United States biological defense program

From Wikipedia, the free encyclopedia

 https://en.wikipedia.org/wiki/United_States_biological_defense_program

The United States biological defense program—in recent years also called the National Biodefense Strategy— refers to the collective effort by all levels of government, along with private enterprise and other stakeholders, in the United States to carry out biodefense activities.

Biodefense is a system of planned actions to counter and reduce the risk of biological threats and to prepare, respond to, and recover from them if they happen. The National Defense Authorization Act (NDAA) of 2016 required high-level officials across the federal government to create a national biodefense strategy together. As a result, in 2018 the National Biodefense Strategy was released by President Donald J. Trump. In essence, the strategy comprises the U.S. biological defense program in that it is the official framework that provides a "single coordinated effort" to coordinate all biodefense activities across the federal government. To execute the strategy, the White House issued a Presidential Memorandum on the Support for National Biodefense, which puts the specific directives and rules in place for carrying out the plans written in the strategy. It is worth noting that the National Biodefense Strategy elevated natural outbreaks as a vital component of the U.S. biological defense program for the first time, mostly because of the significant risk that natural outbreaks pose to civilian, animal and agricultural populations across the country.

The U.S. biological defense program began as a small defensive effort that parallels the country's offensive biological weapons development and production program, active since 1943. Organizationally, the medical defense research effort was pursued first (1956-1969) by the U.S. Army Medical Unit (USAMU) and later, after publicly known discontinuation of the offensive program, by the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). Both of these units were located at Fort Detrick, Maryland, where the U.S. Army Biological Warfare Laboratories were headquartered. The current mission is multi-agency, not exclusively military, and is purely to develop defensive measures against bio-agents, as opposed to the former bio-weapons development program.

In 1951, due to biological warfare concerns arising from the Korean War, the US Centers for Disease Control and Prevention (CDC) created the Epidemic Intelligence Service (EIS), a hands-on two-year postgraduate training program in epidemiology, with a focus on field work.

Since the 2001 anthrax attacks, and the consequent expansion of federal bio-defense expenditures, USAMRIID has been joined at Fort Detrick by sister bio-defense agencies of the U.S. Department of Health and Human Services (NIAID's Integrated Research Facility) and the U.S. Department of Homeland Security (the National Biodefense Analysis and Countermeasures Center and the National Bioforensic Analysis Center). These—along with the much older Foreign Disease Weed Science Research Unit of the U.S. Department of Agriculture—now constitute the National Interagency Confederation for Biological Research (NICBR).

Broadly defined, the "United States biological defense program" now also encompasses all federal-level programs and efforts to monitor, prevent, and contain naturally occurring infectious disease outbreaks of widespread public health concern. These include efforts to forestall large-scale disasters such as flu pandemics and other "emerging infections" such as novel pathogens or those imported from other countries.

Overview

Biological agents have been used in warfare for centuries to produce death or disease in humans, animals, or plants. The United States officially began its biological warfare offensive program in 1941. During the next 28 years, the U.S. initiative evolved into an effective, military-driven research and acquisition program, shrouded in secrecy and, later, controversy. Most research and development was done at Fort Detrick, Maryland, while production and testing of bio-weapons occurred at Pine Bluff, Arkansas, and Dugway Proving Ground (DPG), Utah. Field testing was done secretly and successfully with simulants and actual agents disseminated over wide areas. A small defensive effort always paralleled the weapons development and production program. With the presidential decision in 1969 to halt offensive biological weapons production—and the agreement in 1972 at the international BWC never to develop, produce, stockpile, or retain biological agents or toxins—the program became entirely defensive, with medical and non-medical components. The U.S. biological defense research program exists today, conducting research to develop physical and medical countermeasures to protect service members and civilians from the threat of modern biological warfare.

Both the U.S. bio-weapons ban and the BWC restricted any work in the area of biological warfare to defensive in nature. In reality, this gives BWC member-states wide latitude to conduct biological weapons research because the BWC contains no provisions for monitoring of enforcement. The treaty, essentially, is a gentlemen's agreement amongst members backed by the long-prevailing thought that biological warfare should not be used in battle.

In recent years certain critics have claimed the U.S. stance on biological warfare and the use of biological agents has differed from historical interpretations of the BWC. For example, it is said that the U.S. now maintains that the Article I of the BWC (which explicitly bans bio-weapons), does not apply to "non-lethal" biological agents. Previous interpretation was stated to be in line with a definition laid out in Public Law 101-298, the Biological Weapons Anti-Terrorism Act of 1989. That law defined a biological agent as:

any micro-organism, virus, infectious substance, or biological product that may be engineered as a result of biotechnology, or any naturally occurring or bio-engineered component of any such microorganism, virus, infectious substance, or biological product, capable of causing death, disease, or other biological malfunction in a human, an animal, a plant, or another living organism; deterioration of food, water, equipment, supplies, or material of any kind ...

According to the Federation of American Scientists, U.S. work on non-lethal agents exceeds limitations in the BWC.

History

1950s

After World War II, and with the onset of Cold War tensions, the US continued its clandestine wartime bio-weapons program. The Korean War (1950–53) added justification for continuing the program, when the possible entry of the Soviet Union into the war was feared. Concerns over the Soviet Union were justified, for the Soviet Union would pronounce in 1956 that chemical and biological weapons would, indeed, be used for mass destruction in future wars. In October 1950, the US Secretary of Defense approved continuation of the program, based largely on the Soviet threat and a belief that the North Korean and Chinese communists would use biological weapons. With expansion of the biological warfare retaliatory program, the scope of the defensive program was nearly doubled. Data were obtained on personnel protection, decontamination, and immunization. Early detection research produced prototype alarms for use on the battlefield, but progress was slow, apparently limited by technology.

The U.S. Army Medical Unit, under the direction of The U.S. Army Surgeon General, began formal operations in 1956. One of the Unit's first missions was to manage all aspects of Project CD-22, the exposure of volunteers to aerosols containing a pathogenic strain of Coxiella burnetii, the etiologic agent of Q fever. The volunteers were closely monitored and antibiotic therapy was administered when appropriate. All volunteers recovered from Q fever with no adverse aftereffects. One year later, the Unit submitted to the U.S. Food and Drug Administration an Investigational New Drug application for a Q fever vaccine.

1960s

In the following decade, the US accumulated significant data on personnel protection, decontamination, and immunization; and, in the offensive program, on the potential for mosquitoes to be used as biological vectors. A new Department of Defense (DoD) Biological and Chemical Defense Planning Board was created in 1960 to establish program priorities and objectives. Preventive approaches toward infections of all kinds were funded under the auspices of biological warfare. As concern increased over the biological warfare threat during the Cold War, so did the budget for the program: to $38 million by fiscal year 1966.

The U.S. Army Chemical Corps was given the responsibility to conduct biological warfare research for all of the services. In 1962, the responsibility for the testing of promising biological warfare agents was given to a separate Testing and Evaluation Command (TEC). Depending on the particular program, different test centers were used, such as the Deseret Test Center at Fort Douglas, Utah, the headquarters for the new biological and chemical warfare testing organization. In response to increasing concerns over public safety and the environment, the TEC implemented a complex system of approval of its research programs that included the U.S. Army Chief of Staff, the Joint Chiefs of Staff, the Secretary of Defense, and the President of the United States.

During the last 10 years of the offensive research and development program (1959–69), many scientific advances were made that proved that biological warfare was clearly feasible, although dependent on careful planning, especially with regard to meteorological conditions. Large-scale fermentation, purification, concentration, stabilization, drying, and weaponization of pathogenic microorganisms could be done safely. Furthermore, modern principles of biosafety and containment were established at the Fort Detrick laboratories which have greatly facilitated biomedical research in general; still today, these are followed throughout the world. Arnold G. Wedum, M.D., Ph.D., a civilian scientist who was Director of Industrial Health and Safety at Fort Detrick, was the leader in the development of containment facilities.

During the 1960s, the US program underwent a philosophical change, and attention was now directed more towards biological agents that could incapacitate, but not kill. In 1964, research programs involved staphylococcal enterotoxins capable of causing food poisoning. Research initiatives also included new therapy and prophylaxis. Pathogens studied included the agents causing anthrax, glanders, brucellosis, melioidosis, plague, psittacosis, Venezuelan equine encephalitis, Q fever, coccidioidomycosis, and a variety of plant and animal pathogens.

Particular attention was directed at chemical and biological detectors during the 1960s. The first devices were primitive field alarms to detect chemicals. Although the development of sensitive biological warfare agent detectors was at a standstill, two systems were, nonetheless, investigated. The first was a monitor that detected increases in the number of particles sized 1 to 5 µm in diameter, based on the assumption that a biological agent attack would include airborne particles of this size. The second system involved the selective staining of particles collected from the air. Both systems lacked enough specificity and sensitivity to be of any practical use.

But in 1966, a research effort directed at detecting the presence of adenosine triphosphate (a chemical found only in living organisms) was begun. By using a fluorescent material found in fireflies, preliminary studies indicated that it was possible to detect the presence of a biological agent in the atmosphere. The important effort to find a satisfactory detection system continues today, for timely detection of a biological attack would allow the attacked force to use its protective masks effectively, and identification of the agent would allow any pre-treatment regimens to be instituted. The US Army also experimented with and developed highly effective barrier protective measures against both chemical and biological agents. Special impervious tents and personal protective equipment were developed, including individual gas masks even for military dogs.

During the late 1960s, funding for the biological warfare program decreased temporarily, to accommodate the accelerating costs of the Vietnam War. The budget for fiscal year 1969 was $31 million, decreasing to $11.8 million by fiscal year 1973. Although the offensive program had been stopped in 1969, both offensive and defensive programs continued to be defended. John S. Foster, Jr, Director of Defense Research and Engineering, responded to a query by Congressman Richard D. McCarthy:

It is the policy of the U.S. to develop and maintain a defensive chemical-biological (CB) capability so that our military forces could operate for some period of time in a toxic environment, if necessary; to develop and maintain a limited offensive capability in order to deter all use of CB weapons by the threat of retaliation in kind; and to continue a program of research and development in this area to minimize the possibility of technological surprise.

On 25 November 1969, President Richard Nixon visited Fort Detrick to announce a new policy on biological warfare. In two National Security Memoranda, the U.S. government renounced all development, production, and stockpiling of biological weapons and declared its intent to maintain only small research quantities of biological agents, such as are necessary for the development of vaccines, drugs, and diagnostics.

Ground was broken in 1967 for the construction of a new, modern laboratory building at Fort Detrick. The building would open in phases during 1971 and 1972. With the disestablishment of the biological warfare laboratories, the name of the U.S. Army Medical Unit, which was to have been housed in the new laboratories, was formally changed to U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) in 1969. The institute's new mission was stated in General Order 137, 10 November 1971 (since superseded):

Conducts studies related to medical defensive aspects of biological agents of military importance and develops appropriate biological protective measures, diagnostic procedures and therapeutic methods.

The emphasis now shifted away from offensive weapons to the development of vaccines, diagnostic systems, personal protection, chemoprophylaxis, and rapid detection systems.

1970s

After Nixon declared an end to the U.S. bio-weapons program, debate in the Army centered around whether or not toxin weapons were included in the president's declaration. Following Nixon's November 1969 order, scientists at Fort Detrick worked on one toxin, Staphylococcus enterotoxin type B (SEB), for several more months. Nixon ended the debate when he added toxins to the bio-weapons ban in February 1970.

In response to Nixon's 1969 decision, all antipersonnel biological warfare stocks were destroyed between 10 May 1971 and 1 May 1972. The laboratory at Pine Bluff Arsenal, Arkansas, was converted to a toxicological research laboratory, and was no longer under the direction or control of the DoD. Biological anticrop agents were destroyed by February 1973. Biological warfare demilitarization continued through the 1970s, with input provided by the U.S. Department of Health, Education and Welfare; U.S. Department of the Interior; U.S. Department of Agriculture; and the Environmental Protection Agency. Fort Detrick and other installations involved in the biological warfare program took on new identities, and their missions were changed to biological defense and the development of medical countermeasures. The necessary containment capability, Biosafety Levels 3 and 4 (BSL-3 and BSL-4) continued to be maintained at USAMRIID.

1980s

In 1984, the DoD requested funds for the construction of another biological aerosol test facility in Utah. The proposal submitted by the army called for BSL-4 containment, although maintaining that the BSL-4 inclusion was based on a possible need in the future and not on a current research effort. The proposal was not well received in Utah, where many citizens and government officials still recalled the secretive projects of the military: the areas on DPG still contaminated with anthrax spores, and the well-publicized accidental chemical poisoning of a flock of sheep in Skull Valley, Utah, in March 1968. Questions arose over the safety of the employees and the surrounding communities, and a suggestion was even made to shift all biological defense research to a civilian agency, such as the National Institutes of Health. The plan for a new facility was revised to utilize a BSL-3 facility, but not before the US Congress had instituted more surveillance, reporting, and control measures on the army to ensure compliance with the BWC.

1990s

In the 1990s, the US medical biological defense research effort (part of the U.S. Army's Biological Defense Research Program [BDRP]) was concentrated at USAMRIID at Fort Detrick. The army maintained state-of-the-art containment laboratory facilities there, with more than 10,000 ft2 of BSL-4 and 50,000 ft2 of BSL-3 laboratory space. BSL-4, the highest containment level, included laboratory suites that are isolated by internal walls and protected by rigorous entry restrictions, air-locks, negative-pressure air-handling systems, and filtration of all out-flow air through high-efficiency particulate air (HEPA) filters. Workers in BSL-4 laboratories also wore filtered positive-pressure total body suits, which isolated the workers from the internal air of the laboratory. BSL-3 laboratories had a similar design, but do not require that personnel wear positive-pressure suits. Workers in BSL-3 suites were protected immunologically by vaccines. U.S. governmental standards provided guidance as to which organisms might be handled under various containment levels in laboratories such as USAMRIID.

The unique facilities available at USAMRIID also included a 16-bed clinical research ward capable of BSL-3 containment, and a 2-bed patient care isolation suite—the Medical Containment Suite (MCS), known as "The Slammer"—where ICU-level care could be provided under BSL-4 containment. Here, healthcare personnel wore the same positive-pressure suits as are worn in BSL-4 research laboratories. The level of patient isolation required depended on the infecting organism and the risk to healthcare providers. Patient care can be provided at BSL-4. There were no patient-care category analogous to BSL-3; humans who are ill as a result of exposure to BSL-3 agents were to be cared for in an ordinary hospital room with barrier nursing procedures.

USAMRIID guidelines were prepared to determine which level of containment would be employed for individual patients who required BSL-4 isolation or barrier nursing care. Staff augmentation for BSL-4 critical care expertise came from the Walter Reed Army Medical Center (WRAMC), Washington, D.C., in accordance with a memorandum of agreement between the two institutions. Patients could be brought directly into the BSL-4 suite from the outside through specialized ports with unique patient-isolation equipment. (The MCS was decommissioned and discontinued in December 2010.)

Additionally, starting in the 1970s USAMRIID maintained a unique evacuation capability known as the Aeromedical Isolation Team (AIT). Led by a physician and a registered nurse, each of the two teams consisted of eight volunteers who trained intensively to provide an evacuation capability for casualties suspected of being infected with highly transmissible, life-threatening BSL-4 infectious diseases (e.g., hemorrhagic fever viruses). The unit used special adult-sized Vickers isolation units (Vickers Medical Containment Stretcher Transit Isolator). These units were aircraft transportable and isolated a patient placed inside from the external environment. The AIT could transport two patients simultaneously; obviously, this was not designed for a mass casualty situation. During the 1995 outbreak of Ebola fever in Zaire, the AIT remained on alert to evacuate any US citizens who might have become ill while working to control the disease in that country.

During this period, some biological defense research also continued at the U.S. Army Medical Research Institute of Chemical Defense, Edgewood Arsenal, Maryland, and the Walter Reed Army Institute of Research (WRAIR), Washington, D.C. USAMRIID and these sister laboratories conducted basic research in support of the medical component of the US biological defense research program, which developed strategies, products, information, procedures, and training for medical defense against biological warfare agents. The products included diagnostic reagents and procedures, drugs, vaccines, toxoids, and antitoxins. Emphasis is placed on protecting personnel before any potential exposure to the biological agent occurs.

In 1997, United States law formally defined weaponizable bio-agents as "Biological Select Agents or Toxins" (BSATs) — or simply Select Agents for short — which fall under the oversight of either the U.S. Department of Health and Human Services or the U.S. Department of Agriculture (or both) and which have the "potential to pose a severe threat to public health and safety".

In 1998, several DoD organizations consolidated to create the Defense Threat Reduction Agency (DTRA), headquartered in Fort Belvoir, Virginia. This agency is DOD's official Combat Support Agency for countering weapons of mass destruction, including bio-agents. DTRA's main functions are threat reduction, threat control, combat support, and technology development. In the US national interest, DTRA supports projects at more than 14 locations around the world, including Russia, Kazakhstan, Azerbaijan, Uzbekistan, Georgia, and Ukraine.

In 1999, a "National Pharmaceutical Stockpile" — renamed Strategic National Stockpile in 2002 — was created under the oversight of DHHS. In the same year, the Laboratory Response Network — a collaborative effort within the US federal government involving the Association of Public Health Laboratories and the Centers for Disease Control and Prevention — was established to facilitate the confirmatory diagnosis and typing of possible bio-agents. Also in 1999, President Bill Clinton issued Executive Order 13139, which provided for experimental anti-WMD drugs to be given to service members at the discretion of the Secretary of Defense only under informed consent; only the President may waive the necessity for informed consent.

2000s

Three secret DoD projects involving countermeasures against anthrax – code named Project Bacchus, Project Clear Vision and Project Jefferson – were publicly disclosed by The New York Times in 2001. (The projects were undertaken between 1997 and 2000 and focused on the concern that the old Soviet BW program was secretly continuing and had developed a genetically modified anthrax weapon.)

Since the September 11 attacks and the 2001 anthrax attacks, the US government has allocated nearly $50 billion to address the threat of biological weapons. Funding for bioweapons-related activities focuses primarily on research for and acquisition of medicines for defense. Biodefense funding also goes toward stockpiling protective equipment, increased surveillance and detection of bio-agents, and improving state and hospital preparedness. Significant funding goes to BARDA (Biomedical Advanced Research and Development Authority), part of DHHS. Funding for activities aimed at prevention has more than doubled since 2007 and is distributed among 11 federal agencies. Efforts toward cooperative international action are part of the project.

A "Select Agent Program" (SAP) was established to satisfy requirements of the USA PATRIOT Act of 2001 and the Public Health Security and Bioterrorism Preparedness and Response Act of 2002. The Centers for Disease Control and Prevention administers the SAP, which regulates the laboratories that may possess, use, or transfer Select Agents within the United States. The Project Bioshield Act was passed by Congress in 2004 calling for $5 billion for purchasing vaccines that would be used in the event of a bioterrorist attack. According to President George W. Bush:

Project BioShield will transform our ability to defend the nation in three essential ways. First, Project BioShield authorizes $5.6 billion over 10 years for the government to purchase and stockpile vaccines and drugs to fight anthrax, smallpox and other potential agents of bioterror. The DHHS has already taken steps to purchase 75 million doses of an improved anthrax vaccine for the Strategic National Stockpile. Under Project BioShield, HHS is moving forward with plans to acquire a safer, second generation smallpox vaccine, an antidote to botulinum toxin, and better treatments for exposure to chemical and radiological weapons. 

This was a ten-year program to acquire medical countermeasures to biological, chemical, radiological and nuclear agents for civilian use. A key element of the Act was to allow stockpiling and distribution of vaccines that had not been tested for safety or efficacy in humans, due to ethical concerns. Efficacy of these agents cannot be directly tested in humans without also exposing humans to the chemical, biological, or radioactive threat being treated. In these cases efficacy testing follows the US Food and Drug Administration Animal Rule for pivotal animal efficacy.

Since 2007, USAMRIID has been joined at Fort Detrick by sister bio-defense agencies of the U.S. Department of Health and Human Services (NIAID's Integrated Research Facility) and the U.S. Department of Homeland Security (the National Biodefense Analysis and Countermeasures Center and the National Bioforensic Analysis Center). These—along with the much older Foreign Disease Weed Science Research Unit of the U.S. Department of Agriculture—now constitute the National Interagency Confederation for Biological Research (NICBR).

2010s

In July 2012, the White House issued its guiding document on the National Biosurveillance Strategy.

2020s

In December 2019, Congress moved forward with a spending package that provided increases for several key U.S. biological defense programs, including the Strategic National Stockpile. The Centers for Disease Control and Prevention was slated to receive $8 billion, a $636 million increase over 2019, with a mandate written in the bill for CDC "to maintain a strong and central role in the medical countermeasures enterprise." Within the CDC budget, the Public Health and Social Services Emergency Fund, which prepares for "all public health emergencies" including bioterrorism and federal efforts against infectious diseases, was funded at $2.74 billion. Another change was a specific item in the budget for the Strategic National Stockpile, which directed $535 million for vaccines, medicines and diagnostic tools to fight Ebola, which has become an emerging threat.

Current status

In August 2019, the U.S. Government Accountability Office (GAO) issued a report that identified specific challenges that the United States faces in protecting the nation against biological events. The report focused on four specific vulnerabilities: assessment of "enterprise-wide threats", situational awareness and data integration, biodetection technologies, and lab safety and security.

Products currently being produced or under development through military research include:

• Vaccines to prevent tularemia, Q fever, Rift Valley fever, Venezuelan equine encephalitis, Eastern and Western equine encephalitis, chikungunya fever, Argentine hemorrhagic fever, the botulinum toxicoses, and anthrax;
• Antitoxins for diseases such as botulism;
• Human immune globulin preparations (passive antibody protection) against various bacteria and viruses; and
• Antiviral drugs against multiple viral agents.

Some vaccines also have applicability for diseases of domestic animals (e.g., Rift Valley fever and Venezuelan equine encephalitis). In addition, vaccines are provided to persons who may be occupationally exposed to such agents (e.g., laboratory workers, entomologists, and veterinary personnel) throughout government, industry, and academe.

USAMRIID also provides diagnostic and epidemiological support to federal, state, and local agencies and foreign governments. Examples of assistance rendered to civilian health efforts by the U.S. Army Medical Research and Materiel Command (USAMRMC) include:

• The massive immunization program instituted during the Venezuelan equine encephalitis outbreak in the Americas in 1971;
• The laboratory support provided to the U.S. Public Health Service during the outbreak of Legionnaire's disease in Philadelphia, Pennsylvania, in 1976;
• The management of patients suspected of having African viral hemorrhagic fever in Sweden during the 1980s;
• International support during the outbreak of Rift Valley fever in Mauritania in 1989;
• Assistance with the outbreak of Ebola infections among monkeys imported to Reston (Virginia) in 1990 (→ Reston virus); and
• Epidemiological and diagnostic support to the World Health Organization–Centers for Disease Control and Prevention field team that studied the Ebola outbreak in Zaire in 1995 (→ Zaire ebolavirus).

The current research effort combines new technological advances, such as genetic engineering and molecular modeling, applying them toward development of prevention and treatment of diseases of military significance. The program is conducted in compliance with requirements set forth by the U.S. Food and Drug Administration (FDA), U.S. Public Health Service, Nuclear Regulatory Commission, U.S. Department of Agriculture, Occupational Safety and Health Administration, and Biological Weapons Convention.

Catalytic reforming

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Catalytic_reforming 

Catalytic reforming is a chemical process used to convert petroleum refinery naphthas distilled from crude oil (typically having low octane ratings) into high-octane liquid products called reformates, which are premium blending stocks for high-octane gasoline. The process converts low-octane linear hydrocarbons (paraffins) into branched alkanes (isoparaffins) and cyclic naphthenes, which are then partially dehydrogenated to produce high-octane aromatic hydrocarbons. The dehydrogenation also produces significant amounts of byproduct hydrogen gas, which is fed into other refinery processes such as hydrocracking. A side reaction is hydrogenolysis, which produces light hydrocarbons of lower value, such as methane, ethane, propane and butanes.

In addition to a gasoline blending stock, reformate is the main source of aromatic bulk chemicals such as benzene, toluene, xylene and ethylbenzene which have diverse uses, most importantly as raw materials for conversion into plastics. However, the benzene content of reformate makes it carcinogenic, which has led to governmental regulations effectively requiring further processing to reduce its benzene content.

This process is quite different from and not to be confused with the catalytic steam reforming process used industrially to produce products such as hydrogen, ammonia, and methanol from natural gas, naphtha or other petroleum-derived feedstocks. Nor is this process to be confused with various other catalytic reforming processes that use methanol or biomass-derived feedstocks to produce hydrogen for fuel cells or other uses.

History

In the 1940s, Vladimir Haensel, a research chemist working for Universal Oil Products (UOP), developed a catalytic reforming process using a catalyst containing platinum. Haensel's process was subsequently commercialized by UOP in 1949 for producing a high octane gasoline from low octane naphthas and the UOP process become known as the Platforming process. The first Platforming unit was built in 1949 at the refinery of the Old Dutch Refining Company in Muskegon, Michigan.

In the years since then, many other versions of the process have been developed by some of the major oil companies and other organizations. Today, the large majority of gasoline produced worldwide is derived from the catalytic reforming process.

To name a few of the other catalytic reforming versions that were developed, all of which utilized a platinum and/or a rhenium catalyst:

• Rheniforming: Developed by Chevron Oil Company.
• CCR Platforming: A Platforming version, designed for continuous catalyst regeneration, developed by Universal Oil Products (UOP).
• Powerforming: Developed by Esso Oil Company, currently known as ExxonMobil.
• Magnaforming: Developed by Engelhard and Atlantic Richfield Oil Company.
• Ultraforming: Developed by Standard Oil of Indiana, now a part of the British Petroleum Company.
• Houdriforming: Developed by the Houdry Process Corporation.
• Octanizing: A catalytic reforming version developed by Axens, a subsidiary of Institut francais du petrole (IFP), designed for continuous catalyst regeneration.

Chemistry

Before describing the reaction chemistry of the catalytic reforming process as used in petroleum refineries, the typical naphthas used as catalytic reforming feedstocks will be discussed.

Typical naphtha feedstocks

A petroleum refinery includes many unit operations and unit processes. The first unit operation in a refinery is the continuous distillation of the petroleum crude oil being refined. The overhead liquid distillate is called naphtha and will become a major component of the refinery's gasoline (petrol) product after it is further processed through a catalytic hydrodesulfurizer to remove sulfur-containing hydrocarbons and a catalytic reformer to reform its hydrocarbon molecules into more complex molecules with a higher octane rating value. The naphtha is a mixture of very many different hydrocarbon compounds. It has an initial boiling point of about 35 °C and a final boiling point of about 200 °C, and it contains paraffin, naphthene (cyclic paraffins) and aromatic hydrocarbons ranging from those containing 6 carbon atoms to those containing about 10 or 11 carbon atoms.

The naphtha from the crude oil distillation is often further distilled to produce a "light" naphtha containing most (but not all) of the hydrocarbons with 6 or fewer carbon atoms and a "heavy" naphtha containing most (but not all) of the hydrocarbons with more than 6 carbon atoms. The heavy naphtha has an initial boiling point of about 140 to 150 °C and a final boiling point of about 190 to 205 °C. The naphthas derived from the distillation of crude oils are referred to as "straight-run" naphthas.

It is the straight-run heavy naphtha that is usually processed in a catalytic reformer because the light naphtha has molecules with 6 or fewer carbon atoms which, when reformed, tend to crack into butane and lower molecular weight hydrocarbons which are not useful as high-octane gasoline blending components. Also, the molecules with 6 carbon atoms tend to form aromatics which is undesirable because governmental environmental regulations in a number of countries limit the amount of aromatics (most particularly benzene) that gasoline may contain.

There are a great many petroleum crude oil sources worldwide and each crude oil has its own unique composition or "assay". Also, not all refineries process the same crude oils and each refinery produces its own straight-run naphthas with their own unique initial and final boiling points. In other words, naphtha is a generic term rather than a specific term.

The table just below lists some fairly typical straight-run heavy naphtha feedstocks, available for catalytic reforming, derived from various crude oils. It can be seen that they differ significantly in their content of paraffins, naphthenes and aromatics:

Typical Heavy Naphtha Feedstocks

Crude oil name ${\displaystyle \Rightarrow }$ Location ${\displaystyle \Rightarrow }$	Barrow Island Australia	Mutineer-Exeter Australia	CPC Blend Kazakhstan	Draugen North Sea
Initial boiling point, °C	149	140	149	150
Final boiling point, °C	204	190	204	180
Paraffins, liquid volume %	46	62	57	38
Naphthenes, liquid volume %	42	32	27	45
Aromatics, liquid volume %	12	6	16	17

Some refinery naphthas include olefinic hydrocarbons, such as naphthas derived from the fluid catalytic cracking and coking processes used in many refineries. Some refineries may also desulfurize and catalytically reform those naphthas. However, for the most part, catalytic reforming is mainly used on the straight-run heavy naphthas, such as those in the above table, derived from the distillation of crude oils.

The reaction chemistry

There are many chemical reactions that occur in the catalytic reforming process, all of which occur in the presence of a catalyst and a high partial pressure of hydrogen. Depending upon the type or version of catalytic reforming used as well as the desired reaction severity, the reaction conditions range from temperatures of about 495 to 525 °C and from pressures of about 5 to 45 atm.

The commonly used catalytic reforming catalysts contain noble metals such as platinum and/or rhenium, which are very susceptible to poisoning by sulfur and nitrogen compounds. Therefore, the naphtha feedstock to a catalytic reformer is always pre-processed in a hydrodesulfurization unit which removes both the sulfur and the nitrogen compounds. Most catalysts require both sulphur and nitrogen content to be lower than 1 ppm.

The four major catalytic reforming reactions are:

1: The dehydrogenation of naphthenes to convert them into aromatics as exemplified in the conversion methylcyclohexane (a naphthene) to toluene (an aromatic), as shown below:

2: The isomerization of normal paraffins to isoparaffins as exemplified in the conversion of normal octane to 2,5-Dimethylhexane (an isoparaffin), as shown below:

3: The dehydrogenation and aromatization of paraffins to aromatics (commonly called dehydrocyclization) as exemplified in the conversion of normal heptane to toluene, as shown below:

4: The hydrocracking of paraffins into smaller molecules as exemplified by the cracking of normal heptane into isopentane and ethane, as shown below:

During the reforming reactions, the carbon number of the reactants remains unchanged, except for hydrocracking reactions which break down the hydrocarbon molecule into molecules with fewer carbon atoms.^[11] The hydrocracking of paraffins is the only one of the above four major reforming reactions that consumes hydrogen. The isomerization of normal paraffins does not consume or produce hydrogen. However, both the dehydrogenation of naphthenes and the dehydrocyclization of paraffins produce hydrogen. The overall net production of hydrogen in the catalytic reforming of petroleum naphthas ranges from about 50 to 200 cubic meters of hydrogen gas (at 0 °C and 1 atm) per cubic meter of liquid naphtha feedstock. In the United States customary units, that is equivalent to 300 to 1200 cubic feet of hydrogen gas (at 60 °F and 1 atm) per barrel of liquid naphtha feedstock. In many petroleum refineries, the net hydrogen produced in catalytic reforming supplies a significant part of the hydrogen used elsewhere in the refinery (for example, in hydrodesulfurization processes). The hydrogen is also necessary in order to hydrogenolyze any polymers that form on the catalyst.

In practice, the higher the content of naphthenes in the naphtha feedstock, the better will be the quality of the reformate and the higher the production of hydrogen. Crude oils containing the best naphtha for reforming are typically from Western Africa or the North Sea, such as Bonny light oil or Norwegian Troll.

Model reactions using lumping technique

Owing to too many components in catalytic reforming process feedstock, untraceable reactions and the high temperature range, the design and simulation of catalytic reformer reactors is accompanied by complexities. The lumping technique is used extensively for reducing complexities so that the lumps and reaction pathways that properly describe the reforming system and kinetic rate parameters do not depend on feedstock composition. In one of the recent works, naphtha is considered in terms of 17 hydrocarbon fractions with 15 reactions in which C₁ to C₅ hydrocarbons are specified as light paraffins and the C₆ to C₈₊ naphtha cuts are characterized as isoparaffins, normal paraffins, naphthenes and aromatics. Reactions in catalytic naphtha reforming are elementary and Hougen-Watson Langmuir-Hinshelwood type reaction rate expressions are used to describe the rate of each reaction. Rate equations of this type explicitly account for the interaction of chemical species with catalyst and contain denominators in which terms characteristic of the adsorption of reacting species are presented.

Process description

The most commonly used type of catalytic reforming unit has three reactors, each with a fixed bed of catalyst, and all of the catalyst is regenerated in situ during routine catalyst regeneration shutdowns which occur approximately once each 6 to 24 months. Such a unit is referred to as a semi-regenerative catalytic reformer (SRR).

Some catalytic reforming units have an extra spare or swing reactor and each reactor can be individually isolated so that any one reactor can be undergoing in situ regeneration while the other reactors are in operation. When that reactor is regenerated, it replaces another reactor which, in turn, is isolated so that it can then be regenerated. Such units, referred to as cyclic catalytic reformers, are not very common. Cyclic catalytic reformers serve to extend the period between required shutdowns.

The latest and most modern type of catalytic reformers are called continuous catalyst regeneration (CCR) reformers. Such units are defined by continuous in-situ regeneration of part of the catalyst in a special regenerator, and by continuous addition of the regenerated catalyst to the operating reactors. As of 2006, two CCR versions available: UOP's CCR Platformer process and Axens' Octanizing process. The installation and use of CCR units is rapidly increasing.

Many of the earliest catalytic reforming units (in the 1950s and 1960s) were non-regenerative in that they did not perform in situ catalyst regeneration. Instead, when needed, the aged catalyst was replaced by fresh catalyst and the aged catalyst was shipped to catalyst manufacturers to be either regenerated or to recover the platinum content of the aged catalyst. Very few, if any, catalytic reformers currently in operation are non-regenerative.

The process flow diagram below depicts a typical semi-regenerative catalytic reforming unit.

Schematic diagram of a typical semi-regenerative catalytic reformer unit in a petroleum refinery

The liquid feed (at the bottom left in the diagram) is pumped up to the reaction pressure (5–45 atm) and is joined by a stream of hydrogen-rich recycle gas. The resulting liquid–gas mixture is preheated by flowing through a heat exchanger. The preheated feed mixture is then totally vaporized and heated to the reaction temperature (495–520 °C) before the vaporized reactants enter the first reactor. As the vaporized reactants flow through the fixed bed of catalyst in the reactor, the major reaction is the dehydrogenation of naphthenes to aromatics (as described earlier herein) which is highly endothermic and results in a large temperature decrease between the inlet and outlet of the reactor. To maintain the required reaction temperature and the rate of reaction, the vaporized stream is reheated in the second fired heater before it flows through the second reactor. The temperature again decreases across the second reactor and the vaporized stream must again be reheated in the third fired heater before it flows through the third reactor. As the vaporized stream proceeds through the three reactors, the reaction rates decrease and the reactors therefore become larger. At the same time, the amount of reheat required between the reactors becomes smaller. Usually, three reactors are all that is required to provide the desired performance of the catalytic reforming unit.

Some installations use three separate fired heaters as shown in the schematic diagram and some installations use a single fired heater with three separate heating coils.

The hot reaction products from the third reactor are partially cooled by flowing through the heat exchanger where the feed to the first reactor is preheated and then flow through a water-cooled heat exchanger before flowing through the pressure controller (PC) into the gas separator.

Most of the hydrogen-rich gas from the gas separator vessel returns to the suction of the recycle hydrogen gas compressor and the net production of hydrogen-rich gas from the reforming reactions is exported for use in the other refinery processes that consume hydrogen (such as hydrodesulfurization units and/or a hydrocracker unit).

The liquid from the gas separator vessel is routed into a fractionating column commonly called a stabilizer. The overhead offgas product from the stabilizer contains the byproduct methane, ethane, propane and butane gases produced by the hydrocracking reactions as explained in the above discussion of the reaction chemistry of a catalytic reformer, and it may also contain some small amount of hydrogen. That offgas is routed to the refinery's central gas processing plant for removal and recovery of propane and butane. The residual gas after such processing becomes part of the refinery's fuel gas system.

The bottoms product from the stabilizer is the high-octane liquid reformate that will become a component of the refinery's product gasoline. Reformate can be blended directly in the gasoline pool but often it is separated in two or more streams. A common refining scheme consists in fractionating the reformate in two streams, light and heavy reformate. The light reformate has lower octane and can be used as isomerization feedstock if this unit is available. The heavy reformate is high in octane and low in benzene, hence it is an excellent blending component for the gasoline pool.

Benzene is often removed with a specific operation to reduce the content of benzene in the reformate as the finished gasoline has often an upper limit of benzene content (in the UE this is 1% volume). The benzene extracted can be marketed as feedstock for the chemical industry.

Catalysts and mechanisms

Most catalytic reforming catalysts contain platinum or rhenium on a silica or silica-alumina support base, and some contain both platinum and rhenium. Fresh catalyst is chlorided (chlorinated) prior to use.

The noble metals (platinum and rhenium) are considered to be catalytic sites for the dehydrogenation reactions and the chlorinated alumina provides the acid sites needed for isomerization, cyclization and hydrocracking reactions. The biggest care has to be exercised during the chlorination. Indeed, if not chlorinated (or insufficiently chlorinated) the platinum and rhenium in the catalyst would be reduced almost immediately to metallic state by the hydrogen in the vapour phase. On the other hand, an excessive chlorination could depress excessively the activity of the catalyst.

The activity (i.e., effectiveness) of the catalyst in a semi-regenerative catalytic reformer is reduced over time during operation by carbonaceous coke deposition and chloride loss. The activity of the catalyst can be periodically regenerated or restored by in situ high temperature oxidation of the coke followed by chlorination. As stated earlier herein, semi-regenerative catalytic reformers are regenerated about once per 6 to 24 months. The higher the severity of the reacting conditions (temperature), the higher the octane of the produced reformate but also the shorter the duration of the cycle between two regenerations. Catalyst's cycle duration is also very dependent on the quality of the feedstock. However, independently of the crude oil used in the refinery, all catalysts require a maximum final boiling point of the naphtha feedstock of 180 °C.

Normally, the catalyst can be regenerated perhaps 3 or 4 times before it must be returned to the manufacturer for reclamation of the valuable platinum and/or rhenium content.

Weaknesses and Competition

The sensitivity of catalytic reforming to contamination by sulfur and nitrogen requires hydrotreating the naphtha before it enters the reformer, adding to the cost and complexity of the process. Dehydrogenation, an important component of reforming, is a strongly endothermic reaction, and as such, requires the reactor vessel to be externally heated. This contributes both to costs and the emissions of the process. Catalytic reforming has a limited ability to process naphthas with a high content of normal paraffins, e.g. naphthas from the gas-to-liquids (GTL) units. The reformate has a much higher content of benzene than is permissible by the current regulations in many countries. This means that the reformate should either be further processed in an aromatics extraction unit, or blended with appropriate hydrocarbon streams with low content of aromatics. Catalytic reforming requires a whole range of other processing units at the refinery (apart from the distillation tower, a naphtha hydrotreater, usually an isomerization unit to process light naphtha, an aromatics extraction unit, etc.) which puts it out of reach for smaller (micro-)refineries.

Main licensors of catalytic reforming processes, UOP and Axens, constantly work on improving the catalysts, but the rate of improvement seems to be reaching its physical limits. This is driving the emergence of new technologies to process naphtha into gasoline by companies like Chevron Phillips Chemical (Aromax) and NGT Synthesis (Methaforming).

Economics

Catalytic reformation is profitable in that it converts long-chain hydrocarbons, for which there is limited demand despite high supply, into short-chained hydrocarbons, which, due to their uses in petrol fuel, are in much greater demand. It can also be used to improve the octane rating of short-chained hydrocarbons by aromatizing them.

Nucleic acid analogue

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Nucleic_acid_analogue 

RNA with its nucleobases to the left and DNA to the right.

Nucleic acid analogues are compounds which are analogous (structurally similar) to naturally occurring RNA and DNA, used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain (PNA can even form a triple helix). Nucleic acid analogues are also called Xeno Nucleic Acid and represent one of the main pillars of xenobiology, the design of new-to-nature forms of life based on alternative biochemistries.

Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.

In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, and by including individual artificial nucleotides in the culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides. The artificial nucleotides featured 2 fused aromatic rings.

Medicine

Main article: Nucleoside analogues

Several nucleoside analogues are used as antiviral or anticancer agents. The viral polymerase incorporates these compounds with non-canonical bases. These compounds are activated in the cells by being converted into nucleotides, they are administered as nucleosides since charged nucleotides cannot easily cross cell membranes.

Molecular biology

Common changes in nucleotide analogues

Nucleic acid analogues are used in molecular biology for several purposes: Investigation of possible scenarios of the origin of life: By testing different analogs, researchers try to answer the question of whether life's use of DNA and RNA was selected over time due to its advantages, or if they were chosen by arbitrary chance; As a tool to detect particular sequences: XNA can be used to tag and identify a wide range of DNA and RNA components with high specificity and accuracy; As an enzyme acting on DNA, RNA and XNA substrates - XNA has been shown to have the ability to cleave and ligate DNA, RNA and other XNA molecules similar to the actions of RNA ribozymes; As a tool with resistance to RNA hydrolysis; Investigation of the mechanisms used by enzyme; Investigation of the structural features of nucleic acids.

Backbone analogues

Hydrolysis resistant RNA-analogues

Chemical structure of Morpholino

To overcome the fact that ribose's 2' hydroxy group that reacts with the phosphate linked 3' hydroxy group (RNA is too unstable to be used or synthesized reliably), a ribose analogue is used. The most common RNA analogues are 2'-O-methyl-substituted RNA, locked nucleic acid (LNA) or bridged nucleic acid (BNA), morpholino, and peptide nucleic acid (PNA). Although these oligonucleotides have a different backbone sugar or, in the case of PNA, an amino acid residue in place of the ribose phosphate, they still bind to RNA or DNA according to Watson and Crick pairing, but are immune to nuclease activity. They cannot be synthesized enzymatically and can only be obtained synthetically using phosphoramidite strategy or, for PNA, methods of peptide synthesis.

Other notable analogues used as tools

Dideoxynucleotides are used in sequencing . These nucleoside triphosphates possess a non-canonical sugar, dideoxyribose, which lacks the 3' hydroxyl group normally present in DNA and therefore cannot bond with the next base. The lack of the 3' hydroxyl group terminates the chain reaction as the DNA polymerases mistake it for a regular deoxyribonucleotide. Another chain-terminating analogue that lacks a 3' hydroxyl and mimics adenosine is called cordycepin. Cordycepin is an anticancer drug that targets RNA replication. Another analogue in sequencing is a nucleobase analogue, 7-deaza-GTP and is used to sequence CG rich regions, instead 7-deaza-ATP is called tubercidin, an antibiotic.

Precursors to the RNA world

Main article: RNA world hypothesis

RNA may be too complex to be the first nucleic acid, so before the RNA world several simpler nucleic acids that differ in the backbone, such as TNA and GNA and PNA, have been offered as candidates for the first nucleic acids.

Base analogues

Nucleobase structure and nomenclature

Purine

 

Pyrimidine

 

Main article: Nucleobase

 

For structures of the analogues that may be mentioned in the literature, see Simple aromatic ring.

Naturally occurring bases can be divided into two classes according to their structure:

• pyrimidines are six-membered heterocyclic with nitrogen atoms in position 1 and 3.
• purines are bicyclic, consisting of a pyrimidine fused to an imidazole ring.

Artificial nucleotides (Unnatural Base Pairs (UBPs) named d5SICS UBP and dNaM UBP) have been inserted into bacterial DNA but these genes did not template mRNA or induce protein synthesis. The artificial nucleotides featured two fused aromatic rings which formed a (d5SICS–dNaM) complex mimicking the natural (dG–dC) base pair.

Mutagens

One of the most common base analogs is 5-bromouracil (5BU), the abnormal base found in the mutagenic nucleotide analog BrdU. When a nucleotide containing 5-bromouracil is incorporated into the DNA, it is most likely to pair with adenine; however, it can spontaneously shift into another isomer which pairs with a different nucleobase, guanine. If this happens during DNA replication, a guanine will be inserted as the opposite base analog, and in the next DNA replication, that guanine will pair with a cytosine. This results in a change in one base pair of DNA, specifically a transition mutation.

Additionally, HNO2, or nitrous acid is a potent mutagen that acts on replicating and non-replicating DNA. It can cause deamination of the amino groups of Adenine, Guanine and Cytosine. Adenine is deaminated to hypoxanthine, which base pairs to cytosine instead of thymine. Cytosine is deaminated to uracil, which base pairs with Adenine instead of Guanine. Deamination of Guanine is not mutagenic. Nitrous acid-induced mutations also are induced to mutate back to wild-type using nitrous acid.

Fluorophores

Main article: Fluorophore

 

Structure of aminoallyl-uridine

Commonly fluorophores (such as rhodamine or fluorescein) are linked to the ring linked to the sugar (in para) via a flexible arm, presumably extruding from the major groove of the helix. Due to low processivity of the nucleotides linked to bulky adducts such as florophores by taq polymerases, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling):

• amine reactive: Aminoallyl nucleotide contain a primary amine group on a linker that reacts with the amino-reactive dye such as a cyanine or Alexa Fluor dyes, which contain a reactive leaving group, such as a succinimidyl ester (NHS). (base-pairing amino groups are not affected).
• thiol reactive: thiol containing nucleotides reacts with the fluorophore linked to a reactive leaving group, such as a maleimide.
• biotin linked nucleotides rely on the same indirect labelling principle (+ fluorescent streptavidin) and are used in Affymetrix DNAchips.

Fluorophores find a variety of uses in medicine and biochemistry.

Fluorescent base analogues

The most commonly used and commercially available fluorescent base analogue, 2-aminopurine (2-AP), has a high-fluorescence quantum yield free in solution (0.68) that is considerably reduced (appr. 100 times but highly dependent on base sequence) when incorporated into nucleic acids. The emission sensitivity of 2-AP to immediate surroundings is shared by other promising and useful fluorescent base analogues like 3-MI, 6-MI, 6-MAP, pyrrolo-dC (also commercially available), modified and improved derivatives of pyrrolo-dC, furan-modified bases and many other ones (see recent reviews). This sensitivity to the microenvironment has been utilized in studies of e.g. structure and dynamics within both DNA and RNA, dynamics and kinetics of DNA-protein interaction and electron transfer within DNA.

A newly developed and very interesting group of fluorescent base analogues that has a fluorescence quantum yield that is nearly insensitive to their immediate surroundings is the tricyclic cytosine family. 1,3-Diaza-2-oxophenothiazine, tC, has a fluorescence quantum yield of approximately 0.2 both in single- and in double-strands irrespective of surrounding bases. Also the oxo-homologue of tC called tC^O (both commercially available), 1,3-diaza-2-oxophenoxazine, has a quantum yield of 0.2 in double-stranded systems. However, it is somewhat sensitive to surrounding bases in single-strands (quantum yields of 0.14–0.41). The high and stable quantum yields of these base analogues make them very bright, and, in combination with their good base analogue properties (leaves DNA structure and stability next to unperturbed), they are especially useful in fluorescence anisotropy and FRET measurements, areas where other fluorescent base analogues are less accurate. Also, in the same family of cytosine analogues, a FRET-acceptor base analogue, tC_nitro, has been developed. Together with tC^O as a FRET-donor this constitutes the first nucleic acid base analogue FRET-pair ever developed. The tC-family has, for example, been used in studies related to polymerase DNA-binding and DNA-polymerization mechanisms.

Natural non-canonical bases

In a cell, there are several non-canonical bases present: CpG islands in DNA (are often methylated), all eukaryotic mRNA (capped with a methyl-7-guanosine), and several bases of rRNAs (are methylated). Often, tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing, in particular in/near the anticodon: inosine can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific than uracil (with a purine). Other common tRNA base modifications are pseudouridine (which gives its name to the TΨC loop), dihydrouridine (which does not stack as it is not aromatic), queuosine, wyosine, and so forth. Nevertheless, these are all modifications to normal bases and are not placed by a polymerase. 

Base-pairing

Canonical bases may have either a carbonyl or an amine group on the carbons surrounding the nitrogen atom furthest away from the glycosidic bond, which allows them to base pair (Watson-Crick base pairing) via hydrogen bonds (amine with ketone, purine with pyrimidine). Adenine and 2-aminoadenine have one/two amine group(s), whereas thymine has two carbonyl groups, and cytosine and guanine are mixed amine and carbonyl (inverted in respect to each other).

Natural basepairs
A GC basepair: purine carbonyl/amine forms three intermolecular hydrogen bonds with pyrimidine amine/carbonyl	An AT basepair: purine amine/- forms two intermolecular hydrogen bonds with pyrimidine carbonyl/carbonyl

The precise reason why there are only four nucleotides is debated, but there are several unused possibilities. Furthermore, adenine is not the most stable choice for base pairing: in Cyanophage S-2L diaminopurine (DAP) is used instead of adenine (host evasion). Diaminopurine basepairs perfectly with thymine as it is identical to adenine but has an amine group at position 2 forming 3 intramolecular hydrogen bonds, eliminating the major difference between the two types of basepairs (Weak:A-T and Strong:C-G). This improved stability affects protein-binding interactions that rely on those differences. Other combination include,

• isoguanine and isocytosine, which have their amine and ketone inverted compared to standard guanine and cytosine, (not used probably as tautomers are problematic for base pairing, but isoC and isoG can be amplified correctly with PCR even in the presence of the 4 canonical bases)
• diaminopyrimidine and a xanthine, which bind like 2-aminoadenine and thymine but with inverted structures (not used as xanthine is a deamination product)

Unused basepair arrangements
A DAP-T base: purine amine/amine forms three intermolecular hydrogen bonds with pyrimidine ketone/ketone	An X-DAP base: purine ketone/ketone forms three intermolecular hydrogen bonds with pyrimidine amine/amine	A iG-iC base: purine amine/ketone forms three intermolecular hydrogen bonds with pyrimidine ketone/amine

However, correct DNA structure can form even when the bases are not paired via hydrogen bonding; that is, the bases pair thanks to hydrophobicity, as studies have shown using DNA isosteres (analogues with same number of atoms), such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z). An alternative hydrophobic pair could be isoquinoline, and the pyrrolo[2,3-b]pyridine

Other noteworthy basepairs:

• Several fluorescent bases have also been made, such as the 2-amino-6-(2-thienyl)purine and pyrrole-2-carbaldehyde base pair.
• Metal-coordinated bases, such as pairing between a pyridine-2,6-dicarboxylate (tridentate ligand) and a pyridine (monodentate ligand) through square planar coordination to a central copper ion.
• Universal bases may pair indiscriminately with any other base, but, in general, lower the melting temperature of the sequence considerably; examples include 2'-deoxyinosine (hypoxanthine deoxynucleotide) derivatives, nitroazole analogues, and hydrophobic aromatic non-hydrogen-bonding bases (strong stacking effects). These are used as proof of concept and, in general, are not utilised in degenerate primers (which are a mixture of primers).
• The numbers of possible base pairs is doubled when xDNA is considered. xDNA contains expanded bases, in which a benzene ring has been added, which may pair with canonical bases, resulting in four possible base-pairs (8 bases:xA-T,xT-A,xC-G,xG-C, 16 bases if the unused arrangements are used). Another form of benzene added bases is yDNA, in which the base is widened by the benzene.

Novel basepairs with special properties
A F-Z base: methylbenzimidazole does not form intermolecular hydrogen bonds with toluene F/F	An S-Pa base: purine thienyl/amine forms three intermolecular hydrogen bonds with pyrrole -/carbaldehyde	An xA-T base: same bonding as A-T

Metal base-pairs

In metal base-pairing, the Watson-Crick hydrogen bonds are replaced by the interaction between a metal ion with nucleosides acting as ligands. The possible geometries of the metal that would allow for duplex formation with two bidentate nucleosides around a central metal atom are: tetrahedral, dodecahedral, and square planar. Metal-complexing with DNA can occur by the formation of non-canonical base pairs from natural nucleobases with participation by metal ions and also by the exchanging the hydrogen atoms that are part of the Watson-Crick base pairing by metal ions. Introduction of metal ions into a DNA duplex has shown to have potential magnetic, conducting properties, as well as increased stability.

Metal complexing has been shown to occur between natural nucleobases. A well-documented example is the formation of T-Hg-T, which involves two deprotonated thymine nucleobases that are brought together by Hg²⁺ and forms a connected metal-base pair. This motif does not accommodate stacked Hg²⁺ in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation. Two thymines across from each other in a duplex do not form a Watson-Crick base pair in a duplex; this is an example where a Watson-Crick basepair mismatch is stabilized by the formation of the metal-base pair. Another example of a metal complexing to natural nucleobases is the formation of A-Zn-T and G-Zn-C at high pH; Co⁺² and Ni⁺² also form these complexes. These are Watson-Crick base pairs where the divalent cation in coordinated to the nucleobases. The exact binding is debated.

A large variety of artificial nucleobases have been developed for use as metal base pairs. These modified nucleobases exhibit tunable electronic properties, sizes, and binding affinities that can be optimized for a specific metal. For, example a nucleoside modified with a pyridine-2,6-dicarboxylate has shown to bind tightly to Cu²⁺, whereas other divalent ions are only loosely bound. The tridentate character contributes to this selectivity. The fourth coordination site on the copper is saturated by an oppositely arranged pyridine nucleobase. The asymmetric metal base pairing system is orthogonal to the Watson-Crick base pairs. Another example of an artificial nucleobase is that with hydroxypyridone nucleobases, which are able to bind Cu²⁺ inside the DNA duplex. Five consecutive copper-hydroxypyridone base pairs were incorporated into a double strand, which were flanked by only one natural nucleobase on both ends. EPR data showed that the distance between copper centers was estimated to be 3.7 ± 0.1 Å, while a natural B-type DNA duplex is only slightly larger (3.4 Å). The appeal for stacking metal ions inside a DNA duplex is the hope to obtain nanoscopic self-assembling metal wires, though this has not been realized yet.

Unnatural base pair (UBP)

Main article: Unnatural base pair

An unnatural base pair (UBP) is a designed subunit (or nucleobase) of DNA which is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP). The two new artificial nucleotides or Unnatural Base Pair (UBP) were named d5SICS and dNaM. More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium E. coli that successfully replicated the unnatural base pairs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.

The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins. Earlier, the artificial strings of DNA did not encode for anything, but scientists speculated they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses. Transcription of DNA containing unnatural base pair and translation of corresponding mRNA were actually achieved recently. In November 2017, the same team at the Scripps Research Institute that first introduced two extra nucleobases into bacterial DNA, reported having constructed a semi-synthetic E. coli bacteria able to make proteins using such DNA. Its DNA contained six different nucleobases: four canonical and two artificially added, dNaM and dTPT3 (these two form a pair). Also, this bacteria had two corresponding additional RNA bases included in two new codons, additional tRNAs recognizing these new codons (these tRNAs also contained two new RNA bases within their anticodons) and additional amino acids, making the bacteria able to synthesize "unnatural" proteins.

Another demonstration of UBPs were achieved by Ichiro Hirao's group at RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions in vitro in transcription and translation, for the site-specific incorporation of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription. Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by in vitro selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins.

Orthogonal system

The possibility has been proposed and studied, both theoretically and experimentally, of implementing an orthogonal system inside cells independent of the cellular genetic material in order to make a completely safe system, with the possible increase in encoding potentials. Several groups have focused on different aspects:

• novel backbones and base pairs as discussed above
• XNA (Xeno Nucleic Acid) artificial replication/transcription polymerases starting generally from T7 RNA polymerase
• ribosomes (16S sequences with altered anti Shine-Dalgarno sequence allowing the translation of only orthogonal mRNA with a matching altered Shine-Dalgarno sequence)
• novel tRNA encoding non-natural aminoacids. See Expanded genetic code