Oligonucleotide synthesis

From Wikipedia, the free encyclopedia

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA only in a 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is, most often, carried out in the opposite, 3' to 5' direction. Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. LNA or BNA.

To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product (see Synthetic cycle below). The process has been fully automated since the late 1970s. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized.^[1] Products are often isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15–25 bases in length.

Oligonucleotides find a variety of applications in molecular biology and medicine. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

History

The evolution of oligonucleotide synthesis saw four major methods of the formation of internucleosidic linkages and has been reviewed in the literature in great detail.

Early work and contemporary H-phosphonate synthesis

Scheme. 1. i: N-Chlorosuccinimide; Bn = -CH₂Ph

In the early 1950s, Alexander Todd’s group pioneered H-phosphonate and phosphate triester methods of oligonucleotide synthesis. The reaction of compounds 1 and 2 to form H-phosphonate diester 3 is an H-phosphonate coupling in solution while that of compounds 4 and 5 to give 6 is a phosphotriester coupling (see phosphotriester synthesis below).

Scheme 2. Oligonucleotide sythesis by the H-Phosphonate Method

Thirty years later, this work inspired, independently, two research groups to adopt the H-phosphonate chemistry to the solid-phase synthesis using nucleoside H-phosphonate monoesters 7 as building blocks and pivaloyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), and other compounds as activators. The practical implementation of H-phosphonate method resulted in a very short and simple synthetic cycle consisting of only two steps, detritylation and coupling (Scheme 2).  Oxidation of internucleosidic H-phosphonate diester linkages in 8 to phosphodiester linkages in 9 with a solution of iodine in aqueous pyridine is carried out at the end of the chain assembly rather than as a step in the synthetic cycle. If desired, the oxidation may be carried out under anhydrous conditions. Alternatively, 8 can be converted to phosphorothioate 10 or phosphoroselenoate 11 (X = Se), or oxidized by CCl₄ in the presence of primary or secondary amines to phosphoramidate analogs 12. The method is very convenient in that various types of phosphate modifications (phosphate/phosphorothioate/phosphoramidate) may be introduced to the same oligonucleotide for modulation of its properties.

Most often, H-phosphonate building blocks are protected at the 5'-hydroxy group and at the amino group of nucleic bases A, C, and G in the same manner as phosphoramidite building blocks (see below). However, protection at the amino group is not mandatory.

Phosphodiester synthesis

Scheme. 3 Oligonucleotide coupling by phosphodiester method; Tr = -CPh₃

In the 1950s, Har Gobind Khorana and co-workers developed a phosphodiester method where 3’-O-acetylnucleoside-5’-O-phosphate 2 (Scheme 3) was activated with N,N'-dicyclohexylcarbodiimide (DCC) or 4-toluenesulfonyl chloride (Ts-Cl). The activated species were reacted with a 5’-O-protected nucleoside 1 to give a protected dinucleoside monophosphate 3. Upon the removal of 3’-O-acetyl group using base-catalyzed hydrolysis, further chain elongation was carried out. Following this methodology, sets of tri- and tetradeoxyribonucleotides were synthesized and were enzymatically converted to longer oligonucleotides, which allowed elucidation of the genetic code. The major limitation of the phosphodiester method consisted in the formation of pyrophosphate oligomers and oligonucleotides branched at the internucleosidic phosphate. The method seems to be a step back from the more selective chemistry described earlier; however, at that time, most phosphate-protecting groups available now had not yet been introduced. The lack of the convenient protection strategy necessitated taking a retreat to a slower and less selective chemistry to achieve the ultimate goal of the study.

Phosphotriester synthesis

Scheme 4. Oligonucleotide coupling by phosphotriester method; MMT = -CPh₂(4-MeOC₆H₄).

In the 1960s, groups led by R. Letsinger and C. Reese developed a phosphotriester approach. The defining difference from the phosphodiester approach was the protection of the phosphate moiety in the building block 1 (Scheme 4) and in the product 3 with 2-cyanoethyl group. This precluded the formation of oligonucleotides branched at the internucleosidic phosphate. The higher selectivity of the method allowed the use of more efficient coupling agents and catalysts, which dramatically reduced the length of the synthesis. The method, initially developed for the solution-phase synthesis, was also implemented on low-cross-linked "popcorn" polystyrene, and later on controlled pore glass (CPG, see "Solid support material" below), which initiated a massive research effort in solid-phase synthesis of oligonucleotides and eventually led to the automation of the oligonucleotide chain assembly.

Phosphite triester synthesis

In the 1970s, substantially more reactive P(III) derivatives of nucleosides, 3'-O-chlorophosphites, were successfully used for the formation of internucleosidic linkages. This led to the discovery of the phosphite triester methodology. The group led by M. Caruthers took the advantage of less aggressive and more selective 1H-tetrazolidophosphites and implemented the method on solid phase. Very shortly after, the workers from the same group further improved the method by using more stable nucleoside phosphoramidites as building blocks. The use of 2-cyanoethyl phosphite-protecting group in place of a less user-friendly methyl group led to the nucleoside phosphoramidites currently used in oligonucleotide synthesis (see Phosphoramidite building blocks below). Many later improvements to the manufacturing of building blocks, oligonucleotide synthesizers, and synthetic protocols made the phosphoramidite chemistry a very reliable and expedient method of choice for the preparation of synthetic oligonucleotides.

Synthesis by the phosphoramidite method

Building blocks

Nucleoside phosphoramidites

Protected 2'-deoxynucleoside phosphoramidites.

As mentioned above, the naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford an expeditious synthetic preparation of oligonucleotides in high yields. The selectivity and the rate of the formation of internucleosidic linkages is dramatically improved by using 3'-O-(N,N-diisopropyl phosphoramidite) derivatives of nucleosides (nucleoside phosphoramidites) that serve as building blocks in phosphite triester methodology. To prevent undesired side reactions, all other functional groups present in nucleosides have to be rendered unreactive (protected) by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired oligonucleotides. Below, the protecting groups currently used in commercially available and most common nucleoside phosphoramidite building blocks are briefly reviewed:

• The 5'-hydroxyl group is protected by an acid-labile DMT (4,4'-dimethoxytrityl) group.
• Thymine and uracil, nucleic bases of thymidine and uridine, respectively, do not have exocyclic amino groups and hence do not require any protection.
• Although the nucleic base of guanosine and 2'-deoxyguanosine does have an exocyclic amino group, its basicity is low to an extent that it does not react with phosphoramidites under the conditions of the coupling reaction. However, a phosphoramidite derived from the N2-unprotected 5'-O-DMT-2'-deoxyguanosine is poorly soluble in acetonitrile, the solvent commonly used in oligonucleotide synthesis. In contrast, the N2-protected versions of the same compound dissolve in acetonitrile well and hence are widely used. Nucleic bases adenine and cytosine bear the exocyclic amino groups reactive with the activated phosphoramidites under the conditions of the coupling reaction. By the use of additional steps in the synthetic cycle or alternative coupling agents and solvent systems, the oligonucleotide chain assembly may be carried out using dA and dC phosphoramidites with unprotected amino groups. However, these approaches currently remain in the research stage. In routine oligonucleotide synthesis, exocyclic amino groups in nucleosides are kept permanently protected over the entire length of the oligonucleotide chain assembly.

The protection of the exocyclic amino groups has to be orthogonal to that of the 5'-hydroxy group because the latter is removed at the end of each synthetic cycle. The simplest to implement, and hence the most widely used, strategy is to install a base-labile protection group on the exocyclic amino groups. Most often, two protection schemes are used.

• In the first, the standard and more robust scheme (Figure), Bz (benzoyl) protection is used for A, dA, C, and dC, while G and dG are protected with isobutyryl group. More recently, Ac (acetyl) group is used to protect C and dC as shown in Figure.
• In the second, mild protection scheme, A and dA are protected with isobutyryl or phenoxyacetyl groups (PAC). C and dC bear acetyl protection, and G and dG are protected with 4-isopropylphenoxyacetyl (iPr-PAC) or dimethylformamidino (dmf) groups. Mild protecting groups are removed more readily than the standard protecting groups. However, the phosphoramidites bearing these groups are less stable when stored in solution.
• The phosphite group is protected by a base-labile 2-cyanoethyl group. Once a phosphoramidite has been coupled to the solid support-bound oligonucleotide and the phosphite moieties have been converted to the P(V) species, the presence of the phosphate protection is not mandatory for the successful conducting of further coupling reactions.

2'-O-protected ribonucleoside phosphoramidites.

• In RNA synthesis, the 2'-hydroxy group is protected with TBDMS (t-butyldimethylsilyl) group. or with TOM (tri-iso-propylsilyloxymethyl) group, both being removable by treatment with fluoride ion.
• The phosphite moiety also bears a diisopropylamino (iPr₂N) group reactive under acidic conditions. Upon activation, the diisopropylamino group leaves to be substituted by the 5'-hydroxy group of the support-bound oligonucleotide (see "Step 2: Coupling" below).

Non-nucleoside phosphoramidites

Non-nucleoside phosphoramidites for 5'-modification of synthetic oligonucleotides. MMT = mono-methoxytrityl,

(4-methoxyphenyl)diphenylmethyl.

Non-nucleoside phosphoramidites are the phosphoramidite reagents designed to introduce various functionalities at the termini of synthetic oligonucleotides or between nucleotide residues in the middle of the sequence. In order to be introduced inside the sequence, a non-nucleosidic modifier has to possess at least two hydroxy groups, one of which is often protected with the DMT group while the other bears the reactive phosphoramidite moiety.

Non-nucleosidic phosphoramidites are used to introduce desired groups that are not available in natural nucleosides or that can be introduced more readily using simpler chemical designs. A very short selection of commercial phosphoramidite reagents is shown in Scheme for the demonstration of the available structural and functional diversity. These reagents serve for the attachment of 5'-terminal phosphate (1), NH₂ (2), SH (3), aldehydo (4), and carboxylic groups (5), CC triple bonds (6), non-radioactive labels and quenchers (exemplified by 6-FAM amidite 7 for the attachment of fluorescein and dabcyl amidite 8, respectively), hydrophilic and hydrophobic modifiers (exemplified by hexaethyleneglycol amidite 9 and cholesterol amidite 10, respectively), and biotin amidite 11.

Synthetic cycle

Scheme 5. Synthetic cycle for preparation of oligonucleotides by phosphoramidite method.

Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues to the 5'-terminus of the growing chain until the desired sequence is assembled. Each addition is referred to as a synthetic cycle (Scheme 5) and consists of four chemical reactions:

Step 1: De-blocking (detritylation)

The DMT group is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene). The orange-colored DMT cation formed is washed out; the step results in the solid support-bound oligonucleotide precursor bearing a free 5'-terminal hydroxyl group. It is worth remembering that conducting detritylation for an extended time or with stronger than recommended solutions of acids leads to depurination of solid support-bound oligonucleotide and thus reduces the yield of the desired full-length product.

Step 2: Coupling

A 0.02–0.2 M solution of nucleoside phosphoramidite (or a mixture of several phosphoramidites) in acetonitrile is activated by a 0.2–0.7 M solution of an acidic azole catalyst, 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. A more extensive information on the use of various coupling agents in oligonucleotide synthesis can be found in a recent review. The mixing is usually very brief and occurs in fluid lines of oligonucleotide synthesizers (see below) while the components are being delivered to the reactors containing solid support. The activated phosphoramidite in 1.5 – 20-fold excess over the support-bound material is then brought in contact with the starting solid support (first coupling) or a support-bound oligonucleotide precursor (following couplings) whose 5'-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage. The coupling of 2'-deoxynucleoside phosphoramidites is very rapid and requires, on small scale, about 20 s for its completion. In contrast, sterically hindered 2'-O-protected ribonucleoside phosphoramidites require 5-15 min to be coupled in high yields. The reaction is also highly sensitive to the presence of water, particularly when dilute solutions of phosphoramidites are used, and is commonly carried out in anhydrous acetonitrile. Generally, the larger the scale of the synthesis, the lower the excess and the higher the concentration of the phosphoramidites is used. In contrast, the concentration of the activator is primarily determined by its solubility in acetonitrile and is irrespective of the scale of the synthesis. Upon the completion of the coupling, any unbound reagents and by-products are removed by washing.

Step 3: Capping

The capping step is performed by treating the solid support-bound material with a mixture of acetic anhydride and 1-methylimidazole or, less often, DMAP as catalysts and, in the phosphoramidite method, serves two purposes.

• After the completion of the coupling reaction, a small percentage of the solid support-bound 5'-OH groups (0.1 to 1%) remains unreacted and needs to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n-1) shortmers. The unreacted 5'-hydroxy groups are, to a large extent, acetylated by the capping mixture.
• It has also been reported that phosphoramidites activated with 1H-tetrazole react, to a small extent, with the O⁶ position of guanosine. Upon oxidation with I₂ /water, this side product, possibly via O⁶-N7 migration, undergoes depurination. The apurinic sites thus formed are readily cleaved in the course of the final deprotection of the oligonucleotide under the basic conditions (see below) to give two shorter oligonucleotides thus reducing the yield of the full-length product. The O⁶ modifications are rapidly removed by treatment with the capping reagent as long as the capping step is performed prior to oxidation with I₂/water.
• The synthesis of oligonucleotide phosphorothioates (OPS, see below) does not involve the oxidation with I₂/water, and, respectively, does not suffer from the side reaction described above. On the other hand, if the capping step is performed prior to sulfurization, the solid support may contain the residual acetic anhydride and N-methylimidazole left after the capping step. The capping mixture interferes with the sulfur transfer reaction, which results in the extensive formation of the phosphate triester internucleosidic linkages in place of the desired PS triesters. Therefore, for the synthesis of OPS, it is advisable to conduct the sulfurization step prior to the capping step.

Step 4: Oxidation

The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. Oxidation may be carried out under anhydrous conditions using tert-Butyl hydroperoxide or, more efficiently, (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). The step of oxidation may be substituted with a sulfurization step to obtain oligonucleotide phosphorothioates (see Oligonucleotide phosphorothioates and their synthesis below). In the latter case, the sulfurization step is best carried out prior to capping.

Solid supports

In solid-phase synthesis, an oligonucleotide being assembled is covalently bound, via its 3'-terminal hydroxy group, to a solid support material and remains attached to it over the entire course of the chain assembly. The solid support is contained in columns whose dimensions depend on the scale of synthesis and may vary between 0.05 mL and several liters. The overwhelming majority of oligonucleotides are synthesized on small scale ranging from 10 nmol to 1 μmol. More recently, high-throughput oligonucleotide synthesis where the solid support is contained in the wells of multi-well plates (most often, 96 or 384 wells per plate) became a method of choice for parallel synthesis of oligonucleotides on small scale. At the end of the chain assembly, the oligonucleotide is released from the solid support and is eluted from the column or the well.

Solid support material

In contrast to organic solid-phase synthesis and peptide synthesis, the synthesis of oligonucleotides proceeds best on non-swellable or low-swellable solid supports. The two most often used solid-phase materials are controlled pore glass (CPG) and macroporous polystyrene (MPPS).

• CPG is commonly defined by its pore size. In oligonucleotide chemistry, pore sizes of 500, 1000, 1500, 2000, and 3000 Å are used to allow the preparation of about 50, 80, 100, 150, and 200-mer oligonucleotides, respectively. To make native CPG suitable for further processing, the surface of the material is treated with (3-aminopropyl)triethoxysilane to give aminopropyl CPG. The aminopropyl arm may be further extended to result in long chain aminoalkyl (LCAA) CPG. The amino group is then used as an anchoring point for linkers suitable for oligonucleotide synthesis (see below).
• MPPS suitable for oligonucleotide synthesis is a low-swellable, highly cross-linked polystyrene obtained by polymerization of divinylbenzene (min 60%), styrene, and 4-chloromethylstyrene in the presence of a porogeneous agent. The macroporous chloromethyl MPPS obtained is converted to aminomethyl MPPS.

Linker chemistry

Commercial solid supports for oligonucleotide synthesis.

 

Scheme 6. Mechanism of 3'-dephosphorylation of oligonucleotides assembled on universal solid supports.

To make the solid support material suitable for oligonucleotide synthesis, non-nucleosidic linkers or nucleoside succinates are covalently attached to the reactive amino groups in aminopropyl CPG, LCAA CPG, or aminomethyl MPPS. The remaining unreacted amino groups are capped with acetic anhydride. Typically, three conceptually different groups of solid supports are used.

• Universal supports. In a more recent, more convenient, and more widely used method, the synthesis starts with the universal support where a non-nucleosidic linker is attached to the solid support material (compounds 1 and 2). A phosphoramidite respective to the 3'-terminal nucleoside residue is coupled to the universal solid support in the first synthetic cycle of oligonucleotide chain assembly using the standard protocols. The chain assembly is then continued until the completion, after which the solid support-bound oligonucleotide is deprotected. The characteristic feature of the universal solid supports is that the release of the oligonucleotides occurs by the hydrolytic cleavage of a P-O bond that attaches the 3’-O of the 3’-terminal nucleotide residue to the universal linker as shown in Scheme 6. The critical advantage of this approach is that the same solid support is used irrespectively of the sequence of the oligonucleotide to be synthesized. For the complete removal of the linker and the 3'-terminal phosphate from the assembled oligonucleotide, the solid support 1 and several similar solid supports require gaseous ammonia, aqueous ammonium hydroxide, aqueous methylamine, or their mixture and are commercially available. The solid support 2 requires a solution of ammonia in anhydrous methanol and is also commercially available.
• Nucleosidic solid supports. In a historically first and still popular approach, the 3'-hydroxy group of the 3'-terminal nucleoside residue is attached to the solid support via, most often, 3’-O-succinyl arm as in compound 3. The oligonucleotide chain assembly starts with the coupling of a phosphoramidite building block respective to the nucleotide residue second from the 3’-terminus. The 3’-terminal hydroxy group in oligonucleotides synthesized on nucleosidic solid supports is deprotected under the conditions somewhat milder than those applicable for universal solid supports. However, the fact that a nucleosidic solid support has to be selected in a sequence-specific manner reduces the throughput of the entire synthetic process and increases the likelihood of human error.
• Special solid supports are used for the attachment of desired functional or reporter groups at the 3’-terminus of synthetic oligonucleotides. For example, the commercial solid support 4 allows the preparation of oligonucleotides bearing 3’-terminal 3-aminopropyl linker. Similarly to non-nucleosidic phosphoramidites, many other special solid supports designed for the attachment of reactive functional groups, non-radioactive reporter groups, and terminal modifiers (e.c. cholesterol or other hydrophobic tethers) and suited for various applications are commercially available. A more detailed information on various solid supports for oligonucleotide synthesis can be found in a recent review.

Oligonucleotide phosphorothioates and their synthesis

S_p and R_p-diastereomeric internucleosidic phosphorothioate linkages.

Oligonucleotide phosphorothioates (OPS) are modified oligonucleotides where one of the oxygen atoms in the phosphate moiety is replaced by sulfur. Only the phosphorothioates having sulfur at a non-bridging position as shown in figure are widely used and are available commercially. The replacement of the non-bridging oxygen with sulfur creates a new center of chirality at phosphorus. In a simple case of a dinucleotide, this results in the formation of a diastereomeric pair of S_p- and R_p-dinucleoside monophosphorothioates whose structures are shown in Figure. In an n-mer oligonucleotide where all (n – 1) internucleosidic linkages are phosphorothioate linkages, the number of diastereomers m is calculated as m = 2^{(n – 1)}. Being non-natural analogs of nucleic acids, OPS are substantially more stable towards hydrolysis by nucleases, the class of enzymes that destroy nucleic acids by breaking the bridging P-O bond of the phosphodiester moiety. This property determines the use of OPS as antisense oligonucleotides in in vitro and in vivo applications where the extensive exposure to nucleases is inevitable. Similarly, to improve the stability of siRNA, at least one phosphorothioate linkage is often introduced at the 3'-terminus of both sense and antisense strands. In chirally pure OPS, all-Sp diastereomers are more stable to enzymatic degradation than their all-Rp analogs. However, the preparation of chirally pure OPS remains a synthetic challenge. In laboratory practice, mixtures of diastereomers of OPS are commonly used.

Synthesis of OPS is very similar to that of natural oligonucleotides. The difference is that the oxidation step is replaced by sulfur transfer reaction (sulfurization) and that the capping step is performed after the sulfurization. Of many reported reagents capable of the efficient sulfur transfer, only three are commercially available:

Commercial sulfur transfer agents for oligonucleotide synthesis.

• 3-(Dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione, DDTT (3) provides rapid kinetics of sulfurization and high stability in solution. The reagent is available from several sources.
• 3H-1,2-benzodithiol-3-one 1,1-dioxide (4) also known as Beaucage reagent displays a better solubility in acetonitrile and short reaction times. However, the reagent is of limited stability in solution and is less efficient in sulfurizing RNA linkages.
• N,N,N'N'-Tetraethylthiuram disulfide (TETD) is soluble in acetonitrile and is commercially available. However, the sulfurization reaction of an internucleosidic DNA linkage with TETD requires 15 min, which is more than 10 times as slow as that with compounds 3 and 4.

Automation

In the past, oligonucleotide synthesis was carried out manually in solution or on solid phase. The solid phase synthesis was implemented using, as containers for the solid phase, miniature glass columns similar in their shape to low-pressure chromatography columns or syringes equipped with porous filters. Currently, solid-phase oligonucleotide synthesis is carried out automatically using computer-controlled instruments (oligonucleotide synthesizers) and is technically implemented in column, multi-well plate, and array formats. The column format is best suited for research and large scale applications where a high-throughput is not required. Multi-well plate format is designed specifically for high-throughput synthesis on small scale to satisfy the growing demand of industry and academia for synthetic oligonucleotides. A number of oligonucleotide synthesizers for small scale synthesis and medium to large scale synthesis are available commercially.

First commercially available oligonucleotide synthesizers

In March 1982 a practical course was hosted by the Department of Biochemistry, Technische Hochschule Darmstadt, Germany. M.H. Caruthers, M.J. Gait, H.G. Gassen, H.Koster, K. Itakura, and C. Birr among others attended. The program comprised practical work, lectures, and seminars on solid-phase chemical synthesis of oligonucleotides. A select group of 15 students attended and had an unprecedented opportunity to be instructed by the esteemed teaching staff.

Along with manual exercises, several prominent automation companies attended the course. Biosearch of Novato, CA, Genetic Design of Watertown, MA, were two of several companies to demonstrate automated synthesizers at the course. Biosearch presented their new SAM I synthesizer. The Genetic Design had developed their synthesizer from the design of its sister companies (Sequemat) solid phase peptide sequencer. The Genetic Design arranged with Dr Christian Birr (Max-Planck-Institute for Medical Research) a week before the event to convert his solid phase sequencer into the semi-automated synthesizer. The team led by Dr Alex Bonner and Rick Neves converted the unit and transported it to Darmstadt for the event and installed into the Biochemistry lab at the Technische Hochschule. As the system was semi-automatic, the user injected the next base to be added to the growing sequence during each cycle. The system worked well and produced a series of test tubes filled with bright red trityl color indicating complete coupling at each step. This system was later fully automated by inclusion of an auto injector and was designated the Model 25A.

History of mid to large scale oligonucleotide synthesis

Large scale oligonucleotide synthesizers were often developed by augmenting the capabilities of a preexisting instrument platform. One of the first mid scale synthesizers appeared in the late 1980s, manufactured by the Biosearch company in Novato, CA (The 8800). This platform was originally designed as a peptide synthesizer and made use of a fluidized bed reactor essential for accommodating the swelling characteristics of polystyrene supports used in the Merrifield methodology. Oligonucleotide synthesis involved the use of CPG (controlled pore glass) which is a rigid support and is more suited for column reactors as described above. The scale of the 8800 was limited to the flow rate required to fluidize the support. Some novel reactor designs as well as higher than normal pressures enabled the 8800 to achieve scales that would prepare 1 mmole of oligonucleotide. In the mid 1990s several companies developed platforms that were based on semi-preparative and preparative liquid chromatographs. These systems were well suited for a column reactor approach. In most cases all that was required was to augment the number of fluids that could be delivered to the column. Oligo synthesis requires a minimum of 10 and liquid chromatographs usually accommodate 4. This was an easy design task and some semi-automatic strategies worked without any modifications to the preexisting LC equipment. PerSeptive Biosystems as well as Pharmacia (GE) were two of several companies that developed synthesizers out of liquid chromatographs. Genomic Technologies, Inc. was one of the few companies to develop a large scale oligonucleotide synthesizer that was, from the ground up, an oligonucleotide synthesizer. The initial platform called the VLSS for very large scale synthesizer utilized large Pharmacia liquid chromatograph columns as reactors and could synthesize up to 75 millimoles of material. Many oligonucleotide synthesis factories designed and manufactured their own custom platforms and little is known due to the designs being proprietary. The VLSS design continued to be refined and is continued in the QMaster synthesizer  which is a scaled down platform providing milligram to gram amounts of synthetic oligonucleotide.

The current practices of synthesis of chemically modified oligonucleotides on large scale have been recently reviewed.

Synthesis of oligonucleotide microarrays

One may visualize an oligonucleotide microarray as a miniature multi-well plate where physical dividers between the wells (plastic walls) are intentionally removed. With respect to the chemistry, synthesis of oligonucleotide microarrays is different from the conventional oligonucleotide synthesis in two respects:

5'-O-MeNPOC-protected nucleoside phosphoramidite.

• Oligonucleotides remain permanently attached to the solid phase, which requires the use of linkers that are stable under the conditions of the final deprotection procedure.
• The absence of physical dividers between the sites occupied by individual oligonucleotides, a very limited space on the surface of the microarray (one oligonucleotide sequence occupies a square 25×25 μm) and the requirement of high fidelity of oligonucleotide synthesis dictate the use of site-selective 5'-deprotection techniques. In one approach, the removal of the 5'-O-DMT group is effected by electrochemical generation of the acid at the required site(s). Another approach uses 5'-O-(α-methyl-6-nitropiperonyloxycarbonyl) (MeNPOC) protecting group, which can be removed by irradiation with UV light of 365 nm wavelength.

Post-synthetic processing

After the completion of the chain assembly, the solid support-bound oligonucleotide is fully protected:

• The 5'-terminal 5'-hydroxy group is protected with DMT group;
• The internucleosidic phosphate or phosphorothioate moieties are protected with 2-cyanoethyl groups;
• The exocyclic amino groups in all nucleic bases except for T and U are protected with acyl protecting groups.

To furnish a functional oligonucleotide, all the protecting groups have to be removed. The N-acyl base protection and the 2-cyanoethyl phosphate protection may be, and is often removed simultaneously by treatment with inorganic bases or amines. However, the applicability of this method is limited by the fact that the cleavage of 2-cyanoethyl phosphate protection gives rise to acrylonitrile as a side product. Under the strong basic conditions required for the removal of N-acyl protection, acrylonitrile is capable of alkylation of nucleic bases, primarily, at the N3-position of thymine and uracil residues to give the respective N3-(2-cyanoethyl) adducts via Michael reaction. The formation of these side products may be avoided by treating the solid support-bound oligonucleotides with solutions of bases in an organic solvent, for instance, with 50% triethylamine in acetonitrile or 10% diethylamine in acetonitrile. This treatment is strongly recommended for medium- and large scale preparations and is optional for syntheses on small scale where the concentration of acrylonitrile generated in the deprotection mixture is low.

Regardless of whether the phosphate protecting groups were removed first, the solid support-bound oligonucleotides are deprotected using one of the two general approaches.

• (1) Most often, 5'-DMT group is removed at the end of the oligonucleotide chain assembly. The oligonucleotides are then released from the solid phase and deprotected (base and phosphate) by treatment with aqueous ammonium hydroxide, aqueous methylamine, their mixtures, gaseous ammonia or methylamine or, less commonly, solutions of other primary amines or alkalies at ambient or elevated temperature. This removes all remaining protection groups from 2'-deoxyoligonucleotides, resulting in a reaction mixture containing the desired product. If the oligonucleotide contains any 2'-O-protected ribonucleotide residues, the deprotection protocol includes the second step where the 2'-O-protecting silyl groups are removed by treatment with fluoride ion by various methods. The fully deprotected product is used as is, or the desired oligonucleotide can be purified by a number of methods. Most commonly, the crude product is desalted using ethanol precipitation, size exclusion chromatography, or reverse-phase HPLC. To eliminate unwanted truncation products, the oligonucleotides can be purified via polyacrylamide gel electrophoresis or anion-exchange HPLC followed by desalting.
• (2) The second approach is only used when the intended method of purification is reverse-phase HPLC. In this case, the 5'-terminal DMT group that serves as a hydrophobic handle for purification is kept on at the end of the synthesis. The oligonucleotide is deprotected under basic conditions as described above and, upon evaporation, is purified by reverse-phase HPLC. The collected material is then detritylated under aqueous acidic conditions. On small scale (less than 0.01–0.02 mmol), the treatment with 80% aqueous acetic acid for 15–30 min at room temperature is often used followed by evaporation of the reaction mixture to dryness in vacuo. Finally, the product is desalted as described above.
• For some applications, additional reporter groups may be attached to an oligonucleotide using a variety of post-synthetic procedures.

Characterization

Deconvoluted ES MS of crude oligonucleotide 5'-DMT-T₂₀ 

(calculated mass 6324.26 Da).

As with any other organic compound, it is prudent to characterize synthetic oligonucleotides upon their preparation. In more complex cases (research and large scale syntheses) oligonucleotides are characterized after their deprotection and after purification. Although the ultimate approach to the characterization is sequencing, a relatively inexpensive and routine procedure, the considerations of the cost reduction preclude its use in routine manufacturing of oligonucleotides. In day-by-day practice, it is sufficient to obtain the molecular mass of an oligonucleotide by recording its mass spectrum. Two methods are currently widely used for characterization of oligonucleotides: electrospray mass spectrometry (ES MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). To obtain informative spectra, it is very important to exchange all metal ions that might be present in the sample for ammonium or trialkylammonium [e.c. triethylammonium, (C₂H₅)₃NH⁺] ions prior to submitting a sample to the analysis by either of the methods.

• In ES MS spectrum, a given oligonucleotide generates a set of ions that correspond to different ionization states of the compound. Thus, the oligonucleotide with molecular mass M generates ions with masses (M – nH)/n where M is the molecular mass of the oligonucleotide in the form of a free acid (all negative charges of internucleosidic phosphodiester groups are neutralized with H⁺), n is the ionization state, and H is the atomic mass of hydrogen (1 Da). Most useful for characterization are the ions with n ranging from 2 to 5. Software supplied with the more recently manufactured instruments is capable of performing a deconvolution procedure that is, it finds peaks of ions that belong to the same set and derives the molecular mass of the oligonucleotide.
• To obtain more detailed information on the impurity profile of oligonucleotides, liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) or capillary electrophoresis mass spectrometry (CEMS) are used.

Peptide synthesis

From Wikipedia, the free encyclopedia

 

Coupling of two amino acids in solution. The unprotected amine of one reacts with the unprotected carboxylic acid group of the other to form a peptide bond. The second reactive group (amine/acid) in each of the starting materials bears a protecting group.

In organic chemistry, peptide synthesis is the production of peptides, compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another. Protecting group strategies are usually necessary to prevent undesirable side reactions with the various amino acid side chains. Chemical peptide synthesis most commonly starts at the carboxyl end of the peptide (C-terminus), and proceeds toward the amino-terminus (N-terminus). Protein biosynthesis (long peptides) in living organisms occurs in the opposite direction.

The chemical synthesis of peptides can be carried out using classical solution-phase techniques, although these have been replaced in most research and development settings by solid-phase methods (see below). Solution-phase synthesis retains its usefulness in large-scale production of peptides for industrial purposes however.

Chemical synthesis facilitates the production of peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.

Solid-phase synthesis

Solid-phase synthesis of a dipeptide using an (amine-functionalized) amide resin. The N-terminal protecting group (PG) can be Fmoc or Boc, depending on the protecting group scheme used (see below). The amino acid side chains (R₁, R₂ etc.) are orthogonally protected (not shown).

The established method for the production of synthetic peptides in the lab is known as solid-phase peptide synthesis (SPPS). Pioneered by Robert Bruce Merrifield, SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support.

The solid support consists of small, polymeric resin beads functionalized with reactive groups (such as amine or hydroxyl groups) that link to the nascent peptide chain. Since the peptide remains covalently attached to the support throughout the synthesis, excess reagents and side products can be removed by washing and filtration. This approach circumvents the comparatively time-consuming isolation of the product peptide from solution after each reaction step, which would be required when using conventional solution-phase synthesis.

Each amino acid to be coupled to the peptide chain N-terminus must be protected on its N-terminus and side chain using appropriate protecting groups such as Boc (acid-labile) or Fmoc (base-labile), depending on the side chain and the protection strategy used (see below).

The general SPPS procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions. The resin can be washed between each steps. First an amino acid is coupled to the resin. Subsequently, the amine is deprotected, and then coupled with the free acid of the second amino acid. This cycle repeats until the desired sequence has been synthesized. SPPS cycles may also include capping steps which block the ends of unreacted amino acids from reacting. At the end of the synthesis, the crude peptide is cleaved from the solid support while simultaneously removing all protecting groups using a reagent strong acids like trifluoroacetic acid or a nucleophile. The crude peptide can be precipitated from a non-polar solvent like diethyl ether in order to remove organic soluble by products. The crude peptide can be purified using reversed-phase HPLC. The purification process, especially of longer peptides can be challenging, because small amounts of several byproducts, which are very similar to the product, have to be removed. For this reason so-called continuous chromatography processes such as MCSGP are increasingly being used in commercial settings to maximize the yield without sacrificing on purity levels.

SPPS is limited by reaction yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically aggregation-prone sequences such as amyloids are difficult to make. Longer lengths can be accessed by using ligation approaches such as native chemical ligation, where two shorter fully deprotected synthetic peptides can be joined together in solution.

Peptide coupling reagents

An important feature that has enabled the broad application of SPPS is the generation of extremely high yields in the coupling step. Highly efficient amide bond-formation conditions are required. The minimization of amino acid racemization during coupling is also of vital importance to avoid epimerization in the final peptide product.

Amide bond formation between an amine and carboxylic acid is slow, and as such usually requires 'coupling reagents' or 'activators'. Activation of the carboxyl group generally involves the formation of an 'active ester' in situ. A selection of peptide coupling reagents are described below.

Carbodiimides

Amide bond formation using DIC/HOBt.

 

Carbodiimides such as dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are frequently used for amide bond formation. The reaction proceeds via the formation of a highly reactive O-acylisourea. This reactive intermediate is attacked by the peptide N-terminal amine, forming a peptide bond. Formation of the O-acylisourea proceeds fastest in non-polar solvents such as dichloromethane.

DIC is particularly useful for SPPS since as a liquid it is easily dispensed, and the urea byproduct (N,N'-Diisopropylcarbodiimide is easily washed away. Conversely, the related carbodiimide 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is often used for solution-phase peptide couplings as its urea byproduct can be removed by washing during aqueous work-up.

HOBt

 

HOAt

 

Neighbouring group effect of HOAt

Carbodiimide activation opens the possibility for racemization of the activated amino acid. Racemization can be circumvented with 'racemization suppressing' additives such as the triazoles 1-hydroxy-benzotriazole (HOBt), and 1-hydroxy-7-aza-benzotriazole (HOAt). These reagents attack the O-acylisourea intermediate to form an active ester, which subsequently reacts with the peptide to form the desired peptide bond. Ethyl cyanohydroxyiminoacetate (Oxyma), an additive for carbodiimide coupling, acts as an alternative to HOAt.

Aminium/uronium and phosphonium salts

Uronium-based peptide coupling reagents

Some coupling reagents omit the carbodiimide completely and incorporate the HOAt/HOBt moiety as an aminium/uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate). Examples of aminium/uronium reagents include HATU (HOAt), HBTU/TBTU (HOBt) and HCTU (6-ClHOBt). HBTU and TBTU differ only in the choice of anion. Phosphonium reagents include PyBOP (HOBt) and PyAOP (HOAt).

These reagents form the same active ester species as the carbodiimide activation conditions, but differ in the rate of the initial activation step, which is determined by nature of the carbon skeleton of the coupling reagent. Furthermore, aminium/uronium reagents are capable of reacting with the peptide N-terminus to form an inactive guanidino by-product, whereas as phosphonium reagents are not.

Solid supports

Cross-linked polystyrene is the most common solid support used in SPPS.

Solid supports for peptide synthesis are selected for physically stability, to permit the rapid filtration of liquids. Suitable supports are inert to reagents and solvents used during SPPS, although they must swell in the solvents used to allow for penetration of the reagents, and allow for the attachment of the first amino acid.

Three primary types of solid supports are: gel-type supports, surface-type supports, and composites. Improvements to solid supports used for peptide synthesis enhance their ability to withstand the repeated use of TFA during the deprotection step of SPPS. Two primary resins are used, based on whether a C-terminal carboxylic acid or amide is desired. The Wang resin was, as of 1996, the most commonly used resin for peptides with C-terminal carboxylic acids.

Protecting groups schemes

As described above, the use of N-terminal and side chain protecting groups is essential during peptide synthesis to avoid undesirable side reactions, such as self-coupling of the activated amino acid leading to (polymerization). This would compete with the intended peptide coupling reaction, resulting in low yield or even complete failure to synthesize the desired peptide.

Two principle orthogonal protecting group schemes exist for use in solid-phase peptide synthesis: so-called Boc/Bzl and Fmoc/tBu approaches. The Boc/Bzl strategy utilizes TFA-labile N-terminal Boc protection alongside side chain protection that is removed using anhydrous hydrogen fluoride during the final cleavage step (with simultaneous cleavage of the peptide from the solid support). Fmoc/tBu SPPS uses base-labile Fmoc N-terminal protection, with side chain protection and a resin linkage that are acid-labile (final acidic cleavage is carried out via TFA treatment).

Both approaches, including the advantages and disadvantages of each, are outlined in more detail below.

Boc/Bzl SPPS

Cleavage of the Boc group

The original method for peptide synthesis relied on tert-butyloxycarbonyl (or more simply 'Boc') as a temporary N-terminal α-amino protecting group. The Boc group is removed with acid, such as trifluoroacetic acid (TFA). This forms a positively charged amino group in the presence of excess TFA (note that the amino group is not protonated in the image on the right), which is neutralized and coupled to the incoming activated amino acid. Neutralization can either occur prior to coupling or in situ during the basic coupling reaction.

The Boc/Bzl approach retains its usefulness in reducing peptide aggregation during synthesis. In addition, Boc/Bzl SPPS may be preferred over the Fmoc/tBu approach when synthesizing peptides containing base-sensitive moieties (such as depsipeptides), as treatment with base is required during the Fmoc deprotection step (see below).

Permanent side-chain protecting groups used during Boc/Bzl SPPS are typically benzyl or benzyl-based groups. Final removal of the peptide from the solid support occurs simultaneously with side chain deprotection using anhydrous hydrogen fluoride via hydrolytic cleavage. The final product is a fluoride salt which is relatively easy to solubilize. Scavengers such as cresol must be added to the HF in order to prevent reactive t-butyl cations from generating undesired products. A disadvantage of this approach is the potential for degradation of the peptide by hydrogen fluoride.

Fmoc/tBu SPPS

Cleavage of the Fmoc group. Treatment of the Fmoc-protected amine with piperidine results in proton abstraction from the methine group of the fluorenyl ring system. This leads to release of a carbamate, which decomposes into carbon dioxide (CO₂) and the free amine. Dibenzofulvene is also generated. This reaction is able to occur due to the acidity of the fluorenyl proton, resulting from stabilization of the aromatic anion formed. The dibenzofulvene by-product can react with nucleophiles such as the piperidine (which is in large excess), or potentially the released amine.

The use of N-terminal Fmoc protection allows for a milder deprotection scheme than used for Boc/Bzl SPPS, and this protection scheme is truly orthogonal under SPPS conditions. Fmoc deprotection utilizes a base, typically 20–50% piperidine in DMF. The exposed amine is therefore neutral, and consequently no neutralization of the peptide-resin is required, as in the case of the Boc/Bzl approach. The lack of electrostatic repulsion between the peptide chains can lead to increased risk of aggregation with Fmoc/tBu SPPS however. Because the liberated fluorenyl group is a chromophore, Fmoc deprotection can be monitored by UV absorbance of the reaction mixture, a strategy which is employed in automated peptide synthesizers.

The ability of the Fmoc group to be cleaved under relatively mild basic conditions while being stable to acid allows the use of side chain protecting groups such as Boc and tBu that can be removed in milder acidic final cleavage conditions (TFA) than those used for final cleavage in Boc/Bzl SPPS (HF). Scavengers such as water and triisopropylsilane (TIPS) are added during the final cleavage in order to prevent side reactions with reactive cationic species released as a result of side chain deprotection. The resulting crude peptide is obtained as a TFA salt, which is potentially more difficult to solubilize than the fluoride salts generated in Boc SPPS.

Fmoc/tBu SPPS is less atom-economical, as the fluorenyl group is much larger than the Boc group. Accordingly, prices for Fmoc amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, enfuvirtide, began in the 1990s, when market demand adjusted the relative prices of Fmoc- vs Boc- amino acids.

Other protecting groups

Benzyloxy-carbonyl

The (Z) group is another carbamate-type amine protecting group, first used by Max Bergmann in the synthesis of oligopeptides. It is removed under harsh conditions using HBr in acetic acid, or milder conditions of catalytic hydrogenation. While it has been used periodically for α-amine protection in peptide synthesis, it is almost exclusively used for side chain protection.

Alloc and miscellaneous groups

The allyloxycarbonyl (alloc) protecting group is sometimes used to protect an amino group (or carboxylic acid or alcohol group) when an orthogonal deprotection scheme is required. It is also sometimes used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side-chain functional group. The Alloc group can be removed using tetrakis(triphenylphosphine)palladium(0).

For special applications like synthetic steps involving protein microarrays, protecting groups sometimes termed "lithographic" are used, which are amenable to photochemistry at a particular wavelength of light, and so which can be removed during lithographic types of operations.

Regioselective disulfide bond formation

The formation of multiple native disulfides remains challenging of native peptide synthesis by solid-phase methods. Random chain combination typically results in several products with nonnative disulfide bonds. Stepwise formation of disulfide bonds is typically the preferred method, and performed with thiol protecting groups. Different thiol protecting groups provide multiple dimensions of orthogonal protection. These orthogonally protected cysteines are incorporated during the solid-phase synthesis of the peptide. Successive removal of these groups, to allow for selective exposure of free thiol groups, leads to disulfide formation in a stepwise manner. The order of removal of the groups must be considered so that only one group is removed at a time.

Thiol protecting groups used in peptide synthesis requiring later regioselective disulfide bond formation must possess multiple characteristics.^{[citation needed][verification needed]} First, they must be reversible with conditions that do not affect the unprotected side chains. Second, the protecting group must be able to withstand the conditions of solid-phase synthesis. Third, the removal of the thiol protecting group must be such that it leaves intact other thiol protecting groups, if orthogonal protection is desired. That is, the removal of PG A should not affect PG B. Some of the thiol protecting groups commonly used include the acetamidomethyl (Acm), tert-butyl (But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and trityl (Trt) groups. Importantly, the NPYS group can replace the Acm PG to yield an activated thiol.

Using this method, Kiso and coworkers reported the first total synthesis of insulin in 1993. In this work, the A-chain of insulin was prepared with following protecting groups in place on its cysteines: CysA6(But), CysA7(Acm), and CysA11(But), leaving CysA20 unprotected.

Microwave-assisted peptide synthesis

Microwave-assisted peptide synthesis has been used to complete long peptide sequences with high degrees of yield and low degrees of racemization.

Synthesizing long peptides

Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment.

A new development for producing longer peptide chains is chemical ligation: unprotected peptide chains react chemoselectively in aqueous solution. A first kinetically controlled product rearranges to form the amide bond. The most common form of native chemical ligation uses a peptide thioester that reacts with a terminal cysteine residue.

Other methods applicable for covalently linking polypeptides in aqueous solution include the use of split inteins, spontaneous isopeptide bond formation and sortase ligation.

In order to optimize synthesis of long peptides, a method was developed in Medicon Valley for converting peptide sequences.^{[citation needed]} The simple pre-sequence (e.g. Lysine (Lysn); Glutamic Acid (Glun); (LysGlu)n) that is incorporated at the C-terminus of the peptide to induce an alpha-helix-like structure. This can potentially increase biological half-life, improve peptide stability and inhibit enzymatic degradation without altering pharmacological activity or profile of action.

Cyclic peptides

On resin cyclization

Peptides can be cyclized on a solid support. A variety of cylization reagents can be used such as HBTU/HOBt/DIEA, PyBop/DIEA, PyClock/DIEA. Head-to-tail peptides can be made on the solid support. The deprotection of the C-terminus at some suitable point allows on-resin cyclization by amide bond formation with the deprotected N-terminus. Once cyclization has taken place, the peptide is cleaved from resin by acidolysis and purified.

The strategy for the solid-phase synthesis of cyclic peptides in not limited to attachment through Asp, Glu or Lys side chains. Cysteine has a very reactive sulfhydryl group on its side chain. A disulfide bridge is created when a sulfur atom from one Cysteine forms a single covalent bond with another sulfur atom from a second cysteine in a different part of the protein. These bridges help to stabilize proteins, especially those secreted from cells. Some researchers use modified cysteines using S-acetomidomethyl (Acm) to block the formation of the disulfide bond but preserve the cysteine and the protein's original primary structure.

Off-resin cyclization

Off-resin cyclization is a solid-phase synthesis of key intermediates, followed by the key cyclization in solution phase, the final deprotection of any masked side chains is also carried out in solution phase. This has the disadvantages that the efficiencies of solid-phase synthesis are lost in the solution phase steps, that purification from by-products, reagents and unconverted material is required, and that undesired oligomers can be formed if macrocycle formation is involved.

The use of pentafluorophenyl esters (FDPP, PFPOH) and BOP-Cl are useful for cyclising peptides.

Impact of health on intelligence

From Wikipedia, the free encyclopedia

Health can affect intelligence in various ways. Conversely, intelligence can affect health. Health effects on intelligence have been described as being among the most important factors in the origins of human group differences in IQ test scores and other measures of cognitive ability. Several factors can lead to significant cognitive impairment, particularly if they occur during pregnancy and childhood when the brain is growing and the blood–brain barrier of the child is less effective. Such impairment may sometimes be permanent, sometimes be partially or wholly compensated for by later growth.

Developed nations have implemented several health policies regarding nutrients and toxins known to influence cognitive function. These include laws requiring fortification of certain food products and laws establishing safe levels of pollutants (e.g. lead, mercury, and organochlorides). Comprehensive policy recommendations targeting reduction of cognitive impairment in children have been proposed.

Improvements in nutrition (often involving specific micronutrients) due to public policy changes have been implicated in IQ increases in many nations (as part of the overall Flynn effect), such as efforts fighting iodine deficiency in the U.S.

Nutrition

Malnutrition may occur during several periods of growth, such as pregnancy, during breastfeeding, infancy, or childhood. It may also happen due to deficiencies of different nutrients, such as micronutrients, protein or energy. This may cause different effects.

Timing

Some observers have argued that malnutrition during the first six months of life harms cognitive development much more than malnutrition later in life. However, a study from the Philippines argues that malnutrition in the second year of life may have a larger negative impact than malnutrition in the first year of life.

Intrauterine growth retardation

Undernutrition during pregnancy, and other factors, may cause intrauterine growth retardation (IUGR), which is one cause of low birth weight. However, it has been suggested that in IUGR the brain may be selectively spared. Brain growth is usually less affected than whole body weight or length. Several studies from developed nations have found that with the exception of extreme intrauterine growth retardation also affecting brain growth, and hypoxic injury, IUGR seems to have little or no measurable effect on mental performance and behavior in adolescence or adulthood. For example, acute undernutrition for a few months during the Dutch famine of 1944 caused a decrease in mean birthweight in certain areas. This was later associated with a change in performance on IQ tests for 18–19 years old Dutch males draftees from these areas compared to control areas. The subjects were exposed to famine prenatally but not after birth. During the famine, births decreased more among those with lower socioeconomic status (SES), whereas after the famine, there was a compensatory increase in births among those with lower SES. Since SES correlates with IQ, this may have hidden an effect caused by the undernutrition.

Breastfeeding

Studies often find higher IQ in children and adults who were breastfed. It has been proposed that omega-3 fatty acids in breast milk, known to be essential constituents of brain tissues, could at least partially account for an increase in IQ.

Recently, however, the longstanding belief that breastfeeding causes an increase in the IQ of offspring was challenged in a 2006 paper published in the British Medical Journal. The results indicated that mother's IQ, not breastfeeding, explained the differences in the IQ scores of offspring measured between ages 5 and 14. The results of this meta-analysis argued that prior studies had not controlled for the mother's IQ. Since mother's IQ was predictive of whether a child was breastfed, the study concluded that "breast feeding [itself] has little or no effect on intelligence in children." Instead, it was the mother's IQ that had a significant correlation with the IQ of her offspring, whether the offspring was breastfed or was not breastfed.

One study found that breastfeeding was linked to raised IQ (as much as 7 points when not controlling for maternal IQ) if the infants had an SNP coding for a "C" rather than G base within the FADS2 gene. Those with the "G" version showed no IQ advantage, suggesting a biochemical interaction of child's genes on the effect of breastfeeding. Other studies have failed to replicate any correlation between the FADS2 gene, breastfeeding and IQ, while others show a negative effect on IQ when combining bottled feeding, and the "G" version of FADS2.

Infancy

Two studies in Chile on 18-year-old high-school graduates found that nutritional status during the first year of life affected IQ, scholastic achievement, and brain volume.

Micronutrients and vitamin deficiencies

Micronutrient deficiencies (e.g. in iodine and iron) influence the development of intelligence and remain a problem in the developing world. For example, iodine deficiency causes a fall, in average, of 12 IQ points.

Policy recommendations to increase availability of micronutrient supplements have been made and justified in part by the potential to counteract intelligence-related developmental problems. For example, the Copenhagen consensus, states that lack of both iodine and iron has been implicated in impaired brain development, and this can affect enormous numbers of people: it is estimated that 2 billion people (one-third of the total global population) are affected by iodine deficiency, including 285 million 6- to 12-year-old children. In developing countries, it is estimated that 40% of children aged four and under suffer from anaemia because of insufficient iron in their diets.

A joint statement on vitamin and mineral deficiencies says that the severity of such deficiencies "means the impairment of hundreds of millions of growing minds and the lowering of national IQs."

Overall, studies investigating whether cognitive function in already iron-deficient children can be improved with iron supplements have produced mixed results, possibly because deficiency in critical growth periods may cause irreversible damage. However, several studies with better design have shown substantial benefits. One way to prevent iron deficiency is to give specific supplementation to children, for example as tablets. However, this is costly, distribution mechanisms are often ineffective, and compliance is low. Fortification of staple foods (cereals, flour, sugar, salt) to deliver micronutrients to children on a large scale is probably the most sustainable and affordable option, even though commitment from governments and the food industry is needed. Developed nations fortify several foods with various micronutrients.

Additional vitamin-mineral supplementation may have an effect also in the developed world. A study giving such supplementation to "working class," primarily Hispanic, 6–12-year-old children in the United States for 3 months found an average increase of 2 to 3 IQ points. Most of this can be explained by the very large increase of a subgroup of the children, presumably because these were not adequately nourished unlike the majority. The study suggests that parents of schoolchildren whose academic performance is substandard would be well advised to seek a nutritionally oriented physician for assessment of their children's nutritional status as a possible etiology.

More speculatively, other nutrients may prove important in the future. Vitamin B12 and folate may be important for cognitive function in old age. Fish oil supplement to pregnant and lactating mothers has been linked to increased cognitive ability in one study.

Another study found that pregnant women who consumed 340 grams of low-mercury containing fish with fatty acids per week have benefits that outweigh the risks for mercury poisoning. They were less likely to have children with low verbal IQ, motor coordination and behavioral problems. However, foods containing high amounts of mercury, such as shark, swordfish, king mackerel and tilefish, might cause mental retardation.

Protein and energy malnutrition

One study from a developing country, Guatemala, found that poor growth during infancy, rather than low birth weight, was negatively related to adolescent performance on cognitive and achievement tests. A later related very long term study looked at the effect of giving 6–24-month-old children in Guatemala a high protein-energy drink as a dietary supplement. A significantly positive and fairly substantial effects was found on increasing the probability of attending school and of passing the first grade, increasing the grade attained by age 13, increasing completed schooling attainment, and for adults aged 25–40 increasing IQ test scores.

Stunting

31% of children under the age of 5 in the developing world are moderately (height-for-age is below minus 2 standard deviations) or severely stunted (below minus 3 standard deviations). The prevalence was even higher previously since the worldwide prevalence of stunting is declining by about half of a percentage point each year. A study on stunted children aged 9–24 months in Jamaica found that when aged 17–18 years they had significantly poorer scores than a non-stunted group on cognitive and educational tests and psychosocial functioning. Giving a nutritional supplementation (1 kg milk based formula each week) to these already stunted children had no significant effect on later scores, but psychosocial stimulation (weekly play sessions with mother and child) had a positive effect.

Toxins

Industrial chemicals

Certain toxins, such as lead, mercury, toluene, and PCB are well-known causes of neuro-developmental disorders. Recognition of these risks has led to evidence-based programmes of prevention, such as elimination of lead additives in petrol. Although these prevention campaigns are highly successful, most were initiated only after substantial delays.

Policies to manage lead differ between nations, particularly between the developed and developing world. Use of leaded gasoline has been reduced or eliminated in most developed nations, and lead levels in US children have been substantially reduced by policies relating to lead reduction. Even slightly elevated lead levels around the age of 24 months are associated with intellectual and academic performance deficits at age 10 years.

Certain, at least previously, widely used organochlorides, such as dioxins, DDT, and PCB, have been associated with cognitive deficits.

A Lancet review identified 201 chemicals with the ability to cause clinical neurotoxic effects in human adults, as described in the peer-reviewed scientific literature. Most of them are commonly used. Many additional chemicals have been shown to be neurotoxic in laboratory models. The article notes that children are more vulnerable and argues that new, precautionary approaches that recognise the unique vulnerability of the developing brain are needed for testing and control of chemicals in order to avoid the previous substantial before starting restrictions on usage. An appendix listed further industrial chemicals considered to be neurotoxic.

Alcohol and drugs

Fetal alcohol exposure, causing Fetal alcohol syndrome, is one of the leading known causes of mental retardation in the Western world.

Current cannabis use was found to be significantly correlated in a dose-dependent manner with a decline in IQ scores, during the effect of the use. However, no such decline was seen in subjects who had formerly been heavy cannabis users and had stopped taking the drug. The authors concluded that cannabis does not have a long-term effect on intelligence. However this is contradicted by the long term longitudinal study, carried out by Otago and Duke universities,which found that regular use of marijuana in teenage years affects IQ in adulthood even when the use stops. The drop in IQ was 8 points. Adults smoking marijuana had no lasting effect on IQ. Effects on fetal development are minimal when compared with the well-documented adverse effects of tobacco or alcohol use.

Maternal tobacco smoking during pregnancy is associated with increased activity, decreased attention, and diminished intellectual abilities. However, a recent study finds that maternal tobacco smoking has no direct causal effect on the child's IQ. Adjusting for maternal cognitive ability as measured by IQ and education eliminated the association between lower IQ and tobacco smoking. But another study instead looking at the relationship between environmental tobacco smoke exposure, measured with a blood biomarker, and cognitive abilities among U.S. children and adolescents 6–16 years of age, found an inverse association between exposure and cognitive ability among children even at extremely low levels of exposure. The study controlled for sex, race, region, poverty, parent education and marital status, ferritin, and blood lead concentration.

Healthcare during pregnancy and childbirth

Healthcare during pregnancy and childbirth, access to which is often governed by policy, also influences cognitive development. Preventable causes of low intelligence in children include infectious diseases such as meningitis, parasites, and cerebral malaria, prenatal drug and alcohol exposure, newborn asphyxia, low birth weight, head injuries, and endocrine disorders. A direct policy focus on determinants of childhood cognitive ability has been urged.

Stress

A recent theory suggests that early childhood stress may affect the developing brain and cause negative effects. Exposure to violence in childhood has been associated with lower school grades and lower IQ in children of all races. A group of largely African American urban first-grade children and their caregivers were evaluated using self-report, interview, and standardized tests, including IQ tests. The study reported that exposure to violence and trauma-related distress in young children were associated with substantial decrements in IQ and reading achievement. Exposure to Violence or Trauma lead to a 7.5-point (SD, 0.5) decrement in IQ and a 9.8-point (SD, 0.66) decrement in reading achievement.

Violence may have a negative impact on IQ, or IQ may be protective against violence. The causal mechanism and direction of causation is unknown. Neighborhood risk has been related to lower school grades for African-American adolescents in another study from 2006.

Infectious diseases

A 2010 study by Eppig, Fincher and Thornhill found a close correlation between the infectious disease burden in a country and the average IQ of its population. The researchers found that when disease was controlled for, IQ showed no correlation with other variables such as educational and nutritional levels. Since brain development requires a very high proportion of all the body's energy in newborns and children, the researchers argue that fighting infection reduces children's IQ potential. The Eppig research may help to explain the Flynn effect, the rise in intelligence noted in rich countries. They also tested other hypotheses as well, including genetic explanations, concluding that infectious disease was "the best predictor". Christopher Hassall and Thomas Sherratt repeated the analysis, and concluded "that infectious disease may be the only really important predictor of average national IQ".

In order to mitigate the effects of education on IQ, Eppig, Fincher & Thornhill (2010) repeated their analysis across the United States where standardized and compulsory education exists. The correlation between infectious disease and average IQ was confirmed, and they concluded that the "evidence suggests that infectious disease is a primary cause of the global variation in human intelligence".

Tropical infectious diseases

Malaria affects 300–500 million persons each year, mostly children under age five in Africa, causing widespread anemia during a period of rapid brain development and also direct brain damage from cerebral malaria to which children are more vulnerable. A 2006 systematic review found that Plasmodium falciparum infection causes cognitive deficits in both the short- and long-term. Policies aimed at malaria reduction may have cognitive benefits. It has been suggested that the future economic and educational development of Africa critically depends on the eradication of malaria.

Roundworms infect hundreds of millions of people. There is evidence that high intensities of worms in the intestines can affect mental performance, but a systematic review in 2000 and a 2009 update found that there was insufficient evidence to show that deworming treatments improve cognitive performance or school performance in children.

HIV infection in children in sub-Saharan Africa affects their motor development, but there is insufficient evidence to show a slowing of language development.

Effects of other diseases

There are numerous diseases affecting the central nervous system which can cause cognitive impairment. Many of these are associated with aging. Some common examples include Alzheimer's disease and Multi-infarct dementia. Many diseases may be neurological or psychiatric and may primarily affect the brain. Others may affect many other organs, like HIV, Hashimoto's thyroiditis causing hypothyroidism, or cancer. According to a 2015 report in The American Scholar, an assortment of neglected tropical diseases as well as some recently identified pathogens such as Pseudo-nitzschia have also been found to erode human intelligence.

Major depression, affecting about 16% of the population on at least one occasion in their lives and the leading cause of disability in North America, may give symptoms similar to dementia. Patients treated for depression score higher on IQ tests than before treatment.

Myopia and hyperopia

A 2008 literature review writes that studies in several nations have found a relationship between myopia and higher IQ and between myopia and school achievement. Several, but not all, studies have found hyperopia to be associated with lower IQ and school achievements. A common explanation for myopia is near-work. Regarding the relationship to IQ, several explanations have been proposed. One is that the myopic child is better adapted at reading, and reads and studies more, which increases intelligence. The reverse explanation is that the intelligent and studious child reads more which causes myopia. Another is that the myopic child have an advantage at IQ testing which is near work because of less eye strain. Still another explanation is that pleiotropic gene(s) affect the size of both brain and eyes simultaneously. A study of Chinese schoolchildren found that after controlling for age, gender, school, parental myopia, father’s education, and books read per week, myopia was still associated with high nonverbal IQ. Nonverbal IQ was a more important explanation than books read per week.

Other associations

Long working hours (55 vs. 40) was associated with decreased scores on cognitive tests in a 5-year study on midlife British civil servants.