Bioconjugation is a chemical strategy to form a stable
covalent link between two molecules, at least one of which is a
biomolecule
A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or development. Biomolecules include large ...
.
Function
Recent advances in the understanding of biomolecules enabled their application to numerous fields like medicine and materials. Synthetically modified biomolecules can have diverse functionalities, such as tracking cellular events, revealing
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
function, determining
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
biodistribution Biodistribution is a method of tracking where compounds of interest travel in an experimental animal or human subject. For example, in the development of new compounds for PET (positron emission tomography) scanning, a radioactive isotope is chemic ...
,
imaging
Imaging is the representation or reproduction of an object's form; especially a visual representation (i.e., the formation of an image).
Imaging technology is the application of materials and methods to create, preserve, or duplicate images.
...
specific
biomarkers
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
, and delivering
drugs
A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via inhalat ...
to targeted cells.
Bioconjugation is a crucial strategy that links these modified biomolecules with different
substrates.
Synthesis
Synthesis
Synthesis or synthesize may refer to:
Science Chemistry and biochemistry
*Chemical synthesis, the execution of chemical reactions to form a more complex molecule from chemical precursors
** Organic synthesis, the chemical synthesis of organ ...
of bioconjugates involves a variety of challenges, ranging from the simple and nonspecific use of a
fluorescent dye
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
marker to the complex design of
antibody drug conjugates.
As a result, various bioconjugation reactions –
chemical reactions
A chemical reaction is a process that leads to the chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the positions of electrons in the forming and breaking ...
connecting two biomolecules together – have been developed to chemically modify proteins. Common types of bioconjugation reactions on proteins are coupling of
lysine
Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −C ...
, of
cysteine
Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile.
When present as a deprotonated catalytic residue, sometime ...
and of
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the Gr ...
amino acid residues
Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers specifically polypeptides formed from sequences of amino acids, the monomers of the polymer. A single amino acid monomer may ...
; and modification of
tryptophan
Tryptophan (symbol Trp or W)
is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α- carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...
amino acid residues and of the
N- and
C- terminus.
However, these reactions often lack
chemoselectivity
Chemoselectivity is the preferential outcome of a chemical reaction over a set of possible alternative reactions.
In another definition, chemoselectivity refers to the selective reactivity of one functional group in the presence of others; often ...
and efficiency, because they depend on the presence of native amino acid residues, which are usually present in large quantities that hinder selectivity. There is an increasing need for chemical strategies that can effectively attach synthetic molecules site specifically to proteins. One strategy is to first install a unique
functional group
In organic chemistry, a functional group is a substituent or moiety in a molecule that causes the molecule's characteristic chemical reactions. The same functional group will undergo the same or similar chemical reactions regardless of the rest ...
onto a protein, and then a
bioorthogonal or
click type
Click consonants, or clicks, are speech sounds that occur as consonants in many languages of Southern Africa and in three languages of East Africa. Examples familiar to English-speakers are the '' tut-tut'' (British spelling) or '' tsk! tsk!'' ...
reaction is used to couple a biomolecule with this unique functional group.
The bioorthogonal reactions targeting non-native functional groups are widely used in bioconjugation chemistry. Some important reactions are modification of
ketone
In organic chemistry, a ketone is a functional group with the structure R–C(=O)–R', where R and R' can be a variety of carbon-containing substituents. Ketones contain a carbonyl group –C(=O)– (which contains a carbon-oxygen double bo ...
and
aldehydes
In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl group ...
,
Staudinger ligation
The Staudinger reaction is a chemical reaction of an organic azide with a phosphine or phosphite produces an iminophosphorane. The reaction was discovered by and named after Hermann Staudinger. The reaction follows this stoichiometry:
:R3P + R' ...
with
organic azide
An organic azide is an organic compound that contains an azide (–) functional group. Because of the hazards associated with their use, few azides are used commercially although they exhibit interesting reactivity for researchers. Low molecular w ...
s, copper-catalyzed
Huisgen cycloaddition of azides, and strain promoted Huisgen cycloaddition of azides.
Common bioconjugation reactions
The most common bioconjugations are coupling of a small molecule (such as
biotin
Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...
or a fluorescent dye) to a protein, or protein-protein conjugations, such as the coupling of an antibody to an enzyme.
Other less common molecules used in bioconjugation are
oligosaccharides
An oligosaccharide (/ˌɑlɪgoʊˈsækəˌɹaɪd/; from the Greek ὀλίγος ''olígos'', "a few", and σάκχαρ ''sácchar'', "sugar") is a saccharide polymer containing a small number (typically two to ten) of monosaccharides (simple sugar ...
,
nucleic acids
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main clas ...
,
synthetic polymers
Some familiar household synthetic polymers include: Nylons in textiles and fabrics, Teflon in non-stick pans, Bakelite for electrical switches, polyvinyl chloride (PVC) in pipes, etc. The common PET bottles are made of a synthetic polymer, polye ...
such as
polyethylene glycol
Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...
, and
carbon nanotubes
A scanning tunneling microscopy image of a single-walled carbon nanotube
Rotating single-walled zigzag carbon nanotube
A carbon nanotube (CNT) is a tube made of carbon with diameters typically measured in nanometers.
''Single-wall carbon nan ...
.
Antibody-drug conjugates
Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. As of 2019, some 56 phar ...
such as
Brentuximab vedotin
Brentuximab vedotin, sold under the brand name Adcetris, is an antibody-drug conjugate medication used to treat relapsed or refractory Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL), a type of T cell non-Hodgkin lymphoma. ...
and
Gemtuzumab ozogamicin
Gemtuzumab ozogamicin, sold under the brand name Mylotarg, is an antibody-drug conjugate (a drug-linked monoclonal antibody) that is used to treat acute myeloid leukemia.
The most common grade 3 and higher adverse reactions that occurred during ...
are also examples of bioconjugation, and are an active area of research in the pharmaceutical industry. Recently, bioconjugation has also gained importance in
nanotechnology
Nanotechnology, also shortened to nanotech, is the use of matter on an atomic, molecular, and supramolecular scale for industrial purposes. The earliest, widespread description of nanotechnology referred to the particular technological goal o ...
applications such as bioconjugated
quantum dots
Quantum dots (QDs) are semiconductor particles a few nanometres in size, having optical and electronic properties that differ from those of larger particles as a result of quantum mechanics. They are a central topic in nanotechnology. When the ...
.
Reactions of lysine residues
The
nucleophilic
In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...
lysine residue is commonly targeted site in protein bioconjugation, typically through
amine
In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen atoms have been replaced by a substituen ...
-reactive
''N''-hydroxysuccinimidyl (NHS) esters
In chemistry, an ester is a compound derived from an oxoacid (organic or inorganic) in which at least one hydroxyl group () is replaced by an alkoxy group (), as in the substitution reaction of a carboxylic acid and an alcohol. Glycerides are ...
.
To obtain optimal number of
deprotonated
Deprotonation (or dehydronation) is the removal (transfer) of a proton (or hydron, or hydrogen cation), (H+) from a Brønsted–Lowry acid in an acid–base reaction.Henry Jakubowski, Biochemistry Online Chapter 2A3, https://employees.csbsju.edu ...
lysine residues, the
pH of the
aqueous solution
An aqueous solution is a solution in which the solvent is water. It is mostly shown in chemical equations by appending (aq) to the relevant chemical formula. For example, a solution of table salt, or sodium chloride (NaCl), in water would be re ...
must be below the
pKa
PKA may refer to:
* Professionally known as:
** Pen name
** Stage persona
* p''K''a, the symbol for the acid dissociation constant at logarithmic scale
* Protein kinase A, a class of cAMP-dependent enzymes
* Pi Kappa Alpha, the North-American so ...
of the lysine
ammonium
The ammonium cation is a positively-charged polyatomic ion with the chemical formula or . It is formed by the protonation of ammonia (). Ammonium is also a general name for positively charged or protonated substituted amines and quaternary a ...
group, which is around 10.5, so the typical pH of the reaction is about 8 and 9. The common reagent for the coupling reaction is NHS-ester (shown in the first reaction below in Figure 1), which reacts with nucleophilic lysine through a lysine
acylation
In chemistry, acylation (or alkanoylation) is the chemical reaction in which an acyl group () is added to a compound. The compound providing the acyl group is called the acylating agent.
Because they form a strong electrophile when treated with ...
mechanism. Other similar reagents are
isocyanates
In organic chemistry, isocyanate is the functional group with the formula . Organic compounds that contain an isocyanate group are referred to as isocyanates. An organic compound with two isocyanate groups is known as a diisocyanate. Diisocyan ...
and
isothiocyanates
In organic chemistry, isothiocyanate is the functional group , formed by substituting the oxygen in the isocyanate group with a sulfur. Many natural isothiocyanates from plants are produced by enzymatic conversion of metabolites called glucosin ...
that undergo a similar mechanism (shown in the second and third reactions in Figure 1 below).
Benzoyl
In organic chemistry, benzoyl (, ) is the functional group with the formula C6H5CO-. It can be viewed as benzaldehyde missing one hydrogen.
The term "benzoyl" should not be confused with benzyl, which has the formula C6H5CH2. The benzoyl group ...
fluorides (shown in the last reaction below in Figure 1), which allows for lysine modification of proteins under mild conditions (low temperature, physiological
pH), were recently proposed as an alternative to classically used lysine specific reagents.
Reactions of cysteine residues
Because free cysteine rarely occurs on protein surface, it is an excellent choice for chemoselective modification. Under basic condition, the cysteine residues will be deprotonated to generate a
thiolate
In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl grou ...
nucleophile, which will react with soft
electrophiles
In chemistry, an electrophile is a chemical species that forms bonds with nucleophiles by accepting an electron pair. Because electrophiles accept electrons, they are Lewis acids. Most electrophiles are positively charged, have an atom that carries ...
, such as
maleimides
Maleimide is a chemical compound with the formula H2C2(CO)2NH (see diagram). This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -C(O)NHC(O)- functional group. M ...
and
iodoacetamide
2-Iodoacetamide is an alkylating agent used for peptide mapping purposes. Its actions are similar to those of iodoacetate. It is commonly used to bind covalently with the thiol group of cysteine so the protein cannot form disulfide bonds. Also used ...
s (shown in the first two reactions in Figure 2 below). As a result, a
carbon-sulfur bond is formed. Another modification of cysteine residues involves the formation of
disulfide bond
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
(shown in the third reaction in Figure 2). The
reduced cysteine residues react with
exogenous
In a variety of contexts, exogeny or exogeneity () is the fact of an action or object originating externally. It contrasts with endogeneity or endogeny, the fact of being influenced within a system.
Economics
In an economic model, an exogeno ...
disulfides, generating new a disulfide bond on the protein. An excess of disulfides is often used to drive the reaction, such as 2-thiopyridone and 3-carboxy-4-nitrothiophenol.
Electron-deficient
alkynes
\ce
\ce
Acetylene
\ce
\ce
\ce
Propyne
\ce
\ce
\ce
\ce
1-Butyne
In organic chemistry, an alkyne is an unsaturated hydrocarbon containing at least one carbon—carbon triple bond. The simplest acyclic alkynes with only one triple bond and no ...
were demonstrated to selectively react with cysteine residues of proteins in the presence of other nucleophilic amino acid residues. Depending on the alkyne substitution, these reactions can produce either cleavable (when alkynone derivatives are used), or hydrolytically stable bioconjugates (when
3-arylpropiolonitriles
3-Arylpropiolonitriles (APN) belong to a class of electron-deficient alkyne derivatives substituted by two electron-withdrawing groups – a nitrile and an aryl moieties. Such activation results in improved selectivity towards highly reactive thio ...
are used; the last reaction below in Figure 2).
Reactions of tyrosine residues
Tyrosine residues are relatively unreactive; therefore they have not been a popular targets for bioconjugation. Recent development has shown that the tyrosine can be modified through
electrophilic aromatic substitution
Electrophilic aromatic substitution is an organic reaction in which an atom that is attached to an aromatic system (usually hydrogen) is replaced by an electrophile. Some of the most important electrophilic aromatic substitutions are aromatic ni ...
s (EAS) reactions, and it is selective for the
aromatic
In chemistry, aromaticity is a chemical property of cyclic ( ring-shaped), ''typically'' planar (flat) molecular structures with pi bonds in resonance (those containing delocalized electrons) that gives increased stability compared to satur ...
carbon adjacent to the
phenolic
Phenolic is an adjective and a substantive (noun) that may apply to :
* Phenol (or carbolic acid), a colorless crystalline solid and aromatic compound
* Phenols, a class of chemical compounds that include phenol
* Phenolic content in wine
* Phenol ...
hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
group.
This becomes particularly useful in the case that cysteine residues cannot be targeted. Specifically,
diazonium effectively couples with tyrosine residues (
diazonium salt
Diazonium compounds or diazonium salts are a group of organic compounds sharing a common functional group where R can be any organic group, such as an alkyl or an aryl, and X is an inorganic or organic anion, such as a halide.
General properti ...
shown as reagent in the first reaction in Figure 3 below), and an
electron withdrawing substituent in the 4-position of diazonium salt can effectively increase the efficiency of the reaction. Cyclic diazodicarboxyamide derivative like
4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) were reported for selective bioconjugation on tyrosine residues (the second reaction in Figure 3 below).
A three-component
Mannich-type reaction with
aldehydes
In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl group ...
and
aniline
Aniline is an organic compound with the formula C6 H5 NH2. Consisting of a phenyl group attached to an amino group, aniline is the simplest aromatic amine
In organic chemistry, an aromatic amine is an organic compound consisting of an aroma ...
s (the last reaction in Figure 3) was also described to be relatively tyrosine-selective under mild optimised reaction conditions.
Reactions of N- and C- termini
Since natural amino acid residues are usually present in large quantities, it is often difficult to modify one single site. Strategies targeting the termini of protein have been developed, because they greatly enhanced the site selectivity of protein modification. One of the N- termini modifications involves the
functionalization of the terminal amino acid. The
oxidation
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
of N-terminal
serine
Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...
and
threonine
Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), a carboxyl group (which is in the deprotonated −COO ...
residues are able to generate N-terminal aldehyde, which can undergo further bioorthogonal reactions (shown in the first reaction in Figure 4). Another type of modification involves the
condensation
Condensation is the change of the state of matter from the gas phase into the liquid phase, and is the reverse of vaporization. The word most often refers to the water cycle. It can also be defined as the change in the state of water vapor to ...
of N-terminal cysteine with aldehyde, generating
thiazolidine
Thiazolidine is a heterocyclic organic compound with the formula (CH2)3(NH)S. It is a 5-membered saturated ring with a thioether group and an amine group in the 1 and 3 positions. It is a sulfur analog of oxazolidine. Thiazolidine is a colorless ...
that is stable at high pH (second reaction in Figure 4). Using
pyridoxal phosphate
Pyridoxal phosphate (PLP, pyridoxal 5'-phosphate, P5P), the active form of vitamin B6, is a coenzyme in a variety of enzymatic reactions. The International Union of Biochemistry and Molecular Biology has catalogued more than 140 PLP-dependent ac ...
(PLP), several N-terminal amino acids can undergo
transamination
Transamination is a chemical reaction that transfers an amino group to a ketoacid to form new amino acids. This pathway is responsible for the deamination of most amino acids. This is one of the major degradation pathways which convert essential a ...
to yield N-terminal
aldehyde
In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl grou ...
, such as
glycine
Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid (carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinogeni ...
and
aspartic acid
Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
(third reaction in Figure 4).
An example of C-termini modification is the
native chemical ligation Native Chemical Ligation (NCL) is an important extension of the chemical ligation concept for constructing a larger polypeptide chain by the covalent condensation of two or more unprotected peptides segments. Native chemical ligation is the most e ...
(NCL), which is the coupling between a C-terminal
thioester
In organic chemistry, thioesters are organosulfur compounds with the functional group . They are analogous to carboxylate esters () with the sulfur in the thioester playing the role of the linking oxygen in the carboxylate ester, as implied by t ...
and a N-terminal cysteine (Figure 5).
Bioorthogonal reactions
Modification of ketones and aldehydes
A ketone or aldehyde can be attached to a protein through the oxidation of N-terminal serine residues or transamination with PLP. Additionally, they can be introduced by incorporating
unnatural amino acids via the
Tirrell method or
Schultz method Schultz is a German surname derived from '' Schultheiß'', meaning village headman or constable/sheriff in the medieval sense (akin to today's office of mayor). It has many variations, such as Schuldt, Schulte, Schulten, Schultes, Schultheis, S ...
.
They will then selectively condense with an alkoxyamine and a
hydrazine
Hydrazine is an inorganic compound with the chemical formula . It is a simple pnictogen hydride, and is a colourless flammable liquid with an ammonia-like odour. Hydrazine is highly toxic unless handled in solution as, for example, hydrazine ...
, producing
oxime
In organic chemistry, an oxime is a organic compound belonging to the imines, with the general formula , where R is an organic side-chain and R’ may be hydrogen, forming an aldoxime, or another organic group, forming a ketoxime. O-substituted ...
and
hydrazone
Hydrazones are a class of organic compounds with the structure . They are related to ketones and aldehydes by the replacement of the oxygen =O with the = functional group. They are formed usually by the action of hydrazine on ketones or aldehydes. ...
derivatives (shown in the first and second reactions, respectively, in Figure 6). This reaction is highly chemoselective in terms of protein bioconjugation, but the
reaction rate
The reaction rate or rate of reaction is the speed at which a chemical reaction takes place, defined as proportional to the increase in the concentration of a product per unit time and to the decrease in the concentration of a reactant per unit ...
is slow. The mechanistic studies show that the
rate determining step
In chemical kinetics, the overall rate of a reaction is often approximately determined by the slowest step, known as the rate-determining step (RDS or RD-step or r/d step) or rate-limiting step. For a given reaction mechanism, the prediction of the ...
is the
dehydration
In physiology, dehydration is a lack of total body water, with an accompanying disruption of metabolic processes. It occurs when free water loss exceeds free water intake, usually due to exercise, disease, or high environmental temperature. Mil ...
of
tetrahedral intermediate
A tetrahedral intermediate is a reaction intermediate in which the bond arrangement around an initially double-bonded carbon atom has been transformed from trigonal to tetrahedral. Tetrahedral intermediates result from nucleophilic addition to a c ...
, so a mild
acidic
In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a sequ ...
solution is often employed to accelerate the dehydration step.
![Figure 6](https://upload.wikimedia.org/wikipedia/commons/0/0a/Figure_6._Bioconjugation_strategies_for_targeting_ketones_and_aldehydes.jpg)
The introduction of nucleophilic catalyst can significantly enhance reaction rate (shown in Figure 7). For example, using
aniline
Aniline is an organic compound with the formula C6 H5 NH2. Consisting of a phenyl group attached to an amino group, aniline is the simplest aromatic amine
In organic chemistry, an aromatic amine is an organic compound consisting of an aroma ...
as a nucleophilic catalyst, a less populated protonated
carbonyl
In organic chemistry, a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom: C=O. It is common to several classes of organic compounds, as part of many larger functional groups. A compound containing a ...
becomes a highly populated protonated
Schiff base
In organic chemistry, a Schiff base (named after Hugo Schiff) is a compound with the general structure ( = alkyl or aryl, but not hydrogen). They can be considered a sub-class of imines, being either secondary ketimines or secondary aldimine ...
.
In other words, it generates a high concentration of reactive electrophile. The oxime ligation can then occur readily, and it has been reported that the rate increased up to 400 times under mild acidic condition.
The key of this catalyst is that it can generate a reactive electrophile without competing with desired product.
![Figure 7](https://upload.wikimedia.org/wikipedia/commons/0/0e/Figure_7._Nucleophilic_catalysis_of_oxime_ligation.jpg)
Recent developments that exploit proximal functional groups have enabled hydrazone condensations to operate at 20 M
−1s
−1 at neutral pH while oxime condensations have been discovered which proceed at 500-10000 M
−1s
−1 at neutral pH without added catalysts.
Staudinger ligation with azides
The
Staudinger ligation
The Staudinger reaction is a chemical reaction of an organic azide with a phosphine or phosphite produces an iminophosphorane. The reaction was discovered by and named after Hermann Staudinger. The reaction follows this stoichiometry:
:R3P + R' ...
of azides and
phosphine
Phosphine (IUPAC name: phosphane) is a colorless, flammable, highly toxic compound with the chemical formula , classed as a pnictogen hydride. Pure phosphine is odorless, but technical grade samples have a highly unpleasant odor like rotting ...
has been used extensively in field of chemical biology. Because it is able to form a stable
amide bond
In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent organic groups or hydrogen atoms. The amide group is called a peptide bond when it is p ...
in living cells and animals, it has been applied to modification of
cell membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
,
''in vivo'' imaging, and other bioconjugation studies.
![Figure 8](https://upload.wikimedia.org/wikipedia/commons/d/d3/Figure_8._Staudinger_Ligation_with_Azides.jpg)
Contrasting with the classic Staudinger reaction, Staudinger ligation is a
second order reaction
In chemistry, the rate law or rate equation for a reaction is an equation that links the initial or forward reaction rate with the concentrations or pressures of the reactants and constant parameters (normally rate coefficients and partial reactio ...
in which the rate-limiting step is the formation of
phosphazide (specific reaction mechanism shown in Figure 9). The
triphenylphosphine
Triphenylphosphine (IUPAC name: triphenylphosphane) is a common organophosphorus compound with the formula P(C6H5)3 and often abbreviated to P Ph3 or Ph3P. It is widely used in the synthesis of organic and organometallic compounds. PPh3 exists a ...
first reacts with the azide to yield an azaylide through a four-membered ring
transition state
In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked wi ...
, and then an intramolecular reaction leads to the
iminophosphorane An ylide or ylid () is a electric charge, neutral Dipole, dipolar molecule containing a formally negatively charged atom (usually a carbanion) directly attached to a heteroatom with a formal positive charge (usually nitrogen, phosphorus or sulfur), ...
intermediate, which will then give the amide-linkage under hydrolysis.
Huisgen cyclization of azides
= Copper catalyzed Huisgen cyclization of azides
=
Azide has become a popular target for chemoselective protein modification, because they are small in size and have a favorable
thermodynamic reaction potential. One such azide reactions is the
+2cycloaddition reaction with
alkyne
\ce
\ce
Acetylene
\ce
\ce
\ce
Propyne
\ce
\ce
\ce
\ce
1-Butyne
In organic chemistry, an alkyne is an unsaturated hydrocarbon containing at least one carbon—carbon triple bond. The simplest acyclic alkynes with only one triple bond and n ...
, but the reaction requires high temperature and often gives mixtures of
regioisomers.
![Figure 10](https://upload.wikimedia.org/wikipedia/commons/f/f8/Figure_10._Copper-catalyzed_cyclization_of_Azides.jpg)
An improved reaction developed by chemist
Karl Barry Sharpless
Karl Barry Sharpless (born April 28, 1941) is an American chemist and a two-time Nobel laureate in Chemistry known for his work on stereoselective reactions and click chemistry.
Sharpless was awarded half of the 2001 Nobel Prize in Chemistry " ...
involves the copper (I) catalyst, which couples azide with terminal alkyne that only give 1,4 substituted 1,2,3 triazoles in high yields (shown below in Figure 11). The mechanistic study suggests a stepwise reaction.
The Cu (I) first couples with
acetylenes
\ce
\ce
Acetylene
\ce
\ce
\ce
Propyne
\ce
\ce
\ce
\ce
1-Butyne
In organic chemistry, an alkyne is an unsaturated hydrocarbon containing at least one carbon—carbon triple bond. The simplest acyclic alkynes with only one triple bond and no ...
, and then it reacts with azide to generate a six-membered intermediate. The process is very robust that it occurs at pH ranging from 4 to 12, and copper (II) sulfate is often used as a catalyst in the presence of a
reducing agent
In chemistry, a reducing agent (also known as a reductant, reducer, or electron donor) is a chemical species that "donates" an electron to an (called the , , , or ).
Examples of substances that are commonly reducing agents include the Earth meta ...
.
= Strain promoted Huisgen cyclization of azides
=
Even though Staudinger ligation is a suitable bioconjugation in living cells without major toxicity, the phosphine's sensitivity to air oxidation and its poor
solubility
In chemistry, solubility is the ability of a substance, the solute, to form a solution with another substance, the solvent. Insolubility is the opposite property, the inability of the solute to form such a solution.
The extent of the solubil ...
in water significantly hinder its efficiency. The copper (I) catalyzed azide-alkyne coupling has reasonable reaction rate and efficiency under physiological conditions, but copper poses significant
toxicity
Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subst ...
and sometimes interferes with protein functions in living cells. In 2004, chemist
Carolyn R. Bertozzi's lab developed a metal free
+2cycloaddition using strained cyclooctyne and azide. Cyclooctyne, which is the smallest stable cycloalkyne, can couple with azide through
+2cycloaddition, leading to two regioisomeric triazoles (Figure 12).
The reaction occurs readily at
room temperature
Colloquially, "room temperature" is a range of air temperatures that most people prefer for indoor settings. It feels comfortable to a person when they are wearing typical indoor clothing. Human comfort can extend beyond this range depending on ...
and therefore can be used to effectively modify living cells without negative effects. It has also been reported that the installation of
fluorine
Fluorine is a chemical element with the symbol F and atomic number 9. It is the lightest halogen and exists at standard conditions as a highly toxic, pale yellow diatomic gas. As the most electronegative reactive element, it is extremely reacti ...
substituents on a cyclic alkyne can greatly accelerate the reaction rate.
Examples of Applied Bioconjugation Techniques
Growth Factors
Bioconjugation of TGF-β to iron oxide nanoparticles and its activation through magnetic hyperthermia in-vitro has been reported. This was done by using 1-(3-dimethylaminopropyl)ethylcarbodiimide combined with N-Hydroxysuccinimide to form primary amide bonds with the free primary amines on the growth factor. Carbon nanotubes have been successfully used in conjunction with bioconjugation to link TGF-β followed by an activation with near-infrared light. Typically, these reactions have involved the use of a crosslinker, but some of these add molecular space between the compound of interest and base material and in turn causes higher degrees of non-specific binding and unwanted reactivity.
See also
*
Immunofluorescence
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specif ...
*
Biomolecular engineering Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemi ...
*
Biotinylation
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of bi ...
*
SpyTag/SpyCatcher
*
Unnatural amino acids
* ''
Bioconjugate Chemistry
''Bioconjugate Chemistry'' is a monthly peer-reviewed scientific journal covering research in bioconjugation and the interface between man-made and biological materials. The journal was established in 1990 and is published by the American Chemical ...
'' journal
References
{{reflist, 30em
Biochemistry
Chemical bonding