CHEM 380 FINAL EXAM REVIEW

CRISPR/CAS9

CAS9 is an enzyme that cuts DNA at the site in which the guide RNA binds. CRISPR is a technique for gene editing utilizing a piece of single stranded RNA containing two sequences. One is complementary to the target gene sequence while the other recruits the CAS9 enzyme to cleave it.

polymerase chain reaction (PCR)

DNA Amplificstion Steps: 1. Denaturation of double stranded DNA into single stranded DNA 2. Annealing with short complementary primer sequences 3. Chain extension using DNA polymerase and dNTPs 4. This process can be repeatedly cycled to access large quantities of DNA

G-protein couples receptors

GPCRs are integral membrane proteins that transmit important intracellular signals after being activated by ligand binding on the outside of the cell. Ligand binding leads to receptor activation through the release of GDP and replacement via the binding of GTP. The activated complex then release subunits that trigger important downstream biological pathways. When responsibilities are complete, GTP is hydrolyzed back to GDP to return to the inactive state.

glycoproteins

Glycoproteins are generally integral membrane proteins residing in membranes onto which carbohydrate groups are attached so that they are displayed on membrane surfaces. Carbohydrates are either attached on the amide side chain of asparagine (N-linked glycoproteins) or via the hydroxyl group of serine or threonine (O-linked glycoproteins).

yeast 2-hybrid

Goal: probe for protein-protein interactions. 1. yeast GAL4 binds to activator sequence and recruits TATA binding protein to trigger transcription. 2. One protein is attached to the GAL4 DNA-binding domain (bait). Another is attached to GAL4 transcriptional activation domain (prey). 3. when 2 target proteins bind together, the two GAL4 components are brought into proximity, activating transcription and a detectable readout.

Solid phase peptide synthesis

Peptides shorter than ~50 residues can be synthesized by linking together the amino and carboxylate groups of prescribed amino acids to create amide bond linkages. protecting groups must once again be used, particularly to dictate which amine groups are available to react. also synthesized by attaching the growing chain onto an insoluble bead, in this case a polymer. This facilitates the removal of impurities/byproducts through washes after each step and also acts as a protecting group for the C-terminal carboxylate.

site-directed mutagenesis

Purpose: allows the expression of proteins in which single amino acids are replaced. Steps: 1. a plasmid encoding for the natural protein is first denatured into single strands. 2. A short "primer" sequence is added containing a modified codon at the point encoding for the amino acid to be modified. This hybridizes in spite of mismatched base pairs. 3. DNA Polymerase is used to extend the primer to produce a chain complementary to the original plasmid. 4. Strands from the original plasmid can then be degraded, leaving only the modified plasmid.

DNA biosynthesis

Purpose: combine base pairs to create DNA strands How it works: DNA Polymerase enzymes extend upon primer strands that are assembled with template strands that dictate the appropriate sequence through complementarity. The reaction works by reacting the 3'-OH of the elongating chain with the triphosphate version of the appropriate nucleotide to be added. This is a substitution reaction in which pyrophosphate acts as the leaving group. Exonuclease enzymes check that the correct match is made and errors are fixed.

reverse transcriptase

Purpose: synthesize a DNA strand complementary to the viral RNA genome. etroviruses such as HIV have single-stranded RNA genomes and use reverse transcriptase enzymes to convert this into DNA that hijacks host cell replication machinery. The resulting single-stranded DNA can then either be used to synthesize a complementary strand via reverse transcriptase with simultaneous destruction of the RNA strand, or the host DNA polymerase can be hijacked to synthesize the complementary strand

protein binding

R + L <=> R*L R (unbound receptor), L (unbound ligand), R*L (receptor-ligand bound complex) Ka = [R*L]/[R][L] Ka (association constant - units 1/M) Kd = [R][L]/[R*L] Kd (dissociation constant - units M) Ka = 1/Kd Kd = 1/Ka

Ribonuclease

Ribonuclease enzymes contain key histidine residues that interact with both the phosphodiester and 2'-hydroxyl to catalyze hydrolysis. This enables selectivity in the hydrolysis of RNA but not DNA. RNA is cleaved by hydrolysis much more easily than DNA due to the additional 2'-hydroxyl

transcription factors

TFs randomly pass over DNA sequences floating through the cell until they find the proper binding partner. Amino acid side chains interact with the DNA surface through a plethora of contacts, hydrogen bonds, and Van der Waals interactions

Sanger dideoxy sequencing

The Sanger method uses DNA strands synthesized in the presence of four chain-terminating 2',3'- dideoxy nucleotide triphosphates, each bearing a fluorescent dye with different properties. A promiscuous DNA polymerase is used that will accept the modified nucleotides. DNA synthesis is performed with both unlabeled and labeled nucleotide triphosphates present. Capillary electrophoresis is used to separate out the different chain lengths that are synthesized. The fluorescence properties of each strand indicates the identity of the last nucleotide.

DNA hybridization

The annealing of a single-stranded DNA molecule to a complementary sequence for the formation of duplex DNA. 2 dan strands with complementary unhybridized sequences termed sticky end can be hybridized.

anomeric position

The anomeric position on a cyclic sugar occurs one carbon away from the cyclic O bond on the right. It can occur in either the equatorial (β) or axial (α) position and can be interconverted between these positions in a mild acid or base without affecting any of the other bonds. the special reactivity of the anomeric position results from the ability to form a resonance- stabilized carbocation only at this position.

DNA ligation

The final step required to stitch together 2 DNA strands into true duplex DNA. Uses DNA ligase enzyme which links the free 5'-phosphate to the neighboring 3'-hydroxyl to produce a phosphodiester linkage.

glycosidases in disease (neuraminidase)

The glycosidase neuraminidase is a target for influenza virus intervention. After budding, new virions remain bound to the host cells through the binding of viral proteins to N- acetylneuraminic acid sugars on the host cell surface. Neuraminidase hydrolyzes this carbohydrate, releasing the virions. Neuraminidase inhibitors therefore block virion release and proliferation.

DNA denaturation

The separation of a double-stranded DNA molecule into complementary single-stranded molecules by heating.

lipid peroxidation

Lipids are prone to oxidative damage that is disadvantageous since it alters the abundance of important signaling molecules such as arachidonic acid. They are prone to oxidative damage since they have a series of non-conjugated double bonds that place single CH2 groups in between two double bonds. Radicals are stabilized at these positions due to resonance with neighboring double bonds.

interconversion

This is what happens when a mild base or acid is used to change the anomeric OH from the axial position to the equatorial position or vise versa.

protein synthesis by means of ribosomes

Translation: the process by which an RNA sequence is converted into a complementary protein, catalyzed by the ribosome. ribosomes are 1/2 protein and 1/2 RNA they all work the same way - a single lone pair, on a single N atom, on a single adenine carries out deprotonation and activation of the aminoacyl-tRNA.

multivalency

Multivalency is a phenomenon in which biomolecules, and particularly proteins, will bind to multiple ligands simultaneously to enhance binding affinity. This is a common binding mode for protein-carbohydrate binding interactions, which exhibit low affinities in the absence of multivalency.

Amino acid stereochemistry

- 19 of the 20 naturally occurring amino acids are chiral and they occur in the L-form. They have enantiomers which would be in the D-form.

ABPP for fishing out and identifying protein targets of small molecules

- Activity-based protein profiling A. Fatty acids containing click chemistry tags looks similar to natural fatty acids and can be incorporated into lipidated proteins using normal enzymatic machinery. This means that only naturally lipidated proteins will contain the click chemistry tag. B. We can then exploit click chemistry to attach a biotin group to only those proteins that contain the tag. C. In affinity chromatography using an avidin column, only the biotinylated (labeled) proteins will stick, allowing their enrichment. D. We then subject the enriched labeled lipidated proteins to mass spectrometry-based proteomics for identification purposes.

Transcriptional control

- DNA contains instructional sequences such as promoters, operators and terminators that control transcription. - Activation occurs through the recruitment of RNA polymerase to the site to begin transcription

biosynthesis using polyketide synthases

- Fatty acids are part of the complex polyketide family of natural products, so named because they are synthesized by polymerization of the 2-carbon ketene group - core reaction in polyketide synthesis is a modified Claisen Condensation using thioester reactive groups - produces a wide range of natural products with complex structures

RNA structure

- Guanosine and adenosine both contain purine bases. - Cytidine and uridine contain pyrimidine bases. - RNA genetic info is a polymer of nucleosides with phosphodiester linkages connecting the 3' and 5' oxygens of 2 nucleosides. - single stranded and can self assemble into a multitude of different ways (single strand, double strand helix, stem and loop/hair-pin loop, bulge loop, interior loop, junctions/multi-loops).

promoters

- Promoter identities have chemical origins since A-T pairs have weaker π stacking interactions that are more easily bent by TATA-binding protein (TBP). - In addition to TBP, specific transcription factors bind to and activate or repress specific sequences - Promoter sequences like the TATA box are present about 25 base pairs prior to sites where transcription is initiated.

terpene biosynthesis

- Terpenes and isoprenoids are built up from the building blocks dimethylallylpyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). - Each building block brings an additional five carbons, and thus products contain multiples of five carbons (monoterpenes - 10 C, sesquiterpenes - 15 C, diterpenes - 20 C, triterpenes - 30 C) - uilding blocks are combined through reactions similar to alkene addition and electrophilic aromatic substitution, again with pyrophosphate acting as the leaving group

Globo-H

- a hexasaccharide linked to ceramics that's over expressed on the surface of prostate cancer cells. - acts as a biomarker for the disease. - There is evidence that treatment with carrier proteins presenting numerous copies of Globo H on the surface can elicit antibodies against Globo H in an immune response. This has been pursued as an avenue towards an anti-cancer vaccine.

ion channels

- activated by neurotransmitters which commonly act as ligands for both GPCRs and ion channels - typically possess 5 transmembrane domains

base pairing

- adenine (A) binds with thymine (T) - cytosine (C) binds with guanine (G) - Hoogsteen H-bonding can lead to triplex DNA (third strand) where A can interact with 2 Ts and G can interact with 2 Cs, but only under acidic pH.

receptor tyrosine kinase function

- bind growth factors and transduce this signal into cell growth. - 20 known subclasses but not all have known ligands that activate the receptor - commonly aberrant in cancer - forms dimers upon ligand binding and possesses kinase domains that cause autophosphorylation - activated RTKs bind and activate messengers proteins that trigger transcription of DNA via at least four separate downstream pathways - embedded within the cell membrane, and thus must activate other proteins that can infiltrate the nucleus and activate transcription.

carbohydrate-protein binding

- binding proteins is a major role of carbohydrates - recruits proteins to cell membrane surfaces - controls important biological processes, such as cell-cell binding interactions -exploited by viruses and toxins to enter cells

carbohydrate microarrays

- carbohydrate microarrays enable high-throughput detection of protein-carbohydrate binding interactions by immobilizing carbohydrates onto surfaces and treating with proteins of interest.

two component signaling pathways

- cell surface receptors are used to respond to chemical signals that are not capable of permeating the cell membrane. - cytokines are small proteins that act in this way, and include the interleukins, interferons and chemokines - cell surface receptors are embedded in the surface, and when activated phosphorylate a messenger protein to form a dimer which enters the nucleus and activates transcription

fatty acid structure

- contain 12-20 carbons and a polar carboxylic acid head group. - can be saturated or unsaturated - natural unsaturated fatty acids contain cis double bonds (the double bonds are not conjugated)

Prostaglandins

- created by the cyclization of arachidonic acid through cyclooxygenase enzymes. - increased prostaglandins causes neuron sensitization, enhancing pain. -pain killers target cyclooxygenase so that prostaglandins are never created in the first place.

antisense DNA

- exploits extensions on DNA sequences to delete mRNA encoding for target proteins . 1. antisense DNA conjugates with target sequence on mRNA 2. This flags the sequence for deletion 3. Ribonuclease H degrades flagged sequence

GPI anchors

- glycophosphatidylinositol anchors - common in infectious organism and can also be used to link proteins to human cell membranes - contain a variable complex sugar linked to a phospholipid scaffold by an inositol group - Inositol is a cyclitol (cyclohexanol) with specific stereochemistry that lacks the oxygen inside the ring that is seen in sugars.

terpene structure

- hydrocarbon products from the synthesis of polymers made of isoprene units - those with functional groups are known as isoprenoids - for simpler structures the diels alder reaction can be used to make terpenes.

arachidonic acid

- important fatty acid that leads into the prostaglandin/eicosanoid inflammation pathway - begins w the cyclization of arachidonic acid by cyclooxygenase (COX) enzymes

phospholipid structure

- key components of cellular membranes that often control important biological processes.

phospholipid signaling process

- key signaling role of lipids involves binding interactions with proteins that anchor the bound protein on the membrane surface and typically activates their function. - ex: the binding of Akt (protein kinase B) to PI(3,4,5)P3. This activates Akt, which turns on numerous cell growth pathways. The enzyme that produces PI(3,4,5)P3 by installing the key 3-phosphate, phosphatidylinostiol 3-kinase, is the second most commonly mutated enzyme in cancer.

steroid biosynthesis

- long chain terpenes are generated through tail-to-tail coupling of building blocks. This is followed by multiple cyclization reactions that results in the four fused ring systems and six stereogenic centers of the core steroid lanosterol. - steroids have trans-fused rings, so they possess rigidity because they cannot undergo cyclohexane chair flips, while cis fused rings can - Trans fused is more stable because all of the ring junctions are in equatorial positions, while cis fused must place some ring junctions in axial positions.

RNA biosynthesis (Transcription)

- performed by RNA polymerase enzymes - synthesized strand is analogous to sense/coding strand of DNA and is complementary to antisense/template strand. -RNA polymerase separates the DNA strand at the target site, and synthesizes an RNA sequence that is complementary to the antisense strand

chitin

- polysaccharide - primary component of the exoskeletons of insects and crustaceans - hydrogen bonding from the amide groups enhances strength and rigidity

cellulose

- polysaccharide - structural component of plant cell walls and primary component of paper - more crystalline than amylose

amylose

- polysaccharide - component of starch along with branched form of amylopectin

DNA structure

- primary genetic storage molecule - nucleosides consist of ribose sugar backbone with nitrogen heterocycle - big difference between DNA and RNA is the lack of 2' -OH in DNA - numbering is used for the backbone (2', 3', 5')

hyaluronic acid

- repeating disaccharide - gel-like martial that reduces friction in joints and makes up cartilage - hygroscopic

lipid self-assembly

- self-assemble into different structures in water such that the polar head groups maximize attractive forces with water solvent, while the hydrophobic lipid tails aggregate so as to avoid unproductive interactions with water solvent - liposome (think phospholipid bilayer) - micelle - inverted hexagonal phase

siRNA

- small interfering RNA - diminishes production of certain proteins by deleting mRNA encoding for that protein. 1. short double stranded RNA (siRNA) is added to cells 2. This triggers assembly of the RISC complex 3. RISC complex seeks out and degrades complementary mRNA sequences.

amino acid acidity/basicity

- terminal carboxylic acids will always be deprotonated - N-terminal amines are 29-86% protonated - cyclic amines will not be protonated - terminal amines (not in main chain) will be protonated - terminal alcohol groups not part of carboxylic acids will not be deprotonated

transition state inhibitor design

- the best enzyme inhibitors mimic the transition state. - neuraminidase inhibitors typically consist of modified sugars possessing planarity at the anomeric carbon by introducing a double bond there. otherwise, they have positively charges groups to bind carboxylates and negatively charged carboxylates to bind carboxylic acids.

tRNA synthetase

- this enzyme loads the correct amino acids onto tRNA.

nuclear receptors

- transcription factors that bind to small molecules - 28 types encoded by humans, only 8 have been matched with high affinity small molecule ligand. remaining 20 are orphan receptors and may bind to more abundant signaling molecules with lower affinity. - many ligands for nuclear receptors are steroids but there is no natural receptor for cholesterol

tRNA

- transfer RNA - the central loop of each tRNA contains an anticodon that is complementary to the codon (which encodes for an amino acid), and the proper amino acid attaches to the 3' end of the tRNA. - hybridization of the codon and anticodon leads to the addition of the correct amino acid to the protein sequence.

Amino acid structure

-fischer projections are always drawn with the carboxylic acid up and amine pointing to the left (this is the naturally occurring L-form) - one achiral amino acid : glycine - one with R configuration : cysteine - all 18 others have S configuration

plasmid DNA

-small circular pieces of DNA produced by bacteria. they are effective at hijacking host machinery for replication. contain defined sequences that dictate transcription, such as the origin of replication (ORI) sequence that instructs enzymes to make copies of the plasmid.

carbohydrate synthesis

1. "glycosylation" reaction: linkage of sugars 2. Synthesis of "Glycosyl donor" 3. Synthesis of Glycosyl acceptor"

PIP3/Akt pathways

1. RTKs (w a few other proteins) recruit phosphatidylinositol 3-kinase (PI-3K) to the membrane surface 2. PI-3K catalyzes the phosphorylation of PI(4,5)P2 to PIP3 3. PIP3 binds Akt and PDK1 and recruits both to the membrane 4. on the membrane surface, PDK1 phosphorylates Akt which activates it 5. Akt then activates numerous cell growth pathways.

PLC/IP3/DAG/Ca+2 pathways

1. RTKs activate the enzyme phospholipase C (PLC) which hydrolyzes the lipid PI(4,5)P2 to DAG and IP3. 2. IP3 causes the release of calcium stores from the endoplasmic reticulum (ER). 3. This activates production of protein kinase C (PKC) enzymes 4. PKCs bind to DAG on the plasma membrane and activate multiple cell growth pathways

interconversion between Fischer/Hayworth/chair forms

1. convert Fischer projection to a Hayworth projection. OH on the right = down in Hayworth, OH on the left = up in Hayworth 2. rotate the penultimate carbon 3. cyclization of the molecule 4. create chair structure trying to keep as many group as possible in the equatorial position.

factors affecting reactivity

1. electronegativity: as electronegativity increases, so does acidity (bc conj. base is more stable) 2. orbital overlap: larger atoms are more acidic because they have less orbital overlap. 3. octet rule: if the product has a full octet, it is more stable so the starting material is more acidic 4. hybridization: as s-character increases, so does acidity. ie sp> sp2> sp3 bc 50%>33%>25% 5. resonance: if the conj. base is resonance stabilized then the molecule is more acidic. if the acid is resonance stabilized then the molecule is less acidic 6. aromaticity: if the acid is aromatic, then its weak. if the acid has an aromatic conjugate base, then its strong. 7. steric: when there is less steric hindrance, the molecule is more reactive.

Engineered tRNA syntheses using nonsense codons

1. identify a tRNA (typically from another organism) that binds to a nonsense codon (one that doesn't encode for amino acids in humans). 2. genetically engineer the tRNA synthetase associated with that tRNA so it will transfer the desired unnatural amino acid onto its tRNA. This can be done by modifying amino acids on the tRNA synthetase enzyme so it will accept the new amino acid (use site-directed mutagenesis or error-prone PCR) 3. to express the modified protein, feed a plasmid containing DNA encoding for the desired protein but also containing a nonsense codon at the site of modification along with a DNA plasmid encoding for the mutant tRNA synthetase to cells. Then they will synthesize the modified protein.

non-covalent binding interactions

1. ion pairing / electrostatic attraction 2. hydrogen bonding 3. disulfide bonds (technically covalent, but reversible) 4. hydrophobic interactions (due to van Der waals) 5. cation-π interactions 6. π-π interactions (π stacking)

unnatural amino acid incorporation

1. modified amino acid analogs that infiltrate normal pathways 2. engineered tRNA syntheses using nonsense codons

Microarrays steps

1. synthesizing DNA and RNA libraries: DNA "libraries" can be synthesized by performing automated DNA synthesis with all 4 deoxynucleotides mixed simultaneously. commonly synthesized with short identical pieces at the beginning to facilitate PCR amplification using common primers. 2. immobilization onto surfaces: can be immobilized onto surfaces via a variety of methods including physical adsorption, covalent attachment and non-covalent deposition. 3. genomic analysis: performed through extraction of RNA, reverse transcriptase generation of DNA, fluorescent labeling, hybridization with a DNA microarray library and fluorescence detection of bound nucleotides. Comparisons between normal and diseased cells are used to probe for disease biomarkers.

Modified amino acid analogs that infiltrate normal pathways

1. use an unnatural amino acid similar to a natural amino acid. 2. load the unnatural amino acids onto tRNA synthetases specific to the similar natural amino acid 3. ensure that the natural amino acid is lowered through inhibition 4. introduce these amino acids inside the cell

Fischer projections

A drawing style that is often used when dealing with compounds bearing multiple chirality centers, especially for carbohydrates. For sugars, Fischer projections are used to show the chiral centers and the form of the molecule (ie L or D)

capillary electrophoresis

capillary electrophoresis improves resolution and throughput while minimizing amount of sample needed by passing samples through a capillary between the anode and cathode. A laser and detector are set up along the capillary for detection of fluorescence-tagged DNA. These can be multiplexed to analyze numerous samples simultaneously.

conformational analysis and chair structure

cyclohexane are very stable and common in natural products. They are drawn in chair confirmations where substituents can either be in axial or equatorial positions. Axial substituents point up or down and equatorial substituents are parallel to the C-C bonds in the ring that are 2 carbons away. Larger substituents should be placed in the equatorial position. this can be achieved by performing a chair flip on the molecule.

double helix formation

due to multiple factors - base pairing from complementary hydrogen bonding - hydrophobic heterocyclic bases project to the inner core of DNA away from water - charged phosphodiester lines the outside of double helix for favorable water interactions - π stacking interactions between heterocyclic bases.

Microarrays

enables high-throughput genomic studies. fabricated by immobilizing libraries of single-stranded DNA sequences at different locations on a microscope slide or microplate. Hundreds of thousands of unique DNA sequences can be presented at distinct locations on a single slide.

recombinant DNA

enables highly specific genetic manipulation as a great tool for testing biological function. allows the combination of DNA from different samples, and the introduction of desired DNA sequences into plasmids for delivery to cells. Restriction endonucleases specifically cleave DNA at palindromic sites that are often shared between different organisms. Cleavage of DNA from different organisms yields complementary sticky ends that can be annealed and ligated to combine different sequences

posttranslational modification

enzymatic modifications of proteins. 1. phosphonylation (forward: kinase enzyme/reverse: phosphatase enzyme) - adds PO3 2- to protein at OH group. 2. sulfurylation (forward: sulfotransferase/reverse: sulfatase) - adds SO3 - to protein at OH group. 3. acetylation (forward: acyltransferase/reverse: deaetylase) - adds an acetyl group to protein at NH3 +. 4. Alkylation (forward: methyltransferase/reverse: demethylase) - adds a methyl group to protein at NH3 +. 5. glycosylation (forward: glycosyltransferase/reverse: glycosylase) - adds a sugar to protein at OH. 6. Lipidation - adds a lipid to protein at thiol (SH) group.

endonuclease enzymes

enzymes that catalyze the hydrolysis of phosphodiester bonds in nature to breakdown DNA. Restriction endonucleases: cleave specific sequences of DNA called restriction sites. Other endonucleases: cleave DNA in a non-specific manner.

Kd/Ka

equals Kd^2 because they are reciprocals of each other Kd/(1/Kd) = Kd^2

stereochemistry

have the same connectivity and only differ based on geometric presentation Enantiomers: mirror images of the same molecule. diasteriomers: only a portion of chiral centers are mirrored. (ie if there are 2 stereocenters, only 1 mirrored would be diastereomers, but both mirrored would be enantiomers)

polysaccharides

polymers composed of repeating units of monosaccharides. These have widely variable properties depending upon the building blocks and linkages.

chemiluminescence

process by which photons are emitted as a product of a chemical reaction without requiring irradiation with light

selective modification

since there is special reactivity at the anomeric position, it enables selective modification. This means that we can add onto the cyclic sugar at the anomeric position.

sugar stereochemistry

sugars, like amino acids have D and L forms that they can take when drawn as Fischer projections. Unlike amino acids however, they occur naturally in D-form where the OH on the lowest chiral carbon points to the right. D and L forms of the same sugar are enantiomers and different sugars are typically diasteriomers of each other. Some oxidized sugars are achiral even though they have chiral centers because they have a mirror plane (these are meso form).

SELEX

systematic evolution of ligands by exponential enrichment 1. random sequence RNA pool is combined with target 2. bound and unbound RNA are put through a nitrocellulose filter and unbound RNA is discarded 3. bound RNA goes through reverse transcription, amplification (using PCR), and in vitro transcription 4. selected and amplified RNA is put through the cycle again with the target.

double helix intercalation

the process by which polyaromatic molecules insert between heterocyclic bases.

glycosidase mechanism

there are two possible mechanisms- one with interconversion at the anomeric position and one without. For the first one, a single SN2 reaction is performed. The reactant has an anomeric OR group in the equatorial position, and the product has an anomeric OH group in the axial position. The water attacks from the bottom, interconverting the position. For the second one, two SN2 reactions are performed and the equatorial anomeric position is retained because the water attacks from above.

gel electrophoresis

used to separate DNA strands and thereby characterize the purity and identity of samples. In gels, DNA migrates towards the positive electrode (anode) due to the negative charge. Shorter DNA strands migrate faster since they can pass through pores in the agarose gel. A "ladder" or "marker" sample with known sequences is run alongside studies as a reference. Many dyes such as ethidium bromide exhibit pronounced fluorescence when intercalated into DNA. These enable the visualization of DNA bands in gel.

pyrosequencing

uses a coupled enzyme assay for extremely sensitive detection of nucleotide incorporation via chemiluminescence without requiring separation. Each natural nucleotide triphosphate is added one at a time in an automated manner, and chemiluminescence is detected, indicating the correct nucleotide.

glycolipids

way to display carbohydrates on the surfaces of cells by attaching them to lipids that are embedded within cellular membranes. Sugars are commonly attached to the lipid ceramide, resulting in a family called glycosphingolipids.

Fischer proof for determining sugar structure

in the 1890s, Emil Fischer came up with a way fro determining sugar structure by performing a series of reactions and studying the stereochemical results. Fischer knew the form of glucose (C6H12O6) which gives 16 different structures, 8 in D form and 8 in L form. He focused solely on the D form structures. Reactions: 1. end group oxidation to carboxylic acids - uses HNO3. Upon oxidation, only chiral products will be optically active, so meso products will be optically inactive. If the molecule you're looking for is optically active, rule out 1 and 7 and continue with the rest. 2. Wohl Degradation truncates chain by removing aldehyde - uses H2NOH, Ac2O, NaOAc, and NaOH to remove top chiral center. You can then rerun the oxidation reaction and rule out any structures that give achiral truncated molecules (2,5,6) - we still have 3, 4, and 8 left. 3. Kilani Fischer chain extension - uses NaCN, NaOH, Na/Hg H2O to add back the top chiral center. you can then rerun the oxidation reaction and rule out any structures that give achiral products (12 can be ruled out because of 7 which rules out 8) - this leaves only 3 and 4 - glucose and mannose 4. glucose and mannose can be differentiated 3 ways. (a) Oxidation of Mannose yields dicarboxylic acid that is not produced by oxidation of any other sugar. (b) Oxidation of Glucose yields same dicarboxylic acid as oxidation of Gulose. (c) perform end interchange reactions: switch the two terminal groups (CHO and CH2OH) and then rotate the molecule 180º clockwise. Mannose will give you the same structure but glucose will give L-gulose.

EC50

indicates the half maximal effective concentration for a molecule that promotes activity.

IC50

indicates the half maximal inhibitory complex for a molecule that works as an inhibitor