iGEM 2024 — Team sehs

{% extends "layout.html" %}
{% block page\_content %}

Notebook

* Experimental Records-Biology
* Experimental records-Chemistry

# Experimental Records-Biology

# Experimental records-Chemistry

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

Engineering

* Cycle 1
* Cycle 2
* Cycle 3

# Cycle 1

## Design Upon examining the crystal structure of firefly luciferase and luciferin, we identified a large binding pocket surrounding the hydroxyl group of luciferin, thereby allowing for the introduction of a cyclic amino group in that region to augment the affinity with luciferase. Simultaneously, the incorporation of this group may enhance the lipid-water partition coefficient and blood-brain barrier permeability, as well as prolong the bioluminescence wavelength and duration in vivo. Considering the above information, we designed cyclic N-aminoluciferins (cyL) as firefly luciferase substrates in bioluminescence imaging. Built Following rational design, we undertook the organic synthesis of cyL molecules. We adopted cycloaminobenzothiazole-2-nitriles (the courtesy gift from Nobel Drugs) as the starting material and dissolved it in a mixture of methanol, dichloromethane, and water. Subsequently, we gradually introduced D-cysteine, anhydrous potassium carbonate, and hydrochloric acid to facilitate the cyclization reaction. Upon completion of the reaction, which occurred 20 minutes later under nitrogen gas protection, we obtained cyL compounds including 5-cyL, 6-cyL, 7-cyL, and 8-cyL. Test Subsequent to the synthesis of compounds, we conducted a variety of biological evaluations. We assessed their bioluminescent characteristics, enzymatic imaging, cellular imaging, imaging of the xenograft tumor model in nude mice, imaging of transgenic mice, and imaging of the brain tumor model in nude mice. We employed a spectrophotometer to evaluate the bioluminescent wavelengths of cyL compounds. In comparison to the substrates D-luciferin and N-aminoluciferin, the bioluminescent wavelengths of all cyL compounds exhibited red-shifted enhancement, hence facilitating imaging application in deep tissues. Meanwhile, the lower kinetic constants Km and Vmax of 5-cyL, 6-cyL, 7-cyL and 8-cyL suggest that the reaction rate and affinity have obviously potential, compared to D-luciferin and aminoluciferin. These results suggest that these compounds may possess high sensitivity as novel substrates for firefly luciferase. Learn In the evaluation of the dependence of enzymatic bioluminescence intensity on ATP and substrate concentrations, we found that while compounds 5-cyL, 6-cyL, and 7-cyL could not surpass the substrates aminoluciferin and D-luciferin, an increase in ATP concentration resulted in a corresponding enhancement of bioluminescent intensity for all substrates, demonstrating a linear correlation. Nonetheless, compound 8-cyL exhibited unreasonable performance; hence, we exclusively implemented compounds 5-cyL, 6-cyL, and 7-cyL in the following experiments.

# Cycle 2

To evaluate cell-based activity, we initially developed a firefly luciferase-labeled human glioblastoma cell
line (U87-Luc) and conducted experiments to determine the relationship between cellular concentrations and the
bioluminescent intensity of substrates. Under equivalent substrate concentration conditions, the
bioluminescent intensity was directly correlated with the cellular concentrations of U87-Luc brain glioma
cells. In comparison to the substrates aminoluciferin and D-luciferin, the compounds 5-cyL, 6-cyL, and 7-cyL
exhibited much higher sensitivity, advantageous cellular permeability, and bioluminescent feasibility.

We administered identical doses of the three compounds, D-luciferin and N-aminoluciferin, at varying
concentrations to nude mice bearing the U87-Luc subcutaneous xenograft tumor. Following bioluminescent imaging
evaluations via the real-time imaging system in animals, we observed that the bioluminescent intensities of
5-cyL, 6-cyL, and 7-cyL at identical concentration levels were much greater than those of the substrates
D-luciferin and aminoluciferin. The highest bioluminescence intensity and longest duration was 7-cyL,
exhibiting a bioluminescent intensity 25 times greater than that of D-luciferin, with an imaging duration of 7
hours.

# Cycle 3

## Built

Following the imaging results from the nude mice xenograft tumor model, we selected 7-cyL to do the imaging
experiment on the FVB-Tg transgenic mice model. Post intravenous administration, we observed that the
bioluminescent intensity of 7-cyL exceeded that of the substrates D-luciferin and N-aminoluciferin by more
than tenfold, exhibiting a comparatively prolonged in vivo luminescence lifetime of three hours. Upon
calculating the ratio of cerebral bioluminescent intensity in FVB-Tg mice to their own bioluminescent
intensity, in comparison to D-luciferin and N-aminoluciferin, compound 7-cyL exhibited a comparatively high
cerebral imaging ratio and superior blood-brain barrier permeability, indicating its potential significant
application value in brain tumor imaging.

## Test

Given the elevated cerebral bioluminescent ratio of compound 7-cyL in FVB-Tg mice bioluminescent imaging,
we developed a brain tumor model to examine the in vivo imaging of 7-cyL. Compound 7-cyL demonstrated a
bioluminescence intensity over 30 times that of D-luciferin, with a steady bioluminescent duration of up to 4
hours, indicating that compound 7-cyL exhibited good permeability across the blood-brain barrier.
Consequently, this advantage would enhance bioluminescent brain tumor imaging capabilities.

## Learn

In this effort, we developed four cyclic N-aminoluciferins (cyL), with compound 7-cyL exhibiting a
bioluminescence intensity exceeding tenfold that of an equivalent dose of D-luciferin, and a bioluminescent
duration of seven hours. The brain tumor imaging of 7-cyL demonstrated a bioluminescent intensity exceeding
that of the natural substrate D-luciferin by over 30 times. We believe that such cyL substrates would expand
the capabilities of existing bioluminescence technologies and stimulate new uses of bioluminescence imaging in
brain tumor research.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

Experiment

* Organic synthesis
* Biological experiment

# Organic synthesis

## Synthesis of 5-CyL, 6-CyL, 7-CyL and 8-CyL

##### Materials:

1. D-cysteine hydrochloride (36 mg，0.20 mmol)
2. potassium carbonate (28 mg，0.20 mmol)
3. the solution of cycloaminobenzothiazol-2-nitrile (0.10 mmol) in 2 mL DCM/MeOH (1:1)
4. 2 mL H2O/MeOH (1:1)
5. 0.1 M aqueous HCl solution

![](https://static.igem.wiki/teams/5414/content/experiment-01.png)

##### Procedures:

**(s)-2-(6-(pyrrolidin-1-yl)benzo[d]thiazol-2-yl)-4, 5-dihydrothiazole-4-carboxylic acid (5-cyL)**

1. Add the mixture of D-cysteine hydrochloride (36 mg，0.20 mmol) and potassium carbonate (28 mg，0.20 mmol) to
   the solution of cycloaminobenzothiazol-2-nitrile (0.10 mmol) in 2 mL DCM/MeOH (1:1).
2. Dissolve it in 2 mL H2O/MeOH (1:1).
3. Stir vigorously for 20 minutes at room temperature.
4. DCM and MeOH were then removed from the flask under low pressure
5. Add 0.1 M aqueous HCl solution until pH reaches 5-6.
6. Precipitate formed immediately.
7. Filter the precipitate off and wash it with ice water.
8. Dry the precipitate under reduced pressure, 28 mg of pure product as a yellow solid is provided.
   (Yield=84%.)

**(S)-2-(6-(piperidin-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid(6-cyL)**

The synthesis was analogous to 5-cyL. The product was obtained as a yellow-white solid. Yield = 68%.

**(S)-2-(6-(azepan-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid(7-cyL)**

The synthesis was analogous to 5-cyL. The product was obtained as an orange-red solid. Yield = 68%.

**(S)-2-(6-(azocan-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (8-cyL)**

The synthesis was analogous to 5-cyL. The product was obtained as a brown solid. Yield = 86%

# Biological experiment

## Molecular level: Bioluminescence emission spectra and in vitro bioluminescence assay

##### Reagent:

1. 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin- 50 mM Tris-HCl (pH7.4) buffer solution- Firefly luciferase solution(20 μg/mL, containing 2 mM ATP)

##### Device:

1. Quartz cuvettes- 2F-2500 FL Spectrophotometer- Xenogen IVIS Spectrum imaging system

##### Procedures:

1. Prepare 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin- Prepare the solution of them by with 50 mM Tris-HCl (pH7.4) buffer solution- Add the firefly luciferase solution(20 μg/mL, containing 2 mM ATP) to these substrates (100 μM, 0.5 mL) in
       quartz cuvettes.- Measure the bioluminescence emission spectra immediately by a F-2500 FL Spectrophotometer.

## Cellular level

##### Reagent:

1. Luciferase-transfected U-78 cells
2. RPMI 1640
3. 10% fetal bovine serum (FBS)

##### Device:

1. CO2 incubator
2. Centrifuge

##### Procedures:

1. Cultured the expressing firefly luciferase cells in RPMI 1640 supplemented with 10% fetal bovine serum
   (FBS)
   (37°C, humidified atmosphere, 5% CO2 incubator. )
2. These cells in logarithmic phase after trypsinization and centrifugation
3. Diluted the cells into RPMI 1640 supplemented with 10% FBS for a cells-suspension (2x 106 /mL)
4. 200 μL cells suspension were added into black 96-well plates (4x 104cells per well)
5. Incubating for 24 h in the incubator
6. Add the different concentrations of 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin (50 μL,
   0.1−20 μM) to each well
7. Acquire the bioluminescence images using a Xenogen IVIS Spectrum imaging system. Images were acquired
   every 1 min for 30 min (3s- exposure per image).
8. 20 minutes later, measure the luciferase activity.
9. Luminescent signal (photons per second) for each well was measured and plotted as average values
   (experiments conducted in triplicate).

## Nude Xenograft Tumor Model Bioluminescence Imaging

### Animal level

##### Reagent:

1. 6-week-old female nude mice
2. luciferase-expressing U-87 cells (1×107)
3. 100 µL of 5-cyL, 6-cyL, 7-cyL, D-luciferin and Aminoluciferin (1 µM to 10 mM)

##### Devices:

1. puncture needle
2. Xenogen IVIS Spectrum imaging system
3. Anesthesia machine

##### Procedures:

1. The 6-week-old female nude mice were inoculated subcutaneously with luciferase-expressing U-87 cells
   (1×107) in normal saline (0.1 mL)
2. When the size of the tumors is 1 cm3, harvest them
3. Cut the tumors into pieces
4. Disinfected the nude mice locally
5. Use the puncture needle to implant 10 mg tumour pieces subcutaneously under the right armpit region
6. Two weeks later, injected i.p. with 100 µL of 5-cyL, 6-cyL, 7-cyL, D-luciferin and Aminoluciferin (1 µM to
   10 mM) to the nude mice with tumor xenografts
7. Anesthetized the mice with isoflurane (2% in 1 l/min oxygen)
8. Acquire the bioluminescence images using the Xenogen IVIS Spectrum imaging system. (Images were acquired
   every 5 min for 500 min)

## FVB-Tg mice Bioluminescence Imaging

##### Reagent:

1. FVB-Tg transgenic nude mice
2. 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin dissolved in saline and DMSO (10:1).

##### Devices:

1. Anesthesia machine
2. Xenogen IVIS Spectrum imaging system.

##### Procedures:

1. FVB-Tg transgenic nude mice is injected i.v. with 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin
   dissolved in saline and DMSO (10:1), respectively.
2. The animals were anesthetized with isoflurane (2% in 1 L/min oxygen)
3. Bioluminescence images were acquired using the Xenogen IVIS Spectrum imaging system.

## Brain Tumor Bioluminescence Imaging

##### Reagent:

1. female nude mice (6 weeks old, 18-20 g)
2. luciferase-expressing U87-Luc cells (8 × 103 cells in 2 µL saline)
3. 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin
4. saline solution
5. DMSO

##### Devices:

1. Xenogen IVIS Spectrum imaging system.
2. Anesthesia machine

##### Procedures:

1. The female nude mice (6 weeks old, 18-20 g) were inoculated with luciferase-expressing U87-Luc cells (8 ×
   103 cells in 2 µL saline) in brain hippocampus. - Establish the brain tumors for 2 weeks- The nude mice with tumors in brain hippocampus were injected i.p. with 100 µL of 1 mM 7-cyL, D-luciferin
       and aminoluciferin dissolved in saline and DMSO (10:1), respectively.- The animals were anesthetized with isoflurane (2% in 1 L/min oxygen) - Bioluminescence images were acquired using the Xenogen IVIS Spectrum imaging system. (Images were acquired
           every 5 min for 120 min)

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

Result

* Result of the Experiment
* Colleague Evaluation

# Result of the Experiment

##### [In vitro imaging of cyL]

In vitro, there is the positive correlation relationship between the bioluminescence intensity and
concentration for our aminoluciferin analogues (**Figure 1**). Meanwhile, the low kinetic constants
Km, Vmax of
5-cyL, 6-cyL, 7-cyL and 8-cyL suggest that the reaction rate and affinity have obviously potential, compared
to
D-luciferin and aminoluciferin (**Table 1**). These results indicate that these compounds maybe have higher
sensitivity as novel substrates for firefly luciferase. In addition, we measured the relationship of the
bioluminescence intensity and concentration of ATP. Like nature substrates D-luciferin and aminoluciferin, the
bioluminescence intensity of cyL compounds have significant positive correlation relationship with the
increase
of ATP concentration, indicating that the analogues could be used to monitor the level of ATP in vivo.

![](https://static.igem.wiki/teams/5414/content/results-01.png)

**Figure 1. In Vitro Bioluminescence Assay.**
1. Substrate dose-response analysis: (1) Total flux(p/s) of six
different luciferases at different substrate concentrations (μM). (2) The light intensity of six luciferases
at
different substrate concentrations(μM); 2. ATP dose-response analysis: (1) Total flux(p/s) of six different
luciferases at different ATP concentrations(μM). (2) The light intensity of six luciferases at different ATP
concentrations(μM).

**Table 1.**The bioluminescence properties of 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin
and aminoluciferin

| Substrates | λmax(nm) | Vmax (s-1) | Km (mol/L) |
| --- | --- | --- | --- |
| dLuc | 560 | 9.68×1010 | 10.9 |
| aLuc | 588 | 1.89×1010 | 6.63 |
| 5-cyL | 598 | 4.67×109 | 0.344 |
| 6-cyL | 610 | 3.69×109 | 0.274 |
| 7-cyL | 605 | 5.90×109 | 0.281 |
| 8-cyL | 613 | 4.367×109 | 0.313 |

##### [Cell bioluminescence imaging]

In order to measure the bioluminescence intensity of these analogues, our team measured bioluminescence
imaging in living cells. The luciferase-expressing in U87 cells were cultured in a conditioned system. After
the incubation period, the medium was removed and we add different concentration of 5-cyL, 6-cyL, 7-cyL and
8-cyL. In the same condition of the substrate concentration, the bioluminescence intense has an obvious
positive correlation with the concentration of U87-Luc cells and the three novel bioluminescence substrates
has high sensitivity, good cell permeability and bioluminescence activity (**Figure 2**).

![](https://static.igem.wiki/teams/5414/content/results-02.png)

**Figure 2. Cell Bioluminescence imaging in U87-Luc cells.**(1) Total flux(p/s) of five different
luciferases at different Cell-concentrations(μM). (2) The light
intensity of five luciferases at different Cell-concentrations(μM).

##### [Tumor Xenografts Mice Imaging]

In a cell-based assay, our compounds exhibited good biological activity. Subsequently, we carried on
further
bioluminescence imaging of well-established mice xenograft tumor models. The 6-weeks-old female nude mice
were
inoculated subcutaneously with luciferase-expressing U87-Luc cells. After tumor xenografts in mice
generated,
the nude mice with tumor xenografts were injected i.p. with 100 μL of 5-cyL, 6-cyL, 7-cyL, D-luciferin and
aminoluciferin (1 µM, 100 µM, 1 mM and 10 mM). The bioluminescence signals were measured by a Xenogen IVIS
Spectrum imaging system (**Figure 3A**). The cyL compounds all yielded a >10-fold higher bioluminescent signal
than D-luciferin at equivalent doses. Especially, 7-cyL showed 25-fold higher bioluminescence intensity
than
D-luciferin (**Figure 3B**). More interestingly, low to 1 µM dose of aminoluciferin analogues still could show
high and detectable bioluminescent signal, while D-luciferin at equivalent concentrations provided either
weak
or no signal. These results fully demonstrated that the three aminoluciferin analogues have higher
sensitivity
for bioluminescence imaging in live mice. Meanwhile, up to 7 h light emission time of aminoluciferin
analogues
were significantly greater than 10 min of D-luciferin and aminoluciferin, beneficial to real-time dynamic
monitoring imaging (**Figure 3C**). In addition, 5-cyL, 6-cyL and 7-cyL have higher Cy5.5 filter (695–770 nm
bandpass) transmittance ratio, contributing to the imaging of deep tissue in live organism.

![](https://static.igem.wiki/teams/5414/content/results-03.png)

**Figure 3.** Bioluminescence imaging of mouse xenograft tumor models with aminoluciferin analogues. a)
Maximum total
photon flux from with nude mice inoculated ES-2-Luc tumors. Mice were injected i.p. with 5-cyL, 6-cyL,
7-cyL,
D-luciferin and aminoluciferin (1 µM, 100 µM, 1 mM and 10 mM) and imaged 10 min after injection. b)
Quantification of the images from (a); error bars, s.e.m. (n = 3 experiments). c) Time–response analysis of
BLI in mouse xenograft tumor models with 1 mM cyL.

##### [FVB-Tg Transgenic Mice Imaging]

In light of in vivo imaging activity, we chose 7-cyL that performed excellent in the nude mice with tumor
xenografts assay for further bioluminescence imaging in transgenic mouse ubiquitously expressing firefly
luciferase (FVB-Tg mice). As expected, when 100 μL 7-cyL (1 mM) injected i.v to FVB-Tg mice, its
bioluminescence intensity presented up to 10-fold higher than that of D-luciferin and aminoluciferin
(**Figure 4A**). Moreover, in the measurement process, our aminoluciferin analogues have not only higher
bioluminescence
intensity but also longer in vivo bioluminescence time (**Figure 4B**). Interestingly, when computing the
ratio of
brain BLI intensity relative to the whole-body bioluminescence signal in FVB-Tg mice, we found that 7-cyL
has
higher brain bioluminescence ratio than D-luciferin and aminoluciferin (**Figure 4C**). As a result, we
believed
that 7-cyL has an excellent ability to penetrate the blood-brain barrier.

![](https://static.igem.wiki/teams/5414/content/results-04.png)

**Figure 4.**Bioluminescence imaging comparison of 7-cyL, D-luciferin and aminoluciferin (1 mM) in
FVB-Tg
mice. a) Photon
flux from FVB-Tg mice injected i.v. with 100 µL of 1 mM 7-cyL、D-luciferin and aminoluciferin. b)
Time–response
analysis of BLI in FVB-Tg mice with 1 mM 7-cyL、D-luciferin and aminoluciferin. c) The ratio of brain BLI
intensity in FVB-Tg mice with 7-cyL, D-luciferin and aminoluciferin (1 mM) (n = 3). Error bars, s.e.m. \*\*\*\*P
< 0.0001 (t-test).

**[Brain tumor bioluminescence imaging]**

As we know, the blood-brain barrier as a protective barrier limits the access of many small molecules to
this
tissue. So, the bioluminescence imaging in the brain is particularly challenging. In view of the real
biological activity of 7-cyL in living mice, we asked whether 7-cyL could improve bioluminescent signals in
the brain. To evaluate the ability of 7-cyL to access the blood-brain barrier, we imaged nude mouse that had
been inoculating with luciferase-expressing U87-Luc cells in brain hippocampus. After two weeks, these nude
mice with tumor xenografts in the brain were injected i.p. with 100 μL of 7-cyL, D-luciferin and
aminoluciferin (1 mM). Significantly, 7-cyL showed 30-fold higher bioluminescence intensity than D-luciferin
(**Figure 5**). It is not difficult to see that 7-cyL can readily cross the blood-brain barrier and
access
deep
brain regions in living mice. Overall, 7-cyL has distinctive advantages in brain bioluminescence imaging.

![](https://static.igem.wiki/teams/5414/content/results-05.png)

**Figure 5.**Bioluminescence imaging comparison of 7-cyL, D-luciferin and aminoluciferin (1 mM) in
brain.
a): Photon flux
from nude mouse inoculated with U87-Luc cells in brain hippo-campus after i.v. injection with 100 μL of 1 mM
7-cyL, D-luciferin and aminoluciferin; b): Maximum bioluminescence emission for mice with 7-cyL, D-luciferin
and aminoluciferin (1 mM) (n = 3). Error bars, s.e.m. \*\*\*\*P < 0.0001 (t-test); c): Time–response analysis of
BLI in nude mouse brain with 1 mM 7-cyL, D-luciferin and aminoluciferin.

# Colleague Evaluation

![](https://static.igem.wiki/teams/5414/content/results-06.jpg)

**Dr. Tony James**

**Professor of Organic Chemistry & Deputy Head of Department**

**Department of Chemistry**

**University of Bath**

<https://researchportal.bath.ac.uk/en/persons/tony-james>

His research interests include many aspects of Supramolecular chemistry, including:

molecular recognition
fluorescent sensor design
fluorescence imaging
theranostic systems
chiral recognition
saccharide recognition
anion recognition
sensors for reactive oxygen species (ROS)
probes for redox imbalance
He received his BSc from the University of East Anglia (1986), PhD from the University of Victoria (1991), and
was a Postdoctoral Research Fellow in Japan with Seiji Shinkai (1992-95). He was awarded a Royal Society
University Research Fellowship at the University of Birmingham (1995-2000) before moving to the University of
Bath in 2000.

He has been a visiting professor at Tsukuba, Osaka, Kyushu and Sophia Universities, an AMADEus invited
professor at the University of Bordeaux and is a guest Professor at East China University of Science and
Technology, Xiamen University, Shandong Normal University, Nanjing University, Shaanxi University of Science
and Technology, Changzhou University, Zhejiang University, Qufu Normal University, Beijing University of
Chemical Technology, Shanghai Normal University, Ewha Womans University, Henan Normal University, Engineering
Research Centre for Hainan Bio-Smart Materials and Bio-Smart Devices, and is a Hai-Tian (Sea-Sky) Scholar at
Dalian University of Technology.

He has wide-ranging experience within the field of supramolecular chemistry having published over 455
publications, including 3 books, 9 book chapters and 443 papers in international peer reviewed journals. He is
also the named inventor on 25 international patents. He has delivered 256 invited lectures within the UK and
internationally. Citation statistics indicate that one of his publications has been cited over 1,000 times,
four over 500, nine over 400, eighteen over 300, twenty-seven over 200 times, forty-eight over 100, and 132
over 50, with a total of > 25,197 citations at a frequency of >55 citations per article. He has an h-index of
86 (ORCID 0000-0002-4095-2191).

He received the Daiwa-Adrian Prize for developing scientific networks with Japan in 2013, the inaugural CASE
Prize for establishing scientific networks with China in 2015, the MSMLG Czarnik Award in 2018, the Frontiers
in Chemistry Diversity Award in 2020, a Royal Society Wolfson Research Merit Award (2017-2022), is an Ewha
Womans University Global Fellow and is listed by Clarivate as a Highly Cited Researcher in Chemistry (2022).

![](https://static.igem.wiki/teams/5414/content/results-07.jpg)

{% endblock %}