Chapter 28 Biomolecules: Heterocycles and Nucleic Acids Based on McMurry’s Organic Chemistry, 6th edition



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Chapter 28 Biomolecules: Heterocycles and Nucleic Acids

  • Based on McMurry’s Organic Chemistry, 6th edition


Heterocycles

  • Cyclic organic compounds are carbocycles or heterocycles

    • Carbocycle rings contain only carbon atoms
    • Heterocycle rings atoms in addition to carbon (N,S,O are common)
  • Heterocycles include many important natural materials as well as pharmaceuticals



28.1 Five-Membered Unsaturated Heterocycles

  • Pyrrole, furan, and thiophene are common five-membered unsaturated heterocycles

  • Each has two double bonds and N, O, or S



Pyrrole

  • Commercially from coal tar or by treatment of furan with ammonia over an alumina catalyst at 400°C.



Furan

  • Made commercially by extrusion of CO from furfural, which is produced from sugars



Thiophene

  • From coal tar or by cyclization of butane or butadiene with sulfur at 600°C



Unusual Reactivity

  • Pyrrole is an amine but it is not basic

  • Pyrrole, furan, and thiophene are conjugated dienes but they undergo electrophilic substitution (rather than addition)



28.2 Structures of Pyrrole, Furan, and Thiophene

  • Pyrrole, furan, and thiophene are aromatic (Six  electrons in a cyclic conjugated system of overlapping p orbitals)

  • In pyrrole  electrons come from C atoms and lone pair on sp2-N



Why Pyrrole is Not a Base

  • The nitrogen lone pair is a part of the aromatic sextet, protonation on nitrogen destroys the aromaticity, giving its conjugate acid a very low pKa (0.4)

  • The carbon atoms of pyrrole are more electron-rich and more nucleophilic than typical double-bond carbons (see comparison with cyclopentadiene)



28.3 Electrophilic Substitution Reactions of Pyrrole, Furan, and Thiophene

  • The heterocycles are more reactive toward electrophiles than benzene



Position of Substitution



28.4 Pyridine, a Six-Membered Heterocycle

  • Nitrogen-containing heterocyclic analog of benzene

  • Lone pair of electrons on N not part occupies an sp2 orbital in the plane of the ring and is not involved in bonding (Figure 28.3).



Electronic structure of pyridine

  • Pyridine is a stronger base than pyrrole but a weaker base than alkylamines

  • The sp2-hybridized N holds the lone-pair electrons more tightly than the sp3-hybridized nitrogen in an alkylamine



28.5 Electrophilic Substitution of Pyridine

  • The pyridine ring undergoes electrophilic aromatic substitution reactions with great difficulty, under drastic conditions



Low Reactivity of Pyridine

  • Complex between ring nitrogen and incoming electrophile deactivates ring with positive charge

  • Electron-withdrawing nitrogen atom deactivates causes a dipole making positively polarized C’s poor Lewis bases



28.6 Nucleophilic Substitution of Pyridine

  • 2- and 4-substituted (but not 3-substituted) halopyridines readily undergo nucleophilic aromatic substitution



Mechanism of Nucleophilic Substitution on Pyridine

  • Reaction occurs by addition of the nucleophile to the C=N bond, followed by loss of halide ion



Addition-Elimination

  • Addition favored by ability of the electronegative nitrogen to stabilize the anionic intermediate

  • Leaving group is then expelled



28.7 Fused-Ring Heterocycles



Quinoline and Isoquinoline

  • Quinoline and isoquinoline have pyridine-like nitrogen atoms, and undergo electrophilic substitutions

  • Reaction is on the benzene ring rather than on the pyridine ring



Indole

  • Has pyrrole-like nitrogen (nonbasic)

  • Undergoes electrophilic substitution at C3 of the electron-rich pyrrole



Purine and Pyrimidine

  • Pyrimidine contains two pyridine-like nitrogens in a six-membered aromatic ring

  • Purine has 4 N’s in a fused-ring structure. Three are basic like pyridine-like and one is like that in pyrrole



28.8 Nucleic Acids and Nucleotides

  • Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of genetic information

  • Nucleic acids are biopolymers made of nucleotides, aldopentoses linked to a purine or pyrimidine and a phosphate



Sugars in DNA and RNA

  • RNA is derived from ribose

  • DNA is from 2-deoxyribose

    • (the ' is used to refer to positions on the sugar portion of a nucleotide)


Heterocycles in DNA and RNA

  • Adenine, guanine, cytosine and thymine are in DNA

  • RNA contains uracil rather than thymine



Nucleotides

  • In DNA and RNA the heterocycle is bonded to C1 of the sugar and the phosphate is bonded to C5 (and connected to 3’ of the next unit)



The Deoxyribonucleotides



The Ribonucleotides



28.9 Structure of Nucleic Acids

  • Nucleotides join together in DNA and RNA by as phosphate between the 5-on one nucleotide and the 3 on another

  • One end of the nucleic acid polymer has a free hydroxyl at C3 (the 3 end), and the other end has a phosphate at C5 (the 5 end).



Generalized Structure of DNA



Nucleic Acid Sequences

  • Differences arise from the sequence of bases on the individual nucleotides



Describing a Sequence

  • Chain is described from 5 end, identifying the bases in order of occurrence, using the abbreviations A for adenosine, G for guanosine, C for cytidine, and T for thymine (or U for uracil in RNA)

  • A typical sequence is written as TAGGCT



28.10 Base Pairing in DNA: The Watson–Crick Model

  • In 1953 Watson and Crick noted that DNA consists of two polynucleotide strands, running in opposite directions and coiled around each other in a double helix

  • Strands are held together by hydrogen bonds between specific pairs of bases

  • Adenine (A) and thymine (T) form strong hydrogen bonds to each other but not to C or G

  • (G) and cytosine (C) form strong hydrogen bonds to each other but not to A or T



H-Bonds in DNA

  • The G-C base pair involves three H-bonds



A-T Base Pairing

  • Involves two H-bonds



The Difference in the Strands

  • The strands of DNA are complementary because of H-bonding

  • Whenever a G occurs in one strand, a C occurs opposite it in the other strand

  • When an A occurs in one strand, a T occurs in the other



Grooves

  • The strands of the DNA double helix create two continuous grooves (major and minor)

  • The sugar–phosphate backbone runs along the outside of the helix, and the amine bases hydrogen bond to one another on the inside

  • The major groove is slightly deeper than the minor groove, and both are lined by potential hydrogen bond donors and acceptors.



28.11 Nucleic Acids and Heredity

  • Processes in the transfer of genetic information:

  • Replication: identical copies of DNA are made

  • Transcription: genetic messages are read and carried out of the cell nucleus to the ribosomes, where protein synthesis occurs.

  • Translation: genetic messages are decoded to make proteins.



28.12 Replication of DNA

  • Begins with a partial unwinding of the double helix, exposing the recognition site on the bases

  • Activated forms of the complementary nucleotides (A with T and G with C) associate two new strands begin to grow



The Replication Process

  • Addition takes place 5  3, catalyzed by DNA polymerase

  • Each nucleotide is joined as a 5-nucleoside triphosphate that adds a nucleotide to the free 3-hydroxyl group of the growing chain



28.13 Structure and Synthesis of RNA: Transcription

  • RNA contains ribose rather than deoxyribose and uracil rather than thymine

  • There are three major kinds of RNA - each of which serves a specific function

  • They are much smaller molecules than DNA and are usually single-stranded



Messenger RNA (mRNA)

  • Its sequence is copied from genetic DNA

  • It travels to ribsosomes, small granular particles in the cytoplasm of a cell where protein synthesis takes place



Ribosomal RNA (rRNA)

  • Ribosomes are a complex of proteins and rRNA

  • The synthesis of proteins from amino acids and ATP occurs in the ribosome

  • The rRNA provides both structure and catalysis



Transfer RNA (tRNA)

  • Transports amino acids to the ribosomes where they are joined together to make proteins

  • There is a specific tRNA for each amino acid

  • Recognition of the tRNA at the anti-codon communicates which amino acid is attached



Transcription Process

  • Several turns of the DNA double helix unwind, exposing the bases of the two strands

  • Ribonucleotides line up in the proper order by hydrogen bonding to their complementary bases on DNA

  • Bonds form in the 5  3 direction,



Transcription of RNA from DNA

  • Only one of the two DNA strands is transcribed into mRNA

  • The strand that contains the gene is the coding or sense strand

  • The strand that gets transcribed is the template or antisense strand

  • The RNA molecule produced during transcription is a copy of the coding strand (with U in place of T)



Mechanism of Transcription

  • DNA contains promoter sites that are 10 to 35 base pairs upstream from the beginning of the coding region and signal the beginning of a gene

  • There are other base sequences near the end of the gene that signal a stop

  • Genes are not necessarily continuous, beginning gene in a section of DNA (an exon) and then resume farther down the chain in another exon, with an intron between that is removed from the mRNA



28.14 RNA and Protein Biosynthesis: Translation

  • RNA directs biosynthesis of peptides and proteins which is catalyzed by mRNA in ribosomes, where mRNA acts as a template to pass on the genetic information transcribed from DNA

  • The ribonucleotide sequence in mRNA forms a message that determines the order in which different amino acid residues are to be joined

  • Codons are sequences of three ribonucleotides that specify a particular amino acid

  • For example, UUC on mRNA is a codon that directs incorporation of phenylalanine into the growing protein



Codon Assignments of Base Triplets



The Parts of Transfer RNA

  • There are 61 different tRNAs, one for each of the 61 codons that specifies an amino acid

  • tRNA has 70-100 ribonucleotides and is bonded to a specific amino acid by an ester linkage through the 3 hydroxyl on ribose at the 3 end of the tRNA

  • Each tRNA has a segment called an anticodon, a sequence of three ribonucleotides complementary to the codon sequence



The Structure of tRNA



Processing Aminoacyl tRNA

  • As each codon on mRNA is read, tRNAs bring amino acids as esters for transfer to the growing peptide

  • When synthesis of the proper protein is completed, a "stop" codon signals the end and the protein is released from the ribosome



28.15 DNA Sequencing

  • The order of the bases along DNA contains the genetic inheritance.

  • Determination of the sequence is based on chemical reactions rather than physical analysis

  • DNA is cleaved at specific sequences by restriction endonucleases

  • For example, the restriction enzyme AluI cleaves between G and C in the four-base sequence AG-CT Note that the sequence is identical to that of its complement, (3)-TC-GA-(5)

  • Other restriction enzymes produce other cuts permitting partially overlapping sequences of small pieces to be produced for analysis



Analytical Methods

  • The Maxam–Gilbert method uses organic chemistry to cleave phosphate linkages at with specificity for the adjoining heterocycle

  • The Sanger dideoxy method uses enzymatic reactions

  • The Sanger method is now widely used and automated, even in the sequencing of genomes



The Sanger Dideoxy Method

  • The fragment to be sequenced is combined with:

    • A small piece of DNA (primer), whose sequence is complementary to that on the 3 end of the restriction fragment
    • The four 2-deoxyribonucleoside triphosphates (dNTPs)


The Dideoxy Nucleotides

  • The solution also contains small amounts of the four 2,3-dideoxyribonucleoside triphosphates (ddNTPs)

  • Each is modified with a different fluorescent dye molecule



The Dideoxy Method - Growing the and Stopping the Copied Chains

  • DNA polymerase is added and a strand of DNA complementary to the restriction fragment begins to grow from the end of the primer

  • Whenever a dideoxyribonucleotide is incorporated, chain extension cannot continue



Dideoxy Method - Analysis

  • The product is a mixture of dideoxy-terminated DNA fragments with fluorescent tags

  • These are separated according to weight by electrophoresis and identified by their specific fluorescence



28.16 DNA Synthesis

  • DNA synthesizers use a solid-phase method starting with an attached, protected nucleotide

  • Subsequent protected nucleotides are added and coupled

  • After the final nucleotide has been added, the protecting groups are removed and the synthetic DNA is cleaved from the solid support

  • The bases are protected from reacting



DNA Synthesis: Attachment

  • Attachment of a protected deoxynucleoside to a polymeric or silicate support as an ester of the 3 OH group of the deoxynucleoside

  • The 5 OH group on the sugar is protected as its p-dimethoxytrityl (DMT) ether



DNA Synthesis: DMT Removal

  • Removal of the DMT protecting group by treatment with a moderately weak acid



DNA Synthesis: Coupling

  • The polymer-bound (protected) deoxynucleoside reacts with a protected deoxynucleoside containing a phosphoramidite group at its 3 position, catalyzed by tetrazole, a reactive heterocycle



DNA Synthesis: Oxidation and Cycling

  • Phosphite is oxidized to phosphate by I2

  • The cycle is repeated until the sequence is complete



DNA Synthesis: Clean-up

  • All protecting groups are removed and the product is released from the support by treatment with aqueous NH3



28.17 The Polymerase Chain Reaction (PCR)

  • Copies DNA molecules by unwinding the double helix and copying each strand using enzymes

  • The new double helices are unwound and copied again

  • The enzyme is selected to be fast, accurate and heat-stable (to survive the unwinding)

  • Each cycle doubles the amount of material

  • This is exponential template-driven organic synthesis



PCR: Heating and Reaction

  • The subject DNA is heated (to separate strands) with

    • Taq polymerase (enyzme) and Mg2+
    • Deoxynucleotide triphosphates
    • Two, oligonucleotide primers, each complementary to the sequence at the end of one of the target DNA segments


PCR: Annealing and Growing

  • Temperature is reduced to 37 to 50°C, allowing the primers to form H-bonds to their complementary sequence at the end of each target strand



PCR: Taq Polymerase

  • The temperature is then raised to 72°C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands



PCR: Growing More Chains

  • Repeating the denature–anneal–synthesize cycle a second time yields four DNA copies, a third time yields eight copies, in an exponential series.

  • PCR has been automated, and 30 or so cycles can be carried out in an hour



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