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Villanueva, Dalton State University (ovillanueva@daltonstate.edu), and Laurel G. Habgood, Rollins
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Antibacterial Reactivity of Ag(I) Cyanoximate Complexes
Adapted from:
Light Insensitive Silver(I) Cyanoximates As Antimicrobial Agents for Indwelling Medical Devices
Inorg. Chem. 2010, 49, 9863-9874
(DOI: 10.1021/ic100830x)
Learning Goals:
After this laboratory experiment, students will be able to:
1.
2.
3.
4.
5.
6.
Prepare a series of Ag(I) cyanoximate complexes and perform appropriate characterization
Measure antibacterial activity in semi-quantitative way
Design and implement approximate controls for antimicrobial assay
Identify most cost effective additive
Predict possible coordination modes using coordination geometry
Express conclusions using tables
Introduction:
Many issues are associated with the rapidly expanding group of U.S. citizens known as the “Baby
Boomers.” Health issues are one of the primary concerns of an aging population and associated with that
is the increasing practice of joint replacement especially of the knee and hip. The use of dental implants
is increasing as those procedures have become less expensive and more practiced resulting in more
implants and fewer uses of removable dental appliances.
Infections may arise from the introduction of implants into the human body due to the presence of
bacteria such as Streptococcus mutans which is a primary cause of dental caries. Other bacterial sources
may be introduced during hip and knee replacements. Bacteria introduced into the body in these
procedures are very difficult to treat with standard systemic administration of antibiotics so it would be
best to introduce some type of antibacterial agent along with the implant or incorporated into the glues
used to secure the implant. You may be familiar with the addition of silver nanocrystals in athletic
clothing to reduce bacterial growth and odor in those applications. Implants, however, require the
antimicrobial agent to be stable at temperatures of sterilization around 100℃ or higher and also be waterinsoluble and light stable, but still impart antimicrobial activity. Cyanoximes with the general formula,
HO-N=C(CN)-R bonded to various metals, specifically Ag(I) represent a class of compounds with the
desired properties. The R-group may be a wide variety of substituent groups, such as heterocycles,
alkanes and aromatic moieties.
Your opportunity in this lab is to select one of four possible R groups, synthesize the cyanoxime and then
react the cyanoxime with silver to form a new complex. Upon successful synthesis, you will then
characterize your compound utilizing standard spectroscopic and melting point techniques. Furthermore,
you will need to test your compound for heat and light stability (what do you know about silver
compounds?) and determine solubility in water. Remember, these compounds will be used under
physiological conditions so chemical stability will be important. Why? Finally, you will need to check
your compound for antibacterial activity. After you have conducted all of your synthesis, characterization
and antibacterial testing, you will present your findings to your instructor in the method your instructor
reated by Kari Young, Centre College (karin.young@centre.edu); Kristy Stensaas, Millsaps College
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Villanueva, Dalton State University (ovillanueva@daltonstate.edu), and Laurel G. Habgood, Rollins
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requires. Make sure your report contains sufficient detail such that your work could be reproduced by a
technically trained person. And, congratulations! You may have made the next wonder drug!
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Timeline:
Period 1 - Synthesize ligand
Period 2 - Characterize ligand. Metalate and set up for rerystallization
Period 3 - Collect and characterize metal complex
-Melting point test for heat resistance
-Qualitative test for solubility
-Set up Antibacterial test: make disc (and control experiment)
Period 4 - Collect antibacterial test data.
-Gather results from classmates.
-Calculate most effective complex per dollar
Procedure:
Part I: Synthesis Ag(I) cyanoximate complexes (Period 1 and 2)
Step A: Cyanoxime ligand synthesis H(1-4) using the Meyer Reaction under acidic conditions






Add 20 mmol of acetonitrile precursor and 22 mmol solid NaNO to a 50 mL 3-necked round
bottom flask.
Add 20 mL of distilled water and sparge the solution with nitrogen to remove air.
Cool the solution to 0°C.
Add 30 mmol of glacial acetic acid dropwise over one hour using a syringe.
After acetic acid has been added, allow reaction mixture to sit at room temperature for one week.
To work up, acidify with HCl to pH ~3 and saturate the solution with solid NaCl. Extract the
aqueous layer with 3x10 mL of diethyl ether. Rotovap combined ether layers to dryness.
2
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Once you have purified and isolated your cyanoxime ligand, collect characterization data before
carrying out the metalation with Ag(I). Characterization of organic ligands will be carried out using H,
C NMR spectroscopy (solutions in DMSO-d ), FT-IR spectroscopy, melting point and Thin Layer
Chromatography (TLC). Before obtaining characterization data of your ligand, you should gather the
characterization data of your cyanoxime ligand from the literature. Check and discuss with your lab
partner to check the validity of your data collected. Share the characterization data of your ligand with
your instructor before continuing to the metalation with Ag(I).
1
13
6
Step B: Cyanoximate synthesis of Ag(1-4) (Period 2 and 3)
Metalation of each corresponding cyanoxime ligand will be carried out using the following procedure:



In a 100 mL round bottom flask, add about 0.500 grams of your assigned cyanoxime ligand and
dissolve it in a mixture of 10 mL ethanol (EtOH) and 10 mL of distilled water.
Heat the cyanoxime ligand mixture to +50 ∘C.
In a separate flask, dissolve 0.5 equivalents (with respect to your cyanoxime ligand) of potassium
carbonate, K CO and dissolve in 10 mL of distilled water (make sure your stoichiometric ratios
of ligand to K CO are as close to 1 to 0.5, respectively, as possible).
Add the hot cyanoxime ligand solution dropwise to flask containing the aqueous solution of
K CO .
The reaction mixture will change colors immediately. Be sure to record all your measurements
and observations in your laboratory notebook.
Place your reaction mixture for about 2 minutes into an ultrasound bath to accelerate the
evolution of CO If an ultrasound bath is not available, allow the reaction to run for longer time
(about 20-30 minutes).
In a separate flask, dissolve 1.0 equivalents (with respect to your cyanoxime ligand) of silver
nitrate, AgNO , in about 10 mL of distilled water.
Add the aqueous AgNO solution to the round bottom flask containing your cyanoximate ligand
and the K CO mixture. Allow the solution to undergo intensive stirring for about 20-30 minutes.
After 20-30 minutes of stirring, collect the colored precipitate in a vacuum filtration set up, wash
with three portions of 10 mL of distilled water, and allow to dry for at least 15 minutes.
 Note: Silver(I) cyanoximates with ligands have a 1:1 stoichiometry and are poorly
soluble in aqueous/alcohol solutions and thus can be conveniently separated from the
reaction mixture. Be sure to collect as much of the colored solid as possible for the
crystallization step.
2
3
2

2


3
3
2.

3

3
2
3
Crystallization: You (and your laboratory partner) will attempt to recrystallize your silver(I) cyanoximate
complex. Carry out a solubility test with any donor solvents available and make a hot saturated solution
of your silver(I) cyanoximate complex. Allow for slowly cooling (at room temperature) and slow
evaporation of solution until next lab period.
o Read more about crystallization to attempt various methods for crystallizing your
silver(I) cyanoximate complex. http://www.iucr.org/education/teachingresources/crystal-growing
Next lab period, collect your crystalline solid using a vacuum filtration, and rinse with 10 mL of distilled
water. Dry under vacuum and obtain a percent yield.
Characterize your complex by H NMR spectroscopy (solutions in DMSO-d ), FT-IR spectroscopy.
1
6
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Villanueva, Dalton State University (ovillanueva@daltonstate.edu), and Laurel G. Habgood, Rollins
College, (lhabgood@rollins.edu) and posted to VIPEr on August 22, 2015. This work is licensed under the
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reated by Kari Young, Centre College (karin.young@centre.edu); Kristy Stensaas, Millsaps College
(stenskl@millsaps.edu); Willis Weigand, Southwestern University (weigandw@southwestern.edu); Omar
Villanueva, Dalton State University (ovillanueva@daltonstate.edu), and Laurel G. Habgood, Rollins
College, (lhabgood@rollins.edu) and posted to VIPEr on August 22, 2015. This work is licensed under the
Creative Commons Attribution-NonCommerical-ShareAlike 3.0 Unported License. To view a copy of this
license visit http://creativecommons.org/about/license/.
Part II. Applications (Period 3 and 4)
Heat and Light Stability Studies
For your compound to perform in its application it must be heat and light stable and be relatively water
insoluble. You should consider what defines heat and light stability in your application and how you
might test for heat and light stability. Think about the physiological environment in which your
compound will exist and what might be the minimum and maximum limits of heat and light
stability. Conduct a melting point on your compound and use that information to help you determine the
heat stability of the complex.
Aqueous Solubility Studies
All compounds are soluble to some extent in water. Chemists typically use K values to provide some
sense of solubility of a compound. You may want to consider if there are ranges of water solubility that
could be tolerated or what might happen if your compound eventually completely dissolves? What if it
permanently remains at the site, might that cause problems?
sp
Antibacterial properties
The antibacterial properties of your silver compounds will be tested using the Kirby-Bauer
technique. You will infuse a piece of filter paper with your compound, and place that piece of paper on a
petri dish growing e. coli bacteria. It is very important that you use proper aseptic technique to avoid
infection.
Assay for antimicrobial activity
1. Prepare 5 mL of a ~10 mg/mL solution of your complex in dimethylformamide (DMF). Weigh
your complex using the analytical balance and prepare your solution in a volumetric
flask. Determine the actual concentration.
2. Pipette 20 uL (Or add one drop) of your Ag(I) solution onto a 12 mm filter paper disk and allow to
dry.
3. Your instructor will provide a petri dish containing agar growth medium on which e. coli. bacteria
are growing. Using aseptic technique, place your dry filter paper disk on the plate and replace the
cover. Label the cover with which complex you are testing, your name, and the date.
4. Incubate the plate at 37°C. During the next lab meeting, measure the diameter of the growth
inhibition zone in millimeters where the opaque bacteria are no longer growing.
Control experiments
One important issue to keep in mind is to have control experiments so that you can determine only the
effects of the complex. You need to consider what other factors may be interfering with your
determination of antibacterial activity. How do we know that your complex and not some other aspect of
your procedure is responsible for the antibacterial activity?
1. Design at least two control experiments to be approved by your instructor. Add them to your
petri dish. Be sure to label above the dish an identifier that lets you know which piece of filter
paper is which. Measure the growth inhibition zone.
Data organization
1. Collect the growth inhibition data for each of the complexes and control experiments that were
completed for all students in your laboratory.
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2. Prepare a table showing the average and standard deviation for experiments that were replicates.
3. Determine which complex is the most effective for bacterial growth inhibition.
Cost Analysis
The dollar costs of the reagents used in this procedure are listed below (as of August 2015). Calculate the
approximate cost of the different complexes you prepared. Use this information to determine which
complex is the most effective per dollar.
Product
CAS
Amount Price
malononitrile
109-77-3
100 g
22.60
cyanoacetamide
107-91-5
100 g
24.00
ethyl cyanoacetate
105-56-6
250 g
41.60
4,4-dimethyl-3-oxopentanenitrile 59997-51-2 100 g
168.50
benzoylacetonitrile
614-16-4
5g
44.10
2-pyridyl acetonitrile
2739-97-1
5g
42.90
sodium nitrite
7632-00-0
500 g
65.60
acetic acid (glacial)
64-19-7
2.5 L
99.70
hydrochloric acid
7647-01-0
2.5 L
92.00
sodium chloride
7647-14-5
500 g
37.40
potassium carbonate
584-08-7
500 g
73.70
silver nitrate
7761-88-8
25 g
116.00
ethanol
64-17-5
1L
112.00
acetonitrile
75-05-8
1L
108.50
pyridine
110-86-1
1L
164.00
2-picoline
109-06-8
1L
57.50
dimethyl sulfoxide
67-68-5
1L
91.50
Part III. Post-lab Directions
Option 1: Communicate your results and interpretations in the style of a paper to be published in
Inorganic Chemistry, the flagship ACS journal in this field. Gather data from your classmates for each of
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the compounds listed above. In order to acknowledge their contributions to your report, you will list them
as coauthors, but put your name as first author. Give your report a catchy and informative title. Your
report should include the following parts:
a. Introduction: In this section, you will pave the way for the experiments that will be reported in the
body of your report. You should find at least one literature source not already given to you to
contextualize the complexes you prepared within the rest of chemistry and prepare your audience for
the topics you will discuss. Certainly one way to do this is to center your discussion around the need
for light- and heat-stable antimicrobial complexes for dental implants, but there may be other
ways. The important thing is that you impart some motivation to your work and highlight important
points that you will want to call on later.
b. Experimental: You should write an experimental section in the format of Inorganic Chemistry for
each of the compounds you discuss. This is often the best section to write first. You should also
gather this information for the compounds you did not prepare from someone else in the
class. Acknowledge their contributions by making them co-authors on your paper. Be sure to report
yields and spectroscopic results. This should be in the style of the experimental section in Inorg.
Chem. See example below:
From Mantel, C.; Baffert, C.; Romero, I.; Deronzier, A.; Pécaut, J.; Collomb, M.-N.; Duboc,
C.; Inorg. Chem., 2004, 43 (20), pp 6455–6463
Synthesis of [Mn(terpy)(SCN) ]. To a stirred solution of terpy (50 mg, 0.214 mmol) in 3 mL
of methanol was added Mn(CH CO ) ·4H O (52.4 mg, 0.214 mmol). The resulting yellow
solution was stirred at room temperature for 10 min and filtered. A saturated solution of
Bu N(SCN) in 5 mL of methanol was added to the yellow solution, and a precipitate was
formed. It was filtered, washed with a small amount of ethanol, and dried in vacuo. Yellow
crystals of 4 were obtained at room temperature by slow evaporation of the yellow precipitate
dissolved in a mixture of CH CN/DMSO (90/10). Yield: 0.082 g (95%). Anal. Calcd for
complex 4 (C H MnN S (404.37)): C, 50.50; H, 2.74; N, 17.32. Found: C, 50.74; H, 2.73;
N, 17.31. IR (cm , KBr): ν = 3059 (m), 2061 (vs), 1596 (s), 1574 (m), 1477 (m), 1452 (s),
1437 (m), 1313 (m), 1245 (m), 1164 (m), 1015 (m), 777 (s), 660 (m), 650 (m), 639 (m), 483
(m), 400 (m).
2
3
2 2
2
4
3
17
11
5
2
-1
c. Results: In this section, you will present and assign your data without interpreting its significance.
Gather the antimicrobial data from your classmates and compute the average and standard deviation
of any replicate measurements. Your results section should include a master data table containing
your results, and you should also summarize your results in words. Do not, however, feel like you
must have a sentence for every spectroscopic signal or every measurement for every
compound. Generalize. Note trends.
d. Discussion: In this section, you will interpret the larger significance of the data you presented in the
results section. What conclusions do you draw from your data? Which is the best Ag(I) complex and
what criteria did you use to evaluate it? How do your data address the issue that you described in the
introduction? Do your data compare to the data presented in the literature? Characterization should
include evidence of both identity and purity. Are the characterization techniques used in this
experiment sufficient evidence that the correct organic ligands and silver complexes were made?
What other characterization techniques that are available to you could you perform on your Ag(I)
cyanoximate complex?
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e. Conclusion: This is often a difficult part because you may feel like you have already said
everything you could possibly say on the topic. Consider the reader who lacks the patience to read all
of the glorious details of your report, but just wants the take-home message. Restate the two or three
big points—the “spoilers” if you will.
Option 2: Prepare a 6 minute oral presentation communicating your results with an accompanying
PowerPoint (or Keynote or Prezi) visual aid. Estimate about 1 slide per minute. You should introduce
the topic and the goal of your study first. Then, explain your general experimental strategy. This is a
short presentation, so you do not need to belabor your synthetic strategy. Then, present your data using
charts or graphs that you interpret as part of your explanation. Use your interpretations to justify your
conclusions about which is the best Ag(I) cyanoximate according to your criteria.
Option 3: Prepare a poster appropriate for presentation in an inorganic chemistry session at a chemistry
conference. Posters are designed to communicate the same information you would receive in a paper, but
quickly and graphically. The star of a poster is your results, which should be front and center. Microsoft
PowerPoint is a good tool for preparing a poster, and templates can be found online. (For example:
http://www.posterpresentations.com/html/free_poster_templates.html)