Photochemistry 2025

PHOTO CHEMISTRY

Empowering Peptide Innovation

With this guiding theme in mind, Iris Biotech’s mission is to support researchers by supplying

• innovative technologies,

• rare compounds,

• as well as a broad portfolio on standard consumables, available in flexible quantities from small scale to bulk quantities. To fulfill our dedication “Empowering Peptide Innovation”, we are attending various conferences, symposia, and exhibitions each year. This allows us to remain in direct contact with scientists all over the world, both from academia and industry, to exchange knowledge, and to gather new ideas to tackle your current challenges.

Guided by our dedication to provide

• competent service,

• as well as novel substances and latest technologies,

Iris Biotech is your trusted partner for the world of peptides, while having strong expertise in associated disciplines. Thus, our portfolio comprises reagents and tools for the synthesis and modification of peptides, e.g., amino acids, resins and solvents but also for related technologies such as drug delivery, linkerology® and life sciences.

Owed to the growing demand for tailor-made compounds, our portfolio is fine-tuned by our custom synthesis service at Iris Biotech Laboratories. Our skilled scientists offer profound expertise in de novo route development,

• upscaling towards larger scale production,

• as well as synthesis optimization for increased efficiency.

Examples are the synthesis of rare chiral building blocks, unnatural amino acid derivatives, sophisticated orthogonal protecting groups, heterocycles, building blocks for nucleotides, PEGs and PEG-analogs as well as specific linkers for controlled drug delivery and release.

Portfolio Overview

Peptide Synthesis and Modification

(Protected) Amino Acids

Standards such as Fmoc-D/L-AAA and Boc-D/L-AAA, Smoc amino acids for peptide synthesis in water, variety of protecting groups (e.g., Pbf, Trt, tBu, Bzl, Acm, Mob, SIT, Phacm, Allocam, Mmt), unusual amino acids, fluorinated derivatives, substituted prolines, arginine analogs

Building Blocks

Amino alcohols, amino aldehydes, diamines and hydrazines, (pseudoproline) dipeptides, polyamines and spermines, fatty acid derivatives, peptide nucleic acids (PNAs)

Reagents

Coupling reagents, solvents and scavengers, protecting groups

Resins

Preloaded resins (e.g., based on Trityl, TCP, TentaGel, Methoxybenzhydryl, Merrifield, PAM, Rink, Wang), scavenger resins, hydrazone resins, poly(acrylamide) resins, Cyclover

Linkerology® and Drug Delivery Life Sciences

Linkers for Solid Phase Peptide Synthesis

Cleavable Linkers

Val-Ala-based, Val-Cit-based, disulfide-based, Dde-helping hands, pH-sensitive linkers

Photo-Activatable Linkers

Functionalized Linkers

Clickable linkers, trifunctional linkers, linkers with maleimide function, cross-linkers, selective N-term acylation and biotinylation, 5HP2O

PROTACs

Ligands, linkers & modules

Fullerenes, Poly(2-oxazolines), Dextrans & Plant-Derived Cholesterol

Superparamagnetic Iron Oxide Nanoparticles

Poly-Amino Acids

Poly-Arg, Poly-Glu, Poly-Lys, Poly-Orn, Poly-Sar

PEGylation

Branched PEGylating reagents, (amino-)PEG-acids, PEG-amines & hydrazides & guanidines, reagents for Click-conjugation, Biotin-PEG-reagents, PEG-thiols, PEG-maleimides, other PEGylating reagents

Biotinylation Reagents

Carbohydrates

Galactose, Glucose, Mannose, Xylose and others

Drug Metabolites

Peptides

Substrates & Inhibitors

E.g., protein kinase inhibitors, substrates for fusion (Halo/ Snap/Clip)-tagged proteins

Natural Products

Dyes and Fluorescent Labels

E.g., ICG, AMC, DAPI

Maillard & Amadori Reaction Products

Large portfolio of derivatives useful as standards for food, pharma and cosmetics industry

Vitamins

Custom Synthesis

Your project requires a compound not listed in our portfolio? Get in contact and inquire about our custom synthesis capabilities.

Our experienced scientists are excited to accept your synthetic challenge! In such cases, your request undergoes the following stages:

Step-by-Step Analysis

Customer’s demands

Process Evaluation

Det ailed literature review

Synthetic possibilities

Strategy Development

Protocol development

Method development and validation

• Customized synthesis

Our Service Promise

Quality Consistency

• Identity confirmation Purity verification

All our services are based on high standards, transparency & documentation, trust, honesty & confidentiality, as well as the required know-how.

High Standards

Values: sustainability & responsibility

State-of-the-art equipment & latest technologies

High quality standards

Qualified suppliers & regular audits

Trust, Honesty & Confidentiality

Intergenerational business valuing partnerships

• Meeting the customer‘s expectations

• Integrity towards our customers

Transparency & Documentation

Talk to our specialists – customer care

Certificates of analysis & origin

Impurity profiling

Safety data sheets

• Analytical and process reports

Our Know-How

One-step reactions & complex multi-step synthesis

• Scalability from mg to kg quantities

• Route scouting

1. Photochemistry in Peptide Synthesis and Bioconjugation

The term photochemistry describes a group of chemical transformations initiated by irradiation with light. Photochemical reactions usually occur at room temperature and normal pressure, and mostly do not require additional reagents or catalysts, with the notable exception of some cases where the presence of a photosensitizer is necessary. Therefore, photochemical transformations are usually orthogonal to classical chemical transformations, a characteristic that renders them a valuable tool for chemists.

Consequently, the applications of photochemistry are numerous. Orthogonality is a trait often sought for in protecting groups and linkers, as it allows for their selective cleavage. Furthermore, photocleavage is a convenient method for selective removal of auxiliaries after their function has been served. The typical moiety that is incorporated into protecting groups, linkers and auxiliaries to facilitate light-induced cleavage is an o-nitrobenzyl group, which undergoes a Norrish-type II reaction upon UV-irradiation.

Another common application of photochemistry is the labeling or crosslinking of biomolecules in vitro and in vivo. The latter is of particular interest as a photochemical reaction is one of the few chemical transformations that can be selectively initiated in living cells.

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Do you require different photoactivated compounds?

Inquire with our Custom Synthesis service and download our booklet for more information on our capabilities!

2. Diazirine Amino Acids for Photo-Crosslinkage in Living Cells

Iris Biotech introduces a comprehensive set of photo-crosslinking amino acids bearing the diazirine moiety. Irradiation of diazirines with UV light (approx. 350 nm – 360 nm) yields a highly reactive carbene species that can undergo insertions into C-C, CH, O-H and X-H (X = heteroatom) bonds of neighboring molecules to irreversibly form a covalent bond. The diazirine moiety is the smallest of all photoreactive groups, so introduction of a diazirine-bearing amino acid into a peptide or protein usually does not impair its biological activity. Further advantages of diazirine crosslinkers are their stability at room temperature, as well as their relative stability to nucleophiles, and to both acidic and basic conditions.

Applications:

Probing protein–protein and protein- peptide interactions by photo-crosslinking (e.g., site-specific incorporation of photo-lysine into glutathione S-transferase allowing covalent crosslinking of the two subunits of the dimeric protein in E. coli).

• Probing protein-peptide interactions in order to identify cellular targets of peptides of interest.

• Studying protein−drug interactions and identifying new drug targets by comparing results obtained from different proteomic setups (e.g., live cells and cell lysates), more putative targets can be identified.

Fig. 1: Diazirine amino acids (right) and their natural counterparts (left).

These amino acids are available in Fmoc- as well as Boc-protected versions for their incorporation into synthetic peptides via standard coupling methods. Furthermore, the unprotected diazirine amino acids are also available for incorporation into expressed peptides and proteins by utilizing the appropriate aminoacyl-tRNA synthetase/tRNA pair. A combination of synthetic and recombinant approaches utilizing NCL has been demonstrated as well. Unnatural amino acids are frequently toxic to cells; however, these photo-amino acids are functional and nontoxic, which allows them to be a premium tool for studying mechanisms and interactions in living cells.

Photochemistry

noncovalent binding covalently bound

Fig. 2: Use of photo-phenylalanine for the identification of angiotensin-II-receptor binding sites; 125I is used as a radiotracer.

HAA3100 H-L-Photo-Leucine*HCl

(S)-2-amino-3-(3-methyl-3H-diazirin-3-yl)propanoic acid hydrochloride

CAS-No. 2421187-96-2

Formula C 5H9 N3 O 2*HCl Mol. weight 143,14*36,45 g/mol

BAA3070 Boc-L-Photo-Leucine*DCHA

(S)-2-(tert-butoxycarbonylamino)-3-(3-methyl-3H-diazirin-3-yl)propanoic acid dicyclohexylamine

CAS-No. 1000770-97-7 net

Formula C10 H17N3 O4*C12H23 N Mol. weight 243,26*181,34 g/mol

FAA4590 Fmoc-L-Photo-Leucine

(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(3-methyl-3H-diazirin-3-yl)propanoic acid

CAS-No. 1360651-24-6

Formula C 20 H19 N3 O4 Mol. weight 365,38 g/mol

HAA3110 H-L-Photo-Lysine*HCl

(S)-2-amino-6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy) carbonylamino)hexanoic acid hydrochloride

CAS-No. 2421187-79-1

Formula C11H20 N4O4*HCl Mol. weight 272,30*36,45 g/mol

Product details

FAA4600 Fmoc-L-Photo-Lysine

(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-6((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonylamino) hexanoic acid

CAS-No. 2250437-42-2

Formula C 26 H30 N4O 6 Mol. weight 494,54 g/mol

BAA3080 Boc-L-Photo-Lysine

(S)-2-(tert-butoxycarbonylamino)-6-((2-(3-methyl-3Hdiazirin-3-yl)ethoxy)carbonylamino)hexanoic acid

CAS-No. 1330088-06-6

Formula C16 H28 N4O 6 Mol. weight 372,42 g/mol

HAA3120 H-L-Photo-Methionine*HCl

(S)-2-amino-4-(3-methyl-3H-diazirin-3-yl)butanoic acid hydrochloride

CAS-No. 2922715-90-8

Formula C 6 H11N3 O 2*HCl Mol. weight 157,17*36,45 g/mol

BAA3090 Boc-L-Photo-Methionine*DCHA

(S)-2-(tert-butoxycarbonylamino)-4-(3-methyl-3H-diazirin-3-yl)butanoic acid dicyclohexylamine

CAS-No. 1002754-75-7 net

Formula C11H19 N3 O4*C12H23 N Mol. weight 257,29*181,34 g/mol

FAA4610 Fmoc-L-Photo-Methionine

(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-methyl-3H-diazirin-3-yl)butanoic acid

CAS-No. 945859-89-2

Formula C 21H21N3 O4 Mol. weight 379,42 g/mol

HAA3490 H-L-Photo-Phe-OH 4-(trifluoromethyldiazirin)-L-phenylalanine

CAS-No. 92367-16-3

Formula C11H10 F 3 N3 O 2 Mol. weight 273,21 g/mol

Photochemistry

BAA1530 Boc-L-Photo-Phe-OH

N-alpha-(t-Butyloxycarbonyl)-4-(trifluoromethyldiazirin)-L-phenylalanine

CAS-No. 92367-17-4

Formula C16 H18 F 3 N3 O4

Mol. weight 373,33 g/mol

FAA5690 Fmoc-L-Photo-Phe-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-4-(trifluoromethyldiazirin)-L-phenylalanine

CAS-No. 133342-64-0

Formula C 26 H20 F 3 N3 O4 Mol. weight 495,45 g/mol

HAA3130 H-L-Photo-Proline*HCl

(S)-1,2,5-triazaspiro[2.4]hept-1-ene-6-carboxylic acid

CAS-No. 2421187-90-6

Formula C 5H7N3 O 2*HCl Mol. weight 141,13*36,45 g/mol

BAA3100 Boc-L-Photo-Proline

(S)-5-(tert-butoxycarbonyl)-1,2,5-triazaspiro[2.4]hept-1ene-6-carboxylic acid

CAS-No. 1266778-55-5

Formula C10 H15N3 O4 Mol. weight 241,24 g/mol

FAA4620 Fmoc-L-Photo-Proline

(S)-5-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2,5-triazaspiro[2.4]hept-1-ene-6-carboxylic acid

CAS-No. 1266778-58-8

Formula C 20 H17N3 O4 Mol. weight 363,37 g/mol

References:

→ Protein-polymer conjugation via ligand affinity and photoactivation of glutathione S-transferase; E. W. Lin, N. Boehnke, H. D. Maynard; Bioconjug Chem 2014; 25: 1902-9. arrow-up-right-from-square https://doi.org/10.1021/bc500380r

→ Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes; H. Shi, C. J. Zhang, G. Y. Chen, S. Q. Yao; J Am Chem Soc 2012; 134: 3001-14. arrow-up-right-from-square https://doi.org/10.1021/ja208518u

→ Probing protein-protein interactions with a genetically encoded photo-crosslinking amino acid; H. W. Ai, W. Shen, A. Sagi, P. R. Chen, P. G. Schultz; Chembiochem 2011; 12: 1854-7. arrow-up-right-from-square https://doi.org/10.1002/cbic.201100194

→ Proteome profiling reveals potential cellular targets of staurosporine using a clickable cell-permeable probe; H. Shi, X. Cheng, S. K. Sze, S. Q. Yao; Chem Commun (Camb) 2011; 47: 11306-8. arrow-up-right-from-square https://doi.org/10.1039/c1cc14824a

→ Aliphatic diazirines as photoaffinity probes for proteins: recent developments; J. Das; Chem Rev 2011; 111: 4405-17. arrow-up-right-from-square https://doi.org/10.1021/cr1002722

→ Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1; P. O. Hetu, M. Ouellet, J. P. Falgueyret, C. Ramachandran, J. Robichaud, R. Zamboni, D. Riendeau; Arch. Biochem. Biophys. 2008; 477: 155-62. arrow-up-right-from-square https://doi.org/10.1016/j.abb.2008.04.038

→ Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker; M. Vila-Perello, M. R. Pratt, F. Tulin, T. W. Muir; J Am Chem Soc 2007; 129: 8068-9. arrow-up-right-from-square https://doi.org/10.1021/ja072013j

→ Photo-leucine incorporation reveals the target of a cyclodepsipeptide inhibitor of cotranslational translocation; A. L. MacKinnon, J. L. Garrison, R. S. Hegde, J. Taunton; J Am Chem Soc 2007; 129: 14560-1. arrow-up-right-from-square https://doi.org/10.1021/ja076250y

→ Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein; T. Durek, J. Zhang, C. He, S. B. Kent; Org Lett 2007; 9: 5497-500. arrow-up-right-from-square https://doi.org/10.1021/ol702461z

→ Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells; M. Suchanek, A. Radzikowska, C. Thiele; Nat Methods 2005; 2: 261-7. arrow-up-right-from-square https://doi.org/10.1038/nmeth752

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3. Photo-Crosslinkers for Various Applications

Many photo-crosslinkers for photoaffinity labeling rely on benzophenone as the crosslinking agent. However, these types of crosslinkers usually show low crosslinking yields and require relatively long irradiation times due to slow reaction rates, which may lead to non-specific labeling. Moreover, the irradiation conditions for benzophenones have been shown to lead to cell damage and cell death.

Conversely, diazirine- and perfluorophenyl-based crosslinkers generate a reactive species (carbene and nitrene, respectively) upon relatively short irradiation with UV light. Consequently, diazirine- and perfluorophenyl-crosslinkers are commonly used in molecular biology and biochemistry.

3.1.

Photo-Crosslinkers

Diazirine-bearing crosslinkers are activated at wavelengths that cause little to no damage to cells. In addition to this, trifluoromethylaryl diazirines show a fast initial reaction rate and rapid termination of reaction. Consequently, they exhibit a highly ligand-dependent reactivity, which renders them ideal probes for ligand binding to low-affinity targets, e.g., for probing carbohydrate-lectin interactions. Moreover, the small size of the diazirine group minimizes the risk of impairing or altering the biological activity of a ligand. Our diazirine crosslinkers are functionalized to react with either carboxyl- or amine-reactive ligands, respectively.

Fig. 3: Three types of photoreactive groups: Perfluorophenyl azides, diazirines and benzophenones.

Another application of diazirine photo-crosslinkers is the surface immobilization of molecules of interest. The linker is bound to a surface through its carboxyl or amino functionality, leaving the diazirine group free to react with any type of molecule. Since this reaction takes place irrespective of available functional groups, it is not necessary to chemically modify molecules of interest prior to immobilization. This virtually ensures that molecules are immobilized without altering their binding properties. By using this approach, it is possible to easily create microarrays of whole libraries of small molecules for rapid screening.

R = H or Tfm

Fig. 4: Immobilization of molecules of interest on a surface (e.g., a glass slide) using a diazirine crosslinker.

/ alkyl-spacer

coupling to amine- or carboxy-reactive probe

/ alkyl-spacer probe

irradiation

aryl- / alkyl-spacer probe

R1 = COOH or CH2NH2 R2 = H or Tfm

Fig. 5: Photoaffinity labeling of a target molecule by a diazirine-functionalized binding probe.

Photochemistry

RL-2890 Photo-Pentanoic acid

3-(3-methyl-3H-diazirin-3-yl)propanoic acid

CAS-No. 25055-86-1

Formula C 5H 8 N2O 2 Mol. weight 128,13 g/mol

RL-2900 Photo-Hexanoic acid

4-(3-methyl-3H-diazirin-3-yl)butanoic acid

CAS-No. 16297-97-5

Formula C 6 H10 N2O 2 Mol. weight 142,16 g/mol

RL-3410 Photo-Click-Heptanoic acid

2-(3-(but-3-ynyl)-3H-diazirin-3-yl)acetic acid

CAS-No. 2049109-24-0

Formula C 7H 8 N2O 2 Mol. weight 152,15 g/mol

RL-3740 Photo-Palmitic acid

9-(3-hexyl-3H-diazirin-3-yl)nonanoic acid

CAS-No. 2100292-41-7

Formula C16 H30 N2O 2 Mol. weight 282,43 g/mol

RL-3720 Photo-Click-Palmitic acid

11-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)undecanoic acid

CAS-No. 2988174-40-7

Formula C16 H26 N2O 2 Mol. weight 278,40 g/mol

RL-3750 Photo-Stearic acid

11-(3-hexyl-3H-diazirin-3-yl)undecanoic acid

CAS-No. 1704120-02-4

Formula C18 H34N2O 2 Mol. weight 310,48 g/mol

RL-3760 Photo-Click-Stearic acid

13-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)tridecanoic acid

CAS-No. 2989205-54-9

Formula C18 H30 N2O 2 Mol. weight 306,45 g/mol

RL-2920 Photo-Benzoic acid

4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzoic acid

CAS-No. 85559-46-2

Formula C9 H 5F 3 N2O 2 Mol. weight 230,14 g/mol

RL-2910 Photo-Butylamine

2-(3-methyl-3H-diazirin-3-yl)ethan-1-amine hydrochloride

CAS-No. 25055-95-2

Formula C 4H9 N3*HCl Mol. weight 99,13*36,45 g/mol

RL-2930 Photo-Benzylamine*HCl

4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzylamine hydrochloride

CAS-No. 1258874-29-1

Formula C9 H 8 N3 F 3*HCl Mol. weight 215,18*36,45 g/mol

References:

→ Hide and seek: Identification and confirmation of small molecule protein targets; A. Ursu, H. Waldmann; Bioorg Med Chem Lett 2015; 25: 3079-86. arrow-up-right-from-square https://doi.org/10.1016/j.bmcl.2015.06.023

→ Development and leading-edge application of innovative photoaffinity labeling; Y. Hatanaka; Chem Pharm Bull (Tokyo) 2015; 63: 1-12. arrow-up-right-from-square https://doi.org/10.1248/cpb.c14-00645

→ Diazirine based photoaffinity labeling; L. Dubinsky, B. P. Krom, M. M. Meijler; Bioorg Med Chem 2012; 20: 554-70. arrow-up-right-from-square https://doi.org/10.1016/j.bmc.2011.06.066

→ Aliphatic diazirines as photoaffinity probes for proteins: recent developments; J. Das; Chem Rev 2011; 111: 4405-17. arrow-up-right-from-square https://doi.org/10.1021/cr1002722

→ Recent Progress in Diazirine-Based Photoaffinity Labeling; M. Hashimoto, Y. Hatanaka; Eur. J. Org. Chem. 2008; 2008: 2513-2523 arrow-up-right-from-square https://doi.org/10.1002/ejoc.200701069

→ Endeavors to make the photophore, diazirine easy to use; Y. Sadakane; Yakugaku Zasshi 2007; 127: 1693-9. arrow-up-right-from-square https://doi.org/10.1248/yakushi.127.1693

→ Photoaffinity labeling coupled to MS to identify peptidebiological partners: Secondary reactions, for betteror for worse? A. Walrant, E. Sachon; Mass Spec Rev. 2024; 1-42. arrow-up-right-from-square https://doi.org/10.1002/mas.21880

Photochemistry

→ Comparison of the reactivity of carbohydrate photoaffinity probes with different photoreactive groups; K. Sakurai, S. Ozawa, R. Yamada, T. Yasui, S. Mizuno; Chembiochem 2014; 15: 1399-403. arrow-up-right-from-square https://doi.org/10.1002/cbic.201402051

→ Identification of a substrate-binding site in a peroxisomal beta-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative; Y. Kashiwayama, T. Tomohiro, K. Narita, M. Suzumura, T. Glumoff, J. K. Hiltunen, P. P. Van Veldhoven, Y. Hatanaka, T. Imanaka; J Biol Chem 2010; 285: 26315-25. arrow-up-right-from-square https://doi.org/10.1074/jbc.M110.104547

→ Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases; E. W. Chan, S. Chattopadhaya, R. C. Panicker, X. Huang, S. Q. Yao; J Am Chem Soc 2004; 126: 14435-46. arrow-up-right-from-square https://doi.org/10.1021/ja047044i

→ Insecticidal and neural activities of candidate photoaffinity probes for neonicotinoid binding sites; K. Matsuda, M. Ihara, K. Nishimura, D. B. Sattelle, K. Komai; Biosci Biotechnol Biochem 2001; 65: 1534-41. arrow-up-right-from-square https://doi.org/10.1271/bbb.65.1534

→ Synthesis of Photoactive α-Mannosides and Mannosyl Peptides and Their Evaluation for Lectin Labeling; M. Wiegand, T. K. Lindhorst; Eur. J. Org. Chem. 2006; 2006: 4841-4851. arrow-up-right-from-square https://doi.org/10.1002/ejoc.200600449

→ A study on photolinkers used for biomolecule attachment to polymer surfaces; D. M. Dankbar, G. Gauglitz; Anal Bioanal Chem 2006; 386: 1967-74. arrow-up-right-from-square https://doi.org/10.1007/s00216-006-0871-x

→ SPR imaging of photo-cross-linked small-molecule arrays on gold; N. Kanoh, M. Kyo, K. Inamori, A. Ando, A. Asami, A. Nakao, H. Osada; Anal Chem 2006; 78 : 2226-30. arrow-up-right-from-square https://doi.org/10.1021/ac051777j

→ Grafting Organic and Biomolecules on H-Terminated Porous Silicon from a Diazirine; S. Wei, J. Wang, D.-J. Guo, Y.-Q. Chen, S.-J. Xiao; Chem. Lett. 2006; 35: 1172-1173. arrow-up-right-from-square https://doi.org/10.1246/cl.2006.1172

→ Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions; N. Kanoh, S. Kumashiro, S. Simizu, Y. Kondoh, S. Hatakeyama, H. Tashiro, H. Osada; Angew. Chem. Int. Ed. 2003; 42: 5584-7. arrow-up-right-from-square https://doi.org/10.1002/anie.200352164

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Download our Linkerology(R) booklet and discover available linkers!

3.2. Tetrafluorophenyl-Azide-based Photo-Crosslinkers

Tetrafluorophenyl-azides follow a principle similar to diazirines. Upon irradiation with UV light (ca. 260 nm), a highly stabilized nitrene is formed. Nitrenes are the nitrogen analogs of carbenes (isoelectronic) and react in a comparable fashion. In terms of crosslinking yield and duration of irradiation, they compare favorably to benzophenones. Moreover, the azido group can also undergo classical copper-catalyzed azide-alkyne cycloadditions. Tetrafluorophenyl-azido crosslinkers are also available with a short PEG-spacer for increased solubility (PEG5000 on page 13), and as Biotin-TEG-ATFBA (PEG2065 on page 13) for applications such as surface functionalization with biotin, or the biotinylation of biomacromolecules.

Fig. 6: Applications of tetrafluorophenyl-azides.

semiconductors

Photochemistry

RL-2035 ATFB

4-Azido-2,3,5,6-tetrafluorobenzoic acid

CAS-No. 122590-77-6

Formula C 7HF4N3 O 2 Mol. weight 235,1 g/mol

RL-2045 ATFB-NHS

N-Succinimidyl 4-azido-2,3,5,6-tetrafluorobenzoate

CAS-No. 126695-58-7

Formula C11H4F4N4O4 Mol. weight 332,17 g/mol

PEG2065 Biotin-TEG-ATFBA

Biotin-triethylenglycol-( p-azido-tetrafluorobenzamide)

CAS-No. 1264662-85-2

Formula C 27H37F4N7 O 6 S Mol. weight 663,68 g/mol

PEG5000 N3

-TFBA-O2Oc

{2-[2-(4-Azido-2,3,5,6-tetrafluorobenzoyl-amino)ethoxy] ethoxy}acetic acid

CAS-No. 1993119-45-1

Formula C13 H12F4N4O 5 Mol. weight 380,25 g/mol

References:

→ Tri- and Tetravalent Photoactivable Cross-Linking Agents; A. Welle, F. Billard, J. Marchand-Brynaert; Synthesis 2012; 44: 2249-2254. arrow-up-right-from-square https://doi.org/doi:10.1055/s-0031-1290444

→ Perfluorophenyl azides: new applications in surface functionalization and nanomaterial synthesis; L. H. Liu, M. Yan; Acc Chem Res 2010; 43: 1434-43. arrow-up-right-from-square https://doi.org/10.1021/ar100066t

→ Photo-click immobilization of carbohydrates on polymeric surfaces--a quick method to functionalize surfaces for biomolecular recognition studies; O. Norberg, L. Deng, M. Yan, O. Ramstrom; Bioconjug Chem 2009; 20: 2364-70. arrow-up-right-from-square https://doi.org/10.1021/bc9003519

→ Photoreactive insulin derivatives for the detection of the doubly labeled insulin receptor; J. Kleinjung, M. Fabry; Peptides 2000; 21: 401-6. arrow-up-right-from-square https://doi.org/10.1016/S0196-9781(00)00164-9

→ Recent Trends in the Evaluation of Photochemical Insertion Characteristics of Heterobifunctional Perfluoroaryl Azide Chelating Agents: Biochemical Implications in Nuclear Medicine; R. S. Pandurangi, S. R. Karra, R. R. Kuntz, W. A. Volkert; Photochem. Photobiol. 1997; 65: 208-221. arrow-up-right-from-square https://doi.org/10.1111/j.1751-1097.1997.tb08547.x

4. Photoactivated Self-Cleaving Linkers and Protecting Groups via Trimethyl Lock

Iris Biotech introduces a series of self-immolative compounds that find application as protecting groups, linkers, or amino acid derivatives (Spr = stimulus-responsive peptide bond cleaving residue). The self-cleavage is induced by irradiation with UV light (ca. 350 – 365 nm) that leads to the unmasking of a hydroxyl group of a 2-alkyl-3,5-dimethyl phenol moiety. The photocleavable group is either o-nitrobenzyl or o-nitroveratryl, which can be cleaved at wavelengths > 350 nm. Since wavelengths above 350 nm tend to be unproblematic for biomacromolecules, this technique is especially interesting for cell-based systems.

The liberated OH-group serves as a nucleophile that intramolecularly cleaves ester (or amide) bonds at neutral pH and room temperature by cyclization via a six-membered transition state. This reaction is greatly accelerated since the sterically unfavorable interaction between the methyl group at the 3-position of the phenol core and the two geminal CH 3 -groups on the alkyl chain (in β-position to the ester or amide carbonyl group) favor conformations that bring the phenyl-OH-group and the neighboring carbonyl function into closer vicinity. This phenomenon is termed the gem-dialkyl effect, for which a theory was first proposed by Thorpe and Ingold in 1915 (“Thorpe-Ingold effect”). An alternative explanation for this effect was posited by Bruice and Pandit in 1960 (“reactive rotamer effect”).

sterically unfavorable interaction

Fig. 7: Principle of photoactivated trimethyl lock; PG = o-nitrobenzyl or o-nitroveratryl; X = O or NH; R1–XH = target molecule; R2 = H or NH-alkyl.

The applications for this approach are numerous. Incorporation of a Spr-residue into a peptide sequence enables the photoactivated self-cleavage of the peptide at the position of said residue.

This technique allows for the intracellular removal of e.g., cell penetrating peptides or localization sequences from a bioactive molecule. The Spr-residue can also be used as a photolabile linker in order to reversibly connect a moiety such as biotin to a molecule of interest. Finally, while o-nitroveratryl itself is a valuable protecting group for sulfhydryl groups, the combination of o-Nv with a trimethyl lock moiety also allows for its use as a protecting group for hydroxyl and amino functions.

Photochemistry

FAA7190 Fmoc-Spr(oNB)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-beta,beta-dimethyl-(2,4-dimethyl-6-(2-nitrobenzyloxy)phenyl) alanine (rac.)

CAS-No. 1032400-98-8

Formula C 35H34N2O 7 Mol. weight 594,66 g/mol

FAA7200 Fmoc-Spr(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-beta,beta-dimethyl-(2-methyl-6-(2-nitroveratryl)phenyl)alanine (rac.)

CAS-No. 1228829-20-6

Formula C 36 H36 N2O 9 Mol. weight 640,68 g/mol

RL-2970 Photo-Trimethyl-Lock

3-(2-Nitroveratryl-4,6-dimethylphenyl)-3-methylbutyric acid

CAS-No. 2095134-25-9

Formula C 22H27NO 7 Mol. weight 417,45 g/mol

References:

→ Invention of stimulus-responsive peptide-bond-cleaving residue (Spr) and its application to chemical biology tools; A. Shigenaga, J. Yamamoto, T. Kohiki, T. Inokuma, A. Otaka; J. Pept. Sci. 2017; 23: 505-513. arrow-up-right-from-square https://doi.org/10.1002/psc.2961

→ Syntheses and kinetic studies of cyclisation-based self-immolative spacers; S. Huvelle, A. Alouane, T. Le Saux, L. Jullien, F. Schmidt; Org Biomol Chem 2017; 15: 3435-3443. arrow-up-right-from-square https://doi.org/10.1039/c7ob00121e

→ Photo-triggered fluorescent labelling of recombinant proteins in live cells; D. Jung, K. Sato, K. Min, A. Shigenaga, J. Jung, A. Otaka, Y. Kwon; Chem Commun (Camb) 2015; 51: 9670-3. arrow-up-right-from-square https://doi.org/10.1039/c5cc01067e

→ Trimethyl lock: A trigger for molecular release in chemistry, biology, and pharmacology; M. N. Levine, R. T. Raines; Chem. Sci. 2012; 3: 2412-2420. arrow-up-right-from-square https://doi.org/10.1039/C2SC20536J

→ Design and synthesis of caged ceramide: UV-responsive ceramide releasing system based on UV-induced amide bond cleavage followed by O–N acyl transfer; A. Shigenaga, H. Hirakawa, J. Yamamoto, K. Ogura, M. Denda, K. Yamaguchi, D. Tsuji, K. Itoh, A. Otaka; Tetrahedron 2011; 67: 3984-3990. arrow-up-right-from-square https://doi.org/10.1016/j.tet.2011.04.048

→ Development and photo-responsive peptide bond cleavage reaction of two-photon near-infrared excitationresponsive peptide; A. Shigenaga, J. Yamamoto, Y. Sumikawa, T. Furuta, A. Otaka; Tetrahedron Lett. 2010; 51: 2868-2871. arrow-up-right-from-square https://doi.org/10.1016/j.tetlet.2010.03.079

→ gem-disubstituent effect: theoretical basis and synthetic applications; M. E. Jung, G. Piizzi; Chem Rev 2005; 105: 1735-66. arrow-up-right-from-square https://doi.org/10.1021/cr940337h

→ The Effect of Geminal Substitution Ring Size and Rotamer Distribution on the Intramolecular Nucleophilic Catalysis of the Hydrolysis of Monophenyl Esters of Dibasic Acids and the Solvolysis of the Intermediate Anhydrides; T. C. Bruice, U. K. Pandit; J. Am. Chem. Soc. 1960; 82: 5858-5865. arrow-up-right-from-square https://doi.org/10.1021/ja01507a023

→ CXIX.—The formation and stability of spiro-compounds. Part I. spiro-Compounds from cyclohexane; R. M. Beesley, C. K. Ingold, J. F. Thorpe; J. Chem. Soc., Trans. 1915; 107: 1080-1106. arrow-up-right-from-square https://doi.org/10.1039/ct9150701080

5. Furfuryl-Alanine for Side-chain Modification and Bioconjugation

2-Furyl-alanine can be incorporated into peptides via SPPS or by using enzymatic approaches. UV-irradiation in the presence of oxygen and a photosensitizer converts furyl-alanine to an intermediate that selectively reacts with certain nucleophiles. This property can be employed for site-specific labeling of peptides and proteins.

Labeling with different tags and reporter groups is a pivotal technique for the elucidation of peptide and protein function.

A novel and innovative approach is the site-specific labeling using the unnatural amino acid 2-furyl-alanine. UV-irradiation in the presence of oxygen and a photosensitizer converts furyl-alanine to an unsaturated dicarbonyl compound. This intermediate selectively reacts with certain nucleophiles such as hydrazine derivatives of dyes or fluorescent labels. This reaction can be used for the site-specific labeling of peptides and proteins and can be carried out in aqueous solution.

, O2, H2O peptide photosensitizer

hν, O2, H2O photosensitizer

Fig. 8: Site-specific labeling of peptides and proteins using 2-furyl-alanine.

Iris Biotech offers Fmoc-L-Ala(2-Furyl)-OH suitable for SPPS, as well as H-L-Ala(2-Furyl)-OH which can be incorporated into proteins using the amber suppression methodology.

Photochemistry

HAA2930 H-L-Ala(2-Furyl)-OH

3-(2-Furyl)-L-alanine

CAS-No. 127682-08-0

Formula C 7H9 NO 3 Mol. weight 155,15 g/mol

FAA4250 Fmoc-L-Ala(2-Furyl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-3-(2-furyl)-L-alanine

CAS-No. 159611-02-6

Formula C 22H19 NO 5 Mol. weight 377,39 g/mol

(S) H2N OH O O

(S) H N OH O O O O

References:

→ Novel furan-oxidation based site-specific conjugation methodology for peptide labeling and antibody drug conjugates; presented at the 6th World ADC meeting October 19 th-22nd 2015, San Diego, CA; An Van Den Bulcke, Eirini Antonatou, Willem Vannecke, Kurt Hoogewijs, Annemieke Madder.

→ Exploiting furan‘s versatile reactivity in reversible and irreversible orthogonal peptide labeling; K. Hoogewijs, D. Buyst, J. M. Winne, J. C. Martins, A. Madder; Chem Commun (Camb) 2013; 49: 2927-9. arrow-up-right-from-square https://doi.org/10.1039/c3cc40588e

→ Sequence specific DNA cross-linking triggered by visible light; M. Op de Beeck, A. Madder; J Am Chem Soc 2012; 134: 10737-40. arrow-up-right-from-square https://doi.org/10.1021/ja301901p

→ Unprecedented C-selective interstrand cross-linking through in situ oxidation of furan-modified oligodeoxynucleotides; M. Op de Beeck, A. Madder; J Am Chem Soc 2011; 133: 796-807. arrow-up-right-from-square https://doi.org/10.1021/ja1048169

→ Furan-modified oligonucleotides for fast, high-yielding and site-selective DNA inter-strand cross-linking with non-modified complements; K. Stevens, A. Madder; Nucleic Acids Res 2009; 37: 1555-65. arrow-up-right-from-square https://doi.org/10.1093/nar/gkn1077

→ From DNA cross-linking to peptide labeling: on the versatility of the furan-oxidation-conjugation strategy; A. Deceuninck, A. Madder; Chem Commun (Camb) 2009; 340-2. arrow-up-right-from-square https://doi.org/10.1039/b817447d

→ Bioorthogonal chemistry: fishing for selectivity in a sea of functionality; E. M. Sletten,C. R. Bertozzi; Angew. Chem. Int. Ed. 2009; 48: 6974-98. arrow-up-right-from-square https://doi.org/10.1002/anie.200900942

→ Structural basis of furan-amino acid recognition by a polyspecific aminoacyl-tRNA-synthetase and its genetic encoding in human cells; M. J. Schmidt, A. Weber, M. Pott, W. Welte, D. Summerer; Chembiochem 2014; 15: 1755-60. arrow-up-right-from-square https://doi.org/10.1002/cbic.201402006

→ Red-light-controlled protein-RNA crosslinking with a genetically encoded furan; M. J. Schmidt, D. Summerer; Angew. Chem. Int. Ed. 2013; 52: 4690-3. arrow-up-right-from-square https://doi.org/10.1002/anie.201300754 . circle-arrow-right

You need more information about furan-crosslinking?

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6. Photocaged Amino Acid Building Blocks

Photocages are a class of substances bearing a photosensitive group (= photoremovable protecting group, PPG) keeping a compound in its inactive (= caged) state until being removed by irradiation with light (=photocleavage) of a defined wavelength. Light as external trigger stands out for a variety of reasons, with the main ones being its non-invasiveness, high spatiotemporal resolution, and ease of dosage control.

Photocages can either be used for synthetic purposes, e.g., during solid-phase peptide synthesis, to introduce another dimension of orthogonality besides, e.g., acid/base labile protecting groups (PGs). Furthermore, PPGs allow to temporarily mask a functional group relevant for the biological activity of a certain drug molecule or to suppress a certain protein-protein interaction. In terms of photocaged drugs, pharmacological activities can be timely and selectively induced at a desired point of action reducing the risk of undesired off-target effects.

The “cage” concept was first introduced in 1978 by Hoffmann et al., who synthesized a caged ATP bearing a o-nitrobenzyl moiety as photosensitive group.

O -nitrobenzyl residues (and derivatives thereof) absorb UV light at about 350 nm. Upon absorption of a photon, a diradical is formed, the group undergoes a Norrish type II reaction, the activated oxygen of the N=O group abstracts a proton from the benzylic position, and in a rearrangement, the bond between the nitrobenzyl residue (where the protected functional group is attached) is broken, and the functional group is released, while an o-nitroso benzaldehyde is formed. This protection/deprotection scheme works well for many functional groups present in amino acids.

To reduce photolysis time and to increase yields, the original nitrobenzyl protection has been improved by the introduction of o-nitroveratryl (Nve, oNV, also named 4,5-dimethoxy-2-nitrobenzyl [DMNB]), and nitrodibenzofuranyl (NDBF) residues. Besides, we also offer the photosensitive 4-methoxy-7-nitroindoline (MNI) group used to cage carboxylates. Photolysis is achieved by irradiation at 365 nm.

Enlargement of the aromatic system allows to trigger photolysis with longer wavelengths which is especially favorable for in vitro and in vivo applications in cells and organisms due to improved penetration depth and reduced cell damaging. Nitrodibenzofuran (NDBF) has a very high quantum yield. NDBF-caged compounds are photolyzed 16-160 times more efficiently than those protected with nitrobenzoyl residues, and thus are accessible by two-photon excitation with infrared light of 800 nm.

Photochemistry

Fig. 9: Variations of the 2-nitrobenzyl photocage: oNB: o -nitrobenzyl, Nve: nitroveratryl, DMNB: dimethoxynitrobenzyl, oNv: o-nitroveratryl, MDNPE: Methyl-o-nitropiperonylethyl (R=Me), NDBF: nitrodibenzofuranyl. R can be H or methyl; Y depicts the remainder of the protected amino acid.

FAA3970 Fmoc-L-Cys(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2-nitroveratryl)-L-cysteine

CAS-No. 214633-71-3

Formula C 27H26 N2O 8 S Mol. weight 538,57 g/mol

FAA8870 Fmoc-L-hCys(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(2-nitroveratryl)-L-homocysteine

Formula C 28 H28 N2O 8 S Mol. weight 552,60 g/mol

FAA7350 Fmoc-N(o Nv)-Gly-OH

N-alpha (9-Fluorenylmethyloxycarbonyl)-N-alpha-(o-nitroveratryl)-glycine

CAS-No. 850859-64-2

Formula C 26 H24N2O 8 Mol. weight 492,48 g/mol

FAA7200 Fmoc-Spr(o Nv)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-beta,beta-dimethyl-(2-methyl-6-(2-nitroveratryl)phenyl)alanine (rac.)

CAS-No. 1228829-20-6

Formula C 36 H36 N2O 9 Mol. weight 640,68 g/mol

Product details

FAA9575 Fmoc-L-Ser(o Nv)-OH

N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine

CAS-No. 628280-43-3

Formula C 27H26 N2O 9 Mol. weight 522,50 g/mol

FAA9580 Fmoc-L-Tyr(o Nv)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3(4-((4,5-dimethoxy-2-nitrobenzyl)oxy)phenyl)propanoic acid

CAS-No. 207727-88-6

Formula C 33 H30 N2O 9 Mol. weight 598,60 g/mol

FAA7230 Fmoc-L-Lys(Nvoc)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-epsilon-(o-nitroveratryloxycarbonyl)-L-lysine

CAS-No. 150571-28-1

Formula C 31H33 N3 O 10 Mol. weight 607,61 g/mol

FAA8420 Fmoc-L-Cys(NDBF)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-S-(1-(3-nitro-dibenzofuran-2-yl)-ethyl)-L-cysteine

CAS-No. 1895883-28-9

Formula C 32H26 N2O 7S Mol. weight 582,62 g/mol

FAA8425 Fmoc-L-Lys(NDBFOC)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-epsilon-(1-(3-nitro-dibenzofuran-2-yl)-ethoxycarbonyl)-L-lysine

Formula C 36 H33 N3 O 9 Mol. weight 651,66 g/mol

HAA9360 H-L-Sec(MDNPE)-OH

Se-(Methyl-o-nitropiperonyl)-selenocysteine

CAS-No. 2235373-47-2

Formula C12H14N2O 6 Se Mol. weight 361,21 g/mol

Photochemistry

HAA9255 H-L-Sec(o Nv)-OH*TFA

Dimethoxynitrobenzyl selenocysteine TFA salt

CAS-No. 1644398-13-9

Formula C12H16 N2O 6 Se*CF 3 COOH Mol. weight 363,24*114,02 g/mol

HAA9320 H-L-Cys(o Nv)-OH

S-(4,5-dimethoxy-2-nitrobenzyl)-L-cysteine

CAS-No. 214633-68-8

Formula C12H16 N2O 6 S Mol. weight 316,33 g/mol

HAA9270 H-L-Cys(MDNPE)-OH

1-[4‘,5‘-(methylenedioxy)-2‘-nitrophenyl]ethyl]-L-cysteine

CAS-No. 1551078-43-3

Formula C12H14N2O 6 S Mol. weight 314,31 g/mol

FAA7945 Fmoc-L-Cys(MDNPE)-OH

N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)-L-cysteine

Formula C 27H24N2O 8 S Mol. weight 536,56 g/mol

HAA9465 H-L-Sec(oNB)-OH*HCl

(R)-2-amino-3-((2-nitrobenzyl)selanyl)propanoic acid

CAS-No. 324582-23-2 net

Formula C10 H12N2O4 Se*HCl Mol. weight 303,18*36,46 g/mol

FAA9345 H-L-Lys(oNB)-OH*HCl

N6-(((2-nitrobenzyl)oxy)carbonyl)-L-lysine

CAS-No. 228564-76-9

Formula C14H19 N3 O 6*HCl Mol. weight 325,32*36,45 g/mol

FAA9350 H-L-Glu(oNB)-OH*HCl

2-amino-5-((2-nitrobenzyl)oxy)-5-oxopentanoic acid

CAS-No. 75707-39-0

Formula C12H14N2O 6*HCl Mol. weight 282,25*36,45 g/mol

FAA9365 Fmoc-L-Lys(oNB)-OH

N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(((2-nitrobenzyl)oxy)carbonyl)-L-lysine

CAS-No. 228564-77-0

Formula C 29 H29 N3 O 8 Mol. weight 547,56 g/mol

HAA9475 H-L-Sec(NPE)-OH*HCl

(2R)-2-amino-3-((1-(2-nitrophenyl)ethyl)selanyl)propanoic acid

Formula C11H14N2O4 Se*HCl Mol. weight 317,02*36,46 g/mol

FAA9555 Fmoc-L-Asp(MNI)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4(4-methoxy-7-nitroindolin-1-yl)-4-oxobutanoic acid

CAS-No. 1799532-96-9

Formula C 28 H25N3 O 8 Mol. weight 531,51 g/mol

FAA9560 Fmoc-L-Glu(MNI)-OH

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(4methoxy-7-nitroindolin-1-yl)-5-oxopentanoic acid

CAS-No. 1799533-00-8

Formula C 29 H27N3 O 8 Mol. weight 545,45 g/mol

Photochemistry

References:

→ Total chemical synthesis of photoactivatable proteins for light-controlled manipulation of antigen–antibody interactions; S. Tang, Z. Wan, Y. Gao, J.-S. Zheng, J. Wang, Y.-Y. Si, X. Chen, H. Qi, L. Liu, W. Liu; Chem. Sci. 2016; 7: 1891-1895. arrow-up-right-from-square https://doi.org/10.1039/C5SC03404C

→ 2-Nitroveratryl as a Photocleavable Thiol-Protecting Group for Directed Disulfide Bond Formation in the Chemical Synthesis of Insulin; J. A. Karas, D. B. Scanlon, B. E. Forbes, I. Vetter, R. J. Lewis, J. Gardiner,F. Separovic, J. D. Wade, M. A. Hossain; Chemistry 2014; 20: 9549-9552. arrow-up-right-from-square https://doi.org/10.1002/chem.201403574

→ Synthesis, stability and optimized photolytic cleavage of 4-methoxy-2-nitrobenzyl backbone-protected peptides; E. C. B. Johnson, S. B. H. Kent; Chem. Commun. 2006; 1557-1559 arrow-up-right-from-square https://doi.org/10.1039/B600304D

→ A red shifted two-photon-only caging group for three-dimensional photorelease. Y. Becker, E. Unger, M. A. H. Fichte, D. A. Gacek, A. Dreuw, J. Wachtveitl, P. J. Walla, A. Heckel; Chem. Sci. 2018; 9: 2797-2902. arrow-up-right-from-square https://doi.org/10.1039/c7sc05182d

→ Nitrodibenzofuran: A One- and Two-Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides. M. M. Mahmoodi, D. Abate-Pella, T. J. Pundsack, C. C. Palsuledesai, P. C. Goff, D. A. Blank, M. D. Distefano; J. Am. Chem. Soc. 2016; 138: 5848-5859. arrow-up-right-from-square https://doi.org/10.1021/jacs.5b11759

→ The nitrodibenzofuran chromophore: a new caging group for ulta-efficient photolysis in living cells.

A. Momotake, N. Lindegger, E. Niggli, R. J. Barsotti, G. C. R. Ellis-Davies; Nat. Methods 2006; 3: 35-40. arrow-up-right-from-square https://doi.org/10.1038/NMETH821

→ Methoxy-Substituted Nitrodibenzofuran-Based Protecting Group with an Improved Two-Photon Action CrossSection for Thiol Protection in Solid Phase Peptide Synthesis. T. K. Bader, F. Xu, M. H. Hodny, D. A. Blank, M. D. Distefano; J. Org. Chem. 2020; 85: 1614-1625. arrow-up-right-from-square https://doi.org/10.1021/acs.joc.9b02751.

→ Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells; D. P. Nguyen, M. Mahesh, S. J. Elsässer, S. M. Hancock, C. Uttamapinant, J. W. Chin; J. Am. Chem. Soc. 2014; 136(6): 2240-2243. arrow-up-right-from-square https://doi.org/10.1021/ja412191m

→ Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines; R. Rakauskaité, G. Urbanavičiūtė, A. Rukšėnaitė, Z. Liutkevičiūtė, R. Juškėnas, V. Masevičiusab, S. Klimašauskas; Chem. Commun. 2015; 51: 8245-8248. arrow-up-right-from-square https://doi.org/10.1039/C4CC07910H

→ Controlled Inhibition of Apoptosis by Photoactivatable Caspase Inhibitors; S. Chakrabarty, S. H. L. Verhelst; Cell Chem Biol. (2020); 27(11) : 1434-1440.e10. arrow-up-right-from-square https://doi.org/10.1016/j.chembiol.2020.08.001

→ Total chemical synthesis of photoactivatable proteins for light-controlled manipulation of antigen–antibody interactions; S. Tang, Z. Wan, Y. Gao, J.-S. Zheng, J. Wang. Y.-Y. Si, H. Qi, L. Liu, W. Liu; Chem. Sci. 2016; 7: 1891-1895. arrow-up-right-from-square https://doi.org/10.1039/C5SC03404C

→ An Fmoc-compatible method for synthesis of peptides containing photocaged aspartic acid or glutamic acid; S. Tang, J.-Y. Cheng, J.-S. Zheng; Tetrahedron Lett. 2015; 56(31): 4582-4585. arrow-up-right-from-square https://doi.org/10.1016/j.tetlet.2015.06.016

→ Rapid photolytic release of adenosine 5‘-triphosphate from a protected analog: utilization by the sodium: potassium pump of human red blood cell ghosts; J. H. Kaplan, B. Forbush III, J. F. Hoffman; Biochemistry 1978, 17(10): 1929–1935. arrow-up-right-from-square https://doi.org/10.1021/bi00603a020

→ Recent progress in studies of photocages; Y. Li, M. Wang, F. Wang, S. Lu, X. Chen; Smart Molecules 2023; 1(1): e20220003. arrow-up-right-from-square https://doi.org/10.1002/smo.20220003

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7. Photoswitches

Photoswitches are a subtype of molecular switch that undergo a reversible structural change upon irradiation with light to adopt a different configuration. Activation of a molecular switch by light offers several advantages. In addition to being traceless, light can be precisely controlled in its intensity (dosage control) and can be focused with sub-micron accuracy with a high temporal and spatial resolution. Consequently, photoswitches ideally lend themselves to the construction of light-responsive pharmaceutical compounds, a field that is described by the term photopharmacology.

The most common motif that is used in this approach is the azobenzene moiety, owing to the large geometrical change that results from adopting a different configuration. Azobenzene-based photo-responsive systems have also been successfully applied in several biological systems. The azobenzene moiety is synthetically accessible via various pathways (Mills reaction, oxidative/reductive coupling, azo coupling), and its photochromic properties can easily be finetuned. The thermodynamically more stable trans isomer adopts an extended planar configuration with a dipole moment close to zero, while the higher energy, metastable cis isomer is more polar and exhibits unfavorable steric interactions.

Fig. 10: Interconversion between the trans and cis isomers of the diazobenzene motif in a photoswitchable peptide.

An additional benefit of the azobenzene motif is its high degree of reversibility. It can undergo approximately 100 switching cycles without detectable photodegradation or loss of responsiveness. However, although the trans isomer can be regenerated 100% by thermal relaxation, full conversion of one or the other isomer by irradiation with visible light is impossible owing to a substantial overlap of the absorption spectra of both isomers.

H-L-Phe(4-N=NPh)-OH*HCl 4-Phenylazo-L-phenylalanine hydrochloride

CAS-No. 2137036-84-9

Formula C15H15N3 O 2*HCl

Mol. weight 269,30*36,45 g/mol

HAA3710

Photochemistry

BAA1660 Boc-L-Phe(4-N=NPh)-OH

N-alpha-t-Butyloxycarbonyl-4-phenylazo-L-phenylalanine

CAS-No. 134816-33-4

Formula C 20 H23 N3 O4

Mol. weight 369,41 g/mol

FAA7010 Fmoc-L-Phe(4-N=NPh)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-4-phenylazo-L-phenylalanine

CAS-No. 210366-26-0

Formula C 30 H25N3 O4 Mol. weight 491,54 g/mol

FAA9180 Fmoc-AMPB-OH

4-(4-(9-Fluorenylmethoxycarbonylaminomethyl)-phenyldiazenyl)benzoic acid

CAS-No. 159790-79-1

Formula C 29 H23 N3 O4 Mol. weight 477,52 g/mol

References:

→ Photopharmacology and Photochemical Biology; A. Deiters, S. Kossatz, O. Vázquez; ChemPhotoChem 2021; 5: 1031-1032. arrow-up-right-from-square https://doi.org/10.1002/cptc.202100216

→ Photoswitchable peptides for spatiotemporal control of biological functions; L. Albert, O. Vazquez; Chem Commun (Camb) 2019; 55: 10192-10213. arrow-up-right-from-square https://doi.org/10.1039/c9cc03346g

→ In Vivo Photopharmacology; K. Hull, J. Morstein, D. Trauner; Chem Rev 2018; 118: 10710-10747. arrow-up-right-from-square https://doi.org/10.1021/acs.chemrev.8b00037

→ Photopharmacological control of bipolar cells restores visual function in blind mice; L. Laprell, I. Tochitsky, K. Kaur, M. B. Manookin, M. Stein, D. M. Barber, C. Schon, S. Michalakis, M. Biel, R. H. Kramer, M. P. Sumser, D. Trauner, R. N. Van Gelder; J Clin Invest 2017; 127: 2598-2611. arrow-up-right-from-square https://doi.org/10.1172/JCI92156

→ Emerging Targets in Photopharmacology; M. M. Lerch, M. J. Hansen, G. M. van Dam, W. Szymanski, B. L. Feringa; Angew. Chem. Int. Ed. 2016; 55: 10978-99. arrow-up-right-from-square https://doi.org/10.1002/anie.201601931

→ Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells; C. Hoppmann, V. K. Lacey, G. V. Louie, J. Wei, J. P. Noel, L. Wang; Angew. Chem. Int. Ed. 2014; 53: 3932-6. arrow-up-right-from-square https://doi.org/10.1002/anie.201400001

→ Reversible photocontrol of biological systems by the incorporation of molecular photoswitches; W. Szymanski, J. M. Beierle, H. A. Kistemaker, W. A. Velema, B. L. Feringa; Chem Rev 2013; 113: 6114-78. arrow-up-right-from-square https://doi.org/10.1021/cr300179f

→ Photoisomerization in different classes of azobenzene; H. M. Bandara, S. C. Burdette; Chem Soc Rev 2012; 41: 1809-25. arrow-up-right-from-square https://doi.org/10.1039/c1cs15179g

→ Azobenzene photoswitches for biomolecules; A. A. Beharry, G. A. Woolley; Chem Soc Rev 2011; 40: 4422-37. arrow-up-right-from-square https://doi.org/10.1039/c1cs15023e

→ Properties of the pyramidal tract neuron system within the precentral wrist and hand area of primate motor cortex; D. R. Humphrey, W. S. Corrie, R. Rietz; J Physiol (Paris) 1978; 74: 215-26. arrow-up-right-from-square https://doi.org/10.1016/j.isci.2021.102771

→ Visible-light-switchable azobenzenes: Molecular design, supramolecular systems, and applications; M. Gao, D. Kwaria, Y. Norikane, Y. Yue; Natural Sciences 2023; 3(1) : e220020. arrow-up-right-from-square https://doi.org/10.1002/ntls.20220020

8. Photocleavable Auxiliary Reagent for Native Chemical Ligation

cleavage from resin

1) iodoacetylation

2) attachment of auxiliary PAA2000

3) Fmoc-removal and PEGylation

4) cleavage from resin

thioester formation

1a) optional: derivatization of side chains

1b) optional: purification by precipitation 2) liberation of SH-group (e.g., with TCEP)

Fig. 11: Native Chemical Ligation utilizing the photocleavable NCL-auxiliary glycine building block (PAA2000).

PEG27

Photochemistry

Native Chemical Ligation (NCL) is one of the most powerful tools for the preparation of complex peptides and small proteins. However, the classical variant of NCL requires an N-terminal cysteine at the ligation site. Iris Biotech presents an innovative auxiliary reagent that can be incorporated in place of a glycine residue.

Since glycine usually occurs several times in a peptide sequence, this approach significantly increases variability regarding the choice of possible ligation sites. In NCL, the auxiliary’s SH-group mimics the action of an N-terminal cysteine’s sulfhydryl group.

Following SPPS, the auxiliary is attached to the N-terminus of a peptide sequence in lieu of a glycine residue. The auxiliary’s Fmoc-protected amino functionality can subsequently be deprotected and functionalized, e.g., with a monodisperse PEG. PEGylation is useful for increasing the solubility of peptide fragments, and for facilitating their purification by precipitation with EtOH/Et 2 O. These properties are especially valuable if the peptide’s amino acid side-chains are supposed to be further derivatized postSPPS, for example by enzymatic glycosylation. Following NCL, the auxiliary can be conveniently removed by irradiation with UV-light (10 min in water or water/acetonitrile). This method is particularly useful for the synthesis of sophisticated peptides such as glycopeptides, where cost- and labor-intensive short sequences can be prepared separately, and subsequently conjugated to long fragments synthesized in a standard manner.

details

PAA2000 t Bu-SS-Photo(Fmoc)-Gly-OH

Photocleavable-NCL-auxiliary-Gly-OH

CAS-No. 1994388-93-0

Formula C 36 H44N4O 9 S 2 Mol. weight 740,89 g/mol

Reference:

→ A PEGylated photocleavable auxiliary mediates the sequential enzymatic glycosylation and native chemical ligation of peptides; C. Bello, S. Wang, L. Meng, K. W. Moremen, C. F. Becker; Angew. Chem. Int. Ed. 2015; 54: 7711-5. arrow-up-right-from-square https://doi.org/10.1002/anie.201501517

Product

9. Photo-Linker for Solid Phase Synthesis of Peptide Amides and Acids

Peptide linkers are usually cleaved under acidic conditions or using two-step procedures. Photocleavage proceeds under neutral conditions using UV light and can either be performed in batch or using flow chemistry.

Fig. 12: Cleavage of photolabile linkers.

Furthermore, photolabile linkers are orthogonal to standard peptide chemistry reaction conditions, thus enabling the use of a wide variety of amino acid protecting groups.

Three different photolabile linkers are available for your convenience:

Photo-linker for the synthesis of C-terminal carboxylic acids

RL-2150 Acid-Photo-Linker

4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid

CAS-No. 175281-76-2

Formula C13 H17NO 7 Mol. weight 299,28 g/mol

Photochemistry

Fmoc-amino photo-linker for the synthesis of peptide amides

RL-1026 Fmoc-Photo-Linker

4-{4-[1-(9-Fluorenylmethyloxycarbonylamino)ethyl]-2methoxy-5-nitrophenoxy}butanoic acid

CAS-No. 162827-98-7

Formula C 28 H28 N2O 8 Mol. weight 520,56 g/mol

Product details

References:

→ Continuous photochemical cleavage of linkers for solid-phase synthesis; M. Hurevich, J. Kandasamy, B. M. Ponnappa, M. Collot, D. Kopetzki, D. T. McQuade, P. H. Seeberger; Org Lett 2014; 16: 1794-7. arrow-up-right-from-square https://doi.org/10.1021/ol500530q

→ Photolytic Mass Laddering for Fast Characterization of Oligomers on Single Resin Beads; K. Burgess, C. I. Martinez, D. H. Russell, H. Shin, A. J. Zhang; J. Org. Chem. 1997; 62: 5662-5663. arrow-up-right-from-square https://doi.org/10.1021/jo970866w

→ Direct Monitoring of Organic Reactions on Polymeric Supports; M. R. Carrasco, M. C. Fitzgerald, Y. Oda, S. B. H. Kent; Tetrahedron Lett. 1997; 38: 6331-6334. arrow-up-right-from-square https://doi.org/10.1016/s0040-4039(97)01456-1

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10. Related Products

10.1. Fmoc-Phe-Aca – a Cell Internalization Reporter, and further Fluorescent Amino Acid Derivatives

The unnatural amino acid Aca (7-amino-coumarin-4-acetic acid) is a coumarin derivative and exhibits fluorescence. When incorporated into a peptide C-terminally of phenylalanine, Aca is a useful reporter group for the successful internalization of CPPs. The phenyl moiety of phenylalanine quenches the fluorescence of Aca. Internalization of the CPP containing Phe-Aca leads to proteolytic cleavage of the Phe-Aca bond and thus to fluorescence. However, the peptide bond formation between phenylalanine and Aca is considered to be a difficult coupling and often leads to low coupling yields. For your convenience, Iris Biotech offers Fmoc Phe-Aca as building block suitable for SPPS. This pseudodipeptide can be coupled to the resin of your choice and subsequently elongated to prepare your target CPP.

Fig. 13: Loading of a solid support, subsequent elongation to CPPs and function as a reporter group for successful CPP internalization.

Photochemistry

In addition to Fmoc-L-Phe-Aca-OH, we offer various 7-amino-4-methylcoumarin (AMC)-functionalized amino acid derivatives. Like Aca, when bound to a peptide, the AMC-amide fluoresces very weakly and excitation/emission wavelengths are shorter (ca. 330/390 nm). When the free AMC-amine is released by proteolytic cleavage, the fluorescence increases by a factor of approx. 700 and excitation and emission wavelengths are red-shifted. Thus, the low fluorescence of the AMC-amide substrate does not interfere with the fluorometric assay. Besides, Iris Biotech offers dipeptide recognition motifs such as Phe-Arg ( ZAA1480 on page 33) or Arg-Arg ( ZAA1470 on page 33) as fluorogenic 7-amido-4-methylcoumarin based protease substrates. The acetic acid derivative of AMC, 7-amino-4-methyl coumarin-3-acetic acid (AMCA) emits in the blue region (440-460 nm) upon activation with UV light of 350 nm.

Further fluorescent amino acids include those functionalized with acridine derivatives (2-Acd and 2-Bacd).

Coumarin Derivatives

BAA3650 Boc-L-Lys-AMC*AcOH

N-alpha-t-Butyloxycarbonyl-L-lysine 7-amido-4-methylcoumarin acetate

CAS-No. 116883-12-6 net

Formula C 21H29 N3 O 5*CH3 CO 2H Mol. weight 403,47*60,05

BAA6410 Boc-L-Lys(Ac)-AMC

tert-butyl (S)-(6-acetamido-1-((4-methyl-2-oxo-2Hchromen-7-yl)amino)-1-oxohexan-2-yl)carbamate

CAS-No. 233691-67-3

Formula C 23 H31N3 O 6 Mol. weight 445,52 g/mol

FDP1240 Fmoc-L-Phe-Aca-OH

7-[N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-phenylalaninyl-amido]-coumarin-4-acetic acid

CAS-No. 2250437-40-0

Formula C 35H28 N2O 7 Mol. weight 588,61 g/mol

Product details

HAA1200 H-L-Ala-AMC

L-Alanine-7-amido-4-methylcoumarin

CAS-No. 77471-41-1

Formula C13 H14N2O 3 Mol. weight 246,27 g/mol

HAA1174 H-L-Ala-AMC*TFA

L-Alanine 7-amido-4-methylcoumarin trifluoroacetate

CAS-No. 96594-10-4

Formula C13 H14N2O 3*CF 3 CO 2H Mol. weight 360,29 g/mol

HAA7630 H-L-Arg-AMC*2HCl

L-Arginine 7-amido-4-methylcoumarin dihydrochloride

CAS-No. 113712-08-6

Formula C16 H21N 5 O 3*2HCl Mol. weight 331,37*72,90 g/mol

HAA7645 Glt-Gly-Arg-AMC.HCl

Glutaryl-glycyl-L-arginine 7-amido-4-methylcoumarin hydrochloride

CAS-No. 103213-40-7

Formula C 23 H30 ClN 6 O 7 *HCl Mol. weight 502,53*36,46 g/mol

HAA3200 H-L-Leu-AMC

L-Leucine-7-amido-4-methylcoumarine

CAS-No. 66447-31-2

Formula C16 H20 N2O 3 Mol. weight 288,34 g/mol

HAA7740 H-L-Pyr-AMC

L-Pyroglutamic acid 7-amido-4-methylcoumarin

CAS-No. 66642-36-2

Formula C15H14N2O4 Mol. weight 286,29 g/mol

Photochemistry

HAA2890 H-L-Asp(AMC)-OH

L-Aspartic acid beta-(7-amido-4-methylcoumarin)

CAS-No. 133628-73-6

Formula C14H14N2O 5 Mol. weight 290,27 g/mol

HAA7971

H-Gly-AMC

Glycine 7-amido-4-methylcoumarin

CAS-No. 77471-42-2

Formula C12H12N2O 3 Mol. weight 232,24 g/mol

RL-1005 AMCA-OSu

7-Amino-4-methyl-3-coumarinylacetyl succinimidyl ester AMCO-NHS

CAS-No. 113721-87-2

Formula C16 H14N2O 6 Mol. weight 330,29 g/mol

RL-1170 Fmoc-ACA-OH

7-(9-Fluorenylmethyloxycarbonylamino)-coumarin-4-acetic acid

CAS-No. 378247-75-7

Formula C 26 H19 NO 6 Mol. weight 441,43 g/mol

ZAA1470 Z-L-Arg-L-Arg-AMC*2HCl

N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin-dihydrochloride

CAS-No. 140686-23-3

Formula C 30 H39 N9 O 6*2HCl Mol. weight 621,69*72,91 g/mol

ZAA1480 Z-L-Phe-L-Arg-AMC*HCl

N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin hydrochlorid

CAS-No. 65147-22-0

Formula C 33 H36 N 6 O 6*HCl Mol. weight 612,69*36,46 g/mol

Acridine Derivatives

BAA1580 Boc-L-Ala(2-Bacd)-OH

N-alpha-t-Butyloxycarbonyl-3-[benzo[b]acridin-12(5H)on-2-yl]-L-alanine

CAS-No. 916834-72-5

Formula C 25H24N2O 5 Mol. weight 432,47 g/mol

BAA1590 Boc-L-Ala(2-Acd)-OH

N-alpha-t-Butyloxycarbonyl-3-{2-[9(10H)-acridonyl]}-L-alanine

CAS-No. 643018-86-4

Formula C 21H22N2O 5 Mol. weight 382,41 g/mol

FAA5830 Fmoc-L-Ala(2-Acd)-OH

N-alpha-(9-Fluorenylmethoxycarbonyl)-3-{2-[9(10H)-acridonyl]}-L-alanine

CAS-No. 1013328-63-6

Formula C 31H24N2O 5 Mol. weight 504,53 g/mol

FAA5880 Fmoc-L-Ala(2-Bacd)-OH

N-alpha-(9-Fluorenylmethoxycarbonyl)-3-[benzo[b] acridin-12(5H)-on-2-yl]-L-alanine

CAS-No. 1157859-85-2

Formula C 35H26 N2O 5 Mol. weight 554,59 g/mol

HAA3570 H-L-Ala(2-Bacd)-OH*HCl

3-[Benzo[b]acridin-12(5H)-on-2-yl]-L-alanine hydrochloride

CAS-No. 933802-95-0 net

Formula C 20 H16 N2O 3*HCl Mol. weight 332,35*36,45 g/mol

Product details

Photochemistry

HAA3580 H-L-Ala(2-Acd)-OH*HCl

3-{2-[9(10H)-acridonyl]}-L-alanine hydrochloride

CAS-No. 854503-32-5

Formula C16 H14N2O 3*HCl

Mol. weight 282,29*36,45 g/mol

References:

→ Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries; J. L. Harris, B. J. Backes, F. Leonetti, S. Mahrus, J. A. Ellman, C. S. Craik; PNAS 2000; 97: 7754-7759. arrow-up-right-from-square https://doi.org/10.1073/pnas.140132697

→ Expedient Solid-Phase Synthesis of Fluorogenic Protease Substrates Using the 7-Amino-4-carbamoylmethylcoumarin (ACC) Fluorophore; D. J. Maly, F. Leonetti, B. J. Backes, D. S. Dauber, J. L. Harris, C. S. Craik, J. A. Ellman; J. Org. Chem. 2002; 67: 910-915. arrow-up-right-from-square https://doi.org/10.1021/jo016140o

→ Synthesis of a New Fluorogenic Substrate for Cystine Aminopeptidase; Y. Kanaoka, T. Takahashi, H. Nakayama, T. Ueno, T. Sekine; Chem. Pharm. Bull. 1982; 30(40) : 1485-1487. arrow-up-right-from-square https://doi.org/10.1248/cpb.30.1485

→ Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates; M. Zimmerman, B. Ashe, E. C. Yurewicz, G. Patel; Anal. Biochem. 1977; 78(1): 47-51. arrow-up-right-from-square https://doi.org/10.1016/0003-2697(77)90006-9.

→ Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins; H. Khalfan, R. Abuknesha, M. Rand-Weaver, R. G. Price, D. Robinson; Histochem J. 1986; 18(9) : 497-499. arrow-up-right-from-square https://doi.org/10.1007/BF01675617

→ A Simple Fluorescent Labeling Method for Studies of Protein Oxidation, Protein Modification, and Proteolysis; A. M. Pickering, K. J. A. Davies; Free Radic Biol Med. 2012; 52(2) : 239-246. arrow-up-right-from-square https://doi.org/10.1016/j.freeradbiomed.2011.08.018

→ Fluorescence imaging of drug target proteins using chemical probes; H. Zhu, I. Hamachi; J. Pharm. Anal. 2020; 10: 426-433. arrow-up-right-from-square https://doi.org/10.1016/j.jpha.2020.05.013

→ A New Fluorogenic Substrate for Chymotrypsin; M. Zimmerman, E. Yurewicz, G. Patel; Anal. Biochem. 1976; 70: 258-262. arrow-up-right-from-square https://doi.org/10.1016/S0003-2697(76)80066-8

→ Determination of Caspase Specificities Using a Peptide Combinatorial Library; N. A. Thornberry, K. T. Chapman, D. W. Nicholson; Method Enzymol 2000; 322: 100-110. arrow-up-right-from-square https://doi.org/10.1016/S0076-6879(00)22011-9 . circle-arrow-right

Interested in dyes? Discover our available rhodamine derivatives!

10.2. Building Blocks for the SPPS of FRET-based Fluorogenic Protease Substrates

The classic FRET pair EDANS and DABCYL is now available linked to Fmoc amino acid building blocks that can be readily used in SPPS. Conveniently synthesize your own custom protease substrates!

Fig. 14: Screening of protease activity using FRET-based fluorogenic protease substrates

Traditionally, fluorogenic protease substrates for the screening of protease activity are prepared by peptide synthesis and subsequent regioselective deprotection and functionalization with a fluorophore/ quencher pair such as EDANS and DABSYL or DABCYL, respectively.

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-epsilon-4-[4‘-(dimethylamino)phenylazo]benzoyl-L-lysine

CAS-No. 146998-27-8

Formula C 36 H37N 5 O 5 Mol. weight 619,73 g/mol

Photochemistry

LS-4180 Dabcyl-KTSAVLQSGFRKM-Glu(Edans)-amide

Dabcyl-lysyl-threonyl-seryl-alanyl-valyl-leucyl-glutaminyl-seryl-glycyl-phenylalanyl-arginyl-lysyl-methionyl-glutamyl(Edans)-amide

CAS-No. 2642630-08-6

Formula C95H142N26 O 23 S 2 Mol. weight 2080,46 g/mol

LS-4670 5HP2O((PEG)2-OH)-(CH2)5-Dansyl

2-(2-(2-hydroxyethoxy)ethoxy)ethyl 3-(1-(5-((5-(dimethylamino)naphthalene)-1-sulfonamido)pentyl)-2hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)propanoate

Formula C 30 H43 N3 O 9 S Mol. weight 621,75 g/mol

LS-4665 H-L-Cys-L-Glu(Dansyl)-OH*TFA

N2-(L-cysteinyl)-N5-(2-((5-(dimethylamino)naphthalene)-1-sulfonamido)ethyl)-L-glutamine bis(trifluoroacetate)

CAS-No. 2941222-88-2

Formula C 22H31N 5 O 6 S 2*2C 2HF 3 O 2 Mol. weight 525,64*228,05 g/mol

FAA1446 Fmoc-L-Lys(Dansyl)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-epsilon-dansyl-L-lysine

CAS-No. 118584-90-0

Formula C 33 H35N3 O 6 S Mol. weight 601,7 g/mol

FAA4410 Fmoc-L-Glu(EDANS)-OH

N-alpha-(9-Fluorenylmethyloxycarbonyl)-5-(2-(5-sulfonaphthalen-1-ylamino)ethylamido)-L-glutamic acid

CAS-No. 193475-66-0

Formula C 32H31N3 O 8 S Mol. weight 617,67 g/mol

FAA9300 Fmoc-L-Asp(EDANS)-OH

N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N4-(2-((5-sulfonaphthalen-1-yl)amino)ethyl)-L-asparagine

CAS-No. 182253-73-2

Formula C 31H29 N3 O 8 S Mol. weight 603,65 g/mol

References:

→ Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique; S. Chen, L. L. Chen, H. B. Luo, T. Sun, J. Chen, F. Ye, J. H. Cai, J. K. Shen, X. Shen, H. L. Jiang; Acta Pharmacol Sin 2005; 26: 99-106. arrow-up-right-from-square https://doi.org/10.1111/j.1745-7254.2005.00010.x

→ Synthesis and evaluation of fluorescent probes for the detection of calpain activity; S. Mittoo, L. E. Sundstrom, M. Bradley; Anal Biochem 2003; 319: 234-8. arrow-up-right-from-square https://doi.org/10.1016/s0003-2697(03)00324-5

→ A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis; L. L. Maggiora, C. W. Smith, Z. Y. Zhang; J Med Chem 1992; 35: 3727-30. arrow-up-right-from-square https://doi.org/10.1021/jm00099a001

→ Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer; E. D. Matayoshi, G. T. Wang, G. A. Krafft, J. Erickson; Science 1990; 247: 954-8. arrow-up-right-from-square https://doi.org/10.1126/science.2106161

→ Design and synthesis of new fluorogenic HIV protease substrates based on resonance energy transfer; G. T. Wang, E. Matayoshi, H. Jan Huffaker, G. A. Krafft; Tetrahedron Lett. 1990; 31: 6493-6496. arrow-up-right-from-square https://doi.org/10.1016/s0040-4039(00)97099-0

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Photochemistry

FAA9300 Fmoc-L-Asp(EDANS)-OH

Photochemistry

Notes

Code of Conduct

As business activity of Iris Biotech GmbH impacts people’s lives and health, it must be operated in ethical and correct manner and act with integrity and responsibility. To ensure high ethical standards and fair business practices, Iris Biotech GmbH applies an integrated policy known as its Code of Conduct.

In 2001 Iris Biotech GmbH was founded just at the beginning of the Biotech movement and the first remarkable breakthrough of biotech pharma products. Although the biotech field is rather young compared to other industries we believe on long-term business, a good partnership between our business partners and Iris Biotech GmbH and a good reputation. It is our duty as well as our responsibility to maintain and to extend this over the next generations – based on the principles of an honourable and prudent tradesman which based upon the concept of honourable entrepreneurship.

This Code of Conduct has been developed following the “Voluntary Guidelines for Manufacturers of Fine Chemical Intermediates and Active Ingredients” issued by AIME (Agrochemical & Intermediates Manufacturers in Europe) and the requirements of some of our business associates.

Iris Biotech GmbH commits to hold this Code of Conduct and to include and apply its principles in the management system and the company policies.

Ethics

Iris Biotech GmbH undertakes business in an ethical manner and acts with integrity. All corruption, extortion and embezzlement are prohibited. We do not pay or accept bribes or participate in other illegal inducements in business or government relationships. We conduct our business in compliance with all applicable anti-trust laws. Employees are encouraged to report concerns or illegal activities in the workplace, without threat of reprisal, intimidation or harassment.

Labour

Iris Biotech GmbH is committed to uphold the human rights of workers and to treat them with dignity and respect. Child labour, workplace harassment, discrimination, and harsh and inhumane treatment are prohibited. Iris Biotech GmbH respects the rights of the employees to associate freely, join or not join labour unions, seek representation and join workers’ councils. Employees are paid and their working timetable is established according to applicable wage and labour laws. Employees are able to communicate openly with management regarding working conditions without threat of reprisal, intimidation or harassment.

General Policies

Contracts and Secrecy Agreements are binding and the confidential information received is only used for intended purposes. Clear management and organizational structures exist to provide efficient normal working and to address problems quickly. Know-how is protected and intellectual property isrespected.

Health and Safety

Iris Biotech GmbH provides a safe and healthy working environment to the employees and protects them from overexposure to chemical and physical hazards. Products are produced, stored and shipped under the guidelines of the relevant chemical and safety legislation. Risks and emergency scenarios are identified and evaluated, and their possible impact is minimized by implementing emergency plans and written procedures. Safety information regarding hazardous materials is available to educate, train and protect workers from hazards. Preventive equipment and facilities maintenance is performed at suitable periods to reduce potential hazards. Employees are regularly trained in health and safety matters and are informed about product properties and risk classification when it is required.

Environment

Iris Biotech GmbH operates in an environmentally responsible and efficient manner, minimizing adverse impacts on the environment. Waste streams are managed to ensure a safe handling, movement, storage, recycling and reuse, before and after being generated. Systems to prevent and mitigate accidental spills and releases to the environment are in place. All required environmental permits and licenses are obtained and their operational and reporting requirements are complied with.

Production and Quality Management

A quality management system following the Good Distribution Practices (GDP rules) of Active Pharmaceutical Ingredients is established covering all the aspects of the worldwide distribution of products. Regular audits are performed to evaluate the efficiency and fulfilling of the quality system. Process controls to provide reproducible product quality are established. There are preventive maintenance procedures to ensure plant reliability and the lowest risk of failure. Staff is trained periodically about GMP and GDP rules. Procedures are established and installations are designed to avoid cross contamination. Batch and analytical records are kept for inspection and audit purposes for suitable periods according guidelines.

Research and Development

Research and development staff education is appropriate to their functional activity and they are trained to develop, optimize and scale-up the processes. Intellectual property is respected and knowhow protected. Development of manufacturing processes reflects the principles of the Green Chemistry according to the American Chemical Society Green Chemistry Institute. Animal testing is not used unless alternatives are not scientifically valid or accepted by regulators. If animal testing is carried out, animals are treated so that pain and stress are minimized.

Terms and Conditions of Sales

All orders placed by a buyer are accepted and all contracts are made subject to the terms which shall prevail and be effective notwithstanding any variations or additions contained in any order or other document submitted by the buyer. No modification of these terms shall be binding upon Iris Biotech GmbH unless made in writing by an authorised representative of Iris Biotech GmbH.

Placing of Orders

Every order made by the buyer shall be deemed an offer by the buyer to purchase products from Iris Biotech GmbH and will not be binding on Iris Biotech GmbH until a duly authorised representative of Iris Biotech GmbH has accepted the offer made by the buyer. Iris Biotech GmbH may accept orders from commercial, educational or government organisations, but not from private individuals and Iris Biotech GmbH reserves the right to insist on a written order and/or references from the buyer before proceeding.

There is no minimum order value. At the time of acceptance of an order Iris Biotech GmbH will either arrange prompt despatch from stock or the manufacture/acquisition of material to satisfy the order. In the event of the latter Iris Biotech GmbH will indicate an estimated delivery date. In addition to all its other rights Iris Biotech GmbH reserves the right to refuse the subsequent cancellation of the order if Iris Biotech GmbH expects to deliver theproduct on or prior to the estimated delivery date. Time shall not be of the essence in respect of delivery of the products. If Iris Biotech GmbH is unable to deliver any products by reason of any circumstances beyond its reasonable control („Force Majeure“) then the period for delivery shall be extended by the time lost due to such Force Majeure. Details of Force Majeure will be forwarded by Iris Biotech GmbH to the buyer as soon as reasonably practicable.

Prices, Quotations and Payments

Prices are subject to change. For the avoidance of doubt, the price advised by Iris Biotech GmbH at the time of the buyer placing the order shall supersede any previous price indications. The buyer must contact the local office of Iris Biotech GmbH before ordering if further information is required. Unless otherwise agreed by the buyer and Iris Biotech GmbH, the price shall be for delivery ex-works. In the event that the buyer requires delivery of the products otherwise than ex-works the buyer should contact the local office of Iris Biotech GmbH in order to detail its requirements. Iris Biotech GmbH shall, at its discretion, arrange the buyer‘s delivery requirements including, without limitation, transit insurance, the mode of transit (Iris Biotech GmbH reserves the right to vary the mode of transit if any regulations or other relevant considerations so require) and any special packaging requirements (including cylinders). For the avoidance of doubt all costs of delivery and packaging in accordance with the buyer‘s requests over and above that of delivery in standard packaging ex-works shall be for the buyer‘s account unless otherwise agreed by both parties. Incoterms 2020 shall apply. Any tax, duty or charge imposed by governmental authority or otherwise and any other applicable taxes, duties or charges shall be for the buyer‘s account. Iris Biotech GmbH may, on request and where possible, provide quotations for multiple packs or bulk quantities, and non-listed items. Irrespective of the type of request or means of response all quotations must be accepted by the buyer without condition and in writing before an order will be accepted by Iris Biotech GmbH. Unless agreed in writing on different terms, quotations are valid for 30 days from the date thereof. Payment terms are net 30 days from invoice date unless otherwise agreed in writing. Iris Biotech GmbH reserves the right to request advance payment at its discretion. For overseas transactions the buyer shall pay all the banking charges of Iris Biotech GmbH. The buyer shall not

be entitled to withhold or set-off payment for the products for any reason whatsoever. Government/ Corporate Visa and MasterCard (and other such credit cards) may be accepted on approved accounts for payment of the products. Personal credit cards are not acceptable. Failure to comply with the terms of payment of Iris Biotech GmbH shall constitute default without reminder. In these circumstances Iris Biotech GmbH may (without prejudice to any other of its rights under these terms) charge interest to accrue on a daily basis at the rate of 2% per month from the date upon which payment falls due to the actual date of payment (such interest shall be paid monthly). If the buyer shall fail to fulfil the payment terms in respect of any invoice of Iris Biotech GmbH Iris Biotech GmbH may demand payment of all outstanding balances from the buyer whether due or not and/or cancel all outstanding orders and/or decline to make further deliveries or provision of services except upon receipt of cash or satisfactory securities. Until payment by the buyer in full of the price and any other monies due to Iris Biotech GmbH in respect of all other products or services supplied or agreed to be supplied by Iris Biotech GmbH to the buyer (including but without limitation any costs of delivery) the property in the products shall remain vested in Iris Biotech GmbH.

Shipping, Packaging and Returns

The buyer shall inspect goods immediately on receipt and inform Iris Biotech GmbH of any shortage or damage within five days. Quality problems must be notified within ten days of receipt. Goods must not be returned without prior written authorisation of Iris Biotech GmbH. Iris Biotech GmbH shall at its sole discretion replace the defective products (or parts thereof) free of charge or refund the price (or proportionate price) to buyer. Opened or damaged containers cannot be returned by the buyer without the written prior agreement of Iris Biotech GmbH. In the case of agreed damaged containers which cannot be so returned, the buyer assumes responsibility for the safe disposal of such containers in accordance with all applicable laws.

Product Quality, Specifications and Technical Information

Products are analysed in the Quality Control laboratories of Iris Biotech GmbH’s production partners by methods and procedures which Iris Biotech GmbH considers appropriate. In the event of any dispute concerning reported discrepancies arising from the buyer’s analytical results, determined by the buyer’s own analytical procedures, Iris Biotech GmbH reserves the right to rely on the results of own analytical methods of Iris Biotech GmbH. Certificates of Analysis or Certificates of Conformity are available at the discretion of Iris Biotech GmbH for bulk orders but not normally for prepack orders. Iris Biotech GmbH reserves the right to make a charge for such certification. Specifications may change and reasonable variation from any value listed should not form the basis of a dispute. Any supply by Iris Biotech GmbH of bespoke or custom product for a buyer shall be to a specification agreed by both parties in writing. Technical information, provided orally, in writing, or by electronic means by or on behalf of Iris Biotech GmbH, including any descriptions, references, illustrations or diagrams in any catalogue or brochure, is provided for guidance purposes only and is subject to change.

Safety

All chemicals should be handled only by competent, suitably trained persons, familiar with laboratory procedures and potential chemical hazards. The burden of safe use of the products of Iris Biotech GmbH vests in the buyer. The buyer assumes all responsibility for warning his employees, and any persons who might reasonably be expected to come into contact with the products, of all risks to person and property in any way connected with the products and for instructing them in their safe handling and use. The buyer also assumes the responsibility for the safe disposal of all products in accordance with all applicable laws.

Uses, Warranties and Liabilities

All products of Iris Biotech GmbH are intended for laboratory research purposes and unless otherwise stated on product labels, in the catalogue and product information sheet of Iris Biotech GmbH or in other literature furnished to the buyer, are not to be used for any other purposes, including but not limited to use as or as components in drugs for human or animal use, medical devices, cosmetics, food additives, household chemicals, agricultural or horticultural products or pesticides. Iris Biotech GmbH offers no warranty regarding the fitness of any product for a particular purpose and shall not be responsible for any loss or damage whatsoever arising there from. No warranty or representation is given by Iris Biotech GmbH that the products do not infringe any letters patent, trademarks, registered designs or other industrial rights. The buyer further warrants to Iris Biotech GmbH that any use of the products in the United States of America shall not result in the products becoming adulterated or misbranded within the meaning of the Federal Food, Drug and Cosmetic Act (or such equivalent legislation in force in the buyer‘s jurisdiction) and shall not be materials which may not, under sections 404, 505 or 512 of the Act, be introduced into interstate commerce. The buyer acknowledges that, since the products of Iris Biotech GmbH are intended for research purposes, they may not be on the Toxic Substances Control Act 1976 („TSCA“) inventory. The buyer warrants that it shall ensure that the products are approved for use under the TSCA (or such other equivalent legislation in force in the buyer‘s jurisdiction), if applicable. The buyer shall be responsible for complying with any legislation or regulations governing the use of the products and their importation into the country of destination (for the avoidance of doubt to include, without limitation, the TSCA and all its amendments, all EINECS, ELINCS and NONS regulations). If any licence or consent of any government or other authority shall be required for the acquisition, carriage or use of the products by the buyer the buyer shall obtain the same at its own expense and if necessary produce evidence of the same to Iris Biotech GmbH on demand. Failure to do so shall not entitle the buyer to withhold or delay payment. Any additional expenses or charges incurred by Iris Biotech GmbH resulting from such failure shall be for the buyer‘s account. Save for death or personal injury caused by negligence of Iris Biotech GmbH, sole obligation of Iris Biotech GmbH and buyer‘s exclusive remedy with respect to the products proved to the satisfaction of Iris Biotech GmbH to be defective or products incorrectly supplied shall be to accept the return of said products to Iris Biotech GmbH for refund of the actual purchase price paid by the buyer (or proportionate part thereof), or replacement of the defective product (or part thereof) with alternative product. Iris Biotech GmbH shall have no liability to the buyer under or arising directly or indirectly out of or otherwise in connection with the supply of products by Iris Biotech GmbH to the buyer and/or their re-sale or use by the buyer or for any product, process or services of the buyer which in any way comprises the product in contract tort (including negligence or breach of statutory duty) or otherwise for pure economic loss, loss of profit, business, reputation, depletion of brand, contracts, revenues or anticipated savings or for any special indirect or consequential damage or loss of any nature except as may otherwise be expressly provided for in these terms. All implied warranties, terms and representations in respect of the products (whether implied by statute or otherwise) are excluded to the fullest extent permitted by law. The buyer shall indemnify Iris Biotech GmbH for and against any and all losses, damages and expenses, including legal fees and other costs of defending any action, that Iris Biotech GmbH may sustain or incur as a result of any act or omission by the buyer, its officers, agents or employees, its successors or assignees, its customers or all other third parties, whether direct or indirect, in connection with the use of any product. For the avoidance of doubt and in the event that Iris Biotech GmbH supplies bespoke or custom product to the buyer‘s design or specification, this indemnity shall extend to include any claim by a third party that the manufacture of the product for the buyer or the use of the product by the buyer infringes the intellectual property rights of any third party.

General

Iris Biotech GmbH shall be entitled to assign or sub-contract all or any of its rights and obligations hereunder. The buyer shall not be entitled to assign, transfer, sub-contract or otherwise delegate any of its rights or obligations hereunder. Any delay or forbearance by Iris Biotech GmbH in exercising any right or remedy under these terms shall not constitute a waiver of such right or remedy. If any provision of these terms is held by any competent authority to be invalid or unenforceable in whole or in part the validity of the other provisions of these terms and the remainder of the provision in question shall not be affected. These terms shall be governed by German Law and the German Courts shall have exclusive jurisdiction for the hearing of any dispute between the parties save in relation to enforcement where the jurisdiction of the German Courts shall be non-exclusive.

Get in Contact Distribution Partners

Iris Biotech GmbH

Adalbert-Zoellner-Str. 1 95615 Marktredwitz Germany

+49 (0) 9231 97121-0

+49 (0) 9231 97121-99

info@iris-biotech.de www.iris-biotech.de

The list contains the current distributors of Iris Biotech in different regions of the world. The latest list of distribution partners and contact details is available at: arrow-up-right-from-square www.iris-biotech.de/distribution-partner

China: Chengdu Yoo Technology Co., Ltd.

Japan:

BizCom Japan, Inc.

Shigematsu & Co., Ltd

Cosmo Bio Co., Ltd.

USA & Canada: Peptide Solutions LLC

India, Bangladesh, Oman, Sri Lanka, United Arab Emirates: Sumit Biosciences Pvt Ltd.

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