• The rates of enzymatically catalysed reactions are typically 106 to 1012 greater than the corresponding uncatalysed reactions.

• The catalysis occurs under relatively mild conditions.

• Enzymes often catalyse their reactions with a high degree of substrate selectivity.

• As enzymes are chiral the active site is “chiral space” allowing differentiation between pro-‐

chiral groups. Enzymes

• Types of enzyme catalysis:

1. Acid-‐base catalysis.

2. Covalent catalysis.

3. Metal ion catalysis.

4. Electrostatic catalysis.

5. Proximity and orientation effects.

6. Preferential binding (stabilisation) of the transition state complex.

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Egg white lysozyme (retaining glycosidase)

• Lysozyme enzymes are glycoside hydrolase or glycosidase enzymes.

• These enzymes catalyse hydrolysis or transacetylation of glycosidic linkage in sugars.

• Egg white lysozyme is a retaining glycosidase, meaning that the configuration of the anomeric centre is the same in substrate and product.

• The retaining glycosidases employ both acid-‐base catalysis and covalent catalysis in their mechanism.

• Chemical tools and crystallography have helped to determine the proposed mechanism of this enzyme.


• Lysozyme enzymes are involved in the destruction of bacterial cell walls.

• These enzymes work by hydrolysing β (1→4) glycosidic linkages from N-‐acetylmuramic acid to N-‐acetylglucosamine.

• Lysozyme occurs mainly in the cells and secretions of vertebrate, where it may function as an antibacterial agent.

• However, few pathogenic bacteria are susceptible to lysozyme alone, suggesting that this enzyme may help dispose of bacteria after they have been killed by other means.

• Hen egg white lysozyme is the most widely studied species of lysosyme, mainly as it is readily available -‐ one egg contains about 5 g.


OOR1HO

OOR2HOretaining glycosidase

R2 OH

+

R1 OH

+

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33



O O

D52retaining glycosidase

retaining glycosidase

OHO

HO

OH

ORHO

O O

D52

OH

O

E35



OHO

HO

OH

HO

O O

D52

O O

E35

O R'H



OHO

HO

OH

HO

O O

E35

H

OR'

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34



• The proposed covalent intermediate in the mechanism of lysozyme had never been observed, as the breakdown of this intermediate must be much faster than the rate of formation, in order for the enzyme to function efficiently.

• A sugar containing a fluoride at the anomeric position should react rapidly with the enzyme

to form a covalent intermediate.

• The additional fluorine at the C2 position of the ring will reduce the rate of the covalent intermediate breaking down.

• Mutation of glutamate 35 to glutamine (E35Q) slows the rate of the reaction further,

meaning that the covalent intermediate with the fluorosugar accumulates and can be observed by X-‐ray crystallography.

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35

Inverting glycosidase

• Inverting glycosidases catalyse the same overall transformation as retaining glycosidases, but the resulting effect on the stereochemistry of the anomeric centre is different.

• The mechanism of inverting glycosidases means that the configuration of the anomeric centre is inverted during the reaction.

• Inverting glycosidases employ acid-‐base catalysis in their mechanism.

• Glycosidases are important in all forms of life, mainly in the metabolism of complex carbohydrates.



OOR1HO

OHO

inverting glycosidase

R2 OH

+

R1 OH

+OR2

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36





OOR

O O

D278

OH

O

E95

OH



O

O O

D278

O O

E95

OH

OH

H



O

O O

D278

O O

E95

OH

OH

H

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37

Serine protease

• Serine proteases (e.g. trypsin, chymotrypsin, elastase) are digestive enzymes that are synthesised in pancreatic acinar cells and secreted into the small intestine.

• These enzymes all catalyse the hydrolysis of amide bonds.

• Different enzymes have different selectivities for the amino acid side chains that flank the amide bond to be cleaved.

• Serine proteases employ both acid-‐base catalysis and covalent catalysis in their mechanism.

• Additionally, the intermediate oxyanion is stabilised in the “oxyanion hole”. Serine protease

serine proteaseR1

HN R2

O

HO R2

OR1 NH2 +

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38

Serine protease

Serine protease

O

OD102

N NH

H57

O

S195

H

N R

OR'

H

serine protease

O

OD102

N NH

H57

O

S195

serine protease

H

N ROR'

H

O

OD102

N NH

H57

O

S195

serine protease

O

R

R' NH

H

HO

HO

OD102

N NH

H57

O

S195

serine protease

O

ROH

H

R

O

O H

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39

Serine protease

Serine protease

• The charged carbonyl carbon of the tetrahedral intermediate is stabilised by the “oxyanion hole”.

• The negatively charged oxygen forms hydrogen bonds with the backbone NH groups of Gly193 and Ser195.

Serine protease

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40

Sulfatase

• Sulfatase enzymes cleave sulfate esters in biological systems.

• These enzymes are involved in regulating the sulfation states that determine the function of may physiologically important molecules.

• The mechanism of sulfatase enzymes involves covalent catalysis, metal (calcium) ion catalysis, acid-‐base catalysis.

• In addition, one of the catalytic residues is post-‐translationally modified to aid the catalytic function of these enzymes.

Sulfatase

Sulfatase

sulfatase

O S OHRO O

HO S OH

O OR OH +

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41

Sulfatase

Sulfatase

sulfatase

O H

FGly51

NN H

115H

H

O

OD317

sulfatase

N

NH

H211

OSO OO

R

H

Ca2+

sulfatase

O O

FGly51

NN

115H

H

O

OD317

sulfatase

N

NH

H211

OSO OO

R

H

Ca2+

H HOH

H

sulfatase

O O

FGly51

NN

115H

H

HO

OD317

sulfatase

N

NH

H211

S O

Ca2+

H

O O

R OH

sulfatase

O H

FGly51

NN

115H

H

HO

OD317

sulfatase

N

NH

H211

Ca2+

SO42-

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42

Phospholipase C

• Phospholipase C catalyses the hydrolysis of the minor membrane phospholipid, phosphatidylinositol 4,5-‐bisphosphate, to give inositol 1,4,5-‐trisphosphate and diacyl glycerol, both of which are intracellular second messengers.

• PLC uses acid-‐base catalysis and metal (calcium) ion catalysis in its mechanism. Phospholipase C

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43

Phospholipase C

Phospholipase C

PLC!

N

N

H

HH311

NN

H356

HH

O H

O PO

OO

Ca2+

B

R

PLC!

N

N

H

HH311

NN

H356

HH

O

O PO

OO

Ca2+

B

R

H

PLC!

N

N

H

HH311

NN

H356

HO

O P

Ca2+

BH

O

OH O H

PLC!

N

N

H

HH311

NN

H356

HO

O P

Ca2+

B

HO

OO

HH

R OH

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44

Phospholipase C

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45

Phospholipase C

• There are few potent and selective inhibitors of PLC.

• In order to develop such compounds, an effective assay for PLC (enzyme) activity is required.

• Huang et al. have developed a compound, based on PtdIns(4,5)P2 that produce a

fluorescent molecule when PLC is functioning.

• This compound allows accurate monitoring of PLC activity inside cells and hence will enable the discovery of new PLC inhibitors.

Phospholipase C

OH

O PO

OHO

HO

H2O3PO HO

H2O3POO

O lipid

lipid

OH

O PO

OOH

OC8H17O

HN

O

N

HO

H2O3PO HO

H2O3PO

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46

Phospholipase C

Phospholipase C

OH

O PO

OOH

HO

H2O3PO HO

H2O3PO

phospholipase C

OH

O PO

OOH

HO

H2O3PO HO

H2O3PO

O NH

O

N

OC8H17

O

O NH

O

N

OC8H17

OOC8H17

O N

O

NH HN

H2N

fluorescentCO2

OH

O PO

OOH

HO

H2O3PO HO

H2O3PO

phospholipase COH

O PO

OOH

HO

H2O3PO HO

H2O3PO

OOC8H17

N

H2N

fluorescentO N

H

O

N

OC8H17

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47

Affinity chromatography

• Many proteins possess the ability to selectively bind small molecules tightly but non-‐covalently.

• This property can be used to assist the purification of proteins using a technique called

affinity chromatography.

• The molecule that binds to the protein (ligand) is covalently attached to a solid support.

• When an impure protein solution is passed through this chromatographic material the desired protein binds to the immobilised material.

• Other substances and proteins are washed through the column with the buffer.

• The desired protein can be recovered (in a highly purified form) by changing the elution

conditions.

• An affinity matrix can also be used to identify which proteins bind to the immobilised ligand.

• Proteins that bind to the matrix and that can be competed off by the presence of the

soluble ligand, demonstrate selective binding to the ligand.

• This technique has been used to understand cellular signalling pathways.

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48



R3OOR4

HO OOHOR5

PO O

OHOR2

OR1

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49


• The various PIPs were synthesised and immobilised onto a solid phase via a terminal amino

group.

• The immobilised PIPs formed an affinity matrix that was used to identify novel PIP-‐binding proteins and to determine their binding selectivity.


• Cell lysates were passed through affinity columns made of the immobilised PIPs.

• The proteins that bound to the matrix were then passed down the column having been pre-‐incubated with soluble ligand.

• Proteins that did not bind in the second case are the proteins that bind to the ligand

selectively.

(NaO)2OPOOPO(ONa)2

HO OOHOPO(ONa)2

PO O

ONaOCOC11H22NH

OCOC15H31O

= solid support

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50


• Protein kinase B (PKB) is known to bind to PtdIns(3,4,5)P3.

• PKB binds most strongly to the natural D-‐form of PtdIns(3,4,5)P3.

• The protein does not bind to unfunctionalised, control, beads. Affinity chromatography

(NaO)2OPOOPO(ONa)2

HO OOHOPO(ONa)2

PO O

ONaOCOC11H22NH

OCOC15H31O

= solid support

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51

Transition state mimics

• One method that enzymes catalyse reactions is by binding preferentially to the reaction transition state rather than the substrate or product.

• Therefore molecules that mimic the transition state could potentially act as competitive

inhibitors of the enzyme. Transition state mimics

O O

D52retaining glycosidase


OHO

HO

OH

ORHO

O O

D52

OH

O

E35



OHO

HO

OH

HO

O O

D52

O O

E35

O R'H



OHO

HO

OH

HO

O O

E35

H

OR'

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52


• The δ-‐lactone above is a mimic of the lysozyme transition state.

• The ester unit (red) is coplanar, similar to the SN2 transition state. Transition state mimics

• Neuraminidases, which are involved in virus proliferation are retaining glycosidases.

• Neuraminidases cleave the budding virus particle from the host cell.

• Inhibition of neuraminidases is potentially of therapeutic value. Transition state mimics

• Both Relenza and Tamiflu, which are marketed as treatments for influenza, are neuraminidase transition state mimics and competitive inhibitors.

• Relenza was one of the first examples of crystal structures being employed in rational drug design.

OCO2H

O glycoconjugateHO

R

HO OH

HO

neuraminidase(retaining glycosidase)

OCO2H

OHHO

R

HO OH

HOO

OHCO2H

HOR

HO OH

HO

O CO2H

HOR

HO OH

HO

O CO2H

HNR

HO OH

HO

H2NNH

CO2H

H2N

OR

O CO2H

HOR

HO OH

HO

LG

Nu

!!"#$%&'()*+'#($&$, -./. 0,1,'2& 3&4)56

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53


Caged compounds

• Caged compounds are biologically active molecules that are rendered biologically inert by the addition of a photolabile protecting group to an important functional group.

• Removal of the protecting group releases (uncages) the active compound.

• This technique is often applicable to the non-‐invasive control of biologically important compounds inside cells.

• A high degree of temporal and spatial control is obtained by using caged compounds.

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54

Caged compounds

Caged compounds

• The dimethoxynitrobenzyl moiety is a commonly used caging group.

• Irradiation with light of wavelength ~355 nm gives efficient release of the caged compound.

OR

NO2

MeOOMe

inactive compounds

light

active compound

O

NO

MeOOMe

H

ROH

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55

Caged compounds

• The first step of photocleavage is the n → π* excitation of the nitro group to the 1st singlet excited state.

• The singlet state then decay, via intersystem crossing, to the triplet state, which behaves as a diradical in the subsequent “dark” reactions.

Photoaffinity labelling

• Photoaffinity labelling employs a photoactivatable but chemically inert ligand analogue.

• Once the ligand is bound to the receptor, activation by light forms a highly reactive species that binds covalently to the protein at the site of interaction.

• Subsequent analysis of the protein can provide information about the site of ligand

interaction.

O

HR

NO O

H3COOCH3

O

HR

NO O

H3COOCH3

O RNO OH

H3COOCH3

O RNO

H3COOCH3

O HOH

H

NO

H3COOCH3

O

ROH

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56




R N N Nlight

R N

N Nlight

R

R

R

R

R N Nlight

RX

light

R

R

R

R

NN

O

R

lightO

R

azide

diazo compounds

diazonium salts

diazirines

benzophenones

nitrene (singlet or triplet)

carbene (singlet or triplet)

carbocation

carbene

carbonyl excited state(triplet or singlet)

R N N Nlight

R NR N N N + N2

NN

12

3

45

6

7

12

3

4

56

7

Nu

H+

HN

Nu

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57


slide 141

Documents

Organic Chemistry Option II: Chemical Biology